Cysteine lactose electrolyte deficient agar cled

Brilliance UTI medium and Cysteine lactose electrolyte deficient agar-CLED (Oxoid, Basingstoke, UK) were used for primary culture of urine. Primary blood culture was inoculated into BacTalert bottles with sampling based on weight-based criteria and incubated in the BacTalert system BacT/Alert system (BioMerieux, Marcy l′Etoile, France) until positive. Sheep blood agar, Chocolate agar, and MacConkey agar were used for other specimens as required. Culture plates were incubated for 18–24 h at 35.5 °C in ambient air (Chocolate agar incubated in 5% CO₂).
The Vitek 2 Compact (bioMerieux, Marcy l'Etoile, France) with VITEK 2 GN ID card was used for bacterial identification and the VITEK 2 AST-280/ VITEK 2 AST-281 cards for susceptibility testing. Clinical and Laboratory Standards Institute (CLSI) M100 30th Edition guidelines³⁴ was used for interpretation of MIC results. E. coli ATCC 25922 was used as control. Multi-drug resistance was defined as resistance to antibiotics in three of more different classes.
Shipped isolates to Thakur Molecular Epidemiology laboratory, North Carolina State University (NC State), USA underwent microdilution assay with the Gram-negative Sensititre™ (CMV3AGNF) plate (Trek Diagnostic Systems, OH). Plates were read using Sensititre ARIS and interpreted according to CLSI M100 28th Edition guidelines³⁵ . E. coli ATCC 25922 was used as a control.

Each urine samples were inoculated onto a Cysteine-Lactose-Electrolyte Deficient agar (CLED) (Oxoid Ltd., England) by using a calibrated, sterile, nonreusable plastic loop 1 μl (0.001 ml) and incubated aerobically at 37°C for 18 to 24 hours to check the growth, and urine cultures were considered as significant bacteriuria when colony forming units (CFUs) were ≥10⁵/ml of voided urine, and a single colony was picked and suspended in nutrient broth and then subcultured onto blood agar plate and MacConkey agar plate, finally incubated at 37°C for 24 hours for further identification. Bacterial identification was then done using standardized biochemical tests, namely, indole production, lactose fermentation, hydrolysis of urea, citrate utilization, lysine decarboxylation, and motility test for Gram-negative bacteria and for Gram-positive bacteria, mannitol fermentation, and catalase and coagulase tests.