Staphylococcus

For Undergraduate MBBS students and Post Graduate MD Microbiology students

1. Staphylococcus Dr. Abhijeet Mane Assistant Professor BVDUMC Pune Thursday, October 13, 2016 1

2. Index Taxonomy Introduction History Staphylococcus aureus Factors predisposing serious infection Virulence factors Pathogenesis Clinical Manifestations Laboratory Diagnosis Detection of MRSA MRSA Control measures Thursday, October 13, 2016 2

3. Taxonomy Staphylococci are Gram positive cocci and classified into 2 families (as per 1986 edn . Of Bergey’s Manual of Systemic Bacteriology): Micrococcaceae (GPC, catalase positive) Streptococcaceae (GPC, catalase negative) Micrococcaceae included 4 genera Planococcus Micrococcus Stomatococcus Staphylococcus Thursday, October 13, 2016 3

4. Newer edition suggests: Staphylococci are Phylum: Firmicutes Genus I Family V : Staphylococcaceae Order I : Bacillales Class III : Bacilli Thursday, October 13, 2016 4

5. Introduction Staphylococci are non motile, non spore forming, catalase positive, GPC Arranged in single cells, pairs, tetrads, short chains but appear predominantly in grape like clusters Facultative anaerobes, except S.aureus subsp. a naerobius and S. saccharolyticus (These are often CATALASE – NEGATIVE) Thursday, October 13, 2016 5

6. Staph: generally found on skin, mucous membrane of humans and other animals Other colonisation sites: intertriginous skin folds, perenium , axillae, vagina Sometimes this association is amazingly specific: S.capitis ssp capitis: normal human flora of skin and seb . Glands of scalp, forehead, neck S.auricularis : external auditory canal S.hyicus : infectious dermatitis in swine Some pathogenic Staphylococci produce – Coagulase – detected in lab S.aureus and CONS ( S.epidermidis , S.saprophyticus ) seen frequently in human infections Thursday, October 13, 2016 6

7. History 1 st observed in pus: von Recklinghausen (1871) 1 st cultured in liquid media: Louis Pasteur (1880) Named ‘Staphylococcus’ (Gr. Staphyle – bunch of grapes, kokkos - berry) by Sir Alexanger Ogston (1880) Rosenbach (1884) – named S.aureus (golden yellow colonies), S.albus (white colonies) Passet (1885) – S.citreus (lemon yellow colonies) Thursday, October 13, 2016 7

8. Thursday, October 13, 2016 8

9. Staphyloccus aureus Catalase – positive, coagulase – positive, facultative anaerobe, non motile, non sporing, occasionally capsulated Spherical cocci, 1 micron diameter, arranged in grape like cluster (due to…) Produces golden yellow pigment on NA and beta hemolytic colonies on SBA Very important in hospital - MRSA Thursday, October 13, 2016 9

10. Thursday, October 13, 2016 10

11. Factors predisposing serious infections Defects in leukocyte chemotaxis, either congenital ( Wiskott AS, Down’s, Job’s syndrome) or acquired (DM, RA) Defects in opsonisation by antibodies secondary to congenital or acquired hypogammaglobulinemia Defects in complement component (esp. C3 & C5) Defects in intracellular killing of bacteria following phagocytosis Thursday, October 13, 2016 11

12. Skin injuries ( eg . Burns, surgical incisions, eczema) Foreign bodies (sutures, i /v lines, prostheses) Infection with other agents ( eg . influenza) Chronic underlying diseases (malignancy, alcoholism, heart disease) Therapeutic or prophylactic AMA administration Thursday, October 13, 2016 12

13. Burns Suture site infection iv line infection Thursday, October 13, 2016 13

14. Virulence factors Capsular polysaccharide Peptidoglycan and Teichoic acids Protein A Enzymes Hemolysin Toxins Superantigens Thursday, October 13, 2016 14

15. Capsular polysaccharide Production of an exopolysaccharide Prevents ingestion of organism by PMN cells Seen by EM studies of – infected pacemaker leads, peritoneal catheters, i /v lines Clinical isolates of SA – classified into 11 types based on capsular polysaccharide immunotyping From significant clinical isolates: capsular serotype 5 or 8 isolated These 2 types also associated with TSST Thursday, October 13, 2016 15

