Optimizing a Screening Protocol for Potential Extended-Spectrum β-Lactamase Escherichia coli on MacConkey Agar for Use in a Global Surveillance Program

Abstract

The increasing prevalence of ESBL-producing Escherichia coli is worrisome. Coordinated efforts to better understand global prevalence and risk factors are needed. Developing LMIC countries need reliable, readily available, and cost-effective solutions for detecting ESBL E. coli to contribute to global surveillance. We evaluated MacConkey agar supplemented with ceftriaxone or cefotaxime as a screening method for accurately detecting and quantifying potential ESBL E. coli. MacConkey from eight manufacturers representing seven countries were prepared with 2 or 4 μg/ml ceftriaxone or cefotaxime, respectively. Four E. coli strains (NC 11, ATCC 25922, CM-13457, and CM-10455) and one K. pneumoniae strain (CM-11073) were grown overnight, serially diluted, and plated in triplicate for enumeration on all media combinations. After recovery was assessed, US-1 MacConkey agar with cefotaxime was used to further evaluate reproducibility and detection of potential ESBL E. coli from poultry cecal (n=30) and water (n=30) samples. Results indicated recovery of E. coli 13457 from four MacConkey agar manufacturers was reduced by up to 4 log CFU/ml, and phenotypic differences in colony size and color were apparent by manufacturer for control E. coli strains. A true ESBL, NC11, was not reduced with 4 μg/ml cefotaxime. From ceca and water, potential ESBL E. coli were only confirmed from MacConkey agar with 4 μg/ml cefotaxime, where 45% and 16.6% of E. coli isolates phenotypically expressed ESBL-production. The quality and reproducibility of MacConkey agar varied by manufacturer, which suggests that a single manufacturer and media type should be selected for global monitoring efforts so that training and interpretation can be standardized.