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This study investigated the genetic environment of bla CTX-M genes and associated resistance genes in seven Proteus mirabilis and six Morganella morganii extended spectrum b-lactamase (ESBL)-positive isolates. The isolates were recovered from hospitalized patients with respiratory or urinary tract infections at the Military Hospital of Tunis, Tunisia. Twenty-one of the 200 strains exhibited non-susceptibility to third generation cephalosporins and, among these strains, the double-disk synergy test confirmed the ESBL phenotype in 13 isolates. These ESBL producers were co-resistant to chloramphenicol, tetracycline and oflaxacin but remained susceptible to ertapenem (MIC,0.25 mg l "1 ). The presence and nature of bla CTX-M-15 , bla CTX-M-8 , bla TEM-24 , bla TEM-1 and bla TEM-2 genes was determined by PCR and sequencing. Chromosomal localization of the bla CTX-M-15 gene was confirmed in all strains, with the exception of M. morganii isolate M-17991, by Southern-blot analysis performed either on chromosomal or plasmid DNA. M. morganii M-12012 and M. morganii M-6019 showed the same PFGE pattern, whereas the remaining CTX-M-15-producing isolates were unrelated. The presence of ISEcp1 was ascertained in CTX-M-15-producing isolates. A class 1 integron with different gene cassettes (dfrA1, orfC and aadB) was found in five P. mirabilis and six M. morganii isolates. INTRODUCTIONCTX-M b-lactamases are the most widespread enzymes, with a growing prevalence in different members of the Enterobacteriaceae family worldwide (Livermore et al., 2007). They are not confined to only human isolates but are also present in animal and environmental bacteria (Nordmann et al., 2008; Díaz et al., 2010; Dolejska et al., 2011). The genes encoding these enzymes have been found in Escherichia coli, Klebsiella pneumoniae and Salmonella enterica. The penetration of bla CTX-M genes to many bacterial populations could be a consequence of the extraordinary dissemination of these genes by mobile elements, including plasmids, transposons and insertion sequences (IS) (ISEcp1 or ISCR1). In several reports, the role of ISEcp1-like sequences in the spread and expression of bla CTX-M genes has been ascertained (Poirel et al., 2003;Nordmann et al., 2008). ISEcp1 mobilizes the downstreamlocated gene by a transposition mechanism and also provides a strong promoter for high-level expression of bla CTX-M (Poirel et al., 2003). In this study, we investigated the genetic elements associated with the bla CTX-M-15 gene and described the surrounding sequences that included three IS elements, ISEcp1, IS26 and IS903 in seven Proteus mirabilis and six Morganella morganii extended spectrum b-lactamase (ESBL)-positive isolates recovered from the Military Hospital of Tunis. METHODSA total of 200 P. mirabilis (n5120) and M. morganii (n580) isolates were obtained from different hospitalized patients in the Military Hospital of Tunis, Tunisia, from May 2005 to June 2009. The isolates were collected from various pathological specimens, which included urine (33 %), blood (24...

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A clinical Providencia stuartii isolate SM662 was recovered from a patient hospitalized in the intensive care unit at the Military hospital, Tunisia. This isolate was resistant to penicillins, cephalosporins, aminoglycosides and fluoroquinolones. A marked in vitro synergy between ceftazidime or cefotaxime and amoxicillin-clavulanic acid on Mueller-Hinton agar plates suggested the presence of an extended-spectrum-β-lactamase. In addition, an unusual synergy was found between cefepime or aztreonam, and cefoxitin or imipenem on a double disk synergy test suggesting a VEB-type extended-spectrum-β-lactamase. The characterization of β-lactamases and associated resistance genes was performed by isoelectric focusing, polymerase chain reaction and nucleotide sequencing. Two β-lactamases bands with pI values of 5.4 and 7.7, which were matched to TEM-1, VEB-1-a and OXA-2-like β-lactamases were detected. The blaVEB-1-a gene was found to be associated with complex genetic structures, including Re elements. These β-lactamases were not transferred by electroporation or conjugation experiments to the transconjugants and electroporants. Hybridization methods showed that the extended-spectrum-β-lactamase encoding gene may have a chromosomal localization. The isolate SM662 produced the quinolone resistance determinants qnrA6 and aac(6')-Ib-cr which were successfully transferred. The detection of an associated chromosomal quinolone resistance revealed the presence of a gyrA mutation at codon 83 (Ser83Ile). This is the first report of the linkage VEB-1-a/OXA-2-like in P. stuartii associated with the description of qnrA6 and aac(6')-Ib-cr genes in this isolate.