The purpose of this study was to describe a putative role for a novel soxS mutation in contributing to multiple-antibiotic resistance in canine fluoroquinolone-associated MDR (FQ-MDR) Escherichia coli. This soxS mutation was discovered in canine faecal E. coli isolates during a study investigating the effect of oral fluoroquinolone administration on faecal E. coli in healthy dogs. We determined via quantitative real-time RT–PCR that both soxS and acrB were overexpressed in the clinical soxS Ala-12→Ser (soxS^A12S) mutants and this may account for their FQ-MDR phenotype. We validated the FQ-MDR phenotype of the clinical isolates by reconstructing the WT and the soxS^A12S mutation in the E. coli soxS null mutant JW4023 (soxS::kn) via allelic exchange. The JW4023 soxS^A12S derivative showed an increase in MICs of ciprofloxacin, enrofloxacin and chloramphenicol compared with the JW4023 derivative in which the WT soxS had been restored. The soxS and acrB genes were overexpressed in the JW4023 soxS^A12S mutant compared with JW4023 with WT soxS. A similar overexpression of efflux pump genes and an increase in antibiotic resistance were observed upon stimulation with paraquat to resemble the phenotype of the clinical soxS^A12S isolates. Our data suggest that the soxS^A12S substitution mutation is selected in clinical isolates when dogs are exposed to a fluoroquinolone and that this mutation contributes to the FQ-MDR phenotype of E. coli isolates.

The emergence of multi-drug resistant (MDR) strains and even pan drug resistant (PDR) strains is alarming. In this study, we studied the resistance pattern of E. coli pathogens recovered from patients with different infections in different hospitals in Minia, Egypt and the co-existence of different resistance determinants. E. coli was the most prevalent among patients suffering from urinary tract infections (62%), while they were the least isolated from eye infections (10%). High prevalence of MDR isolates was found (73%) associated with high ESBLs and MBLs production (89.4% and 64.8%, respectively). bla_TEM (80%) and bla_NDM (43%) were the most frequent ESBL and MBL, respectively. None of the isolates harbored bla_KPC and bla_OXA-48 carbapenemase like genes. Also, the fluoroquinolone modifying enzyme gene aac-(6′)-Ib-cr was detected in 25.2% of the isolates. More than one gene was found in 81% of the isolates. Azithromycin was one of the most effective antibiotics against MDR E. coli pathogens. The high MAR index of the isolates and the high prevalence of resistance genes, indicates an important public health concern and high-risk communities where antibiotics are abused.

Purpose. Multidrug-resistant Klebsiella pneumoniae is a common nosocomial pathogen that plays an important role in ventilator-associated pneumonia (VAP). This study aimed to define the clonal relatedness of K. pneumoniae strains isolated from paediatric VAP in addition to those isolated from environmental samples. Methodology. This study included 19 clinical and 4 environmental K. pneumoniae isolates recovered from the paediatric intensive care unit (PICU) in Assiut University Children's Hospital. The K. pneumoniae isolates were confirmed by biotyping using API strips and subjected to antimicrobial susceptibility testing. The genes coding K1 and K2 capsular types were detected by PCR. The clonal relationships between the K. pneumoniae isolates were determined by pulsed-field gel electrophoresis (PFGE). Results. Ten resistotypes were detected among all the K. pneumoniae isolates, while PFGE identified seventeen K. pneumoniae pulsotypes. Similar PFGE patterns were found between environmental and clinical isolates and between isolates recovered from different patients, suggesting the circulation of K. pneumoniae pathogens in the PICU and the role of the environment in the spread of infection. No correlation was found between the resistotypes and pulsotypes of the K. pneumoniae isolates. PFGE showed higher discriminatory power for the typing of nosocomial K. pneumoniae [Simpson’s diversity index (DI)=0.96] than resistotyping (DI=0.72). Conclusion. As far as we know, this is the first report of the isolation of the same multidrug-resistant (MDR) K. pneumoniae pulsotype from patients and environmental samples in the same hospital ward in Egypt. This study provides a step on the way to understanding the genotyping and epidemiology of MDR K. pneumoniae for enhanced prevention of bacterial transmission.

