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.

Disease transmission among wild boars, domestic animals and humans is a public health concern, especially in areas with high wild boar densities. In this study, fecal samples of wild boars (n = 200) from different locations of the Metropolitan Area of Barcelona were analyzed by PCR to explore the frequency of β-lactamases and extended cephalosporin and carbapenem resistance genes (ESBLs) in Escherichia coli strains and the presence of toxigenic Clostridioides difficile. The prevalence of genes conferring resistance to β-lactam antimicrobials was 8.0% (16/200): bla_CMY-2 (3.0%), bla_TEM-1b (2.5%), bla_CTX-M-14 (1.0%), bla_SHV-28 (1.0%), bla_CTX-M-15 (0.5%) and bla_CMY-1 (0.5%). Clostridioides difficile TcdA+ was detected in two wild boars (1.0%), which is the first report of this pathogen in wild boars in Spain. Moreover, the wild boars foraging in urban and peri-urban locations were more exposed to AMRB sources than the wild boars dwelling in natural environments. In conclusion, the detection of E. coli carrying ESBL/AmpC genes and toxigenic C. difficile in wild boars foraging in urban areas reinforces the value of this game species as a sentinel of environmental AMRB sources. In addition, these wild boars can be a public and environmental health concern by disseminating AMRB and other zoonotic agents. Although this study provides the first hints of the potential anthropogenic sources of AMR, further efforts should be conducted to identify and control them.

Wildlife has been suggested to be a good sentinel of environmental health because of its close interaction with human populations, domestic animals, and natural ecosystems. The alarming emergence of antimicrobial resistance (AMR) in human and veterinary medicine has activated/triggered the awareness of monitoring the levels of AMR pollution in wildlife. European hedgehogs (Erinaceus europaeus) are common wild species habiting urban areas in Europe. However, there are few studies conducted in hedgehogs as reservoirs of AMR bacteria or genes. The aim of this study was to assess the occurrence and distribution of ESBL, AmpC, and carbapenem-resistant enterobacteria and AMR genes in wild European hedgehogs in Catalonia, a densely populated region of NE Spain. A total of 115 hedgehogs admitted at the Wildlife Rehabilitation Center of Torreferrussa were studied. To our knowledge, this is the first description of β-lactam resistant enterobacteria in wild hedgehogs. Interestingly, 36.8% (42/114) of the animals were detected as carriers of β-lactamase/carbapenemase resistance genes. Klebsiella spp. (59.6%), and specifically K. pneumoniae (84.6%), were the bacteria with the highest proportion of resistance genes, followed by E. coli (34.6%) and C. freundii (5.8%). The most frequently detected genetic variants were blaCTX-M-15 (19.3%), blaSHV-28 (10.5%), blaCMY-1 (9.7%), blaCMY-2 (8.8%), and blaOXA-48 (1.7%). In addition, 52% (27/52) of the isolates presented a multidrug resistance (MDR) phenotype and 31% had an extended drug resistance (XDR) profile. No clustering of animals with AMR genes within the study region was shown in the spatial analysis, nor differences in the proportion of positive animals among regions, were detected. The results of this study suggest that wild European hedgehogs could be good sentinels of AMR environmental pollution, especially in areas with a high human population density, because they either inhabit and/or feed in an anthropogenic environment. In conclusion, it is crucial to raise awareness of the strong interconnection between habitats and compartments, and therefore this implies that AMR issues must be tackled under the One Health approach.

Escherichia coli is considered one of the most common agents associated with neonatal diarrhea in piglets. The aim of this work was to characterize the pathogenic and antimicrobial resistance (AMR) profiles of 122 E. coli strains isolated from pigs suffering diarrhea (n = 94) and pigs without diarrhea (n = 28) of 24 farms in Spain. Virulence factors, toxins and AMR (ESBL and colistin) genes and AMR phenotypes of E. coli isolates were analyzed. Low prevalence of pathogenic E. coli strains (26%) was found in both groups. However, ETEC and VTEC strains were more frequently isolated from diarrheic piglets. Irrespectively of diarrhea occurrence, 97.5% of the strains showed a multidrug-resistance (MDR) profile to aminopenicillins, sulfonamides and tetracyclines. It was found that 22% of E. coli was CTX-M+, with CTX-M-14 being the principal allelic variant. Remarkably, 81.5% of CTX-M+ strains were isolated from diarrheic animals and presented an extended MDR profile to aminopenicillins, quinolones and aminoglycosides. Finally, low frequencies of colistin resistance genes mcr-1 (4/122) and mcr-4 (1/122) were found. MDR E. coli strains are circulating in pig farms of Spain, representing a serious threat to animal and public health. More appropriate diagnostic approaches (genetic and AMR phenotypic analysis) should be implemented in animal health to optimize antibiotic treatments.