16. Peptidoglycan and teichoic acid SA cell wall contain peptidoglycans Crossed linked polymers of N-acetyl-glucosamine and N-acetyl- muramic acid Thursday, October 13, 2016 16

17. Teichoic acids function : specific adherence of SA to mucosal surfaces Provide rigidity and resilience to cell wall Activate complement, enhance chemotaxis of PMN cells, elicit production of IL-1 by monocytes, stimulate production of opsonic Abs Several other proteins including adhesins, fibronectin binding proteins, collagen binding proteins, clumping factor covalently incorporated in structure of SA peptidoglycan Thursday, October 13, 2016 17

18. Protein A MW – 42 kDa Found on cell surface and in growth medium Able to bind with Fc region of all human IgG subclasses except IgG3 Interferes with opsonisation , ingestion of organism by PMN cells, activates complement, elicits immediate and delayed hypersensitivity reactions It is immunogenic; Abs are found in pt with serious SA infections Presence of protein A provides basis for Co-agglutination reaction (used to identify gonococci, streptococcal grouping) and detection of bacterial Ags in body fluids Thursday, October 13, 2016 18

19. Enzymes Catalase: Function to inactivate toxic H2O2 and free radicals formed by myeloperoxidase system within phagocytic cells after ingestion of SA Clumping factor: Cell bound material able to bind fibrinogen, responsible for binding of SA to both fibrin & fibrinogen Thursday, October 13, 2016 19

20. Coagulase: Can exist in free or bound form Binds to prothrombin – becomes active Catalyses conversion of fibrinogen to fibrin Leading to coat bacterial cell with fibrin, rendering them more resistant to opsonisation and phagocytosis Fibrinolysin Breaks down fibrin clots and facilitate spread of infection to contiguous tissues Thursday, October 13, 2016 20

21. Hyaluronidase Hydrolyses intercellular matrix of acid mucopolysaccharides in tissues – spread organisms to adjacent areas in tissues Lipases Seen in chronic furunculosis pt Spread organism in cutaneous and sub cut tissues Thursday, October 13, 2016 21

22. Phosphatidylinositol – specific Phospholipase C Associated with ARDS and DIC pt Tissues become more susceptible to damage & destruction by bioactive complement components and products during complement activation Nuclease and phosphodiesterase Having exonuclease and endonuclease activity Thursday, October 13, 2016 22

23. Beta-lactamase enzymes: Inducible (produced only in presence of beta lactam AMA) or Constitutive (produced continually) Resistant to penicillin and ampicillin Plasmid coded Resistance to several AMA like erythromycin and tetracycline Resistance may be transferred by Transformation and Transduction Thursday, October 13, 2016 23

24. Hemolysin : Alpha hemolysin Lethal effects on human PMNs cells Lyse rabbit RBCs MW – 33 kDa Secreted in medium : late logarithmic growth phase Mech. Of Action : Individual monomers interact on target cell membrane to form cylindrical heptamers with central pore Pores allow rapid efflux of potassium ions and influx of Sodium and Calcium ions Leads to osmotic swelling and rupture of cell Also dermonecrotic on s/c inj Neurotoxicity: demyelination of both rabbit and murine models Zone of hemolysis on SBA Thursday, October 13, 2016 24

25. Beta hemolysin A sphingomyelinase Exotoxin, MW – 35 kDa Secreted toward end of logarithmic growth phase Exhibits “hot-cold phenomenon”: Maybe due to initial disruption of cohesive forces within membrane by toxin and subsequent phase separation within membrane itself as temp is lowered Synergy – with CAMP factor; identify Gr. B Streptococci Thursday, October 13, 2016 25

26. Thursday, October 13, 2016 26

27. Delta hemolysin MW – 3 kDa Secreted toward end of exponential growth phase Produced by > 97% SA strains MOA: Acts as surfactant; disrupts cell membrane Forms channels; leakage of cellular contents Thursday, October 13, 2016 27

28. Gamma hemolysin : Has 3 protein fragments which act together with Leukocidin to exhibit hemolytic activity MOA: Exerted on PMNs membranes causing degranulation of cytoplasm Cell swelling leading to cell lysis Formation of pores seen Thursday, October 13, 2016 28

