One hundred extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae were recovered from the intensive care unit and the urology ward of the University Hospital of Sahloul in Tunisia between May 2005 and May 2006. The majority of strains showed a high level of resistance to cefotaxime and ceftazidime. Double-disk synergy test and E-test strips were used to confirm production of ESBLs. The molecular analysis revealed that the majority of strains (91%) carried genes encoding CTX-M-15. SHV-12 and SHV-2a were produced, respectively, by 9% and 3% of the strains. Pulsed-field gel electrophoresis of ESBL-producing Klebsiella pneumoniae isolates revealed four different clonal groups and three for Escherichia coli , showing the absence of spread of any epidemic clone. The CTX-M-15 ESBL-producing E. coli of the major clonal group belong to the B2 phylogenetic group, to the sequence type 131, and has a high virulence potential. In conclusion, CTX-M-15 ESBLs accounted for the overwhelming majority of ESBL types among Enterobacteriaceae from our hospital. This study confirms the high rate of ESBLs in Tunisia and further demonstrates the worldwide spread of genes coding for CTX-M-15 enzymes in clinical isolates.

The aim of this study was to describe the distribution of the trimethoprim–sulfamethoxazole resistance genes and their association with class 1 integrons in a collection of clinical isolates of Enterobacteriaceae recovered at the University Hospital Sahloul in Tunisia. A total of 80 isolates of Enterobacteriaceae were studied, including six different species. There were 35 extended-spectrum beta-lactamases (ESBL)-producing isolates. Resistance to trimethoprim–sulfamethoxazole was assessed by the disk diffusion method. Polymerase chain reaction (PCR) with primers specific for sul1 , sul2 , and sul3 was used to detect the three known sulphonamide resistance genes. The presence of class 1 integrons in the studied isolates was detected using PCR and the resistance gene cassettes were characterized by directly sequencing the PCR products obtained with 5′conserved segment (5′CS) and 3′conserved segment (3′CS) primers. The int1 gene was found in 68 out of 80 enterobacterial isolates. The sul1 gene was found in 22 isolates (27.5%), sul2 gene in 5 isolates (6.25%), and both genes in 49 isolates (61.25%). Eight of the studied isolates had no dfr alleles, and in the remaining 72 isolates, 7 dfr genes were identified. The most prevalent were dfrA7 (40%) and dfrA17 (33%). Class 1 integrons were found to be an important genetic element of resistance to trimethoprim–sulfamethoxazole among the clinical isolates of Enterobacteriaceae. The types, combinations, and frequency of the gene cassettes in integrons provide useful data for the surveillance of antimicrobial resistance in our hospital and for the prescription practice of cotrimoxazole.

Molecular features of the first carbapenem-resistant Klebsiella pneumoniae isolates (KP1 and KP2) from the University Hospital Tahar Sfar, Tunisia, were investigated. Antimicrobial susceptibility testing, multilocus sequence typing, S1 nuclease pulsed-field gel electrophoresis, Southern blot, and polymerase chain reaction (PCR)-based replicon typing were performed. Extended-spectrum β-lactamases and carbapenemase genes were detected by PCR and sequencing. Both isolates were multidrug resistant. KP1 was of sequence type (ST) ST101 and exhibited bla _CTX-M-15 and bla _TEM-1 on an untypeable plasmid and bla _OXA-48 on an IncL/M plasmid. KP2 was genetically unrelated to KP1 (ST147) and harbored an IncA/C plasmid carrying bla _CMY-4 and the bla _OXA-48 derivative gene: bla _OXA-204 . This study reports the second case worldwide of an OXA-204-producing K. pneumoniae isolate from the same country, however, in a different genetic background.

This study was conducted to identify the molecular mechanisms of imipenem resistance in a Klebsiella pneumoniae (Kp16137) isolate recovered in August 2008 at the University Hospital Sahloul, Sousse, Tunisia. The strain was identified with the API 20E system; antibiotic-containing disks were used for detection of antibiotic susceptibility by a disk diffusion assay. We investigated the presence of β-lactamases by PCR, using specific primers for bla _TEM , bla _SHV , bla _CTX-M , bla _OXA , bla _CMY , bla _ACC , bla _FOX , bla _IMP , bla _KPC , bla _VIM , and by sequencing. Extraction of plasmid DNA from Kp16137 and the transconjugant was performed by the method of Kado. Southern transfer was performed on nylon. The membrane was hybridized with a specific probe for the bla _CMY-2 gene. Outer membrane proteins were isolated and were examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 12% polyacrylamide gel. K. pneumoniae Kp16137 was resistant to all available β-lactams, including third generation cephalosporins and carbapenems. The screening of β-lactamases showed the presence of three β-lactamases: TEM-1, SHV-61, and CMY-4. The CMY-4 β-lactamase was located on an 80-kb plasmid. An analysis of the outer membrane proteins of this isolate revealed that it lacked a porin of 42 kDa. The loss of this outer membrane protein band correlated with imipenem resistance in this strain. In K. pneumoniae 16137, synthesis of a plasmid-mediated β-lactamase: AmpC CMY-4, together with alteration in permeability led to resistance to all available β-lactams and carbapenems.

Purpose: To study the resistance to third-generation cephalosporins in Providencia stuartii strain isolated from hospitalized patient in Tunisia and to identify the responsible genes Materials and Methods: This strain was analysed by PCR and sequencing to identify the genes responsible for the β-lactamase resistance phenotypes. The transferability of the phenotypes was tested by conjugation to Escherichia coli J53. The isoelectric point was determinate by isoelectrofocalisation. Results: This resistance was carried by a 60 kb plasmid that encoded a β-lactamase with a pI of 5.4. This β-lactamase revealed identity with the blaTEM-1 gene encoding the TEM-1 β-lactamase, except for a replacement of the Val residue at position 84 by Ile, and the Ala residue at position 184 by Val. These two mutations were encountered in TEM-116 β-lactamase. Conclusion: This study demonstrates the first description of TEM-116 in the P. stuartii species in the world and the first one in a Tunisian hospital.