JAC Advance Access originally published online on May 4, 2007
Journal of Antimicrobial Chemotherapy 2007 60(1):132-135; doi:10.1093/jac/dkm126
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A highly carbapenem-resistant Pseudomonas aeruginosa isolate with a novel blaVIM-4/blaP1b integron overexpresses two efflux pumps and lacks OprD
1 Department of Microbiology, Medical School, University of Thessaly, Larissa, Greece 2 Department of Obstetrics and Gynaecology, Medical School, University of Thessaly, Larissa, Greece
* Correspondence address. Department of Medical Microbiology, University Hospital of Larissa, Mezourlo, 411 10 Larissa, Greece. Tel: +30-6976-881203; Fax: +30-2410-681570; E-mail: pournaras{at}med.uth.gr
Received 15 December 2006; returned 1 March 2007; revised 13 March 2007; accepted 8 April 2007
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Abstract |
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Objectives: A Pseudomonas aeruginosa clinical isolate that exhibited high-level carbapenem resistance and produced metallo-ß-lactamase (MBL) was recovered from a Greek patient. This study was conducted to determine the underlying mechanisms that conferred the carbapenem resistance phenotype.
Methods: MICs were determined by Etest and Etest MBL. PCR assays were performed for identification of blaVIM-type, other antibiotic resistance and efflux pump genes and mapping of class 1 integrons. Expression of efflux pump genes was quantified by real-time PCR. Nucleotide sequencing was used to determine the blaVIM allele. The location of the MBL allele was investigated by mating experiments, plasmid analysis and hybridization studies.
Results: The isolate was highly carbapenem-resistant (MICs of imipenem and meropenem were 512 and 128 mg/L, respectively) and multidrug-resistant. It harboured the ß-lactamase genes blaVIM-4 and blaP1b in a novel class 1 integron named InV4P1, and a second integron with aac(6’)-Ib and blaOXA-35 gene cassettes. The isolate was deficient in porin OprD and overexpressed efflux pumps MexAB-OprM and MexXY-OprM. Conjugation experiments failed to detect transferable MBL determinants, plasmids were not visualized and blaVIM was detected by PCR in the chromosomal band.
Conclusions: Multiple carbapenem resistance mechanisms are demonstrated to coexist in a single P. aeruginosa isolate and might confer the high-level carbapenem resistance.
Keywords: PSE-1 , InV4P1 , P. aeruginosa
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Introduction |
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Carbapenems are commonly used as last resort drugs for treatment of infections caused by multiresistant Pseudomonas aeruginosa isolates. However, pseudomonads often possess mechanisms to overcome their activity, including metallo-ß-lactamase (MBL) production, decreased outer membrane permeability due to OprD deficiency and intrinsic tripartite efflux systems MexAB-OprM and MexXY-OprM.1 P. aeruginosa producing acquired MBLs are increasingly implicated in nosocomial infections. Among them, two distinct MBL types, IMP and VIM, encoded by gene cassettes in class 1 integrons, predominate worldwide.1
Studies indicate that VIM-producing microorganisms are endemic in various Mediterranean countries, including Greece, as well as in the Far East.2,3 VIM-4 is a point mutant of the VIM-1 enzyme originally identified in Larissa, Greece, and thereafter in several other regions.1 Currently, isolates with a phenotype indicative either of MBL production or efflux pump overexpression account for the majority of hospital-acquired pseudomonal infections in our region (A. N. Maniatis, unpublished data). We report the detection of a highly carbapenem-resistant P. aeruginosa that carries a blaVIM-4 MBL gene in a novel class 1 integron along with a second integron, overproduces efflux pumps MexAB-OprM and MexXY-OprM and is deficient in porin OprD.
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Materials and methods |
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Strain P. aeruginosa 140 (PA140) was isolated in August 2005 from a surgical wound infection of a 75-year-old female who was hospitalized in the gynaecological ward of the University Hospital of Larissa. Species identification was performed by the Vitek 2 automated system (bioMérieux, Marcy l’Étoile, France) according to the manufacturer’s instructions and was confirmed by API 20NE (bioMérieux). Susceptibility to antipseudomonal antimicrobials (amikacin, ciprofloxacin, cefepime, ceftazidime, gentamicin, aztreonam, kanamycin, ofloxacin, piperacillin and piperacillin/tazobactam) was performed by disc diffusion. MICs of these antipseudomonal agents were determined by agar dilution. All susceptibility testing methods were performed according to CLSI 2006. The isolate was also screened by Etest MBL (AB Biodisk, Solna, Sweden) for possible MBL production. P. aeruginosa ATCC 27853 was used as a control in all susceptibility assays.