29. Toxins: Exfoliatin or epidermolytic toxin Made up of 2 proteins ET-A and ET-B ET-A – thermostable, structural gene chromosomal ET-B – heat labile, plasmid origin Proteolytic, dissolve mucopolysaccharide matrix of epidermis Results in intra epidermal splitting of cellular linkages in the stratum granulosum SSSS Thursday, October 13, 2016 29

30. Enterotoxin: A, B, C, D, E, H and I Heat stable Seen in staphylococcal food poisoning Increases intestinal peristalsis Consuming food products (bakery goods, custards, potato salad, ice cream) having preformed toxins Self limited (24-48 hours); supportive therapy Thursday, October 13, 2016 30

31. Superantigens Staphylococcal enterotoxins and TSST -1 Share 3 biologic characteristics Pyrogenicity Superantigenicity Ability to enhance lethal effects of minute amounts of endotoxin in rabbits upto 100000 fold Superantigenicity : ability to stimulate proliferation of T-lymphocytes without regard for their antigenic specificities Thursday, October 13, 2016 31

32. Pathogenesis Colonisation Introduction into tissues Minor abrasion Instrumentation Invasion Serine protease, hyaluronidase, lipase, etc Evasion of host defences Metastatic spread Hematogenous Thursday, October 13, 2016 32

33. Clinical manifestations Skin and soft tissue infection Folliculitis Furuncle (boil) Carbuncle Impetigo Hidradenitis suppurativa Botryomyocosis Thursday, October 13, 2016 33

34. Musculoskeletal infection Septic arthritis – knee, shoulder, hip, phalanges Osteomyelitis – children: long bones, adults: vertebrae Pyomyositis – skeletal muscle infection – in tropics and HIV infected people Abscess – psoas abscess and epidural abscess Thursday, October 13, 2016 34

35. Respiratory tract infections VAP in adults Septic pulmonary emboli Post viral pneumonia (influenza) Empyema and Pneumothorax Pneumomatocele – neonates : MCC : SA Thursday, October 13, 2016 35

36. Bacteremia and complications Sepsis, septic shock CLABSI Metastatic foci of infection involving kidney, joints, bone and lung Infective endocarditis Native valve prosthetic i /v drug assoc endocarditis Thursday, October 13, 2016 36

37. UTI SA UTI and pyelonephritis occur secondary to bacteremia Following instrumentation – rare Toxin mediated TSS Food poisoning SSSS Thursday, October 13, 2016 37

38. Laboratory diagnosis Sample collection Direct smear Microscopy Culture Biochemicals Typing of SA ABST Thursday, October 13, 2016 38

39. Sample collection Infection Specimen Suppurative lesion Pus, wound swab Respiratory infection Sputum UTI Mid stream urine PUO, Bacteremia Blood Food poisoning Feces, Vomitus, food Carriers Nasal and perianal swab Thursday, October 13, 2016 39

40. Direct smear Microscopy SA appear as GPC 0.5 to 1.5 microns Singly, in pairs, short chains or clusters Both within and outside PMNs Reports should include quantitation of cell types and microorganisms Eg . Many PMNs, moderate GPC seen If Gram stain more typical, report “ GPC resembling Staphylococci seen” Note: Gm stain cant be used to differentiate staphylococci from micrococci and related genera or from planococci Thursday, October 13, 2016 40

41. Thursday, October 13, 2016 41

42. Culture Overnight incubation at 37 deg C NA 1-3 mm, circular, smooth, convex, opaque, easily emulsifiable , butyrous Most strains produce non diffusible Golden yellow pigment Pigmentation can be enhanced – Tween Agar NA slope Confluent growth, Oil paint appearance Thursday, October 13, 2016 42

43. SBA Colonies similar to those on NA; in addition surrounded by a narrow zone of beta-hemolysis Liquid medium – Uniform turbidity MA Small pink colonies due to Lactose fermentation Thursday, October 13, 2016 43

