Occurrence of multidrug resistant Gram-negative bacteria and resistance genes in semi-aquatic wildlife - Trachemys scripta, Neovison vison and Lutra lutra - as sentinels of environmental health.

Emergence of antimicrobial resistance (AMR) in bacterial pathogens has been recognized as a major public health concern worldwide. In the present study, antimicrobial resistant Gram-negative bacteria (AMRGNB) and AMR genes were assessed in semi-aquatic wild animals from a highly populated and intensive farming region of Spain, Catalonia. Cloacal/rectal swab samples were collected from 241 animals coming from invasive species Trachemys scripta (n = 91) and Neovison vison (n = 131), and endangered-protected species Lutra lutra (n = 19). Accordingly, 133 (55.2%) isolates were identified as AMRGNB. Escherichia coli and Pseudomonas fluorescens were among the bacteria most frequently isolated in all animal species, but other nosocomial agents such as Klebsiella pneumoniae, Salmonella spp. or Citrobacter freundii, were also prevalent. The phenotypic susceptibility testing showed the highest resistance to β-lactams (91%). Molecular analysis showed 25.3% of turtles (15.4% ESBL/Ampc genes), 21% of Eurasian otters (10.5% ESBL/Ampc genes) and 14.5% of American minks (8.4% ESBL/Ampc genes) were positive to AMR genes. The genotyping frequency was tetM (20.6%), blaCMY-2 (13%), ermB (6.1%), blaCMY-1 (4.6%), blaCTX-M-15 (3.1%) and mcr-4 (0.8%). Turtles had a larger prevalence of AMRGNB and AMR genes than mustelids, but American mink carried mcr-4 colistin-resistance gene. Moreover, cluster analysis of AMR gene distribution revealed that an ESBL/AmpC cluster in a highly populated area comprising big metropolitan regions, and another tetM/emrB cluster in an expended area with highly intensive livestock production. Although the mcr-4 positive case was not included in those clusters, that case was found in a county with a high pig farm density. In conclusion, semi-aquatic wild animals are a good sentinel for environmental contamination with AMRGNB and AMR genes. Therefore, One Health Approach is urgently needed in highly populated regions, and with intensive livestock production like Catalonia.