Quantification of β-lactamase producing bacteria in German surface waters with subsequent MALDI-TOF MS-based identification and β-lactamase activity assay
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 3rd gen-
eration 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 environ-
mentally relevant strains with Pseudomonas, Flavobacterium, and Janthinobacterium being pre-
dominant 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 (blaTEM, blaCMY-2, blaOXA-48, blaVIM-2,
blaSHV, and blaNDM-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 carbapen-
emase enzymes in oligotrophic environmental bacteria that have to be considered as a reservoir of
ARGs and contribute to the spread of antibiotic resistance
