Introduction Enterococci commonly inhabit the gastrointestinal tract of both human and animals; however, they have emerged as a leading cause of several infections with substantial morbidity and mortality. Their ability to acquire resistance combined with intrinsic resistance to various antimicrobials makes treatment of enterococcal infections challenging. Materials and methods The aim of the study was to evaluate the antimicrobial resistance pattern, and assess the prevalence of multidrug resistance (MDR) and extensive drug resistance (XDR) among enterococcal isolates, collected from different clinical sources, in Mansoura University Hospitals, Egypt. Results Antibiotic sensitivity testing revealed elevated levels of resistance among enterococcal clinical isolates (N=103). All E. faecium (N=32) and 74.6% of E. faecalis isolates (N=71) were MDR, while two E. faecalis and four E. faecium isolates were XDR. High level gentamicin resistance was detected in 79.6%, most of them carried the aac(6’)-Ie-aph(2’’)-Ia gene. High level streptomycin resistance was seen in 36.9%, of which 52.6% carried the ant(6’)-Ia gene. Resistance to macrolides and lincosamides were mediated by ermB (92.2%) and msrA/B (42.7%). tetK , tetL , and tetM genes were detected among tetracyclines resistant isolates. Resistance to vancomycin was detected in 15.5%, where vanB and vanC 1 gene clusters were detected in VRE isolates. Ten isolates (9.7%) were resistant to linezolid, eight of which harbored the optrA gene. Vancomycin and linezolid resistant enterococci were more likely to exhibit strong/moderate biofilm formation than vancomycin and linezolid sensitive ones. Conclusion Elevated levels of resistance to different classes of antimicrobial agents and emergence of MDR and XDR strains pose a major threat with limited therapeutic options for infections caused by this emerging pathogen.

The incidence of fluconazole-resistant Candida albicans has been increasing worldwide. Both biofilm and fungal morphogenesis are main virulence factors of C. albicans cells. Extracellular fungal prostaglandins are synthesized during biofilm adhesion and development and through yeast-hypha conversion. Hence, we targeted prostaglandin synthesis with various cyclooxygenase (COX) inhibitors (aspirin, diclofenac, ketoprofen, tenoxicam, and ketorolac) and assessed their effect on fungal adhesion, biofilm formation, and yeast-hypha conversion in clinical isolates of Fluconazole resistant C. albicans. Significant reduction in fungal adhesion and detachment of mature biofilm was attained down to 1 mM concentrations of anti-inflammatory agents. Microscopical examination of fungal cells in the presence of the tested drugs showed significant reduction of germ tube formation. Therefore, COX inhibitors have a significant effect on reduction of Candida adhesion and biofilm development in correlation with fungal morphogenesis. Moreover, inhibition of C. albicans by COX inhibitors gave synergistic activity with fluconazole suggesting that combination therapeutic strategies may be fruitful for management of infection of Fluconazole resistant C. albicans.

β-lactam resistance represents a worldwide problem and a serious challenge for antimicrobial treatment. Hence this research was conducted to recognize several mechanisms mediating β-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 β-lactamases (ESBLs), AmpC β-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 β-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. β-lactam resistance in K. pneumoniae and E. coli isolates in our hospitals is due to a combination of β-lactamase activity and porin loss/alteration. Hence more restrictions should be applied on β-lactams usage to decrease the emergence of resistant strains.

Uricase is an important microbial enzyme that can be used in the clinical treatment of gout, hyperuricemia, and tumor lysis syndrome. A total of 127 clinical isolates of Pseudomonas aeruginosa were tested for uricase production. A Pseudomonas strain named Ps43 showed the highest level of native uricase enzyme expression. The open reading frame of the uricase enzyme was amplified from Ps43 and cloned into the expression vector pRSET-B. Uricase was expressed using E. coli BL21 (DE3). The ORF was sequenced and assigned GenBank Accession No. KJ718888. The nucleotide sequence analysis was identical to the coding sequence of uricase gene puuD of P. aeruginosa PAO1. We report the successful expression of P. aeruginosa uricase in Escherichia coli. E. coli showed an induced protein with a molecular mass of about 58 kDa that was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. We also established efficient protein purification using the Ni-Sepharose column with activity of the purified enzyme of 2.16 IU and a 2-fold increase in the specific activity of the pure enzyme compared with the crude enzyme.

