staph - flashcards

i left out some morphology stuff so review those slides !

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

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

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

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

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