Antimicrobial Resistance and Comparative Genome Analysis of Klebsiella pneumoniae Strains Isolated in Egypt.

Klebsiella pneumoniae is an important human pathogen in both developing and industrialised countries that can causes a variety of human infections, such as pneumonia, urinary tract infections and bacteremia. Like many Gram-negative bacteria, it is becoming resistant to many frontline antibiotics, such as carbapenem and cephalosporin antibiotics. In Egypt, K. pneumoniae is increasingly recognised as an emerging pathogen, with high levels of antibiotic resistance. However, few Egyptian K. pneumoniae strains have been sequenced and characterised. Hence, here, we present the genome sequence of a multidrug resistant K. pneumoniae strain, KPE16, which was isolated from a child in Assiut, Egypt. We report that it carries multiple antimicrobial resistance genes, including a blaNDM-1 carbapenemase and extended spectrum ß-lactamase genes (i.e., blaSHV-40, blaTEM-1B, blaOXA-9 and blaCTX-M-15). By comparing this strain with other Egyptian isolates, we identified common plasmids, resistance genes and virulence determinants. Our analysis suggests that some of the resistance plasmids that we have identified are circulating in K. pneumoniae strains in Egypt, and are likely a source of antibiotic resistance throughout the world.

Ketorolac-fluconazole: A New Combination Reverting Resistance in Candida albicans from Acute Myeloid Leukemia Patients on Induction Chemotherapy: In vitro Study.

BACKGROUND AND OBJECTIVES: Candida albicans is a significant source of morbidity and mortality for patients with acute myeloid leukemia (AML). Prolonged use of fluconazole as empirical antifungal prophylaxis in AML patients leads to overexpression of efflux pump genes that resulted in the emergence of azole-resistant species. Consequently, the introduction of a new strategy to improve the management of C. albicans infections is an urgent need. Nonsteroidal anti-inflammatory drug (NSAID) ketorolac is associated with a reduction in cancer relapses. The present study was performed to investigate the use of ketorolac-fluconazole combination to reverse fluconazole resistance in C. albicans isolated from AML patients on induction chemotherapy. PATIENTS AND METHODS: One hundred and seventy AML patients were evaluated. Fifty C. albicans were isolated and subjected to disc diffusion assay and broth microdilution for fluconazole alone and combined with different concentrations of ketorolac. Efflux pump gene (CDR1, CDR2, and MDR1) expressions were quantified by real-time PCR. RESULTS: The tested ketorolac acted synergistically with fluconazole against resistant C. albicans with the minimum inhibitory concentration (MIC) of fluconazole decreased from >160 µg/mL to 0.3-1.25 µg/mL in (93.8%) of resistant isolates with fractional inhibitory concentration index (FICI) value of 0.25. The majority of the resistant isolates overexpressed CDR1 (71.1%) and MDR1 (60%). CONCLUSION: Ketorolac-fluconazole in vitro combination would be a promising strategy for further clinical in vivo trials to overcome fluconazole resistance in AML patients on induction chemotherapy.

Antimicrobial resistance and gene regulation in Enteroaggregative Escherichia coli from Egyptian children with diarrhoea: Similarities and differences.

Enteroaggregative Escherichia coli (EAEC) is a common diarrhoeagenic human pathogen, isolated from patients in both developing and industrialized countries, that is becoming increasingly resistant to many frontline antibiotics. In this study, we screened 50 E. coli strains from children presenting with diarrhea at the outpatients clinic of Assiut University Children's Hospital, Egypt. We show that all of these isolates were resistant to multiple classes of antibiotics and identified two as being typical EAEC strains. Using whole genome sequencing, we determined that both isolates carried, amongst others, blaCTX-M and blaTEM antibiotic resistance genes, as well as many classical EAEC virulence determinants, including the transcriptional regulator, AggR. We demonstrate that the expression of these virulence determinants is dependent on AggR, including aar, which encodes for a repressor of AggR, Aar. Since biofilm formation is the hallmark of EAEC infection, we examined the effect of Aar overexpression on both biofilm formation and AggR-dependent gene expression. We show that whilst Aar has a minimal effect on AggR-dependent transcription it is able to completely disrupt biofilm formation, suggesting that Aar affects these two processes differently. Taken together, our results suggest a model for the induction of virulence gene expression in EAEC that may explain the ubiquity of EAEC in both sick and healthy individuals.

IP-10 Levels in Patients Infected with HBV Predicts The Response to Treatment with Entecavir.

