Massive culture-based approach for the screening of AmpC-, ESBL- and carbapenemase producers from complex samples

ABSTRACTGram-negative bacilli producing beta-lactamases are major causes of difficult-to-treat infections, especially the AmpC, ESBL, and carbapenemase types. Their spread within and outside hospital settings demands effective detection and monitoring in various environments, but current methods for this purpose often neglect important groups of beta-lactamases or are expensive and time consuming. We aimed to develop and test a massive culture approach to detect and differentiate between beta-lactamase producers from complex samples. The method involved an enrichment step on MacConkey agar supplemented with ceftriaxone to select for AmpC, ESBL, and carbapenemase producers, followed by replica plating under selective pressures (cefoxitin, cefepime, imipenem) to differentiate them. The massive culture approach effectively differentiated strains producing different beta-lactamases in mixed cultures. In tests with rectal swabs, our method demonstrated 100% sensitivity, higher specificity, and greater accuracy for ESBL detection compared to the reference method. Additionally, it identified a broader spectrum of beta-lactamase producers, including AmpC and carbapenemase. The massive culture approach is a promising tool for detecting and differentiating gram-negative bacilli producing beta-lactamases from rectal swabs. Due to the additional time required to produce results, this method is most suitable for central and research laboratories and enhances surveillance capabilities for antimicrobial resistance. IMPORTANCEThe dissemination of multidrug-resistant bacteria producing beta-lactamases in healthcare and community settings demands effective detection and monitoring. Existing methods may miss key beta-lactamase groups or are costly and time-consuming. In this study, a comprehensive culture-based approach using replica plating was developed and used to detect and differentiate beta-lactamase producers from rectal swabs. It showed higher specificity and greater accuracy for ESBL detection compared to the reference method, while identifying a broader range of beta-lactamase producers, including AmpC and carbapenemase. Despite requiring additional processing time, this method is suitable for central and research laboratories, enhancing antimicrobial resistance surveillance.