1. Lecture 4 Vancomycin resistance VRE VISA / hVISA / VRSA Thursday – 1/17/2008

2. Enterococcus Gr+ Cocci (in chains). Two species infecting human: E. faecium and E. faecalis. Initially considered harmless GI commensal. Infection typically follows GI colonization. Low pathogenicity Yet, can cause high mortality in patients with bacteremia.

3. Enterococcus infections E. faecalis vs. E. faecium Nosocomial infections, mostly in debilitated patients Common cause of nosocomial urinary tract infection Currently 3 rd leading cause of bloodstream infections. Serious complication: endocarditis

4. Nosocomial transmission Mode of transmission: –HCW –Environmental sources (medical devices) Carriage: –GI tract –Duration: months - years

5. Enterococcus and antibiotic resistance Enterococci intrinsically resistant to various antimicrobial classes –Low level aminoglycoside (low ability to penetrate cell wall) –Relative resistance to β-lactams (Cephalosporines + penicillin) (PBP5) –macrolides (low level) –TMP-SMX Acquire high level antibiotic resistance through horizontal transfer of relevant genes. 1980’s: emergence of beta-lactam and high gentamycin resistance. 1986 fist report of VRE in Europe, 1989 first report in US. Currently: alarming situation in US hospitals, rare in Europe

6. Glycopeptides vancomycin, teicoplanin, telavancin Antibiotic class used to treat Gram positive bacteria. Mode of action: disruption of peptidoglycan polymerization Bind to the amino-acids within the cell wall. (D-ala-D-ala). Prevent the addition of new units to the peptidoglycan.

7. VRE - mechanism of resistance 1.Modifying enzymes 2.Degrading enzymes 3.Target Change 4.Efflux pumps

8. Cell wall synthesis in enterococci:

9. Mechanism of resistance Operons that encode enzymes for –synthesis of low-affinity precursors (D-ala-D-lac vs. D-ala-D-ala). –Elimination of normal high-affinity precursors (removing the vancomycin-binding target) Operon encoded on a transposon VanA operon

10. Glycopeptide resistance gene operons: vanA-vanE and vanG Enterococci – acquisition of VanA, B, D, E and G phenotypes. VanC – affords intrinsic resistance, and is chromosomal (arises in less virulent enterococci). VanD, E, G: reported only sporadically. VanC and VanE: D-Ala-D-Ser VanA & VanD: resistant to vancomycin + teicoplanin. VanA & VanB: most common The phenotype is accomplished by multiple proteins in gene clusters.

11. The vanA gene cluster Adapted from Courvalin et al. 2006 vanA (ligase) and vanH (dehydrogenase) are responsible for the synthesis of the modified depsipeptide (D-Ala-D-Lac). vanX (D,D-Dipeptidase) and vanY (D,D-Carboxypeptidase) cleave normal peptidoglycan substrates.

12. VanA type: inducible high levels of vanco-R and teicoplanin-R (Tn1546) –VanH - dehydrogenase (pyruvate->lactate) –VanA - ligase catalyses bond of D-Ala-D-Lac. –VanX and VanY - remove c-terminal D-Ala to eliminate normal precursor VanB different in its regulation (and no vanZ, additional vanW - function unknown).

13. vanB operon Phenotype: Acquired inducible resistance to various levels of vancomycin and susceptible to teicoplanin Cluster of genes: vanR B, vanS B, vanY B, vanW, vanH B, vanB and vanX B. Operon located on transposons: Tn1547, Tn1549, Tn5382. Most cases chromosomally located on transposon (~65Kbp). Or transposon carried on plasmid. Has been described in other Streptococcal sp. and gut anaerobes.

14. Transformation Plasmid transfer Genetic Mechanisms of Resistance Acquisition Mutation

15. Genetic Mechanisms of VRE Resistant genes clustered in operons Operons located on transposons Transposons transmitted – Directly to chromosome – via plasmids

16. Rapid spread of VRE Tn1546 Transposon (vanA) A small mobile genetic element (6625 bp) More mobile than a plasmid Plasmids that carry Tn1546 - highly efficient conjugative plasmids (~65 Kbp)

17. Virulence genes associated with VRE Enterococcal surface protein variant (esp) hyl EFM

18. Variant esp gene (esp - / esp Efm ) enterococcal surface protein Enhanced adherence Associated with hospital infections (VREF-100% and VSEF~50%). Prevalent in E. faecalis strains related with infections. Absent in community isolates. Not more virulent in mouse model.

