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MICROBIOLOGY
-is a science that deals with the study of microorganism and their activities. It is concerned with
their form, structure, reproduction, physiology, metabolism and identification. The science also
includes the studyof their distribution in nature, their relationship to each other as well as to other
livimg things, their beneficial and detrimental effects on men and the physical and chemical
changes they make in their environment.
SCOPE OF THE MICROBIOLOGY:
1. Bacteriology- concentrates on the study of structure functions and activities of bacteria
2. Phycology- is the study of various type of algae
3. Mycology- is the study of fungi
4. Protozoology- is the study of protozoa and their activities.
5.Virology- encompasses in the study of viruses and their effect on living cells.
6. Immunology
Virologist and cell biologist may become genetic engineers who manipulate genetic
materials (DNA) from one cell to another.
Prions and viroids are infectious agents smaller than viruses.
Division of Microbiology
1.GeneralMicrobiology- the study and classification of microorganism and how they function. It
encompasses all areas of microbiology.
2. MedicalMicrobiology- involves the study of pathogens, the disease they cause and the body
defense against disease.
3. VeterinaryMicrobiology- study of the spread and control of infection disease among animals.
4.AgriculturalMicrobiology- studies of both beneficial and harmful of microorganism in soil
formation, fertility and disease in plants.
Food microbiology- concerned with the production. Processing, storage serving of food,
prevention of food spoilage, food poisoning and toxicity.
Dairy microbiology- oversees the grading, pasteurizing and processing of milk and cheeses.
5. SanitaryMicrobiology- includes the processing and disposal of garbage and sewage wastes and
purification of water. Inspect food processing installations and eating establishments.
6.IndustrialMicrobiology- deals with the proper growth and maintenance of certain microbes to
produce beers, wines, alcohol and antibiotics.
7. MicrobialPhysiologyandGenetics- deals with the study and structure of DNA ang genetics.
8. EnvironmentalMicrobiology or microbialecology- encompasses the areas of soil, air, water
sewage, food and dairy.
vaccine for
4. Modern vaccines are prepared from isolated components of pathogens and by recorbinant
DNA techniques.
Birth of Modern Chemotherapy: Dreams of a magic bullet
1. Chemotherapy is the chemical trertment of a disease.
2. Two types of chemotherapeutic agents are synthetic drugs (chemically prepared in the
laboratory) and antibiotics (substance produced naturally by bacteria and fungi to inhibit the
growth of microorganism.
3. Paul Ehrlich introduced an arsenic containing chemical called salvarsan to treat syphilis.
(1910)
4. Alexander Filerniag observed that the mold penicilliumnotalium inhibited the growth of a
bacterial culture. He be name the active ingredient penicillin.
5. Penicillin has been used clinically as antibiotic since 1940s.
6. In 1939, Rene Dubos discovered two antibiotics produced by bacterium Bacillus.
7. Researches are tackling the problems of drug resistant microbes.
8.Domagle- prohtosil
Development of Microbiology
1. Bacteriology is the study of bacteria, mycology is the study of fungi and parasitology is the
study of parasitic protozoa and worms.
2. The study of AIDS, analysis of the action of interferon and the development of newer vaccines
are among the current research inherent in immunology.
3.New techniques in molecular biology and electron microscopy have provided tools for the
advancement of your knowledge in virology.
4.The development recombinant DNA TECHNOLOGY has helped were all areas of
microbiology.
CLASSIFYINGTHE MICROORGANISM
1.In a nomenclature system of designed by carolusbuinaeus (1935), each living organism is
assigned two names.
2. The two names consist of genus and a specific specie both in which are underlines and
italicized.
THE DIVERSITY OF MICROORGANISM
1. Bacteria are unicellular organism beacausthey have no nucleus prokaryotic cells.
2. The three major basic shapes of bacteria are bacillus coccus and spirili.
3. Meat has pychoglycan cell wall, they devide by binary
4.. cell wall is made up of chitin.
Protozoa-priotine.
1. are unicellular and rytes and are classified according bto their
2.obtain nourishment by absorption or ingestion through specified structures.
Algae- is isolation word for seaweeds
1.unicellular and multicellular eukaryotes that obtain nourishment by photosynthesis.
2. produce oxygenandcarbohydratesthatare used by oraganism.Cellwall is made up of cellulose.
Viruses
1. Non cellular entities that are parasites of the cells, no capacity to synthesized their own food.
2. consist of nucleic acid (DNA or RNA) surrounded by protein coat and envelope may surround
the coat.
Multicellular Animal Parasites
-These are flat forms and the round worms called Helminthes.
USES OF MICROORGANISM
1. Microorganism degrade dead plants and animals and recycle chemical element to be used by
living plants and animals.
2. Bacteria are used to decompose organic matter in sewage.
3.Bioremedation processes used bacteria to clean up toxic wastes.
4. Bacteria that cause disease in insects are being used as biological controls of insect pest.
Biologicalcontrol are specific for thepestiside harm the environment.
5.Using microbes to snake products such as foods and chemicals is called biochenolopy.
6. Using recombinant DNA, bacteria can produced important substances or missing genes into
human cells.
8. Genetically engineered bacteria are used in agriculture protect plants from host and insects
that shelf life of produce.
hAaerc-nodpygtil w.
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CHAPTER1
THEGNRALCSIOFB
Bacterimnu,logshpdbyarif.
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BACTERZLSINDFOM
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mhopeiluas-t
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2.Cociu(ns,glar)-phedm
3.Spirla(sumn,g)-edochplrivyabsgmndote.hrical
ocbail-E.Lstermyng i
nAmgraet
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singly-oaremt
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1)Planeofdivs
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2n.Chsai-trepco,blSyTBdhia
3p.Gelikracust-ohyS
4.Groupfd-tegsa(Pc)yn
5k.Petsacofiguhb-dl(nSr)
6.Palisde(png)-ormthcvbyside
g.(eyConbractium)
7n.Chesiarctp(g)-omdbnif
bacilus-rget
vdison.exCbyuactrmhp
OpticalhModes
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Theligtmocrpsundav wbeofhtlngiduR.sv(powherbtlyangidjcs.)hWotwelfavng4u0mi ocrpysetlax0.2umThfgnicorpsetadubigylnmo90pwerjctvhsa1uln,mygifpec90ts.Par2undmhfoegibt0.asmcylrveFuhgniftowdarsleucthbvi(fd)Barlsenoyutpdgicrlqnoasehtfivrcxd.
PhaseContrMicpy
Itisahfcglwvepnrou bjt,ascmgidfephnortsmalugwhiceyp.Wotsm,rcnvehdifpoastmucerpadknthos.vIlgreifabctuvnls.
kDdfiearMlocpys
hInistecqupmobjralyingtedsckou.Aparbldietmncsghauoidtelrn,whbjcvs.Titequanfylodmrhpisetc,wafulbvymndgh.
(2)
TheElctronMips
Bacteriluswdmonyhfvpetrasiolcnm(TEMh),wvegpbroufa0t1.m2sihlcpneTraotmsiydvlgufrheaboctnsiy(pgw)merdhltconb.aSpisufx,edTrhntalovibcesu zdythrowangivespcu.Tmfrolditnhgaye bjcptaofmelnisvcu.Thbdrtoqy,aejcsui"hwntrmfoades.Whlcnbmitparougedshlcnmipaotverdfg" h,nimsoalectdv.Ng uronsehyamtli,cpugohsaermnybdlt,ifcwuohesampvrdltn,yfiuoces.
Otherimpnoaqculsdf mbeatrnizgospcuwhdyelqnitr(fzg)cpvsdoauenbytilgr.
ThescanigomrSpEM()tlfunab0.5m,orivdesthfTEMSpucarm-doenisglbyt90afcdroensmi.thpualfbordenymict.a
oglynxc-"suarteEPS() H.inflz-mgts
yposachlride n
BACTERILSUh.anrtcisD-glumd
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oFunctis:
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h2.wmityulerdapong0c(-6)."sit
pPtidoengly-camsfhr uito
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o1n.csitdpfgely,arumbohscide
layers,
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pCylasmt-eiquodrnghc
-H20,enzymOCpidls
(4)
oCyplasmticMebrn
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v-serathconl
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FUNOCTSI
1.vSelctiprmyabndsouf
2.Electonspradvxihy,bec.
3.oExcretnifhydlzms
4.Bearingthysmzdcuolfebhnist
ofDNA,celwaypmrsndbi.
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osenrydtaucim.BlDNA-gerof
deribnlstaofDNAcpm.
NUCLESPlasmid-exorch unwt s
Transtio-mRchegl(f)
thegnomribs.
Thoepkayrticunsl mbdpar
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egsintakbymohr.
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of2apritebclns
CYTOPLASMGIRNUEu(nsiocl)
Moetachmriuvnlgsd fer.
Examples: o*ribmse-tf
1.Babes-ErntgulofCycimhdp snet
2.Muchgranlesofybti pkc-70Srsme
bRosmipe-y"rltfCHONnhukac80S"
heryomtnaciu50Sbs
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SPOREND rate
yHighresltanucopdb Bis
nhdaetrobicgusClm,w venromentalodcisryf,hpu.Tetanc
bisatduroehynpcflmiate.
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foprmulictan. bRse-dofprna
BacilusndoCtrm ehfu7ano0.(8st)
ClasoifcntdAgr . (6)
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PilorFmbae
ing-bacter
Hairlkebmcofusypdgtnaive
bacteriosvlynmp.
onglerTypsadFucti:
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another. (3)
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hpnogaticebr.
Parts: CloifcndAgNumbeatM:s(')7
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arouhndetlw3.Ampics-gfaoeuSrl
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aspeci u5.FnorltbdyhqAT()
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axilnfmtse-gp
CMROISPEXANTFB
nLivgStae
A.DirectWMounbslhfamgy
1.Spnecimkluts,xdafro nvglische
ndayurisemtclpohf.
(8)
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o3.Avcerslipadthmnwxquobfre
dgehswaitu.
B.nHgaodihpmretbsvfly
1.Adropfliqutcesanhv
. p2Aecisaldwthonr u
3.Thpedrsiongwtaflmuhe
dupesiownvrthclP.(mjy)
4.Thendtirslowvcpyquk hagnetdrofl
ohmfretvcslipndw .
dFixSeta
nExamtiofsedrlhu pvmtinofca
ofbacteri.
PrepationfSm
1.Materiolbdsnpq(fu)weohtsrac
cleany,dgrsi.
2.Asterilndopymubaf ltchreso
slidhewcntmufarop slie.
3m.Thearsoidwltny
(9)
4h.Itiaxeydfrnbpgslou amevrtis
orbyvelingwth95%macf1u.
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Stainsrecomdpf+-.Thl ore
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n+ioacd"-
NSTAIG
Theprocsfatilyngm wders.
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Ahuxormce(sp.ftaing!)
Purpose:
1.Tovbnesrpdacith fmg.
2.Todiferntamcsgup onther.
p3.Hnselthidfcaomrg uset.
dMouanrhbt-sxwilfe
Clasoifcnt:
A.Simpnlest-auzgyodr hcsmeatofy.
(egy.Cstalrvio)
qeou-asrlc.ntifgbdy 10()
B.nDifertalso-cmhdyvertpswianuc
diferntaobylcswhp(uvgerintos)
1.TheGramnS-tiflxosuBcg
Thnisdtferalvmobcpwughs:taek
hetbasicdy,rlovwu zm(g-natie).
nGeralRus:
a.oAlciregmvpxstN,VnadBhel.
b.AlaciregmntvpxoMy,CbiadlsrBcu
Listera,yEopxhlcbAnmu,Priate
Mobuilns,cBfd-aterm
c.oAlsphiretagmnv
dProuce:
a.Stindfxsmerwhcyolvp()1uit.
bh.Wwasiter
c.dMoanrtwihGm'slue(ybkanodhtcwl)
or1fminute.
dh.Wwasiter
e.oDcrizlhw95t%aumxf(70nd)ceo3lr
10-3second
f.hWwasiter (1)
g.Countersaiwhf10-3cd
h.Wwasiter,ndyxmuol.Mcbaterikwyg+
oSpchirets-g
RESULT:Gram-positvelu Myamcn-o(ewl)
mGra-negtivpkod heg+roThriesnGamStRc
a.gMunmesiRboclAdThry
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ompcundytreasilbh.
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subthmadgeclnrpkiosydatge.
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b.nBiaeoThry
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mgrapositvebcnlhx.
c.Snteardsohy
hTyeoistnrbadlcp.Gmvihe
lowerisctnphmad boreyfiwlt
basicdye. 12)(
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whicupontreaml dsciperbmyileatofhcwsunkd t.
e.hTociadnt
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positvebacrymlnhdgofstvie.
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a.mRovelfMgnbypitrcwhs
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c.AnaidusotlgGrm'e
dh.Tnecialrov(zt)
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a.oInmpcletdriz
bk.Thsmicear
pRidaNon-StmyseDrTuGRc
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toudcheanylfgrmvib
b.3%KOHTest
2.TheAcidFastSn
Adiferntalsmpoyhguc frn
acidfst.
nPciprle:
dAfacigstomnryevh,bu adto
odlecriz.Thanfstupomylcdrhx
hmoxetyacidwslngf50(-9rbmt.
Cobrynacteihs fdougntrem
acidfst,Norpenmxly50bashctre
slightyacdf.Mobervn mliacdfst
adistnocperyhwmkf.
1%HNOo3dlecriz
1%H2SOo4dlecriz
hToiceadtgsnrm,p
orfsedgiclntampy. 14()
Gram+ Layers3l
10%5-dpeoti
pidl -LPS
oncte
nodexit
Listera
porin hwt
bvulnerasitoyzm
penicladtk
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a.Stemingprocs
nbcre.Iasigoth fpdlu
c.oPrngiltheafswm
do.Anitfwegas(rl)hu,
hModetsfAciFaSng
a.hZlienNsMtod(gpcur)
dProuce
i.mFxeasrbyht
i.Coverwthucabnlf,smgy5 die.
i.hWwaster (15)
ivo.Dlecnrzadhuytfpkmis.
vh.Wwasiter
vi.Countersa10f-3cdwhmylb
vhi.Wwaster,ndyxmAcfogpa
rednagstiubkloc.
b.nKyoui'smethd(cl)
dProuce:
xi.Fsmearbyht
i.Sntao(hecsry)wkublf
i.nCoutheaswZl-Nmdri
Malchitegrnd.Afsomp kbuacn.
c.pPnaheim'sModt
ThismaybdeutonfrgM. lcsi
gM.smeati(lco)drzhbyxusfani
oalhcringtbuewM.sdzlaminr
d.uBmgartenh'sMo
ThdisutoefrnaM.blcmp is
easilyntdbucohfgrwileM.ts
doesnytrakiluph b.
Specialtns-duomhzr faen(16)
norgismaS.pe(dxA)
1.CelWnaSti
a.DyhrModet
2.pCsuahlrModet
a.HhisModet-fnubcpl
bn.Gh'sMiodet-rga
c.hAnoyt'sMdeu-apligrkbcn
dh.W'selcMot-buap\vi
e.Tylr'smthod
f.Muir'sethod-caplb,
gd.Whswoa'rtme
hN.Mealc
i.wLsona
3. Stain for Metachromatic Granules
a. LAMB ( LoefflersAlk. Methylene Blue)- deep blue granules, light blue bodies MB+KOH
b. Neissers
c. Alberts- blu black granules, cytoplasm green
d. Ljubinsky Method- deep purple granules, brown bodies
e. Gohar
4. Spore Stain
a. Acetic Acid Method
b. Dorners Method- 10% nequosine- red spores(carbolfuchsin), colorless background
c. Wirtz and Conklin
d. Schaeffer and Fulton -(most commonly used)are green and safranin as counterstain
(17)
5. Flagellar Stain
a. Grays
d. Casares - Gils
precipitate with
e. Van Ermengens
the flagella.
D. Indirect/ Relief/ Negative Staining a staining process in which the microorganisms appear as
bright colorless objects against a dark background.
1. India Ink (Burris Method)
2. Nigrosin Method
Mordant
Decolorizer
Alcohol/aceton
e
Acid alcohol
Acid alcohol
Counterstain
Positive
Negative
Safranin
Purple
Pink
Methylene
blue
Km0
Pink
Blue
Orangefluoresc
e
No fluoresce
Rhodamine
Calcofluor
10% KOH
Bluish
white No fluoresce
with fluoresce
(18)
CHAPTER II
BACTERIAL GROWTH AND CULTIVATION
Bacterial Growth- increase in bacterial numbers nor increase in the size of individual
Growth is the orderly increase of all chemical constituents of the cell. A process that entails the
replication of all cellular structures, organelles and protoplasmic components from the nutrients
present in the surrounding environment. Under favorable conditions almost all bacteria are able
to reproduce rapidly. The average time required for an organism to double its number is referred
to as the generation time or doubling time. ( e.g., E. coli 20 minutes)
Binary Fission divides into 2 equal parts. Steps:
1.
2.
3.
4.
GROWTH REQUIREMENT
Nutrient Requirements
A Carbon- is the structural backbone of living matter
Classification:
1. Autotrophs (lithotrophs)- require only water, inorganic salts, and carbon dioxide for
growth; they dont require organic nutrient for growth.
2. Heterotrophs (organotrophs)- require an organic form of carbon for growth from CHON,
CHC
a. Parasites-requires living organism matter for growth.These includes pathogens.
b. Saphrophytes- those which live dead organic matter.
B
Nitrogen
Nitrogen is a major component of proteins and nucleic acids of a typical bacterial cell. The
end product produce it from amines.
Sulfur- used to synthesis S containing a vit.like thiamine
Phosporus- essential for the synthesis of nucleic acid and phospholipids
(19)
C.
Growth Factors
Growth factors are organic compounds necessarily needed by a bacterium in order to grow.
These substances are usually provided in the culture medium.
Classification:
1. Prototrophics- do not require an exogenous source of growth factor since they synthesize
their own.
2. Auxotrophics- require the addition of growth factor to culture media for growth to occur.
Examples of Growth Factors :
1.
2.
3.
4.
5.
6.
B- complex vitamins
Amino acids
Purines
Pyrimidines
Pentoses
Fatty acids
F. Carbon Dioxide
Some organisms such as Neisseria, Brucella, etc. require for growth a higher concentration
(3-10%) of CO2. These organisms are called capnophiles.
G. Moisture
This is indispensable for bacterial growth. It serves as a solvent for food and forms the major
portion of the protoplasm. Organisms requiring an increased moisture content are termed
humidophilic.
Physical Requirements
A. Temperature
Every has an optimal temperature, the temperature at which the organism grows best.
Classification:
1. Psychrophilic (cold-loving)- grows at 10-20C
2. Mesophilic- grows at 20-40C (most pathogens grows at 37C)
3. Thermophilic (heat-loving)- grows at 50-60C
4. Thermodiuric- important in organic composed piles.
Minimum growth temperature- lowest temperature at which the specie will grow
Optimum growth temperature- the temperature at which the specie grow best
Maximum growth temperature-highest temperature at w/c growth is possible
Refrigeration- slow down growth of most spoilage microorganisms and will entirely prevent the
growth of all
Freezing- no significant growth
(21)
C. Osmotic Pressure
Organism requiring high osmotic pressures are called osmophilic.
Plasmolysis-osmotic loss of H2O(shrinking of cells plasma membrane)
(22)
Temperature
Dessication
Lights
Electric current
Agesetation
Pressure
Vibration
(23)
BACTERIAL CULTIVATION
THE CULTURE MEDIUM
A culture medium is any material containing the necessary nutritional and environmental
requirements for bacterial growth.
CLASSIFICATION OF CULTURE MEDIA
A. According to Physical State and Consistency:
1. Agar- red algae polymer (derived from a marin algae)
2. Liquid- without any trace of agar or solidifying substances
3. Semisolid- contains 0.5-1.0% of agar (e.g, SIM)
4. Solid- contains 2-3% of agar (e.g, BAP, CAP, TSI)
B. According to Composition
1. Synthetic- exact chemical composition of the ingredients is known (e.g, commercially
prepared culture media).
According to Use:
1. Simple or Supportive- contains nutrients that allow most nonfastidious organisms to grow
at their natural rates, without providing a growth advantage to any particular organism ;
utilize only for routine cultivation of microorganisms ( e.g., nutrient agar, brain heart
infusion agar)
2. Enrichment- designed to enhance the growth of particular organisms( usually pathogens
and suppress the growth of normal flora in a specimen (e.g.,
Selenite, GN, and Tetrathionate Broths)
Sheep Blood Agar preferred blood because it will give a good hemolytic creation.
3. Differential- distinguishes organisms growing together by their differences in cultural
characteristics (e.g., Blood agar)
CAP- fastidious isolates hemo
BAP- most isolation protocol
4. Selective- contains one or more agents inhibitory to all organisms except the organism
being sought (e.g., bismuth sulphite agar, phenylethyl alcohol and Salmonella-shigella
agar)
Some of the inhibitory agents added to media:
a.
b.
c.
d.
e.
f.
g.
5. Selective and Differential- some media are both selective and differential like the media
used in the primary isolation of enteric gram negative organisms (e.g., Endo agar,
MacConkey agar, XLD agar and Eosin methylene blue agar)
6. Special-specially prepared to support the growth of specific microorganisms
Examples:
a. Middlebrook 7II-10 agar M. tuberculosis
(25)
b. Fletcher medium Leptospira
c. W medium Brucella
d. Bordet-Gengou agar Bordetella pertussis
e. Thayer-Martin Neisseria
f. McBride Listeria monocytogenes
g. Dieudonnes medium V. cholera
General Steps in Preparation of Culture Medium (Tubed Medium)
1. Weighing weigh the different ingredients and then place in clean, dry containers
2. Dissolving- add the exact amount of solvent to the ingredients and then dissolve by
heating
3. Titration- adjustment to the right ph (Ph7.2)
4. Distribution- distribute in the test tubes
5. Sterilization
NOTE: PLATED MEDIUM- sterilized first before distribution.
Special Media:
Inoculation of Media
A. Inoculation of Tubed Media
1. Liquid Medium
a. With the use of a sterile Pasteur pipet
b. Inoculate by shaking a previously heated wire loop or needle.
2. Slant Medium
With the use of a wire loop or needle, transfer the inoculum to the bottom of the slant
and streak in a zig-zagging manner across the entire surface toward the mouth of the
tube.
3. Butt Medium
Just stab the medium with an inoculating needle.
4. Butt/Slant Medium
Inoculate the butt first by stabbing the needle to the bottom of the medium and then
streak the surface in a zig-zagging manner toward the mouth of the tube.
B. Inoculation of Plated Media
1. Streak Plate Technique
a. Purpose: To isolate organisms in pure culture.
(26)
b. Procedure:
I.
With a sterile loop, deposit the inoculum at one edge and streak back and forth
over the area progressing across the agar until one fourth of the plate surface is
covered.
II.
Flame the loop, streak at right angles to the originally inoculated agar, and
cover the second quarter of the plate.
III.
Flame loop again and repeat procedure until third and fourth quadrants are
covered.
IV.
Turn plate upside down (to prevent contamination by water condensation) and
incubate at 35-37 degree Celsius under 5-10 CO2
Note: 5-10% is not recommended for media in which Ph alteration is used to
differentiate colony types (e.g, XLD agar, Hektoen enteric agar).
V.
Examine initially after 18-24 hours of incubation.
2. Pour Plate
a. Purpose:
i. To determine the approximate no. of viable organisms in a liquid such as water, milk , urine, or
broth/culture. Data are expressed as the no. of colony-forming units per milliliter (CFU) of the
substances examined.
ii. To determine the type of hemolysis produced by streptococci.
b. Procedure:
i. prepare serial dilutions of the specimen.
ii. transfer a prescribed volume of diluted specimen into tubes of melted agar.
iii. mix the tubes and pour them into petri dish.
THE CULTURE
Types of culture
1. Pure (axenic) culture- made up of organisms belonging to one specie.
2. Mixed culture- made up of organisms belonging to different species.
3. Stock Culture- is a pure culture of organisms used as a soured of supply for industry,
reseach, and students use
4. Contaminated culture- is culture accidentally contains more than , species of bacteria
Methods of obtaining pure culture:
1.streak plate method
2.pour plate method
3. use of selective media and media containing antibiotics
BACTERIAL COLONY
Colonies are visible macroscopic masses of growth on a solid culture medium as a result
of repeated divisions of one or few microorganisms.
TYPES OF COLONY
A. Mucoid (M) Colony
1. exhibits a water-like, glistening, confluent appearance.
2. Characteristic of organisms that form slimes or well-developed capsules.
3. ex. K. pneumonia, S. pneumonia, H. influenza, etc.
