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Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
Mrsa seminar final draft 2642012
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Mrsa seminar final draft 2642012

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  • This is great information on staph. I think it is destined to be a much bigger problem than it already is, due to the issue of resistant drugs. Unfortunately my father has been the victim of staph, and if it wasn't for the work of Microbiologist, Michelle Moore's treatment, I don't think he would have made it. Anyone who needs support, like my dad did (he's fully healed), may want to visit her information site at http://www.staph-infection-resources.com Great information here. Keep up the great work!!
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  • 1. Seminar 4 MRSA Methicillin resistant S. aureus Mon – 4/30/201212/30/12 1
  • 2. Outlines• Introduction• Abbreviation• The basic Biology of Staphylococcus aureus• History of MRSA• Molecular basis for virulence factors and resistance• Epidemiology• Clinical Manifestations• Laboratory Diagnosis• Treatment• prevention and control12/30/12 2
  • 3. Abbreviations/Definitions• MRSA = methicillin resistant Staphylococcus aureus• MSSA = methicillin sensitive S. aureus• HA = healthcare associated• CA = community associated• Colonization = organism is on or in the body but not causing disease – 30% of people are carriers of MSSA – 1.5% of people are carriers of MRSA• Infection = organism is present and causing signs and symptoms of disease12/30/12 3
  • 4. S. aureus – the pathogen• Microbiology – Gr+ cocci with many virulent factors (toxins and enzymes)• Frequent nosocomial- and community-acquired pathogen• Mode of transmission – contact• Clinical manifestations: – Skin and soft tissue infections – Pneumonia – Osteomyelitis / Arthritis – Bacteremia / Sepsis – Endocarditis – Toxin-mediated disease: TSS, Food poisining12/30/12 4
  • 5. Summary of Virulence Determinants Of Staphylococcus aureus• http://textbookofbacteriology.net/staph.html http://textbookofbacteriology.net/staph.ht ml12/30/12 5
  • 6. What Is MRSA? • MRSA is “Methicillin Resistant S. aureus • Is a bacteria that is resistant to a synthetic penicillin- methicillin. • MRSA causes a variety of disseminated, lethal infections in humans. • Has the ability to easily transfer resistant genes to other species directly and indirectly • Overuse of antibiotics imposes selective pressures which mediates the acquisition of resistance12/30/12 6
  • 7. Microbiology of MRSA• Caused by changes in penicillin binding proteins• Generally resistant to all B-lactam antibiotics• May be resistant to TMP/Sulfa, Tetracyclines, Quinolones• Vancomycin resistance has been reported (VISA, GISA)12/30/12 7
  • 8. How “Tough” is MRSA?• Staphylococci can survive many extreme environmental conditions.• The bacteria can be cultured from dried clinical material after several months, are relatively heat resistant, and can tolerate high salt media. So, “What Do we Do?”• You can not get rid of MRSA; you can only control it.12/30/12 8
  • 9. Risk factors for MRSA CA-MRSA HA-MRSA• Skin, soft tissue infection • Previous contact with• ??? health care system • Longer hospitalization • ICU admission or invasive procedures • Ab Rx.12/30/12 9
  • 10. CA-MRSA: 1996-2008 Changing definitions • No contact with health-care facilities in prior 6-12 m. – Maybe more than 1y. X X • Resistant only to b-lactams, but not to other classes. – Resistant to quinolones, macrolides and others • SCCmec IV – and V … and VI…12/30/12 10
  • 11. How is MRSA spread? 1. Direct contact with infected or colonized host -human-to-human contact 2. Contaminated intermediate surfaces -hand towels -faucets -tub/shower 3. Airborne fluid droplets How did CA-MRSA evolve? •Recent evolution of CA-MRSA from common MSSA? •“Hospital escape” of unsuccessful HA-MRSA12/30/12 11
  • 12. MRSA – a nosocomial pathogen Until ~199612/30/12 12
