staph - flashcards

i left out some morphology stuff so review those slides !

facultative anaerobes

facultative anaerobes

staph and rothia

what do these GPC have in common?

• all are normal flora of skin and mucous membranes

• non motile

• catalase producing

what test should we do when we see a GPC?

catalase!

transmission/predisposing conditions - s. aureus

• unwashed hands, inanimate objects, traumatic introduction

• hospital outbreaks in nurseries, burn units, surgical pts

• indwelling devices, skin injuries, immune response defects, chronic infections

s. aureus diseases

• skin/wound infections

• scalded skin syndrome (ritter’s)

• TSS

• food poisoning

• pneumonia (after influenza A; v deadly)

• bacteremia, endocarditis (via IV injection)

• osteomyelitis (secondary to bacteremia)

• septic arthritis

skin and wound infections - s. aureus

folliculitis, furunculosis, carbunculosis, bullous impetigo

scalded skin syndrome (ritter’s)

• infants/children

• exfoliative toxin: body-wide exfoliative dermatitis

• profuse peeling of epidermis

TSS - s. aureus

• toxin TSST-1: super Ag stimulates t-cells = cytokine storm

• multisystem illness

• fever, trunk rash, dehydration, hypotension, shock, death

• high absorbent tampons and post op infections

food poisoning - s. aureus

• infected food handler

• rapid (2-8hr): nausea, vomiting, abd pain/cramps, diarrhea, headaches (NO FEVER)

• enterotoxin: present in food (formed outside body)

  • heat stable (A-E, G-J); super Ags

list the s. aureus virulence factors

• cytolytic (alpha, beta, gamma, delta)

• exfoliative toxin

• TSST-1

• enterotoxins

• protein A

• enzymes

alpha hemolysin - s. aureus

lyses rbcs, damages plts, macros, tissue

beta hemolysin (sphingomyelinase C) - s. aureus

attacks sphingomyelin in rbc membrane = lysis

gamma hemolysin - s. aureus

• associated with PVL

  • toxic to PMNs preventing phagocytosis; associated with severe primary skin infections and necrotic pneumonia

delta hemolysin - s. aureus

usually less toxic than alpha/beta hemolysins

protein A - s. aureus

• binds Fc portion of IgG neutralizing Ab

• found in cell wall

• blocks phagocytosis

virulence enzymes used by s. aureus

• protease, hyaluronidase, lipase: destroy tissue and spread infection

• staphylocoagulase

s. aureus colony morphology

• large zones of beta hemolysis

• appear sl yellow after long incubation

• larger colonies than CoNS

list the CoNS

• epidermidis

• saprophyticus

• lugdunensis

• haemolyticus

• pseuodintermedius

diseases - s. epidermidis

• hospital acquired (indwelling devices, immunotherapy)

  • catheters, shunts, prosthetics, implants (biofilms)

• leading cause of nosocomial UTIs

list the 2 virulence factors of s. epidermidis

• slime biofilm layer

• poly-gamma-DL-glutamic acid: adherence and protection against host defenses

s. epidermidis colony morphology

small-medium white/gray; non hemolytic (gamma)

disease - s. saprophyticus

• UTIs in young sexually active women

• adheres better to epi cells in urinary tract than other CoNS

• resistant to novobiocin

s. saprophyticus colony morphology

can appear yellowish

diseases - s. lugdunensis

• infective endocarditis, septicemia, meningitis, skin/soft tissue infections, UTIs, septic shock

• more virulent (like s. aureus)

• can be resistant to oxacillin (mecA)

• aggressive endocarditis = requires valve replacement; higher mortality

s. lugdunensis colony morphology

often beta hemolytic

diseases - s. haemolyticus

• wound infections, bacteremia, UTIs, endocarditis

• some resistant to vancomycin

s. haemolyticus colony morphology

medium; weak/mod beta hemolysis

s. pseudintermedius

• zoonotic: seen in pet owners/vet staff

• surgical site infections, rhinosinusitis, catheter-related bacteremia

• oxacillin resistance

what requires a full ID regarding staph spp?

• s. aureus

• sterile specimens (non sterile = CoNS okay)

methods to ID staphylococcus spp

• biochemicals (cat, coagulase, latex)

• antimicrobials (novobiocin)

• selective/differential media (mannitol salt, DNase, purple broth w glucose)

• maldi tof

catalase test

• collect colony carefully from BAP (agar will react = FP)

• catalase + H2O2 = water and O2

• bubbles = pos

• no bubbles = neg

coagulase test principle

• enzyme converts FIB = fibrin = fibrin clot

  • pos in s. aureus

  • neg in CoNS

• rabbit plasma

slide coagulase test

• detects bound coagulase (clumping factor)

  • on bacterial surface directly converting FIB to fibrin

• some lugdenensis/schleiferi isolates are pos

  • neg for tube test

• must be confirmed by tube coagulase

tube coagulase test

• detects free coagulase (staphylocoagulase)

  • reacts with coagulase-reacting factor forming thrombin-like complex indirectly causing clot formation

• inoculate/incubate 37C

• at 2 & 4 hrs examine for clot, if neg = let incubate at RT for 24 hrs

latex agglutination assay

• uses latex particles coated with plasma/Abs

  • detects clumping factor/protein A

• more sensitive/specific than slide coagulase test

• pos = clumping = s. aureus

  • some saprophyticus/hominis/haemolyticus appear pos

• neg = no clumping

CLSI - presumptive ID of s. aureus

• must have: GPC in clusters, cat pos, tube/slide coag pos OR latex pos

• additional tests: beta hemolytic on BAP

  • tube coag not needed if beta hemolytic

• report as “presumptive s. aureus”

mannitol salt agar

• selective/differential, nutritive with 7.5% NaCl, mannitol, phenol red

  • high salt conc = inhibits all except staph and micrococcus

• yellow colonies = s. aureus (mannitol fermenter)