IMPORTANT DESCRIPTION OF CULTURE CHARACTERISTIC
Form: circular , filamentous, pinpoint irregular
(28)
FUNGI
PROTOZOA
-PH-6.8
-aerobic, heterotrophs
-amino acids/melanin/CHO
(29)
QUALITY CONTROL: is a system used to monitor the quality of performance and result in the
cli. Lab
2. PROCEDURE MANUAL- through organized current procedure manual that contains the
policy and regulations procedures, location of reagents supplies
3. CULTURE MEDIA AND RGT.: each new shioment of C.M should be tested w/ control
org. by known results stock org. should be clean so both g+ & g- can be observed
4. PERSONNEL- properly educated & attend continuing education courses to human
updated
5. SAFETY POLICIES
CHAPTER III
BIOCHEMICAL TESTS
These are tests designed to demonstrate the physiological and chemical char. of microo.
w/c enables the bacteriologist by elimination.
CARBOHYDRATE FERMNETATION
Fermentation is the anaerobic breakdown of subs. (CHO) resulting often in the production
of large amounts of organic acids.
B.Durham Tube:
This consist of a tests tube w/a small inner tube.
(31)
C.Principle:
Organisms that ferment the CHO in the broth produces acid thus, changing the
color of the medium (depending upon the indicator used) to the acidic color. Gas formation may
accompany this reaction in some micro.
D.Procedure
1. Inoculate broth lightly.
2. Incubate overnight at 37C.
3. Examine daily for 4-5 days
Confusing tests:
1. Schick-C. diptheriae
2. Dick-susceptability to scarlet fever
3. Schuloz Charlton-scarlet fever
4. Alik-confum toxin prod. For C. diptheriae in vitro.
E.Result:
1. Positive-purple to yellow (bromcresol purple)
- pale yellow to reddish pink (Andrade indicator)
-red to yellow (phenol red)
-gas formation in the inverted insert tube
2. Negative-color of medium remains the same (e,g. , red w/ phenol red)
A.CHO Content
1. TSI-glucose (0.1%), Lactose (1%), and sucrose (1%)
2. KIA-glucose (0.1%) and lactose (1%)
B.pH indicator: phenol red
C.H2S Indicator: ferrous ammonium sulfate (black deposit)
D.Principle:
1.Glucose utilization by certain micro. Occurs both aerobically on the slant where oxygen is
available and anaerobically in the butt. 6 hrs of incubation.
2.After depletion of the limited glucose, some org. utilize lactose or sucrose and continue making
acid end product. The slant and butt will remain yellow after 18-24 hrs incubation.
3.Some org. are not able to utilize lactose but instead breakdown the peptone in the medium.
After 18-24 hrs incubation the TSI will thus show a red slant and a retained yellow butt. The
reaction is called Alkaline over acid, signifying that only glu. Was fermented.
4.Glucosenonfermenters may also produce alkaline products from peptone utilization.
5.Some org. have the ability to produce gas from the fermentation sugar while others produce
large amount of hydrogen sulfide gas.
E.Procedure:
1.inoculate medium and incubate overnight.
(33)
INDOLE TEST
Principle:
Organisms that produce the enzyme tryptophase are able to degrade tryptophan into
indole, w/c can be detected by its ability to combine w/ certain aldehydes to form a colored
compd.
Methods
1.Kovacs Method-Pink to deep red color (+ result)
a.Medium: tryptone broth or trypticasebroth
b.indoleindicator:Kovacsrgt. (p-methylaminobenzaldehyde)
c.procedure and result:
(34)
b.IndoleIndicator:Erlichsrgt. (p-methylaminobenzaldehyde)
c.Procedure and result:
i.add 1 ml xylene to a 48 hr old broth culture.
ii.shake the tube well and allow to stand for a few mins. Until the solvent rises to the surface.
iii.gently add 0.5 ml of erlichsrgt. Down the slidesof the tube and observe bet. The solvent and
the medium.
3.SpotIndole Test-Blue or blue green (+ result)
This is a rapid method for the detection of indole.
a.Indole Indicator:1% p-dimethylaminobenzaldehyde
b.Procedure and result:
i.saturate a filter paper in the bottom of a petri dish w/ the rgt.
ii.With a wooden stick or loop, rub a portion of the colony on the filter paper and observe for the
rapid development of a blue or blue green color.(+ result).
Some organisms produce large amounts of acid from dextrose while others produce
less.
D.Procedure:
Add 5 drops of methyl red to 5 ml of a 48 hr old broth culture and observe for the
results.
E.Result:
1.Positive-red color (pH 4.5 or below)
(35)
VOGES-PROSKAUER TEST
Differentiate
Enterobacter and K. pneumoniae (+) from E. coli (-)
Moraxella (+) from actinobacter (-)
A.Medium:MR-VP medium
B.Rgts.:
1.5% alpha-naphthol in 95% ethyl alcohol.
2.40% KOH in distlled water (provides alkalinity)
3.0.3% creatine in distilled water (prevents false negative results)
C.Principle:
Some bacteria have the abilty to produce acetoin (acetylmethylcarbinol) from
glucose.w/c reacts with guandine compd. Present in the broth to give a red-colored complex.
D.Procedure and result:
1.Inoculate NR-VP broth and incubate at 35c to 37 for 48 hrs.
2.to 1 ml of the culture broth, add 2 drops creatine solution, addition of each rgt.
3.Observe for a pink to cherry red color w/c is the (+) result.
(36)
Some organisms can utilize citrate as their sole source of CO2 producing acetate and
other alkaline carbonate end products in the process. These products change the color of the
indicator from green to blue.
D.Procedure:
Inoculate Simmons citrate slant incubate for 24 hrs or up to 4 days at 35-37C
E.Result:
*NOTE:The test should not be performed with colonies grown on blood agar (red blood cells
contains catalase thus, give a positive test).
OXIDASE TEST
A.Principle:
Some organisms produce cytochrome oxidase which in the presence of air, acts on
certain aromatic amines to produce colored compd.
(37)
B.Methodes:
1.Spot Oxidase Test (Kovacs Method)-blue or black purple
a.rgt.:1% tetramethyl-p-phenylenediaminedihydrochloride
b.Procedure and result:
i.Place a filter p[aper into a petri dish and moisten it wuth several drops of the rgt.
ii.With a platinum wire or wooden stick ruba 24 hr old colony on the moistened filter paper.
iii.Observe for color-change to blue or dark purple w/c is a (+) result.
2.Oxidase Test for Neiserria-Pink to red and to purple black
a.rgt.:1% dimethyl-p-phenylenediamine hydrochloride.
b.Procedure:
Add several drops of the rgt. Onto the colony on a soilidmrdium or add 1 drop
of the reagent to a filter paper and spread the colony onto it .
c.Positive Result:
The colonies become pink then red and finally purple black.
3.IndophenolMethod:Intense blue
a.rgt.:
i.p-aminodimethylaniline hydrochloride (or oxalate)
ii.alpha-naphthol
b.Procedure:
i.To an 18-4 hrs old culture grown on a nutrient agar slant add 2-3 drops of the above rgt.
ii.Tilt the tube to mix the rgts. And flow over the growth.
c.Positive result: intense blue color within 2 mins.
(38)
UREASE TEST-Hydrolyze urea to ammonia by urease enzyme
MLC)
e. Uses:
A good research laboratory technique for testing anaerobic bacteria.
(39)
The principle of this method is identical to that of the broth dilution metjod.The utilizes
small plastic microtiter trays which contain a final volume of organism-antimicrobial
suspension 0.1 ml
This test utilizes a filter paper disk impregnated with a specifief amount of antimicrobial
agent which is applied onto an agar surface that has been freshly inoculated with a
microorganism. The use of this test is largely limitef to rapidly growing aerobic and
favultatively anaerobic bacteria.
The Kirby-Bauer Technique - this is a paper disc method of sensitivity that is indicated for those
microorganism whose susceptible cannot be predicted after their identity is known.
1. Procedure:
a. With a sterile wireloop, pick (4-5) pure isolated colonies of organism and make a
subculture using MHB, or any other suitable broth medium.
b.Incubate at 35C for 2-8 hours(up to 18 hours) then compare the turbidity of the
subculture to a McFarland 0.5 barium sulfate standard. If the turbidity is greater than
the 0.5 bacterial suspension) by adding sterile NSS and if lesser than the standard
principle of this method is the basis of some rapid, semi automated systems(Autobac MS-2or
Avantage) used for susceptibility testing of aerobic and facultatively anaerobic bacteria. Disk
elution is used also in susceptibility testing of anaerobic bacteria and Mycobacteria.
Interpretation of Results:
A. Susceptible
(41)
This implies that an infe tiondue to an organisms can be appropriate treated with the
recommended dosage of antimicrobial agent for that of infectionand infecting organism, unless
otherwise contraindicated
B. Moderately Susceptible
This implies that the amount of the antimicrobial agent given to patient can be increased
beyond or recommended dosages without serious risk of adverse reaction to obtain higher
levels in the infection site.
C. Intermediate
Also known as the intetmediate category , indicates that the test result is equivocal and that
susceptibility of that particular organisms cannot be predicted. This is generally applied to
antimicrobial agents a narrow toxic to therapeutic ratio in which higher dosages are like be
associated with adverse side effects.
D. Resistant
This implies that an infection due to an organism is unlikely to respond or will not reliably
respond to the antimicrobial agent.
SENSITIVITY TESTING
DISCUSSION:
Antibiotics differ from disinfectants and antiseptic in that they are chemicals that are
biosynthesized. Biosynthesized refers to the production of substances by living organisms.
These antibiotic substances are of no use to the microbe so we call them by products of
microbial metabolism. The genera of microbes that produce the largest numbers of
antibioticsuseful to humans are bacillus, penicillium and streptomyces. Antibiotics also differ
from disinfectants and antiseptics in that they are administered internally and travel in the blood
stream to all parts of the body. This means that the antibiotic must have low toxicity to body cells
while
being
toxic
or
destructive
to
the
parasitic
invader.
(42)
Other antimicrobial chemicals used therapeutically are artificially synthesized in the
laboratory. Because of their artificial synthesis they are often called as chemotherapeutic
agents. More commonly they are reffered to a narrow and or broad spectrum antibiotic.
In vitro is the Latin term for "in glass", as opposed to in vivo meaning "in life" or in a
living animal.
The kirby -Bauer method for determining bacteridical sensitivity to antibiotics is
employed in diagnostic laboratories because it is a standardized and therefore gives more
accurate results. It involves growing pure colonies of the etiological agent in a special broth,
diluting the broth culture to match a comparative standard, and inoculating a specific medium
before the sensitivity disks are added. A certain size zone of inhibition for each different
antibiotic has been empirically determined to be effective therapeutically for each bacterial type
and an organism is not considered to be sensitive to the antibiotic unless its zone of inhibition is
as large as longer than the predetermined zone for that antibiotic.
The kirby Bauer method has the advantage in that it standardizes MOST OF THE
FACTORS THAT INFLUENCE INHIBITION ZONE SIZE EXCEPT bacteticidal susceptibility.
These factors are inoculum size, pH of medium, depth of medium, concentration of agar, rate of
diffusion of drug (a function of the molecular weight of the antibiotic) concentration of antibiotic
impregnated disks, and incubation.
The mutant resistant strains of bacteria often arise because of indiscriminate use of
antibiotics or from undertreatment with the proper antibiotics. Therefore, it is important for
the patient to take all of the correctly prescribed dosage. In the case of antibiotic therapy,
undertreatment can be more harmful than overtreatment. In low dosage it is possible that the
antibiotic can act as a selective agent by killing the normally susceptible bacteria thereby
Millimeter scale or vernier or dial caliper scale- usd to measure the zone of zone of inhibiyion
0.5 McFarland standard (Barium sulfate turbidity standard) - 10^8org/m
The preaparation
* 150mm petridish
* 60ml of MH
* 0.5 ml of 1%BaSO4
* 99.5 ml of 1%H2SO4
Action of Antimicrobial
(44)
ex. Polymuxin B
(45)
Agar Culture
C. Blood Culture
1. Most basic blood culture media contain a nutrient broth and an anticoagulant ( SPS).
Examples of blood culture bottles are:
a. Trypticase soy broth
b. brain heart infusion agar (BHIA)
c. Supplemental peptone
d. thioglycollate broth
2.
e.Columbia broth
a. Brucella broth
b. Castaneda medium ( biphasic medium-agar slant with broth)
3. Penicillinase may be added to blood culture media to specifically inactivate penicillins
and other beta-lactam antibiotics present in the patients blood.
4. The ideal dilution factor is 1:10 ( 1 part blood and 10 parts medium). This combines the
largest feasible amount of blood collected (usually 10 ml) with the smallest amount of
medium that can still encourage growth of bacteria and dilute out the antibacterial
components of the system.
5. The use of 2 different bottles of blood culture media, one vented ( aerobic- TSB) and the
other unvented (anaerobic-thioglycollate) is recommended for optimal recovery of
pathogens.
5. Blood culture should be incubated at 35 oC examined visually daily (with subcultures
done at intervals) for 7 days before reporting as negative or no growth. But in patients
suspected of having endocarditis, brucellosis and fungemia, incubation should be
prolonged for 2-4 weeks
6. Growth is usually indicated by the following:
a. Hemolysis of red cells
B. Processing of CSF
glucose
satts
white cells
1. Specimens are centrifuged and decanted, using the sediments for smears (Gram
stain, India ink) and culture (specially of H. influenza , Neis-seria meningitides, S.
pheumoniae, and Crytococous neoformans). The supernatant can be used to test for
the presence of antigens or for chemistry examinations.
Examples of Serological Tests;
bacterial
fungal
multiple sclerosis
B. Handling
1. Urine should be cultured within 2 hours after collection.
(51)
CPU/ ml of urine
b. Calibrated loop method
A calibration loop is designated to deliver a known
Volume, either 0.01 0r 0.001 ml of urine
insert the calibrated loop vertically onto the urine specimen
place the loop at the center of the plate and spread the inoculum in a line across the
diameter of the plate
\without flaming, draw the loop across the entire plate, crossing the first inoculum streak
numerous times to produced isolated colonies.
(52)
Count the colonies and multiply by 1000 (if using 0.001 ml loop) or count the colonies
and multiply by 100 (if using 0. 01 ml loop)
161- 0.00ml X 1000
0.145 Colonies x 1000
145, or 1.45 x 10 CFU/ml
100000- infection
1000-1000000- contaminated
3. interpretation of culture results:
Colony counts of >105 CFULml (100,000 CFU/ml) for 1 or 2 bacterial species are
clinically significant of infection
Colony conts of > 105 CFU/ml for 3 or more bacterial species most likely represent
contamination
A colony count of >10 3/ml of bacterial specie from a midstream urine of a male is also
considered clinically significant
A colony count of 102 10 3/ml of 1 or 2 bacterial species from females wiyh lower
urinary tract symptoms may also be clinically significant
Colony count of < 50, 000 CFU/ml are seen in contaminated specimens aspiration is
significant.
Anaerobic Culturette
Bio-Bag
Anaerobic Pouch
GasoPak Pouch
(53)
1. Swabs
Voided urine
Sputum
Flees,gastric,iodin,small intestine
C. Never use a dry swab or a regular aerobic transport media
Anaerobic Culture Methods
A.Requirements
Exclusions of oxygen
Low redox potential ( E reducing capacity of certain culture media ;
It becomes more negative as the hydrogen ion concentration decreases)
With water added to the CO2 and H2 generator envelope and oxygen catalyzed with H2 to
water via the pellets, anaerobiosis is achieved
Indications of anaerobiosis:
-Production of heat
-Moisture inside jar
-decolorization of indicator strip (white or colorless)
(54)
CHAPTER VII
HOST- PARASITE RELATIONSHIP
DEFINITONS
1. Host an animal or plant that harbors or nourishes another organism
2. Parasite an organism which dependent on another organism for food and shelter
3. Infection- the process by which a parasitic organism enters into a prolonged relationship with
the host.
(55)
4. Parasitism a relationship whereby one organism derives benefits at the expense of another.
5. Commensalism a relationship wherein the parasite derives benefits from the host with out
causing injury or harm to the host.
6. Symbiosis- refers to a mutually beneficial relationship between the two species.
7. Pathogens organisms capable of producing disease
8. Vectors are arthropods or invertebrates which serve as a means by which infectious agents
are transmitted from one host to another.
Mechanical Transmission- feel a blies biological bites
9. Carriers are individuals who harbor the infectious agents without manifesting symptoms but
who serve as a means of patnogenicity of a particular microorganism
Comprised host- is one whose resistance is impaired by disease or burns
ABILITIES OF MICROORGANISMS TO CAUSE DISEASE
1. Pathogenicity the ability of the organism to produce disease
2. Invasiveness- the ability of the organism to enter host tissues, multiply and spread further.
3. Toxigenecity the ability of the organismto produce toxins (poisonous substances produced
by some organisms)
DIFFERENCES BETWEEN THE TYPES OF TOXINS
(56)
ENDOTOXINS
EXOTOXINS
2. Part of the cell wall and produced only when 2. Produced extracellularly even though the cells are
the cells are lysed.
intact and viable.
3. Lipopolysacharide in nature.
3. Proteins in nature.
Erythrogenic
Bolulinum toxin
TOXOIDS (ANATOXINS)\
These are non-poisonous forms of toxins which can be used for vaccination. The following
are ways of converting toxins to toxoids:
a. by aging
b. by exposure to heat
c. by exposure to 50% alcohol, formaldehyde and delute acids
(57)
In tracing the source of outbreaks for certain bacterial diseases, bacteriophage or serologic
typing have been to be useful. For example: bacteriophage typing of S. aureus amd serotyping of
Salmonelle.
2. Pordormal
3. Period of illness
4. Peiod of decline
5. Period of convalescence regain strength
clostridium botulin
flaccid
clostridium prefigenes
Food poisoning
3. diphtheria
C. diphteria
Staph. aureus
Food poisoning
5. Cholesa
dehydration
6. scarlet fever
Vibrio cholera
strep. Pyogenes
7. Travelers fever
dehydration
rashes
enterogenes
(58)
E. coli
8. Tetanus
muscle
C. Titani
uncontrolabe
Contraction
(59)
MIDTERM LECTURE
PERIOD OF INFECTION:
1. Incubation period- the time between the entry of organisms into the body and the onset of
the first sign and symptoms. An average is usually 7-10 days
Ex. Tetanus 4-21 days, rubella is 10-14 days
2. Period of illness - incubation period up to the first appearance of illness or sickness.
3. Period of convalescence extends from the time the symptoms start to abate until the person
returns to a normal state of health. healing stage
FACTORS OF INFECTION :
1. number of organisms
2. virulence
3. resistance of the host
4. portal of entry
5. toxigenicity
A. Endotoxin these are synthesize components of the cell structure that are extremely
poisonous. Ex. Salmonella typhosa , Neisseria gonorrheae and Myobacterium tuberculosis
B .exotoxins comes from toxigenic bacteria that form toxic proteins that diffuse out the from
the cell as waste product. Ex. Corynebacterium diptheriae and Staphylococcus aureus
diphtheria toxin- is an exotoxin that injuries the kidney, nervous tissue and heart
tetanus toxin- injuries certain nerves thus produced spasms of lockjaw. Enterotoxin refers to the
toxin that acts in the intestine.
C. Susceptibility to infection
1. anatomical defense
2. chemical defense
3. inflammation
4. phagocytosis
5. immune system
MANNERS OF TRANSMISSION
Portal of entry
(61)
1. mucous membrane
2. skin
Mortality rate- the number of the death resulting from a disease on a population
Etiology cause
Pathogenesis development
Epidemiology is the study of the transmission, incidence and frequency of the disease
1. Acute infection- are those which develop rapidly and usually result a high fever and severe
sickness. Most severe stage of a disease: usually short duration or period of time.
Ex. Acute appendicitis
2. chronic infection- develops more slowly with milder but long lasting symptoms usually
undetected
Ex. Tuberculosis and leprosy
3. Sub- acute between 1 and 2 case reporting best way to establish the chain of transmission
4. Talent disease- C.A remain inactive classification of infectious disease
1. local infection- is one in which the causative agent is limited to one locality of the body.
Ex. Boil, diphtheria
2. systemic infection is one in which the infecting agent spreads throughout the body
Ex. Typhoid fever
1. Communicable spread from one host to another either directly
Contagious are disease that easily spread from one person to another
2. Non communicable- do not spread from one person to another
(63)
PRIMARY AND SECONDARY INFECTION
FACTORS
THAT
MAY
MICROORGANISMS:
CONTRIBUTE
TO
THE
PATHOGENICITY
OF
1. Capsule for protection and surrounds the cell wall: prevent phygocytosis
Ex. Streptococcus pnumoniae
2. enzymes
a.hemolysins bring about the lysis of the host red blood cells
b. leukocidins- kills the host leukocytes
c. hyaluronidase- reffered to as the spreading factor various pathogenic bacteria
excretes
enzymes capable of breaking down hyaluronic acid, the intracellular material of
connective tissue.
d. streptokinase- causes the dissolution of blood clots and thus allows the spread of the
organisms.
e. coagulase- organisms producing coagulase causes the plasma to clot.
f. collagenase is formed by some clostridia that cause gas gangrene. It causes the
breakdownof collagen
3. endotoxins
4. exotoxins
5. fimbrane for attachment/adherence
2 TYPES OF IMMUNITY
(64)
Phagocytic Cells
a. neutrophils
b. monocytes
c. fixed macrophages found in the tissue
Kupffers cells- liver
Alveolar macrophages lungs
Microglial cell- brain
Wandering macrophages- roam around the tissues and gather around the sites of
infection or inflammation
Mechanisms of Phagocytosis
2. inflammation is the defensive local response of the body against tissue injury
4 signs of inflammation
1. swelling (RUBOR) Edema due to the movement of blood to tissue
2. redness ( erythema) - due to the increased blood flow to the site of injury
3. pain (dolor) due to the release of lactic acid
Crisis- the skin becomes warm and the person begins to sweat, indicates that the body
temperature is falling
Increased temperature:
1. intensifies the effect of interferon, an antiviral agent
2. helps set up the production of T lymphocytes
3. speed up body reaction like tissue repair
4. decrease the amount of iron available for the microorganisms
Complication of fever:
1. tachycardiac rapid heart rate which may compromised elderly with cardiopulmonary
2. increased metabolic rate which may produced acidosis
3. dehydration
4.electrolytes imbalance
5. convulsion- seizures in young children
6. delirium
7. coma
Antimicrobial substance
a. complement these are serum proteins that causes bacterial cell to lyze
b. interferon antiviral agent produced in response to viral infections
3 types of interferon
(68)
Active Acquired Immunity - immune system produced the antibodies, long lasting immunity.
a. natural active acquired immunity acquired from an infection and recovery
b. artificial active acquired immunity vaccination of the attenuated microorganisms
(69)
vaccination or immunization introduces specially prepared antigens called vaccines into the
thereby stimulating the immune system to produce the specific antibodies
2. Passive acquired Immunity- antibodies is produced by another animal, short time immunity
a. natural passive acquired immunity colostrums, placental transfer of antibodies
b. artificial passive acquired immunity- antiserum
antiserum blood derived fluid containing antibodies produced by other animals or individual.
Types of Immunity
In the presence of antigen B cell differentiate into memory B cells and plasma cells
Plasma cells are differentiated B lymphocytes and the one in the production of antibodies
Cell Mediated Immunity- involves specialized lymphocytes called T lymphocytes that act against
the foreign organisms or tissues.
Tcells produced the cylokines these are the chemical messengers with in the immune system
Types of T cells
1. T helper cells released the cytokinase that will active the B cells to undergo
differentiation
2. Cytotoxic T cells destroys target cells in contact
3. Suppressor T cells turns off the production of antibodies if the antigen is already
eliminated
4. Memory T cells
Antigen ( immunogen) any substance that causes antibody formation and reacts only
with each specific antibodies.
Characteristics of antigen
1. Foreign to the host
2. High molecular weight
3. Lipid and nucleic acid contents
2 parts of an Antigen
1. Epitopose or antigenic determinant region of antigen that recognizes the
antibodies
2. Hapten which cannot stimulate antibody production unless attach to a carrier
molecule
Antibody (immunoglobulins) a Y shaped protein produced by the body in response to the
antigen and capable of combining specifically with that antigen.
2 Parts of an Antibody
Antigen binding site fragment binds with the antigenic determinant to crystalline fragment
Of Immunoglobulin
Diagnostic Immunology
Adenovirus SQ
Vesicella Var SQ
Yearly
(72)
- Japanese encephalitis SQ
- Hepatitis A M
Anthrax SQ
Meningococcal SQ
Meningitis (H Influenzae)Hib IM Pneumococcal pneumonia IM or SQ
Pertusis IM
2,4,6,12-15 mos
Nucleic acid vaccine newest and the most promising vaccine but still no commercial vaccine for
human has been introduced
TYPES OF HYPERSENSITIVITY
a.
b.
c.
d.
Autoimmunity is an action of the immune system in response to self antigen and causes
damage to
ones own organs.
