Cefotaxime

We isolated strain BP-1(T) (bedside rail) and BP-2 (nurse call button) from an intensive care room at a tertiary care hospital in Pakistan with the Eswab collection device and transport system (Becton Dickenson & Company, Franklin Lakes, NJ) in June 2016. Surfaces were swabbed in triplicate during an ongoing yearlong longitudinal sampling study. Swabs were sent to the US at room temperature and arrived in the US within ~3 days of sampling. Samples from Pakistan were received by the Burnham lab under approval of the Washington University IBC Protocol number 6,572. Swabs were cultured within 24 h of arrival in the US laboratory site. The isolates were recovered on MacConkey agar with 5 μg/ml cefotaxime (Hardy Diagnostics, Santa Maria, CA) and re-streaked on blood agar using the four-quadrant method to recover distinct single colonies. The isolates were unidentified by the MALDI-TOF VITEK MS IVD v2.3.3 (bioMerieux, Durham, NC) and Bruker BioTyper (Bruker Daltonics, Billerica, MA) mass spectrometry systems. For subsequent genomic analysis, frozen stocks of each isolate were made by creating a dense suspension of the isolate in Tryptic Soy Broth (Sigma Aldrich, St. Louis, MO) with 15% Glycerol (Hardy Diagnostics, Santa Maria, CA).

All of the samples above, excluding the tap water, were plated on R2A, Middlebrook 7H11 (Remel, Lenexa, KS), Pseudosel (Becton Dickinson, Franklin Lakes, NJ), Chromagar KPC (Chromagar, Paris, France), and MacConkey with 2 mg/L cefotaxime (Hardy Diagnostics, Santa Maria, CA). R2A plates were incubated at 25 °C for 7 days; 7H11 Middlebrook plates were incubated at 30 °C and counted at 3, 5, and 7 days (all samples were CPC treated as described above prior to plating on 7H11 Middlebrook medium); Pseudosel plates were incubated at 35 °C for 24 hrs and then at 25 °C for an additional 24 hrs; Chromagar KPC plates were incubated for 24 hours at 35 °C; and MacConkey with 2 mg/L cefotaxime plates were incubated at 35 °C for 72 hrs. Volume plated was maximum 5 ml (concentrated on a filter), making the limit of detection 6 CFU/ml given a countable plate definition of 30–300 colonies. Final bacterial counts factored in dilutions and the original volume collected or surface area sampled.