1. Current issues in Vancomycin-resistant enterococci Wee Gyo Lee, MD, PhD Department of Laboratory Medicine, Ajou University School of Medicine, Suwon, Korea

2. Background Mechanisms ▫Biochemical ▫Genetic Epidemiology ▫Global ▫Molecular Current issues

3. Background Enterococci: - major nosocomial pathogens - Vancomycin-resistant enterococci (VRE) 1986, first VRE in France ≥ 15 - 20% of enterococci - exchange genetic information among themselves and with other genera - in 2002, VRSA with vanA gene  11 cases

4. Biochemical Mechanism Alternative pathway for peptidoglycan synthesis 1. Synthesis of low-affinity precursors: D-Ala-D-Lac or D-Ala-D-Ser 2. Elimination of precursors normally produced in the chromosome by the host

5. Cell membrane Cell membrane VanSVanS Vancomycin ATP PP ADPVanRVanR ORF1ORF1 ORF2ORF2vanRvanRvanSvanSvanHvanHvanAvanAvanXvanXvanYvanYvanZvanZ Lactate D-Ala-D-Lac UDP-MurNAc-L-Ala-D-Glu-L-Lys- vanH vanA vanX Pyruvate D-AlaD-Ala-D-Ala Cell wall synthesis

6. Genetic Mechanism

7. Global Epidemiology Differences between Europe & USA Europe ▫widespread use of avoparcin (VM analogue) ▫High VRE colonisation rate: pigs, calves, turkeys ▫Asymptomatic carriage among healthy people is common ▫Only a few nosocomial outbreaks have been reported

8. USA ▫Glycopeptides: never been used in the farm animals industry ▫VRE colonisation among healthy people is extremely rare. ▫VRE appear to have first emerged as nosocomial infection. ▫The greater use of vancomycin in hospitals ▫The establishment of VRE endemicity  Outbreaks of single strain  monoclonal nature  multiple enterococcal strains  polyclonal endemicity

9. Korea ▫widespread use of avoparcin (VM analogue) ▫VRE colonisation in animals is common. ▫No transmission of VRE between human & poultry ▫Rare reservoir of VRE among healthy people ▫The spread of VRE: similarly to USA ▫The greater use of vancomycin ▫Polyclonal endemic state in Korea

10. ChromosomeTn1546 with vanA gene cluster Tn typing PFGE, MLST, MLVA Molecular Epidemiology

11. Transposon typing ▫vanA gene cluster is carried as a part of Tn1546 ▫Analysis of genetic variation of Tn1546 ▫Majority of the variations – integrations of insertion sequences with or without a deletion at the insertion site ▫Horizontal transfer Strain typing ▫PFGE, MLST, MLVA ▫Clonal spread, genetic evolution

12. Strain Typing MLST (Multilocus sequence typing) ▫Identifying alleles from DNA sequences of internal fragments of 7 house keeping genes ▫Allellic profile:  the alleles at each of the seven housekeeping genes  E. faecium 1-2-3-3-2-2-1 ▫Sequence type (ST):  Defined by the allelic profile.  ST36: 1-2-3-3-2-2-1 ▫Objective & readily comparable between Lab.

13. E. faeicum ▫adk (adenylate kinase) ▫atpA (ATP synthase) ▫ddl (D-alanine-D-alanine ligase) ▫gyd (glyceraldehyde-3-phosphate dehydrogenase) ▫gdh (glucose-6-phosphate dehydrogenase) ▫purK (phosphoribosylaminoimidazol carboxylase ATPase subunit) ▫pstS (phosphate ATP-binding cassette transporter)

19. MLST of VRE in the world

20. Current Issues Clonal Complex 17 ▫“Epidemic” genetic complex-17 : derived from secondary founder ST-17 ▫Epidemic-virulent, hospital-adapted strains ▫Ampicillin resistance (MICs ≥ 16mg/l): uniformly ▫Higher levels of quinolone resistance ▫Presence of the E. feacium pathogenicity island carrying the esp gene: majority ▫Either resistant to glycopeptides  Such strains were already spread before they acquired the van gene

21. “Genetic capitalism” – hospital adapted strain capable of surviving in hospital settings 1 st : ampicillin R – selective advantage in hospitals  acquisition of PAI – facilitated transmission  horizontal transfer of vanA/B genes  has emerged on several continents

22. 1, animal isolates; 2, human community surveillance isolates; 3, surveillance (feces); 4, clinical isolates; 5, isolates from documented hospital outbreaks Global distribution of complex-17 isolates

23. Clonal complex-17 VRE : Ko. JCM 43, 2005 Lee. Letters in AM 43, 2006 국내 E. faecium 분포

24. VanB phenotype-vanA genotype Impaired resistance to Teicoplanin (I or S) Mechanism: still not clear 1. Point mutations of vanS - Three point mutations in the putative sensor domain of vanS 2. Lacked vanY or vanZ - not affected to Vm resistance - vanZ: required for Teicoplanin resistance

25. East asian area: Japan, Korea, & Taiwan Japan & Taiwan ▫First reported by Hashimoto et al. 2000, Japan. ▫In Japan & Taiwan: mutation of vanS ▫Gu et al. 2009, Taiwan, 52.2% of VRE  No mutation of vanS  Impairment of vanY/vanZ  MLST: different genetic background? Korea ▫Eom et al 2004: mutation of vanS ▫Lee et al 2004, Song et al 2006: Impairment of vanY/vanZ Recently, increased prevalence

26. VanC VRE with vanA gene VanC VRE ▫Intrinsic constitutive resistance ▫Species-specific vanC1 : E.gallinarum vanC2 : E.casseliflavus vanC3 : E.flavescens ▫Located in the chromosome ▫Peptidoglycan precursors : D-Ala-D-Ser ▫MIC : Vm 2-32 mg/L, Te  1 mg/L

27. Clinical significance Not required special infection control measures Emerging vanA containing VanC VRE ▫VanA phenotype ▫Potential reservoirs of transmissible van genes ▫Require infection control ▫Genotypic testing

28. Thank You