Micro - Unit 1: Staphylococcus and Streptococcus

What Gram stain shape is shared by Staphylococcaceae, Streptococcus, and Enterococcaceae?

Gram positive cocci

What arrangements are seen with Gram positive cocci?

Pairs, clusters, tetrads, and chains

What atmospheric requirement do Staphylococci have?

Facultative anaerobe

What CO2 concentration do most clinical labs use for incubation?

3-5% CO2

What is the mechanism of beta lactam antibiotics?

They interfere with cell wall synthesis (peptidoglycan production)

What does the beta lactam ring structure target?

Cell wall synthesis

What is the first choice of antimicrobials for Staphylococci?

Penicillins, oxacillin, ampicillin, cephems

What does CLSI stand for?

Clinical and Laboratory Standards Institute

What does CNISP stand for?

Canadian Nosocomial Infection Surveillance Program

What is the purpose of CNISP?

To monitor rates and trends of healthcare-associated infections, enable comparison of rates, and provide data to develop national guidelines

What agency runs CNISP?

Public Health Agency of Canada

What was the former family name of Staphylococcus?

Micrococcaceae

What is the Gram stain and arrangement of Staphylococcus?

Gram positive cocci in grapelike clusters

What is the atmospheric requirement of most Staphylococcus?

Facultative anaerobe

What is the colony morphology of Staphylococcus?

Smooth, butyrous, cream coloured/white after 18-24 hrs growth

What is the hemolysis of most Staphylococcus aureus strains?

Beta hemolytic

What percentage of the population carries Staphylococcus as normal flora?

20-40%

What is the primary reservoir of Staphylococcus?

Nares

What are other reservoirs of Staphylococcus?

Axillae, vagina, pharynx, skin

Where is Staphylococcus carriage higher?

Healthcare professionals/hospital personnel

What are the two types of Staphylococcus infections?

Suppurative (invasive) or toxin mediated

What skin infections does Staphylococcus aureus cause?

Boils (furuncle, carbuncle), folliculitis, cellulitis

What organ infections does Staphylococcus aureus cause?

Pneumonia, endocarditis, osteomyelitis, septicemia

What causes Staphylococcus aureus food poisoning?

Ingestion of toxin - heat stable enterotoxin (A-E)

How soon after ingestion do Staphylococcus aureus food poisoning symptoms occur?

2-8 hours

What foods are associated with Staphylococcus aureus food poisoning?

Meat or dairy products

What causes Scalded Skin Syndrome?

Pyogenic exfoliating toxin

What is the mortality rate of Toxic Shock Syndrome?

2-5%

What are the symptoms of Toxic Shock Syndrome?

Fever, chills, vomiting, diarrhea, aches, rash, progressing to hypotension and shock

What does Staphylococcus epidermidis cause?

Nosocomial infection from catheters (Foley) and prosthetic devices via biofilm production

What patients are susceptible to Staphylococcus epidermidis?

Immuno-compromised patients, diabetic, cancer, pregnancy

What does Staphylococcus saprophyticus cause?

UTI; 2nd most common cause of female cystitis

What is Staphylococcus lugdunensis?

Commensal (Normal flora, Mixed indigenous flora ) opportunistic pathogen causing endocarditis, osteomyelitis, septicemia

What are the predisposing factors for Staphylococcus infection?

Defects in leukocyte chemotaxis, opsonization by antibodies, intracellular killing after phagocytosis, skin injuries, foreign bodies, sutures, IVs, infection with another agent, chronic underlying disease, therapeutic or prophylactic antibiotic treatment

What is the virulence factor of Enterotoxins?

Stable exotoxin to 100 degrees C

What is the virulence factor of Toxic shock syndrome toxin-1?

Superantigen that elicits an aggressive immune response

What is the virulence factor of exfoliative toxin?

Infected bacteria causes skin to slough off

What is the virulence factor of cytolytic toxins?

Affects RBCs (alpha, beta) and WBCs

What are the virulence factor enzymes of Staphylococcus?

Catalase: Present in all Staph. species

Coagulase: Staph. aureus

Hyaluronidase: Hydrolyzes hyaluronic acid

Protease: Breaks down proteins/peptides

What is the overall effect of Staphylococcus enzymes?

Destruction of tissues and allows spread of infection

What is Protein A?

Cell wall of Staphylococcus aureus; blocks phagocytosis

What is the function of capsule formation in Staphylococcus?

Resist phagocytosis

What is ESS?

Extracellular slime substance; produced by all Staphylococcus sp; biofilms and love of plastic

What is the catalase test reaction?

2H2O2 -> 2H2O + O2 (catalase)

What does a positive catalase test look like?

Bubbling appears immediately

What does the catalase test differentiate?

Staphylococcus (positive) from Streptococcus (negative)

What is the reagent for coagulase testing?

EDTA rabbit plasma

What does slide coagulase detect?

Clumping factor (bound coagulase)

What is the procedure for slide coagulase?

