Staphylococci - Staphylococcus aureus - dr.somesh 2015 - Bacteriology - Microbiology -

Staphyloccus aureus - NOSOCMIAL pathogen - SECOND MBBS STUDENTS - UNDERGRADUATE STUDENTS - MEDICAL COLLEGE - POWERPOINT

1. STAPHYLOCOCCI Dr. Someshwaran rajamani , md

2. Introduction Gram positive cocci arranged in grape-like clusters Ubiquitous in nature Most common cause of suppurative lesions in humans Potential pathogen due to its ability to develop antibiotic resistance 1871 - Von Recklinghausen first observed Staphylococci in human pyogenic lesions January 28, 2015 2

3. Introduction (Cont.) 1880 - Pasteur obtained liquid cultures of the cocci from pus and produced abscess by inoculating into rabbits 1880 - Sir Alexander Ogston (Scottish surgeon) named Staphylococci Greek word: Staphyle - Bunch of grapes, Greek word: kokkos - grain or berry Pathogenic Staphylococci produce golden yellow colonies Non-pathogenic Staphylococci – white colonies January 28, 2015 3

4. Genus Staphylococcus - Gram positive cocci: In singles, pairs or irregularly as grape-like clusters - Facultative anaerobe - Catalase positive bacteria - Comprises 40 species and subspecies today January 28, 2015 4

5. Genus Staphylococcus Family - Micrococcaceae Genus - Micrococcus and Staphylococcus Species - Staphylococcus aureus, S.citreus , S.albus etc., January 28, 2015 5

6. Genus Staphylococci Divided into 2 sub-groups: Enzyme coagulase 1. Coagulase positive Staphylococci 2. Coagulase Negative Staphylococci (CONS) January 28, 2015 6

7. Genus Staphylococcus A.Coagulase positive Staphylococci: Staphylococcus aureus - Rosenbach – 1884 Staphylococcus intermedius Staphylococcus hyicus January 28, 2015 7

8. Genus Staphylococcus B.Coagulase negative Staphylococci: Staphylococcus albus (Staphylococcus epidermidis) - Rosenbach - 1884 Staphylococcus citreus – Passet – 1885 Staphylococcus saprophyticus Staphylococcus hominis , Staphylococcus capitus Staphylococcus hemolyticus January 28, 2015 8

9. STRUCTURE and PHYSIOLOGY Gram positive cocci in grape like clusters b’coz the cells divide along different planes and the daughter cells remain attached to one another Non-motile Facultative anaerobes Salt-tolerant and dessication tolerant (survival on fomites) January 28, 2015 9

10. Morphology Gram positive spherical cells, mostly arranged in irregular grape like clusters Polysaccharide capsule is rarely found on cells Peptidoglycan layer is the major structural component of the cell wall. Teichoic acid is present. January 28, 2015 10

11. Morphology (Cont.) Protein A is the major protein component of the cell wall. Released into the culture medium during the cell growth. Unique property of Protein A – Ability to bind to Fc part of IgG3 (Not an antigen-antibody specific reaction) January 28, 2015 11

12. Staphylococcus aureus Morphology: Spherical cocci 1µm in diameter, arranged in a grape like clusters (Cluster formation- cell division in 3 planes with daughter cells in close proximity) Non motile, nonsporing , mostly small amount of capsular material seen in non-capsulated bacteria January 28, 2015 12

13. Culture characteristics Grow readily on ordinary media Optimum: 37 ⁰C, pH 7.4-7.6 – Aerobe and Facultative anaerobe Colony morphology: Large 2-4mm, circular, convex, smooth, opaque an easy emulsifiable. Most strains produce golden yellow colonies on nutrient agar, some also white, orange, yellow pigments. January 28, 2015 13

14. Culture characteristics Pigment production is enhanced with 1% Glycerol monoacetate or milk Pigment: Lipoprotein allied to carotene Nutrient agar slope: Confluent growth – ‘Oil-paint apperance ’ Blood agar: Hemolytic or non-hemolytic colonies January 28, 2015 14

15. Culture characteristics Most strains incubated with 20-25% CO2 are Hemolytic. Marked hemolysis on Rabbit or sheep blood Minimum hemolysis on horse blood agar Mac Conkey’s medium: Small pink lactose fermenting colonies. Liquid media: uniform turbidity January 28, 2015 15

16. Selective media Ludlam’s media- Lithium chloride and Tellurite Mannitol salt agar Milk salt agar or broth – 8-10% NaCl Baird – Parker agar Agar containing P olymyxin B Primary isolation: Sheep Blood Agar Plate (S-BAP) January 28, 2015 16

