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Extended-spectrum cephalosporinase producing (ESC) E. coli are regarded as key indicator microorganisms of antimicrobial resistance (AMR), calling for a One Health integrated global surveillance strategy. Wildlife is exposed to antibiotic contaminants and/or resistant bacteria that have been released into the environment, potentially acting as reservoirs and spreaders of resistance genes as well as sentinels of anthropogenic pressure. Monitoring AMR in wildlife has become crucial in determining anthropogenic environmental impacts as well as transmission routes. In this study, we determined the occurrence and potential sources of ESC E. coli in 218 Eurasian griffon vultures (Gyps fulvus) foraging regularly on human waste disposed at a dumpsite in north-eastern Spain. Minimal inhibitory concentration for 14 different antimicrobials was performed to evaluate the phenotype of the isolates, and whole genome sequencing was carried out to investigate lineages and plasmids harbouring ESC genes. Our sequences were compared to previously published Spanish sequences of human, animal, and wildlife origin. We report a high prevalence of CTX-M-15, as well as the presence of other resistance genes such as OXA-10, CTX-M-27, and CTX-M-65 which are rarely described in European livestock, suggesting a human origin. The isolates also carried a diverse range of additional AMR genes for a broad spectrum of drug families, with the majority being multi-drug resistant. The phylogenomic analyses suggests the transmission of high-risk lineages from humans to vultures, with 49 % of our isolates matching the most common extraintestinal pathogenic E. coli (ExPEC) lineages described in humans worldwide, including ST131, ST10 and ST58. We conclude that anthropogenically altered habitats, such as landfills, are hotspots for the acquisition and spread of high-risk ESC E. coli lineages associated with hospital infections. Measures must be implemented to limit their spread into natural environments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Objectives: To describe the susceptibility of Escherichia coli to medically important antibiotics, collected over four periods (2004-2006, 2008-2009, 2013-2014, 2017-2018), from food-producing animals at slaughter., Methods: Intestinal contents from cattle, pigs and broilers were randomly sampled (5-6 countries/host; ≥4 abattoirs/country; one sample/animal/farm) for isolation of Escherichia coli; antimicrobial susceptibilities were centrally determined by CLSI agar dilution. Clinical breakpoints (CLSI) and epidemiological cut-off values (EUCAST) were applied for data interpretation., Results: In total, 10 613 E. coli strains were recovered. In broilers, resistance percentages were the lowest (P ≤ 0.01) in the latest time period. A significant decrease in MDR over time was also observed for broilers and a tendency for a decrease for pigs. Resistance to meropenem and tigecycline was absent, and resistance to azithromycin was 0.2%-2.0%. Also, low resistance to third-generation cephalosporins (1.1%-7.4%) was detected in broilers. Resistance to colistin varied between 0.1%-4.8%. E. coli from broilers showed high resistance to ciprofloxacin (7.3%-23.3%), whereas for cattle and pigs this was 0.2%-2.5%. Low/moderate resistance to chloramphenicol (9.3%-21.3%) and gentamicin (0.9%-7.0%) was observed in pigs and broilers. The highest resistance was noted for ampicillin (32.7%-65.3%), tetracycline (41.3%-67.5%), trimethoprim (32.0%-35.7%) and trimethoprim/sulfamethoxazole (27.5%-49.7%) from pigs and broilers, with marked country differences. MDR peaked in pigs and broilers with 24 and 26 phenotypes, with 21.9%-26.2% and 18.7%-34.1% resistance, respectively., Conclusions: In this pan-EU survey antibiotic susceptibility of commensal E. coli varied largely between antibiotics, animal species and countries. Resistance to critically important antibiotics for human medicine was absent or low, except for ciprofloxacin in broilers and ampicillin in pigs and broilers., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

This study assessed the dynamics of cephalosporin resistant (CR) E. coli populations during the life cycle of pigs treated early in life with ceftiofur or tulathromycin. The study was conducted at eight conventional pig farms; four for each treatment with ceftiofur or tulathromycin. At each farm, 70 7-day-old piglets were divided into two groups: a control group (n = 30) and a treatment group (n = 40). Faecal samples were collected on day 0 and on days 2, 7 and 180 post-treatment. Sows were also sampled on day 0. CR E. coli were selected on MacConkey agar with ceftriaxone. On five farms, 7-day-old piglets excreted CR E. coli before treatment associated with the presence of CR E. coli in sows. The occurrence of CR E. coli positive animals decreased with increasing piglet age. The remaining three farms tested negative for CR E. coli during the study period. Results