Expression of the Fluoroquinolones Efflux Pump Genes acrA and mdfA in Urinary Escherichia coli Isolates

Escherichia coli is one of the most frequent causes of urinary tract infections. Efflux system overexpression is reported to contribute to E. coli resistance to several antibiotics. Our aim in this study was to investigate the relation between antibiotic resistance and the expres­sion of the efflux pump genes acrA and mdfA in E. coli by real-time reverse transcription-PCR. We tested the in vitro susceptibilities to 12 antibiotics in 28 clinical isolates of E. coli obtained from urine samples. We also determined the minimum inhibitory concentrations of levofloxacin to these samples. We then revealed significant correlations between the overexpression of both mdfA and acrA and MICs of levofloxacin. In conclusion, we demonstrated that the increased expression of efflux pump genes such as mdfA and acrA can lead to levofloxacin resistance in E. coli. These findings contribute to further understanding of the molecular mechanisms of efflux pump systems and how they contribute to antibiotic resistance

Prevalence of the colistin resistance gene <i>MCR-1</i> in colistin-resistant <i>Klebsiella pneumoniae</i> in Egypt

Klebsiella pneumoniae is a nosocomial pathogen with high morbidity and mortality rates in hospitalized patients. The emergence of multidrug-resistant K. pneumoniae has become more challenging to treat, with the prevalence of colistin-resistance. Therefore, reliable methods for detecting colistin resistance are required. Many plants' essential oils have antimicrobial activity and have been used to combat multiple antibiotic resistances. This study aimed to investigate the characterization and prevalence of the colistin resistance gene mcr-1 in K. pneumoniae in Egypt, evaluate rapid polymyxin NP test, determine the transferability of mcr-1 gene, and study the synergistic activity of eugenol combined with colistin against K. pneumoniae isolates. Eighty-two K. pneumonia isolates were collected from different human samples, followed by antibiotic susceptibility testing, rapid polymyxin NP test, and detection of the mcr-1 gene and its transfer frequency. Determination of the MICs of colistin alone and in combination with eugenol was performed, then mcr-1 gene expression was determined in the presence of eugenol. Thirty-two isolates (39%) were colistin-resistant. Rapid polymyxin NP test failed to detect resistant isolates with MICs below 32 µg/mL. Detection of mcr-1 gene was made in 27 (84%) of colistin resistant isolates. The rest isolates possess alteration in the mgrB gene which probably causes colistin resistance. The mcr-1 gene was transferred by conjugation to other sensitive isolates. MIC of eugenol ranged from 416 to 1664 µg/mL, and FICI ranged from 0.265 to 0.75. Results also revealed suppression of mcr-1 gene expression in the presence of sub MIC of eugenol. Our results demonstrated a high prevalence of mcr-1 in Egypt and its ability to transfer to other strains. Difficult determination of colistin-resistant isolates with low values with rapid polymyxin NP test was apparent. Eugenol exerted a synergistic effect with colistin and improved its antimicrobial activity

Genotyping and molecular investigation of plasmid-mediated carbapenem resistant clinical Klebsiella pneumoniae isolates in Egypt

Klebsiella pneumoniae is a multidrug-resistant nosocomial pathogen. Carbapenem resistance is mediated mainly by enzymes carried on transmissible plasmids causing their dissemination among other members of Enterobacteriaceae. This study aimed to molecularly detect carbapenem resistance genes in K. pneumoniae clinical isolates, genotype them using ERIC-PCR, and investigate plasmid transformation of resistant genes by using ERIC-PCR and sequencing. Methods: Antimicrobial resistance of sixty carbapenem-resistant K. pneumoniae strains was evaluated by using the disc diffusion method. Five carbapenemases' genes were amplified by conventional PCR. Genotyping was performed using ERIC-PCR. Gene transformation was performed for the five genes to sensitive isolates. Wild and transformed isolates were genetically investigated using ERIC-PCR and sequencing. Results: Carbapenem resistance in our isolates was associated with high resistance to all tested antibiotics. The 60 K. pneumoniae isolates were divided into 6 resistor types. The prevalence of KPC, IMP, VIM, NDM, and OXA-48 genes were 17%, 63%, 93%, 85% and 100%, respectively. Dendrogram analysis showed 57 distinct patterns, arranged in three clusters. The five genes were transformed successfully into sensitive isolates. ERIC profiles of wild and transformed isolates showed cluster A contained all the wild isolates, and cluster B contained all transformed isolates. Genetic sequences of the 5 genes reflected high genetic similarity with the GenBank reference genes before plasmid transformation; however, a distinguishable decrease of genetic similarity was observed after transformation. Conclusion: Plasmid-mediated carbapenem resistance in K. pneumoniae and its dissemination among different strains is a real threat to public health

Prognosis of Human Cytomegalovirus in Cancer Patients Undergoing Chemotherapeutic Treatment in Egypt and an Emergent Prevalence of Glycoprotein B-5