  • 13. Pets’/Animals’ Roles in MRSA Transmission• In several studies, it has been found that farm owners, veterinary clinicians, and pet owners are at greater risk for MRSA infection• In particular, there have been documented MRSA transmission to humans from: – Dogs – Cats – Horses – Pigs• It is often noted that animals, especially pets, can serve as “reservoirs of infection”12/30/12 13
  • 14. Antimicrobial resistance of S. aureus - history SA genome sequence, Kuroda ‘01 CA-MRSA Cloning of mecA SCCmec sequenced sequence, Baba ‘02 MRSA single clone theory Lacey & Grinsted, ‘73 Matsuhashi ‘86 Ito ‘99 1960 1970 1980 1990 2000 2003 Epidemic spread of 2nd wave of epidemic MRSA, Europe, India, MRSA (MDR), USA, Increasing reports Australia, USA Australia, Ireland - CA-MRSA 1st MRSA isolate ‘61 CA-MRSA Worldwide in Australia disseminationIntroduction ofMethicillin – ‘59 1st VISA, Japan ‘97 1st VRSA, USA ‘02 12/30/12 14
  • 15. S. aureus Nasal Colonization National Health and Nutrition Examination Survey 2001-02 50 45 S. aureus: 32.4% = 89.4 M people 40 35 Prevalence (%) 30 Male 25 Female 20 15 MRSA: 0.8% = 2.3 M people 10 5 0 1--5 6--11 12--19 20--29 30--39 40--49 50--59 60--69 70+ Age (years)MRSA colonization associated with age >= 60 years & being female12/30/12 15
  • 16. Prevalence of MRSA 2006 Grundmann, Lancet 2006
  • 17. Genetic Mechanisms Horizontal vs. Vertical transmission Mutation Plasmid transfer Transformation12/30/12 17 Large genetic mobile elements (cassettes)
  • 18. Horizontal Gene Transfer-Another Mechanism For Resistance http://www.bioteach.ubc.ca/Biodiversity/AttackOfT12/30/12 18 heSuperbugs
  • 19. MRSA – mechanism – I • Horizontally transferred DNA element - SCCmec. • Site specific recombination. • mecA gene encodes PBP2a. • PBP2a = 78 KDa PBP - capable of cell wall synthesis. • PBP2a has low affinity for12/30/12 all β-lactams. 19
  • 20. MRSA - mechanism of resistance 1. Modifying enzymes 2. Degrading enzymes 3. Target Change 4. Efflux pumps12/30/12 20
  • 21. MRSA – mechanism-II• mecA is part of a large, mobile, genetic element – Staphylococcal cassette chromosome mec (SCCmec) 12/30/12 21
  • 22. SCCmec cassette Mec complex (class B) ccr complex (type2) orfX ♠ mecA IS431mec♠ ΨIS 1272 ∆mecR1• A unique class of mobile genetic element (21-67kb)• Resembles a pathogenicity island, but with no virulence genes.• Ccr complex: ccrA & ccrB encode recombinase A & B enable SCCmec to integrate into the chromosome in correct orientation.• Mec complex: encodes β-lactam resistance and its inducible regulation + transposons + integrated copies of plasmids that carry various resistance genes (non-b-lactam)12/30/12 22
  • 23. Origin of SCCmec and the mec gene• Single clonal origin theory• Hiramatsu et al. 1996: Clonal diversity: different strains developed independently• Origin of mecA gene - horizontal transfer from: – SCN – S. scuiri – Enterococcus hiriae12/30/12 23
  • 24. Clonal spread of MRSA• Spread is mainly clonal. Only few clones are the cause of most infections.• Major cause for clonal spread: lapses in IC• Yet - role of Ab pressure:…12/30/12 24
  • 25. mecA gene carried on SCCmec MSSA acquired the mecA gene encodes a PBP with low affinity for penicillin (PBP2a) Carried on a large chromosomal element (SCCmec) Integrates into chromsome in orfX site specific int 20 kb-60kb orfX mecA (complex A, B and C)ccr recombinase genes12/30/12 25(allotypes A, B, C and complex 1,2,3,4,5,)
  • 26. Currently identified SCC mec types in S. aureus strainsSCCmec types ccr gene complexes mec gene complexes strainsI 1 (A1B1)* B NCTC10442, COL N315, Mu50, Mu3, MRSA252,II 2 (A2B2) A JH1, JH9III 3 (A3B3) A 85/2082 CA05, MW2, 8/6-3P, 81/108, 2314, cm11, JCSC4469, M03-IV 2 (A2B2) B 68, E-MRSA-15, JCSC6668, JCSC6670 WIS(WBG8318), TSGH17,V 5 (C1) C2 PM1,VI 4 (A4B4) B HDE288VII 5 (C1) C1 JCSC6082VIII 4 (A4B4) A C10682, BK20781IX 1(A1B1) C2 JCSC6943X 7(A1B6) C1 JCSC694512/30/12 26XI 8(A1B3) E LGA251
  • 27. 12/30/12 27
  • 28. http://www.sccmec.org/Pages/SCC_ClassificationEN.html12/30/12 28