The increasing incidence of β-lactam resistance due to AmpC β-lactamases in Egypt necessitated this study which aimed to evaluate four different phenotypic methods for detection of AmpC β-lactamases among some clinical isolates of Enterobacteriaceae and compare these results with those obtained using polymerase chain reaction. The distribution of five AmpC β-lactamases genes (AmpC, CIT-M, Fox-1, ACC-1, ACT-1) were determined among the clinical isolates. Among 180 clinical isolates of Enterobacteriaceae, only 57 isolates were AmpC producers by phenotypic methods and 108 were AmpC producers by polymerase chain reaction. Phenotypic methods adapted in this study gave variable results with the most discriminatory results given by direct inoculation of both the enzyme extract and the bacterial culture in the wells. Of these, the best results were given by enzyme inoculation methods where 43 isolates exhibited positive result by this method. The distribution of AmpC β-lactamases gene among the clinical isolates showed that AmpC gene predominated in Escherichia coli. Fox gene was predominantly present in Enterobacter cloacae and E. coli isolates. ACT-1 predominated in E. cloacae. In contrast, enzymes from CIT-M and ACC-1 group were rarely present in Enterobacteriaceae.

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.

Escherichia coli is a multifaceted microbe since some are commensals, normally inhabiting the gut of both humans and animals while others are pathogenic responsible for a wide range of intestinal and extra-intestinal infections. It is one of the leading causes of septicemia, neonatal meningitis, urinary tract infections (UTIs), cystitis, pyelonephritis, and traveler’s diarrhea. The present study aims to survey the distribution and unravel the association of phylotypes, virulence determinants, and antimicrobial resistance of E. coli isolated from different clinical sources in Mansoura hospitals, Egypt. One hundred and fifty E. coli isolates were collected from different clinical sources. Antimicrobial resistance profile, virulence determinants, and virulence encoding genes were detected. Moreover, phylogenetic and molecular typing using ERIC-PCR analysis was performed. Our results have revealed that phylogroup B2 (26.67%) with the greatest content in virulence traits was the most prevalent phylogenetic group. Different virulence profiles and varying incidence of virulence determinants were detected among tested isolates. High rates of resistance to different categories of antimicrobial agents, dramatic increase of MDR (92.67%), and emergence of XDR (4%) were detected. ERIC-PCR analysis revealed great diversity among tested isolates. There was no clustering of isolates according to resistance, virulence patterns, or phylotypes. Our research has demonstrated significant phylogenetic diversity of E. coli isolated from different clinical sources in Mansoura hospitals, Dakahlia governorate, Egypt. E. coli isolates are equipped with various virulence factors which contribute to their pathogenesis in human. The elevated rates of antimicrobial resistance and emergence of MDR and XDR mirror the trend detected globally in recent years. Key points • Clinical E. coli isolates exhibited substantial molecular and phylogenetic diversity. • Elevated rates of antimicrobial resistance and emergence of XDR in pathogenic E. coli. • B2 Phylogroup with the highest VS was the most prevalent among pathogenic E. coli.

Extensive use of antibacterials in clinical practice has been associated with increasing frequency of multi-resistant E. coli strains. Genetic elements such as Class 1 integrons have an important role in resistance development. In the current study, a total of 84 E. coli clinical isolates from Mansoura hospitals patients in Egypt were screened for antibacterial susceptibility against 12 different antibacterials. High resistance rates were identified for Ampicillin (92.9%) and sulfamethoxazole/ trimethoprim (84.5%). Class 1 integron was investigated in E. coli isolates by PCR. As a result, Class 1 integron was identified in 51.2% of these isolates. The contents of amplified integron varriable regions, were digested by Alu I restriction endonuclease. Cluster analysis of Class 1 integron digested varriable regions revealed that RFLP digested fragments generated could be classified into 9 different patterns, namely A, B, C, D, E, F, G, H and I. The most prevalent genotype was identifed in Group D. PCR and sequencing were used for detection of antimicrobial resistance genes harbored in integrons of Group D. As a result, main phylogenetic group identified harbored integron cassette carrying resistance gene for two antimicrobial groups namely aminoglycoside and trimethoprim. Multiresistance profiles in Group D exhibited association between antimicrobial resistance and integron presence. These findings suggest that the strategy for treatment of patients with E. coli infections needs to be revised. Furthermore, the high prevalence of Class 1 integron carrying gene confirms the importance of integron-mediated antimicrobial gene cassettes.