Interferon--inducible protein-10 (IP-10), is an inflammatory cytokine produced by different subsets of the immune cells and induces chemotaxis, apoptosis, growth of cells and angiostasis after binding to its receptor CXCR3. Inflammatory disorders, involving infectious diseases, immune dysfunction, and tumour growth have been linked to changes in CXCL10 levels. We aimed to investigate serum levels of IP-10 in chronic HBV infected patients undergoing treatment with entecavir and possible correlation with response to therapy. A total of 53 chronic HBV infected patients and 25 healthy controls were enrolled in this study. Patients included 20 with cirrhosis and 33 non-cirrhotic individuals. All patients received 0.5 mg/day entecavir and serum IP-10 level was determined by ELISA at baseline and at week 24 of treatment. mRNA expression of CXCR3 of PBMC was assessed by real-time polymerase chain reaction (RT-PCR). Response to therapy was achieved in 27/33 (81.8%) non-cirrhotic and 14/20 (70%) cirrhotic patients. Mean serum IP-10 levels was higher in patients than healthy controls, and cirrhotic patients had higher IP-10 than non-cirrhotic patients (520 vs 293.5 pg/ml; P<0.005). Response to treatment was associated with decreased IP-10 levels. Before treatment, the mean level in non-cirrhotic patients was 235±54 pg/ml, which decreased to 95±34 pg/ml (P<0.005) at week 24 of treatment. Similarly, in the cirrhotic group, IP-10 decreased from 458±42 pg/ml to 354±25 pg/ml (P <0.05) after 24 weeks of treatment. On the other hand, no change in IP-10 levels was observed for patients who did not respond to treatment. Interestingly, IP-10 levels correlated with PBMC's expression of CXCR3 mRNA (r= 0.448, P = 0.004), ALT level (r=0.273, P =0.048), liver fibrosis score 4 (FIB-4) (r=0.664, P = 0.01) and HBV DNA level (r=0.762, P =0.0001). In conclusion; IP10 may be used to predict response to therapy in HBV-infected patients.

Regulatory T Cells, IL10 and IL6 in HCV Related Hepatocellular Carcinoma after Transarterial Chemoembolization (TACE).

Hepatocellular carcinoma (HCC) is a primary malignancy of the liver. Tumors can recruit and promote the expansion of regulatory T cells (Tregs) to suppress antitumor immune responses for survival and progression. Furthermore, there is a strong evidence for the potential roles of cytokines in promoting HCC carcinogenesis and progression. We aimed to evaluate the frequency of Treg cells and serum levels of IL6 and IL10 before and after transarterial chemoembolization (TACE). We carried out a cross-sectional study at Assiut University hospitals that included 34 HCC patients and 10 matched apparently healthy controls. Peripheral Treg frequency was evaluated by Flow cytometry. IL6 and IL10 serum levels were evaluated by ELISA before and after TACE. HCC patients had a significantly higher level of IL6 and IL10 when compared to the control group (P=0.0002, P < 0.0001), respectively. However, after treatment, there was an elevation in the levels of IL6 and IL10 followed by a decrease to the baseline levels. Patients with large tumors (≥5 cm) showed higher levels of both IL 6 and IL 10 than those with smaller tumors. Moreover, HCC patients showed a higher frequency of Treg cells in comparison to the controls (P=0.002). No significant correlation was observed between the frequency of Treg cells and IL10 before and after treatment (r=0.38, P=0.30). In conclusion, HCC patients have significantly higher levels of IL 6, IL 10 and a higher percentage of Tregs than control individuals. Treg levels are altered after chemoembolization. IL 6 have a potential in reflecting the patient's condition after treatment, thus, can help in monitoring therapy.

Patterns of Candida biofilm on intrauterine devices.

Biofilms are colonies of microbial cells encased in a self-produced organic polymeric matrix and represent a common mode of microbial growth. Microbes growing as biofilm are highly resistant to commonly used antimicrobial drugs. We aimed to screen and characterize biofilm formation by different isolates of Candida on removed intrauterine devices (IUDs), to perform experimental biofilm formation with isolated strains, and to examine biofilm by the crystal violet and XTT reduction assays and scanning electron microscopy (SEM). A total of 56 IUDs were examined for biofilm formation using Sabouraud's dextrose chloramphenicol agar. Suspected colonies were identified by different methods. Antifungal susceptibility testing with fluconazole (FLU) and amphotericin B for the isolated strains and in vitro experimental biofilm formation was carried out. The biofilm was quantified by crystal violet, XTT reduction assay and SEM. Among the 56 IUDs investigated, 26 were Candida positive (46.4 %). Candida albicans was recovered from 15 isolates. The biofilm MIC of FLU was increased 64 to 1000 times compared to the MIC for planktonic cells. The XTT method results were dependent on the Candida species; biofilm formation was highest in Candida krusei and Candida glabrata strains, followed by C. albicans and Candida tropicalis. SEM of Candida biofilm revealed a heterogeneous thick biofilm with a mixture of micro-organisms. The main conclusion from this study was non-albicans Candida represents more than a half of the Candida biofilm. Better understanding of Candida biofilms may lead to the development of novel therapeutic approaches for the treatment of fungal infections, especially resistant ones among IUD users.