  • 29. One last Mechanism• MRSA has shown variability in its resistance to erythromycin by two mechanisms, one of which can be transferred to clindamycin. • 1. Efflux pump - encoded by msrA gene; confers resistance of e-mycin, but not clindamycin • 2. erm gene - erythromycin ribosomal methylase; mutation to clinda resistance may develop during clinda therapy 12/30/12 29
  • 30. S. aureus - Epidemiology • Epidemiologic niche: – Nasal carriage (anterior nares) – GI tract (rectal) – Perineal – Throat • Nasal carriage – 30% of adults – 20% Persistant carriers – 60% Transient carriers – 20% Never carriers • Nosocomial transmission – transient hand carriage12/30/12 30
  • 31. Frequency of Staphylococcus aureus colonization in carriers on various body sites Nose 100%Axilla 19%Skin chest 45% Forearm 45% Hand 90%Ankle 10% Perineum 60%12/30/12 S. Importance of environment in Methicillin Resistant Staphylococcus aureus acquisition: the case 31 Dancer for hospital cleaning Lancet 07:70241-4
  • 32. Intracellular Persistence• MRSA may survive within host cells such as phagocytes. May be found in chronic infections such as osteomyelitis, prosthetic joints, and skin infections.12/30/12 32
  • 33. Predisposing Factors Of Susceptibility• Integument injury• Burns and trauma• Foreign objects• A history of chronic Infections• Hormonal changes and stress• Immunocompromised12/30/12 33
  • 34. Risk groups with high carriage rates• Diabetes Mellitus• Dialysis patients• HIV• Chronic skin diseases• IV Drug abusers• Health care workers (?)12/30/12 34
  • 35. Who Is At Risk? – 5”C” • The 5 C’s – Crowding – Contact (frequent) – Compromised skin – Contaminated surfaces and shared items – Cleanliness or lack of12/30/12 35
  • 36. Factors that Facilitate Transmission Crowding Frequent Contact Antimicrobial Use Contaminated SurfacesCompromised Skin and Shared Items Cleanliness
  • 37. Laboratory Diagnosis And Susceptibility Testing Of Methicillin-Resistant Staphylococcus aureus (M.R.S.A.)12/30/12 37
  • 38. Conventional approaches for:o Identification of S.aureus o Routine microscopy, culturing & susceptibility testing o Slide coagulase test o Tube coagulase test o Latex agglutination tests o DNase and heat-stable nuclease tests o Commercial biochemical tests o Molecular testso Methicillin/oxacillin susceptibility testing o Dilution methods (MICs) o Breakpoint methods o Disc diffusion o Agar screening method o E-test method (Diffusion & dilution) 12/30/12 38
  • 39. Detection of MRSA in screening samples Conventional approaches Solid agar media e.g (MSA), contains o An indicator to distinguish S. aureus o Inhibitory substances o NaCl, ciprofloxacin, polymyxin-B, aztreonam o And methicillin, oxacillin or,more recently, cefoxitin to select methicillin-resistant strains12/30/12 39
  • 40. Enrichment Enrichment broths have commonly been used to increase the sensitivity of screening by allowing small numbers of MRSA to grow during overnight incubation before subculture on a screening agar medium. Enrichment media generally contain additional NaCl and may also contain methicillin, oxacillin or, more recently, cefoxitin to add selectivity12/30/12 40
  • 41. Culture Detection of MRSA• Clinical and Laboratory Standards Institute (CLSI) recommends: – Cefoxitin disk screen test – Latex agglutination test for PBP2a – 6 ug/ml oxacillin in Mueller-Hinton agar with 4% NaCl12/30/12 41
  • 42. D-zone test for Inducible Clindamycin Resistance E CC-Perform on erythromycin-resistant, clindamycin-susceptible S. aureus isolates-Clinical implications unclear, but treatment failures have occurred-Does not require pre-treatment or co-treatment with erythromycin in vivo 12/30/12 42
  • 43. erm gene Detection• The D test - place a pair of erythromycin and clindamycin disks on a plated dish.• Flattening of the clindamycin zone confers a positive test, i.e. inducible clinda resistance 12/30/12 43
  • 44. 12/30/12 44
  • 45. Molecular Identification and Strain Typing of MRSA12/30/12 45