demonstrated great variability in the frequency of CR E. coli positive animals between farms, independent of treatment. Treatment with ceftiofur resulted in a transitory increase in the counts of CR E. coli after 48 h. However, other risk factors including the presence of CR E. coli in sows and animal age were more important than antimicrobial treatment. Accordingly, intervention strategies targeting sows would likely have a beneficial effect in reducing the occurrence of antimicrobial resistance in primary pig production., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Avian pathogenic Escherichia coli (APEC) are the major cause of colibacillosis in poultry production. In this study, a total of 22 E. coli isolated from colibacillosis field cases and 10 avian faecal E. coli (AFEC) were analysed. All strains were characterised phenotypically by susceptibility testing and molecular typing methods such as pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). The presence of 29 virulence genes associated to APEC and human extraintestinal pathogenic E. coli (ExPEC) was also evaluated. For cephalosporin resistant isolates, cephalosporin resistance genes, plasmid location and replicon typing was assessed. Avian isolates belonged to 26 O:H serotypes and 24 sequence types. Out of 22 APEC isolates, 91% contained the virulence genes predictors of APEC; iutA, hlyF, iss, iroN and ompT. Of all strains, 34% were considered ExPEC. PFGE analysis demonstrated a high degree of genetic polymorphism. All strains were multi-resistant, including those isolated from healthy animals. Eleven strains were resistant to cephalosporins; six contained blaCTX-M-14, two blaSHV-12, two blaCMY-2 and one blaSHV-2. Two strains harboured qnrA, and two qnrA together with aac(6')-Ib-cr. Additionally, the emergent clone O25b:H4-B2-ST131 was isolated from a healthy animal which harboured blaCMY-2 and qnrS genes. Cephalosporin resistant genes were mainly associated to the presence of IncK replicons. This study demonstrates a very diverse population of multi-drug resistant E. coli containing a high number of virulent genes. The E. coli population among broilers is a reservoir of resistance and virulence-associated genes that could be transmitted into the community through the food chain. More epidemiological studies are necessary to identify clonal groups and resistance mechanisms with potential relevance to public health.

Flies may act as potential vectors for the spread of resistant bacteria to different environments. This study was intended to evaluate the presence of Escherichia coli strains resistant to cephalosporins in flies captured in the areas surrounding five broiler farms. Phenotypic and molecular characterization of the resistant population was performed by different methods: MIC determination, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and phylotyping. The presence of extended-spectrum beta-lactamase (ESBL) genes, their plasmid location, and the mobile genetic elements involved in their mobilization were studied. Additionally, the presence of 35 genes associated with virulence was evaluated. Out of 682 flies captured, 42 yielded ESBL-producing E. coli. Of these isolates, 23 contained bla(CTX-M-1), 18 contained bla(CTX-M-14), and 1 contained bla(CTX-M-9). ESBL genes were associated mainly with the presence of the IncI1 and IncFIB replicons. Additionally, all the strains were multiresistant, and five of them also harbored qnrS. Identical PFGE profiles were found for E. coli isolates obtained from flies at different sampling times, indicating a persistence of the same clones in the farm environment over months. According to their virulence genes, 81% of the isolates were considered avian-pathogenic E. coli (APEC) and 29% were considered extraintestinal pathogenic E. coli (ExPEC). The entrance of flies into broiler houses constitutes a considerable risk for colonization of broilers with multidrug-resistant E. coli. ESBLs in flies reflect the contamination status of the farm environment. Additionally, this study demonstrates the potential contribution of flies to the dissemination of virulence and resistance genes into different ecological niches., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The aim of this study was to evaluate if the treatments with ceftiofur and amoxicillin are risk factors for the emergence of cephalosporin resistant (CR) E. coli in a pig farm during the rearing period. One hundred 7-day-old piglets were divided into two groups, a control (n = 50) group and a group parenterally treated with ceftiofur (n = 50). During the fattening period, both groups were subdivided in two. A second treatment with amoxicillin was administered in feed to two of the four groups, as follows: group 1 (untreated, n = 20), group 2 (treated with amoxicillin, n = 26), group 3 (treated with ceftiofur, n = 20), and group 4 (treated with ceftiofur and amoxicillin, n = 26). During treatment with ceftiofur, fecal samples were collected before