Candida albicans Genotyping and Relationship of Virulence Factors with Fluconazole Tolerance in Infected Pediatric Patients.

Purpose: Candida albicans of different genotypes is a common cause of fungal infection in pediatric setting. This cross sectional study was designed to investigate ABC genotypes and the relationship between virulence factors and fluconazole tolerance among C. albicans isolates from infected pediatric patients. Materials and Methods: C. albicans isolates were identified by germ tube test and ABC typing using PCR. Antifungal susceptibility testing was done according to Clinical Laboratory Standard Institution recommendations. Testing for proteinase and phospholiase production were done using bovine serum albumin agar and egg yolk agar, respectively. All isolates were tested for biofilm formation. Fluconazole tolerance was detected by reading the fluconazole susceptibility testing after 48 hours. Candida albicans isoltes were considered as fluconazole tolerant if they exhibited a susceptible minimum inhibatory concentration (MIC) after 24 hours of incubation and a resistant MIC following 48 hours of incubation. Results: A total of 88 C. albicans isolates were collected. Genotype A was the most prevalent (46 isolates, 52.3%). Biofilm formation, proteinase and phospholipase enzymes activity were detected in 76.1% 77.3% and 65.9% of the C. albicans isolates, respectively. Fluconazole resistance was found in 36.4% of the isolated C. albicans. Fluconazole tolerance was detected in 29 isolates (33%). Fluconazole tolerance has significant positive correlation with proteinase production and biofilm formation. Conclusion: Genotype A was the most prevalent genotype. Biofilm and hydrolytic enzymes production are important Candida albicans virulence determinants in pediatric infections. Fluconazole tolerance has significant positive correlation with biofilm formation and proteinase production in C. albicans. More studies are recommended to investigate the molecular relationship between fluconazole tolerance and C. albicans virulence determinants. Also, to identify the effect of fluconazole tolerance on the clinical outcome of virulent Candida albicans infections.

Activity of imipenem/relebactam on Klebsiella pneumoniae with different mechanisms of imipenem non-susceptibility.

BACKGROUND AND OBJECTIVES: Imipenem/relebactam (IMP/R) is a newly FDA approved ß-lactam/ß-lactamase inhibitor combination. Relebactam ability to restore IMP activity could differ according to the cause of imipenem non-susceptibility. Therefore, we investigated the in-vitro activity of IMP/R against Klebsiella pneumoniae with different mechanisms of imipenem non-susceptibility. MATERIALS AND METHODS: Imipenem-nonsusceptible (IMP-NS) K. pneumoniae isolates were collected and characterized for ß-lactamase encoding genes by multiplex PCR. For IMP-NS carbapenemase-negative isolates, study of Ompk35 & Ompk36 gene expression was performed by reverse transcription-PCR while efflux pump activity was studied by minimum inhibitory concentration (MIC) reduction assay using efflux pump inhibitor. Susceptibility testing of K. pneumoniae to IMP and IMP/R were achieved by broth microdilution (BMD) method. RESULTS: During the study period, 140 isolates of IMP-NS K. pneumoniae were collected. BMD method showed that relebactam restored IMP susceptibility in 100%, 60% and 49% of isolates that only harbor AmpC, extended spectrum beta lactamase (ESBL) and carbapenemases, respectively. IMP/R was most potent against all bla KPC and 50% of bla OXA-48 _producing isolates. No demonstrable activity of IMP/R against K. pneumoniae harboring metallo-ß-lactamases (MBLs). Out of 18 isolates with IMP non-suceptibility due to porins loss with overproduction of ESBL and/or AmpC, 14 (77.7%) isolates were IMP/R susceptible. IMP/R showed no activity against isolates with only efflux pump hyperactivity. CONCLUSION: Relebactam could restore IPM activity in KPC or AmpC-producing IMP/NS K. pneumoniae but with no activity against MBL- producing isolates. Relebactam activity against isolates harbouring-bla OXA-48 or with altered Ompk35 & Ompk36 gene expression and efflux pump hyperactivity need further studies. Therefore, using IMP/R antibiotic in the treatment of infections caused by IMP/NS K. pneumoniae should be based on its molecular profile of IMP resistance to optimize the utility of IMP/R.

Colistin-heteroresistance in carbapenemase-producing Enterobacter species causing hospital-acquired infections among Egyptian patients.