  • 46. Typing Techniques1. Plasmid Profile Analysis – Several limitations1. Enzymatic Restriction (REA) – Difficult (RFLP)1. Hybridisation with a Nucleic Acid Probe (Southern Blotting) – Ribotyping yields good reproducibility and discriminatory power for epidemiological characterizations of MRSA1. Pulsed-Field Gel Electrophoresis (PFGE) ex; ( SmaI-PFGE) – The discriminatory power is equal to or superior to phenotypic techniques as well as to genotypic techniques such as ribotyping, RAPD, PCR-RFLP and inter-IS 256 PCR – has been sug-gested to be a gold standard for typing of MRSA1. Polymerase Chain Reaction (PCR) – SCCmec typing – Spa typing – RAPD (Random Amplified Polymorphic DNA) • discriminatory power will still remain poor – Repetitive Palindromic Extragenic Elements PCR (Rep-PCR) high discriminatory power1. Multilocus Sequence Typing (MLST)12/30/12 46
  • 47. Advantages of MRSA PCR• Rapid Identification of carriers – 2 hours PCR – 24-72 hours Culture• Swift implementation of appropriate interventions• Limits unnecessary preemptive isolation• Limits unnecessary antibiotic use• Identifies MRSA in mixed populations – Methicillin resistant coagulase negative Staph – mecA negative Staph12/30/12 47
  • 48. Molecular methods • To detect genes which identify strains as S. aureus – (nuclease (nuc), coagulase (coa), protein A (spa), femA and fem B and mecA) • Generally only applicable to identification of purified cultures of staphylococci, and therefore they should not be used directly on samples. • Most molecular methods for identification of S. aureus have been PCR based.12/30/12 48
  • 49. Why Utilize Molecular Testing? • Rapid diagnosis • Cost effective • Highly specific • Improved patient care • Provides ultimate resolution • No outside interference from medication or treatments can alter the testing results12/30/12 49
  • 50. MRSA PCR Procedure• Specimen preparation• Concentration• Lysis-DNA extraction• Reconstitution of molecular reagents• Real-time PCR• Automated results• Full run time < 2 hours12/30/12 50
  • 51. 12/30/12 51
  • 52. MRSA PCR vs. Culture Method Sensitivity % Specificity % Culture 78-80% 99% PCR 93% 96% PPV: 94% NPV: 98% Detection limit: 325 MRSA per swab12/30/12 52
  • 53. MRSA Strain Typing• MRSA is a clonal organism• Very useful for epidemiology – Strain types between patients – Monitor trends in types – Track seasonal outbreaks – Database of samples for long term studies• Trace infection source – Patient-Patient / Patient-Caregiver• Determine if HA or CA MRSA12/30/12 53
  • 54. Resolution of Typing Methods12/30/12 54
  • 55. Pulsed Field Gel Electrophoresis12/30/12 55
  • 56. PFGE Results12/30/12 56
  • 57. Approach to Laboratory identification of MRSA• Sensitivity Testing: a) Conventional methods b) Automated systems c) Quenching fluorescence method• Serological Identification• Genotyping Identification (Molecular method)• Direct identification of MRSA in blood cultures• Identification of MRSA in endotracheal aspirates and other clinical samples12/30/12 57
  • 58. Dilution methods Broth micro dilution:o No single set of test conditions is suitable for detection of all resistant strains.o The CLSI(NCCLS)method :o The CLSI method, which requires the use of MH broth with 2% NaCl, an inoculum of 5 x105 cfu/mL and incubation at 33–35°C for 24 h, is the only defined methodo Tray should include o a growth control (no antimicrobial agent), o a sterility control (not inoculated at all) & +ve control o positive MRSA control strain = Staph.aureus NCTC 6571o An oxacillin MIC of ≤ 2 μg /ml indicates that the strain is susceptible and > 2 μg /ml resistant.12/30/12 58
  • 59. Interpretive Criteria (in μg /ml) for Oxacillin MIC Tests Susceptible Intermediate Resistant S. aureus ≤ 2.0 μg /ml N/A ≥ 4.o μg /ml CoNS ≤ 0.25 μg /ml N/A ≥ 0.5 μg /ml N/A = not applicable12/30/12 59
  • 60. Agar dilution Method • BSAC method: o M-H agar or Columbia agar with 4% NaCl o CLSI (NCCLS) only M-H agar with 2 % NaCl o Spot inoculum of 104 cfu/mL o Sensitivity at 24 hrs incubation is variable, 48hrs incubation often required for an o acceptable result. o The BSAC requires incubation at 30°C, while the NCCLS requires 33–35°C. o An oxacillin MIC of ≤ 2 μg /ml indicates that the strain is susceptible and > 2 μg /ml resistant. o In the BSAC method a methicillin MIC of o ≤ 4μg /ml indicates that the strain is susceptible and > 4μg /ml resistant,12/30/12 60