-
Antigens of hypersensitivity
Reaction
Type1 (anaphylactic)
Time before
Clinical signs
Less than 30 min
(75)
characteristics
EXAMPLES
Drug injection
Insect venom
High fever
Asthma
Type2(cytotoxic)
5-12hours
Type3(immune
complex)
308hours
Type4(delayed)
24-48 hours
histamine
Antigen
causes
formation of IgM and
IgG antibodies that
binds to target cells
when combined with
complement will lyze
target cells
Antigen
and
antibodies
formed
complexes that cause
damaging
inflammation
Antigen active T
cytotoxic cells to kill
the target cells
Transfusion reaction
Rh incompatibility
Hemolytic disease of
Newborn (HDN)
Arthus reaction
Serum sickness
Rheumatoid arthritis
Organ
rejection
contact dermatitis like
poison ivy tuberculin
test
(76)
CHAPTER VIII
GRAM POSITIVE COCCI
STAPHYLOCOCCI
GENERAL CHARACTERISTICS
1. Nonmotile, spherical, gram-positive organisms, usually arranged in irregular grapelike
clusters (may also be seen singly in pairs or in tetrands)
2. Nonencapsulated except for a few strains that produce a capsule or slim layer
3. Aerobic or facultatively anaerobic.
4. Catalase positive (this test separates staphylococcus from streptococcus
5. Often present in solid, water and skin of humans
6. Differences between stapgylococcus and micrococcus genera
Genus
Resistant to
Lysostaphin
(200 ug/ml)
modified oxidase
and benzidin
Resistant to
bactericin
(0.04 U)
Micrococcus
Staphylococcus
Staphylococcus aureus
-
The most pathogenic among the staphylococcus, produce exotoxin, grows in the
presence of a high concentration
A . Cultural Characteristics
1.
2.
3.
4.
Grow well in most routin laboratory media like nutrient agar or trypticase soy agar
For primary isolation from clinical specimens, sheep blood agar is recommended
Colonies on solid media are round, smooth, opaque and butyrous
Most strains of S. aureus produce gray to deep golden yellow colonies about 2-3mm in
decimeter entire edge
5. On blood agar a zone of beta hemolysis surrounds the colonies
(77)
*NOTE: Pigmentation and hemolysis are variable characteristics of the staphylococcus
genera
B . Antigenic structure
1. Teichoic acid s. aureus contains ribitol teichoic acid in their cell wall.
2. Peptidoglycan together with teichoic acid it protects the organisms from lysis a d
probably aids in adherence. Elecits the hemoral cellular response
3. Protein A this is a group specific antigen unique to s aureus strains; it prevents
antibody-mediated phagocytosis by PMNS
4. Clumping factor a component on the cell wall of S. aureus responsible for the clumping
of the while staphylococci in the presence of plasma
5. Capsular polysaccharide protects the organism from phagocytosis
C. Determinants of pathogenecity
1. Surface antigens
a. Polysaccharids
b. Protein receptors (e.g., fibronectin, fibrinogen, IgG
2. Extracellular enzymes
a. Coagulase the single most reliable chac of the ID of S. aureus
b. Lipase
c. Hyaluronidase- facilitates the spread of infection
d. Staphylocokinase ( Fibrinolysin)
e. Phosphatase (DIS, RDS)
f. Thermostable deoxyribonuclease
g. Gelatinase
h. Protease
3. Toxins
A. Cytolytic toxins
i. Alpha toxins (alpha-hemolysin) lethal and dermonecrotic
ii. beta toxin (staphylococcal sphingomyelinase) lethal and dermonecrotic ; produces
hot cold
(78)
iii. delta toxin
iv. gamma toxin
v. leukocidin ( Panton- valentine) cause cell lysis
(79)
Staphylococcus Saprophyticus
1. Present on the normal skin and in theperiurethral and urethral flora.
2. Common cause of the urinary tract infections in young sexually active women.
3. Closely resembles S. Epidermidis.
4. Distinguished from S. Epidermidis by virtue of its resistance to novo-biocin and nalidixicacid
and absence of phosphatase production.
5. Coagulase (-) and mannitol fermentation (+).
6. Common cause of UTI
7. Second most common cause of cystitis after E. coli
8. Only Staphylococcus resistant to novobiocin.
Streptococci
General characteristics
1. Spherical to avoid, catalase (-) , gram (+) organisms that tend to occurs in pairs or chains
when grown in liquid media.
2. Nonsporeformer and generallynonmotileexcept
streptococci.
Pyogenic group
-grows at neither 10C nor at 45CExample: S. Pyogenes
Viridans group
-grow at 45C butnot at 10C
Example: S. mutan( cause of dental plague )S. sanguis , S. mitis , S. salivarius and S.
anginosus
- resists classification by Lancefield precipitation test.
Enterococcus
grows at both 10C and 45C ; can stands temperature above 60C.
( Enterococcus0faecalis ( part of the normal faecal flora )
Example:
S.
(83)
Lactic group
grow at 10C but not at 45C
Example: S. Lactis
1. Beta-hemolytic Streptococci Produce a clear colorless zone around the colony indicating a
complete hemolysis of the red blood cell (e.g. , S. pyogenes, S. aga-Lactiae
2. Alpha-hemolytic Streptococci Produce a zone of partial hemo-lysis with a greenish or
brownish discoloration (e.g. ,viridans species, S. pneumoniae)
Gamma-hemolytic or non-hemolytic or indifferent streptococci-colonies do not produce any
hemolysis (e.g. , S. faecalis)
C.Lancefield Classification
(Based on the antigenic characteristics of group-specific C substance, which is a cell-wall
polysaccharide.
1. Group A Streptococci: Streptococcus pyogenes.
A.Diseases Produced
Sore throat, pharyngitis, tonsillitis, skin infection- pyoderma, impetigo, cellulitis and scarlet
fever.
(84)
B.Cultural characteristics
Colonies are transparent to translucent, convex or domed entire, c
Lipotechoic acid
present in the cell wall; responsible for the adherence of the bacteria to respiratory epithelial
cells.
Protein
major virulence factor of group A streptococci which renders the orgarnisms resistant to
phagocytosis.
Hyaluronic acid capsule
assists the organisms in avoiding phagocytosis.
Hemalysins
Streptolysin O (SLO): Oxygen labile,
antigenic toxin
: involve in ASO titer test
: responsible for subsuraface
hemolysis.
Streptolysin S (SLS) : Oxygen
stable, non antigenic.
: responsible for surface hemolysis
Erythrogenic (pyrogenic) toxins-responsible for the characteristics rash seen in scarlet fever
Hyaluronidase
spreading factor
Streptokinase
enzyme that dissolve cloths.
(85)
Identification
2. PYR test
Based on the presence of the L pyroglutamylaminopeptidase enzyme which hydrolyzes
an amide substrate to form the free beta-naphthylamire, which when reacted with a
cinnamaldehyde reagent (PYR reagent) forms a brightened end product (+)
Aside from S. pyogenes, the enterococcus species also posses the aminopeptodase
enzyme enzyme.
3. Confirmatory Test
Lancefield Precipitin Test
Direct FA Test
Coagglutination (Phadebact)
Cultural characteristics
(86)
Colonies are large, mucoid, more translucent to opaque, whitish gray, soft , smooth
colonies surrounded by a smaller zone of beta-homolysis
Identification
(+) result is an arrowhead-shaped zone of enhanced hemolysis at the juncture between the
(+) streptococci and the staphylococcus.
S. aureus streak
(+) arrowhead-shapedhemolysis
Group C . Streptococci
(87)
Group D Streptococci
Traditionally divided into two groups, the enterococcal species (E. faecalis , E. faecium,
E. durans) and the nonenterococci (S. quinus& S. bovis). They both grow in the presence of 40%
bile and hydrolyzeesculin. The enterococci are differentiated from the nonenterrococci by their
ability to grow in the presence of 6.5% sodium chloride.
Group G Streptococci
This group does not have species designations. Colonies are large with a wide zone of
beta-hemolysis resembling S. pyogenes.
Mode of Transmission
(88)
Impetigo
Cellulitis
Erysipelas
Wound infections
Puerperal sepsis
Scarlet fever
Control Arm
(-)
Based on the neutralization of erythrogenic toxins when an anti toxin is injected into the skin of a
patient with scarlet fever skin rasher fade or blanch (+)
Used to diagnose whether the skin rashes are due to scarlet fever or not.
(89)
Treatment
Intramuscular benzathine penicillin as single dose
Oral penicillin V for 10 days
For penicillin-allergic patients- erythromycin, clindamycin and cephalexin.
Streptococcus
Colonies
Large, round, white,
yellow/gold.
Small, round, surface
colonies,
lancetshaped in polar plate
Small, round surface
colonies, round in
pour plate
Hemolysis
Beta type
Morphology
Clusters
Alpha type
Treatment
Penicillin G- antibiotic of choice
Erythromycin ekloramphenicol, cephalosporins, vancomycin, Imipenem and clindamycin
Group C Streptococci
Disease Produced
The members of this group are primarily animal pathogens but they may infect humans as well:
A. S. equi
Causes disease in horses
S. equisimilis
May cause pharyngitis, puerperal sepsis, endocartisis, bacteremia, osteomyelitis, brain
abscess, post-operative wound infection and pneumonia in humans.
Source of streptokinase used in thrombolytic therapy in humans.
Treatment
Similar to group A streptococci
(91)
Normal flora
Skin
Upper respiratory
Gastrointestinal
Genitourinary tracts.
Diseases Produced
Frequent cause of bacterial endocarditis in the elderly
Urinary and biliary tract infections
Septicemia
Wound infection and intra-abdominal abscesses
Treatment
Enterococcal strains
Generally resistant to penicillin G, ampicillin and penicillinase-resistant penicillins
Penicillin in combination with an aminoglycoside (gentamicin or streptomycin)
For penicillin allergic patients- vancomycin and erythromycin
Nonenterococcal strains- penicillin G.
(92)
Thioglycollate
Staphylococcus
Pneumococcus
Streptococcus
Viridans Streptococci
Normal flora
Growth
Abundant
Inhibited
Abundant
Optochin Test
Bile Solubility
(+) Sensitive
(-) Resistant
(+) Soluble
(-) Not Soluble
Nasopharynx
Mouth
Gingival crevices, gastrointestinal tract and female genital tract. They are opportunistic
pathogens thought to be of low virulence.
Diseases Produced
Major etiologic agent of bacterial endocarditis especially S. sanguis
Major etiologic agent of dental caries (plague)- S. mutans
Cellulitis and wound infection
Meningitis
Sinusitis
Biliary or intra-abdominal infections
Treatment
Aqueous penicillin G or with the additional of gentamicin
For penicillin allergic patients- vancomycin
(93)
LABORATORY DIAGNOSIS
Specimens: threat swabs, vesicular or pustular fluids, skin lessions, aspirated tissue fluids, blood,
etc.
A. Stained smears
B. Culture
Sheep blood agar is preferred for primarily isolation because it is inhibitory to the
growth of Haemophilushaemolyticus( a normal throat commensal whose colonies may be
mistaken for beta haemolytic streptococci).
Other culture media used are chocolate agar, Columbia agar with colistin and nalidixic
acid (CAN), phenylethyl alcohol agar (PEA) and Todd-hewitt broth.
C. Bacitracin disk test, PYR test, hippurate hydrolysis, CAMP test, bile-esculin test, growth in
6.5% NaCI
Serological Tests
a. Lance field precipitin test
confirmation test wherein the cell wall antigens are extracted either physically be heating
or chemical or enzymatic extraction of a cell suspension grown overnight in Todd-hewitt broth
b. Direct flurescent antibody test
c. Coagglutination (Phadebact)
d. Enzyme-linked immunosorbent assay (ELLISA)
e. Latex agglutination test
f. ASO titer for group A streptococcal infection
(94)
Cultural Characteristics
1. On blood agar plates young colonies are circular , glistening, dome-shaped while some are
larger and more mucoid. As the colonies becomeolder, autolytic changes result in a collapse of
the center of the colony (with raised margins and depressed centers), giving it an umbilicate or
doughnut appearance ( checker or nailhead colonies).
2. Colonies incubated aerobically produce a zone of alpha hemolysis. Colonies incubated
anaerobically produce a zone of beta hemolysiswhich is due to the oxygen-labile pneumolysin 0.
Determinants of Pathogenicity
1. Polysaccharide capsule
2. Adherence
3. Enzymes
Neuramidase- enzyme that degrades surfaces surface structures of host tissue.
Proteases-enzymes that facilitate bacterial colonization on mucosal surfaces by
eliminating immunoglobulins such as IgA, IgG and IgM.
4. Pneumolysin 0- an oxygen-sensitive toxin that is cytolytic for cells.
5. Autolysin facilitates the release of pneumolysin 0 and other toxic proteins or inflammatory
substances from cells.
6. C-subtance- a component of the cell wall of pneumococci which is a teichoic acid that reacts
with C-reactive proteins (CRP) resulting in the activation of some nonspecific host immune
responses.
(95)
Diseases Produced
Most common cause of bacterial pneumonia (lobar)
Second most common cause o bacterial meningitisis
Otitis media, purulent sinusitis and occasionally peritonitis
Laboratory Diagnosis
Specimens: Sputum, swabs, pus and blood
Polysaccharide capsule
Adherence
Enzymes
neuraminidase enzymes that degrades surface structures of host tissue
proteases enzymes that facilitates bacterial colonization on mucosal surface by
eliminating immunoglobins such as IgA, IgG and IgM.
4. Pneumolysin O an oxygen-sensitive toxin that is cytolytic for cells
5. Autolysin facilitates the release of pneumolysin O and other toxic proteins or
inflammatory substances from cells
6. C-substances a component of the cell wall of pneumococci which is a teichoic acid that
reacts with C-reactive protein (CRP) resulting in the activation of some nonspecific host
immune responses
D. Diseases Produced
1. Most common cause of bacterial pneumonia (lobar) (rusty sputum)
2. Second most common cause of bacterial meningitis
3. Otitis media, purulent sinusitis and occasionally peritonitis, bacteremia
E. Laboratory Diagnosis
Specimens: sputum, swabs, pus and blood
(97)
1. Gram-stained smears
2. Culture
3. Optochin sensitivity test Taxo P
4.
5.
a.
b.
c.
6.
a.
b.
c.
7.
a. Most widely used presumptive test for differentiating pneumococci from other alphahemolytic streptococci (e.g. , the viridans streptococci)
b. The optochin disk or Taxo P (ethylhydrocupreine hydrochloride) is placed on a blood
agar plate heavily inoculated with suspected organisms and incubated overnight
c. (+) reaction is a 14-16 mm zone of inhibition depending on the size of optochin disk
(6&10 mm)
Bile solubility test
- Based on the presence in pneumococci of an autolytic amidase which when activated
by bile results in the lysis of the organisms.
Mouse virulence test
The mouse is particularly sensitive to even a small inoculum of pneumococci
Sputum containing pneumococci is injected intraperitoneally to a mouse which
eventually dies within 16-48 hours
The heart blood of the dead mouse harbors a pure culture of pneumococci
Neufeld/ quelling or Capsular precipitation reaction
The most useful, specific and rapid method for the identification of S. pneumoniae
Test is performed by mixing on a slide of a loopful of emulsified sputum with a loopful
of antipneumococcal serum and methylene blue
(+) reaction capsule appears swollen due to a change in refractive index which in turn is
due to a serological reaction
Other serologic test like pneumoslide
F. Treatment
1. Penicillin drug of choice
2. Cephalosporins or erythromycin (for pneumonia) and chloramphenicol ( for meningitis)
for patients allergic to penicillin
Table 8-1 Tests Differentiating Pneumococci and Streptococci
TESTS
Bile Solubility Test
Inulin Fermentation Test
Capsular Swelling Test
Quinidine Test
Optochin Test
Mouse Virulence Test
PNEUMOCOCCI
Bile soluble
Fermenter
Swelling of capsule
Susceptible
Susceptible
Mouse dies within
hours
STREPTOCOCCI
insoluble
Non- fermenter
No swelling
Resistant
Resistant
16-48 Wont die
Francis Test skin test for determining the presence of antibodies against pneumococci
(98)
marked surface. These species are normal flora of the mouth, urogenital tract and gastrointestinal
tract but may cause pleuropulmonary disease, brain abscess and gynecologic infections.
a) Peptococcus commonly known as the anaerobic staphylococcus, catalase + and in
clusters
b) Peptostreptococcus commonly known as the anaerobic streptococcus
c) Gaffykatetragena arranged in packets of 4 or tetrads
d) Sarcinalutea occurs in cubical packets of 8s, 16s, 32s or more. Also known as the
saphrophyticinterococci. Produce a bright yellow pigment. Contaminants in the lab.
(99)
CHAPTER IX
GRAM NEGATIVE COCCI
NEISSERRIA
There are two species of this genus that are major of medical importance; they are N.
meningitidis (causative agent of meningococcal meningitis) and N. gonorrhea (causative agent of
gonorrhea). Humans are the only known reservoir of these species.
General Characteristics
1. Gram (-) kidney or coffee bean shaped with adjacent sides flattened.
2. Most are encapsulated; nonmotile and nonsporeformer.
3. Aerobic or facultatively anaerobic organisns requiring an increased CO2 tension for
growth (3-10% CO2).
4. Highly susceptible to drying, chilling and exposure to unfavorable pH or to sunlight.
5. Most species can grow on blood agar except for N. gonorrhea which requires the added
nutrients of chocolate agar or special enrichments.
6. Catalase and oxidase (+)
Cultural Characteristics
1. Fastidious organisms requiring complex nutritional growth and requirements.
2. They appear as translucent, grayish, convex, shiny colonies with entire margins that are
nonhemolytic and nonpigmented.
Culture Media
1. For normally sterile specimens (blood, CSF, synovial fluid, etc.)
- Blood agar and chocolate agar
2. For contaminated specimens (discharges and swabs)
a. Thayer-Martin medium (Specific, Selective)
- Enriched chocolate agar with supplement B or Isovitale X and antibiotics:
I.
Vancomycin inhibits gram (+) organisms
II.
Colistin inhibits gram (-) rods
III.
Nystatin inhibits yeast (fungi)
b. Modified Thayer-Martin medium
- With the addition of hemoglobin solution and trimethoprim lactate which inhibits the
spreading of proteus
c. Transgrow medium
- A Thayer-Martin medium with glucose, 2% agar, trimethoprim lactate and CO2
incorporated in bottle
d. Martin-Lewis medium
- A modified Thayer-Martin medium with the substitution of anisomycin, an antifungal
agent with a longer half-life for nystatin
e. New York City medium
- Also a modified Thayer-Martin medium with the substitution of amphotericin B for
nystatin.
Neisseria gonorrhea
Gonorrhea is the most common sexually transmitted disease (STD) seen in adults, children and
infants.
Clinical Manifestation
1. In men, it usually presents as acute urethritis with dysuria and purulent urethral discharge,
although up to 25% have only minimal discharge and some have no symptoms.
2. In women, between 20% to 80% are asymptomatic, though there may be increased
vaginal discharge, dysuria menstrual abnormalities, and lower abdominal pain and
physical examination may show a friable erythrematous cervix with mucopurulent
discharge.
3. Pharyngeal gonorrhea occurs in both sexes and is usually asymptomatic.
4. Gonococcal arthritis-dermatitis syndrome is the most common manifestation of
disseminated gonococcal disease which is heralded by fever, chills, malaise, intermittent
bacteremia, polyarticular arthritis or tenosynovitis and development of typical skin
lesions.
5. Gonorrhea may be seen in sexually abused children but in infants it usually results from
contamination during passage through an infected birth canal. The infant develops
gonococcal opthalmianeonatrum, an eye infection which cause blindness. This is
prevented by Credes prophylaxis (1-2 drops of 1% silver nitrate), erythromycin or
penicillin drops instilled in the eyes of all newborn babies. Children may also acquire the
disease from fromites.
Laboratory Diagnosis
Specimens: discharges from urethra, cervix and anal canal; specimens from the oropharynx,
skin lesions, inflamed joints, prostate and blood; swabs of eye discharge
1. Stained smear
- Typical gram (-) diplococci, intracellular (within PMNs) and/or extracellular (outside
PMNs); intracellular existence is indicative of active infection
2. Culture
Four colony types:
a. T1 and T2 produced on 1 culture; small and dome-shaped colonies; piliated thus
virulent (pili or fimbriae principal virulence factor)
b. T3 and T4 produced on subsequent cultures; larger and flatter colonies; piliated thus
avirulent
3. Presumptive Identification Test
4. Confirmatory Identification Test
a. Sugar degradation
i.
CTA (cystinetrypticase agar)
ii.
Gonobio Test a rapid 4 hour micro method
iii.
Gonochek II
*N. gonorrheae ferment only glucose producing acid without gas
b. Direct fluorescent antibody technique
5. Antibody-based particle agglutination
a. GonoGen
b. Phadebact
c. Meritec GC
6. ELISA (Gonozyme) for direct detection of gonococcus in specimens
7. Limulus test rapid screening test for N. gonorrhea
8. Superexol test (+) production of gas bubbles from 30% H2O2
Treatment
1. Amoxicillin, ampicillin and aqueous procaine penicillin G
(101)
2. Ceftrimoxazole most commonly recommended for uncomplicated cases due to
increasing incidence of penicillinase-producing N. gonorrhea (PPNG) or chromosomally
mediated resistant N. gonorrhea (CMRNG)
3. Other alternatives are spectionomycin, trimethoprim-sulfamethoxazole, ciprofloxacin and
cefuroxime
Neisseria meningitidis
Disease Produced: Epidemic meningococcal meningitis/ cerebrospinal fever/ spotted fever (due
to the presence of petechial lesions)
Clinical Manifestation:
1. Meningococci enter the body thru the upper respiratory tract including the conjunctiva
and establish on the mucous membrane of the nasopharynx. Then dissemination occurs
via the bloodstream
2. Signs and symptoms include mild fever, meningitis, pharyngitis, prostration,
erythematous macular rash usually superseded by a petechial eruption. This vasculitic
purpura is the hallmark of meningococcal disease.
3. Hemorrhage into the adrenal tissue with resultant hypoadrenergic state is known as the
Waterhouse-Friedrichsen syndrome ( a highly fatal fulminating meningococcemia).
4. Sequelae include eight nerve deafness, CNS damage, skin or tissue necrosis due to
vascular thrombosis.
Laboratory Diagnosis:
Specimens: blood, CSF, synovial fluid, pleural fluid, aspirates of petechiae, and nasopharyngeal
or throat swabs (for carrier)
1.
2.
3.
4.
5.
a.
b.
6.
Stained smears
Culture
Presumptive oxidase test
Confirmatory test: CHO fermentation test (CTA)
- N. meningitidis ferments glucose and maltose
Rapid detection of capsular polysaccharide antigen in urine, serum, CSF and synovial
fluid by:
Countercurrent immunoelectrophoresis (CIE)
Particle agglutination techniques such as latex and coagulatination
Limulus test for lipopolysaccharide endotoxin
Treatment
1. Penicillin drug of choice
2. Chloramphenicol and ceftriaxone for penicillin sensitive patients
Prophylaxis for carriers
1. Rifampicin and minocycline primarily
2. Ciprofloxacin
Neisseria cinerea
A pathogen associated with bacteremia, conjunctivitis, nosocomial pneumonia and proctitis. It
has a biochemical resemblance to N. gonorrhea by its ability to grow on Mueller-Hinton agar (N.
gonorrhea unable to grow on MH) and inability to grow on Thayer-Martin medium.
(102)
Branhamella (subgenus of Moraxella)
Branhamellacatarrhalis
-
Tiny, strictly anaerobic gram (-) cocci that occur in masses or as diplococci which are part
of the normal flora of the mouth.
Produce small, convex, translucent to transparent colonies with entire edge which may
show red fluorescence under long-wave ultraviolet light
Fatty acid end products are propionic and acetic acids
Other anaerobic gram-negative cocci commonly found in human clinical
infections are Megasphaera and Acidaminococcus.
(103)
CHAPTER X
AEROBIC, NONSPOREFORMING GRAM POSITIVE BACILLI
MYCOBACTERIA
General Characteristics
1. Gram positive, acid fast, strictly aerobic, nonmotile, nonencapsulated, slender, slightly
curved or straight rods
2. Often have a beaded appearance due to the presence of much granules which are non-acid
fast, gram (+) granules.
3. May have palisades or X, Y, V & L (snapping) formation.
4. Stain with difficulty, but once stained, they are resistant to decolorization.
A. Mycobacterium tuberculosis
1. Morphology same as above
2. Cultural characteristics
a. able to grow on simple synthetic medium but for primary isolation, a more
complex medium containing either an egg-pota
b. to base or serum-agar based is required
c. the very slow growth of the organisms requires an increased CO2 tension (5-10%
CO2) at an optimal temperature of 37c; it takes 10-20 days before growth can be
visualized
d. growth first appear as small (1-3mm), dry, friable colonies that are rough, warty,
granular and buff-colored which then become typical colonies having a flat
irregular margin and a cauliflower center after several weeks
e. the growth is luxuriant enhanced by glycerol thus termed eugenic; other
mycobacteria are dysgonic (with smooth scanty growth)
3. Viability and Pathogenicity
a. Tubercle bacilli can resist adverse environmental conditions such as drying, heat
and chemical agents; this is due to the lipids present in the cell wall which are
responsible for the hydrophobic nature of the organism
b. In the body, the organisms are protected due to their intracellular existence (since
they are phagocytosed by alveolar macrophages)
c. Does not produce any toxin but have the following:
i. Cord factor (trehalose-6, 6-dimycolate) glycolipid present in virulent
strains of M. tuberculosis responsible for the formation of tight serpentine
cords in smears and in cord media
ii. Sulfatides glycolipids responsible for the neutral red reacting associated
with virulent strains of M. tuberculosis
Laboratory Diagnosis
(104)
sputolysin
Used for
specimen
cholorox
Procedure:
a.
effective inhibition
with
low
toxicity
can
also
2%
specimen
of
to
NaOH
digestant
mix and stand at room
temperature
for several minutes centrifuge
decant sediments for smears and cultures
i. Ziehl-Neelsen
- scan at least 100 fields before reporting smear as
negative
or
sputum
positive
specimen
for
are
reported
as
acid
fast
acid
fast
bacilli,
bacilli
seen
semiquantitation
recommended:
decolorizer
permanganate
(105)
-
acid
alcohol;
counter-stain
potassium
i.
ii.