treatment (day 0) and at days 2, 7, 14, 21, and 42 posttreatment, whereas with amoxicillin, the sampling was extended 73 days posttreatment. CR E. coli bacteria were selected on MacConkey agar with ceftriaxone (1 mg/liter). Pulsed-field gel electrophoresis (PFGE), MICs of 14 antimicrobials, the presence of cephalosporin resistance genes, and replicon typing of plasmids were analyzed. Both treatments generated an increase in the prevalence of CR E. coli, which was statistically significant in the treated groups. Resistance diminished after treatment. A total of 47 CR E. coli isolates were recovered during the study period; of these, 15 contained blaCTX-M-1, 10 contained blaCTX-M-14, 4 contained blaCTX-M-9, 2 contained blaCTX-M-15, and 5 contained blaSHV-12. The treatment with ceftiofur and amoxicillin was associated with the emergence of CR E. coli during the course of the treatment. However, by the time of finishing, CR E. coli bacteria were not recovered from the animals., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Postweaning diarrhea (PWD) is a multifactorial disease caused by different aetiological agents, like viruses or bacteria and where the role of the microbiota remains unclear. The aim of this study was to assess differences between healthy and diarrheic weaned pigs concerning the prevalence of pathogens and changes in the intestinal microbiota. Eighteen farms with PWD were selected and 277 fecal samples were collected (152 diarrheic vs 125 healthy). Presence of Rotavirus A (RVA), B (RVB), C (RVC) and Porcine Epidemic Diarrhea Virus (PEDV), virulence factors of Escherichia coli and Clostridioides difficile were analyzed by PCR. Finally, the microbiota composition was also study by 16 S rRNA sequencing on 148 samples (102 diarrheic vs 46 healthy). RVA (53.95 % vs 36 %, p=0.04) and RVB (49.67 % vs 28.8 %, p<0.001) were more frequent in diarrheic animals. Furthermore, RVA viral load was higher in diseased animals. VT2 toxin was significantly associated with diarrhea, whereas other virulence factors were not. Presence of C. difficile and PEDV was almost negligible. Regarding microbiota changes, Fusobacteriota phylum was more frequent in diarrheic samples and Ruminococcaceae family in healthy penmates. During the first week postweaning, Enterobacteriace and Campylobacteria were enriched in animals presenting diarrhea. Furthermore, Lactobacillus was detected in those individuals with no RVA infection. In conclusion, RVA seems to play a primary role in PWD. Classic E. coli virulence factors were not associated with diarrhea, indicating the need for revising their implication in disease. Moreover, Lactobacillus was found frequently in animals negative for RVA, suggesting some protective effect. • Rotavirus A (RVA) was the main pathogen implicated in postweaning diarrhea. • Classical Escherichia coli virulence factors were not linked to diarrhea, indicating the need to update this information. • Ruminococcaceae was more abundant in healthy animals, while Fusobacteriota was more frequent in those with diarrhea. • Lactobacillus was associated with lower prevalence of RVA, indicating a possible protective action. [ABSTRACT FROM AUTHOR]

Colistin has become the last-line antimicrobial for the treatment of multidrug resistant (MDR) Enterobacterales in human medicine. To date, several colistin resistance genes have been described. Of them mcr -1 is disseminated worldwide in Escherichia coli of human and animal origin. The aim of this study was to characterize mcr -mediated resistance plasmids from E. coli of animal origin in Spain. From our strain collection, 70 E. coli of pig origin collected between 2005 and 2014 (10 per year, except for years 2009–2010–2013) were randomly selected and screened for the presence of mcr -genes. Additionally, 20 E. coli isolated in 2011 from white storks (Ciconia ciconia) from the same urban household waste landfill associated colony were also included. Whole genome sequencing of mcr -positive isolates was carried out on a MiSeq (Illumina). Hybrid whole genome sequencing strategy combining nanopore and Illumina technologies were performed in a selection of isolates to close the genomes and plasmids and identify the presence of antimicrobial resistance genes. Minimum inhibitory concentration (MIC) was used to assess the susceptibility to colistin. Mating experiments were carried out to evaluate transferability of the mcr -genes. A total of 19 mcr -1 and one mcr -4 positive isolates were detected, 15 from pigs distributed during the study period, and five from storks collected in 2011. No other mcr -variants were found. The MICs for colistin ranged between 4 and >4 mg/L. High diversity of STs were detected among the mcr-1 positive E. coli isolates, with only ST-10 shared between pigs and white storks. Except for one isolate, all were genotypic and phenotypically MDR, and five of them also harbored cephalosporin resistance genes (bla CTX–M–14, bla SHV–12, and three bla CMY–2). mcr -1 genes were mobilizable by conjugation, associated with IncX4, IncHI2, and IncI2 plasmids. In our study, mcr -1 genes have been circulating in pig farms since 2005 harbored by a variety of E. coli clones. Its persistence may be driven by co-selection since plasmids containing mcr -1 also exhibit resistance to multiple drugs used in veterinary medicine. Furthermore, this is the first report of the presence of mcr -1 gene in isolates from white storks in Spain. This finding highlights the potential importance of wildlife that forage at urban household waste landfills in the transmission and spread of colistin resistance genes. [ABSTRACT FROM AUTHOR]

