JAC Advance Access originally published online on May 8, 2007
Journal of Antimicrobial Chemotherapy 2007 60(1):136-139; doi:10.1093/jac/dkm138
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Transmission in the community of clonal Proteus mirabilis carrying VIM-1 metallo-ß-lactamase
1 Department of Microbiology, Medical School, University of Athens, Athens, Greece 2 Department of Medical Microbiology, University of Thessaly, Larissa, Greece 3 Department of Microbiology, General Hospital of Serres, Serres, Greece
* Corresponding author. Tel: +30-210-746-2010; Fax: +30-210-746-2249; E-mail: atsakris{at}med.uoa.gr
Received 11 March 2007; returned 5 April 2007; revised 11 April 2007; accepted 12 April 2007
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Abstract |
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Objectives: Several infections among patients attending our outpatient clinic were caused by imipenem-resistant Proteus mirabilis that were phenotypically metallo-ß-lactamase (MBL)-positive. The aim of the study was to investigate this extrahospital dissemination and the mechanisms of resistance to carbapenems.
Methods: During a 1 year period (December 2005–December 2006), the characteristics of the outpatients with infections caused by isolates of P. mirabilis with reduced susceptibility to imipenem (MIC > 4 mg/L) were prospectively collected. The isolates were tested by agar dilution MICs, phenotypic MBL testing and enterobacterial repetitive intergenic consensus PCR. PCR assays and nucleotide sequencing were used for the identification of bla gene types and mapping of the integron carrying the MBL gene. The location of the MBL allele was investigated by mating experiments, plasmid analysis and hybridization of the Southern-blotted plasmid extract with a blaVIM-1 probe.
Results: During the study, 12 MBL-positive P. mirabilis isolates were recovered from urinary tract infections of community patients. In all cases, the patients had a previous hospitalization in a Greek regional hospital and had received fluoroquinolones and/or aminoglycosides and ß-lactams. MICs of imipenem ranged from 32 to >128 mg/L, whereas those of meropenem ranged from 1 to 8 mg/L and those of ertapenem ranged from 0.5 to 4 mg/L. The isolates originated from the same clonal strain and harboured a blaVIM-1 gene in a common integron structure. Conjugation experiments, plasmid analysis and hybridization assays indicated the chromosomal location of the blaVIM-1 gene.
Conclusions: This is the first study that documents transmission in the extrahospital setting of acquired MBL-producing Gram-negatives causing community-onset infections.
Keywords: MBLs , P. mirabilis , blaVIM-1
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Introduction |
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Metallo-ß-lactamases (MBLs) constitute a growing class of ß-lactamases that readily hydrolyse all ß-lactams except aztreonam. During the last decade, acquired MBLs have emerged among nosocomial Pseudomonas aeruginosa isolates and more recently have effectively diffused into other Gram-negative pathogens posing serious consequences in antimicrobial treatment.1 MBL genes normally reside within class 1 integrons of various compositions of gene cassettes, which may be located in the chromosome or in the plasmid DNA.1,2 In the European countries, the increase in MBL production in Gram-negatives is primarily due to the spread of VIM-type MBLs, suggesting a large reservoir of the respective blaVIM gene cassettes.2,3 However, the dissemination of these enzymes in the community setting has not been described in Europe or elsewhere. Preliminary susceptibility data in our clinical laboratory indicated that several infections in patients attending our outpatient clinic were caused by imipenem-resistant Proteus mirabilis that were phenotypically MBL-positive and exhibited cross-resistance to almost all alternative antimicrobials. The similar antimicrobial susceptibility patterns of these isolates prompted a prospective investigation into their community dissemination and carbapenem resistance mechanisms. We report the transmission in the community of clonal blaVIM-1-producing P. mirabilis isolates that caused urinary tract infections (UTIs).
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Materials and methods |
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A total of 155 P. mirabilis isolates were obtained during a 1 year period (December 2005–December 2006) from clinical specimens taken from separate patients with community-onset clinical infections in the region of Serres General Hospital, Greece. This hospital is an acute care hospital serving a population of approximately 200 000 habitants. Identification and susceptibility testing was performed with the Microscan system (Dade Behring Inc., West Sacramento, CA, USA). Among isolates with reduced susceptibility to imipenem (MIC > 4 mg/L), phenotypic detection of MBL production was performed using the imipenem-EDTA double-disc synergy test (DDST) and the Etest MBL assay (AB Biodisk, Solna, Sweden). Antibiotic susceptibility testing of the DDST-positive isolates for ß-lactams (aztreonam, ceftazidime, cefepime, ertapenem, imipenem, meropenem and piperacillin), ß-lactam/ß-lactamase inhibitor combinations (ampicillin/sulbactam and piperacillin/tazobactam), aminoglycosides (amikacin, gentamicin, netilmicin and tobramycin), ciprofloxacin and trimethoprim was performed by the agar dilution method using the CLSI interpretative criteria4 and Escherichia coli ATCC 25922 as control.
To type the ß-lactamase genes carried by the DDST-positive isolates, PCR was performed using specific primers and amplification conditions for blaIMP, blaVIM, blaSPM, blaOXA-23-like, blaOXA-24-like, blaTEM, blaSHV, blaCTX-M, blaGES and blaCMY.5–7 Integron mapping was performed by using PCR assays, combining primers specific for conserved 5'-CS and 3'-CS sequences with primers specific for blaVIM, aacA, dfrA, aadA, qacE
1 and sul genes. PCR products were purified using ExoSAP-IT reagent (USB Corporation, Cleveland, OH, USA) and used as templates for nucleotide sequencing on both strands with an ABI Prism 377 DNA sequencer (Perkin-Elmer, Applied Biosystems, Foster City, CA, USA).
