Gram-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.
