ß-lactam resistance associated with ß-lactamase production and porin alteration in clinical isolates of E. coli and K. pneumoniae.

ß-lactam resistance represents a worldwide problem and a serious challenge for antimicrobial treatment. Hence this research was conducted to recognize several mechanisms mediating ß-lactam resistance in E. coli and K. pneumoniae clinical isolates collected from Mansoura University hospitals, Egypt. A total of 80 isolates, 45 E. coli and 35 K. pneumoniae isolates, were collected and their antibiotic susceptibility was determined by the Disc diffusion method followed by phenotypic and genotypic detection of extended-spectrum ß-lactamases (ESBLs), AmpC ß-lactamase, carbapenemase enzymes. The outer membrane protein porins of all isolates were analyzed and their genes were examined using gene amplification and sequencing. Also, the resistance to complement-mediated serum killing was estimated. A significant percentage of isolates (93.8%) were multidrug resistance and showed an elevated resistance to ß-lactam antibiotics. The presence of either ESBL or AmpC enzymes was high among isolates (83.75%). Also, 60% of the isolated strains were carbapenemase producers. The most frequently detected gene of ESBL among all tested isolates was blaCTX-M-15 (86.3%) followed by blaTEM-1 (81.3%) and blaSHV-1 (35%) while the Amp-C gene was present in 83.75%. For carbapenemase-producing isolates, blaNDM1 was the most common (60%) followed by blaVIM-1 (35%) and blaOXA-48 (13.8%). Besides, 73.3% and 40% of E. coli and K. pneumoniae isolates respectively were serum resistant. Outer membrane protein analysis showed that 93.3% of E. coli and 95.7% of K. pneumoniae isolates lost their porins or showed modified porins. Furthermore, sequence analysis of tested porin genes in some isolates revealed the presence of frameshift mutations that produced truncated proteins of smaller size. ß-lactam resistance in K. pneumoniae and E. coli isolates in our hospitals is due to a combination of ß-lactamase activity and porin loss/alteration. Hence more restrictions should be applied on ß-lactams usage to decrease the emergence of resistant strains.

The effect of EDTA in combination with some antibiotics against clinical isolates of gram negative bacteria in Mansoura, Egypt.

Extensive use of antibiotics in clinical practice has been associated with increasing frequency of resistant microorganisms. So new strategy is needed to treat the resistant pathogens. Hence this study was conducted to determine the effect of Ethylenediaminetetraacetic acid (EDTA) in increasing the inhibition effect of some antibiotics on multi-drug resistant (MDR) gram-negative bacteria. For this purpose, 40 E. coli isolates, 40 K. pneumoniae isolates and 50 P. aeruginosa isolates were collected from different University's hospitals in Mansoura, Egypt. Antibacterial susceptibility pattern against 9 different antimicrobials were studied by disc diffusion method. Also the effect of two sub-inhibitory concentrations of EDTA (1 and 2 mM) on the inhibition zones of antibiotic discs against the highly multidrug resistant (MDR) isolates was determined. Checkerboard method was used for testing the activity of gentamicin/EDTA and cefotaxime/EDTA combinations on the highly MDR isolates. Additionally, the effect of EDTA on the expression of efflux pump genes was tested by real time-PCR. Most of the clinical isolates were found to be resistant to the tested antibiotics except imipenem and high prevalence of MDR isolates was recorded. 34 isolates were selected as those showed the highest multi-drug resistance and were tested to specify their MIC for EDTA as EDTA showed strong antibacterial activity with MIC ranging 4-8 mM. The addition of sub-MIC of EDTA (1or 2 mM) to the agar plate resulted in changing the 11 tested E. coli isolates from resistant to sensitive to ceftazidime, gentamicin, rifampin, ampicillin, erythromycin and vancomycin, the tested K. pneumoniae isolates were turned also from resistant to sensitive to gentamicin and ceftazidime, additionally the tested P. aeruginosa isolates became sensitive to gentamicin, ceftazidime and ciprofloxacin. Indifference to additive activity was observed for tested combinations and MIC value of cefotaxime or gentamicin in combination with EDTA was less than antibiotic alone in the most tested isolates. Moreover, significant reduction (P < 0.01) in the expression of all tested efflux pump genes in treated E. coli, K. pneumoniae and P. aeruginosa isolates with EDTA compared to untreated isolates was observed. In conclusion, these results suggest that the combination of antibiotic especially gentamicin with EDTA may be fruitful for management of resistant gram negative infections.