PCR assays for genes blaVIM-like and aacA4 and conserved segments of class 1 integrons, as well as PCR mapping of integronic gene cassette arrays were performed with previously described primers and amplification conditions.4,5 Both strands of all PCR products were sequenced with the use of an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA, USA). Plasmid isolation was performed using an alkaline lysis protocol and Escherichia coli 39R861 as a standard plasmid control. Mating experiments were performed by mixed broth mating using E. coli 26R793 (rifampicin-resistant) and P. aeruginosa PU21 (rifampicin-resistant) as recipients. Selection of transconjugants was made on Mueller–Hinton agar plates containing rifampicin (100 mg/L) and imipenem (2 mg/L) or ceftazidime (2–4 mg/L). The location of the blaVIM allele was tested by PCR on the plasmid extract bands after gel electrophoresis, followed by Southern blotting and gene-specific hybridization using a digoxigenin-labelled blaVIM probe.6 As a negative control we used the plasmid extract of E. coli 39R861 and as a positive control we used an E. coli transconjugant strain carrying blaVIM-1 in a conjugative plasmid.7
Total cellular RNA was extracted with TRI Reagent (Ambion Inc., Austin, TX, USA) in the late logarithmic phase of the bacterial culture. RNA abundance was determined spectrophotometrically at 260 nm and contaminating DNA was removed with DNaseI treatment (Promega, Madison, WI, USA). Reverse transcription (RT) of 1 µg of total RNA was performed with the ThermoScrirt RT–PCR System (Invitrogen Corporation, Carlsbad, CA, USA). Quantitative real-time RT–PCR (QRT–PCR) was performed in an MX3005P instrument (Stratagene, La Jolla, CA, USA) with Brilliant SYBR Green (Qiagen, Hilden, Germany) to detect transcripts of genes mexB, mexY and ampC corresponding to MexAB-OprM and MexXY-OprM pumps and AmpC cephalosporinase, respectively.8,9 Relative expression levels of the tested genes in strain PA140 were compared against those in the known efflux pump overproducer strain PA5.10 The single-copy housekeeping gene rpsL11 was used for normalization and also control RT–PCRs of untranscribed RNA were included. Cycling parameters for all tested genes were one cycle of 95°C for 10 min followed by 40 cycles of 95°C for 30 s, 55°C for 60 s and 72°C for 90 s.
Outer membrane protein (OMP) analysis for the study strain PA140 and, as a control, an imipenem-susceptible strain (PA32) from our collection, used in previous studies,3,10 was performed. Bacterial cells were grown in Mueller–Hinton broth for 18 h, centrifuged at 3700 g for 15 min at 4°C and solubilized with 3 mL of 10 mM phosphate buffer, pH 7.2. To disrupt, the solubilized cells were sonicated eight times for 90 s each, with 2 min intervals. Unbroken cells were removed by centrifugation at 3700 g for 15 min at 4°C and the supernatant was further centrifuged at 22 200 g for 45 min at 4°C. The pellet containing the cell envelopes was solubilized with 1 mL of 2% sodium lauryl sarcosinate in 10 mM phosphate buffer, pH 7.2, and incubated at room temperature for 45 min. After centrifugation at 10 000 g for 60 min at 4°C, the insoluble material was resuspended in 200 µL of 62.5 mM Tris–HCl buffer, pH 6.8. Protein concentration was calculated by UV spectrophotometry at 280 nm. Electrophoresis of 100 µg of protein extract was performed using a NUPAGETM 4% to 12% Bis–Tris Gel 1.0 mm Kit (Invitrogen) according to the manufacturer’s instructions. Staining was achieved with Coomassie using SimplyBlueTM SafeStain reagent (Invitrogen).
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Results |
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The isolate PA140 was resistant to all tested antimicrobials except aztreonam and colistin. Agar dilution MICs of imipenem and meropenem were 512 and 128 mg/L, respectively. MICs of amikacin, gentamicin, tobramycin, cefepime and piperacillin/tazobactam were > 128 mg/L, whereas the MIC of ciprofloxacin was > 64 mg/L. MICs of colistin, ceftazidime and aztreonam were 2, 32 and 8 mg/L, respectively. The patient had been treated with ciprofloxacin prior to the isolation of the organism. The infection was resolved with surgical debridement of the wound, being left to heal by secondary intention. Etest MBL exhibited a marked decrease in MICs of imipenem compared with imipenem–EDTA (>256 and 1.5 mg/L, respectively), implying the production of MBL.