  • 61. Breakpoint methods  Breakpoint methods include both agar and broth methods  Similar to dilution MIC methods but test only the breakpoint concentration (2 ug /ml oxacillin, 4 ug /ml methicillin)  The breakpoints for S. aureus are different from those for coagulase-negative staphylococci (CoNS).  Susceptibility and resistance breakpoints  Methicillin : ≤ 4 and ≥ 16 ug /ml  Oxacillin & nafcillin: ≤2 and ≥ 8 ug /ml12/30/12 61
  • 62. Disc diffusion• Breakpoint methods include both agar and broth methods and are essentially similar to dilution MIC methods but test only the break point concentrationo Tests batteries : o 1µg oxacillin disk o 5µg methicillin disko “Cefoxitin” (30ug) disc diffusion tests are more reliable than those with oxacillin.o No special medium or incubation temperature is required.o Many different combinations of conditions have been recommended for disc diffusion.12/30/12 62
  • 63. Discs tests for methicillin Resistance Solid agar Media: • Iso-sensitest agar with the ‘1st line’ set of antibiotics, including 5ug methicillin disc. 30°C incubation for a full 24 hrs. • Use M-H agar at 35°C for 24 hrs. some batches of MH not perform reliably ( Qc with control strains) • Use M-H or Columbia agar added with 5% NaCl. Incubate at 35°C for 40hrs. • Use IST agar with a final salt conc. of 4%. Incubate at 30°C for 18-24 hrs. • Run a quality control batch with known sensitive & resistant strains. Ref : Mackie & McCartney 4th edition12/30/12 63
  • 64. Disc diffusion cont…. Interpretation: o Most true methicillin-/oxacillin-resistant isolates give no zone. ( Homo-resistance) o Sometime isolates give borderline suscp (small zones) due to hyper- production of B-lactamase resulting in inactivation of oxacillin. (sometimes give no zone and will therefore be falsely reported as MRSA.) o Heterogeneously resistant to penicillinase-stable penicillins, o Only 1 daughter cell out of 104 to 106 cells appears phenotypically resistant by routine antimicrobial susceptibility tests. o For this reason, methicillin or oxacillin resistance is not detected well by some susceptibility testing systems.12/30/12 64
  • 65. 12/30/12 65
  • 66. Interpretive Criteria (in mm) for Oxacillin Disk Diffusion Tests Susceptible Intermediate ResistantS. aureus ≥ 13 mm 11-12 mm ≤ 10 mm N/ACoNS ≥ 18 mm ≤ 17mm N/A = not applicable Interpretive Criteria (in mm) for Cefoxitin Disk Diffusion Test Susceptible* † ResistantS. aureus ≥ 20 mm ≤ 19 mmCoNS ≥ 25 mm ≤ 24 mm † There is no intermediate category with the cefoxitin disk diffusion test12/30/12 66
  • 67. ???? M-H agar plate Although the zone of inhibition is at the Left plate : A methicillin (oxacillin)- borderline for resistance (12 mm); the susceptible S. aureus (zone of inhibition presence of small colonies within the zone within susceptibility range) of inhibition (yellow arrows) indicates the presence of heteroresistant strains. Right plate : A methicillin (oxacillin)- The interpretation here, therefore, is resistant S. aureus (zone of inhibition is "methicillin-resistant" staphylococci less than the susceptibility range)12/30/12 67
  • 68. Chromogenic agar medias  Recently developed chrom agars with cefoxitin as a selective agent. o MRSA Select CHROMagar MRSA o MRSA ID Bio-Connections o Bio-Rad BioMerieux  Ciprofloxacin-resistant MRSA medium This medium shows improved specificity when compared with traditional media. Sensitivity is also improved but requires 48hrs incubation to achieve >85%.12/30/12 68
  • 69. MRSA on MRSA ID medium MRSA grow as distinct denim blue colonies (Oxoid Chromogenic MRSA Agar). CHROMagar™ Staph aureus •S.aureus – • mauve • Other bacteria – • blue, colorless or inhibited12/30/12 69