17. Biochemical reactions Indole test: Negative Urease test: positive Methyl Red test: positive Voge-Prauskuer test: positive Catalase test – positive Modified Oxidase test – Negative (It is positive for Micrococci) January 28, 2015 17

18. Biochemical reactions Urea hydrolysis test - positive Gelatin liquefaction test – positive Lipolytic – dense opacity on egg agar Phosphatase test – positive (Phenolphthalein diphosphate on nutrient agar – exposed to ammina vapour – turn pink color due to free Phenolphthalein) January 28, 2015 18

19. Staphylococcus aureus Coagulase positive, Ferment mannitol Clear hemolysis on Blood agar Golden yellow pigment L iquefy gelatin; Produce phosphatase Potassium tellurite medium: Black colonies Produce thermostable nucleases January 28, 2015 19

20. Penicillin resistane – 3 types Betalactamase production – P enicillinase A,B,C,D Changes in bacterial surface receptors binding of beta lactam antibiotics Development of tolerance to penicillin January 28, 2015 20

21. Pathogenicity and Virulence Cell Associated polymers: Polysaccharide peptidoglycan Teichoic acid Capsular polysaccharide January 28, 2015 21

22. Peptidoglycan Half of the cell wall weight is peptidoglycan Subunits of peptidoglycan is N-Acetyl muramic acid (NAM) and N-Acetyl Glucosamine (NAG) Unlike gram negative cell wall – Gram positive cell wall has many cross-linked bridging layers which makes the cell wall more rigid (RIGID CELL WALL) January 28, 2015 22

23. Teichoic acid Species-specific Phosphate containing polymers Bound covalently to peptidoglycan layer or through lipophobic linkage to the cytoplasmic membrane ( Lipo -teichoic acid) It mediates attachment to mucosal surfaces through its specific binding to Fibronectin January 28, 2015 23

24. Capsule or polysaccharide slime layer Commonly believed to be found in-vivo Occasionally found when cultured in-vitro Eleven capsular serotypes identified in Staphylococcus aureus. Serotypes 5 and 7 – accounts for major infection. January 28, 2015 24

25. Capsule or polysaccharide slime layer Protects by inhibiting chemotaxis and phagocytosis by polymorphonuclear leukocytes; Also inhibits proliferation of mononuclear cells Facilitates the adherence of bacteria to catheters and other synthetic metrials January 28, 2015 25

26. Protein A Surface of Staphylococcus aureus but not CONS is specially coated with Protein A – covalently linked to Peptidoglycan layer. Has a unique affinity for binding Fc receptor of Immunoglobulin IgG. Protein-A detection is one of the specific test to detect Staphylococcus aureus. Protein-A coated Staphylococci used as non-specific carrier of antibodies directed against other antigens like Streptococci (Serology: Co-agglutination test). January 28, 2015 26

27. Pathogenicity and Virulence Cell Surface proteins: Protein A Clumping factor January 28, 2015 27

28. Cytoplasmic membrane Made up of complex proteins, lipids and small amount of carbohydrates Serves as osmotic barrier for the cell Provides anchorage for cellular biosynthetic and respiratory enzymes January 28, 2015 28

29. Pathogenicity and Virulence Extracellular enzymes: January 28, 2015 29

30. Coagulase and other surface proteins Coagulase Reacting Factor (CRF) in Plasma Clumping factor (Bound coagulase) on the outer surface Binds Fibrinogen – converts it into insoluble fibrin – Clumping or aggregates. Primary test in identifying Staphylococcus aureus Others: Collagen binding protein, Elastin binding protein, Fibronectin binding protein January 28, 2015 30

31. Staphylococcal enzymes - Coagulase: Triggers blood clotting - Hyaluronidase: breaksdown Hyaluronic acid – enables the bacteria to spread between cells - Staphylokinase : Dissolves fibrin threads in blood clots – allows to free itself from blood clots January 28, 2015 31

32. Staphylococcal enzymes (Extra cellular) Coagulase , Catalase Hyaluronidase Fibrinolysin Lipase, Nuclease Penicillinase Betalactamase January 28, 2015 32

33. Staphylococcal enzymes - Lipases : Digests lipids – aloow them to grow on skin surfaces and in cutaneous oil glands - Beta lactamase: breaks down penicillin – resistant to beta lactam antibiotics like penicillins and Cephalosporins January 28, 2015 33

