Interes'ng

Topics on Microbiology

Department of Microbiology Faculty of Medicine Siriraj Hospital

Scope
Review of bacterial & fungal iden'ca'on Issues with drug-resistant bacteria

Bacterial & Fungal Identification

Gram-positive cocci
Clusters
Staphylocccus spp.
(Catalase-Pos) (+) (-)

Chains

Catalase test
H2O2 H2O + O2

Streptococcus spp. Enterococcus spp.

(Catalase-Neg)

Staphylococci
Coagulase test
Slide test

(+) S. aureus (-) CNS

Tube test

(-) (+) (-) (+)

Streptococci
Beta-hemolysis Group A streptococci (S. pyogenes) Group B streptococci (S. agalac3ae) Alpha/gamma-hemolysis (Non-groupable) S. pneumoniae Viridans streptococci

Hemolysis Bacitracin-Group A Optochin-S. pneumo S. aureus

PosiGve

CAMP-posiGve Latex-grouping [Group B Strep]

Gram-Positive Rods Aerobe / Facultative anaerobe Spore-former Bacillus B. anthracis -anthrax (3 forms) -anthrax toxin B. cereus -Food poisoning -Enterotoxin -Intoxication Regular rods AF negative Listeria L. monocytogenes -meningitis -brain, placenta -CMI defect Erysipelothrix E. rhusiopathiae -erysipeloid -occupation -CMI defect AF positive Mycobacterium Slow grower M. tuberculosis -TB -intracellular Rapid grower M. fortuitum comp -skin infection Uncultivable M. leprae -leprosy Non-spore-former Coryneform Corynebacterium C. diphtheriae -diphtheria -diphtheria toxin Spore-former Clostridium C. perfringens -gas gangrene (alpha toxin) -enteritis necroticans (beta toxin) C. difficile Anaerobe Non-spore-former Actinomyces A. israelii -mycetoma -tissue abscess
Propionibacterium

Coccobacilli Rhodococcus R. equi -pneumonia -occupation -CMI defect

Branching filament Gram-variable coccobacilli Gardnerella G. vaginalis -bacterial vaginosis -clue cell -Whiff s test MAF negative Actinomyces A. israelii -mycetoma -tissue abscess -anaerobe! MAF positive Actinomycetes Nocardia N. asteroides -abscess -CMI defect

P. acnes -antibiotic-associated -skin flora diarrhea -acne vulgaris -toxin A & B C. botulinum Lactobacillus -botulism -botulinum toxin L. rhamnosus -vaginal flora C. tetani -chorioamnionitis -tetanus -tetanus toxin

AF = acid-fast stain MAF = modified AF stain

Pathogenic Bacteria Worksheet 1 By Pattarachai Kiratisin

Spot Diagnosis

Gram-negative rods
Coccobacilli Enterobacteriaceae
(Oxidase-neg)

Non-fermenters

Curved rods

Gram-Negative Rods
Aerobe / Facultative anaerobe Coccobacilli Moraxella M. catarrhalis -pneumonia -acute in COPD Small pleomorphic coccobacilli Haemophilus H. influenzae -capsule: Hib -pneumonia -meningitis H. ducreyi -chancroid Bordetella B. pertussis -pertussis -pertussis toxin Brucella B. melitensis -brucellosis -zoonosis Francisella F. tularensis -tularemia -zoonosis Pasteurella P. multocida -wound infection -zoonosis Microaerophile Helicobacter H. pylori -gastritis -urease Campylobacter C. jejuni -enterocolitis -animal reservoir Anaerobe Bacteroides B. fragilis -GI flora -mixed infection Porphyromonas P. gingivalis -oral flora -mixed infection Prevotella P. melaninogenica -oral flora -mixed infection Fusobacterium F. nucleatum -oral flora -vincent angina Capnocytophaga C. canimorsus -oral flora (dog) -wound infection

Regular rods Glucose fermenter (Enterobacteriaceae) Escherichia

Fastidious Legionella L. pneumophila -Legionaires -water, cooler

E. coli Non-fermenter (NF) -UTI -diarrhea Pseudomonas -meningitis P. aeruginosa -several toxins & -wound infection adhesins -exotoxin A Klebsiella K. pneumoniae -pneumonia -capsule Salmonella S. enterica -typhoid fever -intracellular Shigella S. dysenteriae -enterocolitis -shiga toxin Burkholderia B. pseudomallei -melioidosis -soil / SEA area Acinetobacter A. baumannii -pneumonia -nosocomial
Stenotrophomonas