1 drop loop saline/water + isolate + coagulase reagent

What is a positive slide coagulase result?

Clumping in <10 seconds

What must be done if slide coagulase is negative?

Tube coagulase test

What does tube coagulase detect?

Free coagulase (thrombin-like substance forms a fibrin clot); both free and bound clumping factor detected

What is the procedure for tube coagulase?

Reaction tube + 0.5 mL EDTA rabbit plasma + loopful of isolate

How often is tube coagulase observed?

Every 30 minutes up to 4 hours at 35 degrees C, then up to 24 hours overnight

What is a positive tube coagulase result?

Any type of clot

What controls must be run with tube coagulase?

Positive and negative controls

What can happen to the clot in tube coagulase?

Some strains lyse clot after formation or produce a weak clot

What should you NOT do while viewing tube coagulase?

Do NOT over shake tube

What can a contaminant produce in tube coagulase?

Fibrinolysin

What is the coagulase result for MRSA?

Can be slide negative, tube weak positive

What other staphylococci can produce clumping factor or staphylocoagulase?

Staphylococci other than S. aureus

What is the rapid latex agglutination method for S. aureus?

Commercial kit of a slide latex detecting protein A and clumping factor

What is the procedure for rapid latex agglutination?

"Drop, drop, rock method"

What is the commercial kit used in lab for S. aureus ID?

Staphaurex

Why is latex agglutination used instead of tube coagulase in lab?

Due to time constraints

What does DNase agar test for?

Enzyme secreted by cell to cleave DNA (DNA hydrolysis)

What is the procedure for DNase agar?

"Spot" inoculate onto media, incubate 18-24 hrs, view for zone of clearing around colony growth

What is the DNase result for S. aureus?

Positive

What is the purpose of novobiocin susceptibility?

For urinary tract isolates; for coagulase negative staphylococci

What is the resistance breakpoint for novobiocin?

R <16, S >=16

What does novobiocin resistance presumptively identify?

Staphylococcus saprophyticus (common UTI clinical isolate)

What are the methods not routinely performed for Staphylococcus?

Glucose OF, Modified oxidase, Bacitracin (Taxo A disk)

What is MALDI-TOF?

Mass spectrometry - matrix-assisted laser desorption-ionization time-of-flight

What are the characteristics of Staph aureus?

Bound or free coagulase positive, DNase positive, 2 mm creamy white/butyrous beta hemolytic, MF, PYR and ODC negative

What are the characteristics of Staph epidermidis?

Bound or free coagulase negative, DNase negative, 1 mm white non hemolytic, NMF, PYR and ODC negative

What are the characteristics of Staph saprophyticus?

Bound or free coagulase negative, DNase negative, 1 mm creamy white non hemolytic, mannitol fermenter, delayed reaction appearing as NMF after 18 hours

What is unique about Staph lugdunensis coagulase?

May produce bound coagulase (positive slide-coagulase) but negative tube-coagulase

What is unique about Staph lugdunensis biochemistry?

Decarboxylates ornithine (ODC+), PYR+

What is the colony morphology of Staph lugdunensis?

Usually beta hemolytic, sticky, yellow or tan, 1-2 mm

Why are most Staph aureus penicillin resistant?

They secrete beta-lactamase (penicillinase)

What gene encodes methicillin resistance?

mecA gene

What is the product of the mecA gene?

Penicillin-binding protein 2a (PBP2a)

What does oxacillin/cefoxitin resistance imply?

Methicillin resistance and resistance to all beta lactams (penicillins, beta lactam/beta lactamase inhibitor combinations, cephalosporins, carbapenems)

Why is methicillin not stable in vitro?

It degrades

What disk is used to detect oxacillin resistance per CLSI?

30 ug cefoxitin (surrogate)

What should NEVER be reported to the doctor?

Cefoxitin and novobiocin

What is reported instead of cefoxitin?

Oxacillin or methicillin

What media is used for oxacillin disk diffusion?

Mueller Hinton agar

What supplement is added to Mueller Hinton agar for hetero-resistant strains?

2% NaCl

What is considered resistance on oxacillin disk diffusion?

Any growth

How should susceptibility media be examined?

With transmitted overhead light; sharp clear borders = S, feathered border or trailing edges = R

What temperature may fail to detect resistance?

Above 35 degrees C

What is oxacillin salt agar screening used for?

Determining MRSA carriers

What are the components of oxacillin salt agar?

Mueller Hinton agar with 4% NaCl and 6 ug/mL oxacillin

How long is oxacillin salt agar incubated?

24 up to 48 hours aerobic

When may a preliminary report be issued for oxacillin salt agar?

At 24 hours

What indicates resistance on oxacillin salt agar?

Any growth

What is the media for CHROM ID?

Cefoxitin 4 mg/L, targets alpha glucosidase

What color are colonies on CHROM ID?

Green

What is the media for ORSAB?

Mannitol salt agar, oxacillin 2 mg/L, polymyxin, aniline blue