Characterization of Enterococci

I) Sample collection

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

II) Identification of Enterococci:

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%)

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

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

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

by Detection of hemolytic activity on the blood agar

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

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

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.

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

VIII) Antibiotic sensitivity test:

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

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

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.