Staphylococcus by nissim

Staphylococcus aureus, COPS, CONS, MRSA, extremely helpful for medical and paramedical students.

1. Staphylococcus Nissim Ghimire M.Sc . Medical Microbiologist Freelance Microbiology Trainer and lecturer.

2. Classification: M icrococcaceae S taphylococcus Micrococcus Stomatococcus COPS CONS S.aureus S.hycus S.intermedius S.saprophyticus S.epidermidis S.haemolyticus Stomatococcus mucilaginosus

3. Staphylococci are gram positive cocci, Occur in grape like clusters , First observed by Von Recklinghausen(1871) in human pyogenic lesions Pasteur(1880) obtained liquid culture from pus and produced abscesses by inoculating them into rabbits Sir Alexender Ogston gave it the name “ STAPHYLOCOCCUS ” In Greek; staphyle - Bunch of grapes Kokkus - Berry Introduction & History

4. Staphylococci strain from: Pyogenic lesions: produce golden yellow colonies (S .aureus) Normal skin: white colonies (S.albus) S.citreus: lemon yellow colonies Human skin: coagulase negative staphylococcus S.aureus :lesser extent Prefered habitat:anterior nares (40%adults carriers) Micrococci :in skin and in environment Stomatococcus ( s.mucilaginosus ):normal human oral flora Contd …

5. Species of Staphylococci found in human skin: S.saprophyticus S.epidermidis S.haemolyticus S.hominis S.warneri S.lugdunensis S.simulans S.xylosus C ontd …

6. Morphology : Gram positive cocci Diameter: 1 µm (approx.) Arranged in grapes like clusters Non-motile Non-spore forming Non capsulated (some strains posseses microscopically visible capsule (young culture)) Staphylococcus aureus

7. Culture: Media used :- i ) Non selective media: Nutrient agar, Blood agar, MacConkey’s agar. ii) Selective media: Salt-milk agar, Ludlam’s medium Robertson’s cooked meat medium with 10% sodium chloride Temperature range :10°C - 42°C (optimum temp: 37°C) pH:7.4-7.6 Aerobes & facultative anaerobes Culture characteristics:

8. Culture characteristics: i ) On nutrient agar: The colonies are : large circular, convex, smooth , shiny , opaque and Easily emulsifiable . Most strains produce golden yellow pigments .

9. ii) On MacConkey’s agar- The colonies are small & pink in colour . (due to lactose fermentation) iii) On blood agar- Most strains produce β - haemolytic colonies . (specially incubated under 20-25% carbondioxide ) iv) On liquid medium: uniform urbidity is produced

10. Biochemical reactions: 1.Catalase test- Positive . 2H 2 O 2  O 2 + 2H 2 O

11. 2) Coagulase test- i ) Slide coagulase test- Positive. ii) Tube coagulase test- Positive. SLIDE COAGULASE TEST TUBE COAGULASE TEST

12. Contd …. 3) Reduces nitrate to nitrite. 4 ) Ferments mannitol anaerobically with acid only. 5 ) Urea hydrolysis test- Positive. 6 ) Gelatin liquefaction test- Positive. 7 ) Produces Lipase. 8 ) Produces Phosphatase. 9 ) Produces Thermostable nuclease. 10) MR & VP positive 11) Indole negative 12) Bacitracin resistant 13)Grow on agar that contains peptone 15)Some are ß-hemolytic

13. Mechanism of antibiotic activity Example of antimicrobial agents 1. inhibition of bacterial cell wall synthesis bacitracin, cephalosporin, imipenem , methicillin, penicillins , vancomycin 2. inhibition of enzymes involved in DNA replication and function flucytosone , metronidazole, novobiocin , quinolones 3. inhibition of tetrahydrofolic acid needed for DNA synthesis sulfonamides , trimethoprin 4. inhibition of transcription by binding to bacterial RNA-polymerase actinomycin , rifampin 5. inhibition of protein synthesis by interfering with ribosome functions aminoglycosides, chloramphenicol, clindamycin, erythromycin, tetracycline 6. inhibition of plasma (cell) membrane activities imidazoles , colistin , polyenes 7. inhibition of protein synthesis initiation oxazolidinones

14. Staphylococci are among the more resistant non-spore forming bacteria Remain viable for 3-6 months (have been isolated from dried pus after 2-3 months May withstand 60°C for 30 minutes (thermal death time:62°C for 30 minutes) some Staphylococci r equire heating at 80°C for 1 hour to be killed Heat resistant strains have ability to grow in higher temperature even at 45°C Resist 1 % phenol for 15 mins . Mercury peroxide 1% solution can kill them in 10 mins . Resistance :