PCR assays suggested the presence of blaVIM-type and aacA4 genes, respectively. Using primers 5'CS and 3'CS, two distinct bands of 
2 and 1.5 kb were produced. PCR mapping with the extracted DNA of the 2 kb band as template gave specific products with primer combinations 5'CS—VIMF/N (800 bp) and VIMB/N—3'CS (1.8 kb). When the 1.5 kb band DNA was used as template, amplicons of 550 bp and 1.5 kb were produced with primer combinations 5'CS—AACA7R and AACA7F—3'CS, respectively. Nucleotide sequencing of the respective PCR products revealed two distinct class 1 integrons. The integron carrying the MBL, designated InV4P1 from its gene content, included the blaVIM-4 allele downstream of the attI1 recombination site, directly followed by the ß-lactamase gene blaP1b coding for PSE-1/CARB-2. The gene cassette of blaVIM-4 with its 59-base element was identical to previously submitted sequences in Klebsiella pneumoniae from Tunisia (GenBank accession number AM181293
[GenBank]
) and P. aeruginosa from Poland (GenBank accession number AM087411
[GenBank]
). The blaP1b gene cassette was identical to those described in Acinetobacter baumannii from China (GenBank accession number DQ857723
[GenBank]
) and P. aeruginosa strains from Portugal (GenBank accession number AY560837
[GenBank]
). The second integron included sequentially genes aac(6’)-Ib and blaOXA-35 coding for the OXA-10-related restricted-spectrum oxacillinase OXA-35, being identical to a previously described integron in P. aeruginosa from France (GenBank accession number AF315786
[GenBank]
). No transconjugants were selected by mating experiments. Plasmid analysis did not reveal any visible plasmids. PCR using as template the chromosomal band shown in the gel electrophoresis of the plasmid extracts was positive for blaVIM and 16S rDNA, possibly suggesting a chromosomal location of the blaVIM-associated integron. A positive signal was also produced with the chromosomal band after hybridization of Southern blots of the plasmid analysis gel with a blaVIM-1 probe.
QRT–PCR revealed increased expression of genes mexB and mexY in strain PA140 compared with strain PA5. In particular, expression of genes mexB and mexY in strain PA140 were 3.2 ± 0.3 and 1.7 ± 0.2, respectively, orders of magnitude higher than PA5. Expression of gene ampC did not differ substantially between strains PA140 and PA5 (Figure 1). Also, OMP analysis revealed a lower intensity of a protein band of 46 kDa, consistent with the OprD2 porin in strain 140 compared with the imipenem-susceptible control strain PA32 (Figure 1).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionTransparency declarationsReferences |
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This study is one of the few to demonstrate multiple carbapenem resistance mechanisms in a single P. aeruginosa isolate. These mechanisms, in combination, probably conferred the very high carbapenem MICs and provide a plausible explanation for the high-level carbapenem resistance that is commonly observed in hospital-acquired pseudomonads in our region.3 In particular, the interplay of VIM with the overproduced MexAB-OprM and MexXY-OprM efflux pumps and the deficient porin OprD would increase the resistance to meropenem and imipenem, respectively.12 Nevertheless, the contribution of other less well-defined mechanisms cannot be excluded.
The novel blaVIM-associated class 1 integron carries two ß-lactamase genes and no aminoglycoside resistance genes. The structure of InV4P1 is the first example where a blaVIM-1-related allele is found in association with blaP1b that was previously reported along with blaVIM-2 and other gene cassettes in integron In100.13 A second unrelated class 1 integron was also carried by the same P. aeruginosa strain and contained a aac(6')-Ib gene that possibly contributed to the aminoglycoside resistance of the strain. These findings further support an extended variability of genetic structures carrying MBLs in the hospital environment.
Nucleotide sequence accession number
The nucleotide sequence of integron InV4P1 has been submitted to GenBank under accession number DQ986304 [GenBank] .
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None to declare.
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1 Walsh TR, Toleman MA, Poirel L, et al. Metallo-ß-lactamases: the quiet before the storm? Clin Microbiol Rev (2005) 18:306–25.
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3
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5
Poirel L, Naas T, Nicolas D, et al. Characterization of VIM-2, a carbapenem-hydrolyzing metallo-ß-lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France. Antimicrob Agents Chemother (2000) 44:891–7.
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Tsakris A, Pournaras S, Woodford N, et al. Outbreak of infections caused by Pseudomonas aeruginosa producing VIM-1 carbapenemase in Greece. J Clin Microbiol (2000) 38:1290–2.
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8
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10
Pournaras S, Maniati M, Spanakis N, et al. Spread of efflux pump-overexpressing, non-metallo-ß-lactamase-producing, meropenem-resistant but ceftazidime-susceptible Pseudomonas aeruginosa in a region with blaVIM endemicity. J Antimicrob Chemother (2005) 56:761–4.
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Quinteira S, Sousa JC, Peixe L. Characterization of In100, a new integron carrying a metallo-ß-lactamase and a carbenicillinase, from Pseudomonas aeruginosa. Antimicrob Agents Chemother (2005) 49:451–3.
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