Staphylococcus aureus : The Superbug

2. Diagnosis of Methicillin Resistant Staphylococcus aureus (MRSA)&Methicillin Susceptible Staphylococcus aureus(MSSA)

4. Staphylococcus aureus: The Superbug

6. Introduction • Staphylococcus aureus has been reported as a major cause of community and hospital acquired infection .

7. Continue… • Methicillin-resistant S. aureus (MRSA) were first reported in 1961 in UK and have since become a major nosocomial pathogen worldwide. • Treatment of infection caused by MRSA has become problematic.

8. Continue… Indiscriminate use of multiple antibiotics especially in developing countries, prolonged hospital stay .intravenous drug abuse ,carriage of MRSA in nose are few implicated risks factors for MRSA actuation.

9. Evolution of Antimicrobial Resistance Penicillin  (1950s) Methicillin  Penicillin-resistant S. aureus Methicillin-resistant S. aureus (MRSA) S. aureus (1960s) Vancomycin (1997) Vancomycin - intermediate resistant S. aureus Vancomycin- Resistant S. aureus (2002)

10. A brief history of vancomycin resistance

11. Epidemiology of MRSA • Overall 20% of S.aureusin Europe are MRSA • In USA hospitals the prevalence of MRSA ranges from 33% to 55%

12. Epidemiology of MRSA In Asian countries : In Taiwan prevalence of MRSA increased 26% in 1986 to 77% in 2001 In Korrean hospitals the prevalence of MRSA was 64% between 1999-2000

13. Epidemiology of MRSAStefania 2012 IJAA

14. The prevalence of MRSA from different studies in Iran

15. MRSA MRSAs are those strains of S. aureus that express mecA Another mechanism of methicillin resistance, such as changes in affinity of penicillin-binding proteins for oxacillin

19. Hospital Acquired-MRSA (HA-MRSA) Community Acquired-MRSA (CA-MRSA)

20. Methicillin (oxacillin ) susceptibility testing • Microbiology laboratories have an important role in detection of MRSA. There are two major methods for detection of MRSA in laboratory. • Phenotypic Methods • Genotypic Methods.

21. Phenotypic Methods for detection of MRSA • Disk diffusion Methods ( Oxacillin and Cefoxitin) • Dilution methods ( for MIC) • E-test method • Agar screening method • Latex agglutination method • MRSA Choromoagar • Automated methods.

22. Genotypic Methods • Accurate routine phenotypic detection of MRSA is difficult by using standard phenotypic methods. This has been ascribed to the heterogeneous expression of Methicillin resistance in many strains of S. aureus. • For this reason detection of mecA or PBP2a by molecular method is very important. Using of PCR for detection of mecAis gold standard method for detection of MRSA

23. Why are oxacillin and cefoxitin tested instead of methicillin Cefoxitin is an even better indicator of the mecA gene, and disk diffusion tests Using cefoxitin gives clearer end points and are easier to read than tests with oxacillin

24. Oxacillin based methods • MICs Methods : • Microdilusion or E-test • Oxacillin Screen Agar • Oxacillin disk Diffusion (Cefoxitin)

25. Continue.. The addition of NaCl ( 4%) is required for both agar and broth dilution testing of oxacillin to improve the detection heterogeneous MRSA. For disk diffusion testing MHA should not be supplemented

26. Cefoxitin –Based Methods The results of tests using Cefoxitin (either broth microdilution or disk diffusion tests using a 30µg Cefoxitin disk).

27. Cefoxitin Forcoagulase-negative staphylococci currently only the cefoxitin disk diffusion test has been validated for predication of mecA –mediated resistance. For disk diffusion testing of S.lugdunensis, only cefoxitin disk should be used.

28. Vancomycin resistance in S. aureus VISA (vancomycin intermediate S. aureus) • First case 1996 (VISA) • MIC 4-8µg/ml • Thick cell wall (reduces vanco penetration through cell wall). • Accumulates multiple mutations that activate pathways for cell wall synthesis & change cell physiology

29. Testing Recommendations Disk diffusion is not recommended for testing vancomycin susceptibility in S. aureus • VRSA isolates are detected by : • Broth microdilution • E-test • Agar dilution • Vancomycin screen agar plates [Brain Heart Infusion (BHI) agar containing 6 µg/ml of vancomycin].

30. Screening of VRSA • Vancomycin Agar Screen Plates • The vancomycin agar screen test uses commercially prepared plates containing brain heart infusion (BHI) agar and 6 μg/ml of vancomycin to screen pure cultures of bacteria for vancomycin resistance.

32. 1- Journal of Clinical Microbiology November 2012 Volume 50 Number 11 (ISI) 2- Cogent Medicine (2016), 3: 1163768 (Pubmed) 3-Asian Pac J Trop Dis 2016; 6(9): 726-731 (Pubmed) 4-Oman Medical Journal (2014) Vol. 29, No. 5:335-339 (Pubmed)

34. دو خوشه ژنی مهم درsccmec : mec-gene کمپلکس و cassette chromosome recombinase (ccr) -gene complex میباشند این نواحی بزرگ DNA بطوراختصاصی درintegration site sequence (ISS) درانتهای 3پریم orfX قراردارند وبه آنها کاست DNA گویند این نواحی بنام staphylococcal cassette chromosome mec یا SCCmec شناسایی میشوند.

35. نواحی اطراف دو خوشه ژنی را نواحی J( برگرفته از ‘junkyard) گویند و سکانس آن برای ساب تایپینگ تیپهای SCCmec بکار میرود. SCCmec بوسیله توالیهای تکراری معکوس اختصاصی وتکرارهای مستقیم که حاوی integration site sequence فعال شونده توسط CCR میباشند محدود میشود درنتیجه اینتگریشن SCC element بوسیله یک جفت سکانس direct repeat (DR) که هردو جایگاههای اینتگریشن برای SCC element بعدی را تشکیل میدهند احاطه میشود

36. SCCmec type با ترکیبی از ccr-gene complex و کلاس mec-gene complex شناسایی میشود. تابحال 11تیپ SCCmec شناسایی شده واحتمالا درآینده بیشتر هم خواهد شد تیپهای I-III، SCCmec type های قدیمی هستند که توسط HA-MRSA حمل میشدند که اندازه بزرگی دارند وچندین شاخص مقاومت آنتی بیوتیکی دارند تیپهای IV , V نسخه های جدیدند که توسط CA-MRSA حمل میشوند کوتاهند ویطور معمول جزmec-gene complex ژن مقاومت آنتی بیوتیکی دیگری را حمل نمیکنند

37. اینتگریشن SCCmec بداخل oriC environ کروموزوم MSSA سبب بوجود آمدن MRSA میشود هرکلون MRSA میتواند براساس ترکیبی از ژنوتیپ MSSA پذیرنده و ژنوتیپ SCCmec اینتگره شده مشخص شود(clonotype) تیپهای 4و5 SCCmec از 3تیپ اولیه که بیمارستانی بودند با ترکیبی از mec-gene complex و ccr-gene complex افتراق داده میشوند سویه های CA-MRSAtype-V SCCmecو-IV را حمل کرده رشد سریعتری دارند بیماریزایی بیشتری دارند وبه کلاسهای انتی بیوتیکی کمتری مقاومند ودرجه پایین تری از مقاومت را به بتالاکتام نشان میدهند(مقاومت هتروژنوس به متیسیلین دارند)