JAC Advance Access originally published online on November 2, 2007
Journal of Antimicrobial Chemotherapy 2008 61(1):221-222; doi:10.1093/jac/dkm420
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Research letters |
Multidrug-resistant Pseudomonas aeruginosa isolate co-expressing extended-spectrum β-lactamase PER-1 and metallo-β-lactamase VIM-2 from Turkey
1 Malatya State Hospital, Clinic Microbiology Laboratory, 44000 Malatya, Turkey 2 UPRES EA3539, South-Paris Medical School, 94275 Le K-Bicêtre, France 3 Firat University Medical Center, Clinic Microbiology Laboratory, 23000 Elazig, Turkey 4 Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris and UPRES EA3539, Faculté de Médecine Paris-Sud, Université Paris XI, 94275 Le K-Bicêtre, France
* Corresponding author. Tel: +33-1-45-21-36-32; Fax: +33-1-45-21-63-40; E-mail: nordmann.patrice{at}bct.aphp.fr
Keywords: P. aeruginosa , ESBLs , MBLs
The emergence of acquired metallo-β-lactamases (MBLs) in Gram-negative bacteria, especially in Pseudomonas spp., gives rise to significant therapeutic challenges for treating nosocomial infections due to multidrug-resistant pathogens.1 The first reports of transferable MBLs reported IMP types at the end of the 1980s in Japan. The most common MBL identified worldwide is now VIM-2,1,2 but only VIM-5 and IMP-1 MBLs have been identified in Turkey to date.1,3 Although clavulanic acid-inhibited extended-spectrum β-lactamases (ESBLs) are rarely reported from Pseudomonas aeruginosa, ESBL PER-1-positive P. aeruginosa are widespread in Turkey.4
In this study, we report a multidrug-resistant P. aeruginosa isolate, which was recovered from a 2-year-old child suffering from pneumonia in an underlying context of cystic fibrosis. The strain was isolated from several endotracheal aspirates and blood cultures and was identified with the API32GN system (bioMérieux, Marcy l'Étoile, France). Disc diffusion and broth microdilution methods were used to determine antibiotic susceptibility, and results were interpreted according to the CLSI criteria.
P. aeruginosa strain YUS was resistant to multiple antibiotics, but remained susceptible to ciprofloxacin and colistin, with MICs of 0.25 mg/L. MICs of imipenem and meropenem were 128 mg/L, and those of ceftazidime, cefepime, aztreonam and piperacillin/tazobactam were 
256 mg/L. MICs of all other agents tested (chloramphenicol, tetracycline, gentamicin, amikacin, sulphonamides and rifampicin) were all 32 mg/L. MBL detection was performed by using Etest MBL strips (AB Biodisk, Solna, Sweden) and gave a positive result. ESBL detection was performed by a synergy test using discs of ticarcillin/clavulanic acid and aztreonam (which is not a substrate for MBLs) and also gave a positive result, thus indicating expression of both ESBL and MBL enzymes.
Whole-cell DNA of strain YUS was extracted as reported previously2 and was used as template for PCR amplification using primers able to amplify the MBL and ESBL genes [primers PER-A (5'-ATGAATGTCATTATAAAAGC-3') and PER-D (5'-AATTTGGGCTTAGGGCAGAA-3') for bla PER-1 and primers VIM-2A (5'-ATGTTCAAACTTTTGAGTAAG-3') and VIM-2B (5'-CTACTCAACGACTGAGCG-3') for screening of bla VIM-2-type genes]. A bla VIM-2 gene was identified as the first gene cassette in an In60 class 1 integron and was located upstream of an aacA4 cassette, which encodes the AAC(6')-Ib aminoglycoside acetyltransferase. In addition, strain YUS also carried a bla PER-1 ESBL-encoding gene inside a Tn1213 composite transposon structure, as previously demonstrated.5 This study is only the second report of co-production of VIM-2 and PER-1 in P. aeruginosa after that of Docquier et al.6 from Italy. Attempts to transfer ceftazidime resistance which could be mediated either by the MBL or by the ESBL determinant to an Escherichia coli or a P. aeruginosa recipient strain2 failed, as did attempts to demonstrate the presence of plasmids in strain YUS.
The locations of the blaPER-1 and blaVIM-2 genes were determined using the endonuclease I-Ceu-I technique.7 PFGE gave four DNA fragments from both P. aeruginosa strain YUS and a P. aeruginosa reference isolate (Figure 1). A DNA probe for 16S rRNA, consisting of a 1504 bp PCR internal fragment obtained with primers A (5'-AGAGTTTGATCHTGGYTYAGA-3') and B (5'-ACGGYTACCTTGTTACGACTT-3'), hybridized with all the fragments, two of these fragments co-hybridized with DNA probes internal to bla VIM-2 and bla PER-1 genes, respectively (Figure 1). Therefore, it was deduced that the two β-lactamase genes were chromosomally located.
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Aztreonam is the only β-lactam that may remain fully active against MBL producers, unless the strain possesses other mechanisms of β-lactam resistance; isolate YUS was resistant to aztreonam because of production of PER-1 ESBL. As colistin is not available in Turkey, the patient was treated with meropenem, amikacin and ciprofloxacin. Unfortunately, she died because of septic complications, probably as a consequence of weak antibiotic activity (only ciprofloxacin was active). Identification of both MBL and ESBL in a single P. aeruginosa isolate may represent the ultimate state of multidrug resistance in that species. Furthermore, the emergence of VIM-2 MBL in Turkey, identified for the first time, represents a significant additional threat when considered in the context of high levels of antibiotic resistance in that country.
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This work was partially funded by a grant from the Ministère de l'Education Nationale et de la Recherche (UPRES-EA3539), Université Paris XI, France and mostly by a grant from the European Community (LSHM-CT-2005-018705). L. P. is a researcher from the INSERM, France.
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None to declare.
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1 Walsh TR, Toleman MA, Poirel L, et al. Metallo-β-lactamases: the quiet before the storm? Clin Microbiol Rev (2005) 18:306–25.
2
Poirel L, Naas T, Nicolas D, et al. Characterization of VIM-2, a carbapenem-hydrolyzing metallo-β-lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France. Antimicrob Agents Chemother (2000) 44:891–7.
3 Aktas Z, Bal C, Midilli K, et al. First IMP-1-producing Klebsiella pneumoniae isolate in Turkey. Clin Microbiol Infect (2006) 12:695–6.[CrossRef][Web of Science][Medline]
4
Nordmann P, Ronco E, Naas T, et al. Characterization of a novel extended-spectrum β-lactamase from Pseudomonas aeruginosa. Antimicrob Agents Chemother (1993) 37:962–9.
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Poirel L, Cabanne L, Vahaboglü H, et al. Genetic environment and expression of the extended-spectrum β-lactamase blaPER-1 gene in Gram-negative bacteria. Antimicrob Agents Chemother (2005) 49:1708–13.
6 Docquier JD, Luzzaro F, Amicosante G, et al. Multidrug-resistant Pseudomonas aeruginosa producing PER-1 extended-spectrum serine-β-lactamase and VIM-2 metallo-β-lactamase. Emerg Infect Dis (2001) 7:910–1.[Web of Science][Medline]
7 Liu SL, Hessel A, Sanderson KE. Genomic mapping with I-Ceu-I, an intron-encoded endonuclease specific for genes for ribosomal RNA in Salmonella spp. Escherichia coli, and other bacteria. Proc Natl Acad Sci USA (1993) 90:6870–4.
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