15. Penicillin resistance 3 types: Production of Beta lactamase Alteration in penicillin binding protein PBP2a Development of tolerance to penicillin Bacterium is only inhibited but not killed

16. Contd … Production of beta lactamase: ( penicillinase ) Inactivates penicillin Staphylococci produces 4 types of penicillinases (A-D) Hospital strains:type A penicillinase Penicillinase : inducible enzyme Production usually controlled by plasmids,which are transmitted by transduction or conjugation Same plasmid: may carry genes for resistance to a range of other antibiotics and heavy metals

17. Alteration in penicillin binding protein(PBP2a) & changes in bacterial surface receptors: reduces binding of beta lactam antibiotics to cells Normally chromosomal in nature Expressed more at 30°C than at 37°C Also extends to cover beta lactamase-resistant penicillins such as methicillin and cloxacillins (MRSA) EMRSA:’epidemic methicillin-resistant Staphylococcus aureus ’ erythromycins,tetracyclines,aminoglycosides and heavy metals Contd …

18. PATHOGENICITY : Source of infection: A) Exogenous: patients or carriers B) Endogenous: From colonized site Mode of transmission: A) Contact: direct or indirect( through fomites) B) Inhalation of air borne droplets Pathogenicity and virulence

19. Virulence factors : These include A) Cell associated factors B) Extracellular factors Contd …

20. A) CELL ASSOCIATED FACTORS : a) Cell associated polymers b) Cell surface proteins a) CELL ASSOCIATED POLYMERS 1. Cell wall polysaccharide 2. Teichoic acid 3. Capsular polysaccharide b) CELL SURFACE PROTEINS: 1. Protein A 2. Clumping factor (bound coagulase)

21. Protein A:present in cell wall of most S.aureus Strains Chemotactic Anti-phagocytic: Binds to F c part of IgG Blocks phagocytosis Anti-complementary Induces platelet damage and hypersensitivity

22. B) EXTRACELLULAR FACTORS a) Enzymes: 1 . Free coagulase 2. Catalase 3. Lipase(infect skin and subcuaneous tissues) 4. Hyaluronidase ( hydrolyse hyaluronic acid in connective tissues) 5. DNAase 6 . Thermonuclease 7. Staphylokinase ( Fibrinolysin ) 8. Phosphatase

23. Contd … b) Toxins: 1.Cytolytic toxins(membrane active substance) ( affects RBC and WBC) i ) Haemolysins Alpha haemolysin ( paradoxic ):toxic to macrophages,lysosomes,muscle tissues,renal cortex and circulatory system Beta haemolysin ( sphingomyelinase ) Shows hot-cold phenomena Gamma haemolysin Delta haemolysin : detergent like effects on cell membreane of erythrocytes,leucocytes,macrophages and platelets ii) Leucocidin (Panton-Valentine toxin)

24. 2. Enterotoxin 3. Toxic shock syndrome toxin (TSST) 4. Exfoliative ( epidermolytic toxin)

25. Staphylococcal diseases Diseases produced by Staphylococcus aureus:2 groups A) Infections B) Intoxications

26. A) INFECTIONS: Mechanism of pathogenesis : Cocci gain access to damaged skin, mucosal or tissue site Colonize by adhering to cells or extracellular matrix Evade the host defense mechanisms and multiply Cause tissue damage

27. Common Staphylococcal infections : Skin and soft tissue : Folliculitis, furuncle (boil), carbuncle, styes , abscess, wound infections, impetigo, paronychia and less often cellulitis . Folliculitis Furuncle (boil)

28. Styes Abscess

29. Impetigo Paronychia Wound infection Cellulitis

30. 2) Musculoskeletal : Osteomyelitis, arthritis, bursitis, pyomyositis . osteomyelitis 3) Respiratory : Tonsillitis, pharyngitis, sinusitis, otitis, bronchopneumonia, lung abscess, empyema, rarely pneumonia.

31. 4) Central nervous system : Abscess, meningitis, intracranial thrombophlebitis. 5 ) Endovascular : Bacteremia, septicemia, pyemia , endocarditis. Endocarditis 6) Urinary : Urinary tract infection.

32. B) INTOXICATIOINS: The disease is caused by the bacterial exotoxins, which are produced either in the infected host or preformed in vitro. There are 3 types- Food poisoning Toxic shock syndrome Staphylococcal scalded skin syndrome

33. 1) Food poisoning: (Enterotoxin) Enterotoxin is responsible for manifestations of staphylococcal food poisoning. Eight types of enterotoxin are currently known, named A, B, C1-3, D, E, and H. It usually occurs when preformed toxin is ingested with contaminated food. The toxin acts directly on the autonomic nervous system to cause the illness, rather than gut mucosa.

