INSTRUCTIONS

j\S you na\-;gatc th is laboratory module, you will notice that many words ha\"c been

bold cd. I r you hover your mouse o\'cr lhese words, a "pop-up" balloon will appear.

These balloons will provide you with a dcfi nilion of tbe term, or an explanation of

It.<; lmpo n ancc.

You will also be able to click {)n cerr-din words and/or images that will then take

rou to additio nal infonnatio n. You may navis-.ne forwa rd and backward within th is

module by clicking o n a " NE"A'T " o r "BAC K" button.

•
Throughout th is module, you will encounter $e\'cml questio ns intended 10 guide
your learn ing. 111 cse l{ucstions will NOT be graded.

Please click on rhe ''NE)''''">' bunoo to be in the Introducrion.

 LABORATORY #3: SKIN AND SOFT TISSUE INFECTIONS

INTRODUCTION

The skin (which covers and protects the human body) constitutes our first line of
defense ag2inst pathogenic microorganisms. T he skin set....es as both an
inhospitable en"ironmcni for palhogen growth, and a physical harrier tn
pathogen cmry. Both of these charactcristics help prc'"cnt infection.

Generally speaking, the skin is not a suitable envi ronment for microorganism
colonizarion and growth because of two main factors: I) the ImtjorilY of [he
skin's surface does not contai n suflicien l amounts of moiHure to sustain bacterial
growlh, and 2) thc secrctions of the skin co ntain a high salt concentration and are
of low pH.

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 INTRODUCTION

Despite these harsh conditions, the human ski n docs support the grO\vth of
.
certain microbes (c.g.' Stttpl?J·loromls tpinmllidi!, XtroSIS,

PropiMibo<lfrilllf' (IntU, t/t, .. ) . These bacteria afC able ((): 1) tolerate the dry. acidic,

high salt environment chardctcristic of the skin, and 2) derive nlnricms from skin

secretions (e.g.: scbum). These highly adapted microbes arc referred to as normal
skin fl ora.

The normal flord. of the skin benefits the host (i.e.: humans) by competing \\-;th

pathogenic microbes for nmrients, and by maintaining the low p H of the skin -

both of which ultimately limit pathogen growth and reduce the risk of infection.

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 INTRODUCTIO N

It ;s important to remember that the skin also acts as a ph}'sical barrier - thereby

prc\'cnting pathogen cnlry into the hody. Fo r the most pan, the skin surface is
impcmlcablc 10 micrDbcs. As such, mOSt skin and soft tissue infections arise
from exogenous pcnclT'J.tion (c.g.: trauma, surgery, bites) .

Howc\'er, certain microort,"2nisms (e.g.: Xlttpllj'/()(O{{//S flllrtl/J) can penetnlte the skin
\';a natural openings presen! on the skin surface (e.g.: sweat pores, or the

passageways that exist between the hair sha ft and hair follicle), also leading tt}

infection.

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 INTRODUCTION

In lecture, you ha\"c learned that a wide range of microorganisms can cause skin

and so ft ti ssue infections. ~Iorcon":r, you have secn that each type o f infection

pOSSCSSCl" itS own ch aracteristic signs and symptoms tbat, when identified, help
facilitate diagnosis.

In thi s laboratory module, you \\~ Il explore some of the specimen collection
methods and laboratory diagnostic techniques used to identify a serious skin and

so ft ti ssue infection ; necrotizing faseiitis.

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 PATIENT SCENARIO

Paul is a 38 year old man \\<;rh 2 yOllng ch ildren. Paul is heal thy and fit and takes
no regular medications. One Saturday afu:moon, Paul notices a splinter in his
left thigh aher playing a football game with some of his friends. He remm"es (he
splinter and thinks nothing more of it.

Please dick on the "NEXT ' bun on [0 continue the pa tient sccnaril).
 PATI ENT SCENARIO

Later in the day, Paul's left thigh begins to ache but he is still able [0 get around

the hOllse and help pm the childre n to bcd. Exhausted, Paul fa lls asleep in front
of rhe rdc,-ision.

i\ few hours later, Paul wakes fedi ng terrible, Paul calls for his wtfe Samantha,

who finds him drenched in sweat and shaking with rigors. Paul complains of

severe leg pain and tells Samantha "rhat he has never experienced pain like rhis in
his life", Worried. Samantha decides [0 Din for an ambulance.

Please d ick on the "NEXT' bUlIon to continue the patient scenario.

