Spread of efflux pump-overexpressing, non-metallo-ß-lactamase-producing, meropenem-resistant but ceftazidime-susceptible Pseudomonas aeruginosa in a region with blaVIM endemicity

S. Pournaras1, M. Maniati1, N. Spanakis2, A. Ikonomidis1, P. T. Tassios2, A. Tsakris2,*, N. J. Legakis2 and A. N. Maniatis1

1 Department of Microbiology, Medical School, University of Thessaly, Mezourlo, Larissa, Greece; 2 Department of Microbiology, Medical School, University of Athens, 11527 Athens, Greece


* Corresponding author. Tel: +30-210-746-2140; Fax: +30-210-746-1489; E-mail: atsakris{at}med.uoa.gr

Received 2 July 2005; returned 24 July 2005; revised 26 July 2005; accepted 28 July 2005


    Abstract
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 Abstract
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 Materials and methods
 Results
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Objectives: To investigate the resistance mechanisms of meropenem-resistant, ceftazidime-susceptible Pseudomonas aeruginosa isolates, in a clinical setting where VIM-2 or VIM-4 metallo-ß-lactamase (MBL)-producing pseudomonads are common.

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 RT–PCR 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. RT–PCR 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 , RT–PCR , MexAB-OprM , MexXY-OprM


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
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Pseudomonas aeruginosa is an opportunistic pathogen that causes a variety of infections in immunocompromised patients. Carbapenems, such as imipenem and meropenem, have a broad antibacterial spectrum and play a fundamental role in the treatment of infections caused by multiresistant P. aeruginosa isolates. However, pseudomonads may develop resistance to carbapenems through mechanisms such as diminished permeability, stable derepression of chromosomal AmpC ß-lactamases, and overexpression of tripartite efflux systems, principally MexAB-OprM and MexXY-OprM.1,2 In recent years, carbapenem resistance in clinical P. aeruginosa isolates has been also attributed to the production of metallo-ß-lactamases (MBLs), which hydrolyse most ß-lactams except aztreonam, and usually confer high-level resistance.2 In our region, highly carbapenem-resistant pseudomonads have become endemic during the past 5 years. The most common mechanism of resistance to carbapenems identified among nosocomial P. aeruginosa isolates from 2001–2002 was the production of VIM-type MBLs.3 These VIM-producing isolates exhibited resistance to all ß-lactams and also to almost all alternative anti-pseudomonal drugs. Recently, however, carbapenem-resistant (MIC ≥ 16 mg/L) P. aeruginosa strains susceptible to oxymino-cephalosporins, such as ceftazidime (MIC ≤ 8 mg/L), have been isolated increasingly, and currently account for almost 40% of the carbapenem-resistant isolates (unpublished data). We therefore decided to investigate the resistance mechanisms of these meropenem-resistant, ceftazidime-susceptible isolates.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
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The study included all meropenem-resistant, ceftazidime-susceptible P. aeruginosa non-repetitive isolates recovered consecutively from clinical infections of separate patients at University Hospital of Larissa, Thessaly, Greece, from May to December 2003. This tertiary care hospital has 550 beds and covers ~1 million population. The isolates were identified at the species level using the Vitek 2 automated system (bioMérieux, Marcy l'Etoile, France) according to the manufacturer's instructions. Identification was confirmed by using the API 20NE system (bioMérieux).

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 Mueller–Hinton 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 RT–PCR 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 RT–PCR 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.


    Results
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 Abstract
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 Materials and methods
 Results
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Thirteen non-repetitive meropenem-resistant, ceftazidime-susceptible P. aeruginosa strains were isolated during the study period, representing 35.1% of the 37 P. aeruginosa that exhibited resistance to either imipenem or meropenem. The MICs of meropenem, imipenem, and ceftazidime and their resistance phenotype against alternative anti-pseudomonal antimicrobials are shown in Table 1. The MBL Etest was negative in the 12 imipenem-intermediate or low-level-resistant isolates and positive in the highly resistant one. The blaIMP and blaSPM genes were not detected in any isolate, whereas blaVIM was detected in the MBL producer that was shown by sequencing to harbour a blaVIM-2 allele. PFGE showed that the 13 ceftazidime-susceptible pseudomonads belonged to seven distinct types (Table 1).


