Linking land use to the likely origins of third-generation cephalosporin-resistant Enterobacterales in freshwater

ABSTRACT Antimicrobial resistance is a major public health threat, as resistant infections are harder to treat and carry greater risks. Using a One Health framework, this survey of freshwater samples investigated the influence of local land use on the occurrence of waterborne third-generation cephalosporin-resistant Enterobacterales (3GC-E) mediated through extended-spectrum β-lactamases (ESBLs) or plasmid-mediated AmpC (pAmpC) genes. A cross-sectional survey of 339 freshwater samples from 49 New Zealand sites was undertaken, encompassing catchments with urban, agricultural (namely dairy; sheep and beef; and mixed dairy, sheep, and beef), avian-impact, and low-impact (exotic/native forest) dominant land uses. 3GC-E ( n = 63), including E. coli , Enterobacter , Citrobacter, and Klebsiella, were isolated from freshwater samples ( n = 35), where urban (32), dairy (1), avian (1), and sheep and beef (1) were identified as the dominant land use. 3GC-E genome assemblies ( n = 43) from freshwater were compared with human ESBL- E. coli ( n = 467) assemblies. Waterborne 3GC- E. coli were phylogenetically diverse, represented by seven phylotypes and 20 sequence types (ST131, ST38, ST68, and ST219) associated with human infections, suggesting contamination with urban wastewater. Two freshwater E. coli harbored the plasmid-associated carbapenemase-encoding bla NDM-5 - ble MBL gene cassette, which was absent from the human isolates. Two further freshwater E. coli and four human E. coli isolates carried pAA plasmids containing agg genes encoding for enteroaggregative E. coli adherence factors. By linking local land use at a national scale with the occurrence of freshwater 3GC- E. coli , we demonstrate the value of a One Health approach for understanding how human activities influence the environmental dissemination of antimicrobial resistance with implications for infectious disease risk. IMPORTANCE Antimicrobial resistance (AMR) threatens public health by making infections harder and more expensive to treat. While AMR is often studied in clinical settings, environmental pathways that spread resistant bacteria remain less understood. Using a One Health approach, we investigated how land use influences the presence of third-generation cephalosporin-resistant Enterobacterales in freshwater across New Zealand. We found that resistant Escherichia coli , including strains carrying plasmid-associated virulence factors, were most frequently detected at sites impacted by urban land use and human fecal contamination. These findings show that human activities shape the environmental distribution of clinically relevant resistance and highlight freshwater as a potential exposure pathway. By linking national-scale land-use patterns with the occurrence of resistant bacteria in the environment, this study demonstrates how integrated environmental and public health surveillance can improve our understanding of AMR dissemination and inform strategies to reduce the spread of resistant pathogens.