In vivo transmission of a plasmid containing the KPC-2 gene in a single patient.

Here we describe a case of in vivo horizontal interspecies transmission of a KPC-2-producing plasmid from a Klebsiella pneumoniae to an Enterobacter aerogenes strain in the same patient. The patient's gut flora initially contained a carbapenem-susceptible E. aerogenes strain and 10 days after admission a KPC-2-positive K. pneumoniae. Three months after admission, a KPC-2-positive E. aerogenes was identified in fecal surveillance cultures. This isolate was isogenic with the initial E. aerogenes and contained a KPC-2-coding plasmid identical to that of the K. pneumoniae. The patient developed bacteraemia by the KPC-2-positive K. pneumoniae 17 days after her first colonization. In vivo horizontal transmission of blaKPC-carrying plasmids between bacterial species underscores the importance of antibiotic stewardship along with implementation of infection control measures for the containment of KPC-producers.

Epidemiology and molecular characterisation of metallo-ß-lactamase-producing Enterobacteriaceae in a university hospital Intensive Care Unit in Greece.

The molecular epidemiology of VIM-producing Enterobacteriaceae isolated at the beginning of an epidemic in the Intensive Care Unit (ICU) of a university hospital in Athens, Greece, was studied. All Gram-negative organisms isolated from March 2004 to November 2005 positive for metallo-ß-lactamase (MBL) production were submitted to polymerase chain reaction (PCR) and sequencing, to repetitive sequence-based PCR (Rep-PCR) for molecular typing, and to S1 nuclease digestion for plasmid DNA characterisation. Conjugation experiments and isoelectric focusing were performed to identify co-existing ß-lactamases. Amongst 23 patients, 12 suffered one or more clinical infections. Eighty-two isolates representing one isolate per clone, source and ICU patient were studied, including Klebsiella pneumoniae (77), Enterobacter cloacae (2), Citrobacter freundii (1) and Pseudomonas aeruginosa (2). High clonal diversity was detected amongst the K. pneumoniae, with 10 distinct clones identified. Conjugation was successful in 54.5% of K. pneumoniae, and five different-sized plasmids were detected. All K. pneumoniae and both E. cloacae isolates shared the same bla(VIM-1)-containing class 1 integron structure also carrying aacA7, dhfrI and aadA1 gene cassettes. The C. freundii isolate carried a different integron that included bla(VIM-1) and aac(6')-IIc. Both P. aeruginosa isolates were positive for bla(VIM-2). It was not possible to identify specific clones with the potential to cause clinical infections. In conclusion, a multiclonal cluster of MBL-producers was responsible for the first cases of colonisation and/or infection in the ICU. A single integron structure, common in Greek hospitals, efficiently disseminated between clones and species, suggesting that the epidemic was mainly the result of successful horizontal transfer of mobile genetic material rather than the result of horizontal transfer of one or a few clones.

In vitro interactions of antimicrobial combinations with fosfomycin against KPC-2-producing Klebsiella pneumoniae and protection of resistance development.

Using time-kill methodology, we investigated the interactions of fosfomycin with meropenem or colistin or gentamicin against 17 genetically distinct Klebsiella pneumoniae clinical isolates carrying blaKPC-2. Synergy was observed with meropenem or colistin against 64.7 and 11.8% of tested isolates, while the combination with gentamicin resulted in indifference. All studied combinations showed improved bactericidal activity, compared to fosfomycin alone and prevented the development of fosfomycin resistance in 69.2, 53.8, and 81.8% of susceptible isolates, respectively.

Efficacy of carbapenems against a metallo-ß-lactamase-producing Escherichia coli clinical isolate in a rabbit intra-abdominal abscess model.

BACKGROUND: Although metallo-ß-lactamases (MBLs) hydrolyse most ß-lactams, including carbapenems, MBL-producing Enterobacteriaceae very often remain susceptible to carbapenems in vitro. We studied the in vivo efficacy of imipenem, meropenem, ertapenem and aztreonam against a carbapenem-susceptible MBL-producing clinical Escherichia coli strain in a rabbit intra-abdominal abscess model. METHODS: Rabbits were inoculated intraperitoneally with 10(8) cfu/mL of VIM-1-positive E. coli and were assigned to receive no treatment (controls) or intravenous imipenem/cilastatin (imipenem) 70 mg/kg/12 h or meropenem 125 mg/kg/12 h or ertapenem 60 mg/kg/12 h or aztreonam 70 mg/kg/12 h. Dosing regimens were chosen on the basis of preliminary pharmacokinetic studies so that T(>MIC) was achieved for ≥50% of the dosing interval for all tested antibiotics. A total of eight doses were administered before sacrifice and the abscesses were harvested and quantitatively cultured. RESULTS: MICs of imipenem, meropenem, ertapenem and aztreonam for the infecting isolate were 1, ≤0.25, 1.5 and ≤0.25 mg/L, respectively. The log(10) cfu/g (mean ±â€ŠSD) viable counts in pus were as follows: controls (n = 16), 8.71 ±â€Š1.34 (P < 0.001 versus all other groups); imipenem (n = 15), 4.89 ±â€Š2.42; meropenem (n = 15), 4.24 ±â€Š2.44; ertapenem (n = 16), 3.17 ±â€Š1.85 (P = 0.022 versus imipenem); and aztreonam (n = 15), 3.62 ±â€Š3.05. Mortality among treated rabbits was significantly reduced compared with controls. Four animals in the aztreonam group (26.7%) had culture-negative pus and no mortality was noted among aztreonam-treated animals. CONCLUSIONS: In the rabbit experimental model, carbapenems were shown to be effective in the treatment of intra-abdominal infection due to an extended-spectrum ß-lactamase-negative carbapenem-susceptible VIM-1-producing clinical E. coli strain, but treatment with aztreonam resulted in a more favourable outcome overall.

