Nosocomial outbreak of imipenem-resistant Pseudomonas aeruginosa producing VIM-2 metallo-β-lactamase in a kidney transplantation unit

Background Twenty four non replicate imipenem resistant P. aeruginosa were isolated between January and November 2008, in the kidney transplantation unit of Charles Nicolle Hospital of Tunis (Tunisia). This study was conducted in order to establish epidemiological relationship among them and to identify the enzymatic mechanism involved in imipenem resistance. Methods Analysis included antimicrobial susceptibility profile, phenotypic (imipenem-EDTA synergy test) and genotypic detection of metallo-β-lactamase (MBL) (PCR), O-serotyping and pulsed-field gel electrophoresis. Results All strains showed a high level of resistance to all antimicrobials tested except to colistin. The presence of MBL showed concordance between phenotypic and genotypic methods. Sixteen isolates were identified as VIM-2 MBL-producers and 13 of them were serotype O4 and belonged to a single pulsotype (A). Conclusions This study describes an outbreak of VIM-2-producing P. aeruginosa in a kidney transplantation unit. Clinical spread of blaVIM-2 gene is a matter of great concern for carbapenem resistance in Tunisia.

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.

Patients, Bacterial strains, Serotyping and susceptibility testing
Twenty four non duplicate imipenem resistant P. aeruginosa isolates were recovered between January and November 2008 in the kidney transplantation unit of Charles Nicolle hospital of Tunis. They were isolated from urine (n = 20), cutaneous pus (n = 3) and blood (n = 1).
All samples were taken for microbial diagnosis from 24 different patients [sex ratio 3.8]. The median age of the patients was 51 years [range: 34 to 80 years]. They were admitted in the urology unit (n = 10) and nephrology unit (n = 14).
A retrospective review was conducted for only 15 patients (6 from urology unit, 9 from the nephrology unit). Date of transplantation was mentioned in Table  1. However, no data concerning kind of transplantation and live donator was collected. All patients presented chronic renal failure associated in 13 cases to at least one underlying co-morbidity [High blood pressure (n = 13), systemic lupus erythematosis (n = 1), diabetes (n = 1), chronic glomerulonephritis (n = 5), nephritic syndrome (n = 1) and renal tuberculosis (n = 1)]. In all patients fever was the major symptom. Ceftazidime combination therapy with colistin accounted for all patients. Attributable mortality was 5% of cases.
Antimicrobial susceptibility was tested with the agar disk diffusion method according to the CLSI guidelines [19]. The MICs of ticarcillin, ticarcillin + clavulanic acid, ceftazidime, aztreonam, cefepime, imipenem and meropenem were determined using the dilution method in Mueller Hinton agar according to the CLSI guidelines [19]. E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as control strains.
To detect MBL production, the imipenem-EDTA synergy test was used [20]. An enlargement of the inhibition zone of imipenem facing the disc of EDTA was considered as a positive test.

PCR amplification
For the detection of bla VIM and bla IMP genes PCR experiments were performed using consensus primers as described previously [21]. Primers specific for bla VIM-2 were also used [22].

Clonal relationship by Pulsed-Field Gel Electrophoresis (PFGE)
Molecular typing of MBL producing isolates was carried out, as described previously by Pulsed-Field Gel Electrophoresis (PFGE) using SpeI restriction endonuclease [23]. Clonal relationships based on PFGE patterns were interpreted according to the criteria proposed by Tenover et al [24].

Results and discussion
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 βlactams, 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 βlactam 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.

Conclusion
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 broadspectrum β-lactams including carbapenems. List of abbreviations MBL: metallo-beta-lactamase; PFGE: Pulsed-Field Gel Electrophoresis.