Vancomycin

During the study period, 1290 patients were admitted in the PICU, out of which 1018 patients stayed >48 h. As calculated sample size was 196, convenient sampling was carried out to achieve the sample size. Rectal swabs were collected after 48 h of admission to PICU and inoculated onto bile esculin sodium azide agar containing 6mg/L vancomycin (Himedia laboratories, Mumbai, India).[5 (link)] Black/brown colonies were presumptively identified as Enterococcus and confirmed to species level based on Facklam and Collins standard biochemical tests.[24 (link)] Minimum inhibitory concentration (MIC) of vancomycin and teicoplanin was determined by agar dilution method for all the enterococcal isolates grown on BEA as per the Clinical and Laboratory Standard Institute guidelines.[25 ] MIC of ≥32 was considered as resistant to both vancomycin and teicoplanin. The antimicrobial susceptibility of the isolates to other antibiotics, namely, ampicillin, high-level gentamicin, tetracycline, and linezolid was also performed by Kirby-Bauer disc diffusion method.[26 ] Association of VRE colonization with demographic features of the patients and various risk factors for VRE colonization were assessed based on previous studies.

All samples from inpatient wards, sent for culture to the Department of Microbiology, were selected according to the criteria proposed in the study methodology. For blood culture, paired samples were inoculated in blood culture bottles (BacT/Alert FA Plus for adults and PF Plus for pediatric patients) and cultured in BacT/Alert systems (bioMérieux, France) for a period of at least 7 days. For urine culture, colony counts of ≥10⁵ colony-forming units per ml (CFU/ml) were evaluated. Selected samples were cultured in conventional media and screened for presumptive vancomycin resistance on a VRE screen agar and prepared using bile esculin azide agar supplemented with 6% (6 μg/ml) vancomycin (HiMedia Laboratories, Mumbai, India).[4 9 (link)10 ] Identification of Enterococcus species by conventional biochemical reactions (growth on potassium tellurite agar, 6.5% sodium chloride (NaCl) broth, 1% sugar fermentation tests, esculin hydrolysis, and arginine hydrolysis) and results interpreted by the identification scheme proposed by Facklam and Collins.[2 (link)3 11 (link)]

Stool samples were collected as early as within 2 days of admission in the ICU irrespective of their previous hospitalization status and then weekly until discharge from all the patients and transported to the microbiology laboratory. Samples were then plated directly on bile esculin azide agar (BEAA) with and without 6 μg/ml vancomycin (HiMedia, Mumbai) and also following enrichment in BEA broth with 6 μg/ml vancomycin for 24 h at 37°C. All the plates were incubated aerobically at 37°C for 48 h. Four to five colonies with dark brown halo morphologically resembling enterococcal colonies were selected and further tested by Gram's stain, PYRase test and growth in 6.5% NaCl. Speciation of the isolates was done based on standardized biochemical classification.[4 (link)] Enterococcus fecalis ATCC 29212 and previously characterized vanA E. faecium strains were taken as controls. Minimum inhibitory concentration (MIC) for vancomycin and teicoplanin were determined by agar dilution method.[5 ] Detection of the resistant determinants were done by polymerase chain reaction amplification of vanA, vanB, and vanC genes.[6 ]