Characterization of Enterococci

Study Overview
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Brief Summary
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Study on Characterization of Enterococci because nowadays it become an important cause of nosocomial infections .detection of the most common two species of Enterococci and most common virulence factors & its genes with determination of antibiotics sensitivity test for the isolated strains

Official Title
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Characterization of Enterococci; Distribution of Virulence Markers, Virulence Genes and Antibiotic Resistance Pattern of the Isolated Species

Conditions
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Samples From Patients With Enterococcal Infections

Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: any patients in ICU has manifestations of urinary tract infections, surgical wound infections, intra-abdominal infections, intrapelvic infections, bacteraemia and infective endocarditis Exclusion Criteria: patients receiving antibiotics in previous 48 hours

Ages Eligible for Study
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Minimum Age: 2 Months
Maximum Age: 90 Years

Sexes Eligible for Study
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All

Study Plan
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How is the study designed?
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What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| I) Sample collection | Samples are collected from intensive care unit (ICU) including urine, pus swabs, sputum, blood, tracheal aspirates and pharyngeal swabs | 2 months | 
| II) Identification of Enterococci: | by inoculation on Bile esculin azide & growing colonies further identified by : microscopically after staining by gram stain catalase test grow on high concentration of NaCl (6.5%) | 2 month | 
| III) Phenotypic detection of virulence marker of Enterococci(Gelatinase activity) | by culture on nutrient agar containing gelatin.Positive results appeared as liquefaction of gelatin | 2 weeks | 
| IV)Phenotypic detection of virulence marker of Enterococci (hemolytic activity) | by Detection of hemolytic activity on the blood agar | 1 week | 
| V) Phenotypic detection of virulence marker of Enterococci(Caseinase production) | by Detection of Caseinase production on Muller hinton agar containing skimmed milk 3%. | 2 weeks | 
| VI) Phenotypic detection of virulence marker of Enterococci(Formation of Slime layer) | Formation of Slime layer by culture on Brain heart infusion agar containing 5% sucrose, plates are incubated for 24 hrs at 37oC. Positive strains gave mucoid and slimy colonies. | 1 week | 
| VII) Phenotypic detection of virulence marker of EnterococcI (Biofilm formation) | using microtiter plate reader. Biofilm formation is scored as nonbiofilm forming (-), weak - (+), moderate - (++), and strong - (+++) corresponding to the A630 values ≤1, 1-≤2, 2-≤3, and>3, respectively | 2 weeks | 
| VIII) Antibiotic sensitivity test: | by 1- disc diffusion test using Vancomycin 30 μg, Teichoplanin 30 μg, Tetracycline 30 μg, Ampicillin 10 μg and Erythromycin 15 μg. Results ,Results are interpreted according to CLSI 2018. E-test: MICs (minimal inhibitory concentrations) of Vancomycin are measured by E-test for confirmation of vancomycin resistance among the isolated Enterococci. Results are interpreted according to CLSI guidelines. | 3 monthe | 
| IX) Molecular Identification of commonest Enterococcus species | by conventional gene specific uniplex PCR for E. faecalis and E. Faecium | 1 month | 
| X) Molecular detection of virulence genes | Identification of virulence genes; gel E (gene for gelatinase), asa1 (gene for aggregation substance), cylA (gene for cytolysin activator), esp (gene for Enterococcal surface protein) Hyl (gene for Hyaluronidase) of E. faecalis and E. faecium was performed by uniplex PCR.The amplified products are visualized on 2% agarose gel stained with ethidium bromide. The stained gels are visualized and documented with a gel documentation system and analyzed visually to determine the size of PCR amplicons of the target genes directly by comparison with 100 bp DNA ladder marker. | 2 months |