44. Selective media Eg . Swabs from carriers, feces MSA – NA + 7.5% NaCl + phenol red Salt milk agar – NA + 6.5% NaCl + 10% skimmed milk Ludlam’s medium – Lithium chloride and tellurite Thursday, October 13, 2016 44

45. Thursday, October 13, 2016 45

46. Biochemicals Catalase : + ve In 3% H2O2 Immediate and vigorous bubbling Conversion of H2O2 to water and O2 gas Ideally, not done from blood containing media Thursday, October 13, 2016 46

47. Hugh and Leifson O-F test Staph – fermentative Micrococci – oxidative Thursday, October 13, 2016 47

48. S.aureus v/s CONS Thursday, October 13, 2016 48

49. Coagulase test Tube test Slide test Detects free coagulase Reacts with CRF Colony of SA is emulsified in 1 ml of diluted plasma (1:6) in a test tube and incubated in water bath at 37C for 4 hrs Positive – clot formation Negative – RT overnight False positive – Citrated plasma (Pseudomonas, Enterococcus) utilise citrate Can be done with Blood culture pellets Rarely, S.intermidius , S.hyicus , S.delphini , S.schleiferi may be tube positive Detects clumping factor (bound coagulase) If Neg , do tube test Not done from media with High salt content – autoagglutinate Note: S.lugdunensis , S.Schleiferi may be positive Thursday, October 13, 2016 49

50. Thursday, October 13, 2016 50

51. DNAse test On DNA agar, a clear halo is produced surrounding the colonies of SA due to its ability to digest DNA Phosphatase test Positive for SA, S.epidermidis , S.xylosus Organism is inoculated on phenolphthalein diphosphate containing media and later colonies are exposed to ammonia vapour Pink colonies Thursday, October 13, 2016 51

52. Typing of S.aureus Epidemiological purpose to trace source of infection Useful in outbreaks like food poisoning in community Phenotypic methods – bacteriophage typing, antibiogram typing Genotypic methods – PCR-RFLP, ribotyping , PFGE and sequence based typing Thursday, October 13, 2016 52

53. Bacteriophage typing Differentiated into subsp level Method Test train inoculated as lawn culture on NA Drops of routine test dose of known set of different phages are spot inoculated Zone of lysis will be produced in those areas where test strain is susceptible to phages applied If strain lysed by phages 29, 52A, 79, but not other phages; it is designated as phage type 29/52A/79 National Reference Centre: MAMC, New Delhi Thursday, October 13, 2016 53

54. Thursday, October 13, 2016 54

55. Antibiotic Sensitivity Testing Production of beta lactamase enzyme Plasmid coded Produced by >90% SA strains Overcome by addition of Beta lactamase inhibitor eg . Clavulanic acid, sulbactam Alternation of PBP Shown by MRSA strains Chromosomally mediated m ec A gene present Alters PBP to PBP-2a CA-MRSA and HA-MRSA Thursday, October 13, 2016 55

56. Significance of MRSA Mechanism of MRSA Which antimicrobials are ineffective on MRSA? Penicillins , Cephalosporins , Monobactams , BL-BLI combinations, Carbapenems . Molecular methods “Gold standard” for detection Thursday, October 13, 2016 56

57. Methods of ABST Divided into types based on the principle applied to each system Advantage of Agar dilution: Multiple isolates can be tested simultaneously Advantage of Broth dilution: Same tubes can be used for MBC detection Diffusion method Dilution method Diffusion & Dilution Stokes method Agar Dilution E-test method Kirby Bauer method Broth Dilution Thursday, October 13, 2016 57

58. Minimum Inhibitory Concentration (MIC) Lowest concentration of an antimicrobial agent that prevents visible growth of a microorganism in an agar or broth dilution susceptibility test Thursday, October 13, 2016 58

59. Dilution methods (contd.) Either broth / agar dilution methods used Series of tubes / plates prepared with broth or agar medium Various concentrations of antimicrobial agents added Tubes / plates inoculated with std. suspension of test organism Incubation at 35 C Tests examined and MIC determined Thursday, October 13, 2016 59

60. Detection of MRSA CLSI recommends 3 screening tests for detection of MRSA (Oxacillin resistance) Oxacillin Resistance - Agar dilution Broth microdilution Disc diffusion Thursday, October 13, 2016 60

