1. MLAB 2434: M ICROBIOLOGY K ERI B ROPHY -M ARTINEZ Staphylococci

2. T AXONOMY Family: Micrococcaceae Genus: Staphylococcus Coagulase positive Coagulase negative Micrococcus

3. G ENUS S TAPHYLOCOCCUS S. aureusS. epidermidis S. saprophyticus S. haemolyticus Coagulase positiveCoagulase negative

4. S TAPHYLOCOCCUS : G RAM R EACTION AND M ORPHOLOGY Gram-positive spherical cells (0.5-1.5  m) in singles, pairs, and clusters Appear as “bunches of grapes” Gram-stained smear of staphylococci from colony Scanning electron micrograph of staphylococci

5. S TAPHYLOCOCCUS : G ENERAL C HARACTERISTICS Nonmotile Non–spore-forming Nonencapsulated Catalase-producing Oxidase: negative Glucose fermenters Primarily aerobic, some facultatively anaerobic

6. S TAPHYLOCOCCUS : G ENERAL C HARACTERISTICS ( CON ’ T ) Bacitracin resistant Grow on agar that contains peptone Inhibited by media that has high bile salt concentration Some are ß-hemolytic Colony morphology: buttery looking, cream or white colored

7. S TAPHYLOCOCCUS AUREUS Primary pathogen of the genus Habitat: Anterior nares (carriers) Colonization: axilla, vagina, pharynx Produce superficial to systemic infections Skin Bacterial sepsis Hospital acquired infections

8. S TAPHYLOCOCCUS AUREUS Mode of transmission Traumatic introduction Direct contact with infected person Inanimate objects Predisposing conditions Chronic infections Indwelling devices Skin injuries Immune response defects

9. S TAPHYLOCOCCUS AUREUS Infection will elaborate inflammatory response with GPC accumulating as pus Pus: mix of active and inactive neutrophils, bacterial cells and extravascular fluid

10. V IRULENCE F ACTORS OF S. AUREUS Enterotoxins Cytolytic toxins Enzymes Protein A

11. V IRULENCE F ACTORS : E NTEROTOXINS Enterotoxins: Heat-stable exotoxins that cause diarrhea and vomiting Exotoxin: protein produced by a bacteria and released into environment Heat stable @ 100 o C for 30 minutes Implications Food poisoning Toxic shock syndrome Pseudomembranous enterocolitis

12. T YPES OF ENTEROTOXINS Exfoliatin Epidermolytic toxin TSST-1: Toxic shock syndrome toxin-1 Multisystem disease Stimulates T cell production & cytokines Cytolytic Toxins Affects RBCs and WBCs Hemolytic toxins: alpha, beta, gamma, delta Panton-Valentine leukocin, lethal to WBCs

13. V IRULENCE F ACTORS : E XTRACELLULAR E NZYMES Hyaluronidase: Hydrolyzes hyaluronic acid in connective tissue allowing spread of infection Staphylokinase : Fibrinolysin which allows spread of infection Coagulase : Virulence marker Lipase : Allows colonization by acting on lipids present on the surface of the skin.

14. V IRULENCE F ACTORS : E XTRACELLULAR E NZYMES ( CON ’ T ) Penicillinase: Confers resistance DNase: Degrades DNA Beta-lactamase: Cuts the beta lactam wall of certain antibiotics

15. V IRULENCE F ACTORS : P ROTEIN A Protein A: Found in cell wall Binds to F c part of IgG Blocks phagocytosis

16. S TAPHYLOCOCCUS AUREUS : C LINICAL I NFECTIONS Skin and wound Impetigo Furuncles/Boils (Infection of hair follicles usually in areas that sweat) Carbuncles (clusters of boils) Surgical wound infections Bullous impetigo

17. S TAPHYLOCOCCUS AUREUS : C LINICAL I NFECTIONS ( CON ’ T ) Skin and wound Scalded skin syndrome= Ritter’s disease Extensive exfoliative dermatitis Young children and newborns Toxic Shock Syndrome Multisystem disease Caused by TSST-1 Affects women, men, and children

18. S TAPHYLOCOCCUS AUREUS : C LINICAL I NFECTIONS Food poisoning Source is infected food handler Enterotoxin A the most common cause Foods affected include meat, dairy products, bakery goods with cream fillings, and salads made with eggs and mayonnaise.

19. C OAGULASE -N EGATIVE S TAPHYLOCOCCI Found as indigenous flora Presence can indicate contamination Seeing an increase due to prosthetic devices, catheters and immunocompromised Abbreviated CNS or CoNS

20. C OAGULASE -N EGATIVE S TAPHYLOCOCCI Habitat: Skin and mucous membranes Common human isolates S. epidermidis S. saprophyticus S. haemolyticus

21. C OAGULASE -N EGATIVE S TAPHYLOCOCCI : S TAPHYLOCOCCUS EPIDERMIDIS Predominantly hospital acquired infections Skin flora gets introduced by catheters, heart valves, CSF shunts Produces a slime layer that helps adherence to prosthetics and avoidance of phagocytosis UTIs are a common result

22. C OAGULASE -N EGATIVE S TAPHYLOCOCCI : S TAPHYLOCOCCUS SAPROPHYTICUS UTIs in young sexually active women Due in part to increased adherence to epithelial cells lining the urogenital tract Rarely present in other skin areas or mucous membranes Urine cultures If present in low amounts, it is still considered significant