  • 70. Agar screening methodo The method recommended by the CLSI requires suspending the test organism to the density of a 0.5 McFarland standard (1.5x108) and inoculating an M-H agar containing 4% NaCl and 6 µg/ml oxacillin or10 µg/ml methicillino Inoculum preparation : o Overnight cultures on blood agar and turbidity adjusted to be equivalent to that of a 0.5 McFarland std (108 cfu/mL) in tryptone Soya broth.o The Inoculum is spotted on Plates incubated at 35°C for 24 h (additional 24 h if no growth)o Any growth other than a single colony is indicative of resistance. 12/30/12 70
  • 71. Enrichment• Enrichment of screening swabs is more sensitive than direct plating and may be particularly useful for screening in some high- risk groups of patients.• Enrichment media generally contain additional NaCl and may also contain methicillin, oxacillin & recently cefoxitin.• Broth bases used have included nutrient broth, tryptone Soya broth, MH broth, BHI broth and Robertsons cooked meat medium.• Indicator-enrichment media (broth containing carbohydrate, indicator and antibiotics) may be useful in providing an indication of the presence of MRSA after overnight incubation.12/30/12 71
  • 72. MIC by E-test method• It gives an MIC result and is affected by test conditions in a similar way to other MIC and diffusion methods• It requires MH agar with 2% NaCl• An inoculum density equivalent to 0.5–1.0 McFarland standards, application of inoculum with a swab & incubation at 35°C for 24 h.• The Etest has an advantage over other MIC methods in that it is as easy to set up as a disc diffusion test. – MRSA the oxacillin MIC is ≥ 16 ug/ml – MSSA isolates the MIC is ≤ 1 ug /ml – Cefoxitin Etest MICs for mec A-positive strains are ≥ 16 ug/ml , while cefoxitin Etest MICs for mec A-negative strains are ≤ 4 ug/ml12/30/12 72
  • 73. 12/30/12 73
  • 74. Automated MethodsAutomated systems commonly used in North America.  Vitek/Vitek2 (BioMérieux)  Phoenix (Becton Dickinson) &  Baxter Microscan (Dade Behring)  All systems include methods based on the principle of micro broth dilution tests for methicillin/oxacillin susceptibility and are generally reported to be reliable for S. aureus although a small number of incorrect results, mostly false resistance, have been reported.12/30/12 74
  • 75. 12/30/12 75
  • 76. Quenching fluorescence method With the Crystal MRSA method (Becton Dickinson) inhibition of growth of an isolate by oxacillin is indicated by the quenching of fluorescence of an oxygen-sensitive fluorescent indicator by oxygen remaining in the broth. In the presence of an actively metabolizing organism oxygen is consumed, which allows the fluorescence to be observed under a UV light source. The method is reasonably reliable but requires several hours of incubation i.e. 4 hrs @ 35°C.12/30/12 76
  • 77. Serological Identification Latex agglutination: Commercially available as a kit from several suppliers Principle: o Based on detection of PBP2a o Extraction of PBP2a from suspensions of colonies & detection of the protein by agglutination with latex particles coated with monoclonal antibodies to PBP2a. o Isolates producing small amounts of PBP2a may give weak agglutination reactions or agglutinate slowly. o Reactions tend to be stronger if PBPa production is induced by growth of MRSA in the presence of a penicillin. o Rare isolates may give negative reactions.12/30/12 77
  • 78. Molecular Methods • Resistance to penicillinase-resistant penicillin is generally related to the presence of the “mecA gene”. A. Nucleic acid hybridization • Earliest Molecular Probe Methods: – P.F.G.E. – Radiolabelled (P32, I125) OR – Digoxigenin (DIG)-labelled DNA probes B. Amplification Methods: • PCR Assays: • Real-Time PCR -Addition of single set of Primers. • Gold standard method • Determine staphylococcal chromosomal cassette carrying the mecA methicillin-resistant gene (SCCmec) type •12/30/12 78
  • 79. Multiplex PCR and gel electrophoresis for identifyingthe mecA gene in Staph. species.Two distinct polymerase chain reactions areperformed: one amplifies a portion of the 16Sribosomal RNA gene unique to Staphylococcusspecies, and the second amplifies a segment of themecA gene. Lane 1: a strain of methicillin-resistant S. aureus isidentified; electrophoretic bands of appropriate sizeappear for the 16S ribosomal DNA (rDNA) andmecA. Lane 2: a methicillin- susceptible S. aureus isidentified; an electrophoretic band for 16S ribosomalDNA is present, but there is no band for mecA.Lane 3: is a negative control (reagents only), andLane 4: contains DNA fragment standards. Bp =base pair.12/30/12 79
