Dissemination and genetic support of broad-spectrum beta-lactam-resistant Escherichia coli strain isolated from two Tunisian hospitals during 2004-2012.

BACKGROUND: The dissemination of extended-spectrum ß-lactamase (ESBL)-producing bacteria presented a great concern worldwide. Gram-negative organisms such as Escherichia coli and Klebsiella pneumoniae are the most frequently isolated pathogens responsible for nosocomial infections. OBJECTIVES: The aim of this study was to investigate and to follow the emergence of resistance and the characterization of Extended-Spectrum Beta-Lactamases (ESBL) among broad-spectrum beta-lactam-Escherichia coli clinical isolates recovered from the military hospital and Habib Thameur hospital in Tunisia. METHODS: A total of 113 E.coli isolates obtained during the period 2004 through 2012 showed a significant degree of multi-resistance. Among these strains, the double-disk synergy test confirmed the ESBL phenotype in 46 isolates. These included 32(70%) strains from Hospital A and 14(30%) from Hospital B. RESULTS: The ESBL was identified as CTX-M-15. The ESBL resistance was transferred by a 60 kb plasmid CTXM-15-producing isolates were unrelated according to the PFGE analysis and characterization of the regions surrounding the blaCTX-M-15 showed the ISEcp1 elements located in the upstream region of the bla gene and 20 of them truncated by IS26. CONCLUSION: ESBL producing E. coli strains are a serious threat in the community in Tunisia and we should take into consideration any possible spread of such epidemiological resistance.

Multidrug-resistant phenotype and isolation of a novel SHV- beta-Lactamase variant in a clinical isolate of Enterobacter cloacae.

BACKGROUND: ESBL-producing bacteria are a clinical problem in the management of diseases caused by these pathogens. Worldwide, systemic infections with BL enzymes are evolving by mutations from classical bla genes in an intensified manner and they continue to be transferred across species. RESULTS: E.cloacae BF1417 isolate and its transconjugants gave positive results with the DDST, suggesting the presence of ESBL. Sequence analysis revealed a bla SHV-ESBL-type gene that differs from the gene encoding SHV-1 by five point mutations resulting in three amino acid substitutions in the coding region: C123R, I282T and L286P. This novel SHV-type enzyme was designated SHV-128. The conjugation tests and plasmid characterization showed that the bla SHV-128 is located on a conjugative plasmid IncFII type. Expression studies demonstrated that the above mutations participated in drug resistance, hydrolysis of extended spectrum ß-lactam and the change of the isoelectric point of the protein. CONCLUSION: These findings underscore the diversity by which antibiotic resistance can arise and the evolutionary potential of the clinically important ESBL enzymes. In addition, this study highlights the need for systematic surveillance of ESBL-mediated resistance as well as in clinical areas and communities.

Nosocomial dissemination of plasmids carrying blaTEM-24, blaDHA-1, aac(6')-Ib-cr, and qnrA6 in Providencia spp. strains isolated from a Tunisian hospital.

The aim of this study is to report the emergence of IncA/C conjugative plasmids harboring blaTEM-24, blaDHA-1, qnrA6, and aac(6')-Ib-cr genes among Providencia spp. isolates recovered in 2008 in Tunisia. The double-disk synergy test confirmed the phenotype extended-spectrum ß-lactamase (ESBL) in 2 Providencia stuartii and 5 Providencia rettgeri. These ESBLs were coresistant to cefoxitin, chloramphenicol, ofloxacin, and sulfonamides but remained susceptible to imipenem. Three ß-lactamases TEM-2, TEM-24, and DHA-1 were detected. blaTEM-24, blaDHA-1, aac(6')-Ib-cr, and qnrA6 genes were successfully transferred to Escherichia coli strain HB101, and they were found located on the conjugatifs IncA/C plasmids. Genetic relatedness showed similar and different patterns among P. stuartii and P. rettgeri strains, respectively.

First characterization of a Providencia stuartii clinical isolate from a Tunisian intensive care unit coproducing VEB-1-a, OXA-2, qnrA6 and aac(6')-Ib-cr determinants.

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-ß-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-ß-lactamase. The characterization of ß-lactamases and associated resistance genes was performed by isoelectric focusing, polymerase chain reaction and nucleotide sequencing. Two ß-lactamases bands with pI values of 5.4 and 7.7, which were matched to TEM-1, VEB-1-a and OXA-2-like ß-lactamases were detected. The blaVEB-1-a gene was found to be associated with complex genetic structures, including Re elements. These ß-lactamases were not transferred by electroporation or conjugation experiments to the transconjugants and electroporants. Hybridization methods showed that the extended-spectrum-ß-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.

Prevalence of quinolone resistance determinant qnrA6 among broad- and extended-spectrum beta-lactam-resistant Proteus mirabilis and Morganella morganii clinical isolates with sul1-type class 1 integron association in a Tunisian Hospital.

OBJECTIVES: The aim of this study was to investigate the prevalence and the emergence of plasmid-mediated quinolone resistance among broad-spectrum beta-lactam-resistant Proteus mirabilis and Morganella morganii clinical isolates recovered in the Military Hospital in Tunisia. METHODS: Of 200 strains examined, 50 exhibited resistance to quinolones. Quinolone resistance determinants (qnr and aac(6')-Ib-cr) were characterized by multiplex PCR and sequencing. Chromosomal quinolone resistance mutations in the quinolone resistance-determining region (QRDR) and class 1 integron characterization were analysed by PCR and sequencing. The clonal relationship between the isolates was studied by pulsed-field gel electrophoresis (PFGE). RESULTS: Fourteen isolates harboured qnrA6 and among them 8 (57%) were extended-spectrum beta-lactamase (ESBL) producers, whilst 12 (85%) isolates harboured blaDHA-1. Mutations in the QRDR were detected in gyrA (Ser83Ile, Glu87Lys), gyrB (Ser464Phe), and parC (Ser80Ile). qnrA6 and blaDHA-1 genes were found embedded in complex sul1-type class 1 integrons. A gene cassette carrying aac(6')-Ib-cr was found located in the class 1 integron upstream of the qacEΔ1 gene. According to the PFGE analysis, the isolates were clonally unrelated. CONCLUSIONS: This is the first description in North Africa of class 1 integrons carrying blaDHA-1, qnrA6 gene, and aac(6')-Ib-cr determinants in clinical strains of Proteus mirabilis and Morganella morganii.