In wildlife, most of the studies focused on antimicrobial resistance (AMR) describe Escherichia coli as the principal indicator of the selective pressure. In the present study, new species of Enterobacteriaceae with a large panel of cephalosporin resistant (CR) genes have been isolated from wildlife in Catalonia. A total of 307 wild animals were examined to determine CR enterobacteria prevalence, AMR phenotypes and common carbapenem and CR gene expression. The overall prevalence of CR-phenotype was 13% (40/307): 17.3% in wild mammals (18/104) and 11.5% in wild birds (22/191) (p<0.01)). Hedgehogs presented the largest prevalence with 13.5% (14/104) of the mammal specimens, followed by raptors with 7.3% (14/191) of the total bird specimens. Although CR E. coli was obtained most frequently (45%), other CR- Enterobacteriaceae spp like Klebsiella pneumoniae (20%), Citrobacter freundii (15%), Enterobacter cloacae (5%), Proteus mirabilis (5%), Providencia spp (5%) and Serratia marcescens (2.5%) were isolated. A high diversity of CR genes was identified among the isolates, with 50% yielding bla CMY -2, 23% bla SHV -12, 20% bla CMY -1 and 18% bla CTX-M -15. Additionally, new CR-gene variants and resistance to carbapenems associated to OXA-48 were found. Most of the CR isolates, principally K. pneumoniae and C. freundii , were multiresistant with co-resistance to fluoroquinolones, tetracycline, sulphonamides and aminoglycosides. This study describes for the first time in wildlife a high prevalence of Enterobacteriaceae spp harbouring a large variety of carbapenem and CR genes frequently associated to nosocomial human infections. Implementation of control measures to reduce the impact of anthropogenic pressure in the environment is urgently needed.

Background/Objectives: Antimicrobial resistance (AMR) in companion animals is an increasing concern within the One Health framework, particularly regarding opportunistic pathogens such as Klebsiella spp. This retrospective study evaluated the epidemiology, antimicrobial susceptibility profiles, and temporal resistance trends of Klebsiella spp. infections in dogs and cats across the Iberian Peninsula. Methods: A total of 809 clinical isolates collected between 2016 and 2024 and submitted to a private diagnostic laboratory in Barcelona were analysed. Results: Klebsiella pneumoniae was the predominant species (70%), more frequently identified in cats (76%) than in dogs (68%). Dermatological and respiratory samples exhibited the highest prevalence of multidrug-resistant (MDR) isolates. Overall MDR prevalence was high, particularly in cats (51.1%; 95% CI 41.1–60.9%) compared with dogs (38.4%; 95% CI 34.1–42.8%) although it was not statistically significant. K. pneumoniae generally exhibited higher resistance rates than K. oxytoca, particularly to amoxicillin/clavulanic acid, first-/second-generation cephalosporins, third-/fourth-generation cephalosporins (3/4th GC), fluoroquinolones, and tetracyclines. In both bacterial species, resistance rates were consistently higher among feline isolates. In contrast, aminoglycosides and phenicols retained high activity against most isolates. Temporal analysis revealed a significant increasing resistance trend to amoxicillin/clavulanic acid, which is particularly concerning given the widespread use of this antimicrobial as a first-line treatment in small animal practice. However, resistance trend to aminoglycosides showed a significant decline. No significant temporal changes were detected for 3/4th GC and fluoroquinolones, suggesting the persistence of resistant populations within companion animals. Resistance to aminoglycosides and phenicols remained comparatively low in this study. Whereas critically important category B antimicrobials, such as 3/4th GC and fluoroquinolones, exhibited low to moderate effectiveness, raising concerns about their empirical use. Conclusions: These findings highlight the substantial AMR and MDR burden of K. pneumoniae in companion animals in the Iberian Peninsula and reinforce the need for prudent antimicrobial use, routine susceptibility testing, and integrated One Health surveillance strategies.