Antimicrobial resistance is a major global health problem that is developed upon exposure of bacteria to antimicrobial agents, and, thus, reducing or eliminating the ability of the currently available antibacterial drugs to eradicate bacterial infections. The aim of the current study was to encapsulate levofloxacin (third generation fluoroquinolones) into chitosan (CS) nanoparticles, to evaluate the antibacterial potency of the nanosized drug, and to characterize the major genetic mutations associated with the exposure of bacteria to the levofloxacin-loaded nanoparticle versus free levofloxacin. Three consecutive mutants were selected by stepwise exposure of one reference and two clinical Escherichia coli isolates to a series of progressively increasing concentrations of levofloxacin and the levofloxacin-loaded nanoparticles. Mutations in quinolone resistance determining region (QRDR) of gyrA and parC and regulators of AcrAB efflux pump ( soxR , soxS and acrR ) for all the selected-mutants were determined using polymerase chain reaction and sequencing. Mutants developed upon exposure to the nanosized drug had higher sensitivity to levofloxacin, compared with the levofloxacin-selected mutants. In addition, mutations in gyrA were observed in the levofloxacin first mutants, but in the nanosized levofloxacin second mutants. In the third mutants, an additional mutation in parC and mutations in the regulators were found only in levofloxacin-selected mutants. Loading of levofloxacin into the CS nanoparticles could increase the antibacterial activity of the drug and decrease the emergence of resistant mutants. To the best of our knowledge, this is the first study to explore the role of antimicrobial-loaded nanoparticles in the reduction of emergence of bacterial resistance.

Background: An increasing pattern of fluoroquinolone resistance (FQR) among bacterial pathogens has been described worldwide. In this study, we compared the patterns of genetic mechanisms that confer FQR for Escherichia coli and Klebsiella pneumoniae isolated from the Assiut University Hospitals in Egypt. Methods: Eighty-seven clinical E. coli and K. pneumoniae isolates were tested for mutations in gyrA , gyrB , parC , and parE genes by polymerase chain reaction (PCR) amplification and DNA sequencing. The presence of plasmid-mediated quinolone resistance (PMQR) genes qnrA , qnrB , qnrS , aac(6′)-Ib , qepA was screened by PCR and characterized by conjugation. Correlations between different FQR mechanisms and ciprofloxacin minimal inhibitory concentration (MIC) levels were determined. Results: A higher number of quinolone resistance-determining region (QRDR) mutations was detected in E. coli , while the number of PMQR determinants was significantly higher in K. pneumoniae . However, K. pneumoniae showed stronger correlations than E. coli between MIC levels and number of mutations in the QRDR per isolate ( r _s = 0.8, p < 0.0001 and r _s = 0.7, p < 0.0001, respectively) as well as between MIC levels and number of plasmids ( r _s = 0.4, p = 0.005 and r _s = 0.3, p = 0.02, respectively). Conclusions: Although we observed a prevalence of chromosomal mutations for E. coli and the presence of plasmid-encoded genes for K. pneumoniae that resulted in FQR phenotype, high levels of FQR appeared to occur as a result of gradual accumulation of mutations in QRDR for both bacteria. To our best of knowledge, this is the first study to report and compare the correlation between FQ MIC levels and different genetic mechanisms for FQR in Enterobacteriaceae.

Despite the increased reporting of Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) in Egypt, there is a paucity of information regarding the molecular characteristics of such strains. Herein, we present the genome sequence of two CR-hvKp strains, K22 and K45, which were isolated from VAP (ventilator-associated-pneumonia) patients admitted to pediatric ICU at Assiut University Children’s Hospital, Egypt. K22 and K45 isolates were subjected to antimicrobial susceptibility testing and whole-genome sequencing. Genomic analysis was performed to characterize each strain, determining their plasmids, antimicrobial resistance (AMR) genes, and virulence determinants. K22 possessed an extensive drug resistance phenotype (XDR), whilst K45 exhibited a multidrug resistance phenotype (MDR), with genome sequencing revealing the presence of a diverse array of AMR genes. Both strains were resistant to the carbapenem antibiotic imipenem, carrying the OXA-48 carbapenemase, with K22 additionally possessing an NDM-1 carbapenemase. Each strain was considered high-risk, with K22 and K45 respectively belonging to sequence types ST383 and ST14 and possessing virulence genes implicated in hypervirulence (e.g., iucABCD-iutA and rmpA). Importantly, both strains carried multiple plasmid replicons, including an AMR/virulence IncHI1B/FIB hybrid plasmid and MDR IncL/M plasmids. This report highlights the critical role of plasmids in the evolution of virulent K. pneumoniae strains and suggests the circulation of an IncHI1B/FIB hybrid plasmid, simultaneously disseminating AMR and hypervirulence, amongst K. pneumoniae strains within Assiut University Children’s Hospital.