Mrsa molecular part


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Mrsa molecular part

  1. 1. MRSA Molecular Part Hafez Alsumairi SUPERVISED BY Prof Dr Ola Abdelkhader12/30/12 1
  2. 2. Genome of S. aureus• 2.8 Mega-base pairs long with• Approximately 2,600 open reading frames, comprising 84.5% of the genome.• It has a G+C content of about 33%. – A- Core genome: 75% , highly conserved. – B- Accessory genome: 25% , MGE12/30/12 2
  3. 3. Virulence factors1. Phages2. Plasmids3. Genomic islands4. Pathogenicy islands5. Staphylococcal cassette chromosome (scc)6. Transposon7. RNA transcripts12/30/12 3
  4. 4. Evolution1. Mutation2. Acquisition of a resistance gene ( mecA)12/30/12 4
  5. 5. – Horizontally transferred DNA element - SCCmec.– Site specific recombination.– Integration of SCCmec is sequence specific at a unique site (attBSCC) located near the S. aureus origin of replication– mecA gene encodes PBP2a.– PBP2a = 78 KDa PBP - capable of cell wall synthesis.– PBP2a has low affinity for all -lactams.– Target Change 12/30/12 5
  6. 6. MRSA• Horizontal gene transfer of mecA. Expresses a modified penicillin-binding protein (PBP2a) with low affinity to methicillin• Penicillin-binding proteins – PBPs are membrane bound DD-peptidases – PBP1-4, involved in the cell wall peptidoglycan assembly as transpeptidases• PBP2 is a bifunctional protein which, in addition to transpeptidase activity,12/30/12 6
  7. 7. mecA1. The origins of mec are obscure2. Single clonal origin theory3. Hiramatsu et al. 1996: Clonal diversity: different strains developed independently4. Origin of mecA gene - horizontal transfer from: – SCN – S. scuiri – Enterococcus hiriae12/30/12 7
  8. 8. Regulation of mecA1. The mecR1 gene encodes a transmembrane inducer of mecA2. The mecI gene encodes a strong repressor of mecA 12/30/12 8
  9. 9. mec-associated DNA• MecA, mecR1, and mecI are encoded by approximately 5 kb of DNA• up to 100 open reading frames• Transposons and insertion sequences are present, including – Tn554 (which contains ermA, the gene encoding inducible erythromycin resistance), located 5 of mecA, and – one to four copies of IS431, – at least one of which, IS431mec, is located 3 of mecA12/30/12 9
  10. 10. 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)12/30/1212/30/12 10 10
  11. 11. SCCmec cassetteTypeSCCmec may be classified into eight major types by combining the class of the mec gene complex with the ccr gene designated by Roman numerals (I-VIII).Common Characteristics1. Carry the mec gene complex (mec);2. Carry the ccr gene complex (ccr);3. Flanked by both inverted repeats and direct repeats; and4. Integrated at the 3′-end of orfX.In the two essential components, mec and ccr, five classes of mec and five types of ccr have been identified12/30/12 11
  12. 12. SCCmec type: defined by the combination of mec and ccr SCCmec type Combination of ccr and mec Type I ccrA1 and B1 and class B mec (1B) Type II ccrA2 and B2 and class A mec (2A) Type III ccrA3 and B3 and class A mec (3A) Type IV ccrA2 and B2 and class B mec (2B) Type V ccrC and class C mec () Type VI ccrA4 and B4 and class B mec (4B) Type VII ccr C2 and C8 and class C1 or C2 Type VIII ccr AB4 and class A (4A)12/30/12 12
  13. 13. 12/30/12 13
  14. 14. SCCmec cassette• SCCmec elements may be further classified into subtypes according to differences in their junkyard regions (J region) DNA• orfX -J3 -mec -J2-ccr -J1- right-flanking chromosomal region12/30/12 14
  15. 15. SCCmec cassette• HA-MRSA (I-III) – Microbiological resistance is via SCCmec III. The virulence factor is unknown and multiple clones exist.• CA-MRSA (IV-V) – Microbiological resistance via SCCmec IV. The prevalence factor is PVL (panton-valentine leukocidin) and two major clones are identified12/30/12 15
  16. 16. Laboratory diagnosis Molecular tests1. Rapid diagnosis2. Cost effective3. Highly specific4. Improved patient care5. Provides ultimate resolution6. No outside interference from medication7. Limits unnecessary preemptive isolation8. Limits unnecessary antibiotic use9. Identifies MRSA in mixed populations10. Methicillin resistant coagulase negative Staph11. mecA negative Staph12. Directly detect the presence of MRSA in patient specimens. These assays are currently approved by the FDA.13. Equivocal results12/30/12 16
  17. 17. MRSA PCR Procedure1. Specimen preparation2. Concentration3. Lysis-DNA extraction4. Reconstitution of molecular reagents5. Real-time PCR6. Automated results7. Full run time < 2 hours 12/30/12 17
  18. 18. Molecular methods:• PCR based with the using an internal control – Targets • nuclease (nuc), • coagulase (coa), • protein A (spa), • femA and femB, • 16S rRNA and • surface-associated fibrinogen-binding protein genes12/30/12 18
  19. 19. Current MRSA typing• MRSA is a clonal organism• Multiplex PCR – Very useful for epidemiology Strain types between patients – 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 19
  20. 20. Sequence-Based Bacterial Genome Typing• Multilocus sequence type (ST),• Single-locus sequence typing• The accessory gene regulator (agr) type• SCCmec type• Pulsed field gel electrophoresis (PFGE)12/30/12 20
  21. 21. References•• Mitchell, David.MRSA.”what’s New”. Inoculum. Volume 8, number 2 (1999) 1-12.•• dept/ps/2007/mid/2006/transcript_02_mid22.pdf•• Foster, Timothy. The staphylococcus aureus “superbug”.J. clin Ivestigation Volume number114 (2004) 1693-1696.••• 20000815/804.html• Journal of Clinical Microbiology, June 2000, p. 2378-2380, Vol. 38, No. 6 0095 1137/04.00+0• (FDA archives)•••• http://www12/30/12 21