19. Another virulent factor: hyl Efm Significant identity with hyaluronidase genes Hyaluronidase: a virulent factor in S. aureus, S. pneumoniae, GAS Predominant in VREF strains Associated with esp Efm

20. Epidemiology of VRE infections

21. Prevalence of VRE among enterococci in nosocomial infections in ICU patients Bonten et al. Lancet Infect. Dis. 2001

22. Vancomycin-resistant enterococci (VRE) E. faecium Rare, but emerging cause of infection (15% ->~30%) Most (>90%) are VRE (VREF) Colonization of hospitalized patients 1.5%-32% E. faecalis Common cause of infection (>90% ->70%). But most not VRE.

23. VRE: European vs. US epidemiology 1986 - first case. Uncommon as nosocomial infection (<3%) only sporadic outbreaks Widely prevalent in European livestock and in healthy people in the community Inciting factor: animal use of glycopeptides –Avoparcin as a growth factor since 1970s, banned in 1997. 1989 – first case. Epidemic spread: –Small outbreaks –Northeast -> West coast –By 1995 – high endemicity in ICUs. Community reservoir – absent. Inciting factor: use of oral vancomycin. Europe USA

24. VRE: European vs. US epidemiology Sporadic nosocomial outbreaks. Highly prevalent in healthy humans and livestock. ‘Non-epidemic strains’. Do not have esp gene. Endemic in US ICUs No community reservoir ‘Epidemic strains’. Contain esp gene. Europe USA

25. Vancomycin use in US vs. Europe Bonten et al. Lancet Infect. Dis. 2001

26. Bloodstream isolates of VREF SENTRY antimicrobial surveillance program Deshpande et al. Diag. Microbiol. Infect Dis. 2007

27. Genetic capitalism (the rich become richer) The success of a highly adaptive clone. Leavis et al. Curr. Opion. Microbiol 2006

28. Emergence of CC17 in the Netherlands / Top et al. JCM 2008 Is the European epidemic following the US epidemic in a 10 y delay?

29. Factors influencing VRE spread: Patient colonization –GI tract –Groin –Skin Colonization pressure –Number/density of colonized patients –Admission/transfer of colonized patients –Proximity of colonized patients –Shared care givers Contaminated environment –Stethoscopes/ BP cuffs, etc.. Antibiotic pressure –Vanco –Cephalosporins –Antianaerobic Bacterial virulence factors –esp gene

30. Suggested strategies for IC of VRE / Bonten et al. Lancet Infec Dis 2001

31. Infection Control and/or Ab control When not clonal spread, strict IC - not efficient. Then probably resriction of Ab classes but which? Studies show: RF for VRE: –Vancomycin (IV - controversial) –Extended-spectrum cephalosporin –Other B-lactam-B-lactam inhibitors (controversial which) –Anti-anaerobic regimens.

32. Vancomycin resistant S. aureus (VRSA) Vancomycin intermediate S. aureus (VISA, hVISA)

33. Vancomycin resistance in S. aureus VISA (vancomycin intermediate S. aureus) First case 1996 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 High fitness cost

34. hetero-VISA (hVISA) hVISA (hetero-VISA) –Appear to be susceptible, but consists of subpopulations that have MIC≥4µg/ml –Difficult to detect.

35. VRSA - yet a very rare event 2002 Michigan – 1 st case vanA mediated since then 6 more cases. vanA resistance in VRSA rare, most occurred in the same geographical area (Michigan) Only very few descriptions of vanA gene cluster in MRSA, though in lab – years ago it was demonstrated. Gene cluster found on a plasmid-specified transposon (Tn1546)

36. Currently most worrisome: Concomitant carriage of VRE and MRSA is increasing.