Middlebrook 7H10} good for antimicrobial susceptibi iii.Middlebrook 7H11} lity test but 7H10 is much preferred
iv. Mitchisons selective 7H11
Egg-based
i.Petragnani more inhibitory, reserved
ii. Lowenstein-Jansen good for niacin test
iii. Dorset egg medium
iv. American Thoracic Society (ATS) medium less inhibitory
- all of the above contains eggs, potato flour, glycerol
with malachite green to inhibit the growth of contaminants.
Liquid
i.Bactec 12B medium
ii.Middlebrook 7H9 Broth for aseptically collected specimens from normaly sterile sites
Note:
Cultures are examined weekly for growth and reported and discarded as negative (no growth) after
8 weeks of incubation.
inc. 37C
1 hour
d.
(106)
a.
b.
INH prophylaxis
Mode of transmission
-
ii.
cultural characteristic
-
iii.
Biochemical characteristic
-
b. Mycobacteriumafricanum
- rarely isolated in the US
- inactive biochemically but urease (+) ; variably (+) growth
in TCH.
(107)
1.Morphology
a.
2.
acid fast rods predominatly in modified mononuclear or epitheloid structure known as lepra cells
b.
c.
Cultivation
a.
b.
mode of transmission
(108)
4. Lepromin Test
A
skin
test
consisting
of
heat-killed
suspension
of
m.
leprae
24-48
hours
Types of reaction
a.
early/
Fernandez
reaction
induration
appears
b. late/ Mitsuda reaction indurated nodule develops after 3-4 weeks.
in
5. Laboratory Diagnosis
- conventional acid fast stains or via wade-fite technique of
Skin lesion, nasal scrapings, ear lobes, tissue secretions,
lymph node, bone marrow and breast milk.
6.
treatment
a. combined regimen of dapsone or DDS (4,4-diaminodiphenyl sulfone), rifampicin and clofazime for lepromatous leprosy
b. combineddapsone and rifampicin for tuberculoid leprosy
7.
Prevention
a. BCG vaccination
1. Biochemical Tests
a.
niacin
principle: accumulation of niacin in the medium due of lack
test
M. tuberculosis and
(109)
i. certain species of mycobacteria are capable of hyrolyzing tween 80, a derivative of sorbitanmonoleate,
with the production of oleic acid
ii. In the presence of the pH indicator neutral red or phenol red
the oleic acid is indicated by a change from amber to pink (positive result)
iii. Runyons groups II and III are positive in 5 days
iv. M. tuberculosis nis positive in 10-20 days
f.
arylsulfatase test
principle
- based on the ability of arylsulfatase to breakdown
phenolphthalein disulfate into phenolphthalein
Procedure and result
i. inoculate test medium and incubate at 35C for 3 days
ii. Add sodium carbonate and observe for color change to
pink or red (+)
g. tellurite reduction black ppt.
Principle
- telluritereductase reduces potassium tellurite to metallic
tellurite, visualized as a black precipitate; M. avium and
rapid growers posses this fast-acting enzyme
(111)
(112)
b. rapid growers (group IV) produce visible growth within less than 7 days.
M. fortuitum- nonchromogenic, strongly arylsufutase (+) grows on MacConkey agar, niacin (-)
nitrate (+)
c. with club shaped swellings and beaded and barred forms (meta-chromatic granules or babes
Ernst bodies- responsible for the beaded appearance and when situated at both ends are called
polar bodies)
Cultivation
a. on loefflers coagulated serum media, they appear as minute, grayish white glistening colonies
b. on modified tinsdale, colonies are black with dark brown halos.
(113)
c. on cystinetellurite media, colonies are gray or black with garlic like odor and are differentiated
into 3 major colonial types.
Major types
colonies on CT-BAP
i. gravis
ii. Mitis
hemolysis
(-)
(+)
(+)
(-)
(-)
(-)
iii. Intermedius
iii. mechanical obstruction of the airway may ensue due to the membrane and accompanying
edema of the larynx and trachea.
iv. Skin ulcers
v. complications involve the cardiovascular and nervous systems
Schicks test
a. susceptibility test for diphtheria
b. test arm is injected intracutanously with 0.1 ml of diphtheria toxin and on the opposite arm
(control arm) 0.1 ml of diphtheria toxoid.
c. results are read after 24-48 hours an after 6 days.
d. the test is positive if redness, induration and necrosis appear at the site of injection (test arm)
which persist up to the 6th day and fade gradually leaving a brownish pigmented area, and no
reaction in the control arm.
(114)
e. Interpretaion of results:
i. (+) = t.a. (+) and c.a. (-) patient is susceptible to diphtheria
ii. (-) = t.a. (-) and c.a (-) indication of immunity
Laboratory diagnosis
specimen: nasopharyngeal and throat swabs
a. stained smears
b. culture
i. BAP
ii. Tinsdale
iii.Pais coagulated egg medium
c. biochemical characteristics
i. nitrate reduction positive except for c. diptheriae var. mitis (+/-)
ii. Urease negative
iii. Catalase positive
iv. Ferments glucose and maltose but not sucrose
v. pyrasinamidase negative
d. virulence tests (for toxin production)
i. in vivo toxigenixity test: animal inoculation
-inject intracutanously 0.2 ml of culture into a guinen pig followed 5 hours after with 500 units
of antitoxin injected intraperitoneally.
-after 30 minutes a second 0.2 ml culture is injected into the control area opposite the test site.
-the test site after 48-71 hours and a pinkish nodule on the control site (there is prevention of
necrosis due to prior administration of antitoxin.
Treatment
(115)
a. antitoxin
b. penicillin G. drug of choice; erythromycin for penicillin allergic patients.
Prevention
a. active immunization with DPT (below 7 y/o) and Td (7y/o and above)
b. prophylaxis for case contacts
i. boosters dose of toxoid- for previously immunized persons within 5 years.
ii. Completion of immunization and antibiotics- unimmunized or inadequately immunized
persons.
iii. Passive immunization with antitoxin- may be employed for nonimmunized persons with
heavy exposure.
otherCorynebacteria( Diphtheriod)
these are opportunistic invaders infecting immunocompromised cysts which are oftenly confused
with C. diptheriae.
C. minutissimum
a. causative agent of erythrasma, a skin disease occurring in the intertriginous areas especially
the axillae, genitocrural crease, and webs between the fourth and fifth toes.
b. skin lesions show a corak re fluorescence when exposed to woods light due to the presence of
prophyrin.
c. ulcerans- is now considered a variant of C. diphtheria
c. pseudotuberculosis- also produce an exotoxin and causes subacute relapsing lymphadenitis
c.xerosis- inhabits skin and pharynx especially the conjunctiva.
c. pseudodiphthericum-may cause endocarditis.
othercorynebacteria species are c. bovis, areanobacteriumhaemolyticum (c. haemolyticum),
actinomycespyogenes (c. pyogenes), rhodococcusequi (c.qui) c.jeikeium (group jk)
CDC d2 A4 and G2, c matruchotii. Etc.
LISERIA ( L.monocytogenes)
Morphology
a. small gram (+) , aerobic to microaerophiliccoccobacilli that are nonacid-fast and
nonsporeformers
b. they occur in pairs and short chains (resembling streptococci) or in typical
diphtheriodpalasides arrangement; they are also confused with haemophilus species on over
decolorized
preparations.
o
o
c. with tumbling motility at room temperature (20 -25 c) due to the presence of 4 peritrichous
flagella; less or nonmotile at 35o-37oc
Cultivation
a. Optimum temperature for growth is at 37 oc but it has the ability to grow at low temperature
(4oc) being the basis for the cold enrichment technique.
b. on BAP smooth, translucent gray colonies with narrow zone of beta hemolysis.
c. on clear tryticase agar or nutrient agar smooth, translucent blue-green colonies.
Disease produced: Listeriosis
a. may present from sepsis and meningitis in neonates and immunocompromised patients to a
nonspecific febrile illness in pregnant women.
(116)
b. transplacental infection is also common.
c. in adults, meningitis is the most common presentation of listeriosis.
Laboratory Diagnosis
Specimen: CSF, blood, amniotic fluid and genital tract secretion.
a. stained smears
b. motility test refers to p.23
-umbrella-shaped motility pattern in semisolid agar
c. culture
i. sheeps blood agar
ii. Tryptose agar
iii.Mcbride agar
iv. PEA agar- for contaminated specimens (inhibits gram negative organism)
ERYSIPELOTHERIX ( E. rhusiopathiae)
Morphology
a. nonsporogeneus, non motile, nonencapsulated, microaerophilic gram positive rods.
b.in acute cases of Erysipelothrix disease, colones are smooth with short slender, straight or
slightly curved organism while in chronic cases, colonies are rough with long-filamentous
organisms.
Cultivation
a.
on
BAPalpha-hemolytic,
small
round
b. on gelatin medius- lamp brush or test
c. tellurite medium- black colonies.
and
tube
grayish
white
colonies
brush type of growth.
a. stained smears
b.culture
i. heart infusion agar with 1% flucose and incubated in 5% co2
ii. BAP etc.
c. Biochemical characteristics.
i. oxidase and catalase negative
ii. Produce H2S in TSI
iii.nitrate (-)
iv. Ferments glucose and lactose
Treatment
a. penicillin- drug of choice
b. erythromycin- for penicillin allergic patients.
c. other alternatives tetracyclines, cephalosporins, clindamycin and chloroampenicol.
NOCARDIA
Morphology
a. aerobic, gram-positive, partially acid fast bacilli that are delicate, multiply branched with
beaded filaments
b. partially acid-fast when stained according to kinyouns method; with 1-2% h2so4 as
decolorizers, most strains of nocardia strain acid fast.
c. the organisms can also be stained with Gomorimethenamine silver stain.
Cultivation
a. grow on the most laboratory media like Lowenstein-jensen, brain-heart infusion, sabouraud
dextrose agar etc.
b. colonies are heaped, irregular, waxy shiny, bumpy or velvety.
c. utilized parafiin as a source of carbon and energy.
Clinical infection: nocardiosis
a. Habitat: soil and water
b. species causing nocardiosis: mainly N. asteroids and rarely N. brasiliensis and N.
otitidiscaviarum. (N. cavaie)
c. mode of transmission
i. inhalation of organism
ii. Inoculation through breaks and the skin.
(118)
d. epidemiology and manifestations
i. infection is chronic but occasionally fulminant and usually see in immunocompromised
patients, particularly following renal or cardiac transplantation.
ii. Begins as chronic lobar pneumonia which may disseminate hematogenously to any
organ but with preference to the central nervous system resulting in single or multiple brain
abscesses.
Laboratory diagnosis
Specimens: sputum, skin lesions, tissue biopics or surgical material
a. stained smears
b. culture
c. biochemical characteristics: urease (+) lysozymes resistant
d. gas liquid chromatography (GLC) for cell wall analysis.
Treatment
a.sulfonamides
b.trimethoprin
c.amakacin and imipenem
OTHER AEROBIC NONSPOREFORMING GRAM-POSITIVE BACILLI
Other aerobic actinomycetes- noncadiopsis, actinomadura (causative agent of
actinomycetoma),dermatiphilus, streptomyces, thermoactinomyces, sacharomonospora and
Micropolyspora
Rothia, kurthia, Oorskovia and lactobacillus.
(119)
CHAPTER XI
AEROBIC SPORE-FORMING GRAM POSITIVE BACILLI
BACILLUS
A. Bacillus anthracis
1. Morphology
a. Facultative, large, square ended, boxcar-shaped, encapsulated, nonmotile, gram
positive rod with an ellipsoidal to oval centrally located spore; sporangium is not
swollen (Bamboo fishing rod)
b. They occur in long chains, giving a bamboo pole appearance.
2. Cultivation
a. Optimal growth temperature is at 35C
b. On BAP, colonies are nonhemolytic, large, raised, opaque, grayish white with
irregular, fringe-like margin and cut glass appearance
c. Comma-shaped outgrowths or tangled masses of long hairlikecurlsare common
giving rise to Medusa or lion head colonies
d. The colonies are tenacious thus when pushed and lifted with an inoculating needle
they stand up like beaten egg whites
e. In aelatine, growth is described as inverted fir or pine tree
3. Determination of Pathogenecity
a. Capsule
b. Anthrax toxin consists 3 components: protective antigens, lethal factor, and
edema factor; responsible for the signs and symptoms.
4. Clinical Infection : Anthrax
a. Anthrax infection is primary a disease of lower animals (sheeps and cattles) which
rarely infects humans
b. Three forms of anthrax:
i.
ii.
iii.
c. Septicaemia can occur in all three forms and may lead to fatal meningitis
5. Laboratory Diagnosis
Specimens: swabs of vesicles and eschar, sputum, stool, blood and CSF(cerebrospinal
fluid)
a.
b.
c.
d.
e. Ascoli test
i.
ii.
B. Bacillus cereus
1. Morphologically similar to B. anthracis but usually motile
2. Cultivation
Colonies are beta-hemolytic, small, shiny, and compact or large, feathery and
spreading.
3. Differential Points Between B. cereus and B. anthracis
(121)
a.
b.
c.
d.
e.
f.
g.
Motility
Capsule
Hemolysis
Growth at 45C
Salicin fermentation
Penicillin sensitivity
Gamma phage
B. cereus
B. anthracis
motile
nonencapsulated
beta-hemolytic
(+)
(+)
resistant
resistant
nonmotile
encapsulated
nonhemolytic
(-)
(-)
sensitive
sensitive
4. Clinical Infection
a. An important cause of food poisoning which are of two types :
i. short incubated type associated with fried rice
ii. long incubated type associated with meat or vegetable dishes
b. Seen in serious infections associated with immunocompromised hosts
5. Treatment
- Susceptible to chloramphenicol, aminoglycosides, clindamycin, erythromycin, and
vancomycin
Bacillus subtilis (Hay bacillus)
(122)
CHAPTER XII
ANAEROBIC GRAM-POSITIVE BACILLI
CLOSTRIDIUM
General Characteristics
1. Most are obligate anaerobes but some are aerotolerant like C. histolyticum and C. tertium
2. All are motile with peritrichous flagella except C. perfringens, C. ramosum, and C.
innocuum.
3. All can readily sporulate in vitro except C. perfringens, C. ramosum, and C. clostridioforme.
4. All have swollen sporangia except C. perfringens and C. bifermentans.
5. All are encapsulated except C. perfringens.
6. Colonies are large with irregular with irregular edges or swarming growth (which occurs only
in anaerobic plate) but some may form small, convex, nonhemolytic colonies with an entire
edge (e.g., C. ramosum, C. clostridioforme.)
7. All are usually catalase negative
8. All are Nagler-negative except C. perfringens, C. baratti, C. bifermentans and C. sordellii.
9. They are widely distributed in soil, dust and water and are common onhabitants of the
intestinal and genital tract.
4. histolyticum
5. C. sordellii
6. C. fallax
1. Morphology
a. Short, plump, boxcar-shaped, gram-positive rod which is encapsulated and
nonmotile
b. Does not produce spores in ordinary media
c. Subterminal spores but impossible to demonstrate
2. Cultural and Biochemical Characteristics
a. On BAP circular and smooth colonies with a characteristic target or double zone
hemolysis resulting from a narrow zone of complete hemolysis due to
theta-toxin and a much wider and a much wider zone of incomplete (123)
hemolysis due to alpha-toxin.
b. In chopped-meat glucose media abundant growth with gas formation
c. In milk media stormy fermentation due to the excessive gas production
d. Lecithinase test some Clostridia species like C. perfringens possess lecithinase
which splits lecithin (a normal component of egg yolk) to
insoluble diglycerides, resulting in an opaque halo surrounding
a colony growing on egg yolk agar.
e. Nagler or Lecithovitellinrection
Procedure
i. Swab one-half of an egg yolk agar plate with C. perfringens type A
antitoxin and allow to dry
ii. Inoculate test organism on both halves of plate
iii. Inoculate anaerobically 24-48 hours at 37C
Result and Interpretation
i. Disappearance or reduction of the opacity on the antitoxin half of the plate
which denotes neutralization of the type a lecithinase (+Nagler test)
ii. The reaction is caused by alpha-toxin (a lecithinase C) which is
specifically inhibited by C. perfringens antitoxin.
f. Ferments glucose, lactose, maltose and fructose but not xylose
3. Clinical Infection
a. Wound and tissue infection caused by Type A strains of C. perfringens ;
symptoms are attributed to alpha-toxin
i. Simple wound contamination
ii.
iii.
iv.
v.
Anaerobic cellulitis
Clostridialmyonecrosis
Uterine infection
Clostridialsepticemina
b. Food poisioning caused by type A strain that produce heat resistant spores
and minimal amounts theta toxin; mode of transmission is
by ingestion of enterotoxin (108 - 103 viable bacteria).
Third most common cause of food poisoning in US.
c. Enteritis necroticans (necrotizing jejunitis or necrotic enteritis)
- caused by type C strains ingested from inadequately
cooked pork; also called pig-bel and Darmbrand :
symptoms are due to beta-toxin.
d. Post abortion sepsis
4. Laboratory Diagnosis
Specimens: wound tissues, aspirates, deep swabs, feces and samples of ingested food.
a. Direct and Gram stained smears
b. Culture and biochemical tests
i.
ii.
iii.
iv.
v.
(124)
c. ELISA
5. Treatment
a. Surgical removal or debridement of necrotic tissues
b. Intensive antibiotic (Penicillin) and antitoxin therapy
Antitoxin
Debridement of wound and removal of foreign bodies
Penicillin, tetracycline or metronidazole for penicillin-allergic patients
Barbiturates and diazepam for control of spasms
Careful control of environment
7. Prevention active and passive immunization. DPT 2,4,6 months and booster 12-15
months 1-6 years
C. Clostridium botulinum (Bacillus botulinus or Canned-good bacillus)
Produces 7 types of botulinum toxins. The most common are A,B,C and F.
1. Morphological and Cultural Characteristics
a.
b.
c.
d.
2. Botulinum Toxin
a. One of the most potent exotoxin known
b. A neurotoxin which attaches to individual motor nerve terminals, blocking the
release of acetylcholine at the nerve endings
3. Types of Botulism
a. Food-borne botulism lethal food poisoning which results from ingestion of
preformed toxin in contaminated food
b. Infant botulism related to ingestion of spores, multiplication of organisms
withinthe gastrointestinal tract and subsequent absorption of
toxin.
c. Wound botulism- the least common which occurs with the introduction of spores
or bacteria into wound.
d. Unclassified botulism- occurs in patients over one year of age who have clinical
symptoms of botulism but with no, identifiable vehicle of
transmission.
4. Laboratory diagnosis
5. Treatment
a.
b.
c.
d.
(127)
ACTINOMYCES
1. Normal flora of the mouth, GIT, and female genital tract.
2. Morphological and Cultural Characteristics
a. Branching and beaded with diptheroid and coccobacillary forms that are nonsporogenous
and nonacid-fast
b. Gram stain of sulfur granules shows tangled mass of long filamentous forms
c. Most are facultative anaerobes, growing best in the presence of CO 2; A. israelii and A.
bovis are anaerobes
d. Generally Actinomyces produce colonies that are smooth, flat to convex, gray-white, and
translucent with entire margins; some may show beta-hemolysis like A. pyogenes and A.
odontolyticus
e. A. israelii with molar tooth colony on brain-heart infusion agar described as white,
heaped, rough and lobate; may also produce breadcrumb-like, raspberry or smooth
colony; tight aggregates (balls) in thioglycollate broth
3. Clinical Forms of Actinomycosis
Actinomycosis is a chronic suppurative and granulomatous infection characterized by
pyogenic lesion with interconnecting sinus tracts that contain granules (also called grains or
sulfur granules). These granules are composed of tissue elements and the organisms.
a. Cervicofacial actinomycosis
- Caused by a trauma to the oral mucosa permitting endogenous bacteria to breach the
normal defenses of the mucosa (e.g., following dental caries and gingival disease)
b. Thoracic actinomycosis
- Acquired by aspiration of the bacteria or as extension from the cervicofacial form
leading to pulmonary infection
c. Abdominal actinomycosis
- Initiated by a traumatic perforation of the intestinal mucosa (e.g., ruptured appendix
and ulcer)
d. Genital actinomycosis
- Seen in women using intrauterine devices (IUD)
4. Laboratory Diagnosis
Specimens: sputum, pus, tissue-sections and cervical exudates
a. Examine specimens for presence of granules
- Yellowish color is due to abundance of macrophages containing lipid vacuoles
b. Stained smears
i.
Gram stain
ii.
Fluorescent antibody stain
(128)
ii.
Indole negative
iii.
iv.
5. Treatment
i. Penicillin G drug of choice
ii. Alternative tetracycline, clindamycin and erythromycin and sulfonamide
BIFIDOBACTERIUM
1. Normal flora of the GIT and oral cavity.
2. Morphology
-
Various shapes, with clubbing or bifurcated ends and may show diphtheroidal and
branching forms
3. Culture
-
B. dentium (B. eriksonii) produces white, convex, shiny colony with irregular edge and
has a diffuse growth in broth; they are acidophilic and grow best on low pH agar (e.g.,
tomato juice agar)
4. Biochemical Characteristics
a. Produces large amounts of acetic and lactic acids in peptone yeast glucose broth, with
acetic acid in much greater quantity than lactic acid
b. Generally indole-, nitrate-, and catalase-negative with rare catalase-positive strain
5. Very rarely causes Actinomycosis
EUBACTERIUM
1. Normal Flora of the GIT and mouth.
2. Morphology
a. Generally pleomorphic rods to coccobacilli occurring in pairs or short chains but also as
straight uniform or curved rods
b. E. alactolyticum sea-gull-wing shape forms
c. E. nodatum beading, filamentous and branching similar to Actinomyces
d. E. lentum (most common Eubacteria) small, straight rod with rounded ends
3. Culture
a. Colonies are raised to convex and transparent to translucent
b. E. nodatum colonies are similar to A. israelii
4. Identification
a. Usually identified by exclusion
b. They are obligate anaerobes, nonmotile and catalase negative
(129)
to
grow
3. Identification
a. produce large amounts of lactic acid with or without smaller amounts of acetic acid
b. nonmotile, indole-, catalase-, and nitrate-negative
4. Clinical Infection
a. pleuropulmonary infections caused by L. catenaforme
b. dental caries, UTI, bacteremia, endocarditis, local suppurative infections, and
chorioamnionitis
5. Treatment: penicillin
PROPIONIBACTERIUM
1. Normal flora of the skin, GIT, upper respiratory tract (ant. Nares), and urogenital tract
2. Morphology and General Characteristics
a. irregular, pleomorphic, and club-shaped with one end round and the other tapered
(anaerobic diphtheroids); but sometimes short branching and beading resembling
Actinomyces
b. colonies of P. acnes are small and white to gray-white becoming yellow as they get
older and are sometimes beta-hemolytic
c. species are aerotolerant but grow better anaerobically
d. grow well in broth especially with Tween 80
3. Identification
a. produce large amount of propionic and acetic acids with lesser amount of lactic,
succinic and isovaleric acids in peptone yeast glucose broth
b. catalase positive except for P. propionicus (Arachnia propionica) and a few other
strains
4. Clinical Infection
a. frequently contaminants of blood culture and other sterile body fluids
b. P. acnes
ENTEROBACTERIACEAE
GENERAL CHARACTERISTICS
1. Small, straight sided, facultative anaerobic, gram negative, non-sporeforming rods
2. All motile members possess peritrichous flagella except Tatumella ptyseos which has polar
flagella. Shigella and Klebsiella are non-motile. Some species of Escherichia, Salmonella and
Yersinia are non-motile too.
3. All members grow luxuriantly on BAP as moist, smooth, gray, shiny, entire, covex and
opaque colonies. Smooth to rough variations can occur. Beta-hemolysis is seen in some
strains.
4. All are catalase positive except for one group of Shigella species and a species of
Xernorhabdus.
5. All members ferment glucose but do not liquify alginate and are oxidase negative.
6. Almost all are able to reduce nitrate to nitrites.
7. Predominant facultative flora in the human bowel.
8. Most species are not intestinal pathogens but opportunistic organisms which are responsible
for the majority of nosocomial infections.
