Nano-ciprofloxacin/meropenem exhibit bactericidal activity against Gram-negative bacteria and rescue septic rat model.

Aim: We hypothesized that simultaneous administration of two antibiotics loaded into a nanopolymer matrix would augment their synergistic bactericidal interaction. Methods: Nanoplatforms of chitosan/Pluronic® loaded with ciprofloxacin/meropenem (CS/Plu-Cip/Mer) were prepared by the ionic gelation method, using Plu at concentrations in the range 0.5-4% w/v. CS/Plu-Cip/Mer was evaluated for antibacterial synergistic activity in vitro and in vivo. Results: CS/Plu-Cip and CS/Plu-Mer with Plu concentrations of 3% w/v and 2% w/v, respectively, exhibited ∼80% encapsulation efficiency. The MICs of pathogens were fourfold to 16-fold lower for CS/Plu-Cip/Mer than for Cip/Mer. Synergy was evidenced for CS/Plu-Cip/Mer with a bactericidal effect (at 1× MIC and sub-MICs), and it significantly decreased bacterial load and rescued infected rats. Conclusion: This study illustrates the ability of CS/Plu nanopolymer to intensify synergy between antibiotics, thereby providing a promising potential to rejuvenate antibiotics considered ineffective against resistant pathogens.

Antibiotics are used to treat bacterial infections. However, the more they are used, the less effective they become, because bacteria develop resistance to them. One strategy to overcome this is to treat bacterial infection with a combination of antibiotics that work well together. The antibiotics ciprofloxacin and meropenem are often given together to treat Pseudomonas aeruginosa, a bacterium which can cause sepsis, a type of blood poisoning. Another strategy to overcome antibiotic resistance is to load them into nanocarriers, which can change their properties. Nanocarrier-loaded antibiotics can reduce toxicity and increase effectiveness. This study investigated whether the effectiveness of this pair could be improved by loading them into nanoparticles. When these nanoparticles were given to rats with sepsis, they were significantly more effective than unloaded ciprofloxacin and meropenem combinations. These nanoparticles were also able to directly kill bacteria, rather than just prevent bacterial reproduction, as with the unloaded combination. This study demonstrates that nanocarrier loading can intensify the enhanced benefit of combined antibiotic treatments. This is a promising strategy to reuse antibiotics that have become ineffective at treating bacteria which have developed resistance.

Nanosized Combined Antimicrobial Drugs Decreased Emergence of Resistance in Escherichia coli: A Future Promise.

Antibiotic combinations remain the frontline therapy for severe infections to reduce mortality. However, conventional antibiotic combinations have some limitations such as the low bioavailability and the rise of resistant strains. Nanoparticles are increasingly used as antibiotic delivery systems to promote bioavailability and hence improve efficacy of antibiotics. In this work, we hypothesize that the simultaneous delivery of two antibiotic-loaded nanoparticles will improve the intracellular bioavailability and thus inhibit emergence of resistance. Accordingly, Chitosan-pluronic nanoparticles were used to construct nanosized ciprofloxacin and meropenem and the antibacterial activity of nanosized combined antibiotics were compared versus unloaded single, unloaded combined, and nanosized single antibiotics. Thirty-six stepwise mutants were selected by exposing two E. coli strains to increasing concentrations of free-unloaded and nanosized antibiotics, and mutants were tested for antimicrobial susceptibilities using broth microdilution and disc diffusion methods. The change in expression levels of acrB efflux pump and porins (ompC and ompF) was assessed by real-time reverse transcription-PCR. The in vitro evaluation of combined ciprofloxacin and meropenem-loaded nanoparticles demonstrated that this nanosystem exhibited enhanced antibacterial effect. Step mutants selected with nanosized combined antibiotics showed higher sensitivity to both drugs, exhibited lower mutation frequencies, and less cross-resistance to other antimicrobial classes. Moreover, for all steps of selection, nanosized combined antibiotic mutants expressed significantly lower levels of acrB as well as higher levels of ompC and ompF (p-value <0.01). In view of these results, the use of nanosized combined antibiotics may be considered among the new promising strategies to combat infections through their potential efficacy in reducing microorganisms' ability to form resistant mutants.

Co-Existence of Certain ESBLs, MBLs and Plasmid Mediated Quinolone Resistance Genes among MDR E. coli Isolated from Different Clinical Specimens in Egypt.

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). blaTEM (80%) and blaNDM (43%) were the most frequent ESBL and MBL, respectively. None of the isolates harbored blaKPC and blaOXA-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.

Clinical and microbial characterization of toxigenic Clostridium difficile isolated from antibiotic associated diarrhea in Egypt.

BACKGROUND AND OBJECTIVES: Clostridium difficile infection (CDI) has become a significant healthcare-associated infection throughout the world and is particularly important in developing countries. This study aimed to investigate clinical characterization and risk factors related to toxigenic C. difficile infection in adult and pediatric patients, antimicrobial susceptibility pattern. Also, to evaluate different diagnostic methods for rapid detection of C. difficile associated diarrhea (CDAD) in Egypt. MATERIALS AND METHODS: Stool samples were collected from 95 pediatric patients and 37 adult patients suffering from antibiotic associated diarrhea and were subjected to direct toxin immunoassay and culture on cycloserine/cefoxitin/fructose agar. The presence of tcdA and tcdB genes was tested by PCR. RESULTS: Toxigenic C. difficile was isolated from pediatric and adult patients at a rate of 17.89% (17/95) and 27% (10/37) respectively. The sensitivity and specificity of direct PCR from stool are (100%, 100% and 82.4%, 100%) in adult and pediatric samples respectively. The susceptibility of C. difficile to vancomycin and metronidazole were found to be 66.7% and 48.2% respectively. CONCLUSION: Diabetes mellitus, prior antibiotic treatment, hematological malignancy on chemotherapy, malnutrition, neutropenia and Ryle feeding are risk factors for development of CDAD. Tight restriction of unnecessary antibiotic uses is necessary in our locality. Direct detection of toxin genes in stool by PCR is sensitive and specific method for early detection of C. difficile.

Patterns of Fluoroquinolone Resistance in Enterobacteriaceae Isolated from the Assiut University Hospitals, Egypt: A Comparative Study.

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 (rs = 0.8, p < 0.0001 and rs = 0.7, p < 0.0001, respectively) as well as between MIC levels and number of plasmids (rs = 0.4, p = 0.005 and rs = 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.

Levofloxacin-Loaded Nanoparticles Decrease Emergence of Fluoroquinolone Resistance in Escherichia coli.

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.

Epidemiological typing of multidrug-resistant Klebsiella pneumoniae, which causes paediatric ventilator-associated pneumonia in Egypt.

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.

A novel alanine to serine substitution mutation in SoxS induces overexpression of efflux pumps and contributes to multidrug resistance in clinical Escherichia coli isolates.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

In vitro selection of resistance to pradofloxacin and ciprofloxacin in canine uropathogenic Escherichia coli isolates.

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.

Molecular mechanisms of antimicrobial resistance in fecal Escherichia coli of healthy dogs after enrofloxacin or amoxicillin administration.

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 ß-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 ß-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.