  • 80. Direct identification of MRSA in blood cultures Branched DNA Method:• A commercially available non-PCR-based method (Chiron Diagnostics, East Wampole)• Detecting the mecA gene directly from blood cultures containing Staphylococcus species.• Amplification of a signal & not target nucleic acid.• Signal generation is proportional to the amount of mecA gene sequences in the sample, which in turn is proportional to the number of staphylococcal cells used in the assay. 12/30/12 80
  • 81. DNA is released from staphylococcal cells and hybridized to both capture probes andtarget probes. bDNA molecules (amplifiers) are then hybridized to target probes.Enzyme-labeled probes are subsequently hybridized to the bDNA amplifier. Achemiluminescent substrate dioxetane is added, and emitted light is measured.12/30/12 81
  • 82. Identification of MRSA in endotracheal aspirates and other clinical samples• Rapid and specific detection of MRSA colonization of the upper and lower respiratory tract is of particular importance for critically ill mechanically-ventilated patients in intensive care units.• A 6 h multiplex PCR procedure (targeting the femA and mecA genes) has been used successfully to identify MRSA in endotracheal aspirates from mechanically-ventilated patients12/30/12 82
  • 83. Clinical Manifestations of MRSA Infection1. A localized, superficial abscess or – Skin/soft tissue infection – Pneumonia1. Invasion of lymphatic, blood, and major organs – Endocarditis – Bone infection – bloodstream infections – pneumonia – meningitis – toxic shock syndrome – surgical wound infection12/30/12 83
  • 84. How do MRSA infections present?12/30/12 84
  • 85. Superficial Infections12/30/12 85
  • 86. Scalded Skin Syndrome: Classic Toxic Shock www.aafp.org/afp/ 20000815/804.html12/30/12 86
  • 87. Systemic S aureus In the Lower spine » .12/30/12 87
  • 88. Systemic Menstrual Toxic Shock By MRSA• Most major organs fail with disseminated MRSA (TSS-1)www.web.net/terrafemme/ cashnightmare.htm12/30/12 88
  • 89. Approach to Therapy• Principles of management: • Obtain specimens for culture of all skin infections • Drain purulent collections and provide thorough wound care • Know the organism resistance patterns for the community • Make reliable plans for reassessment12/30/12 89
  • 90. Approach to Therapy• Selecting antibiotic therapy: • Is the infection suspected to severe or non severe? • What can I empirically treat with until I have culture-proven MRSA?12/30/12 90
  • 91. Approach to Treatment• Empiric treatment of non-severe illness (outpatient tx): • dicloxacillin, cephalexin, augmentin or clindamycin • bactrim - avoid if suspecting skin infection by other pathogens, specifically group A Streptococcus• Culture-proven MRSA: • Clindamycin if D-test is negative, bactrim, minocycline and linezolid 12/30/12 91
  • 92. Biotechnology: Current Drug Treatments For MRSA• MRSA Drugs of Choice:• Linezolid-Protein synthesis inhibitor Daptomycin- Causes membrane depolarization in bacteria-so no membrane transport• Vancomycin-Acts by interfering with the construction of cell wall. Still works well with other antibiotics• Alternatives: Synercid, Rifampin• Third-Line agents: TMP-SMX (Sulfa)12/30/12 92
  • 93. Biotechnology: Drugs In Development• Oritavancin-Binds to normal cell wall precursors• Tigecyclin-Works on efflux pumps• Dalbavancin- Bacteriacidal12/30/12 93
  • 94. Biotechnology: A Novel Vaccination For S. aureus• Development of StaphVAX®, a polysaccharide conjugate vaccine against S. aureus infections• The results of the phase 3 clinical trials of the vaccine (Staph VAX) will be presented 2006 according to the NIH.12/30/12 94
  • 95. Prevention and Control in Hospitals12/30/12 95
  • 96. Standard precautions• Hand hygiene• PPE• Laundry/linen• Waste disposal• Cleaning/disinfection• Respiratory hygiene/cough etiquette• Safe injection practices12/30/12 96
