Quantification of β-lactamase producing bacteria in German surface waters with subsequent MALDI-TOF MS-based...

Environmental oligotrophic bacteria are suspected to be highly relevant carriers of antimicrobial resistance (AMR). However, there is a lack of validated methods for monitoring in the aquatic environment. Since extended-spectrum β-lactamases (ESBLs) play a particularly important role in the clinical sector, a culturing method based on R2A-medium spiked with different combinations of β-lactams was applied to quantify β-lactamase-producing environmental bacteria from surface waters. In German surface water samples (n = 28), oligotrophic bacteria ranging from 4.0 × 10³ to 1.7 × 10⁴ CFU per 100 mL were detected on the nutrient-poor medium spiked with 3^rd generation cephalosporins and carbapenems. These numbers were 3 log₁₀ higher compared to ESBL-producing Enterobacteriales of clinical relevance from the same water samples. A MALDI-TOF MS identification of the isolates demonstrated, that the method leads to the isolation of environmentally relevant strains with Pseudomonas, Flavobacterium, and Janthinobacterium being predominant β-lactam resistant genera. Subsequent micro-dilution antibiotic susceptibility tests (Micronaut-S test) confirmed the expression of β-lactamases. The qPCR analysis of surface waters DNA extracts showed the presence of β-lactamase genes (bla_TEM, bla_CMY-2, bla_OXA-48, bla_VIM-2, bla_SHV, and bla_NDM-1) at concentrations of 3.7 (±1.2) to 1.0 (±1.9) log₁₀ gene copies per 100 mL. Overall, the results demonstrate a widespread distribution of cephalosporinase and carbapenemase enzymes in oligotrophic environmental bacteria that have to be considered as a reservoir of ARGs and contribute to the spread of antibiotic resistance.