Characterization of Enterococci

Primary outcomes

1. I) Sample collection

   Time frame: 2 months

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

2. II) Identification of Enterococci:

   Time frame: 2 month

   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%)

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

   Time frame: 2 weeks

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

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

   Time frame: 1 week

   by Detection of hemolytic activity on the blood agar

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

   Time frame: 2 weeks

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

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

   Time frame: 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.

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

   Time frame: 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

8. VIII) Antibiotic sensitivity test:

   Time frame: 3 monthe

   • 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.

9. IX) Molecular Identification of commonest Enterococcus species

   Time frame: 1 month

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

10. X) Molecular detection of virulence genes

    Time frame: 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.