Effect of Sub-Minimum Inhibitory Concentrations of Tyrosol and EDTA on Quorum Sensing and Virulence of Pseudomonas aeruginosa.

INTRODUCTION: Pseudomonas aeruginosa is considered a dangerous pathogen, as it causes many human diseases, besides that it is resistant to almost all types of antibacterial agents. So, new strategies to overcome P. aeruginosa infection have evolved to attenuate its virulence factors and inhibit its quorum-sensing (QS) activity. PURPOSE: This study investigated the effect of tyrosol and EDTA as anti-quorum-sensing and antivirulence agents against P. aeruginosa PAO1. METHODS: Anti-quorum activity of sub-minimum inhibitory concentrations (sub-MICs) of tyrosol and EDTA was tested using Chromobacterium violaceum (CV 12,472) biosensor bioassay. Miller assay was used to assess the inhibition of QS signal molecules by ß-galactosidase activity determination. Also, their effects on the production of protease, lipase, lecithinase, and motility were tested. The inhibitory effects of these molecules on QS regulatory genes and exotoxins genes expression were evaluated by real-time PCR. RESULTS: Tyrosol and EDTA at sub-MICs inhibited the production of violacein pigment. Both compounds inhibited QS molecules production and their associated virulence factors (protease, lipase, lecithinase, and motility) (P≤ 0.05). Besides, the expression levels of QS regulatory genes (lasI, lasR, rhƖI, rhIR, pqsA, and pqsR) and exotoxins genes (exoS and exoY) were significantly reduced (P≤ 0.05). CONCLUSION: Both tyrosol and EDTA can be used to fight P. aeruginosa infection as anti-quorum-sensing and antivirulence agents at their sub-MICs.

The genetic diversity of metronidazole susceptibility in Trichomonas vaginalis clinical isolates in an Egyptian population.

Trichomoniasis is the most common curable sexually transmitted disease worldwide. Resistance to metronidazole in treating trichomoniasis is a problematic health issue. We aimed to determine the minimum lethal concentration (MLC) of metronidazole for Trichomonas vaginalis isolates detected in Mansoura, Egypt and studied the genotypic profile of these isolates. Vaginal swab specimens were obtained from 320 symptomatic and 100 asymptomatic females, for whom clinical examination, vaginal discharge wet mount, Giemsa stain, and culture in modified Diamond's media were performed. Metronidazole susceptibility testing by an aerobic tube assay was performed. Both sensitive and resistant isolates were examined by PCR amplification followed by restriction fragment length polymorphism (RFLP). Trichomonas vaginalis was identified in 49/420 (11.7%) using either culture or PCR, while wet mount and Giemsa stain detected the parasite in 8.1 and 7.6% of participants, respectively. After 48 h incubation, most isolates were sensitive to metronidazole with a minimal lethal concentration (MLC) of 1 µg/ml. Mild resistance was observed in two isolates with MLCs of 64 µg\ml and mild to moderate resistance was observed in an additional two isolates with MLCs of 128 µg/ml. The four isolates that demonstrated low to moderate metronidazole resistance displayed a unique genotype band pattern by RFLP compared to the other 45 samples that were metronidazole sensitive. Our results highlight the presence of in vitro metronidazole tolerance in a few T. vaginalis isolates in Mansoura, Egypt that may lead to the development of drug resistance as well as the possibility of an identifying RFLP pattern in the isolates.