Curved rods Vibrio V. Cholerae -cholera toxin V. parahaemolyticus V. vulnificus Aeromonas A. hydrophila -diarrhea -water reservoir Plesiomonas P. shigelloides -diarrhea

S. maltophilia -pneumonia -nosocomial

Pathogenic Bacteria Worksheet 2 By Pattarachai Kiratisin

Fungi
Yeast
Small rounded form Budding or ssion

Mold
Filamentous form (hyphae / mycelium) ElongaGon

Fungi
Yeast Budding unicellular Mold Non-septate hyphae

37c

25c

Septate hyphae Hyaline mold General Dermatophyte Dimorphic fungi DemaGaceous mold

Spot Diagnosis

Spot Diagnosis

Drug-Resistant Bacteria

MRSA
Mediated by mecA (PBP2a) Disk diusion test: cefoxi'n recommended (cefoxiGn only for CNS, no oxacillin zone) Heteroresistance (1:10^6) may be missed Full 24-h incubaGon required for tesGng Report Resistant to all b-lactams (incld. carbapenems) & BLBI regardless of test results

VISA / VRSA
Mediated by vanA Disk diusion test: not available Vancomycin MIC breakpoint = 2 ug/mL (R, if 16) (for CNS = 4 ug/mL, R if 32) hVISA (1:10^6, MIC = 4) can be detected by MIC MIC 32 cannot be detected by disk diusion Full 24-h incubaGon required for MIC tesGng Report Need to conrm for MIC and van gene

Inducible clindamycin resistance

D-zone test If R to erythromycin and S/I to clindamycin Test for staphylococci and b-hemolyGc streptococci If posi've (D-zone or hazy growth near Clin), report clindamycin R
Erythromycin Clindamycin

Inducible clindamycin resistance

Resistance mechanism Macrolides only: Eux (msrA: staph, mef: strep) Inducible clindamycin in macrolide-R: MLSB (erm: methylaGon of 23S rRNA)

PRSP (Pen-R S. pneumoniae)

Use oxacillin disk for tes'ng Zone 20 mm (=MIC 0.06) - S to Pen, Amp, Amp-sul, Cefuroxime, Imi, Mer Zone 19 mm -Must do MIC for Pen + Cefo / Cefri / Mer -Above MIC be rouGnely reported for CSF isolate PGS MIC Breakpoint -MeningiGs = 0.06 ug/mL -Non-meningiGs = 2 ug/mL

PRSP (Pen-R S. pneumoniae) Disk diusion not reliable for (must do MIC):
-Amp, Amox -Cefuroxime, Cefriaxone, Cefotaxime, Cefepime -Ertapenem, Imipenem, Meropenem

Other streptococci
Not recommended for tes'ng of groups A & B B-lactam test: if clinical response unsaGsed - S to Pen covers all approved b-lactams Macrolide test: if allergic to Pen -Test for inducible clindamycin if indicated Viridans streptococci B-lactam test: Pen disk method not reliable -Test MIC from sterile sites

Enterococci
Pen/Amp Resistance: -Commonly due to low-anity PBPs -Rarely due to b-lactamase Pen S >>> Amp S (no need to test Amp) Amp S >>> Need to test Pen B-lactamase test-Posi've >>> All Pens R Synergy with Aminoglycosides if Pen/Amp S -No R to high-level gentamicin/streptomycin

VRE
Van non-suscep'ble by disk (I, R) must do MIC Full 24-h incubaGon required Van MIC breakpoint = 4 If MIC 8 / 16 (I): conrm idenGcaGon

E. gallinarum & E. casseliavus: intrinsic (I) due to VanC E. faecium & E. faecalis: acquired (R) due to VanA / VanB

ESBL Extended-Spectrum Beta-Lactamase

Screening test: Cepodoxime (10 ug) Cefazidime (30 ug) Cefotaxime (30 ug) Cefriaxone (30 ug) 17 mm, or 22 mm for P. mirabilis 22 mm, or 27 mm, or 25 mm (not for P. mirabilis)

ESBL
Combina'on disk test (conrmatory test) Cefazidime / Cefazidime-clav and Cefotaxime / Cefotaxime-clav

5-mm increase of zone when combined with clav

= ESBL

Non-ESBL

ESBL

ESBL
RouGnely for E. coli, K. pneumoniae, K. oxytoca & clinical signicant isolate of P. mirabilis

Report as R for all penicillins and cephalosporins (regardless of in vitro results)

ESBL
New guideline (CLSI 2010) New cephalosporin breakpoints Drug
Cefazolin

MIC
8/32 1/4

Inhibi'on zone
14/18 None! 14/23 22/26 13/21 19/23

Cefotaxime 8/64 1/4 Cefriaxone 8/64 1/4

Cefazidime 8/32 4/16 14/18 17/21

ESBL
New guideline (CLSI 2010) Apply to all Enterobacteriaceae If adopt new breakpoints, no need to test ESBL!

