Massive culture-based approach for the screening of AmpC, ESBL, and carbapenemase producers from rectal swabs.

Gram-negative bacilli-producing beta-lactamases are major causes of difficult-to-treat infections, especially the AmpC, extended-spectrum beta-lactamases (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 includes enrichment on MacConkey agar supplemented with ceftriaxone to select for AmpC, ESBL, and carbapenemase producers, followed by replica plating under selective pressures (cefoxitin, cefepime, and 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 intestinal tract is a major reservoir of multidrug-resistant gram-negative bacilli, and surveillance of colonization is essential to understand resistance dissemination in both community and healthcare settings. However, standard culture-based methods typically target specific resistance mechanisms, often overlooking the coexistence of distinct beta-lactamase-producing strains within a single host. This limits our understanding of colonization dynamics and resistance evolution. To address this gap, we developed a culture-based approach that assesses the growth of strains under different selective pressures from a single rectal swab. By combining enrichment with replica plating, our method enables phenotypic discrimination between AmpC-, extended-spectrum beta-lactamases-, and carbapenemase-producing bacteria without depleting sample material across multiple media. Although validated for rectal swabs, the approach may be adapted for other complex samples such as urine, blood, soil, or water, expanding its utility in diverse clinical and environmental investigations. This strategy enhances the detection of diverse resistance profiles and supports a more comprehensive view of colonization and antimicrobial resistance dynamics.