OBJECTIVES: Colistin is the last resort for treating carbapenemase-producing Enterobacter. Colistin-heteroresistance is a new concern as it may cause treatment failure. Our study aimed to detect colistin-heteroresistance among carbapenemase-producing Enterobacter species causing hospital-acquired infections in patients at Mansoura University Hospitals (MUHs). METHODS: Sensitivity of recovered Enterobacter species to imipenem, meropenem and colistin was estimated by the broth dilution method. Carbapenemase production was detected with the Carba NP test and confirmed with multiplex PCR. Population analysis profile (PAP) was performed to assess colistin-heteroresistance. Enterobacter isolates with colistin MIC≤2µg/mL had subpopulations growing at colistin concentration>2µg/mL were considered heteroresistant. Isolates with subpopulations growing at colistin concentrations two times higher than MIC but ≤ 2 µg/mLwere considered heterogeneous. RESULTS: Of 115 Enterobacter isolates collected during the period of the study, 61 (53%) were cabapenem-resistant. Of these, 49 isolates (42.6%) were carbapenemase-producers, including Enterobacter cloacae complex (37; 75.5%) and Enterobacter aerogenes (12; 24.5%). The most prevalent carbapenemase gene was blaNDM (20 isolates; 40.8%). Seven isolates were colistin-resistant (7/115; 6.1%). Seventeen isolates (34.7% of carbapenemase-producers) were colistin-heteroresistant and two isolates had heterogeneous profiles. Most of these isolates were E. cloacae complex (12/17) and from bloodstream infection (10/17). The frequency of heteroresistant subpopulations ranged from 1 × 10-5 to 5.5 × 10-4. CONCLUSION: Carbapenem-resistant Enterobacter is a common resistant pathogen in the hospital setting. Colistin-heteroresistance among carbapenemase-producing Enterobacter is a growing serious medical problem as colistin is considered the last hope for treating infections caused by these multidrug-resistant pathogens.

Molecular study of human astrovirus in Egyptian children with acute gastroenteritis.

INTRODUCTION: Human astrovirus (HAstV) has been increasingly identified as an important cause of acute gastroenteritis in young children. Limited information is available about the prevalence and genotype distribution of classic HAstV causing acute gastroenteritis in Egyptian children. METHODS: Stool samples were collected from 100 infants and children attending the gastroenterology outpatient clinic in Mansoura University Children Hospital and suffering from acute gastroenteritis during the period extending from January 2018 to January 2019. Samples were tested for HAstV using reverse transcription PCR. Genotyping was performed using type-specific reverse transcription nested PCR. RESULTS: Among 100 children included in this study, the detection rate of HAstV was 11% (11 patients). There was a significant difference regarding age between cases positive and negative for HAstV (p=0.005). There was a higher prevalence of HAstV in children aged one year or younger. Significant association was detected between HAstV positive cases and rural residence (p=0.002), summer season (p=0.025) and fever (p=0.017). The HAstV genotypes detected were HAstV-8 (8/11, 72.7%), HAstV-3 (2/11, 18.2%) and HAstV-2 (1/11, 9.1%). CONCLUSIONS: This study suggests that HAstV is a common pathogen causing gastroenteritis in Egyptian children especially in rural areas. The most frequent HAstV genotype in our study was HAstV-8.

Vancomycin heteroresistance in coagulase negative Staphylococcus blood stream infections from patients of intensive care units in Mansoura University Hospitals, Egypt.

BACKGROUND: Vancomycin heteroresistance in coagulase negative Staphylococci (CoNS) is a recent health concern especially in serious infections like bloodstream infections as it may lead to failure of therapy. Little information is available about the prevalence vancomycin heteroresistance in CoNS causing bloodstream infections in intensive care units (ICUs) patients of Mansoura University Hospitals (MUHs). METHODS: This prospective study enrolled 743 blood samples collected from ICUs patients presented with clinical manifestations of bloodstream infections over the period extending from January 2014 to March 2016. Samples were processed, coagulase negative Staphylococci were identified by routine microbiological methods and the absence of coagulase activity. Species were identified by API Staph 32. Oxacillin resistant CoNS were identified by cefoxitin disc diffusion method. Susceptibility testing of isolated CoNS to vancomycin was carried out using vancomycin agar dilution method. Mec A gene detection by PCR was done for oxacillin resistant isolates. Screening for vancomycin heteroresistance was done on brain heart infusion (BHI) agar containing 4 µg/mL vancomycin. Confirmation of vancomycin heteroresistance was carried out by population analysis profile (PAP). RESULTS: A total of 58 isolates were identified as CoNS from patients of clinically suspected bloodstream infections. The identified species were 33 (56.9%) Staphylococcus epidermidis, 12 (20.7%) Staphylococcus capitis, 7 (12.1%) Staphylococcus haemolyticus, and 3 isolates (5.2%) Staphylococcus lugdunesis. Three isolates were unidentified by API Staph 32. Forty-four (75.9%) isolates were oxacillin resistant. Mec A gene was detected in all oxacillin resistant isolates. All isolates had susceptible vancomycin MICs by agar dilution. Nine isolates (15.5%) could grow on BHI agar containing 4 µg/mL vancomycin. These isolates showed heterogeneous profile of resistance to vancomycin by population analysis profile. CONCLUSIONS: Vancomycin heteroresistant CoNS causing bloodstream infections is growing unrecognized health hazard in ICUs patients. These isolates have susceptible vancomycin MICs. Screening methods are recommended and should be considered to improve clinical outcome in these high risk patients.