Multidrug resistance of Pseudomonas aeruginosa has been attributed to both intrinsic and acquired antibiotic-resistance mechanisms. Multidrug-resistant (MDR) P. aeruginosa isolates have become a serious healthcare problem worldwide because they are resistant to almost all β-lactams, aminoglycosides, and quinolones. Production of zinc-dependent metallo-β-lactamases (MBLs) has been identified as the most significant mechanism among carbapenem-resistant P. aeruginosa isolates [1]. MBLs are of particular clinical concern because of their broad-spectrum activities, and Imipenemase (IMP)-, Verona Integron-Encoded Metallo-β-lactamase (VIM)-, Sao Paulo metallo-β-lactamase (SPM)-, Germany imipenemase (GIM)-, and New Delhi Metallo-β-lactamase (NDM)-type MBLs have been identified in P. aeruginosa worldwide [2]. Forty-six variants of VIM enzymes have been identified to date (http://www.lahey.org/Studies/other.asp). VIM-38 was recently identified in P. aeruginosa isolates in Turkey and was shown to differ from VIM-5 by a single substitution (Ala316Val) [3]. In P. aeruginosa, VIM-type MBLs have been reported within mobile genetic elements such as integrons, which contribute to the dissemination of antibiotic resistance [3].

We here report a new clinical P. aeruginosa strain isolated from a blood sample on January 2015 at Rize State Hospital in Turkey and identified by using the API 32GN system (bioMerieux, Marcy-l'Etoile, France). Minimal inhibitory concentrations were determined on a VITEK system for the following antibiotics: piperacillin/tazobactam, ceftazidime, cefepime, amikacin, netilmicin, ciprofloxacin, levofloxacin, imipenem, meropenem, cefoperazone-sulbactam, and inducible β-lactamase. 16S rDNA sequencing was used for molecular identification, performed according to Cicek et al [4].

The P. aeruginosa isolate was screened for β-lactamase-encoding genes and the class 1–class 2 integrases conserved region by PCR. The primers used for detection of β-lactamase-encoding genes and class 1 and class 2 integron gene cassettes are listed in Table 1 [45678].

The positive PCR product of the class 1 integron was cloned into pGEM-T easy vector (Promega, Madison, WI, USA) and then sequenced by Macrogen (Amsterdam, The Netherlands). Sequencing results were analyzed by using the BLAST alignment search tool (http://www.ncbi.nlm.nih.gov/BLAST) and the multiple sequence alignment program CLUSTALW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/).

Transferability of antibiotic resistance was tested according to the previously defined protocol [9], by using the rifampin-resistant Escherichia coli K-12 strain J53-2 as a recipient [4]. Susceptibility testing of the MBL-producing integron-positive P. aeruginosa isolate showed that it was resistant to imipenem, meropenem, piperacillin/tazobactam, ceftazidime, cefepime, and cefoperazone-sulbactam. PCR analysis showed that the isolate did not harbor any of the antibiotic resistance genes listed in Table 1, except for bla_VIM-type MBL. Sequence analysis of the bla_VIM-variant identified it as bla_VIM-38. The P. aeruginosa isolate contained a class 1 integron gene cassette, but not a class 2 integron gene cassette. The bla_VIM-38-harboring class 1 integron gene cassette was sequenced and was found to be 3,239 bp long. DNA sequence analysis revealed that bla_VIM-38 MBL was located on the class 1 integron gene cassette together with AAC(6´)-Ib/EmrE/aadA1 (Fig. 1). The conjugation assay revealed that the class 1 integron cassette is not transferable.

The bla_VIM-38 gene was identified in P. aeruginosa isolates in Turkey in 2014, and found to be located in a class 1 integron containing only two gene cassettes (bla_VIM-38/orfD) [3]. This genetic structure has also been associated with the bla_VIM-5 gene in a clinical isolate of Enterobacter cloaceae from Turkey [9]. Moreover, steady-state kinetic analyses in a study on the enzymatic properties of VIM-38 showed that VIM-38 hydrolyzed all of the tested penicillins, cephalosporins, and carbapenems [10].

In the present study, the class 1 integron included four gene cassettes with bla_VIM-38 followed by AAC(6´)-Ib, EmrE (multi-drug transporter), and aadA1. This is the first report in Turkey of the bla_VIM-38/AAC(6´)-Ib/EmrE/aadA1 gene cassette array. Therefore, we report a novel gene cassette array with an MBL gene in a P. aeruginosa clinical isolate, and this is the second report for the detection of VIM-38 in a P. aeruginosa isolate in Turkey with different hospitalization and isolation times.

In conclusion, the presence of class 1 integrons in P. aeruginosa leads to increased resistance to antibiotics. The present study demonstrates the emergence of VIM-producing MDR P. aeruginosa strains harboring class 1 integrons and a gene cassette in Turkey. In particular, the bla_VIM-38 MBL gene appears to be spreading among P. aeruginosa isolates in Turkey.