34. The common food items responsible are - milk and milk products, meat, fish and ice cream. Source of infection- food handler who is a carrier. Incubation period- 2 to 6 hours. Clinical symptoms- nausea, vomiting and diarrhoea . The illness is usually self limited, with recovery in a day or so. Contd …

35. 2) Staphylococcal Toxic shock syndrome (STSS): STSS is associated with infection of mucosal or sequestered sites by TSST( formerly known as enterotoxin type F) producing S.aureus . It is fatal multisystem disease presenting with fever, hypotension, myalgia, vomiting, diarrhoea , mucosal hyperemia and erythematous rash which desquamates subsequently.

36. 2 types of STSS known : i ) Menstrual associated STSS: Here colonization of S.aureus occurs in the vagina of menstruating woman who uses highly absorbent vaginal tampons. ii) Non menstrual associated STSS: Here colonization of S.aureus occurs in other sites like surgical wound.

37. 3) Staphylococcal scalded skin syndrome (SSSS): Exfoliative toxin produced by S.aureus is responsible for this. It is a skin disease in which outer layer of epidermis gets separated from the underlying tissues.

38. Types of SSSS : Severe form Milder form In new born - Ritter’s disease - Pemphigus neonatorum In older patients - Toxic epidermal - Bullous necrolysis impetigo

39. Ritter’s disease Pemphigus neonatorum Toxic epidermal necrolysis Bullous impetigo

40. LAB DIAGNOSIS: 1.Specimens : Depends on the type of infection. Suppurative lesion- Pus, Respiratory infection- Sputum, Bacteremia & septicemia- Blood, Food poisoning- Feces, vomit & the remains of suspected food, For the detection of carriers- Nasal swab.

41. 2.microscopy : Direct microscopy with Gram stained smear is useful in case of pus, where cocci in clusters are seen. This is of no value for specimen like sputum where mixed flora are normally present.

42. 3.Culture: Specimens plated in blood agar Staphylococcal colonies appear after overnight incubation Specimens where staphylococci are expected to be scanty and outnumbered by other bacteria are inoculated on selective media Salt-milk agar, Ludlam’s medium Robertson’s cooked meat medium with 10% sodium

43. 1.Gram staining: Smears are examined from the culture plate and reveals Gram positive cocci(1 μ m in diameter) arranged in grape like clusters . 3.Identification:

44. Differentiation between staphylococci micrococci and stomatococci property staphylococcus micrococcus stomatococcus Anaerobic growth + _ + Carbohydrate utilization F O F catalase + + Weak oxidase _ + _ Bacitracin R S S lysostaphin S R R Adherence to agar _ _ +

45. Differentiation of S.aureus from CONS Contd … test S.aureus S.epidermidis S.saprophyticus Growth on manitol salt agar + _ _ Colonial pigmentation Golden yellow white White Coagulase test + _ _ DNAase Test + _ _ Hemolysis in blood agar beta _ _ Novobiosin sensitivity S S R

46. Staphylococci are primary parasites of human beings and animals. Hospital infections caused by staphylococci deserve special attention because of their frequency & they are caused by strains resistant to various antibiotics. Staphylococci are the common cause of postoperative wound infection and other hospital cross infections. Epidemiology:

47. M ethicillin- R esistant S taphylococcus A ureus Epidemic strains of these; MRSA are usually resistant to several other antibiotics Characteristic of MRSA is its ability to thrive in presence of penicillin like antibiotics which normally prevent bacterial growth by inhibiting cell-wall synthesis. MRSA

48. Methicillin -resistant Staphylococcus aureus (MRSA) Mechanism MRSA contains the mecA gene which is responsible for the production of an altered plasma (cell) membrane-bound enzyme, penicillin-binding protein 2a (PBP- 2a.) , penicillin-binding protein 2’ Penicillin-binding proteins are enzymes that participate in the production of a major component of the bacterial cell wall The altered PBP 2a while able to perform its cell-wall synthesis function, has a low affinity for and does not bind to beta- lactam antibiotics mecA , stops β-lactam antibiotics from inactivating the enzymes (trans-peptidases) critical for cell wall synthesis.