34. Toxins of S. aureus Alpha toxin, beta toxin, delta toxin, gamma toxin, Panton-Valentine toxin, E xfoliative toxins A,B ( Exfoliative dermatitis / Staphylococcal Scalded skin syndrome SSSS, Food poisoning – preformed toxin 2-6 hours, self limiting) Super antigens: A.Enterotoxins - 8 (A-E, G-I), B.Toxic Shock Syndrome Toxin (TSST-1) – super antigen – activates a number of T cells January 28, 2015 34

35. Staphylococcal diseases Skin and soft tissue infections: Folliculitis, furuncle (boil), Abscess ( Particlarly breast abscess), wound infection, carbuncle, impetigo, paronychia, less often cellulitis. Musculoskeletal: Osteomyelitis, Arthritis, Bursitis, Pyomyositis Respiratory: Tonsillitis, pharyngitis, sinusitis, otitis, broncho-pneumonia, lung abscess, empyema, rarely pneumonia January 28, 2015 35

36. Staphylococcal diseases Central nervous sytem : Abscess, meningitis, intracranial thrombophlebitis Endovascular: Bacteremia, Septicemia, Pyemia, Endocarditis Urinary: Uncommon un UTI, S.saprophyticus – females common ( Novobiocin resistant) January 28, 2015 36

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38. Bacteriophage typing ( Staphylococcus aureus ) International basic set of phages for typing Staphylococcus human (origin) Group I - 29, 52, 52A, 79, 80 Group II - 3A, 3C, 55, 71 Group III - 6, 42E, 47,53, 54, 75, 77, 83A Group IV - NIL Group V - 94, 96 Not allocated - 81, 95 January 28, 2015 38

39. Epidemiology Primary parasites of humans Colonise skin glands and mucus membranes Human patients and carriers – potent source of infection Animals and inanimate objects – less important 10-30% healthy population – nasal carriers 10% perineal carriers, 10% Hair carriers 5-10% Vaginal carriers (during menses – tampons - TSS) January 28, 2015 39

40. Staphylococcal carrier state Starts early in life Colonisation of umblical cord – neonates Shedders: these carriers disseminate large numbers for prolonged period. Cocci shed by patients- contaminate fomites like Hand kerchiefs, bed linen, blankets – persists for days-weeks January 28, 2015 40

41. Mode of infection A. Exogenous infection B. Endogenous infection Mode of transmission: A. Direct contact B. Indirect contact (fomites) C. Dust D. Droplet nuclei infection January 28, 2015 41

42. Nosocomial infection HEALTHCARE OR HOSPITAL ACQUIRED INFECTION MULTIDRUG RESISTANT BUGS MRSA- Methicillin resistant Staphylococcus Aureus Alteration of PBP 2a (Penicillin binding protein 2 to 2a) Mutation of Mec A gene – Short arm of Chromosome 6 CA-MRSA (20%); HA-MRSA (80%) - E-MRSA January 28, 2015 42

43. CHARACTERISTICS OF CA-MRSA and HA-MRSA--------------- FEATURES CA-MRSA HA-MRSA Definition Community acquired MRSA Hospital acquired MRSA Prevalence 20% 80% Persons affected Young and healthy persons with no recent exposure to health care Previous contact with health care settings like hospitals, nursing homes, hemo-dialysis centers Type of infection Mild to moderate severity causing skin and soft issue infection and more common Severe invasive disease in hospitalized patients or by frequent contact with health care Site infected Skin and soft tissues, lungs Blood stream, lung, surgical site, prosthetic implant SCC type Type IV / Type V Type II PFGE type USA 300, USA 400 USA100, USA 200 PVL gene Common Rare Antibiotic resistance pattern Susceptible to many antibiotics except betalactam antibiotics Multi drug resistance observed SCC- Satellite Cassette Chromosome, PFGE- Pulsed field Gel Electrophoresis, PVL- Panton Valentine Leukocidin --------- January 28, 2015 43

44. What are the risk factors for MRSA infections? Injection drug abusers Skin trauma (lacerations, abrasions, tattooing etc ) Higher body mass index Cosmetic body shaving Physical contact with a person with draining lesion or a MRSA carrier Incarceration Previous skin infection with MRSA Previous antibiotic use Homosexuals Military recruits and Mental asylum   January 28, 2015 44

