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Objective: The aim of this study was to characterize the O25b/ST131 clone in ciprofloxacin-resistant Escherichia coli isolates from Yemen. Materials and Methods: A total of 41 ciprofloxacin-resistant E. coli strains were collected from clinical samples of inpatients and outpatients from Sana'a (Yemen) from January to December 2013. Antimicrobial susceptibility testing, polymerase chain reaction amplification, and sequencing were used for detection of plasmid-mediated quinolone resistance determinants, extended-spectrum beta-lactamases genes and mutations in the quinolone resistance-determining regions of the target genes gyrA and parC. Genetic relatedness of E. coli isolates was determined by pulsed-field gel electrophoresis (PFGE). O25b/ST131 clone detection was performed using polymerase chain reaction of O25b rfb and allele 3 of the pabB gene and by a multilocus sequence typing. Results: All E. coli isolates contained the aac(6')Ib-cr gene associated with blaCTX-M-15 and qnrS genes in 63.4% and 12.2%, respectively. A rate of 36.6% (15/41) of O25b/ST131 E. coli isolates were identified belonging to the H30-Rx subclone producing both CTX-M-15 and Aac(6')Ib-cr enzymes and carrying two substitutions in GyrA (Ser83Leu/Asp87Asn) and two substitutions in ParC (Ser80Ile/Glu84Val). Most of them were uropathogenic unrelated E. coli isolates recovered from outpatients. Conclusion: This is the first report of a high prevalence of E. coli O25b/ST131 from Yemen.

The objective was to characterize a group of clinical isolates of fluoroquinolone-resistant Haemophilus parainfluenzae collected in Northern Spain (March-December 2014).Twelve clinical isolates of H. parainfluenzae were studied by performing antimicrobial susceptibility testing and PCR amplification and nucleotide sequencing of the QRDR (quinolone resistance-determining region) of gyrA, parC, gyrB, and parE genes. Screening for plasmid-mediated quinolone resistance (PMQR) was also studied. Pulsed-field gel electrophoresis (PFGE) was used for molecular typing.Antimicrobial susceptibility testing showed that all the isolates were resistant to the fluoroquinolones tested (ciprofloxacin, levofloxacin, norfloxacin, and moxifloxacin). Analysis of the QRDR demonstrated that all the isolates presented mutations in gyrA and parC. A Glu88Lys substitution in ParC is reported for the first time in H. parainfluenzae. No PMQR gene was detected. PFGE results showed that isolates were not clonally related.Multiple H. parainfluenzae fluoroquinolone-resistant isolates grouped in the same area in a short period of time showed diverse substitutions in QRDR of gyrA/parC and were not clonally related, indicating individual emergence. In addition, we described the first report of Glu88Lys substitution in ParC.

The objective was to evaluate the cytotoxic effect associated with overexpression of multiple Qnr-like plasmid-mediated quinolone resistance (PMQR) mechanisms in Escherichia coli.Coding regions of different PMQR genes (qnrA1, qnrB1, qnrC, qnrD1, qnrS1, and qepA2) and efsqnr were cloned into pET29a(+) vector and overexpressed in E. coli BL21. E. coli BL21 with and without an empty pET29a(+) vector were used as controls. The cytotoxic effect associated with PMQR mechanism overexpression was determined by transmission electron microscopy and viability assays.Overexpressed qnr genes produced loss of bacterial viability in the range of 77-97% compared with the controls, comparable with loss of viability associated with EfsQnr overexpression (97%). No loss of viability was observed in E. coli overexpressing QepA2. In transmission electron microscopy assays, signs of cytotoxicity were observed in E. coli cells overexpressing EfsQnr and Qnr proteins (30-45% of the bacterial population showed morphological changes). Morphological changes were observed in less than 5% of bacterial populations from the control strains and E. coli overexpressing QepA2.Overexpression of qnr genes produces a cytotoxic cellular and structural effect in E. coli, the magnitude of which varies depending on the family of Qnr proteins.

The objective was to assess the prevalence of plasmid-mediated quinolone resistance (PMQR)-producing isolates in a collection of quinolone-resistant Enterobacteriaceae of community origin isolated in Bejaia, Algeria.A total of 141 nalidixic acid-resistant Enterobacteriaceae community isolates were collected in Bejaia (Northern Algeria) and screened for PMQR genes using polymerase chain reaction (PCR). For PMQR-positive strains, antimicrobial susceptibility testing was performed by broth microdilution and disk diffusion. Mutations in the quinolone resistance-determining regions of the target genes, gyrA and parC, were detected with a PCR-based method and sequencing. Southern blotting, conjugation and transformation assays and molecular typing by pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing were also performed.The prevalence of PMQR-producing Enterobacteriaceae isolates was 13.5% (19/141); 11 of these isolates produced Aac(6')-Ib-cr and 8 were qnr-positive (4 qnrB1-like, 2 qnrS1-like, and 2 qnrD1-like), including the association with aac(6')-Ib-cr gene in three cases. PMQR gene transfer by conjugation was successful in 6 of 19 isolates tested. PFGE revealed that most of the PMQR-positive Escherichia coli isolates were unrelated, except for two groups comprising two and four isolates, respectively, including the virulent multidrug-resistant clone E. coli ST131 that were clonally related.Our findings indicate that PMQR determinants are prevalent in Enterobacteriaceae isolates from the community studied. We describe the first report of the qnrD gene in Algeria.

QepA is a plasmid-mediated quinolone resistance determinant of low prevalence described worldwide, mainly in Enterobacteriaceae. This study describes, for the first time in Algeria, two clonally related, QepA-producing Escherichia coli clinical isolates positive for CTX-M-15. The clonal spread of these multidrug-resistant isolates is a major public health concern.