This study explored and compared the mechanisms and selective concentration of resistance between a 3rd (pradofloxacin) and 2nd (ciprofloxacin) generation fluoroquinolone. Pradofloxacin- and ciprofloxacin-resistant mutants were selected by stepwise exposure of Escherichia coli (E. coli) to escalating concentrations of pradofloxacin and ciprofloxacin. The sequence of the quinolone resistance determining region (QRDR) and the transcriptional regulator soxS were analyzed, and efflux pump AcrAB-TolC activity was measured by quantitative real-time reverse transcription-PCR (qRT-PCR). First-step mutants reduced the fluoroquinolone sensitivity and one mutant bore a single substitution in gyrA. Four of six second-step mutants expressed ciprofloxacin resistance, and displayed additional mutations in gyrA and/or parC, while these mutants retained susceptibility to pradofloxacin. All the third-step mutants were fluoroquinolone resistant, and each expressed multidrug resistance (MDR) phenotypes. Further, they displayed resistance to all antibacterials tested except cefotaxime, ceftazidime and meropenem. The number of mutations in QRDR of gyrA and parC correlated with fluoroquinolone MICs. Mutations in parC were not common in pradofloxacin-associated mutants. Moreover, one second- and one third-step ciprofloxacin-associated mutants bore both mutations at position 12 (Ala12Ser) and 78 (Met78Leu) in the soxS gene, yet no mutations in the soxS gene were detected in the pradofloxacin-selected mutants. Altogether, these results demonstrated that resistance emerged relatively more rapidly in 2nd compared to 3rd generation fluoroquinolones. Point mutations in gyrA were a key mechanism of resistance to pradofloxacin, and overexpression of efflux pump gene acrB played a potential role in the emergence of MDR phenotypes identified in this study.

Background: There are no large epidemiological studies of Clostridium difficile-associated disease (CDAD) in hospitalized children. Aim: To describe the frequency, demography, clinical features and outcome of nosocomial CDAD in children admitted to Assiut University Children's Hospital, Egypt. Patients and Methods: In this descriptive cross-sectional study, 72 children developed nosocomial diarrhoea between April 2010 and March 2011. A medical history, clinical assessment and culture for Clostridium difficile and direct toxin detection from stool samples by enzyme immuno-assay were undertaken in all patients. Results: CDAD was diagnosed in 17 (23•6%) patients. Those aged ≤12 months were the most commonly affected (eight, 47%). The main cause of admission was respiratory disorders (eight, 47% of cases), followed by surgical problems (three, 17•7%). Ten patients (58•8%) had severe symptoms. There were no statistically significant differences between any of the demographic or laboratory data for children with CDAD and children with other causes of nosocomial diarrhoea. None of the patients developed complications. Seven children with CDAD (41•2%) had recurrence. Conclusion: CDAD is an important cause of nosocomial diarrhoea in children in Assiut University Children's Hospital. Established guidelines should be followed in all hospitals to minimise exposure to the pathogen. Physicians can do much to reduce the risk of a severe outcome in children by early identification and rapid management. Further research should be undertaken to identify the risk factors for recurrence.

Background: There are no large epidemiological studies of Clostridium difficile-associated disease (CDAD) in hospitalized children. Aim: To describe the frequency, demography, clinical features and outcome of nosocomial CDAD in children admitted to Assiut University Children's Hospital, Egypt. Patients and Methods: In this descriptive cross-sectional study, 72 children developed nosocomial diarrhoea between April 2010 and March 2011. A medical history, clinical assessment and culture for Clostridium difficile and direct toxin detection from stool samples by enzyme immuno-assay were undertaken in all patients. Results: CDAD was diagnosed in 17 (23•6%) patients. Those aged ≤12 months were the most commonly affected (eight, 47%). The main cause of admission was respiratory disorders (eight, 47% of cases), followed by surgical problems (three, 17•7%). Ten patients (58•8%) had severe symptoms. There were no statistically significant differences between any of the demographic or laboratory data for children with CDAD and children with other causes of nosocomial diarrhoea. None of the patients developed complications. Seven children with CDAD (41•2%) had recurrence. Conclusion: CDAD is an important cause of nosocomial diarrhoea in children in Assiut University Children's Hospital. Established guidelines should be followed in all hospitals to minimise exposure to the pathogen. Physicians can do much to reduce the risk of a severe outcome in children by early identification and rapid management. Further research should be undertaken to identify the risk factors for recurrence.