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Table 1. MICs, synergy test results and PFGE types of the 13 P. aeruginosa isolates in the study

 
A degree of synergy between CCCP and meropenem was observed by agar dilution or disc diffusion, in all blaVIM-negative isolates (Table 1), and between CCCP and aztreonam (data not shown) in nine of the isolates. The inhibition halos around discs of imipenem, ceftazidime, ciprofloxacin, ofloxacin, amikacin, and kanamycin were similar in agar plates containing CCCP, compared with those observed in CCCP-free medium (data not shown).

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. RT–PCR for mexB showed a hyperexpression of the gene in 10 isolates, while in two isolates a product was not visible. The RT–PCR 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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Figure 1. RT–PCR products showing expression of the 16S rRNA (a), mexB (b) and mexY (c) genes of representative P. aeruginosa isolates. Lanes L, 100 bp DNA ladder. The index numbers of the isolates are those listed in Table 1.

 

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 Materials and methods
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The findings of the present study clearly show that carbapenem resistance of P. aeruginosa, which was in our hospital commonly due to the production of VIM-2 or VIM-4 MBLs,3 has now also emerged in strains expressing the tripartite efflux systems MexAB-OprM and MexXY-OprM.

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 (8–16 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.


    References
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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1. Kohler T, Michea-Hamzehpour M, Epp SF et al. Carbapenem activities against Pseudomonas aeruginosa: respective contributions of OprD and efflux systems. Antimicrob Agents Chemother 1999; 43: 424–7.[Abstract/Free Full Text]

2. Livermore DM. The impact of carbapenemases on antimicrobial development and therapy. Curr Opin Investig Drugs 2002; 3: 218–24.[Medline]

3. Pournaras S, Maniati M, Petinaki E et al. Hospital outbreak of multiple clones of Pseudomonas aeruginosa carrying the unrelated metallo-ß-lactamase gene variants blaVIM-2 and blaVIM-4. J Antimicrob Chemother 2003; 51: 1409–14.[Abstract/Free Full Text]

4. Senda K, Arakawa Y, Ichiyama S et al. PCR detection of metallo-ß-lactamase gene (blaIMP) in gram-negative rods resistant to broad-spectrum ß-lactams. J Clin Microbiol 1996; 34: 2909–13.[Abstract]

5. Poirel L, Magalhaes M, Lopes M et al. Molecular analysis of metallo-ß-lactamase gene blaSPM-1-surrounding sequences from disseminated Pseudomonas aeruginosa isolates in Recife, Brazil. Antimicrob Agents Chemother 2004; 48: 1406–9.[Abstract/Free Full Text]

6. Yan J-J, Hsueh P-R, Ko W-C et al. Metallo-ß-lactamases in clinical Pseudomonas isolates in Taiwan and identification of VIM-3, a novel variant of the VIM-2 enzyme. Antimicrob Agents Chemother 2001; 45: 2224–8.[Abstract/Free Full Text]

7. Quale J, Bratu S, Landman D et al. Molecular epidemiology and mechanisms of carbapenem resistance in Acinetobacter baumannii endemic in New York City. Clin Infect Dis 2003; 37: 214–20.[CrossRef][ISI][Medline]

8. Masuda N, Sakagawa E, Ohya, S et al. Substrate specificities of MexAB-OprM, MexCD-OprJ, and MexXY-OprM efflux pumps in Pseudomonas aeruginosa. Antimicrob Agents Chemother 2000; 44: 3322–7.[Abstract/Free Full Text]

9. Yoneda K, Chikumi H, Murata T et al. Measurement of Pseudomonas aeruginosa multidrug efflux pumps by quantitative real-time polymerase chain reaction. FEMS Microbiol Lett 2005; 243: 125–31.[CrossRef][ISI][Medline]

10. Llanes C, Hocquet D, Vogne C et al. Clinical strains of Pseudomonas aeruginosa overproducing MexAB-OprM and MexXY efflux pumps simultaneously. Antimicrob Agents Chemother 2004; 48: 1797–802.[Abstract/Free Full Text]