CULTURE MEDIA
1. For fecal specimens (including rectal swabs)
a. Differential media
i.
MacConkey agar divides Enterobacteriaceae into:
- Lactose fermenters pink or reddish colonies or bile lactose media and
are usually non-pathogenic
- nonlactose fermenters colorless, translucent colonies and are usually
pathogenic
ii.
Eosin methylene blue (EMB) also divides Enterobacteriaceae into lactose
and nonlactose fermenters; aside from
lactose it also contains sucrose
iii.
Desoxycholate agar
b. Selective media
i.
Hektoen enteric agar (HE) contains lactose, sucrose and salicin; able to
detect H2S production
ii.
Xylose lysine deoxycholate (XDL) contains lactose, sucrose and xylose;
able to detect H2S production
iii.
Salmonella-Shigella agar
iv. Desoxycholate citrate agar
2. For other specimens like material from wounds, respiratory tract secretions, aspiration from
abscesses, sterile body fluids, urines, tissue, and blood cultures
a. BAP or chocolate agar (supportive medium to allow growth of all enterobacteriaceae) and
b. McConkey agar (differential medium for gram negative bacilli)
(131)
Enrichment broths
a. selenite broth
b. G-N broth
c. tetrathionate broth
CHEMICAL TEST (refer to Chapter III)
2. Determinants of Pathogenecity
a.
b.
c.
d.
e.
(133)
Edwardsiella
E.tarda
1. Type specie of this genus
2. Isolated from humans and associated with diarrhea, wound infections and sepsis.
3. Biochemical Characteristics
a. MacConkey nonlactose fermenter
b. TSI- K/A + H2S +gas
c. IMVIC - ++-d. Lysine decarboxylase (+)
e. Ornithine decarboxylase (+)
Shigella
1. General Characteristics
a. Slender, aerobic, nonmotile, nonencapsulated, gram negative rods
b. Generally nonlactose fermenters and do not produce H2S
c. Do not produce gas from glucose (anaerogenic)
d. All species can cause bacillary dysentery and are the major cause of it.
2. Classification
a. According to mannitol fermentation
Nonmannitol fermenters
S. dysenteriae (Dysentery of Shiga Bacillus) type specie of the genera
Mannitol fermenters
i.
Nonlactose fermenters
S. flexneri (Strongs Bacillus)
S.boydii (Boyd Bacillus or S. ambigua)
ii.
b.
(134)
According to serogrouping
i. Group A S. dysenteriae
ii. Group B - S. flexneri
iii.
Group C S. boydii
iv. Group D sonnei
3. Factors contributing to Virulence
a. Smooth lipopolsaccharide (LPS) structure may be the one responsible for the
organisms ability to resist gastric acidity
b. Invasiveness
c. Shiga toxin interferes with protein synthesis and is neurotoxin, cytotoxic and
enterotoxic
4. Clinical infection (Bacillary dysentery or Shigellosis)
a. S. sonnei is the most common isolate worldwide followed by S. flexneri and to a
lesser extent S. boydii and S. dysenteriae, however in developing countries S.
dysenteriae and S. boydii are more frequently isolated, followed by S. flexneri
then S. sonnei.
b. Mode of transmission: fecal-oral route
c. Clinical manifestations
i.
Salmonella
1. General characteristics
a. Nonencapsulated, nonsporeforming, gram (-) rods
b. All members are nonlacyose fermenters
c. All are motile except S.pullorum and S. gallinarum
d. All produce gas from glucose except S. typhi and S. gallinarum
e. Most strains produce H2S from thiosulfate
2. Classifications
a. According to Ewing and coworkers (based on O & H antigens known as the
Kauffmann-White antigenic scheme) there were only three species of Salmonella:
S. typhi (S.typhosa or Eberths bacillus) : all other species or serotypes were
identified as serotypes \s. enteritidis like:
i. S. paratyphi A
ii. S. paratyphi B or S. schottmuelleri
iii.
S. paratyphi C or S. hirschfeldii
iv. S. typhimurium
v. S. sendai
vi. S. pullorum
vii. S. gallinarum
viii.
S. derby
b. According to DNA hybridilization studies all Salmonella and organisms formerly
referred to as genus Arisona belong to the same species, S. enterica and now
classified as:
i.
ii.
iii.
iv.
v.
vi.
vii.
2,
salamae
3a,
arizonae
3b,
diarizonae
4,
houtenae
5,
bongori
6,
indica
Majority of human isolates belong to subgroup L.
3. Antigens produced
a. O antigen somatic antigen
b. H antigen flagellar antigen which exist in 2 phases:
i. Phase 1 (specific phase) shared by a few organisms and
react with homologous antisera
ii. Phase 2 (nonspecific phase) - shared by many organisms
and react with heterologous antisera
c. Vi (Virulence) antigen a type of capsular (K) antigen found in serotype typhi
which may prevent the intracellular destruction of the organism.
4. Factors Contributing to Virulence
a. Surface antigen
i. O antigen - somatic
ii. Vi antigen virulence
b. Invasiveness
c. Endotoxin
d. Enterotoxin similar to both the heat-labile (LT) and heat-stable enterotoxin (ST)
of E.coli
e. Cytotoxin
5. Clinical infection: Salmonellosis
a. Mode of transmission
i. Typhoid fever is transmitted by ingesting food or water contaminated with
the feces of a carrier, often a chronic carrier (person who has recovered
from typhoid fever ut harbors the organism asymptomatically in the
gallbladder for long periods of time) : silent carriers (asymptomatic
carriers without ever suffering the disease) also transmit the disease and
contribute to continued episodes of infections: humans carrier is the sole
source of the organism (S. typhi)
ii. Nontyphoidal salmonellosis is transmitted by ingestion of contaminated
food (usually eggs, poultry and beef products) and water; fecal-oral
routes : aside from the human source, animals, and animal products are the
major sources.
*NOTE: the mean infective dose to produce infection is 105-103 organisms
b. Three distinct clinical entities
i. Gastroenteritis
- most commonly causedby S. serotype typhimurium
- characterized by diarrhea, fever and abdominal pain
- self-limiting, last from 2-5 days
ii. Typhoid fever and other enteric fevers
- typhoid fever is the most severe enteric fever which is caused by
serotype typhi\
- other Salmonella, particularly serotypes paratyphi A and B, can also
cause enteric fevers with milder symptoms
- signs and symptoms lethargy, fever, malaise, constipation and body
aches and pain during the first week followed by sustained fever 104F,
tender abdomen with rose-colored spots and diarrhea during the 2nd and
3rd week
- complications intestinal perforation, severe bleeding, thrombophlebitis,
cholecystitis, pneumonia, and abscess formation
iii.
8. Control
a. Proper cooking and storage of food
b. detection and treatment of carriers
c. oral vaccine of attenuated typhoid bacillus
Arizona (Salmonella enterica subgroup 3a-arizonae)
1.
2.
3.
4.
C. amalonaticus
+
+
-
C. freundii
+
+
+
C.diversus
+
+
Klebsiella
General Characteristics
- genus consist of 5 species: K.pneumoniae, K.oxytoca, K.planticola, K. terrigena, and K.group
47: two former species, K.ozanae and K. rhinoscleromatis, are biochemically inactive strains of
K.pneumoniae.
- all are encapsulated and nonmotile
- all are lactose fermenter except K. ozanae and K.rhinoscleromatis
- colonies are large, moist and mucoid
- they possess O and K antigen
- urease (+) and ornithine decarboxylase (-)
1. K.pneumoniae (Friedlanders bacillus or baciluusmucosuscapsulatus)
a. Type specie of the genera
b. Disease produced
i.
Community-acquired and nosocomial pneumonia
ii.
Can also cause UTI, wound infections, bacteremia, and meningitis
iii.
Tropical sprue caused by an enterotoxigenic strain
c. Laboratory diagnosis
i.
Stained smears
ii.
iii.
I.
Culture large, oist, mucoid colonies on EMB, MacConkey and XL, that
have a tendency to coalesce( (+) to strings test)
Biochemical characteristics
TSI = A/A + gas
IMVIC = - - + +
Urease (+)
Grows in KCN medium
Quellang reaction for presence of capsule
Serotyping
iv.
v.
2. K. oxytoca
- Resembles K.pneumoniae in its disease spectrum
- IMVIC = + - + +
3. K.ozanae
- May contribute to the condition called ozaena which is a chronic atrophic
rhinitis characterized by a fetid odor
4. K.rhinoscleromatis
- Causative agent of rhinoscleroma, a chronic, granulomatous infection of the
nasal passages, pharynx and larynx
5. K.planticola UTI, has been implicated in human urinary tract and wound infections
6. K.terrigena isolated only from the environment
7. K.group 47 isolated from resoiratory tract and blood
Enterobacter (formerly Aerobacter)
1. General Characteristic
a. Inhabits soils and water and , to a lesser extent , the large , the large bowels of man and
animals
b. Oftenly confused with klebsiella
c. Most species are rapid lactose fermenters and urease (+)
d. All are motile and with the exception of E. agglomerans ,
e. All are ornithine decarboxylase (+)
2.
UTI
Bacteremia
Tropical sprue caused by enterotoxigenic E. cloacae
Meningitis and brain abscesses
Wound infections
Serratia
1. General characteristic
a. Motile gram- negative rods which are nonlactose fermenters but sucrose fermenters
b. Found in soil and water and are associated with plants and animals
c. Majority of S. rubidaea and some strains of S. marcescens produce a non water soluble
red to pink pigment (Prodigiosin)
d. S. orodifera produces a very musty , pungent odor
2. Biochemical Reactions
a. TSI = K/A or A/A
b. IMVIC = - - + +
c. With the exception of S. fonticola , all are extracellular Dnase , lipase and
gelatinase positive and resistant to colistin and cephalotin
3.
Proteus
1. General Characteristic
a. Motile , pleomorphic , gram- negative rods which are nonlactose fermenters
b. All are urease and phenylalanine deaminase (+)
c. All produce a bluish gray confluent surface growth or translucent sheet of growth on
moist blood agar that gives off a burnt gun powder odor
2. Other Biochemical Reactions
a.TSI = K/A + GAS + H2S
b. IMVIC
i
P. vulgaris = + + - V (indole (+) member)
ii.
P. mirabilis = -+ V V
Ornithine decarboxylase
i.
P. vulgaris negative
ii.
P. mirabilis positive
3. Dienes Phenomenon
Different strains of proteus when inoculated separately on a culture media swarm
towards each other but they do not merge and are separated by a narrow demarcation
line between them.
4. Antigenic Structure
a. All posses O, H and K antigens
b. P. vulgaris have the same antigenic structure as rickettsia such that O antigens (OX19 , OX-K , OX-2) of some strains are used to detect the rickettsial antibodies in the
the Weil-Felix test.
5. Clinical Infections
a. Majority of human infections are caused by P. mirabilis.
b. It is the second leading causes of community- acquired UTI and is a major cause of
nosocomial infections.
6. Treatment : sensitive to ampicillin and cephalosporins
K.
Providencia
1. Biochemically related to proteus.
2. Consists of four species :
a.
b.
P. stuartii
c.
d.
p. rustigianii
4. Biochemical Reactions
a. TSI = K/A
b.
IMVIC = + + - +
L.
Morganella
1. M. morganii (Proteus morganii) is the only species.
2. Biochemical Reactions
a. TSI = K/A + gas
b. IMVIC = + + - c. Phenylalanine deaminase (+)
d. Urease (+)
M.
2.
Cultivation
a. Ca2+ dependency when deprived on Ca2+ the bacteria are stimulated to produce V
& W antigens and several outer membrane proteins (Yops)
b. V & W antigens render the organism less susceptible to intracellular killing
c. Outer membrane proteins
d. F-1 envelope antigen may resist phagocytosis
e. Pigment binding and iron-regulated surface proteins
f. Pesticin I (a bacteriocin) , coagulase and plasminogen activator promotes
invasiveness
g. Murine toxin , endotoxin , etc.
Y.pestis is primarily pathogenic to rodents and humans are accidental host only
b. Clinical forms
i.
bubonic plague
- characterized by infection and swollen lymph nodes (called buboes) which occur
most commonly in the groin and less frequently in the axillary and cervical nodes
ii.
septicemic plague
-
Highly fatal
iii.
5. Laboratory Diagnosis
Specimens: aspirates from buboes , pus from flea bites area , sputum , throat swabs or
blood; these specimens should be placed in Cary- Blair transport medium
i. TSI = K/A
ii. IMVIC = - + - iii. urease (-)
2. Disease Produced :
a. Yersiniosis a term denoting infection with Yersenia species other than Y. pestis ;
enteritis that mimics acute appendicitis
b. Mode of transmission : accidentally acquired via fecal-oral route
c. Clinical manifestation :
i.
involvement
ii.
iii.
3. Laboratory Diagnosis
Specimens : mesenteric lymp nodes , feces , blood , effusions , from serous cavities and
organ specimens
a. Culture
i. noncontaminated samples blood or nutrient agar
ii. for selective enrichment and holding specimens are placed in isotonic saline with
or without potassium tellurite and promptly refrigerated
iii. CIN agar for Y. enterocoloitica (cefsulodin-irgasan-novobiocin)
b. Biochemical test
c. Widal type agglutination test most specific serologic test wherein a titer of 1:160 or
greater is indincative of yersiniosis
4. Treatment
i. aminoglycosides and cotrimoxazole
ii. correction of fluid and electrolyte imbalance
iii. supportive theraphy
CHAPTER XIV
NONFERMENTATIVE GRAM-NEGATIVE BACILLI & COCCOBACILLI
Basis of classification
1. This group of organisms is differentiated from the Enterobacteriaceae in the
way they utilize carbohydrates. They do not ferment sugars (in the absence of
air ) but utilize them oxidatively (in the presence of air ) producing tiny
amounts of acid.
ii.
To detect acid production in these organism Hugh and Leifson
developed a low-peptone medium (0.2%) , the oxidative-fermentative
mediem (O-F medium)
i.
c. interpretation of results
ii. oxidizers- organism that give a yellow reaction only in the open tubes
indicating oxidative utilization of glucose
iii.
nonoxidizers- do not utilize glucose , either fermentatively or
oxidatively.
PSEUDOMONAS
A. Pseudomonas aeruginosa
1. Morphology
a. Aerobic , motile with a single polar flagellum , gram negative rods that
occur singly , in pairs and occasionally in short chains
b. Produces an extracellular slime layer, similar to a capsule usually seen
in mucoid strains isolated from patients with cystic fibrosis
c. Frequently posses pili that promote attachment to host cell surfaces
2. Cultural Characteristics
i. Muller-Hinton agar
ii. Pseodomonas P. Agar
iii. Tech agar
f.
3. Virulence Factor
a. Motility
b. Presence of glycocalyx (slime layer)
c. Pili of fimbriae
d. Endotoxin
e. Extracellular enzymes including elastases, proteases , and 2 hemolysin; a heat
labile phospholipase C and a heat-stable glycolipid
f. Exotoxin A- causes tissue necrosis
g. Exoenzymes S- play a role in necrotic injury
h. Cytotoxin and enterotoxin
4. Diseases Produced
P. aeruginosa inhibits soil and water and causes disease in human with impaired host
defenses.
5. Laboratoty Diagnosis
a. Flagella media
b. Culture (isolation of organism)
i. selective media
= Pseudosel agar- containing cetrimide (cetrimethyl ammonium bromide)
=irgasan agar
=30 ug/ml C-390
ii. media enhancing fluorescent pigment production
=Pseudomonas F agar
=GNF agar
=Flo agar
c. biochemical reactions
i. TSI-K/K with a metallic sheen on slant surface
ii. open OF medium (+) & closed OF medium (-) utilizing glucose oxidatively
iii.oxidase and catalase (+)
iv. unable to oxidize lactose, sucrose and maltose
v. unable to decarboxylate lysine or ornithine
vi. able to dihydrolyze arginine
d. serotyping of O antigen, bacteriophage typing and pyocin typing
- for epidemiologic purposes
6. Treatment
a. aminoglycosides (amikacin, gentamicin,& tobramycin)
b. extendedspectrum penicillins (azlocillin, carbenicillin)
c. 3rd generation cephalosporins (ceftazidime and cefoperazone)
d. quinolones and carbapenems
B.Pseudomonas cepacia
-most commonly isolated from cystic fibrosis patents and has been associated with
endocarditis,
septicemia and wound and urinary tract infections in immunocompromised patients
-colonies are yellow-yellow green, rough with serrated edges which do not fluoresce
-strains are motile and able to oxidize glucose, lactose, maltose and mannitol; lysine
decarboxylase
(+)
-resistant to polymyxin B and sensitive to chloramphenicol, cotrimoxazole and ceftazidime
-causes
contact
through skin abrasions and inhalation of organism
-they are small, nonmotile, aerobic, pleomorphic, cocoid to rod shaped organisms which are
oxidase negative
-colonies on heart-infusion agar are grayish white and translucent later becoming
yellowish and
opaque
-Straus test: test use to diagnose glanders, wherein male guinea pigs are inoculated
intraperitoneally with suspected material; within 2-3 days the animals develop
orchitis (tender swollen scrotum), with tumefaction and purulent inflammation of
testicles
-glanders is treated effectively with tetracycline plus an aminoglycoside
D.Pseudomonaspseudomallei(Whitmores bacillus)
F. Other Pseudomonas
-represent contaminants or colonizers but may cause opportunistic infections
ALCALIGENES
1. Opportunistic pathogens isolated from blood , respiratory secretions, infected wounds, urine
and other sources
3. General Characteristics
ACINETOBACTER
1. Commonly found in soil and water but occasionally found on skin and mucous membranes of
healthy persons.
2 biovars:
a. A. calcoaceticus var. anitratus (Herelleavaginicola) - oxidizer
b. A. calcoaceticus var. Iwoffi- ( Mimapolymorpha) nonoxidizer
3. General Characteristics
4. Occasionally causes nosocomial infection and may be involved in wound infections, UTI,
sepsis and other infections
MORAXELLA
General Characteristics
B. Moraxellaosloensis
1. Small coccoid bacilli that are asaccharolytic, oxidase and catalase positive.
D. Moraxella phenylpyruvica
3.Cultivation
a. blood is required for growth and chocolate agar is the most supportive medium.
b. colonies are tiny with pitting (ability of organism to corrode the agar), yellowish, opaque and
consist of three distinct zone of growth:
4. Biochemical Reactions
6. Susceptibility
FLAVOBACTERIUM
2. Morphology
-Long, thin, filamentous, nonmotile gram negative bacilli with occasional swollen ends.
3.Cultivation
WEEKSELLA(W. zoohelcum)
CHAPTER XV
GRAM-NEGATIVE FACULTATIVELY ANAEROBIC BACILLI
1. Morphology
2. Cultavation
i. glucose-cysteine-blood agar
ii. peptone-cysteine agar
iii. cystine heart agar
iv. chocolate agar
v. rarely on Thayer-Martin agar
3. Virulence Factors
Strains of F. tularensis
biovartularensisbiovarpalaearctica
i. or Jellison type A
i. or Jellisons type B
of rabbits
iv. exhibitcitrullineureidaseactivity
v. ferment glycerol
of rodents
iv.do not exhibit citrullineureidase activity
v. seldom ferment glycerol
5. Disease Produced:
A disease of rodents (particularly rabbits) that is usually seen in hunting men. It is also
one of the most common laboratory acquired infections.
a. mode of transmission
b. clinical forms
ii. ulceroglandular most common form; with ulceration on the site of inoculation and
regional lymphadenitis; with systematic manifestation such as anorexia,
back pain, headache, chills, and fever, sweating and prostration;
pneumonia may also occur.
iii. oculoglandularsimilar to ulceroglandular with the conjunctival sac as its primary source
of inoculation.
6.Laboratory Diagnosis
Specimens: blood, pharyngeal secretion, gastric aspirates, pleural fluid, tissue secretions,
exudates, etc.
a. generally smears and culture are not contributory and the diagnosis relies on serological
tests.
b. serologic tests
i. direct and indirect fluorescent antibody staining best technique for rapid specific
diagnosis from exudates or tissues impressions
ii. agglutination test (Foshays antiserum test) commonly used serologic method
iii. ELISA
iv. lymphocyte stimulation assay
v. tularemia skin test
7. Treatment
8. Prevention
a. avoidance from infected animals, protection from biting insects and provision of clean
watersupplies
b. immunization (by multiple puncture) with live attenuated strain of F. tularensis for all
laboratory personnel handling culture of organisms
BRUCELLA
1. Pathogenic Specie
Preferred
CO2
H2S
Host
Req.
Prod.
B. abortus
cattle
(1:50,000)
goats, sheep
B. suis
swine
B. canis
dogs
(Bangs bacillus)
B melitensis
2. Normal flora of genital and urinary tracts of animals such as goats, sheeps, pigs, cows and
dogs.
3. Morphology
4. Cultural Characteristics
-colonies are small, convex, smooth, translucent and slightly yellow to opalescent which may
become brownish with age.
a. mode of transmission
i. contact with infected tissues of animals (common among abattoir workers, farmers and
veterinarians
ii. ingestion of infected milk and milk products
b. pathogenicity
c. clinical manifestations
i. gradual onset of nonspecific systemic symptoms including fever, malaise, chills, sweats,
8. Laboratory Diagnosis
Specimens: blood (specimen of choice), bone marrow, material from lymph nodes, other
tissue, CSF, and urine.
a. Culture
i.
ii.
Cultures should be incubated in 5-10% CO2 humidified atmosphere at 3537C and should be retained for 3-4 weeks before being discarded as
negative.
Culture Media
- Castaeda biphasic medium (for blood and other body fluids)
- Trypticase soy agar
- Tryptose agar
b. Serologic Test
i.
particle agglutination test with antismooth Brucella serum (most rapid
presumptive identification test)
ii.
ELISA
iii.
2-mercaptoethanol test
iv.
Complement fixation
v.
Fluorescent antibody staining
9. Treatment
a. Doxycycling plus rifampicin for 30 days- for adults
b. Cotrimoxazole for 3 weeks and gentamicin IM for 5 days- in children less than
8 years old.
c. Doxycycling or oxytetracycline for 3 weeks with gentamicin- more than 8 years
old.
10. Prevention
a. Animal immunization
b.
BORDETELLA
Bordetella pertussis (Bordet-Gengou bacillus)
1. Morphology
a. Slightly aerobic, nonmotile, small, gram-negative coccobacilli that occurs
singly, in pairs and in small clusters.
b. Capsules are produced and demonstrated only by special stains.
c. Bipolar metachromatic granukes can be demonstrated with toluidine blue
stains.
2. Cultural Characteristics
Colonies are pinpoint, smooth, convex, glistening with a pearly luster, resembling
mercury drops and show a diffuse zone
of hemolysis.
3. Virulence Test
a. Portussis toxin - an exotoxin promotes lymphocytosis
-role in adhesion to ciliated epithelial cells.
b.
c.
Adenylate cyclase
d.
e.
f.
g.
Pertactin
h.
ii.
paroxysmal stage
5. Laboratory Diagnosis:
Specimens: nasopharyngeal swab (most commonly
nasopharyngeal aspirates, cough droplets expelled onto a cough plate.
recommended),
a. Culture Media
i.
Bordet-Gengou agar (potato-glycerol-blood agar)
ii.
Jones-Kendrick charcoal agar- for transport and cultivation
iii.
Regan-Lowe (half-strength charcoal agar medium with horse blood and
cephalexin)- as transport and selective enrichment medium
iv.
Buffered-charcoal-yeast-extract
v.
Cold casein hydrolysate medium
vi.
Casamino acid broth
vii.
modified Stainer- Scholte agar with cyclodextrin and cephalexinpreferred for 1 isolation
b. direct fluorescent antibody staining
c. Slide agglutination test
d. ELISA
e. biochemical reactions
i.
oxidase (+)
ii.
urease (-)
6. Treatment
a. erythromycin- drug of choices
b. tetracycline, chloramphenicol or cotrimoxazole- alternative
7. Prevention
-Active immunization with killed phase I B. pertussis combined with diphtheria
and tetanus toxiods (DPT)
B. Bordetella parapertussis
-Nonmotie, urease and oxidase positive
-Can cause mild respiratory disease similar to B.pertussis
C. Bordetella bronchiseptica
-Motile with lateral flagella
-Urease and oxidase negative
-Can cause mild respiratory disease similar to B. pertussis
HAEMOPHILUS
General Characteristics
Small, pleomorphic, nonmotile, aerobic, gram- negative bacilli.
Require the following factors present in the blood adequate growth:
2. Cultural Characteristics
a. colonies usually are very small and dewdrop-like, transparent and colorless
with a distinct mousy or bleachlike
odor.
b. exhibits the satellite phenomenon wherein colonies of H. influenza grow
luxuriantly next to the S. aureus streak
(due to the production of V factor)
3. Virulence Factors
a. capsule- associated with H. influenza type b invasiveness
b. outer membrane proteins- responsible for attachment, invasiveness and
resistance to phagocytosis
c. lipooligosaccharide (LOS)- exerts a paralyzing action on the ciliated respiratory
epithelium & promotes
proliferation of the organism
d. adherence
e. IgA proteases- hydrolyze human IgA
4. Clinical Infection
a. Mode of transmission
d. Porphyrin or ALA (delta-aminolevulinic acid) test for X factor requirementpresence of brick red to orange
fluorescence indicates that porphyrins
were produced and that the organism
does not require X factor.