Background: There are no large epidemiological studies of Clostridium difficile-associated disease (CDAD) in hospitalized children. Aim: To describe the frequency, demography, clinical features and outcome of nosocomial CDAD in children admitted to Assiut University Children's Hospital, Egypt. Patients and Methods: In this descriptive cross-sectional study, 72 children developed nosocomial diarrhoea between April 2010 and March 2011. A medical history, clinical assessment and culture for Clostridium difficile and direct toxin detection from stool samples by enzyme immuno-assay were undertaken in all patients. Results: CDAD was diagnosed in 17 (23•6%) patients. Those aged ≤12 months were the most commonly affected (eight, 47%). The main cause of admission was respiratory disorders (eight, 47% of cases), followed by surgical problems (three, 17•7%). Ten patients (58•8%) had severe symptoms. There were no statistically significant differences between any of the demographic or laboratory data for children with CDAD and children with other causes of nosocomial diarrhoea. None of the patients developed complications. Seven children with CDAD (41•2%) had recurrence. Conclusion: CDAD is an important cause of nosocomial diarrhoea in children in Assiut University Children's Hospital. Established guidelines should be followed in all hospitals to minimise exposure to the pathogen. Physicians can do much to reduce the risk of a severe outcome in children by early identification and rapid management. Further research should be undertaken to identify the risk factors for recurrence.

Multidrug resistance (MDR) is associated with fluoroquinolone (FQ) resistance in companion animal Escherichia coli (E. coli). In this study, gyrA, gyrB, parC, and parE quinolone resistance determining regions (QRDR) were sequenced among uropathogenic E. coli isolates with different resistant phenotypes. Also determined were porin, efflux pump and regulatory gene expression based on quantitative real-time reverse transcriptase PCR (qRT-PCR), the impact of efflux pump inhibition (Phe-Arg-β-naphthylamide) and the presence of plasmid-mediated quinolone resistance (PMQR). Using enrofloxacin as the prototypic FQ, we found that (i) the number of mutations in target genes correlate well with minimum inhibitory concentrations (MICs). A single mutation (Ser83Leu) in gyrA increases FQ MIC in susceptible isolates; subsequent mutations result in resistance that increases from low (enrofloxacin MICs 4-16 μg/ml) to high level (enrofloxacin MICs≥128 μg/ml) with each progressive mutation. (ii) as MIC increase, acrB activity and the number of drug classes contributing to the MDR phenotype increases; (iii) a consistent relationship between regulatory gene expression and MIC could not be identified; and (iv) qnrS and aac(6')-Ib-cr gene were detected in 14 and 5 ENR(R)-MDR isolates containing the target mutation, respectively. Of 13 isolates expressing PDR isolates, 10 (77%) were positive for qnrS gene, and 4 (40%) carried both qnrS and aac(6')-Ib-cr gene. These findings demonstrated that MDR-associated FQ resistance in canine and feline uropathogenic E. coli reflects a combination of point mutations, enhanced efflux pump activities, and PMQR mechanisms. Point mutations in DNA gyrase, however, are necessary to achieve a clinical level of FQ resistance.

Escherichia coli respond to selective pressure of antimicrobial therapy by developing resistance through a variety of mechanisms. The purpose of this study was to characterize the genetic mechanisms of antimicrobial resistance in fecal E. coli after the routine use of 2 popular antimicrobials. Fourteen resistant E. coli isolates, representing predominant clones that emerged in healthy dogs' feces after treatment with either amoxicillin (11 E. coli isolates) or enrofloxacin (3 E. coli isolates), were tested for mutations in DNA gyrase (gyrA and gyrB) and in topoisomerase IV (parC) and for the presence of β-lactamases (bla(TEM), bla(SHV), bla(PSE-1) and bla(CTX-M)) and plasmid-mediated quinolone resistance (qnrA, qnrB, qnrS, aac(6')-Ib, and qepA), by polymerase chain reaction. Escherichia coli isolates cultured following amoxicillin therapy only expressed single-drug resistance to β-lactams, while the isolates cultured from dogs receiving enrofloxacin therapy expressed multidrug resistance (MDR). The use of RND efflux pump inhibitors increased the susceptibility of the 3 MDR E. coli isolates to doxycycline, chloramphenicol, enrofloxacin, and ciprofloxacin, which indicates a role of the efflux pump in the acquisition of the MDR phenotype. Amplification and sequencing of AcrAB efflux pump regulators (soxR, soxS, marR, and acrR) revealed only the presence of a single mutation in soxS in the 3 MDR isolates.