• red colonies = other

DNase test agar

• differential to ID bacteria that produce DNase (breaks down DNA)

  • used to confirm equivocal coagulase test

  • pos = s. aureus

  • neg = CoNS

• methyl green:

  • pos = clear zone neg = remains green

• toluidine blue:

  • pos = pink/purple/red zone neg = remains blue

purple broth with glucose

• differential

• staph spp produce acid from glucose (dextrose) in anaerobic conditions

• inoculate tube, overlay surface to ensure

  • pos = yellow

  • neg = no change (purple)

novobiocin disk test

• helps ID s. saprophyticus

• perform on urine cultures that are GPC clusters, cat pos, coag neg

• zone <12mm = resistant (saprophyticus)

• zone >12 mm = susceptible (other CoNS)

what susceptibilities do we test for staphylococcus spp?

• MRSA

• inducible clindamycin resistance (D test)

• beta lactamase production (nitrocefin)

• VRSA/VISA

beta lactamase production

• most s. aureus/lugdunensis strains are resistant to penicillins due to beta-lactamase production

  • ***any colonies that test susceptible to penicillin = test for beta lactamase production***

• rub colony on nitrocefin disk

  • pos = pink (beta lactamase producer)

  • neg = no color change

inducible clindamycin resistance - D test

• certain MRSA/CoNS are susceptible to clindamycin but over course of treatment could become resistant

  • we can test for this beforehand

• streak lawn and place erythromycin and clindamycin disks 15-26mm apart

  • pos = flattened edge adjacent to erythromycin disk (resistant)

  • no flattened edge = no inducible clindamycin resistance (but can be resistant due to other means)

methods to detect MRSA

• oxacillin screen (agar/disk)

• cefoxitin screen

• RTPCR

• CHROMagar MRSA

• latex agglutination test

• film array BCID/verigene assay

MRSA detection

report as resistant for all beta lactam antimicrobials (penicillins, cephems, monobactams, penems)

what other bacteria do oxacillin and cefoxitin testing apply to?

s. lugdunensis and other CoNS but with different pos thresholds

oxacillin disk method

• s. aureus only

• streak lawn and place disk

• zone <10 mm = resistant = MRSA

oxacillin agar method

• s. aureus only

• mueller hinton with 4% NaCl and 6ug/ml oxacillin

• suspend colonies in broth or saline to 0.5McF turbidity and inoculate plate, incubated 24 hrs

  • light film of growth/ >1 colony = oxacillin resistant = MRSA

cefoxitin screen

• disk test

• zone <21 = mecA pos = oxacillin resistant = MRSA

• also used to determine methicillin resistance of lugdunensis

CHROMagar MRSA

• inoculate plate

• MRSA = mauve colored colonies

• non MRSA = normal colonies

latex agglutination test - MRSA

• like staph test but target is PBP-2

• agglutination = pos = MRSA

• no agglutination = neg = MSSA

what is the gold standard for MRSA detection?

• mecA gene detection via PCR or molecular nucleic acid probes

• RTPCR: IDes MRSA/MSSA; used in infection control practices to reduce MRSA via rapid test results (ICU)

mecA gene

• most common cause of oxacillin resistance

• codes for PBP2a (altered) so that oxacillin does not bind (ineffective)

how to detect MRSA from blood cultures directly?

• filmarray BCID and verigene blood culture assay

  • detected 1-2hrs after blood Cx flags pos

VRSA screening test - agar

• inoculate brain-heart infusion + 6ug/ml vancomycin in same manner as oxacillin plate

• incubate for 24hrs and examine for light film OR >1 colony using transmitted light

• if pos:

  • perform broth microdilution test to determine VRSA or VISA and review before reporting

micrococcus characteristics

• normal skin flora; can be opportunistic in immunocomp pts

• usually a contaminant

• can be resistant to numerous different treatments

micrococcus morphology

• strict aerobes

• poor CHOC growth

• small/medium bright yellow colonies

micrococcus biochemicals

• cat pos, coag neg

• anaerobic acid production from glucose neg

• microdase/lysostaphin pos

• bacitracin/polymixin B disk susceptible

microdase (modified oxidase test)

• disk has TMPD and DMSO

• TMPD reacts with oxidase and cytochrome c = indophenol (dark blue/purple)

• rub colonies onto dry disk and examine for 2 min

  • pos = blue/purple

  • neg = no color change (yellow)

microdase reagents

• tetramethyl-p-phenylenediamine: detects cytochrome c

• dimethyl sulfoxide: enhances permeability of bacterial cells

lysostaphin test

• lyses staph but not micrococcus

• add soln to tube with bacterial suspension

• incubate for 3 hrs then examine for turbidity

  • pos = turbid = micrococcus

  • neg = clear = staph spp

bacitracin A disk test

• 0.04 U disk differentiates micrococcus from staph spp

• zone >10mm = susceptible (micrococcus)

• zone 6mm = resistant (staph spp)

• zone 7-9mm = repeat test (usually susceptible)

polymixin B disk test

• differentiates multiple species

• zone <10 mm = resistant = s. aureus, lugdunensis, epidermidis

• zone >10 mm = susceptible = micrococcus, other CoNS

rothia characteristics

• normal flora of URT

• opportunistic in immunocomp pts, drug users

  • endocarditis, septicemia

• rothia (stomatococcus) mucilanginosa

rothia morphology

• small/medium gray

• very sticky: will come off agar completely if pulled

  • can appear like clumping on slide coagulase/staph latex tests!

rothia biochemicals

• cat pos, coag neg

• anaerobic production of acid from glucose pos

• bacitracin susceptible

• polymixin B resistant

• typically susceptible to penicillin