61. Screen test Oxacillin Resistance mecA mediated Oxacillin resistance (Using Cefoxitin) Organism group S.aureus S.aureus S.aureus Test Method Agar dilution Disk diffusion Broth microdilution Medium MHA with 4% NaCl MHA CAMHB Antimicrobial Concentration 6µg/mL Oxacillin 30 µg Cefoxitin disk 4µg/mL Cefoxitin Inoculum Direct colony suspension to obtain 0.5McFarland turbidity Using 1 - µL loop that was dipped in the suspension, spot an area 10-15 mm in diameter. Alternatively, using a swab dipped in the suspension and expressed, spot a similar area or streak an entire quadrant Standard disk diffusion procedure Inoculum preparation by Direct colony suspension 2. Inoculation of plates 3. Application of discs 4. Reading plates & interpretation Standard broth microdilution procedure Incubation conditions 33 to 35 C; ambient air. (Testing at temperatures above 35 C may not detect MRSA ) Incubation length 24 hours; read with transmitted light 24 hours 24 hours Thursday, October 13, 2016 61

62. Screen test Oxacillin Resistance mecA mediated Oxacillin resistance (Using Cefoxitin) Organism group S.aureus S.aureus S.aureus Test Method Agar dilution Disk diffusion Broth microdilution Results Examine carefully with transmitted light for >1 colony or light film of growth. >1 colony = Oxacillin resistant ≤ 21 mm = mecA positive ≥ 22 mm = mecA negative >4µg/mL = mecA positive ≤ 4 µg/mL = mecA negative Further testing and reporting Oxacillin resistant staphylococci are resistant to all β-lactam agents; other β-lactam agents should be reported as resistant or should not be reported. Cefoxitin is used as a surrogate for mecA -mediated Oxacillin resistance. Isolates that test as mecA positive should be reported as Oxacillin (not cefoxitin ) resistant; other β-lactam agents, except those with anti MRSA activity, should be reported as resistant or should not be reported.   QC recommendations- Routine S.aureus ATCC 29213 – Susceptible (with each test day) S.aureus ATCC 25923 – mecA negative (Cefoxitin zone 23-29mm ) S.aureus ATCC 29213 – mecA negative (Cefoxitin MIC 1-4 µg/mL) QC recommendations – Lot / Shipment S.aureus ATCC 43300 – Resistant S.aureus ATCC 43300 – mecA positive (zone ≤ 21 mm) S.aureus ATCC 43300 – mecA positive (MIC >4 µg/mL) Thursday, October 13, 2016 62

63. QC recommendation – Routine Test negative (susceptible) QC strain: With each new lot/shipment of testing materials Weekly if the test is performed at least once a week and criteria for converting from daily to weekly QC testing have been met Daily if the test is performed less than once per week and /or if criteria for converting from daily to weekly QC testing have not been met QC recommendation – Lot/shipment Test positive (resistant) QC strain at minimum with each new lot/shipment of testing materials. Thursday, October 13, 2016 63

64. Chromogenic medium used HiCrome MeReSa Agar ( HiMedia , Mumbai) Ingredients: Casein enzymic hydrolysate, Yeast extract, Beef extract, Agar, Sodium chloride Sodium pyruvate Chromogenic mixture Final pH ( at 25°C) 7.0±0.2 MeReSa Selective Supplement (FD229) & Cefoxitin Supplement (FD259 ) is added Incubation at 30-35°C for 18-48 hours Result: MRSA gives bluish green coloured colonies Study : 3.3% (10/300) were MRSA carriers. All isolates Cefoxitin resistant. Thursday, October 13, 2016 64

65. Chromogenic media (contd.) CHROMagar MRSA (Paris, France ) BBL CHROM agar (BD, Diagnostics, USA ) MRSA Select ( BioRad , USA ) BioSynth AG (Switzerland) Thursday, October 13, 2016 65

66. Control Measures Proper Hand washing Screening of MRSA carriers Treatment of Carriers Topical 2% mupirocin – nasal carrier Chlorhexidine – skin carriers Stoppage of misuse of AMA Bundling - Thursday, October 13, 2016 66

67. Thank you!!! Thursday, October 13, 2016 67