49. Methicillin -resistant Staphylococcus aureus (MRSA) Thus, the presence of the mecA gene confers resistance to all beta- lactam antibiotics such as methicillin . The mec A gene along with several other virulence and/or antibiotic resistance genes is carried on a movable segment or unit of the bacterium’s chromosome called the “staphylococcal cassette chromosome mec ” ( SCCmec ). Currently, there are 6 different types; I, II, III, IV, V and VI, and all of which vary in size The larger the SCCmec type, the more room there is for resistance and other genes Hospital-acquired MRSA strains usually contain types I, II, or III, while community-associated MRSA strains contain types IV, and V. Mechanism

50. The first MRSA strain, ST250 MRSA-1 originated from SCC mec  and ST250-MSSA integration. Historically, major MRSA clones: ST2470-MRSA-I, ST239-MRSA-III, ST5-MRSA-II, and ST5-MRSA-IV were responsible for causing hospital-acquired MRSA (HA-MRSA) infections. ST239-MRSA-III, known as the Brazilian clone, was highly transmissible compared to others and distributed in Argentina, Czech Republic, and Portugal. Methicillin -resistant Staphylococcus aureus (MRSA)

51. Treatment Methicillin -resistant Staphylococcus aureus (MRSA) Both CA-MRSA and HA-MRSA are resistant to traditional anti-staphylococcal beta-lactam antibiotics, such as cephalexin CA-MRSA has a greater spectrum of antimicrobial susceptibility to sulfa drugs (like co- trimoxazole (trimethoprim/sulfamethoxazole),  tetracyclines  (like doxycycline and minocycline) and clindamycin (for osteomyelitis) MRSA can be eradicated with a regimen of linezolid, though treatment protocols vary and serum levels of antibiotics vary widely person to person and may affect outcomes

52. Linezolid  belongs to the newer  oxazolidinone class of antibiotics which has been shown to be effective against both CA-MRSA and HA-MRSA. The effective treatment of MRSA with linezolid has been successful in 87% of people. Linezolid is more effective in soft tissue infections than vancomycin. This is compared to eradication of infection in those with MRSA treated with vancomycin. Treatment with vancomycin is successful in approximately 49% of people The  Infectious Disease Society of America   recommends vancomycin, linezolid, or clindamycin (if susceptible) for treating those with MRSA pneumonia. Methicillin -resistant Staphylococcus aureus (MRSA)

53. Methicillin -resistant Staphylococcus aureus (MRSA) Treatment Vancomycin remains the drug of choice for treatment of infections caused by MRSA, although it is intrinsically less active than the antistaphylococcal penicillins Combinations of vancomycin with beta-lactam antibiotics may be synergistic in vivo against MRSA strains, including those with intermediate susceptibility to vancomycin Given the increasing prevalence of MRSA in hospitals and in community settings, alternative approaches are needed for treatment of infections caused by MRSA.

54. Methicillin -resistant Staphylococcus aureus (MRSA) Treatment Vancomycin and  teicoplanin  are  glycopeptide antibiotics used to treat MRSA infections Teicoplanin  is a structural congener of vancomycin that has a similar activity spectrum but a longer  half-life Because the oral absorption of vancomycin and  teicoplanin  is very low, these agents can be administered intravenously to control systemic infections . Treatment of MRSA infection with vancomycin can be complicated, due to its inconvenient route of administration.

55. Several newly discovered strains of MRSA show   antibiotic resistance even to vancomycin and  teicoplanin . These new strains of the MRSA bacterium have been dubbed vancomycin intermediate-resistant  Staphylococcus aureus  (VISA).Linezolid,  quinupristin / dalfopristin ,  daptomycin ,  ceftaroline , and  tigecycline  are used to treat more severe infections that do not respond to glycopeptides such as vancomycin. Current guidelines recommend  daptomycin  for VISA bloodstream infections and endocarditis.

56. PREVENTION : Isolation & treatment of MRSA patients. Detection of carriers among hospital staff, their isolation & treatment. Avoid indiscriminate usage of antibiotics . Following strict aseptic technique Hand washing ,oldest simplest and most effective method of previnting hospital cross-infection.

57. Other coagulase positive Staphylococci: Staphylococcus aureus  subsp.  Anaerobius S. a. aureus S . hyicus S . intermedius and   Staphylococcus schleiferi  subsp.  coagulans .

58. Co agulase N egative S taphylococci( CoNS ): Two species of coagulase negative Staphylococci can cause human infections- 1. Staphylococcus epidermidis 2. Staphylococcus saprophyticus

59. S. Epidermidis : It is a common cause of stitch abscesses. It has predilection for growth on implanted foreign bodies such as artificial valves, shunts, intravascular catheters and prosthetic appliances leading to bacteraemia . In persons with structural abnormalities of urinary tract, it can cause cystitis. Endocarditis may be caused, particularly in drug addicts.

60. S.saprophyticus : It causes urinary tract infections, mostly in sexually active young women. The infection is symptomatic and may involve the upper urinary tract also. Men are infected much less often. It is one of the few frequently isolated CoNS that is resistant to Novobiocin

61. Other coagulase negative staphylococci: S.haemolyticus S. saprophyticus   S . warneri , S.hominis , S.epidermidis S . caprae and  S.lugdunensis

62. THANK YOU