Report all penicillins / cephalosporins as it appears (no need to change results)

KPC Klebsiella pneumoniae carbapenemase

Hydrolyze all b-lactams including carbapenems Screening Zone diameter MIC Ertapenem 19-21 mm (S 19) 2 Meropenem 16-21 mm (S 16) 2-4 Imipenem *Perform poorly 2-4

KPC
Conrmatory test: Modied Hodge Test
Lawn of E. coli ATCC 25922 Ertapenem/meropenem disk (10 ug) Test isolates

KPC
Report If MHT+ but tested S to carbapenems: Report carbapenem MIC without interpretaGon
With noGcaGon carbapenemase producGon was demonstrated: no established clinical ecacy of carbapenems for treatment

If I or R to carbapenems, no need to do MHT

AmpC b-lactamase
Resistant to penicillins and most cephalosporins (including cephamycins) Susceptible to cefepime, cefpirome and carbapenems Not inhibited by clavulanic acid

Chromosomal & plasmid-mediated

Note: Bolded = dierent from ESBL

Beta-lactamases Molecular class Ac've site Member

A Serine ESBL, KPC, most penicillinases & cephalosporinases Metallo-b-lactamase (VIM, IMP) AmpC OXA

B C D

Zinc Serine Serine

Pseudomonas aeruginosa Resistance mechanisms

Mechanism Eect
(HyperproducGon aects Imi, not Mer)

Increased AmpC Ceph & anG-pseudomonal Pen MexAB-OprM (ConsG. Expr.) MexCD-OprJ MexXY-OprM* MexEF-OprN Porin (OprD) loss

Ceph & anG-pseudomonal Pen FQ, Tet, Chloram, Mer & Erta (not Imi) Pip (not Carben), Cefepime (not Cefaz), FQ, Mer & Erta (not Imi), *also AG FQ, Trimethoprim, Chloram Imi (less for Mer, not Erta)

Metallo-b-lactamase All b-lactams

Acinetobacter baumannii Resistance mechanisms (*Intrinsic)

Mechanism
AmpC* ESBL, OXA, metallo- PBP change* AdeABC eux* AbeM eux Tet eux (TetA, B) Porin (CarO) loss* Membrane alter*

Eect
3G Ceph (hyperproducGon) Ceph, Carbapenems Pen & Ceph Ceph, Tigecycline FQ (high R with gyrA/parC mutaGon & porin loss) FQ, AG Tet, Mino Ceph ColisGn, Polymyxin B

BLNAR H. influenzae
Beta-Lactamase NegaGve, Ampicillin Resistance R to Amox-clav, Amp-sul, Pip-tazo & Cefuroxime (regardless of in vitro results) B-lactamase test posi've: R to Amp & Amox -B-lactamase test required for all isolates CSF isolate: -Report only Amp, a 3GC, Mero, Chloram

Issues with Some Antibacterial Agents

Doripenem

No CLSI guideline for tes'ng & interpreta'on

Daptomycin
Divalent ca'ons (Ca2+) -Insucience: cause false-resistance

Only suscepGble breakpoint [no I/R] available: -Staphylococci & streptococci, not S. pneumoniae (1) -Enterococci (4) *Disk diusion method not reliable

Issues with Some Antibacterial Agents

Colis'n

CLSI guideline -Disk method only available for P. aeruginosa -MIC method available for P. aeruginosa & Acinetobacter spp. MIC breakpoint -P. aeruginosa: [S] 2 / [I] =4 / [R] 8 -Acinetobacter: [S] 2 / no [I] / [R] 4

Issues with Some Antibacterial Agents

Tigecycline

No CLSI guideline -Issues with caGons e.g. Mn2+

Medium (Mn concentra'ons) Medium 1 Medium 2 Medium 3 (2.5 ug/mL) (2.5 ug/mL) (629 ug/mL) MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 0.5 1 0.38 0.75 0.75 1.5 1.5 2 0.5 1 1 1 4 4 2 3 2 4

A. baumannii ESBL (+) E. coli ESBL (+) K. pneumoniae