Heterogeneity of VanA phenotype glycopeptide-resistant enterococci in Scotland
Brown, Alan Reid
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Although traditionally perceived as being of little clinical significance, enterococci have emerged as major nosocomial pathogens, primarily due to the emergence of multi-drug resistant strains. In cases of serious enterococcal infection, the optimal therapeutic option is the synergistic combination of a cell wall-active agent with an aminoglycoside. By the late 1980s, high-level resistance to both penicillin and the aminoglycosides was increasing in prevalence amongst clinical enterococcal isolates, thus compromising this synergistic combination. It was the emergence of glycopeptide resistance in the late 1980s, however, that truly rendered some strains untreatable by proven therapeutic agents.There are currently five distinct phenotypes of glycopeptide resistance in enterococci, the most prominent being VanA, which is characterized by high-level, inducible resistance to vancomycin and teicoplanin, and is conferred by the Tnirelated transposon, Tn1546. Whilst glycopeptide-resistant enterococci (GRE) are a significant nosocomial pathogen, their epidemiology is extremely complex and is not restricted to the hospital setting. In Europe, GRE can be readily isolated from a variety of environmental and animal sources, as well as from healthy nonhospitalized individuals.The aim of this study was to investigate the epidemiology of VanA GRE in Scotland, and to determine the degree of diversity amongst the Tn/54d-related elements conferring resistance. GRE were collected over a five-year period from eight hospitals across Scotland. Fifty-five GRE isolates were collected in total, 48 of which were found to be vanA-positive. In addition, a five-month survey of enterococcal epidemiology was performed, during which time 94 enterococcal isolates were collected, two of which were found to be v<m4-positive.All isolates were examined by PFGE analysis. Twenty-six of the 55 nosocomial GRE isolates collected during the five-year study period were isolated during the course of an outbreak of GRE infection in the renal unit of the Royal Infirmary of Edinburgh in 1995. Many isolates arising from the outbreak were clonal in nature, with an outbreak strain of VanA E. faecium being identified. However, outwith the outbreak situation, GRE isolates were more diverse in nature, with only small clusters of related isolates being identified, and many deemed to be unrelated. PFGE analysis of the 94 isolates from the five-month study of enterococcal epidemiology revealed a heterogeneous enterococcal population, with only small clusters of related isolates recognized. Whilst the two VanA isolates arising from this study were unrelated to the nosocomial strains, some glycopeptide-sensitive enterococci were shown to be related to nosocomial GRE isolates.Despite the ready transfer of glycopeptide resistance from the majority of the 48 nosocomial VanA GRE, attempts to identify plasmids harbouring Tn1546 were unsuccessful. Despite this, the results of PFGE and hybridization studies suggested that many of the VanA E. faecium isolated from Edinburgh harbour a Tn1546- bearing plasmid of approximately 160-kilobases in size.The Tn/54<5-related elements, responsible for conferring resistance in the 48 nosocomial VanA GRE, were assessed for diversity. Whilst complete characterization was not possible for all VanA elements, the majority could be assigned to one of ten different Tn/545-types on the basis of insertion and deletion events. Different Tn/545-types varied in their geographical distribution. The insertion sequence IS1542 and an IS/2/<5F-related element were identified within the orf2-vanR and vanX-vanY intergenic regions respectively. Further variation, at least partly attributable to the presence of an IS/2/bF-related element, was common within the orfllorf2 regions of the transposon. A hybrid promoter located upstream of vanR was identified, being generated by the insertion of IS/542 immediately adjacent to the native -10 (TATAAT) box of the vanR promoter. This hybrid promoter is thought to be responsible for moderate-level constitutive expression of the glycopeptide resistance genes that was seen in those isolates harbouring the IS/542 insertion within the VanA transposon. This hybrid promoter supplements, rather than replaces, the normal inducible expression from the native vanR promoter that follows glycopeptide challenge. A media-dependent increase in the level of teicoplanin resistance was seen in those strains harbouring the hybrid promoter. Whilst it is thought that this increase is attributable to the hybrid promoter, the exact mechanism is unclear.In summary, this study reveals a heterogeneous GRE population within Scottish hospitals, both in terms of chromosome analysis by PFGE, and of genotyping of the VanA resistance transposon. In so doing, a novel mechanism of van gene expression is described, which may, under certain circumstances, enhance the level of teicoplanin resistance.