8. Treatment
a. chloramphenicol alone or in combination with ampicillin- for meningitis and
epiglotittis
b. ampicillin or amoxicillin- drug of choice for otitis media in children;
alternatives are cotrimoxazole, cefaclor or
penicillin with sulfonamide.
c. ampicillin- for sinusitis
d. passive immunotherapy
9. Prevention
a. active immunization with purified capsular polysaccharide
b. passive immunization with intramuscular human hyperimmunoglobulin
C. H. parainfluenzae
- normal flora of mouth and nasopharynx
- infection after dental disease, dental procedures or other oral trauma
- ampicillin alone or with gentamicin is the recommend treatment
D. H. ducreyi
- sexually transmitted
1. Morphology: pleomorphic gram-negative coccobacilli which may occur extracellularly or
intracellularly.
2. Cultivation
a. requires X-factor for growth
b. media: chocolate agar medium containing vancomycin
4. Transmission: sexually
5. Clinical Manifestations
a. male ragged ulcer in the genitalia
- with enlarged painful inguinal lymph node
- multiple lesions occur by autoinoculation
b. female- asymptomatic or with mild vaginitis
6. Laboratory Diagnosis
a. Gram or Giemsa-stained smears of ulcer exudates or bubo aspirate showing the classic
microscopic appearance of a school of a
red fish
b. Ducreyis skin test
- a nonspecific test which is positive 1-2 weeks after injection and may remain
positive for years.
7. Treatment
- Peniciliin- drug of choice
FIGURE 15.1
Potential Properties of Haemophilus Species that colonize Humans
Species
V
Factor X
Factor Increased
Req.
Req.
Co2 Req.
Hemolysis
Influenzae
Aegyptius
+
+
+
+
+
-
Hemolyticus
Ducreyi
+
-
+
+
Parainfluenzae
Parahaemolyticus
+
+
+
+
+
+
-
Paraphrohaemolyticu
s
Hrophilus
Paraphrophilus
H.Agnis
+
-
+
+
+
-
+
+
+
-
ACTINOBACILLUS ( A. actinomycetemcomitans)
Most members are pathogens and commensal organism in domestic mammals birds. Only
A. actinomycetemcomitans is a human parasite.
2. Cultivation
a. grows on BAP and CAP but not on McConkey Agar
b. growth is enhanced in a CO2 incubator or candle jar.
c. in blood cultures bottles- microcolonies may be seen as tiny puffballs growing on the
blood cell layer or as tiny puffs on the sides of
the bottle.
d. on BAP- rpugh and sticky colonies surrounded by a light greenish tinge
e. on brain-heart infusion agar- four to six pointed starlike configuration in the center of
the colony.
3. Diseases Produced
a. often found in actinomycosis
b. severe periodontal disease in adolescents
c. endocarditis
d. abscesses, osteomyelitis and other infections.
4. Treatment
a. tetracycline or chloramphenicol usually
b. penicillin G, ampicillin or erythromycin sometimes
PASTEURELLA
Primarily parasites of domestic and wild animals and birds but may also produce disease
in humans. P. multocida is the most frequently associated with human infections.
1. Morphology
a. small, nonmotile, facultatively anaerobic, coccobacillary or rod shaped
organisms that occur singly, in pairs or in short chains and often exhibit
bipolar staining.
b. some strains show pleomorphism
c. virulent organisms produce capsule
2. Cultural Characteristics
a. does not grow in MacConkeys agar
b. on BAP colonies are small and translucent with a brownish discoloration of
the medium and amiting a musty or mushroom-like
3. Virulence Factors
a. capsule- major virulence factor
b. somatic antigens
c. endotoxin
d. neuraminidase and hyaluronidase
e. fimbriae
4. Clinical Infection
a. mode of transmission
i.
majority are through dog or cat bite or scratch
ii.
possibly inhalation
b. diseases produced
i.
wound infection from animal bites that may progress to
pyarthrosis, necrotizing synovitis and osteomyelitis
ii.
infection of lung in patients with preexisting chronic pulmonary
disease (usually lower respiratory tract disease)
iii.
other foci of disease that are secondary to septicemia like
meningitis, cerebellar abscess and infectious endocarditis.
5. Laboratory Diagnosis
Specimens: Sputum, bronchial washing, nasal swabs, purulent exudates from
animal bites, spinal fluid, blood, joint fluid, biopsy specimens from osteomyelitis
patients, etc.
a. stained smears
b. culture- most important
c. biochemical reactions
i.
catalase and oxidase (+)
ii.
ferments glucose, mannitol and sucrose
iii.
nitrate and spot indole (+)
6. Treatment
a.
b.
c.
d.
e.
f.
g.
h.
i.
j.
k.
l.
m.
n.
i.
all are oxidase (+) and non motile and able to ferment
ii.
glucose
o.
p.
q.
r.
s.
t.
u.
v.
w.
x. -120y.
z. 4.
Biochemical reactions
aa.
a.
there are 3 species: C. ochracea (formerly bacteriodes), C.
sputigena, and C. gingivalis
b.
they are oxidase, catalase and indole(-)
c.
glucose and sucrose fermenters
d.
urease, OD and LD(-)
ab. 5. Virulence Factors
ac.
a. proteases
b. heat-stable factor that alters polymorphonuclear neutrophil
chemotactic activity
ad. 6.
Diseases Produced
ae.
a.
periodontal disease; periodontal lesions in juvenile diabetes
patience
b.
sepsis in leukemia and granulocytopenic patients
c.
osteomyelitis, septic arthritis, endocarditis and soft tissue
infections
af. 7.
Treatment
ag.
a.
susceptible to penicillin, carbenicillin, clindamycin, and
erythromycin
b.
resistant to aminoglycosides
ah. CARDIOBACTERIUM (C. hominis)
ai. 1.
Normal flora of the upper respiratory tract, mouth and maybe
other mucous membranes
aj. 2.
Morphology
ak.
-pleomorphic, microaerophilic, non motile, gram-negative
bacillus that tends to form rosette clusters
or a serpentine pattern
al. 3.
Cultivation
am.
a. grows well on BAP, CAP, trypticase soy agar or heart
infusion agar
b. does not grow in McConkey agar
c. cultures are incubated at 37C in 5% CO
d. colonies are slightly alpha-hemolytic, smooth, round,
glistening and opaque
an. 4.
Biochemical Reactions
ao.
ap.
aq.
ar.
as.
at.
au. -121-
by.
de.
df.
dg.
dh.
b. F. necropharum
-umbonate colonies that may be alpha- or beta-hemolytic
4. Identification Characteristics
a. both produce large amounts of butyric acid
b. both are resistant to vancomycin and susceptible to
kanamycin and colistin
c. both are indole positive
d. F. necropherum is lipase (+)
5. Treatment
- susceptible to penicillin G, cephalosporins, metronidazole and
chloramphenicol
CHAPTER VII
VIBRIONACEAE
GENERAL CHARACTERISTICS
1. Gram-negative facultative organisms that are oxidase positive (except V. metschnikovii)
2. Most are motile with a polar flagella.
3. Naturally found in seawater (more commonly, Vibrio) and fresh water (more frequently,
aeromonas and
plesiomonas)
4. Causes sporadic cases or diarrhea and soft tissue infections.
VIBRIO
The two most important members of this genus are V. cholera and V. parahaemolyticus.
A. Vibrio cholera (V. comma or spirillum cholera asiaticae)
1.
Biotypes
TESTS
a.
b.
c.
d.
e.
soluble hemolysin
Voges-Preskeur
chicken red cell agglutination
polymycin B sensitivity
bacteriophage IV sensitivity
+
+
+
-
*el tor biovars display the typical beta-hemolysis (several years ago) however,
recent epidemics
have been caused by nonhemolytic strains
3.
4.
Morphology
a. small, facultative, gram-negative rods that are slightly curved or comma shaped
b. shows a characteristic darting motility by means of a single thick, sheathed polar
flagellum
5. Cultural Characteristics
a. requires an alkaline medium for growth
b. growth on TOBS agar shows medium-sized, smooth, opaque, thin-edged yellow
colonies which on
prolonged incubation turns green especially if the organism is the el tor biotype
6. Virulence Factors
Laboratory Diagnosis
Specimens: stool and rectal swabs
-rice water stools
a. Gram stained smears
b. culture
i. transport media
- Amies
- Cary-Blair modification of Stuarts medium
ii. selective media
- alkaline peptone water (enrichment broth)
- tellurite taurocholate gelatin agar (TTGA)
- thiosulfate citrate bile sucrose agar (TCBS)
- Gohar, Dieudonnes, Monsur and Aronson media
c.
not specific since it also gives a positive result to indole (+) and nitrate (+) organisms
(
Enterobacteriaciae )
4. Laboratory Diagnosis
Specimens: feces and rectal swabs
a. Staining smears
b. Culture ( similar to V. cholera )
c. Kanagawa phenomenon
- Wherein pathogenic strain of V. parahemolyticus produce hemolyis on a
special high salt medium ( Wagatsuma Blood Agar) distinguishing them from
non pathogenic strains.
d. Biochemical reactions
i. oxidase ( + ), ornithine and lysine decarboxylase ( + )
ii. ONPG ( - ) and sensitive to 0/129
iii. urease ( + )
5. Treatment
C. Other Vibrios
1. V. vulnificus
a. halophilic vibrio from seawater
b. cause intense skin lesions and occasionally enteritis, bacteremia and death in erderly
or debilitated persons.
2. V. mimicus causes diarrhea after ingestion of raw oysters.
3. V. holicas and V. fluvialis cause diarrhea
4. V. alginolyticus - causes eye, ear or wounf infection after seawater exposure.
5. V. damsel causes wound infections.
AEROMONAS
1. Morphology
a. straight sided, medium sized, nonpleomorphic, gram negative bacilli.
b. motile species possess a single polar flagellum
2. Cultivation
a. grow on common laboratory media like BAP and McConkey agar
b. many are beta- haemolytic
3. Virulence Factors
a. heat labile enterotoxin similar to LT of E. Coli and V. cholera enterotoxin
b. heat stable cytotoxic enterotoxin similar to that of shigella dydenteriae
c. extracellular enzymes like protease, amylase, lipase, nucleases, etc.
d. hemolysins
e. adherence
4. Clinical Infection
Aeromonas species are generally considered freshwater organism that are pathogenic to
cold blooded animals ( frogs and snakes ). Human infections are usually caused by motile
species ( A. hydrophilia, A. caviae, A. sobria, A. schubertii and A. veronii )
a. diseases produced
i. gastroenteritis
ii. wound infections after exposure to water or soil.
iii. opportunistic infections of blood or other body sites
b. risk factors for gastroenteritis acquisition
i. derinking untreated water
ii. current gastrointestinal or liver disease
iii. reduced stomach acidity
iv. recent antibiotic administration
5. Laboratory Diagnosis
Specimens: feces, sterile body fluids, tissues and oxidates from wounds
a. Gram stained smears
b. Culture media
i. trypticase soy agar with 5 % sheep blood and 10 or 30 ug/ml ampicillin.
ii. combination of ampicillin blood agar and CIN agar
c. Biochemical reactions
i. all are oxidase ( + ) and resistant to 0/129
ii. they ferment glucose
6. Treatment
a. susceptible to aminoglycosides, tetracycline, cofamandole and trimethoprim
sulfamethoxazole
b. resistant to penicillin, ampicillin, cephalothin and carbonicillin
PLESIOMONAS ( P. Shigelloides )
CHAPTER XVIII
CAMPYLOBACTER, HELICOBACTER AND SPIRILLUM
CAMPYLOBACTER
General Characteristics
1. Organisms are small, slender, helicallycurved, microaerophilic, gram negative rods
that may the following morphologic forms: spirals, S shape, commas, seagull winged
and coccoid shapes.
2. Most have a singleunipolar or bipolar flagellum exhibiting a distinctive corkscrewdarting motility on phase contrast or darkfield microscopy.
3. They require a low oxygen tension ( 5% O 2) and an increase3d CO2 level ( 10% CO2)
for growth.
4. C. jejuni, C. coli, and C. laridis are thermophilic with an optimum tempaerature of
420C.
5. 5. Most of the pathogenic species are oxidase and catalase positive.
6. They do notr ferment or oxidize sugars.
7. 7. Most are susceptible to cephalosporins.
A. Campylobacter jejeuni
1. Morphology
- same as with other Campylobacter species,moves with a single polar flagellum.
2. Cultivation
a. plates are incubated at 420C in an atmosphere containing 5% O2, 10 CO2, and 85% N2
b. culture media
i. Butzlers medium
ii. Skirrows medium
-contains blood agar base, 5% lysed horse blood, vancomycin, polymixin, B and
Trimethoprim, cephalothin and ampothericin B.
iii. campy thio medium
-
c. colonies are gray topinkish or yellowish gray, slightly mucoid looking and
spreading or round and convex ; some may exhibit a tailing effect along the
streakline.
3. Virulence Factors
a. invasiveness
b. cytotoxin
c. enterotoxin
4. Clinical Infections
C. jejuni have birds, dogs, cats , sheep and cattle as its reservoir.
a. mode of transmission
i. ingestion of contaminated milk, water and food
ii. person to person spread via fecal-oral route.
b. diseases produced
i. mainly enteritis
ii. occasionally with systemic invasion leading to bacteremia
Nitrate
420C
Sensitivity to
Reduction
Cephalothin
Nalidixic
a. C. coli
b. C. fenelliae
c. C. fetus
-/+
+/-
e. C. jejuni
f. C. laridis
subsp. fetus
d. C. hyointestinalis
Human Disease
a. C. oli
pigs
diarrhea
b. C. laridis
seagulls
diarrhea
c. C. cinaedi, C.
hyointestinalis
homosexual men
and C.finelliae
HELICOBACTER ( H. Pylori, formerly Campylobacter pylori )
1. Morphology
a. curved, spiral shaped or bizarre U-shaped microaerophilic gram negative rod
b. with sheathed tuft or polar flagella exhibiting a corkscrew motility
2. Cultivation
a. plates are incubated at 35-370C for 1 week, however, growth is also seen at 420C
b. culture media
i. nonselective agar media like chocolate agar and brucella agar with 5% shhep blood
ii. selective agar media like Skirrows agar
iii. modified Thayer-Martin medium
d. Colonies are small, circular, translucent and nonpigmented.
3. Disease Produced
H. pylori is associated with antral gastritis and of antral gastritis with gastric and
duodenal ulcers.
4. Laboratory
Specimens: antral biopsy specimens ( H. Pylori is seen on the surface of gastric antral
epithelium)
a. Stained smears of tissue ( histologically )
i. Gram stain
ii. Giemsa
iii. Special liver stains
b. Culture
c. Biochemical reactions
i.
Oxidase and catalase ( + )
ii.
Rapid urease ( + ) can be detected by placing biopsy specimens directly into
urea broth
iii.
Cephalothin sensitive and nalidixic acid resistant
iv.
No sugars are metabolized
5. Treatment
a. bismuth compounds alone or with antibiotics
Spirillum ( S. minor)
1. Morphology
a. Short, thick, helical gram-negative organism with tapering endsand two or three
spirals that resembles Campylobactermore than spirochetes.
b. Wit polytrichous polar flagella.
2. Cultivation : non culturable on artificial media
3. Disease Produced: Rat- bite fever ( Soduku fever)
The disease is an acute bacteremic infection caused by S. minus and streptobacillus
moniliformis, both of which are present in the normal oropharingeal flora of rodents. The
Disease is primarily seen in wild rats.
a. Mode of transmission : bite of an infected animal ( usually rats )
b. Clinical manifestations
i.
Local lesions chancre like indurated ulcerwith black crust.
ii.
Regional gland swelling
iii.
Skin rashes- purplish maculopapular eruption
iv.
Relapsing type of fever
4. Laboratory Diagnosis
Specimens: blood, exudates from initial lesion, serum from exanthematous patches,
lymph node aspirates or ground up tissue from lesions.
a. Stained smears
i.
Gram stain
ii.
Giemsa or wrights stains- for blood smears
iii.
Silver imp[regnation methods like Fontana-Tribondeau ( flagella stain )
b. Darkfield or phase contrast microscopy
c. Isolation by animal inoculation, either mice or guinea pigs.
5. Treatment
a. Penicillin drug of choice
b. Streptomycin - alternative
CHAPTER XIX
SPIROCHETES AND CURVED RODS
SPIROCHETES
General Characteristics
1.
2.
3.
4.
TREPONEMA
Treponema palidum
1. Morphology
a. Contains numerous tight, rigid coils which are more or less regular.
b. Motility is sluggish, with a drifting motion and flexous movements.
c. Obligate anaerobes.
d. Very hard to stain but best observed by darkfield microscopy.
2. Cultivation
Primary stage
- typically a single dry lesion, nontender and firm, with a clean surface, raised
border and reddish color.
- lesions known as hard chancre or Hunterian chancre appear on the
genitalia or within the anal canal.
- systemic signs or symptoms are absent but lymph nodes are frequently
enlarged and tender.
-lymphadenopathy
ii.
Secondary stage
- fever, sore throat, generalized lymphadenopathy, headache and rash.
- lesions can be found on other parts of the body; may appear as white mucous
patches on mucous membranes.
- secondary lesions are called condylomas(condylomata acuminata) which
occur around moist areas like the vagina and anus.
- all secondary lesions are highly infectious.
iii.
Latent stage
- patients have no signs and symptoms of
seroactive.
iv.
v.
vi.
Tertiary stage
- involvement of the deep organs of the body.
EXAMPLES:
GUMMAS non progressive localized lesions of the dermal elements or
supporting structures of the body; also known as tertiary lesions
(benign tertiary syphilis)
NEUROSYPHILIS involvement of the CNS
CARDIOVASCULAR SYPHILIS commonly involved organs are the
great vessels of the heart.
Congenital syphilis results from transplacental infection of the developing
fetus and is often very severe mutilating form of the
disease.
Application of Methods
a. Primary syphilis dark field identification provides the most definite and earliest means
of diagnosis.
b. Secondary syphilis ( STS) serologic tests or treponemal tests for syphilis are almost
always (+).
c. Tertiary syphilis fluorescent antibody test is the most specific test for syphilis.
5. Treatment
a. Penicillin drug of choice
b. Erythromycin, tetracycline and cephaloridine alternative drugs
Treponema pertenue
1. Morphology
- closely related to T. pallidum; serologically and morphologically indistinguishable and
are differentiated by the type of lesions produced in experimental animals.
2. Clinical Infection
- Yaws (Frambesia) is a spirochetal disease of the tropics caused by T. pertenue
- endemic, particularly among children, in many humid, hot tropical
countries
a. Mode of transmission
- direct contact (person to person) other than sexual contact in children under
age 15
b. clinical manifestations
i.
ii.
iii.
3. Laboratory diagnosis
- no serologic test distinguishes human yaws from syphilis
Treponema carateum
2. Clinical infection: Pinta is a disease of tropical areas of Central and South America
a. Mode of transmission
i.
ii.
b. Clinical manifestations
i.
3. Laboratory Diagnosis
4. Treatment: penicillin
BORRELIA
A. Relapsing Fever
Spirochetes of the genus Borrelia cause the disease in humans known as
relapsing fever. An acute infection characterized by febrile episodes that
subside spontaneously but tend to recur over a period of weeks.
1. Morphology
a. Helical organisms which are loosely coiled with coarser and irregular
spirals
b. The only spirochete demonstrated by direct stain (Wright Giemsa) in the
peripheral blood (blood spirochetes)
c. Microaerophilic and actively motile with a corkscrew-like motion have
11-30 periplasmic flagella per cell end
a. Mode of transmission
i.
ii.
Transplacental transmission
iii.
Infected blood
b. Types of diseases
i.
B. anserina
- B.turicatae
B. hermsii
- B. parkeri
ii.
c. Clinical manifestations
i.
Relapsing fever
ii.
iii.
iv.
Respiratory symptoms
v.
CNS involvement
3. Laboratory diagnosis
Kellys medium
ii.
Chick embryo
iii.
4. Treatment
a. Tetracycline drug of choice
b. Erythromycin and penicillin alternatives
B. Lyme Disease
An illness associated with a characteristic skin rash, erythema chronicum migrans
(ECM) caused by Borrelia burgdorferi.
1. Morphology
b. Vectors : ( ticks)
i.
ii.
iii.
iv.
c. Clinical manifestations
i.
ii.
iii.
3. Laboratory Diagnosis
ELISA
ii.
4. Treatment
a. Phenoxymethylpenicillin or tetracycline drug of choice
b. Penicillin and amoxicillin alternatives
LEPTOSPIRAE
Leptospirae are tightly wound spirochetes about the size of treponemes, often shaped like a
shepherds crook. They are saprophytes and animal pathogens, only accidentally infecting man.
1. Morphology
a. Helicoidal, tightly coiled, thin, flexible spirochetes with very fine spirals in which one
or both ends is often bent forming a hook
b. With active rotational motion (corkscrew like)
c. Obligate aerobes, oxidase (+), catalase (-) or peroxidase positive or both
d. The diaminopimelic acid content of leptospira serves to differentiate these organisms
from Treponema and Borrelia, which instead contain ornithine.
2. Characterization of species
3. Clinical Infection: Leptospirosis ( Swineherds disease, Fort Bragg Fever, Pretibial Fever,
Weils disease, Canicola fever, Autumnal fever)
a. Mode of transmission
i.
Direct and indirect contact with infected urine of an animal (the proximal
convoluted renal tubules of infected animals harbor the leptospira organisms
which are then passed in the urine)
ii.
iii.
Infected soil, food and water enter the body through mucus membrane or
breaks in the skin
b. Clinical manifestations
i.
Infecting serogroups
- severe icteric disease L. icterohemorrhagiae (most common cause of
disease)
- less severe & anicteric disease L. australis, L. pyrogenes
- mild L. canicola, L.ballum, L. Pomona
ii.
- 1st stage variable febrile onset with jaundice, hemorrhage and nitrogen
retention.
4. Laboratory Diagnosis
Fletchers medium
ii.
Noguchis medium
iii.
Stewarts medium
iv.
5. Treatment
CHAPTER XX
CHLAMIDIAS, MYCOPLASMA AND RICKETSIAE
CHLAMYDIAE
A. General Characteristics
1. They are obligate intracellular parasites closely related to gram negative bacteria with
a tropism columnar epithelial cells lining the mucous membranes.
2. They cannot generate high-energy phosphate bonds (ATPs) and are totally dependent
on eukaryotic cells for energy thus restricting them to an intracellular existence.
3. All species exhibit two morphologically distinct forms:
a. Elementary body (EB) Small, dense spherical body which is the infectious form
b. Reticulate bod (RB) intracellular, metabolically active form that divides by
binary fission
4. All are non-motile and non-piliated but possesses unusual cylindric surface
projections arranged in a hexagonal array.
5. All share a common genus-specificor group antigen and multiply in the cytoplasm of
their host cells by a distinctive developmental cycle.
6. All require a living cells for growth.
7. They are rapidly inactivated by heat and lose their infectivity completely after 10
minutes at 60C.
B. Developmental Cycle
1. Phase I. Attachment and penetration of the EB
a. EB attaches to the surface of a susceptible host cell;
2.
3.
4.
5.
C.trachomatis
1. Host range
Humans, mice
2. Inclusion
Morphology
Oval, vacuolar
C.psittaci
Birds, humans
Lower animals
Variable, dense
Humans
Oval, dense
3. Elementary body
Morphology
4. Glycogen in
inclisions
5. Number of
Serovars
6. Folate bioSynthesis
7. Susceptiblility
To sulfonamides
& D-cycloserine
8. Plasmid DNA
Round
(+)
Round
(-)
Pear-shaped
(-)
15
NA
(+)
(-)
(-)
(+)
(-)
(-)
(+)
(-)
(+)
Trachomatis
A.Biovars and Serovars
a. Biovar LGV- consists of 3 serovars (LGV1-LGV3)
b. Biovar trachoma- have 12 serovars
i.
Serovars A, B, Ba, & C- isolated primarily from eyes of trachoma patients in
endemic
Area
ii.
Serovars D to K- isolated most often from genital tracts and eyes of trachoma
patients in
Non-endemic areas
B. Diseases Produced
a. Endemic trachoma
i.
A chronic keratoconjuctivitis that begins with acute inflammatory changes in the
conjunctiva and cornea progressing to scarring and blindness
ii.
Caused by serotypes A, B, Ba, or C
iii.
Seen in poverty-stricken families (overcrowding)
iv.
Transmitted by droplets, hands and contaminated clothing from eye to another
b. Inclusion conjunctivitis
i.
Caused by serotypes D through K; historically referred to s the TRIC (trachomona
inclusion conjunctivitis) agent
ii.
Infants acquire the infection from passage through an infected birth canal; does
not lead to blindness
iii.
Neonatal pneumonia may also be seen
iv.
Adults acquired the infection via self-inoculation of genital secretions or
following contamination of un-chlorinated swimming pools
c. Sexually transmitted chlamydial disease
i.
Caused by oculogenital serotypes D through k
ii.
Includes nongonococcal urethritis, cervicitis, bartholinitis, epididymitis, proctitis,
salpingitis, etc.
iii.
Associated with Reithers syndrome
d. Lymphogranuloma venereum (LGV)
i.
Other names are lymphogranuloma inguinale, climatic bubo, tropical bubo and
esthiomene
ii.
Sexually transmitted disease caused by serotypes LGV1, LGV2, LGV3
iii.
Presents a painless, small, inconspicuous and vesicular primary lesion with painful,
firm and enlarged matted inguinal ad femoral lymph nodes
iv.
Elephantiasis of the vulva (called esthiomene) may occur 2 to lymphatic obstruction
C. Laboratory Diagnosis
a. Specimens: scrapings of epithelial cells from urethra, cervic, vagina, conjunctiva, rectum
and nasopharynx; throat specimens; salpinx and epididymis aspiration biopsies; pus and
buboes
1. Cytological experimentation of cell scraping fror the presence of inclusion bodies
which are stained with iodine, fluorescent antibody and Giemsa stains
2. Rapid antigen detection methods
i.
Direct fluorescent antibody staining (e.g., Micro Trak DFA)
ii.
ELISA (e.g., Chlamydiazyme assay)
iii.
RNA-directed DNA probe conjugated to a chemiluminescent marker (e.g.,
PACE, Gen-Probe)
b. Isolation
i.
Inoculation into embrayonated eggs (chick embrayo)
ii.
Isolation of experimanetal animals (usually mice, inoculated intracerebrally)
iii.
Selected tissue culture cell lines like McCoy, Hela 229, BHK-21, L 929 and
buffalo green monkey which are inoculated by centrifugation;
iii.a. this is the most sensitive ad specifi diagnostic method
iii.b. cells are pretreated with cycloheximide, cytochalasin B or idoxuridine to
enhance
chlamydia rplication and allow easier recognization of inclusions
iii.c. inclusions are visualized using the above mentioned stains,
immunofluorescence
being the most sensitive
iii.d. cycloheximide-treated McCoy cells have been shown to produce higher
inclusion
counts than other cell lines
c. Serologic testing
i.
Compliment fixation (detects antibody to a genus-specific antigen)- used
commonly for LGV
ii.
Microimmunofluorescence (micro-IF) technique (detects type specific antibodies)
- used mainly in ocular and genital infections and neonatal pheumonia
- may also be used inLGV
iii.
Frei test
- An intradermal skin test used in the diagnosis of LGV which detects a delayed
hypersensitivity response to chlamydia antigen
D. Treatment
- Susceptible to tetracyclines, sulfonamides and erythromycin
C.psittaci
1. Disease produced: Psittacosis or Ornithosis
Primarily a disease of birds especially the psittacine birds (parrots, parakeets,
cockatoos, etc.), which is occasionally transmissible to humans.
a. Mode of transmission
i.
Inhalation of organisms from infected birds (aerosols) and their droppings
ii.
Person to person transmission (rare)
b. Clinical manifestation
i.
Pneumonia
ii.
Severe headache and changes in mentation
iii.
Hepatosplenomegaly
2. Laboratory Dignosis
Specimens: Blood, sputum and biopsy tissue specimens
a. Isolation
i.
Yolk sacs of embrayonated eggs
ii.
Mice, inoculated intracerebrally or subcutaneously
iii.
Grows best in L 929 cells
b. FA test using monoclonal antibody
C.pneumoniae
1. History
C.pneumoniae was initially considered to be a psittacosis strain since its inclusion bodies
produced in cell culture resemble that of C.psittaci. the strain was named TWAR, an acronym
reflecting the history of the first two isolates, Taian and acute respiratory.
2. Diseases Produced
a. Associated with pneumonia, bronchitis, pharyngitis, sinusitis and a fluke illness
b. Pneumonia is transmitted from human to human and has been seen in grouped young
adults such as military camps and college campus.
3. Laboratory Diagnosis
Specimen:swabs of the posterior oropharynx
a. Ahdlk
i. kdh
ii. HeLa 229 cells and heteroploid cell
b. FA test using monoclonal antibody
- Used as research tool
c. Serologic testing
i. Complement fixation
ii. micri-IF test- more reliable
4. Treatment: sensitive o tetracycline and erythromycin
A selective biphasic medium containing broth over agar is preferred with the
addition of enicillin and thallium to inhibit overgrowth of other organisms.
i. SP-4 Mycoplasma medium- for primary isolation
ii. Edward-Hayflick agar
- inoculated with subculture coming from SP-4 medium
- colonies are spherical, grainy, yellowish and embedded in the agar with
thin
Outer layer
- The growth is confirm by a small zone of beta-hemolysis around the
colonies
After a thin layer of agar containing 5% sheep or guinea pig erythrocytes
was
Overlaid on the medium and subsequently re-incubated for 24 hours.
c. Serologic tests
i. cold agglutination reaction
ii. complement fixation
iii. growth inhibition test
iv. immunofluorescence
v. passive hemagglutination
vi.RNA-dorected DNa probe test
vii. ELISA
viii. hemdsorption
ix.radioimmunoprecipitation tests
d. Dienes staining method for PPLO colonies
i.
uses
azure
II
and
methylene
ii. colony stain light blue at the periphery and bright deep blue at the center
blue
2. laboratory disease
Specimens: urethral or genital discharges collected on swabs and inflammatory exudates.
a. Microscopic examination: useless
b. Culture
i.
Use of transport media such as modified Stuarts, 2-SP or trypticase soy broth
with 0.5% albumin and 400 U/ml penicillin
ii.
Shepards A7-B agar
iii.
Ureaplasma agar plate and broth
c. Serologic tests
i.
ELISA
ii.
RNA-directed DNA probes (PACE system, Gen Probe)
iii.
Indirect hemagglutination
iv.
Growth inhibition
v.
Nucleic acid dot-blot hybridization methods for M. genitalium
3. Treatment: susceptible to tetracyclines and erythromycin
RICKETSIAE
The family Ricketsiaceae is composed of three tribes, Ricketsiae, Erlichieae and
Wolbachieae. The tribe Ricketsieae has 3 genera that affect man, Ricketsia, Rochalimaea and
Coxiella while genus Ehrlichia has two species, E. sennetsu and E. canis
Morphology and Physiology
1. Small, nonmotil, peromorphic, gram-negative coccobacilli that are obligate intracellular
parasites.
2. They occur singly, in pairs, in short chains or in filaments.
3. They stain poorly with Grams stain but appear blue with Giemsas stain and red with
Machiavellos stain.
Cultivation
1. All require living cells for growth, except Rochalimaea Quintana which can be cultivated
in cell free media
2. All multiply by binary fission intracellularly in the cytoplasm after escaping from
phagosomes, except for C. burnetii which multiplies within vacuoles; occasionally R.
ricketsii (spotted fever group) multiplies also within the nucleus
3. Growth is enhanced in the presence of sulfonamides
Resistance
1. All are quickly destroyed by heat, drying and chemical agents except for C. burnetii
2. C. burnetiis resistance to heat and drying may be due to the formation of endospore-like
structures
Pathogenesis
1. Ricketsiae (except C. burnetii) multiply in endothelial cells of small blood vessels
causing endothelial proliferation and perivascular infiltration leading to leakage and
thrombosis
2. They have the ability to invade and damage vascular smooth muscle cells and the
endothelium of larger blood vessels
3. Centripetal spread from capillaries to arterioles and veins may occur resulting in
widespread infectious vasculitis with rash and organ dysfunction
4. A ricketsial toxin is currently suggested (?)
5. Coxiella is inhaled into the alveoli, picked up by macrophages and carried to lymph
nodes, from which it disseminates into the blood stream; granulomatous inflammation of
the liver (hepatits) and heart (endocarditis) may occur
Clinical infection
Rickettsial infections (except Q fever, in which no rash appears) are characterized by
fever, chills, headache, malaise, myalgia and skin rash
i.
Clinical manifestations
i.
ii.
iii.
Laboratory Diagnosis
Specimens: Primarily blood
Sputum and urine for Q fever
Vesicular fluid for Ricketsial pox
1. Stained Smears using:
a. Giemsa stain
b. Machiavello stain
c. Gimenez stain the best
2. Isolation using:
a. Embryonated hens eggs
Ehrlichia
E. sennetsu
1. Disease Produced
a. Sennetsuricketsiosis
b. other names: infectious mononucleosis, glandular fever, hyuganetsu and
kagaminetsu
2. Transmission: unknown (probably by ticks)
3. Clinical findings:
a. fever
b. lymphadenopathy
c. atypical lymphocytosis
4. Laboratory Diagnosis
Specimens: blood, lymph nodes and bone marrow
a. Intraperitoneal mice inoculation
b. Specific immunoserologic technique
5. Treatment: Tetracycline
E. canis
1. Disease Produced: Canine ehlichiosis/Tropical canine pancytopenia
2. Transmission: tick bites
3. Clinical Findings:
a. resembles RMSF but only a few cases have rashes
b. leukopenia and thrombocytopenia
4. Laboratory Diagnosis
DISEASE
OX-19
OX-2
OX-K
_______
______
______
______
Epidemic typhus
++
+-
Nurine typhus
++
Scrub typhus
RMSF
Boutonneuse fever
Rickettsial pox
Q fever
Trench fever
Ehrlichiosis
CHAPTER XXI
MISCELLANEOUS PATHOGENIC BACERIA
1. Morphology
a. Medium to long, facultatively anaerobic, motilie, gram-negative bacilli with rounded
ends which are occasionally slightly curved.
b. Possesses a single polar flagellum and lateral flagella.
2. Cultivation
a. Grows best at 25C but can also grow at 37C
b. Colonies are violet on media containing tryptophan (5% sheep BA) due to the
pigment violacein.
c. Cultures may have smell of ammonium cyanide, since the organism produces HCN
d. They also grow on McConkeys agar.
3. Clinical Infection
a. Mode of transmission
a. The organism is on inhabitant of soil and water
i.
ii.
iii.
Punctured wounds
Wounds in contact with soil or contaminated water
Ingestion of large amounts of water in near-drowning cases
c. Biochemical test
i.
Oxidase (-) and catalase (-)
ii.
Hydrolyzes sodium hippurate
iii.
Fements starch and raffinose
d. Sniff test
- Fishy odor of discharge material after addition of 10% KOH
e. Direct wet mount of vaginal discharge
- Shows masses of bacilli located on the surface of squamous epithelial cells,
giving the cell a stippled appearance called clue cells
f. pH determination of vaginal secretions
- over pH 4.5 (normal pH is less than 4.5)
5. Treatment
a. metronidazole drug of choice
b. susceptible to ampicillin, carbenicillin, oxacillin, penicillin and vancomycin
STREPTOBACILLUS (S. moniliformis)
1. Morphology
a. Extremely pleomorhic, nonencapsulated, nonmotile, facultatively anaerobic, gramnegative rods that occur in long chains and filaments
b. Elongated bulbous swellings may be produced giving the appearance of a string of
beads or necklace-like arrangement
c. The most distinguishing characteristic is the spontaneous development of L forms
(without cell walls)
d. L form colonies when stained with Dienes or acridine orange stain yield
coccobacillary or bipolar staining coccoid forms
e. Highly fastidious
2. Cultivation
a. Requires the presence of blood, ascitic fluid or serum for growth
b. Plates are incubated in a humid environment with 5-10% CO2 at 37C
c. On brain-heart infusion agar with 20% horse serum, colonies are small, smoothn
grayish or colorless, glistening (butyrous) with irregular edge
d. In thioglycollate medium enriched with ascitic fluid, growth is described as fluff
balls, cotton balls, puff balls, or bread crumbs near the bottom of the tube.
e. L-phase colonies are smaller and exhibit at a fried-egg appearance with a dense or
dark center that extends into the agar and a flattened lacy edge
f. Growth is inhibited by SPS
3. Clinical Infection
a. Mode of transmission
i.
Bite of wild or laboratory rats wherein S. moniliformis is part of the normal
flora of their nasopharynx
ii.
Animal bite from mouse, cat and dog
iii.
Less commonly, ingestion of contaminated milk, food or water
b. Disease Produced
i.
Rat-bite fever
- a systemic febrile disease acquired by direct contact with rats or other
small rodents (another causative agent is Spirillum minor)
ii.
Haverhill fever
- acquired by consumption of contaminated milk
c. Clinical Manifestations
i.
Acute onset of chills, fever, vomiting, headache and severe joint pains
ii.
With a characteristic rash with a morbilliform, maculopapular or petechial
involving the palms, soles and extremities
iii.
Complications include endocarditis, septic arthritis & pneumonia
4. Laboratory Diagnosis
Specimens:
a. Stained smears
i.
Dienes&acridine orange stains for L-forms
ii.
Grams stain with carbolfuchsin as a counterstain &Giemsa stain
b. Culture
c. Serologic test
5. Treatment
a. Penicillin- drug of choice
b. Susceptible to tetracycline, streptomycin, chloramphenicol & erythromycin
c. Resistant to sulfonamides
CALYMMATOBACTERIUM (C.granulomatis)
1. Morphology
a. Pleomorphic, nonmotile, heavily encapsulated, gram-negative rod which exhibits a
single or bipolar condensation of chromatin giving rise to a safety pin appearance
b. Because of its capsular polysaccharide C. granulomatis is antigenically similar to
klebsiella species\
2. Cultivation
a. Initial isolation is very difficult
b. The organism can be cultured on fresh egg yolk media or in the yolk sac of
embryonated chicken eggs
3. Clinical Infection:Donovanosis/Granulosainguinale
a. Mode of transmission
i.
Sexually, however, infectivity is low
ii.
It is an inhabitant of the intestinal tract & skin disease may be contracted
through poor hygiene & abrasions of the skin in the anal and perineal areas
b. Clinical Manifestations
i.
Initial lesions occur in pubic areas and begin as a painless papules that
develop into spreading erythematous, granulomatous ulcerating lesions which
may bleed and become secondarily infected (sometimes mistaken for
neoplasms)
ii.
Lesions spread by direct extension or by contact of skin area with another
(between scrotum and thigh)
iii.
Inguinal lymphadenopathy is common
iv.
If untreated, genital elephantiasis may ensue.
4. Laboratory Diagnosis
a. Demonstration of Donovan bodies (numerous encapsulated bacilli within
mononuclear endothelial cells) by direct examination of biopsy material stained with
i.
ii.
Wright or
Giemsa stain
5. Treatment
a. Tetracycline or ampicillin drugs of choice
b. Susceptible to gentamicin, chloramphenicol, trimethroprim-sulfamethoxazole and
erythromycin
LEGIONELLA (L.pneumophila type species)
1. Morphology
2. Cultivation
a. Culture media
Buffered charcoal yeast extract agar (BCYE) the best
i.
- Charcoal serves t to detoxify the medium , remove carbon dioxide &
modify the surface tension to allow the organism to proliferate more easily
-buffered with N-(2-acetamido)-2-aminoethanesulfonic acid (ACES)
- with growth supplements cysteine (required by Legionella), yeast extract,
alpha-ketoglutarate and iron
- can add antibiotics for selectively (polymyxin B, anisomycin and
cefamandole) and dyes for differentiation (bromthymol blue and bromcresol
purple)
- colonies are gray-white to iridescent pink or blue-green glistening, convex
or flat, circular and may exhibit a cut-glass sort of internal granular speckling
ii.
b.
c.
d.
e.
3. Clinical Infection
a. Epidemiology
i.
Legionella was discovered in the summer of 1976 as a cause of a fulminant
pneumonia among persons at attending an American Legion convention (thus,
Legionnaires disease)
ii.
Has been isolated from natural ( lakes, streams) and tap waters, air
conditioning cooling towers or evaporative condensers and water supplies of
hospitals & hotels (hot shower or whirlpools), often in association with bluegreen algae and free-living protozoa.
iii.
Risk factors
- smokers, chronic pulmonary disease, high alcohol consumption.
Immunosuppressed patients (patients on corticosteroids, renal transplant
patients, CA patients, patients on dialysis, etc)
iv.
Transmission
- airborne exposure (breathing in aerosols)
- person to person spread has not been documented
b. Virulence factors
i.
Extracellular enzymes phosphatase, lipase & nucleases
ii.
Extracellular toxin - may contribute to the intracellular survival
iii.
Ability to survive within phagocytic host cells primary virulence mechanism
c. Clinical Manifestations
i.
Legionnaires diseases ( Broad street pneumonia)
- a multisystem disease manifested primarily as severe, consolidated
pneumonia, which has a significant mortality
- may produce CNS symptoms, diarrhea and renal problems
ii.
Pontiac fever (also caused by L.feeleii)
- a multisystem disease with respiratory symptoms, fever, myalgia &
headache but without pneumonia
4.Cell membrane
5.Antibiotic susceptibility
6.Dimorphism
7.Chromosome
8.Sedimentation coefficient
9.Cultivation
10.Temperature
Bacteria
-no membrane
-absent
-no glucose & mannose
polymers with muramic &
teichoic acid
-with respiratory enzymes
-vice versa of fungi
-None
-only one but not CHON
associated
-7OS
-pH 7.2-7.6
c. Chlamydospores (chlamydoconidia)
i. formed by enlargement of a hyphal cell in which there is a concentration of protoplasm
and nutrient material
ii. large, round, thick-walled resistant spores (e.g., C. albicans)
iii. types
iii.a. intercalary (within hypha)
iii.b. sessile (to side of hypha)
iii.c. terminal (at end of hypha)
2. Conidia
- Asexual spores produced singly or in groups by specialized vegetative hyphal
strands called conidiophores
a. Microconidia
i. borne directly on the hyphal strand or at the end of a long or short conidiophores
ii. small, unicellular, round, elliptical or pyriform in shape
b. Macroconidia (fuseaux)
i. usually borne on a short to long conidiophores
ii. large usually multiseptate, and club-or spindle-shaped that may be smooth-or roughwalled
3. Other Complex Methods of Sporulation
a. Some conidiophores terminate in a swollen vesicle from which short phialides (flaskshaped projections) form and in turn produce radiating chains of conidia
(phialoconidia)
Classification of Fungi
1. Ascomycotina (the ascomyces)
Sexual fusion result in a sc, or ascus, containing the meiotic products as four or eight
spores (ascospores). Asexual spores (conidia) are borne externally at the tips of hyphae.
Ex. Trichophyton (Arthroderma), Microsporum (Nannizzia), Blastomyces (Ajellomyces).
2. Basidiomycotina (the basidiomyces)
Sexual fusion results in formation of a club-shaped organ called a basidium, on the
surface of which are borne the four meiotic products (basidiospores). Asexual spores
(conidia) are borne externally at the tips of hyphae. Ex. Cryptococus neoformans
Filobasidiella neoformans.)
3. Deuteromycotina (the imperfect fungi)
This is not a true phylogenic group but rather an artificial class into which are
temporarily placed all forms in which the sexual process has not yet been observed. Most
of the members resemble ascomyces morphologically. Ex. Epidermophyton, Sporothrix,
Candida species.
4. Zygomycotina (the phycomyces)
Mycelium usually non-septate; asexual spores produced in indefinite numbers
within a structure called a sporangium. Sexual fusion results in formation of a resting,
thick-walled cell termed a zygospore. Ex. Rhizopus nigrans (opportunistic pathogens
only.)
CHAPTER XXIII
LABORATORY IDENTIFICATION OF FUNGI
3. most fungi are detected within the first 4 days of incubation, however, for Histoplasma
Capsulatum it may take 10-14 days for recovery.
D. Hair, Skin and Nail Scrapings
1. Usually submitted for dermatophyte culture.
2. Lesions from skin and nails are scalped with a scalpel blade or microscope slide while
Infected hairs are plucked with forceps.
3. Specimen should be placed in a sterile petri dish or paper envelope prior to culturing
and should not be refrigerated.
4. Specimens are either placed on Mycobiotic or Mycosel agar which contain
chloramphenicol and cycloheximide.
E. Urine
1. Samples are centrifuged and the sediment cultured using loop to adequate isolation of
colonies.
2. Antibacterial agents should be added to media since urine is often contaminated with
gram negative bacteria.
F. Tissue, Bone Marrow and Sterile Body Fluids
1. All tissues are processed by mincing or grinding or placement in a Stomacher prior to
Culturing at least 1 ml of specimen is inoculated.
2. Bone marrow is directly plated to media.
3. Sterile body fluids are concentrated first by centrifugation before culturing and at least
I ml is inoculated.
NOTE: All specimens should be cultured as soon as possible.
Cultivation
A. Requirements
1. A battery of media must be used with the following recommendations:
a. media with and without blood enrichment (since certain fungi have a requirement for
blood 5% sheep blood)
b. media with and without cycloheximide (since some pathogenic fungi such as
C. neoformans, Candida krusei and other species of Candida, Trichosporon biegelii,
Pseudallescheria boydii and species of Asperigillus are partially or completely inhibited
by this compound)
c. all media should contain antibacterial agents
2. Cultures should be incubated at 30C at a 40%-50% relative humidity for 30 days before
Discarding as negative.
B. Fungal Culture Media
1. Primary Recovery Media
a. Brain-heart infusion agar
i. primary recovery of saprobic and pathogenic fungi
ii. isolates and maintains yeast phase of systemic fungi at 27C
b. Brain-heart infusion agar with antibiotics
i. primary recovery of pathogenic fungi exclusive of dermatophytes
c. Brain-heart infusion biphasic blood culture bottles
i. recovery of fungi from blood
ii. isolation and maintenance of yeast phase of systemic fungi at 35C
d. Dermatophyte test medium (DTM)
i. primary recovery of dermatophytes, recommended as screening medium only
ii. most dermatophytes produce a red color on this medium due to a phenol red indicator
e. Inhibitory mold agar
i. primary recovery of pathogenic fungi exclusive of dermatophytes
f. Mycosel or Mycobiotic agar
i. primary recovery of dermatophytes
ii. with chloramphenicol to inhibit bacterial growth and cycloheximide to inhibit growth
of saprophytic fungi
g. SABHI agar (Sabouraud dextrose & brain-heart infusion)
i. primary recovery of saprobic & pathogenic fungi like Blastomyces and Histoplama.
b. Cornmeal agar
i. Stimulates chlamydospore production for C. albicans
ii. 1% glucose can be added to differentiate T. rubrum from T. mentagrophytes by amount
of pigment formation
iii. Stimulates fungal sporulation- used for slide culture
c. Cornmeal agar with Tween 80 and try pan blue
i. Identification of C. albicans by chlamydospore production; identification of Candida by
microscopic morphology
ii. Tween 80 reduces surface tension of medium and promotes optimal production of hyphae
and blastospores
iii. Trypan blue aids visual observation
d. Cottonseed conversion agar
mold-yeast
i. Conversion of dimorphic fungus B. dermatitis from mold to yeast form
e. Czapek's agar
i. Isolation and identification of Aspergillus sp.
f. Niger seed agar (Birdseed agar or Staib's medium)
i. Identification of C. neoformans which produces a brown pigment indicating the presence
of phenol oxidase
ii. Thisle (Guizotia abyssinica) seeds are used
g. Nitrate reduction medium
i. Detection of nitrate reduction in confirmation of Cryptococcus sp.
h. Potato dextrose agar
i. Demonstrates pigment production by T. rubrum
ii. Preparation of microslide cultures
i. Rice medium
i. Identification of M. audounii differentiating it (negative) from M. canis (positive)
j. Trichophyton agars 1-7
i. Identification of members of Trichophyton genus
k. Urea agar
i. Detection of Cryptococcus sp.
ii. Differentiates T.
rubrum (delayed or no utilization)
iii. Detection of Trichosporon sp.
l. Yeast fermentation broth
7. Acridine orange
a. For tinea versicolor
An-an
Superficial mycosis
Skin
b. Green fluorescent fungal elements and orange epithelial cells
TECHNIQUES FOR MICROSCOPIC OBSERVATION OF FUNGI
A. Wet Mount
1. A small portion of an isolated colony from a point intermediate between the cents and the
periphery is removed containing a small amount of the supporting agar with a wire bent at a 90
angle.
2. This portion is placed on a slide and a drop of Lactophenol cotton or aniline blue is added.
3. A coverslip is applied with a gentle pressure using a pencil eraser or other object to disperse
the growth and agar.
4. The preservation is examined microscopically.
B. Scotch or Cellophane Tape Preparation
1. Touch the adhesive side of a small length of transparent tape to the surface of the colony.
2. Adhere the length of tape to the microscope slide surface to,which has been added a drop of
Lactophenol cotton blue or aniline blue.
3. Observe under the microscope for the characteristic shape and arrangement of the spores.
C. Microslide Culture
Original state
1. This technique allows one to observe microscopically the fungus growing directly underneath
the coverslip.
2. Slide cultures should not be made on slowly growing organisms suspected of being dimorphic
pathogens like H. capsulatum, B. dermatitidis, C. immitis, P. brasiliensis or S. schenckii.