Does the activity of the combination of imipenem and colistin in vitro exceed the problem of resistance in metallo-beta-lactamase-producing Klebsiella pneumoniae isolates?

Using time-kill methodology, we investigated the interactions of an imipenem-colistin combination against 42 genetically distinct Klebsiella pneumoniae clinical isolates carrying a bla(VIM-1)-type gene. Irrespective of the imipenem MIC, the combination was synergistic (50%) or indifferent (50%) against colistin-susceptible strains, while it was antagonistic (55.6%) and rarely synergistic (11%) against non-colistin-susceptible strains (with synergy being observed only against strains with colistin MICs of 3 to 4 microg/ml). The combination showed improved bactericidal activity against isolates susceptible either to both agents or to colistin.

Molecular characterization of an Escherichia coli clinical isolate that produces both metallo-beta-lactamase VIM-2 and extended-spectrum beta-lactamase GES-7: identification of the In8 integron carrying the blaVIM-2 gene.

OBJECTIVES: We have previously reported the isolation of a VIM-2-producing Escherichia coli clinical isolate and the coexistence of blaVIM-2 and blaGES-7 in the same isolate. The aim of this study was to elucidate the genetic environment of these genes. METHODS: PCR and sequence analysis were used to identify and analyse the blaVIM-containing integron. The location of the blaVIM-2 and blaGES-7 genes was identified by hybridization experiments on I-CeuI-digested genomic DNA after PFGE. RESULTS: Only the blaVIM-2 gene has been found on a class I integron, designated In8 (1474 bp). Hybridization with the blaVIM-2, the blaGES-7 and the 16S-23S rRNA probes confirmed the chromosomal location of both genes. The blaVIM-2 probe hybridized with a 710 kb fragment whereas the blaGES-7 probe hybridized with a 144 kb fragment of genomic DNA of the E. coli isolate. CONCLUSIONS: This report provides evidence for the chromosomal location of both blaVIM-2 and blaGES-7 genes carried by an E. coli clinical isolate. In8, containing blaVIM-2, presents an emerging threat of carbapenem resistance among E. coli isolates in Greece.

Characterization of a new integron containing bla(VIM-1) and aac(6')-IIc in an Enterobacter cloacae clinical isolate from Greece.

OBJECTIVES: A clinical isolate of Enterobacter cloacae exhibiting reduced susceptibility to imipenem and a positive EDTA-disc synergy test was studied for carbapenemase production. MATERIALS AND METHODS: MICs were determined with standard procedures as well as using a higher inoculum. Isoelectric focusing of cell extracts was used for detection of beta-lactamases. PCR assays with primers specific for the bla(VIM) gene and the conserved segments of class 1 integrons and sequence analyses were carried out to identify the gene and to map the metallo-beta-lactamase encoding integron. Transferability of the gene was assessed with conjugation experiments using the filter mating technique. To identify the location of the bla(VIM-1) gene, Southern hybridization was carried out in genomic DNA using an internal fragment of the bla(VIM-1) gene as a probe, amplified by PCR. RESULTS: The isolate was resistant to extended-spectrum beta-lactams. The MICs of carbapenems were below the resistance breakpoints but rose above resistance breakpoints when an inoculum of 10(8) cfu/mL was used. Isoelectric focusing detected a beta-lactamase with a pI of 6.1, which exhibited imipenem-hydrolysing activity in a microbiological assay. Ceftazidime and imipenem resistance were not transferable by conjugation. PCR assays identified the bla(VIM-1) gene in the variable region of a class 1 integron which also carried the aac(6')-IIc gene. The bla(VIM-1) probe hybridized with an approximately 130 kb fragment of genomic DNA, suggesting a chromosomal location of the gene. CONCLUSION: We describe a novel class 1 integron containing bla(VIM-1) and aac(6')-IIc genes in an E. cloacae clinical isolate.