MLAB 2434: Microbiology Keri Brophy-Martinez

1. MLAB 2434: MicrobiologyKeri Brophy-Martinez Staphylococci

2. Taxonomy • Family: Micrococcaceae • Genus: • Staphylococcus • Coagulase positive • Coagulase negative • Micrococcus

3. Genus Staphylococcus Coagulase positive Coagulase negative • S. aureus • S. epidermidis • S. saprophyticus • S. haemolyticus

4. Staphylococcus: Gram Reaction and Morphology • Gram-positive spherical cells (0.5-1.5 mm) in singles, pairs, and clusters • Appear as “bunches of grapes” Gram-stained smear of staphylococci from colony Scanning electron micrograph of staphylococci

5. Staphylococcus: General Characteristics • Nonmotile • Non–spore-forming • Nonencapsulated • Catalase-producing • Oxidase: negative • Glucose fermenters • Primarily aerobic, some facultatively anaerobic

6. Staphylococcus:General Characteristics (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. Staphylococcus 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. Staphylococcus 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. Staphylococcus aureus • Infection will elaborate inflammatory response with GPC accumulating as pus • Pus: mix of active and inactive neutrophils, bacterial cells and extravascular fluid

10. Virulence Factors of S. aureus • Enterotoxins • Cytolytic toxins • Enzymes • Protein A

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

12. Types 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. Virulence Factors: Extracellular Enzymes • 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. Virulence Factors: Extracellular Enzymes (con’t) • Penicillinase: • Confers resistance • DNase: • Degrades DNA • Beta-lactamase: • Cuts the beta lactam wall of certain antibiotics

15. Virulence Factors: Protein A • Protein A: • Found in cell wall • Binds to Fc part of IgG • Blocks phagocytosis

16. Staphylococcus aureus: Clinical Infections • 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. Staphylococcus aureus: Clinical Infections (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. Staphylococcus aureus: Clinical Infections • 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. Coagulase-Negative Staphylococci • Found as indigenous flora • Presence can indicate contamination • Seeing an increase due to prosthetic devices, catheters and immunocompromised • Abbreviated CNS or CoNS

20. Coagulase-Negative Staphylococci • Habitat: • Skin and mucous membranes • Common human isolates • S. epidermidis • S. saprophyticus • S. haemolyticus

21. Coagulase-Negative Staphylococci: Staphylococcus 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. Coagulase-Negative Staphylococci: Staphylococcus 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. Coagulase-Negative Staphylococci: Staphylococcus Haemolyticus • Habitat: skin and mucous membranes • Rarely implicated in infections • Associated with wound infections, bacteremia, and endocarditis

24. Break Time!!!

25. Laboratory Diagnosis: Specimen Collection and Handling • 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. Laboratory Diagnosis: Direct Smear Examination Microscopic Examination • Gram reaction • Gram-positive cocci • Cell arrangement • Pairs and clusters • Presence/Absence of PMNs • Numerous polymorphonuclear cells (PMNs) Insert Figure 10-1

27. Laboratory Diagnosis: Cultural Characteristics- • 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. Laboratory Diagnosis: Cultural Characteristics • S. epidermidis • Smooth, creamy, white • Small-to medium- sized, usually non-hemolytic • S. saprophyticus • Smooth, creamy, may produce a yellow pigment

29. Identification Tests: Catalase • Principle: tests for enzyme catalase 2 H2O2 2 H2O + O2 • Procedure • Smear a colony of the organism to a slide • Drop H2O2 onto smear • Observe

30. Catalase Test: Interpretation • Presence of bubbles • Positive • Staphylococci • Absence of bubbles • Negative • Streptococci

31. Identification 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. Identification Tests: Coagulase Test • 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. Identification Tests: Rapid Coagulase Test • 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. Novobiocin Susceptibility Test • Test to differentiate coagulase-negative staphylococci from S.saprophyticus from urine samples • S. saprophyticus is resistant (top) • Other CNS are susceptible

35. Micrococcus • 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. Antimicrobial Susceptibility • 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. Methicillin-Resistant Staphylococci MRSA Methicillin-resistant S. epidermidis MRSE Infection control Barrier protection Contact isolation Handwashing Treat with vancomycin Test for susceptibility with cefoxitin disk

39. Methicillin-Resistant Staphylococci (Cont’d) mecA gene Encodes penicillin-binding proteins (PBPs) Causes drug ineffectiveness Gold standard Nucleic acid probe or PCR for the mec A gene

40. Vancomycin-resistant staphylococci • VRSA= vancomycin resistant Staphylococcus aureus • VISA= vancomycin intermediate Saphylococcus aureus • Detection • Vancomycin screening media

41. Antimicrobial Susceptibility • 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. Summary Micrococcaceae

43. References • 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/micrococcaceae_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.