SPECIAL TESTS
A. Hair Perforation or Baiting Test
1. Place a filter paper disk into the bottom of a sterile Petri dish.
2. Cover the surface of paper disk with sterile distilled water.
3. Add a small portion of sterilized prepubertal hair into the water.
4. Inoculate the portion of the colony to be studied directly onto the hair.
5. Incubate at 25C for 10-14 days.
6. Observe hairs at regular intervals by placing them into a drop of water on a glass slide.
Position a coverslip and examine microscopically for the presence of conical perforations of the
hair shaft.
T. mentagrophytes = ( + )
T. rubrum = ( - )
:-*^3^:-* 172 :-*<3:-*
B. Exoantigen Test
A serological test
1. Principle
Antibodies developed against particular mycelial antigens will react specifically in a gel
immunodiffusion precipitin test. The mold forms can be identified definitively by an antigenantibody reaction, hence the conversion the yeast phase is not needed.
2. Procedure
l l - positive
/\ - negative
a. A concentrated merthiolate extract of mature fungus culture used in the microdiffusion
tests
b. The extract is placed into wells punched into a plate of buffered phenolized agar adjacent
to the control antigen well and is tested against positive control antiserum
c. The test is read after 24 hours for the presence of precipitin hands of identity after
incubation at 25
C. Rapid Urease Test
1. Principle
Ability of some bacteria to hydrolyze urea into ammonia, water and CO2 by means of an
enzyme urease.
a. The same as urease test.
2. Procedure
Pink color = (+)
Purple color = (-)
a. Use either a UREA-R broth (Difco) or Urea Base (BBL)
b. Transfer a heavy inoculum of a yeast colony (excluding pink yeasts) to a well of a
microdilution plate containing the urea broth
c. Include positive C. neoformans and negative C. albicans controls
C. albicans - yellow after 4 hours
d. Seal the wells with plastic tape and incubate 4 hours at 37C
e. Observe for a pink to purple color as positive result
D. Rapid Nitrate Reductase Test
1. Principle
Similar to test on page 23 except that benzalkonium chloride is added to test medium
to disassociate the cell wall of yeasts to allow the release of nitrate Reductase (Reductase
enzyme).
2. Procedure
Red color = (+)
a. Sweep the tip of an applicator impregnated with the nitrate reduction test reagents across 2 or
3 colonies of yeast
b. Swirl the applicator containing the yeast against the bottom of an empty test tube to get
the yeast cells in contact with the contact with the cotton fibers
c. Incubate the tube and swab at 45C for 10 minutes
d. Remove the swab and add two drops each of N-naphthylenediamine and sulfanilic acid
reagents to the tube and replace the applicator; a change in color to red indicator a positive test; if
no color appears after 10 minutes, add a pinch of zinc dust and observe for a red color indicating
the presence of previously unreacted nitrate and a negative test
>.<>.<>.< 173 >.<>.<>.<
E. Levodopa-Ferric Citrate Test
1. Principle
Formation of melanin
C. neoformans is known to produce the enzyme phenol oxidase which when made to
react with dihydroxyphenylalanine in the presence of ferric nitrate forms a dark pigmented
compound, melanin (for rapid identification of C. neoformans)
2. Procedure
a. The test is available commercially:
i. G/N - screen (Flow Laboratories)
ii. L-DOPA Ferric citrate reagent disks (Difco)
b. Smear test organism onto the surface of reagent disks which were moistened beforehand
with 2-3 drops of distilled water in a Petri dish
c. Incubate disks at 37C and examine at 30 minutes interval for up to 6 hours.
F. Germ Tube Test
1. Specifically used to identify C. albicans.
2. Procedure
a. Suspend a very small inoculum from an isolated yeast colony in 0.5 mL of serum
b. Incubate tubes at 35-37C for 3 hours
c. Place a drop of the suspension on a glass slide and examine under low-power
magnification for the presence of germ tubes (appendages half the width and 3-4 times the length
of the yeast cell from which they arise)
G. Carbohydrate Utilization Tests
1. Yeasts and yeast-like fungi are inoculated onto a carbohydrate-free medium
5. Laboratory Diagnosis
Specimen: skin scrapings
a. Direct microscopic examination
i. 10% KOH
ii. Calcofluor white stain
appearance: blunt-ended short hyphae and clusters of spherical spores that form
spaghetti and meatballs pattern
b. Wood's light (ultraviolet radiation of lesions in a darkened room)
- lesions Fluoresce golden yellow or brownish
c. Culture
i. not required to establish a diagnosis
ii. M. furfur grows on Sabouraud's medium overlaid with olive oil a related species M.
pachydermatis does not require added lipid for growth
:-o:-o:-o 175 :-o:-o:-o
6. Treatment
a. Topical application of 2.5% selenium sulfide for 10 minutes daily for 7 days
b. Topical miconazole and ketoconazole
c. Folliculitis - treated with oral ketoconazole
B. Tinea nigra (Tinea nigra Palmaris)
Blackish-brown palms, soles or elsewhere
1. Clinical Features
a. Blackish-brown macular patches resembling faded silver nitrate stain occurring most
frequently on the palms but also on the soles or elsewhere
b. Lesions are not elevated or scaly
2. Causative Agent: Phaeoannellomyces werneckii (Cladosporium or Exophiala werneckii)
A dimatiacous fungi hydrolysis casein
3. Laboratory Diagnosis
Specimens: skin scrapings
a. Direct KOH or calcofluor white examination
i. Olive or brown-pigmented, branched, septate hyphae and budding yeast cells that are
one-or two-celled
ii. Older colonies exhibit one-or two-celled conidia produced by annelides, that bear
successive rings (annelations)
Safety pins
b. Culture (Sabouraud's medium with and without antibiotics)
2.
White Piedra
These are fungal infections involving the superficial keratinized tissue of the body such
as the skin, hair, and nails. They are caused by a group of fungi commonly called dermatophytes
which breakdown and utilize keratin as a source of nitrogen but are incapable of penetrating the
subcutaneous tissue. The three genera associated with dermatomycoses include:
Dermatophytes
Colonial Morphology
Growth Rate
Micromorphology
E. Floccosum
1 week
Macroconidia large,
to be folded and is
thin smooth-walled,
mutiseptate, cla-
yellow; reverse
ly or in clusters of
observable folds
M. audouinii
2 weeks
(epidermiecopitis
nal chlamydospores,
ectorix; apple
favic chandeliers,
green in woodlight;
10-21 days)
macroconidia rarely
seen- bizarre
shaped
microconidia
absent
is
1 week
seen;
rare or
M. canis
Thick-walled, spin-
worm; zoophilic;
periphery; center of
ectotrix)
rough-walled, macro-
over orange-yellow;
lemon-yellow or
curved-tip; micro-
yellow-orange apron
177
M. gypseum
Cinnamon-colored
1 week
(geophilic-soil;
powdery-colony;
elliptical multi-
ectotrix; wood-
septate macroconidia;
light (-)
Thick-walled rough,
microconidia few or
absent
T. mentagrophytes
Different colonial
7-10 days
(scanty pigment;
bose microconidia
spiral hyphae;
in grapelike clusters
lates; macroconidia
to reddish brown
are thin-walled,
smooth, club-shaped
& multiseptate; numerous or rare depending upon strain
T. rubrum
2 weeks
Microconidia, usually
(anthrophilic;
woodlight (-);
smooth, thin-walled,
and pencil-shaped
T. tonsurans
7-14 days
or rust, suede-like to
drop or club-shaped
powdery; wrinkled
worm; alopecia)
dermatophytes;
balloon forms-aged
pleomorphic microconidia macroconidia
usually rare
T. schoenleinii
Irregularly heaped,
2-3 weeks
(slow grower;
smooth white to
Mousy odor;
chandeliers)
T. violaceum
(no fluorescence;
2-3 weeks
Branched, tortuous
hyphae that are sterile
chlamydospores
appearance)
with waxy-glabrous
commonly aligned in
chains
be lost on subculture
T. verrucosum
Glabrous to velvety
(swollen hyphae;
2-3 weeks
Microconidia rare;
large and teardrop
concentrate rings
of lesions Kerion)
conidia extremely
characteristics
ratsurface
1.
Epidermophyton floccosum
2.
b. M. canis
i. infected hairs fluoresce bright yellow-green using a Woods lamp
ii. grows well on sterile rice grains
c. M. gypseum
i.
3.
The most common species recovered in the clinical laboratory are T. rubrum and T.
mentagrophytes hence, they should be differentiated (for colonial and microscopical
morphologies see table 24-1)
a. T. rubrum
b. T. mentagrophytes
i. produces urease
ii. perforates hair in vitro (hair baiting test)
iii. teardrop/pencil-shaped
iv. Kerion common cause of tinea pedis
v. Permanent. Alopecia
c. T. tonsurans
i. growth enhanced by thiamine
ii. Black dots Endotrix
iii. club-shaped or flat bottoms, batton-form
d. T. verrucosum
i.
radiating grooves
ii. scutula extensive alopecia, hair breaks off above the scalp
(favic antler type) last for lifetime
f. T. violaceum
i.
B. Clinical Infections
Anthropophilic
Zoophilic
Geophilic
(humans)
(animals)
(soil)
E. floccosum
M. audouinii
T. mentagrophytes var
T. mentagrophytes var
interdigitale
T. rubrum
T. schoenleinii
M. gypseum
mentagrophytes
(rodents)
T. verrucosum
(cattle)
T. tonsurans
T. violaceum
2. Transmission
3. Predisposing Factors
Tinea capitis
a. site of lesion
i. scalp
ii. endothrix:
iii. ectothrix:
c. manifestations
i.
a. site of lesions
i. scalp
ii. torso
c. manifestations
i.
a. site of lesions
i. T. rubrum
ii. T. verrucosum
c. manifestations
i.
Tinea corporis
a. site of lesions
i.
arms
ii. legs
iii. torso
i. T. rubrum
ii. M. canis
iii. T. mentagrophytes
c. manifestations
i.
circular patches with advancing red, vesiculated border and central scaling
ii. pruritic
c. manifestations
c. manifestations
Tinea tonsurans
c. manifestations
Tinea unguim
c. manifestations
5.
Laboratory Diagnosis
i. Microsporum species:
iii. T. schoenleinii:
c. culture
i. Mycosel
ii. DTM
iii. cornmeal agar for production of macroconidia
iv. Inhibitory mold agar
v. Sabourauds agar with antibiotics
6.
Treatment
SUBCUTANEOUS MYCOSES
These are infections that involve the skin and subcutaneous tissue which may rarely
become systemic or disseminated.
A.
1. causative agent:
a. Mycelial phase
i. Colonies are usually small, moist, and white to cream colored which on
further incubation become membrabous and coarsely matted, wrinkled, with
the color becoming irregularly dark brown or black and the colony becoming
leathery
ii.
one-celled conidia which are borne bouquet-like, in clusters from the tips of
single conidiospores. Flowerettes daisy-like
b. Yeast phase
3. Transmission
4. Clinical Manifestation
b. chronic sporotrichosis
i. After the primary lesion, multiple subcutaneous nodules develop along the
lymphatic channels and become hard and cordlike
ii. the course is the same and if untreated becomes chronic, but may heal
spontaneously
c. fixed sporotrichosis
5. Laboratory Diagnosis
Specimens:
a. PAS
ii. methenamine silver stain
iii. fluorescent antibody stain
c. culture
i.
ii.
iii.
others
d. serology
6.
i.
ii.
sporotrichin
Treatment
a. oral solution of saturated potassium iodide (SSKI) treatment of choice for cutaneous
sporotrichosis (can be administered topically also)
b. amphotericin B, ketoconazole, dihydroxystilbamidine, griseofulvin and flucytosine
B.
Chromomycosis (Chromoblastomycosis)
This is a chronic fungal infection acquired thru traumatic inoculation of spores, primarily
in the lower extremities. It is characterized by the development of a papule at the site of the
injury that spreads to form warty or tumor like lesions (cauliflower-like). Secondary infection
and ulceration may ensue. Rarely, brain abscess may occur. Elephantiasis and Lymph stasis can
result to secondary infection.
1.
Causative agents
a. Cladosporium carrionii
b. Phialophora verrucosa
c. Fonsecae pedrosoi and F. compacta
2.
Cultural Characteristics
3. Microscopic Apperance
a. Cladosporium species:
b. Phialophora species:
c. Fonsecae species:
4. Laboratory Diagnosis
a. Microscopic examnination
i.
scrapings from crusted areas added to 1-% KOH show presence of muriform
or sclerotic bodies, which appear rounded brown resembling copper
pennies that exhibit transverse septations
5. Treatment
C.
Phaeohyphomycosis
1. Causative Agents
a. Exophiala jeanselmei
i.
grows early as a black yeast with dark budding yeast forms seen on
microscopy of culture
ii.
develops long slender conidiophores with tapered tips and conidia arising from
growth rings (annelids)
i.
ii.
i.
ii.
2. Laboratory Diagnosis
Actinomycotic mycetoma:
Eumycotic mycetoma:
a. P. boydii is a hyaline mold (sexual stage) that grows rapidly as white fluffy colony
that changes to a brownish-gray (mousey) mycelium; the reverse of the colony is
black
b. the asexual stage is called Scedosporium apiospermum and microscopically produces
elliptical (sperm-shaped), single celled conidia borne singly from the tips of long or
short conidiophores (annelophores); clusters of conidiophores with conidia produced
at the ends are called coremia
c. P. boydii, microscopically exhibit cleistothecia, which are saclike structures that
contain asci and ascospores; the ascospores are oval and delicately pointed at each
end resembling the condia of the asexual form. SDA + Cycloheximide
2. Laboratory Diagnosis
3. Treatment
E. Rhinosporidiosis
1. Laboratory Diagnosis
i.
ii.
large thick-walled sporangia; the cell wall of the spherule is mutilayered and
stains with mucicarmine. Mature spores 7-9 um appear lobulated by globular
bodies
2. Treatment
1. Laboratory Diagnosis
i.
large, spherical or oval yeasts that exhibit multiple budding and form short
chains of three to six or more yeast cells which are multinucleated and thick
walled
b. histologic examination
i.
2. Treatment
a. Sulfa drugs
b. Surgical excision
These are fungal infections that may involve any of the internal organs of the body as
well as lymph nodes, bone, subcutaneous tissue and skin. They are caused by inhalation of the
thermally dimorphic fungi which exist in two phases of growth:
Traditionally, the definitive diagnosis of a dimorphic fungus has been made by observing
both the mold and yeast form. But now most laboratories consider the exoantigen test as the most
conclusive method.
OPPORTUNISTIC MYCOSES
These are caused by fungi that usually do not induce disease and occur almost
exclusively in immunocompromised patients who are often on treatment with corticosteroids,
cytotoxic drugs, or other immuno-suppressive agents
Infection
Coccidioidomycosis
(valley fever or
Etiologic Agent
Habitat
Clinical Implication
Coccidioides
soil
immitis
2-21
infection, osteomyelitis,
meningitis, arthritis,
disseminated infection
desert rheumatism)
Histoplasmosis
Histoplasma
soil enriched
(intracellular mycoses
capsulatum
or bat guano
- mimic
nitis, endocarditis
brain abscess, disseminated
infection
5-45
Blastomycosis
Blastomyces
not clearly
(Gilchrists dse.,
dermatitidis
defined
5-30
infection, oropharyngeal
ulceration, osteomyelitis,
American blastomycosis)
prostatitis, arthritis,
CNS infection, disseminated
Infection
Paracoccidioidomycosis
Paracoccidioides
not clearly
(south American
brasiliensis
defined
21-28
infection, oropharyngeal
blastomycosis)
189
Cultural characteristics at 30 degree C
Microscopic Morpho-
Blood-enriched
logical Features
Medium
:
:
Medium Without
Blood
Medium
: taining Medium
of Tissue Form
Coccidioidomycosis
Colonies may be
Colonies appear
Round, nonbudding
delicate, cobweb-like
thick-walled sphe-
to greenish on
rules 30-60 um in
blood-enriched
diameter containing
be pigmented gray,
2-5 um endospores
orange. brown, or
are characteristic
like, heaped,
yellow; mycelium is
wrinkled, and
membranous
Histoplasmosis
Hyphae 1-2 um in
Young cultures
diameter are
usually have a
to spherical bud-
led, yeastlike,
fluffly to glabrous;
predominance
aggregated in
tan or pink in
ropelike clusters;
walled macro-
color; tuft of
sporulation is
conidia that
in colonial morpholo-
colonies
gy occur
rare
of smooth
seen inside of
mononuclear cells
190
Cultural characteristics at 30 degree C
Microscopic Morpho-
Blood-enriched
logical Features
Medium
:
:
Medium Without
Blood
Medium
: taining Medium
of Tissue Form
Blastomycosis
Hyphae 1-2 um in
Hyphae 1-2 um in
to tan, soft
moist, wrinkled,
pyriform conidia
double-contoured
waxy, flat to
aggregated in
are produced on
ropelike clusters
are produced on
cytoplasmic granu-
like; tufts of
sporulation is
short to long
lation is often
surface
rare
conidiophores
obvious
(lollipop); some
colonies (prick-
cultures produce
ly state) (BAP)
few conidia
Paracoccidioi-
domycosis
1-2 um resembling
a mariners wheel
may be present;
by a narrow neck
191
Probable Recovery
Sites
Confirmatory Tests
Serologic Tests
Treatment
for Identification
Coccidioido-
Respiratory secretions
Compliment fixation
Amphotericin B,
mycosis
Immunodiffusion
miconazole
TP bands
Latex agglutination
ketoconazole
gastric washings
triazole
fluconazole
Histoplasmosis
Respiratory secretions,
Same as above
tive with H, M, or
treatment;
H and M band
Amphotericin B-DOC
most require no
ketoconazole
Blastomycosis
Respiratory secretions
1 Broad-based budding
skin, oropharyngeal
Complement fixation
Amphotericin B and
Immunodiffusion
ketoconazole
exhibit A bands
Paracoccidioidomycosis
Respiratory secretions,
oropharyngeal lesions,
gastric washings, skin,
nose
192
Complement fixation
Ketoconazole-DOC;
Amphotericin B,
ulfa drugs
A.
1. Clinical Classification
b. systemic candidiasis
a. Candida albicans and other Candida species which are members of the normal flora
of the skin, mucous membranes and GIT
Specimens:
a. Microscopic examination
b. Culture germ tube
c. Serology (immunodiffusion, CIE, latex agglutination)
d. Skin test
e. CHROM agar medium: 48 hours incubation
C. albicans green
C. tropecalis blue gray
4. Treatment
B.
Cryptococcosis
1. Clinical Forms
a. Pulmonary cryptococcosis
b. Disseminated cryptococcosis
- Causing meningitis, may also occur in skin and viscera
a. Cryptococcus neoformans which is found in the soil and in avian fecal material
(pigeon droppings)
b. Transmitted via inhalation
Specimens: spinal fluid, aspirates from skin lesions, sputum, urine, serum, tissue
II. Human fetal diploid(HFD)- useful in isolation of varicella-zoster virus & HSV, anderivirus,
picornavirus and RSV & are the only cells in which CMV virus and RSV & are the only cells in
which CMV is recovered.
III. Hep-2 continuous cell line- derived from human epithelial cancer cells; excellent for
recovering adenovirus, HSV and especially RSV.
IV. Hela cells- derived from human cervical cancer cells.
V. African green monkey kidney (AGMK), Verocells, baby hamster kidney (BHK),
Human embryonic kidney (HEK), Human embryonic lung (HEL). Etc.
B. Signs of growth
I. Cytophatic effects (CPES)
-evidence of host cell infection and damage or morphological changes in the cells due
to viral proliferation that can be readily observed in unfixed, unstained cell cultures under
LPO.
II. Plaque formation
-small zones of cell destruction or areas of clearness that can be see with the naked
eye.
III. Production ogHemagglutinins
-detected by hemadsorption/ hemagglitination of guinea pig erythrocytes.
IV. Foci formationdo not destroy cells
- Many tumor viruses do not destroy cells but rather cause them to change
morphology and multiply at a faster rate (called transformed cells)
- Colonies of transformed cells develop into foci that can be visualized with the
naked eye.
G. Serologic tests
-includes the following: complement fixation (CF), neutralization hemagglutation
inhibition (HI), passive hemagglutination (PHA), indireectctfluorescent antibody (FA), Immune
adherence
hemagglutination
(IAHA),
immunoelectronmicscopy
(IEM),
counterimmunoelectrophoresis (CIE), radioimmunoassay (RIA), enzyme-linked immunisorbent
assay (ELISA), Flourescent-focus inhibition (FFI), Anti compliment immunofluorescence
(ACIF), and single radial hemolysis (SRH).
HUMAN IMMUNODEFICIENCY VIRUS (HIV)
-HIV is a retrovirus of the lentovirus group causing acquired Immune Defficiency
Syndrome (AIDS).
1. In 1983 and 1984 retrovirus isolates were described by motagnier and coworkers from
hemophiliac patients with lymphadenopathy (called lymphadenopathy-associated virus or LAV1) and from patients with franki AIDS (called ImmunoDefficiency-associated Virus or IDAV
).
2. In 1984 Gallo and Associated isolated HTLV-III (Human T-cells Lymphocytic Virus type
III) From Homosexual and hemophiliac patients with AIDS and AIDS-related while late inh
1984 levy and colleagues isolated AIDS-realted retrovirus or ARV.
3. Subsequent investigations revealed that LAV-1, HTLV-III, IDAV and ARV are all isolated of
the same virus, now called Human immune Defficiecy Virus type 1 (HIV-1)
4. In 1986 Motagnier isolated LAV-2 Which is now a member of a family of human lentivirus
called HIV-2 (this virus can also cause AIDS but limited to west and central Africa and has not
demistrated the virulence of HIV-1)
MODE OF TRANSMISSION
1. Sexual contact
2. Blood transfusion
3. Sharing of contaminated needles between IV Drug abusers
4. Trans placental transmission
5. HIV has been isolated from blood, semen, mothers milk, tears, CSF and saliva; however, to
date, transmission by fluids other than the first 3 has not been reported.
Risk groups
1. Homosexual and bisexual men
2. IV Drug abusers
3. Hatians
4. Recepients of transfused blood and blood products
5. Hemophiliacs who received factor VIII Concentrate
6. Sexual contacts and children of persons with HIV infection.
D. Incubation Period: 5-65 months
E. Pathogenesis
1. HIV has a strong affinity for the CD4 antigen which serves as a receptor for entering and
infecting cells.
2. In AIDS, HIV Selectivity adheres to T-lymphocytes which carry the CD4 antigen on their
surface (formely T Helper cells with T4 surface antigen). This leads to depletion of CD4 T-cells
resulting in severe lymphopenia.
3. Aside from CD4+ T-cells, HIV also infects all CD4+ Cell types which include peripheral
blood monocytes, monocytoid cell lines and tissue macrophages (e.g, follicular dendritic cells or
micrpglial cells in brain).
4. An important characteristics of HIV is that its replication in some cells in vivo appears
restricted, resulting in maintenance of HIV in alatent form.
F. Clinical Manifestation
1. AIDS
a. It is obvious that the primary site of cellular destruction is the host immune system, thus
patients suffer from numerous opportunistics infections, the most common are the following.
Bacterial
i. Mycrobacteriumavium- intracellular are complex
ii. M. Tuberculosis
iii. Other mycobacteria
Fungal
i. Esophangeal&Disseminated candidiasis
ii. Disseminated aspergillosis
iii. Cryptococcosis
Parasitic
i. chronic crptosporidiosa (Enteritis)
ii. Pneumocystis carinii pneumonia
iii. Intestinal and disseminated strongyloidosis
iv. Toxoplasmosis (pneumonia or CNS infection)
Viral
HEPATITIS VIRUSES
A. Hepatitis A virus (HAV)
-a picornavirus, classified as enterovirus 72
1.Disease Produced: Hepatitis A or infection hepatitis (short incubation hepatitis)
2. Transmission: Predominantly fecal-oral route
3. Incubation Period: 15-45 days (avg. 25-30 days)
4. Laboratory Diagnosis:
a. demonstration of the virus by electron microscopy
b. detection of anti HAV (antibody to HAV) present at onset of symptoms which persist
throughout lifetime.
c. detection of IgM anti- HAV indicating recent infection with Hepatitis A which is
positive up to 4-6 months after infection.
B.Hepatits B virus (RBV)
-A HEPADNAVIRUS conraining the infection dane particle
1. Disease produced: Hepatitis B or serum Hepatitis (long incubation hepatitis); can end to liver
CA and Cirrhosis.
2. TRANSMISSION
a. Predominantly parenteral route
b. sexual contact
c. transplacental
d. mucous membrane exposure
3. Incubationperiod: 50-180 days (avg., 60-90 days)
c.Anti-HBc- Indicates active HBV infection cannot be excluded; a recent HBV infection can be
confirmed by examining the sample for high titers of IgM anti HBC.
d.HBeA- indicates active hepatitis infection,
e. Anti-HBe- when present in HBsAg carrier blood is potentially less infectious.
f.HBcAg (Hepatitis B core antigen)- no test available for routine use.