Pseudomonas aeruginosa is a frequent nosocomial pathogen that causes a wide range of opportunistic infections and nosocomial outbreaks 1 2 3 . Its high intrinsic resistance to antibiotics and ability to develop multidrug resistance pose serious therapeutic problems. Four broad-spectrum β-lactams, such as carbapenems, are potential drugs for the therapy of infections caused by P. aeruginosa 4 5 . However, the increasing use of these compounds has resulted in the emergence of carbapenem-resistant P. aeruginosa isolates, limiting treatment options 6 7 . Most carbapenem resistance is due to impermeability, which arises via loss of the OprD (D2) porin, but carbapenem hydrolysing metallo-β-lactamases (MBLs) are increasingly reported 8 . Genes encoding MBLs are located as cassettes in integrons that provide them with the potential for expression and dissemination 9 10 . To date, nine MBL types, namely, IMP-like 9 , VIM-like 9 , SPM-1 11 , GIM-1 12 , SIM-1 13 , AIM-1 14 , KHM-1 15 , NDM-1 16 and DIM-1 17 , have been identified in Gram negative bacilli. Worldwide, the IMP and VIM types are the most commonly detected MBLs in P. aeruginosa 9 10 . VIM-type MBLs are predominant in the Mediterranean region 9 10 .

At Charles Nicolle hospital of Tunisia, since November 2002, VIM-2 producing P. aeruginosa has been isolated, mainly in surgery and intensive care unit 18 . In 2008, an increasing rate of imipenem resistance in P. aeruginosa was observed in the kidney transplantation unit. The aim of the present study was to determine the occurrence of MBL genes among imipenem resistant isolates and to establish epidemiological relationship among them.

Carbapenems are potent agents against multiresistant Gram negative bacilli, including P. aeruginosa, but their efficacy is increasingly compromised by the emergence and the worldwide dissemination of carbapenem resistance strains, which are implicated in large outbreaks as described in many countries 1 25 . In Tunisia, frequencies of imipenem resistant P. aeruginosa varies between 16% and 37.6% 26 27 28 . At Charles Nicolle Hospital of Tunis, their frequency was stable until 2004 (1%), but increased dramatically from 2005 (25%). They were mainly isolated in surgery and intensive care unit 18 , but in 2008, an increasing rate of multidrug-resistant P. aeruginosa was observed in the kidney transplantation unit. All strains exhibited a multidrug-resistant phenotype; they were resistant to antipseudomonal β-lactams (including aztreonam), aminoglycosides and fluoroquinolones. They remained susceptible only to colistin which is used for the treatment of our kidney transplanted patients despite its renal toxicity 29 . MICs results are shown in table 1. All strains showed a high level of resistance to all β-lactams, particularly to carbapenems (> 512 μg/ml). Only 16 strains (67%) were positive according to the imipenem-EDTA synergy test, suggesting the presence of MBLs. The acquisition of a MBL gene alone does not necessarily confer elevated level of resistance to carbapenems. Indeed, secondary changes in regulatory system of MBL gene expression, outer membrane permeability, active efflux systems in bacterial membrane, and/or multiplication of structure gene might well be implicated in acquisition of high-level carbapenem resistance 30 31 . Aztreonam is the only β-lactam that may remain fully active against MBL producers 8 , however all our strains were resistant to this β-lactam, suggesting the occurrence of other mechanisms of β-lactam resistance 32 . The eight MBL negative strains were also resistant to all antibiotic tested (Table 1), but the mechanism involved in the resistance has not been further examined.

The 16 MBLs-producer strains were positive for bla_VIM-2 gene and none strain harboured the bla _IMP gene (Table 1). In Tunisia, the most common MBL identified VIM-2 in accordance with the actual situation worldwide 9 33 34 . Historically, the first reports of MBLs genes were VIM-2 and VIM-4 types in P. aeruginosa 18 35 and K. pneumoniae 36 respectively.

Serotyping identified 3 serotypes: O4 (n = 16), O11 (n = 4) and O12 (n = 1) (table 1). Only 2 strains were nontypeable with monovalent antisera. In Tunisia 37 as well as in many European countries 38 , it has been repeatedly demonstrated over the past 20 years that serotypes O11 and O12 dominate among multiresistant P. aeruginosa isolates. The 16 MBL-positive strains were divided into 3 pulsotypes designed A (n = 13), B (n = 2) and C (n = 1) (Table 1). The 13 strains of pulsotype A were of serotype O4. These results imply that the dissemination of VIM-2 in our kidney transplantation unit was mainly due to the of spread clonal strains, however, unrelated VIM-2-harboring strains occurred. Outbreaks of VIM β-lactamase-producing P. aeruginosa have been also reported in Greece 3 , Italy 2 and Kenya 39 , but there is still very limited knowledge on the epidemiology of MBLs in Africa.

The emergence of acquired MBLs among P. aeruginosa represents an epidemiological risk for at least two reasons: firstly, MBLs confer resistance not only to carbapenems but to virtually all β-lactams and are frequently associated with resistance to aminoglycosides; and secondly, genes encoding for MBL enzymes are most commonly carried on mobile genetic elements (integrons, plasmids, transposons) that can spread horizontally among unrelated strains 9 18 .

Indeed, the bla _VIM-2 gene was found in strains of different genotypes (A, B and C), reflecting its ability to transfer from one bacterium to another.

In conclusion, we found an outbreak of VIM-2-producing P. aeruginosa in a kidney transplantation unit. Thus, bla _VIM-2 gene may have been spreading in Gram negative rod in Tunisia. This emphasizes the necessity of early recognition of MBL producing isolates, rigorous infection control, and restricted clinical use of broad-spectrum β-lactams including carbapenems.

MBL: metallo-beta-lactamase; PFGE: Pulsed-Field Gel Electrophoresis.

The authors declare that they have no competing interests.

SH designed the study and wrote the manuscript. IBBB, RG, MS, SA and SBR performed critical reading of manuscript and supervision. All authors have read and approved the final manuscript.