23. C OAGULASE -N EGATIVE S TAPHYLOCOCCI : S TAPHYLOCOCCUS H AEMOLYTICUS Habitat: skin and mucous membranes Rarely implicated in infections A ssociated with wound infections, bacteremia, and endocarditis

24. B REAK T IME !!!

25. L ABORATORY D IAGNOSIS : S PECIMEN C OLLECTION AND H ANDLING Samples must be taken from the actual site of infection Prevent delay in transport of collected material from infected sites Transport in appropriate collection device that would prevent drying and minimize growth of contaminating organisms

26. L ABORATORY D IAGNOSIS : D IRECT S MEAR E XAMINATION Microscopic Examination o Gram reaction o Gram-positive cocci o Cell arrangement o Pairs and clusters o Presence/Absence of PMNs o Numerous polymorphonuclear cells (PMNs) Insert Figure 10-1

27. L ABORATORY D IAGNOSIS : C ULTURAL C HARACTERISTICS - Staphylococcus aureus Colony morphology Smooth, butyrous, white to yellow, creamy Grow well @ 18-24 hours S. aureus may produce hemolysis on blood agar S. aureus

28. L ABORATORY D IAGNOSIS : C ULTURAL C HARACTERISTICS S. epidermidis Smooth, creamy, white Small-to medium- sized, usually non- hemolytic S. saprophyticus Smooth, creamy, may produce a yellow pigment

29. I DENTIFICATION T ESTS : C ATALASE Principle: tests for enzyme catalase 2 H 2 O 2 2 H 2 O + O 2 Procedure Smear a colony of the organism to a slide Drop H 2 O 2 onto smear Observe

30. C ATALASE T EST : I NTERPRETATION Presence of bubbles Positive Staphylococci Absence of bubbles Negative Streptococci

31. I DENTIFICATION TEST : SLIDE COAGULASE TEST Differentiates members within the Staphylococci Detects clumping factor found in S. aureus Procedure Place a drop of sterile water on a slide and emulsify a colony Add a drop of rabbit plasma to the suspension Observe Agglutination = Positive No agglutination= Negative

32. I DENTIFICATION T ESTS : C OAGULASE T EST Detects the extracellular enzyme “free coagulase” or staphylocoagulase Causes a clot to form when bacterial cells are incubated with plasma Procedure Inoculate rabbit plasma with organism and incubate at 35-37 0 C Observe at 30 minutes for the presence of a clot Continue for up to 24 hours, if needed

33. I DENTIFICATION T ESTS : R APID C OAGULASE T EST Latex Agglutination Assays Detects cell-bound “clumping factor,” protein A or a combination of both Procedure Varies depending on kit type Positive reaction demonstrated by agglutination

34. N OVOBIOCIN S USCEPTIBILITY T EST Test to differentiate coagulase- negative staphylococci from S.saprophyticus from urine samples S. saprophyticus is resistant (top) Other CNS are susceptible

35. M ICROCOCCUS Rarely produces disease Found in environment and indigenous skin flora Catalase + Coagulase = Produces yellow pigment Microdase disc differentiate between Staph & Micrococcus

36. Schematic Diagram for Identifying Staphylococcal Species

37. A NTIMICROBIAL S USCEPTIBILITY For non–beta-lactamase producing S. aureus Use pencillin Penicillinase-resistant synthetic penicillins (methicillin, nafcillin, oxacillin, dicloxacillin) Beta-lactamase producers break down the beta-lactam ring of penicillin so it inactivates antibiotic before it acts on bacterial cells

38. M ETHICILLIN -R ESISTANT S TAPHYLOCOCCI MRSA Methicillin-resistant S. epidermidis MRSE Infection control Barrier protection Contact isolation Handwashing Treat with vancomycin Test for susceptibility with cefoxitin disk

39. M ETHICILLIN -R ESISTANT S TAPHYLOCOCCI (C ONT ’ D ) mec A gene Encodes penicillin-binding proteins (PBPs) Causes drug ineffectiveness Gold standard Nucleic acid probe or PCR for the mec A gene

40. V ANCOMYCIN - RESISTANT STAPHYLOCOCCI VRSA= vancomycin resistant Staphylococcus aureus VISA= vancomycin intermediate Saphylococcus aureus Detection Vancomycin screening media

41. A NTIMICROBIAL S USCEPTIBILITY Macrolide Resistance Clindamycin sensitivity often requested by physician to treat Staph skin infection. Referred to as “D” test Clindamycin resistance is often inducible meaning it only is detectable when bacteria are also exposed to erythromycin

42. S UMMARY M ICROCOCCACEAE

43. R EFERENCES Engelkirk, P., & Duben-Engelkirk, J. (2008). Laboratory Diagnosis of Infectious Diseases: Essentials of Diagnostic Microbiology. Baltimore, MD: Lippincott Williams and Wilkins. http://archive.microbelibrary.org/ASMOnly/Details.asp?ID=2037 http://brawlinthefamily.keenspot.com/gallery/2009-10-18-breaktime/ http://ericaandkevin.pbworks.com/w/page/5827086/Gram-Stain-and-Other-Tests http://faculty.matcmadison.edu/mljensen/111CourseDocs/111Review/Unit2Reviews/micrococcace ae_answers.htm http://jeeves.mmg.uci.edu/immunology/Assays/LatexAgglut.htm Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders.