MRSA

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MRSA

  1. 1. METHICILLIN RESISTANTSTAPHYLOCOCCUS AUREUS Dr.Riyaz Sheriff 1
  2. 2. What is MRSA?• Methicillin Resistant Staphylococcus aureus demonstrates resistance to semisynthetic penicillins – Methicillin, Cloxacillin & Oxacillin.• MRSA also exhibit resistance to cephalosporins, monobactams and carbepenamases Dr.Riyaz Sheriff 2
  3. 3. • Hospital associated MRSA (HA-MRSA)  MRSA strains that circulate and are transmitted to individuals in health care facilities• Risk factors for HA-MRSA – Isolation of MRSA after 48hours of hospital admission – History of Hospitalization, Surgery, Dialysis – History of long term admission within one year of the MRSA culture date – Presence of indwelling catheter or percutaneous device at the time of culture or previous to isolation of MRSA• Community associated MRSA (CA-MRSA)  – MRSA isolates obtained from individuals in community who have not had recent exposure to the health care system – Patients in health care facilities in whom the infection was present or incubating at the time of admission Dr.Riyaz Sheriff 3
  4. 4. Methicillin / Meticillin• Narrow spectrum antibiotic of penicillin class introduced in 1959• Has ortho-dimethoxy phenyl group attached to side chain carbonyl group of penicillin nucleus.• Has been renamed as meticillin to have a common international nonproprietary name. Dr.Riyaz Sheriff 4
  5. 5. PENICILLIN METHICILLIN Dr.Riyaz Sheriff 5
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  7. 7. Why MRSA?• Methicillin was initially used to detect resistance to beta lactamase stable penicillins hence the name MRSA.• Oxacillin is used as alternative to methicillin  ORSA (oxacillin resistant Staphylococcus aureus) Dr.Riyaz Sheriff 7
  8. 8. Mechanism of resistance to penicillins• Presence of mecA gene – mecA gene is carried on Staphylococcal cassette chromosome mec (SCCmec) – SCC have mecA and ccr (cassette chromosome recombinase) and attach it to the 3’ end of staphylococcal chromosome – Codes for modified penicillin binding protein 2a (PBP 2a OR PBP 2’) – Has low affinity for Beta lactams Dr.Riyaz Sheriff 8
  9. 9. Mechanism of resistance to penicillins• Production of Beta lactamase enzyme – Some strains produce excessive Beta lactamase which makes it appear borderline resistant to oxacillin (BORSA). Difficult to detect. – MRSA with minor alterations to existing PBP  Moderately resistant Staphylococcus aureus (MODSA) Dr.Riyaz Sheriff 9
  10. 10. Virulence factors of Staphylococcus aureus• Capsular polysacchrides • Hemolysins• Peptidoglycan and Teichoic acids – Alpha Hemolysin• Protein A – Beta Hemolysin• Enzymes – Gamma Hemolysin – Catalase – Delta Hemolysin – Clumping factor – Panton-Valentine Leukocidin – Coagulase • Toxins – Fibrinolysins – Epidermolytic toxins – Hyaluronidase – Enterotoxins A,B,C,D,E,H & I – Lipases • Superantigens – Phosphatidylinositol specific – Toxic shock syndrome toxin I phospholipase C – Nuclease Dr.Riyaz Sheriff 10
  11. 11. Virulence Factors in CA-MRSACategory ToxinCytolytic toxins Panton-Valentine leukocidin S and F Fibronectin binding proteins A and B Leukocidin RSuperantigenic toxins Enterotoxins (A to J) Epidermolytic toxins Toxic shock syndrome toxin -1Enhanced growth and survival Arginine catabolic mobile element Dr.Riyaz Sheriff 11
  12. 12. Nomenclature of MRSAUS pulse field Canadian Multi locus SCCmec type PVL presencegel MRSA sequenceelectrophoresi typingsUSA 100 2 5 II NEGATIVEUSA 200 4 36 II NEGATIVEUSA 300 10 8 IVa POSITIVEUSA 400 7 1 IVa POSITIVEUSA 500 5,9 8 IV NEGATIVEUSA 600 1 45 II NEGATIVEUSA 700 72 IVa NEGATIVEUSA 800 2 5 IV NEGATIVEUSA 1000 59 IV POSITIVEUSA1100 30 IV POSITIVE Dr.Riyaz Sheriff 12
  13. 13. Detection of MRSA• Phenotypic detection systems – Disc diffusion test • Colony suspension prepared from 5colonies and plated on muller hinton agar containing 2-4% NaCl at neutral pH. • Oxacillin disc (1ug) placed and incubated at 35°C for 24 hours • <10mm is considered resistant, >13mm is considered sensitive. • For isolates with intermediate results – Test for mec A, PBP2a – Cefoxitin disc test – Oxacillin MIC test or – Oxacillin salt agar screen test may be performed. • Any growth within the zone of inhibition indicates oxacillin resistance Dr.Riyaz Sheriff 13
  14. 14. Other detection methods• Agar dilution test – 0.5 McFarland preparation of isolate is spot inoculated on MHA with 2% NaCl containing 256 – 0.125 ug oxacillin/ml in serial doubling dilutions – MIC of >4ug/ml is considered resistant , MIC <2ug/ml is considered susceptible• Broth microdilution – Muller hinton broth inoculated with inoculum density of 5 X 10 5 cfu/ml• Breakpoint methods – Includes both agar and broth methods but test only the breakpoint concentration (2mg/L oxacillin, 4mg/L methicillin)• E-test oxacillin MIC test – Easy to set up as a disc diffusion test Dr.Riyaz Sheriff 14
  15. 15. • Oxacillin screen agar – MHA with 4%NaCl and 6ug/ml oxacillin – Inoculated with 10uL of 0.5Mcfarland preparation, streaked in one quadrant and incubated at 35°C for 24-48 hours – Any growth after 24hrs is considered oxacillin resistant• Cefoxitin disc diffusion test – Cefoxitin – potent inducerof mecA regulatory system – Used as a surrogate marker for detection of mecA gene mediated methicillin resistance – Inducible resistance to methicillin is better seen with cefoxitin than oxacillin. This is due to enhanced induction of PBP 2a by cefoxitin. – 30ug cefoxitin disc is used.( < 21mm is resistant and > 22mm susceptible)• PBP 2a latex agglutination kit – PBP2a extacted from suspension of colonies and react with latex particles coated with monoclonal antibody against PBP 2a – Visible agglutination indicates positive result and presence of PBP 2a Dr.Riyaz Sheriff 15
  16. 16. Chromogenic Screening mediumMedia Components/ action Colony colourCHROM ID CEFOXITIN 4MG/L GREEN TARGETS ALPHA GLUCOSIDASEORSAB MANNITOL SALT AGAR BLUE OXACILLIN 2MG/L POLYMYXIN ANILINE BLUEMRSA INDENT AGAR CEFOXITIN RUBY COLOURED CHROMOGENIC PHOSPHATASE SUBSTRATEDENIM BLUE AGAR CEFOXITIN DENIM BLUE COLONIES PHOPHATIDASE ACTIVITY Dr.Riyaz Sheriff 16
  17. 17. • Molecular methods – Detection of mecA gene by PCR is considered gold standard • DNA extraction • mecA gene amplified using specific primers – 30 cycles of denaturation at 94°C for 45seconds – Anneling at 50°C for 45 seconds – Extension at 72°C for 1 minute – Final extension step at 72°C for 3mins • PCR products visualized on 2%agarose gel with ethidium bromide dye under U-V transluminator Dr.Riyaz Sheriff 17
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  19. 19. Community associated meticillin-resistant Staphylococcus aureusstrains as a cause of healthcare- associated infection J.A Otter, G.L. French Journal Of Hospital Infection 79 (2011) 189-193 Impact factor 3.393 Dr.Riyaz Sheriff 19
  20. 20. Introduction• CA-MRSA infections were first reported in early 1990s from western Australia, New Zealand and in America.• CA-MRSA seems to have evolved from MSSA acquiring SCCmec cassettes.• Have the ability to affect younger, healthy people and spread rapidly in community settings.• CA-MRSA are generally susceptible to non Beta Lactam antibiotics, Have small SCCmec cassettes (type IV or V), Many of them produce PVL Dr.Riyaz Sheriff 20
  21. 21. Dr.Riyaz Sheriff 21
  22. 22. CA-MRSA in Hospital outbreaks• Nosocomial outbreaks reported from 2003 from North America, Germany, Israel, Switzerland, Greece and UK.• Most outbreaks have been caused by single cross infecting strain• Infections in health care workers and transmission to their household contacts have occurred in several of these outbreaks• PVL + CA-MRSA has been isolated in most of the outbreaks but one outbreak in Israel and two outbreaks in UK were caused by PVL negative strains  CA-MRSA do not need PVL to cause nosocomial infections Dr.Riyaz Sheriff 22
  23. 23. Control Measures for CA-MRSA• Isolation of affected patients• Screening other patients in the unit for asymptomatic carriage• Reinforcement of standard infection control procedures – Hand hygiene – Screening of staff members for colonization – Swabbing of environmental surfaces and equipments – Improved environmental cleaning• Closure of unit to new admissions• Screening of household contacts of health care workers and appropriate management during CA- MRSA outbreaks Dr.Riyaz Sheriff 23
  24. 24. CA-MRSA role in endemic HAICA-MRSA is overtaking HA-MRSA as a cause of HAIInitially CA-MRSA was reported in new born infectionsand post operative prosthetic joint infectionsRecent reports suggest CA-MRSA as a cause of HAbacteraemias.16% of hospital onset infections and 22% of healthcare associated infections are caused by CA-MRSAMany countries have reported 10fold increase in CA-MRSA from 1999 to 2006In Greece PVL positive CA-MRSA accounted for 45% ofHA-MRSA infections at several hospitals during 2001-2003 Dr.Riyaz Sheriff 24
  25. 25. CA-MRSA implications CA-MRSA is emerging as a cause of hospital associated infections in Asia, Africa & South America. The reason for this emergence is not clearly understood. CA-MRSA have the ability to affect healthy individuals CA-MRSA strain with PVL are on the rise resulting in higher virulence. Studies have shown that CA-MRSA behave like HA-MRSA once inside the hospitals but cause more invasive infections than uncomplicated SSTI. CA-MRSA exposed to antibiotics is more likely to acquire resistance genes and become MDR strains like HA-MRSA Definitions for HA-MRSA and CA-MRSA become more confused. Dr.Riyaz Sheriff 25
  26. 26. CA-MRSA implications (Cont’d) Control of MRSA in hospital settings will be more difficult. With lack of clear knowledge on epidemiology of CA-MRSA the current infection control policies may not be successful and needs to be modified. Hospitals should type MRSA strains involved in outbreaks and there should also be periodic assessment of the antibiotic resistance profiles among CA-MRSA strains. The success of CA-MRSA in spreading in various subsets of the community is a major concern. Finally there is an urgent need to measure the prevalence and epidemiology of CA-MRSA and also develop systems to identify and control these organisms in the community, hospitals and other health care facilities. Dr.Riyaz Sheriff 26
  27. 27. Conclusion CA-MRSA has emerged as a Nosocomial pathogen in the recent years and are beginning to overtake HA-MRSA in hospital infection. CA-MRSA infections puts a wider group of people at risk. This includes hospitalized patients, Health workers and their community contacts Classification of CA-MRSA and HA-MRSA is becoming more confusing Western countries report a MRSA incidence ranging from 10.4% to 40% In Tamil Nadu MRSA prevalence in clinical samples is estimated to be around 31.1% in clinical and 37.9% in carrier samples MDR among MRSA in clinical isolates was estimated around 63.6% and 23% carrier samples There is an urgent need to measure the prevalence and epidemiology of CA-MRSA and also develop systems to identify and control these organisms in the community, hospitals and other health care facilities. Dr.Riyaz Sheriff 27
  28. 28. References• Koneman’s atlas and textbook of diagnostic microbiology• Essentials of medical pharmacology KD tripathi 5th edition• Michelle barton et al Guidelines for prevention and management of community - associated methicillin - resistant Staphylococcus aureus: a perspective for Canadian health care practitioners, Canadian journal of infectious disease and medical microbiology vol 17, supplement C 2006 page 4C-24C• Rajadurapandi et al prevalence and antimicrobial susceptibility pattern of methicillin resistant Staphylococcus aureus: a multicentre study, IJMM (2006) 24 (1): 34-38• Benjamin etal Reduced Vancomycin Susceptibility in Staphylococcus aureus, Including Vancomycin-Intermediate and Heterogeneous Vancomycin- Intermediate Strains: Resistance Mechanisms, Laboratory Detection, and Clinical Implications, CMR 2010 p.99-139 Dr.Riyaz Sheriff 28
  29. 29. Dr.Riyaz Sheriff 29

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