BackgroundTwenty 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.MethodsAnalysis 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.ResultsAll 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).ConclusionsThis 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.

From 2002 to 2006, 35 of 73 multidrug-resistant Pseudomonas aeruginosa isolates from different wards at Charles Nicolle hospital of Tunis were positive for class B carbapenemase (using the imipenem-EDTA test), owing to a bla(VIM-2) gene cassette in a class 1 integron. Twenty-three isolates additionally produced the extended-spectrum beta-lactamase SHV2a. DNA sequences immediately surrounding bla(SHV2a) shared extensive identity with a Klebsiella pneumoniae plasmid sequence. Despite belonging to the same chromosomal type, as shown by pulsed-field gel electrophoresis (PFGE), the VIM-2 producing P. aeruginosa isolates prevalent at Charles Nicolle hospital displayed a diversity of VIM-2-carrying integrons.

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We used capillary electrophoresis–single-strand conformation polymorphism (CE-SSCP) analysis of PCR-amplified 16S rRNA gene fragments for rapid identification of Pseudomonas aeruginosa and other gram-negative nonfermenting bacilli isolated from patients with cystic fibrosis (CF). Target sequences were amplified by using forward and reverse primers labeled with various fluorescent dyes. The labeled PCR products were denatured by heating and separated by capillary gel electrophoresis with an automated DNA sequencer. Data were analyzed with GeneScan 672 software. This program made it possible to control lane-to-lane variability by standardizing the peak positions relative to internal DNA size markers. Thirty-four reference strains belonging to the genera Pseudomonas,Brevundimonas, Burkholderia,Comamonas, Ralstonia,Stenotrophomonas, and Alcaligenes were tested with primer sets spanning 16S rRNA gene regions with various degrees of polymorphism. The best results were obtained with the primer set P11P-P13P, which spans a moderately polymorphic region (Escherichia coli 16S rRNA positions 1173 to 1389 [M. N. Widjojoatmodjo, A. C. Fluit, and J. Verhoef, J. Clin. Microbiol. 32:3002–3007, 1994]). This primer set differentiated the main CF pathogens from closely related species but did not distinguishP. aeruginosa from Pseudomonas alcaligenes-Pseudomonas pseudoalcaligenes and Alcaligenes xylosoxidans from Alcaligenes denitrificans. Two hundred seven CF clinical isolates (153 of P. aeruginosa, 26 of Stenotrophomonas maltophilia, 15 ofBurkholderia spp., and 13 of A. xylosoxidans) were tested with P11P-P13P. The CE-SSCP patterns obtained were identical to those for the corresponding reference strains. Fluorescence-based CE-SSCP analysis is simple to use, gives highly reproducible results, and makes it possible to analyze a large number of strains. This approach is suited for the rapid identification of the main gram-negative nonfermenting bacilli encountered in CF.

In 2009, out of the 66 nonrepetitive Enterobacter cloacae collected at Charles Nicolle hospital in Tunisia, 44 were extended spectrum β-lactamase (ESBL) producers. The aim of the current study was to detect and characterize the genes encoding the ESBLs including blaTEM, blaSHV, and blaCTX-M groups by polymerase chain reaction and sequencing. Pulsed-field gel electrophoresis (PFGE) analysis was used to determine the genetic relatedness between isolates. All strains were susceptible to carbapenems. They were resistant to fluoroquinolones, gentamicin, tobramycin, and trimethoprim+sulfamethoxazole but variably resistant to netilmicin, amikacin, and tetracyclines. Sequence analysis of the polymerase chain reaction products revealed the presence of blaCTX-M-15 (39 strains), blaSHV-12 (6 strains), and blaSHV-27 (1 strain). The coexistence of two ESBLs was observed in two isolates harboring, respectively, SHV-12+CTX-M-15 and SHV-27+CTX-M-15. PFGE revealed 36 unrelated profiles. Diffusion of E. cloacae producing CTX-M-15 ESBL in our hospital is the consequence of dissemination of identical or related plasmids harboring the CTX-M-15 gene.