  • 97. Prevention • Draining infections must be kept covered • Talk to your physician about wound management techniques • Wash hands frequently with soap and water • Avoid sharing personal items • Wipe objects down with alcohol. • Advise health care workers to wash their hands before touching you or your hospital equipment12/30/12 97
  • 98. Prevention• 57% of patients receive inappropriate antiobiotic treatment of the index MRSA infection• “Good judgment comes from experience, and a lot of that comes from bad judgment.” Will Rogers12/30/12 98
  • 99. Prevention• Surveillance cultures• Barrier precautions• Hand washing• Single bed facilities• Isolation of infected patients 12/30/12 99
  • 100. How do we control MRSA? • Hospitals: – Infection control!!! – Antibiotic control?? • Community: – ?????12/30/12 100
  • 101. Inappropriate Use of Antibiotics• Encourage over growth of resistant organisms – now have access to nutrients and space• MRSA infections associated with B-lactam and quinolones allow rapid proliferation of an organism that may have been merely colonizing12/30/12 101
  • 102. Changes In Practice • Must rein in use of antibiotics • 55% of all RX for URI in US unnecessary • Avoid broad spectrum antibiotics • How long to treat? • Decrease antibiotic use in agricultural industry.12/30/12 102
  • 103. Control of MRSA in hospitals1. Isolate patient who is colonized or infected in a private room. Cohort if no private room available2. Contact precautions: wear gown, gloves upon entry to the room.3. Hand Hygiene4. Dedicated equipment, decontamination of environment5. Screening for carriage: not universal6. Decolonization with mupirocin, chlorhexidine wash: uncommon7. Use Device bundles: CVC bundles, pneumonia bundle8. Leadership and organizational culture.12/30/12 103
  • 104. Antibiotic pressureColonized/infected patients Susceptible patients “RESERVOIR” LONG TERM CARE12/30/12 Community 104
  • 105. Transmission in Healthcare• Patients colonized and infected with MRSA are the reservoir• Transmitted from one patient to another via HANDS of healthcare worker (HCW) due to transient colonization• Contaminated environmental surfaces also source of transmission but less important than hands of HCW12/30/12 105
  • 106. Clinical cultures focus here MRSA infected MRSA colonized We continue to ignore a major source of the problem12/30/12 106
  • 107. Invasive MRSA In the US• The estimated number of people developing serious infection (i.e. invasive) in 2005 was estimated to be 94,360 infections• Approximately 18,650person died during their hospitalstay related to these serious infections 86% 14%• Overall rates of disease werehighest – among persons (aged >65), – black, – males Community-Associated• Most of the infections are Healthcare-Associatedhealthcare related12/30/12 Klevens et al JAMA 2007;298:1763-71 107
  • 108. That’s All Folks!! Any Questions???? Staph cells attaching photo courtesy of Dr. Sharon Peacock- University of Oxford12/30/12 108
  • 109. References• 1 Mitchell, David.MRSA.”what’s New”. Inoculum. Volume 8, number 2 (1999) 1-12.• 2 textbookofbacteriology.net/resantimicrobial.html• 3 healthsciences.columbia.edu/ dept/ps/2007/mid/2006/transcript_02_mid22.pdf• 4 http://www.bioteach.ubc.ca/Biodiversity/AttackOfTheSuperbugs• 5. Foster, Timothy. The staphylococcus aureus “superbug”.J. clin Ivestigation• Volume number114 (2004) 1693-1696.• 6. www.channing.harvard.edu/4a.htm• 7. ww.ncbi.nlm.nih.gov.• 8. www.aafp.org/afp/ 20000815/804.html• 9. Journal of Clinical Microbiology, June 2000, p. 2378-2380, Vol. 38, No. 6 0095-1137/04.00+0• 10. www.FDA.com (FDA archives)• 11.www.postgradmed.com/issues/2001/10_01/hoel.htm 12. www.cdc.gov/ncidod/hip/aresist/mrsa_CDCactions.htm• 13. www.medscape.com• 14 http://www.nabi.com/images/factsheets/fsStaphVAX.pdf12/30/12 109
  • 110. 12/30/12 110
  • 111. MRSA Infection Control Strategies • contact precautions • screening • decolonization
  • 112. The End

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