 PATI E NT SCENA RI O

At the hospiml, physical examination of Paul's left thigh re\'eals an area of purple
discoloratio n at the si te of the remm'cd splinter, and surn)unding ery thema wi th
no raised margins and two hemorrhagic bullae. Paul tells the nurse that his

thigh is cXl.juisi tcly tender and "cry painful, c\'cn though the skin "doesn't look

too bad - only blistered and bruised",

The nurse takes Paul's vitals, and records the fo llowing: blood pressure 90/50,

heart rate 110. temperarure ·IO"e, respiratory rare of 28 ,virh an oxygen sa ruration
lc" eI of98% on room ai r.
 PATIENT SCENARIO

Erythema

Dusl..-y purple
di scolo uration
orlhe skin ............
_ Hemorrhag ic
Bullae

Pho tographi c im age of Paul's left thigh

 Q UESTION

J\l this point in the patient scenario, we know th at Paul has the following signs

and symptoms:

• Hypotension • Purple discoloration & c'1'lhema surrounding

.
• Fcycr puncture sue

• Tachycardia • SC\>ere pai n, disproportionate tn apparent

tissue damage

Gi\Oen these findings, which diagnosis do you suspect?

• Necrotizing fascii tis • Erysipelas

• Folliculi tis
 PATI ENT SCENARIO

Based on Paul's signs and sym plOms, rhe em ergency mom physician makes a

prcsumpu\-c diagnosis of necrotizing fasciitis, and requests a consult with both

the infenious disease specialist and surgeon on call. Two sets of Palil's blood are

drawn and sent to the medical microbiology lab.

 PATI ENT SCE NAR IO

I\ ftc r examining Paul's thigh, the infCClious disease special ist si milarly suspeCtS

necrotizing fasciitis and o rders the following:

1) Intnwenous antibiotics

• Penicillin G

• 4 mi ll ion unit.<1, e:"e:ry 4 hours

• Clind;Imycin

• 900 mg e ' "ery 8 hours

2) Intravenous immuno~lo bulin (IVIG)

 PATIENT SCENARIO

'Inc surgeon also concurs with [he prcsumpoyc diagnosis of necroo zing fasciiri s,

and takes Paul [0 [he opcrating room for wound exploranon and surgical

debridem ent .

During the procedure, the surgeon find s that both the soft tissue and underlying

fa scia in Paul's thigh arc necro tic, and subsclJue ntly dcbridc!; all the dead and

diseased tissue. A samplc of the necrotic tissuc is sent to the medical

mic robiology lab for culmrc.

 PATIENT SCENARIO

j\ftcr the procedure, a surgical resident meets with Paul's wife Samantha and
con firms the diagnosis of necrotizing fasciitis.

'1111:: resident explains to Samantha that although thc), were able to remO\'c all of

the necrotic tissue in P:I.UI's thigh, there is a possibility that the infection may

progress (despite antibiotic the r.lpy) and thllt sllr'b>1cal reexploration may be

necessary.

He IIlso explains that once the infection is resolved, Paul ,,,il\ need to undergo a

number of s kin grafts to repair the debrided a TC\lS on hi s thigh.

 PATIENT SCENA RIO

Understa ndably, Paul's \\~fc is vcry upset. She \vams to know how Paul gOl this
infection, and why it made him so ill.

The res ident tells Samanlha that n nee they n:cei\'c the results fro m the medical
microbioiugy lab, the)' will know what type of bacteria is responsible fo r the
infection. '111is information will provide them with a ocncr understanding of
Paul's prognosis, and rna)' also explain how Paul Contt"dcte d this infection. At the
moment, they suspeCt that the infection originated from Paul 's splinter.
INSIDE THE MEDICAL MICROBIOLOGY
LABORATORY

SKIN & SOFT TISSUE INFECTIONS

 Q UESTION

\\:.'h ich of the following types of clinical specimens arc appropriate for diagnosing

necrotizing fasditis?

• Blood

• Swab of the infected area

• Tissue (removed during surgical debridement)

 BLOOD SPECIMENS - GRAM STAIN

A Gram stain of Paul's blood sample is performed. \'('hal type of baC[cria do y Oll
see in the image helow?

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 BLOOD SPECIMENS· GRAM STAIN

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'Ille Gram st<t;n or Paul's

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 PATlENT SCE NARIO

Based on the results of the Gram stain pcrfonncd on Paul's blood, the lab
reports the presence of Gram posilin~ cocci in pairs and chains.

Gi,'cn the extent of the necrosis obsen'cd in Paul's thigh during surgery, the lab
repom [hat Streptoc:occ:us pyogencs (a.k.a. Group i\ strt!ptococcus) is likely
responsible for Paul's infection. The physician is infonned that the identity of the
pathogen will be confirmed once the results of the tissue culture are obtained.

