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

IV)Phenotypic detection of virulence marker of Enterococci (hemolytic activity)

时间窗: 1 week

by Detection of hemolytic activity on the blood agar

V) Phenotypic detection of virulence marker of Enterococci(Caseinase production)

时间窗: 2 weeks

by Detection of Caseinase production on Muller hinton agar containing skimmed milk 3%.

VIII) Antibiotic sensitivity test:

时间窗: 3 monthe

1. 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. 2. 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.

IX) Molecular Identification of commonest Enterococcus species

时间窗: 1 month

by conventional gene specific uniplex PCR for E. faecalis and E. Faecium

X) Molecular detection of virulence genes

时间窗: 2 months

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.

VII) Phenotypic detection of virulence marker of EnterococcI (Biofilm formation)

时间窗: 2 weeks

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

II) Identification of Enterococci:

时间窗: 2 month

by inoculation on Bile esculin azide \& growing colonies further identified by : 1. microscopically after staining by gram stain 2. catalase test 3. grow on high concentration of NaCl (6.5%)

I) Sample collection

时间窗: 2 months

Samples are collected from intensive care unit (ICU) including urine, pus swabs, sputum, blood, tracheal aspirates and pharyngeal swabs

III) Phenotypic detection of virulence marker of Enterococci(Gelatinase activity)

时间窗: 2 weeks

by culture on nutrient agar containing gelatin.Positive results appeared as liquefaction of gelatin

VI) Phenotypic detection of virulence marker of Enterococci(Formation of Slime layer)

时间窗: 1 week

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.