The human cytomegalovirus (HCMV) is a global opportunistic β-herpes virus causing severe diseases in immune-compromised patients, such as malignant tumor patients, especially those undergoing chemotherapeutic treatment. This study aimed to determine the prevalence of HCMV-DNA in chemotherapeutic treatment naive cancer patients, and after chemotherapy, to compare between conventional nested PCR and ELISA techniques for the detection of HCMV, and to detect glycoprotein B genotypes. Plasma and serum samples before and after three chemotherapy cycles were collected from 49 chemotherapy-naive cancer patients. DNA was extracted from plasma samples using QIAamp® DNA Mini kit. HCMV-DNA was detected using a nested PCR technique. Multiplex nested PCR was used for HCMV-glycoprotein B (gB) genotyping. HCMV-IgG and -IgM were detected using ELISA technique. Thirty one (63.3 %) of the 49 plasma samples of the chemotherapy-naïve cancer patients were positive for HCMV-DNA; 21 of which remained positive after chemotherapy. However, 18 samples were negative of which 16 became positive after chemotherapy. gB-5 was the most common glycoprotein genotype detected (80.6 %), followed by gB-1, gB-3, gB-4, and gB-2. HCMV IgG was detected in the 49 serum samples of chemotherapy-naïve patients, and after exposure to chemotherapy. HCMV-DNA is commonly identified in cancer patients. Its detection after chemotherapy exposure may suggest HCMV reactivation. The most common genotype detected in cancer patients in Egypt is gB-5 in contrast to earlier research. IgG was detected in all patients. This indicates that HCMV is endemic in Egypt, necessitating the development of public awareness campaigns about HCMV infection and preventive strategies

Interspecies Interaction between Pseudomonas aeruginosa, Staphylococcus aureus and E. coli in vitro

Microbial interactions are frequently categorized according to how they affect each population in a binary system. We aimed to determine the interaction between P . aeruginosa, S . aureus, and E . coli in-vitro. In this experimental hospitalized patients’ sputum, urine, and blood samples were used to collect a total of 90 clinical isolates for the study in Damanhour Medical National Institute, Behira, Egypt, followed by accurate identification and testing for antibiotic sensitivity. To examine the effect of the supernatant of P. aeruginosa on S. aureus and E. coli determined MIC using broth microdilution method. We also measured the activity of lasA protease by assessing the S. aureus cell lysis potential of P. aeruginosa culture supernatants. Extraction of pyocyanin was made to determine the change in the cell nature of S. aureus upon exposure to pyocyanin by using a scanning electron microscope and the shape of colonies on the culture media was determined. Finally, we detect lasA, operon phz, phzM, phzS and rhlAB genes for P. aeruginosa. P. aeruginosa showed a great impact on S. aureus isolates resistant to different antibiotics as it facilitates their killing and may drive the normal colonies of S. aureus into SCVs. The ability to form biofilm by S. aureus and E. coli decreased in the presence of Pseudomonas supernatant

Study of some potential biomarkers in Egyptian hepatitis C virus patients in relation to liver disease progression and HCC

Abstract Background Egypt has the greatest prevalence of hepatitis C worldwide according to the WHO reports, accounting for 13% of the global HCV infections. HCV is a substantial precursor for fibrosis, cirrhosis, and hepatocellular carcinoma. This study aimed to investigate the potential relevance of some cytokines, miR-122 and miR-221 for the diagnosis of liver disease progression associated to HCV infection. Methods One hundred and twenty blood samples were collected from patients with chronic liver disease, HCC, and healthy individuals. Total bilirubin, alanine aminotransferase, aspartate aminotransferase, platelet count, albumin, and creatinine were measured. Serum level of selected cytokines was conducted by ELISA. Serum miRNA expression was detected by RT-PCR. Results IL-2R was higher among HCC patients and the mean concentration of both TNF-αRII and IL-6R was higher among cirrhotic patients. The expression of miRNA-122 showed a little fold decrease in all studied groups; the highest level was observed in HCC patients. The expression of miRNA-221 showed a significant fold increase in HCC and cirrhotic groups. Conclusions This study revealed that there is no difference in liver disease progression in patients regarding sex and age. Routine liver function tests performed poorly in terms of early diagnosis of liver disease progression; however, serum total bilirubin gave somewhat useful guide for discrimination between fibrotic, cirrhotic and HCC cases. IL-2R showed a significant consistent increase in its level with disease progression. The miR-221 serum level showed significant fold increase with liver disease progression. Therefore, making miR-221 a potential non-invasive biomarker for liver disease progression in the diagnostic setting is recommended

Identifying Different Mutation Sites Leading to Resistance to the Direct-Acting Antiviral (DAA) Sofosbuvir in Hepatitis C Virus Patients from Egypt

The hepatitis C virus (HCV) is a major global health challenge and a leading cause of morbidity and mortality. Many direct-acting antivirals (DAAs) target essential macromolecules involved in the virus’ life cycle. Although such DAAs achieve great success in reducing the viral load in genotype 1 infections, other genotypes demonstrate different levels of response. This study focused on mutation sites associated with patients with genotype 4a infections that failed to respond to treatment with sofosbuvir. The genotyping of HCV samples from patients with virological failure, and responder patients, was conducted using Geno2Pheno webserver-based full NS5B sequences. We constructed 3D structural models for all the samples and used structural analysis to investigate the effect of amino acid substitution on the observed resistance to SOF-based treatment, and the docking of sofosbuvir into the active sites of the 10 models was performed. Finally, 10 molecular dynamic (MD) simulation experiments were conducted to compare the stability of the 3D models of the resistant samples against the stability of the 3D models of the responder samples. The results highlighted the presence of HCV subtype 4a in all ten samples; in addition, an amino acid (aa) substitution in the palm region may hinder HCV polymerase activity. In this study, we provide evidence that a mutation in the NS5B gene that induces resistance to sofosbuvir in patients with the S282T/C/R mutant virus is present in the Egyptian population. Overall, the docking and MD results support our findings and highlight the significant impact of the identified mutations on the resistance of HCV NS5B RNA-dependent RNA polymerase to direct-acting antivirals (DAAs)