1 Department of Microbiology, Medical School, University of Thessaly, Mezourlo, Larissa, Greece; 2 Department of Microbiology, Medical School, University of Athens, 11527 Athens, Greece
Received 2 July 2005; returned 24 July 2005; revised 26 July 2005; accepted 28 July 2005
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Abstract |
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Methods: During May to December 2003, 13 consecutive meropenem-resistant, ceftazidime-susceptible P. aeruginosa isolates were recovered from separate patients at the University Hospital of Larissa, Thessaly, Greece. The isolates were studied by Etest MBL, PCR for blaVIM, blaIMP and blaSPM genes and PFGE. Experiments were performed to detect synergy between meropenem or other antimicrobials and the efflux pump inhibitor carbonyl cyanide-m-chlorophenylhydrazone (CCCP). The isolates were also tested by PCR and RTPCR for the expression of the genes mexB and mexY, which encode the efflux pumps MexAB-OprM and MexXY-OprM.
Results: Twelve of the isolates, belonging to six distinct PFGE types, gave negative results in the MBL Etest and lacked genes encoding MBLs but exhibited synergy between meropenem and CCCP, indicating that efflux pump activity contributed to the meropenem resistance. All 12 isolates were positive for mexB and 11 were also positive for mexY genes. RTPCR showed that 10 and five isolates over-expressed mexB and mexY, respectively. One isolate was blaVIM-2-positive and did not show synergy with CCCP, or harbour mexB or mexY.
Conclusions: In our hospital, where MBL-producing P. aeruginosa were previously prevalent, meropenem resistance due to the overexpression of efflux pumps has also now emerged. Early recognition of this resistance mechanism should allow the use of alternative ß-lactams, such as ceftazidime, which would be inactive even against phenotypically susceptible MBL producers.
Keywords: efflux pump inhibitor , CCCP , ceftazidime , RTPCR , MexAB-OprM , MexXY-OprM
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Introduction |
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Materials and methods |
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MICs of meropenem, imipenem and ceftazidime were determined by the agar dilution method using a final inoculum of 104 cfu/spot, while susceptibility testing against other antimicrobials (amikacin, ciprofloxacin, cefepime, gentamicin, kanamycin, ofloxacin, piperacillin, piperacillin/tazobactam) was performed by the disc diffusion method. The isolates were also tested by MBL Etest (AB Biodisk, Solna Sweden) for possible MBL production.
The carriage of blaVIM, blaIMP and blaSPM MBL genes was tested by PCR using published primers and amplification conditions.35 Nucleotide sequencing of both strands of the PCR products was performed on amplicons derived using primers designed to amplify the total blaVIM gene.6 PFGE of SpeI-digested genomic DNA of P. aeruginosa isolates was performed with a CHEF-DRIII system (Bio-Rad, Hemel Hempstead, UK), as previously described3, and banding patterns were compared visually.
Synergy experiments were performed using meropenem and the efflux pump inhibitor carbonyl cyanide-m-chlorophenylhydrazone (CCCP). CCCP was incorporated in MuellerHinton agar at concentrations of 12.5 µM and meropenem susceptibility testing by disc diffusion and agar dilution was performed in parallel in agar plates with and without CCCP.7 Disc diffusion testing of all blaVIM-negative isolates against aztreonam, ceftazidime, ciprofloxacin, imipenem, amikacin and kanamycin, was also assessed in the presence or absence of 12.5 µM CCCP. The latter synergy test was performed in order to check the contribution of efflux pumps to the resistance to these drugs that are selectively extruded by pumps commonly found in pseudomonads.8
The isolates were tested by PCR and RTPCR for the presence and expression of the mexB and mexY genes, encoding MexAB-OprM and MexXY-OprM, respectively, using previously described primers.9 Total RNA extraction was performed using RNAwiz reagent (Ambion Inc, TX, USA). The RNA extract was treated with RNase-free DNase (Promega, Madison, WI, USA) (1 U of enzyme per microgram of RNA for 60 min at 37°C). The RTPCR of total RNA was performed by using the ImProm-II Reverse Transcription System kit (Promega) following the manufacturer's instructions. The mRNA of the constitutively expressed 16S rRNA gene was amplified using primers P891F (TGG AGC ATG TGG TTT AAT TCG A) and P1033R (TGC GGG ACT TAA CCC AAC A) which amplify a 161 bp product. For the gene-specific PCR amplification that was performed simultaneously and under the same conditions for all products, 20 pmol each primer was used per reaction (final volume 50 µL), which involved 5 min at 94°C, and 35 cycles of 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C, and a final extension step of 7 min at 72°C.
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Results |
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The mexB gene was amplified by PCR in all 12 blaVIM-negative isolates, whilst mexY was amplified in 11. The blaVIM-positive isolate was negative for both genes. RTPCR for mexB showed a hyperexpression of the gene in 10 isolates, while in two isolates a product was not visible. The RTPCR bands showed that five isolates strongly expressed the mexY gene and two isolates had a weak expression, while four isolates did not express the gene. All 12 isolates expressed either mexB or mexY (Table 1; Figure 1).
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Discussion |
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These efflux pumps have broad substrate specificity, extruding many antibiotic classes including ß-lactams, quinolones and aminoglycosides. Among ß-lactams however, imipenem and ceftazidime are least affected, if at all, and remain active against pseudomonads.8,10 The susceptibility to ceftazidime of all efflux pump-expressing isolates, implies the absence of other resistance mechanisms against this drug. Although retention of susceptibility to ceftazidime by the blaVIM-producing isolate may seem unexpected since MBLs hydrolyse this compound, similar observations have been reported previously,4 indicating that MBL production alone does not always suffice to significantly elevate ceftazidime MICs in the absence of other resistance mechanisms. In contrast, the synergy observed between CCCP and meropenem suggests that its selective extrusion by the above efflux systems contributes to the resistance. However, although meropenem is recognized and ejected by the up-regulated efflux pumps, a mutation in OprD protein is also deemed necessary to achieve resistance,1 and this probably explains the relatively high meropenem MICs (816 mg/L) even after CCCP inactivation of the efflux pumps. It is presumed that porin mutations are probably also the reason for the MBL-negative isolates exhibiting intermediate or low-level resistance to imipenem, which is not affected by efflux pumps.8 Finally, the absence of synergy between quinolones or aminoglycosides and CCCP indicates the presence of alternative resistance mechanisms such as gyrA/parC gene mutations and aminoglycoside-modifying enzymes, respectively.
In conclusion, an efflux pump overexpression mechanism confers meropenem resistance in unrelated P. aeruginosa strains. The reduced usage of expanded spectrum cephalosporins, such as ceftazidime, and the increased consumption of carbapenems against ESBL-producing Enterobacteriaceae during the last few years, may have contributed to the emergence of this mechanism. A possible further accumulation of carbapenem-resistant P. aeruginosa is threatening. The early recognition of the efflux pump mechanism by the introduction of a phenotypic disc synergy test, and its distinction from the MBL mechanism could, therefore, allow the timely use of alternative ß-lactams, such as ceftazidime, which would be inactive even against apparently susceptible MBL producers.
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References |
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