Epidemiología molecular de les betalactamases AmpC plasmídiques de enterobacteris aïllats a l' Hospital de la Santa Creu i Sant Pau i la seva difusió horitzontal

Abstract

Plasmid-mediated AmpC -lactamases (pACBL) were first described in the late 1980s. The phenotype of these enzymes is indistinguishable from the phenotype of hyperproducing chromosomal AmpC -lactamases. Thus, pACBL confer resistance to penicillins, first, second and third generation cephalosporins, monobactams and -lactamase inhibitors, and they are only susceptible to fourth generation cephalosporins and carbapenems. Detecting pACBL is a great challenge for microbiological laboratories because standardised phenotypic methods are lacking. The results of a proficiency study that we developed and conducted in Spain showed that the ability of microbiological laboratories to detect and report extended-spectrum -lactamases (ESBL) in Klebsiella pneumoniae and Escherichia coli was greater than the ability to detect and report AmpC enzymes in these isolates. Commercial methods have recently been developed to detect the pACBL phenotype in the laboratory. Their main problem is that they do not allow differentiation between chromosomal and acquired AmpC enzymes. Nevertheless, the presence of scattered colonies near the edge of the inhibition zones of cefoxitin, cefotaxime, ceftazidime and aztreonam has been described as a possible indicator of the presence of these enzymes in Escherichia coli. This was the only phenotypic tool that led us to suspect the presence of a pACBL in a chromosomal AmpC producer, thus, allowing the detection of a pACBL in a S. marcescens for the first time. Findings in this study also suggested in vivo horizontal transfer of a 70 kb IncL/M plasmid coharbouring blaDHA-1 and qnrB resistance genes between S. marcescens and E. coli isolates. The increasing number of pACBL-producing isolates and the few studies of prevalence based on these enzymes in Spain led us to conduct a study to determine the prevalence of pACBL in Enterobacteriaceae isolates lacking inducible chromosomal ampC genes. These isolates were collected from 1999 to 2007 at Hospital de la Santa Creu i Sant Pau in Barcelona, Spain (Annex III). Although the overall prevalence was 0. 4%, we observed a significant increase, from 0. 06% in 1999 to 1. 3% in 2007. Proteus mirabilis showed the highest prevalence (1%). Among the 117 pACBL characterised during this period, CMY-2 was the most predominant enzyme (67%), followed by DHA-1 (26%). Less commonly found enzymes were ACC-1, CMY-4, CMY-25, CMY-27 and CMY-40. The latter three CMY-2-variants mentioned are reported in this study for the first time. The continuous increase in the prevalence of AmpC enzymes is mainly due to the spread of ampC genes by horizontal transfer. Nowadays, there is little information available among the kind of vectors involved in their spread. For this reason, we characterized vectors mobilising ampC genes. Results show that ampC genes were mobilised by plasmids in 84% (98/117) of the strains, whereas 6% (7/117) of cases were mobilised through integrative conjugative elements (ICE) belonging to the SXT/R391 family. Plasmid analyses revealed a close relationship between the plasmid-mediated ampC gene and the specific plasmid family involved. In six percent of the cases in which ampC genes were mobilised by an ICE, all of them were CMY-2 producing P. mirabilis. The fact that an ICE was responsible for mobilising blaCMY-2 genes in 37% of P. mirabilis isolates and that a CMY-2 producing P. mirabilis strain was recently described in Japan suggests that these elements play an important role in the dissemination of blaCMY-2, at least in this species in recent years. The Tn10 transposon seems to be responsible for mobilising blaCMY-2 inside the ICE. The regions surrounding blaCMY-2,-4,-25,-27,-40 and blaACC-1 were highly conserved. In all cases they were associated with the ISEcp1 mobile element. The regions surrounding blaDHA-1 were more variable and mainly associated with the conserved genes of the 3'CS of class 1 integrons, qacEΔ1 and sul1, and the IS26 mobile element. As occurs for ESBL-producing strains, plasmids carrying ampC genes usually contain other genes that confer resistance to other antibiotic families. This limits the therapeutic options even further. Susceptibility testing results displayed a high level of resistance to most non--lactam agents tested in clinical isolates. The nalidixic acid resistance determinant was transferred by conjugation in the 62% of DHA-1-producing Enterobacteriaceae. A clear association between blaDHA-1 and qnr genes (genes that confer a low level resistance to quinolones) have been reported in recent years. In most cases both genes are located on the same plasmid, associated with integrons or composite transposons. Co-localization of qnrB and blaDHA-1 genes on broadhost- range plasmids, all but one belonging to the IncL/M group, was demonstrated in all the studied isolates. Analysis of the genetic environment of one of the isolates revealed that both genes were mobilised through an IS26 composite transposon. This is the first time that this genetic structure is detailed in an E. coli strain.

Date of Award	25 Jul 2011
Original language	Catalan
Supervisor	Beatriz Mirelis Otero (Director) & Ferran Navarro Risueño (Director)