45. Lab diagnosis Samples to be collected; Transport and storage Isolation: BAP, MAC, NA; Selective media- Ludlam’s Nutrient agar – Golden yellow pigment Biochemical Tests to be performed: Catalse , Gram stain , Tube Coagulase , Mannitol fermentation, Phosphatase, I, MR, VP, Urea hydrolysis January 28, 2015 45

46. Gram stain January 28, 2015 46

47. Nasal carriers January 28, 2015 47

48. AST: Cefoxitin screen for MRSA January 28, 2015 48

49. Treatment of MRSA Hand washing, Chlorheximide sprays, Mupirocin (topical) Vancomycin , Linezolid, Teicoplanin , Ceftabipirole , Ceftaroline (5 th generation cephalosporins ) January 28, 2015 49

50. Topical liniments January 28, 2015 50

51. Conrol measures 1. Isolation of patient with lesions 2. Detection among carriers of infection in Health care providers 3. Strict aseptic technique 4. Handwashing January 28, 2015 51

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53. What is the five C’s strategy suggested by CDC for MRSA prevention? 1. Avoid Crowding; 2 . Avoid Contact (skin-to-skin); 3. Protect Compromised skin (cuts and scrapes); 4. Clean Contaminated items and surfaces; 5. Prevent Lack of cleanliness January 28, 2015 53

54. Which is the national and international reference center for bacteriophage typing of Staphylococcus aureus ?   - National reference center for Staphylococcal phage typing is Maulana Azad medical college, New Delhi, India. - International reference center for Staphylococcal phage typing is Centers for Disease Control and Prevention (CDC), Atlanta, United States of America. January 28, 2015 54

55. What method would you employ to diagnose MRSA in the laboratory ? Disc diffusion by Kirby Bauer method with cefoxitin 30µg antibiotic disc, where cefoxitin is the surrogate marker for oxacillin resistance Minimum Inhibitory Concentration (MIC) determination of cefoxitin (Resistant if MIC ≥8µg/mL as per CLSI 2014) by E-test, Broth dilution method or automated system like Vitek-2 are employed Oxacillin screen agar is employed to detect oxacillin sensitivity. If resistant (MRSA), Vancomycin screen agar is used to identify resistance to Vancomycin . Polymerase chain reaction (PCR) and DNA probes for mecA gene detection and confirmation of MRSA January 28, 2015 55

56. What is the drug of choice and alternatives available for treating MRSA? Vancomycin is effective against life threatening MRSA infections Chlorhexidine, Bacitracin, Mupirocin ointment Note: Linezolid is used in case of Vancomycin resistance (VRSA/VISA) Teicoplanin is the choice for Linezolid resistant Staphylococcus aureus Fifth generation cephalosporins like Ceftaroline , Ceftabipirole have anti-MRSA activity and are also effective against Vancomycin Resistant Enterococci (VRE)   January 28, 2015 56

57. What is the role of Maggot therapy/Bio-surgery for MRSA ? Maggot Debridement Therapy (MDT) for chronic infected wounds by introduction of sterile medicinal larvae of specific necrotic tissue eating common blow fly or green bottle is a suitable bio-surgery aid for MRSA.   January 28, 2015 57

58. What is the clinical significance of MRSA infections with regards to antibiotic selection? MRSA is resistant to beta lactam antibiotics like penicillins,cephalosporins and other beta lactam antibiotics. January 28, 2015 58

59. CONS Micrococci MS-CONS MR-CONS S. epidermidis –stitch abscess, Endocarditis in drug addicts S. saprophyticus – UTI – Novobiocin resistant Hugh- Leifson test (Oxidative) Modified oxidase positive January 28, 2015 59

60. Staphylococcus aureus Antibotic Susceptibility Test: MSSA- Penicillin, Ampicillin, Amoxy clav , Cloxacillin , Ceftriaxone Cefoxitin – surrogate marker for MRSA Kirby – Bauer’s Disc diffusion method, MIC by E-strip method, Vitek Novobiocin sensitive, Bacitracin resistant, Furazolidone sensitive. January 28, 2015 60

61. Take home message Summary MSSA, MRSA, MS-CONS, MR-CONS Hand washing Diseases produced Lab diagnosis Culture – selective media Mec A gene detection by PCR Bacteriophage typing January 28, 2015 61

62. QUIZ Name three coagulase positive Staphylococci? January 28, 2015 62

63. QUIZ Name three coagulase positive Staphylococci? 1. Staphylococcus aureus 2. Staphylococcus intermedius 3. Staphylococcus hyicus January 28, 2015 63

64. Got it??? January 28, 2015 64

65. THANK YOU January 28, 2015 65