Most of the studies focused on antimicrobial resistance (AMR) performed in wildlife describe Escherichia coli as the principal indicator of the selective pressure. In the present study, several species of Enterobacterales 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 the prevalence of CR enterobacteria, AMR phenotypes and the presence of common carbapenem and CR genes. 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 showed the highest prevalence (13.5% of 104) of the mammal specimens, and raptors the highest in bird specimen (7.3% of 191). Although CR E. coli was the most frequently isolated (45%), other CR- Enterobacterales like Klebsiella pneumoniae (20%), Citrobacter freundii (15%), Enterobacter cloacae (5%), Proteus mirabilis (5%), Providencia spp (5%) and Serratia marcescens (2.5%) were also isolated. A high diversity of CR genes was identified among the isolates, with 50% yielding blaCMY-2, 23% blaSHV-12, 20% blaCMY-1 and 18% blaCTX-M-15. Additionally, resistance to carbapenems associated to OXA-48 gene was found. Most of the CR isolates, principally K. pneumoniae and C. freundii, were multi-resistant with co-resistance to fluoroquinolones, tetracycline, sulphonamides and aminoglycosides. This study reports high prevalence of Enterobacterales harbouring a variety of CR genes and OXA-48 mediated-carbapenem resistance, all of them frequently associated to nosocomial human infections, for the first time in wild mammals and wild birds. Implementation of control measures to reduce the impact of anthropogenic pressure in the environment is urgently needed. [ABSTRACT FROM AUTHOR]

Simple Summary: The wild boar (Sus scrofa) has been identified as a reservoir of zoonoses and food-borne pathogens. Wild boar populations are growing worldwide, also near urban centers such as Barcelona (Catalonia, Spain). The aim of this study was to assess the presence of zoonotic agents in the wild boar populations from the Metropolitan Area of Barcelona (MAB). The detection of Escherichia coli carrying critical antimicrobial resistance genes to β-lactams and, for the first time in Spain, the detection of toxigenic Clostridioides difficile strains in wild boars foraging in urban areas show the value of this game species as a sentinel of antimicrobial-resistant bacteria (AMRB) and zoonotic agents in the environment. 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. The responsible health agencies should undertake specific actions to fully assess the potential implications for the human population and/or environmental health. Zoonotic diseases and hazards beyond the compulsory surveillance diseases should be specifically targeted in the growing human–wildlife interface of urban environments such as the MAB under the One Health approach. 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): blaCMY-2 (3.0%), blaTEM-1b (2.5%), blaCTX-M-14 (1.0%), blaSHV-28 (1.0%), blaCTX-M-15 (0.5%) and blaCMY-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. [ABSTRACT FROM AUTHOR]

Colistin use has mostly been stopped in human medicine, due to its toxicity. However, nowadays, it still is used as a last-resort antibiotic to treat hospital infections caused by multi-drug resistant Enterobacteriaceae. On the contrary, colistin has been used in veterinary medicine until recently. In this study, 210 fecal samples from pigs (n = 57), calves (n = 152), and the farmer (n = 1) were collected from a farm where E. coli harboring mcr-1–mcr-3 was previously detected. Samples were plated, and mcr-genes presence was confirmed by multiplex-PCR. Hybrid sequencing which determined the presence and location of mcr-1, other antibiotic resistance genes, and virulence factors. Eighteen colistin resistant isolates (13 from calves, four from pigs, and one from the farmer) contained mcr-1 associated with plasmids (IncX4, IncI2, and IncHI2), except for two that yielded mcr-1 in the chromosome. Similar plasmids were distributed in different E. coli lineages. Transmission of mcr-1 to the farmer most likely occurred by horizontal gene transfer from E. coli of calf origin, since plasmids were highly similar (99% coverage, 99.97% identity). Moreover, 33 virulence factors, including stx2 for Shiga toxin E. coli (STEC) were detected, highlighting the role of livestock as a reservoir of pathotypes with zoonotic potential. [ABSTRACT FROM AUTHOR]