In light of this infom13tion, the physician continues Paul on Penicillin G,

d inclamycin, and rV1G since necrotizing in fections caused by Streptococcus
pyogenes respond well to this combination of ther-dpies.
 T ISSUE SP EC IM.ENS - CULTURE

Suspected cases of SUcptococcal infection arc culrured o n blood ag:lr (Q help identify
which Strcplococcal spc<:ics is responsible for lhe infection. T his is because
Slrcptococcal species dcmonsU"alc different types of hemolysis on blood agar.

• Bcta"hemolysis appears as a complete clearing o f the agar (complete lys.is of

c~'lhrocyrcs) wherever Ihe bacteria is p resent - this is characlcristic of 5JrrplO£o«f1J

pyo,gmu.

• t\ lpha-hcmolrsis appears as an incomplete clearing of the agar, producing a greenish

discolouration - this is characteristic of SITPPlococfllJ P"IIIHlOllit".

• G:l.nuna-hcmolysis refers to an absence of hemolysis, rhcrcforc Ihere is no cffcci on

the blood agar - this is characteristic of r:.,'ltrrJ«)«(IIJjfll(lUI.
TISSUE SPECIMENS - CULTURE

l 11C results of Paul's tissue

culture show:

- - - - Beta-hemolysis

This fi nding confirms the

identity of the pathogen
I causing Paul's infection:

StreptococclIS pyogcncs
 PATIENT SCENARIO

"Inc physician infomlS Paul and Samantha that hi s infec tion was caused by

JJrrpl(K()({7/] PJogtnU; the same bacterial species that causes Streptococcal

ph:l'1'ngitis (a.k.a. Strcp. throat). li e then goes on to explain that Paul likely

contracted thi s organism from one of hi s ch ildren, since c hildren arc more likely
to be colonized \\~ [h th is type of bacteria (han adults.
 PATIENT SCE NARIO

Upon learning this news, Samantha becomes vcry agitated, and c"presses concern
that theiT child ren Imy be at fisk for de\'eloping th is type of infection. \,\'hilc the

rest of the family is not at immediate risk, [he p hysician explains that there arc

several ap p roaches to managing the rest of the fami ly;

• Anyone in the fami ly widl a sore throat or skin infection should be treated

\\~ lh :l;mibimics specific for this pathogen (e.g. ampici llin)

• Fam ily members with no symplOms sho uld be cultured to see if they arc

colonized \v;th J/rrploro((JfJ P.J'o gmfl, and then treated with ampicillin if they are
shown (0 be positi\-c.
 A CHALLENGE IN MICROBIOLOGY

In the prescnt patlem sccnano, Paul's infection was caused by a singular

pathogen.

Howevcr, many infecoons are caused by a mixture of bancria. As a result, many

different Iypes of bacteria can grow in a single cul tu re. I.n such cases, it may be
difficult to identify each type of bacteria that grows in culture. The next sl ide
demonstrates this concept.
I low many differem rypes of bacterial colonies on this blood agar can you idemify?
There are at leasl 4 diffcrem Iypes of bacteria prescnt on th is blood agar media.

How does the medical microbiology lab identify each of them?

 A SO LUTION TO THE PROBLEM

\'.;Ihen a mix of bacteria arc pre~('nt in a ~ pecimen. differcm types of culture med ia nlly he
helpful in identifying the t}1:leS of bacteria pre~em in the sample. In the pl:ue shown below,

the s:lmc specimen is cultured on CNA media (which o nl y grows Gram positin: b:lClcri:l.).
On CNA media. only b:J.cteri:J. 3 & 4 nne :J.ble to gt"OW. Thi~ ~ ugge~t~ th:!! bacteria 1 & ? afe

Gram negative.
 A SOLUTION TO THE PROBLEM - PART 2

On the next pl ate, the sanle specimen is cuhured on i\ lacConkcy agar media,
Only Gf'am negative bacteria arc able to grow o n this type of agar.

Using this type of media, Gt'.Im negati\'c bacteria are further identified based on
their ability to ferment lactose .

• Lac(O~ fermen lel1' cause a drop in p H , producing a pink-red appearance

• Non-lac[Ose ferm entcrs remai n clear and colourless

 A SOLUTION TO THE PROBLEM

O n this media, only oocted :!. I & 2 :!.re :!.ble (0 grow, confirming that the~e tWO b:tcterb :!.re

ti f:l..m nqr.tive. Fu rthermo re, this culture shows b:!.cteria I is a lactose fe mlente r and
bacteri:!. 2 is not. T his inform:!.tion will :I...(Si51 the microbio logy bb tech nician idemi!}' the

organism~ responsible for the infec tion.

You have completed the Skin and So ft Tissue l nfections

Laboratory "M odule .