The epidemiological relationship was analysed by enterobacterial repetitive intergenic consensus (ERIC)-PCR with primers ERIC2 and ERIC1R. The potential for conjugational transfer of imipenem resistance was examined in diparental filter matings using E. coli 20R764 (lac+ rifr) as the recipient. Transconjugants were selected on MacConkey agar plates containing rifampicin 100 mg/L and imipenem in concentrations of 0.5–2 mg/L or ceftazidime 2 mg/L. Plasmid extraction was performed with two different lysis methods using E. coli 39R861 as a control. The chromosomal location of the blaVIM-1 allele was detected by Southern blotting after electrophoresis of the plasmid extract and gene-specific hybridization using digoxigenin-labelled blaVIM-1 probe.7
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During the study period, 12 (7.7%) P. mirabilis isolates were collected from community-onset infections that exhibited reduced susceptibility to imipenem. MICs of imipenem ranged from 32 to >128 mg/L, whereas those of meropenem ranged from 1 to 8 mg/L and those of ertapenem ranged from 0.5 to 4 mg/L (Table 1). All but one of the isolates were susceptible to amikacin, gentamicin and aztreonam (MICs from 4 to 16 mg/L, 2 to 4 mg/L and 4 to 8 mg/L, respectively), but highly resistant to other ß-lactams, ciprofloxacin, netilmicin, tobramycin and trimethoprim; the remaining isolate was pandrug-resistant. All isolates were recovered from urine samples of patients (average age 72.8 years; range, 61–82 years) who admitted to the outpatient department with symptoms and signs of UTI. In all cases, the patients had been hospitalized in our hospital for genitourinary pathology (six had a surgical urologic intervention), 4 months preceding the isolation of the imipenem-resistant isolate (Table 1). During their hospitalization, none of them was infected with any imipenem-non-susceptible microorganism. The average time of the hospital discharge before presentation with the UTI at the outpatient clinic was 43.7 days (range, 11–124 days). During their hospitalization, they had taken fluoroquinolones alone or received intravenous treatment (amikacin plus ticarcillin/clavulanic for 1–5 days) and subsequently fluoroquinolones. Aztreonam was the only potent ß-lactam and administered as aetiological therapy with successful results in all UTIs.
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ERIC-PCR with primer ERIC2 or ERIC1R showed common banding patterns for all 12 isolates, implying a single underlying genotype. The profiles of this genotype were different from those of several clinically unrelated control P. mirabilis isolates, validating the results of our typing methods (Figure 1). The production of MBL was detected in all isolates with both phenotypic methods. Application of the phenotypic methods in a random sample of 40 susceptible (MIC
4 mg/L) community P. mirabilis isolates, collected during the same period, did not reveal any additional MBL-producing isolate. PCR and sequencing analysis identified a blaVIM-1 allele in all 12 MBL-positive isolates. In all cases, PCR mapping of the implicated integron revealed that the blaVIM-1 allele was located downstream of the aatI1 recombination site and upstream of gene cassettes for aacA7, dfrA1 and aadA1 alleles, similarly to previous integron structures of blaVIM-1-bearing nosocomial Gram-negatives in Greece.3,5 The P1 and P2 promoters were strong and inactive (no GGG insertion), respectively. PCR testing for other bla genes, including various MBLs, extended-spectrum ß-lactamases (ESBLs) and AmpC genes, was negative in all cases. Conjugation experiments failed to detect that the MBL determinant could be transferred and plasmid DNA was not visualized by either of the extraction methods in agarose gel electrophoresis. These findings, along with the hybridization of the chromosomal band with the blaVIM-1 probe, indicated the chromosomal location of the blaVIM-1 gene.
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Discussion |
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MBL-carrying isolates have been almost exclusively found in hospital settings, possibly suggesting a degree of confinement within this environment, where the antimicrobial usage is regularly heavy.1 To the best of our knowledge, clusters of acquired MBL-producing community isolates have not been reported previously. The present study describes the first transmission in the extrahospital setting of MBL-producing Gram-negative bacteria that cause community-onset infections. The most efficient route of extrahospital diffusion of VIM producers could be the prolonged carriage of such strains by our patients after a hospitalization during which they might have acquired them. It is of interest that since March 2006, blaVIM-1-producing P. mirabilis isolates of the same clonal type and carrying the same integron have also been sporadically identified among patients hospitalized in various wards of our hospital (data not shown). This further supports that the VIM-1-encoding integron structure has been acquired by an index hospital strain that became established in this setting and then colonized patients who were discharged from the hospital.
The observed carbapenem resistance phenotype, with high-level imipenem resistance and susceptibility or intermediate susceptibility to other carbapenems, despite the similar hydrolytic activity of VIM-1 for carbapenems, can imply the relatively lower activity of imipenem against Proteus species.8 However, in comparison with other blaVIM-1-bearing nosocomial P. mirabilis clones in our country,9 the clonal isolates of our study exhibited much higher carbapenem MICs, implicating that cofactors such as the diminished outer membrane permeability and/or the reduced affinity of PBP28,10 possibly contribute to these levels of carbapenem resistance.
The apparent dissemination of VIM-1-producing P. mirabilis could represent a substantial barrier in the treatment of community-acquired infections. Previous hospitalization and exposure to antimicrobials were found predictive of MBL-producing P. mirabilis in the extrahospital setting of our region. Consistent with our findings, hospitalization in the preceding 3 months has been frequently linked with the dissemination of ESBL-producing pathogens in the community.11,12 Moreover, genitourinary pathology, a finding common in all our patients, has been identified as an independent risk factor for ESBL community-onset infections.13 Screening for colonization at discharge and continuous surveillance of community isolates with characteristics of MBL-bearing isolates are essential in order to recognize MBL producers and to prevent their dissemination by indicating the proper treatment.
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None to declare.
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2
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