Antimicrobial susceptibility and virulence traits of Escherichia coli in healthy South American camelids in Germany

Abstract

South American camelids (SACs) are gaining popularity in the leisure sector, leading to human contact. However, data on pathogenic, zoonotic, and antimicrobial-resistant (AMR) bacteria in SACs remain limited. This study investigated the occurrence of AMR and virulence determinants in Escherichia coli isolated from feces of healthy alpacas and llamas in Germany. Between April 2021 and February 2023, longitudinal sampling was conducted on up to 20 animals per farm from 10 farms across six German federal states, with four sampling rounds at 6-month intervals. One random E. coli isolate per sample was obtained using a non-selective approach and tested for susceptibility to 14 antibiotics by broth microdilution, following EUCAST guidelines. In parallel, selective screening was performed to detect colistin- and carbapenem-resistant strains as well as ESBL/AmpC β-lactamase producers. Whole-genome sequence data from non-selectively obtained isolates mainly from round A and selectively obtained isolates from all rounds were used to identify AMR determinants and virulence-associated markers. Overall, 706 non-selectively isolated E. coli showed low resistance rates: sulfamethoxazole (4.5%), trimethoprim (1.8%), tetracycline (1.6%), ampicillin (1.4%), enrofloxacin (0.4%), cefotaxime (0.4%), colistin (0.1%), amoxicillin/clavulanate (0.1%), and piperacillin/tazobactam (0.1%). Eleven isolates exhibited multidrug-non-wild-type (MD-non-WT) phenotypes to ≥3 antimicrobial classes. Selective screening identified 18 mcr-1 and 70 cefotaxime non-WT isolates, but no carbapenem non-WT isolates. Resistance genes included blaCTX-M-1/14/15/27/55 (n = 54), blaDHA-1 (n = 4), blaCMY-2 (n = 4) and ampC promoter mutations (C42T) (n = 2). All colistin-resistant isolates carried mcr-1.1 on an IncI2 plasmid and originated from a single farm. Multilocus VNTR analysis revealed high genetic diversity with farm-specific clustering and temporal shifts. Although overall AMR prevalence was low, localized presence of ESBL- and AmpC-β-lactamase-producing and colistin-resistant E. coli indicates that SACs may represent AMR sources. Virulence profiling revealed few pathogenic E. coli except Shiga toxin-producing E. coli. These included low-virulence stx subtypes (stx1c, stx2b, and stx2f) and the high-virulence subtype stx2d, associated with enterohemorrhagic E. coli (EHEC) causing hemorrhagic colitis and hemolytic uremic syndrome in humans. Notably, detection of an EHEC isolate belonging to the O63:H6/ST583 lineage, a serotype/sequence type previously associated with severe clinical outcomes, highlights a zoonotic risk associated with SACs.