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The use of antimicrobials in agricultural, veterinary and medical practice exerts selective pressure on environmental microbiota, promoting the emergence and spread of antimicrobial resistance (AMR), a global concern for the One Health Initiative Task Force (OHITF). Honeybees have been studied as bioindicators of AMR in the environment, but little is known about beehive products like honey and pollen. The aim of this study was to assess the prevalence of AMR genes (ARGs) in beehive products and investigated their origins. Specifically, possible associations between ARGs, microbiota and other characteristics of different honey and pollen samples, including country of origin, flower type, type of commercial distribution and environmental factors, such as land use, weather and composition of the environment surrounding the beehives were investigated. We found that beehive products harboured ARGs conferring resistance to β-lactams, macrolides, (fluoro)quinolones and polymyxins. Most samples possessed resistance to multiple antimicrobial classes, with honey and pollen showing similar ARG profiles. Even if Lactobacillus and Acinetobacter genera were common in the microbial communities of both honey and pollen, Bacillus, Clostridium, and Bombella defined honey microbiota, while Pseudomonas and Vibrio were enriched in pollen. ErmB and blaTEM-1 co-occurred with Lactobacillus and Fructobacillus, while positive associations between β-lactams and macrolides and anthropogenic environments (i.e. industrial and commercial areas and non-irrigated arable lands) were found. Altogether, our findings suggest that ARGs in honey and pollen might originate from the honeybee foraging environment, and that the beehive products can be used as bioindicators of the AMR environmental contamination.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis, the disease with the highest economic loss for the broiler industry. However, studies focusing on the prevalence and population structure of APEC in the broiler production pyramid are scarce. Here, we used genotyping and serotyping data to elucidate the APEC population structure and its changes in different broiler production stages along with whole-genome sequencing (WGS) in a subset of APEC isolates to determine transmission patterns amongst dominant APEC sequence types (STs) and characterize them in detail. Comparison of genotypes encountered in both APEC and avian fecal E. coli (AFEC) provided further insights. Overall, APEC-related mortality, as the proportion of the total sampled mortality in the broiler production, was high (35%), while phylogroup C and serogroup O78 were predominant amongst APEC isolates. We found a low (34.0%) and high (53.3%) incidence of colibacillosis in chicks and end-cycle broilers, respectively, which may be related to a shift in APEC genotypes, suggesting a trend from commensalism to pathogenicity across different broiler production stages. Despite considerable APEC genotypic diversity, there was substantial genotype overlap (40.9%, overall) over the production stages and convergence of STs to the four clusters. Within these clusters, WGS data provided evidence of clonal transmission events and revealed an enriched virulence and resistance APEC repertoire. More specifically, sequenced APEC were assigned to defined pathotypes based on their virulence gene content while the majority (86%) was genotypically multi-drug resistant. Interestingly, WGS-based phylogeny showed that a subset of APEC, which are cephalosporin-resistant, may originate directly from cephalosporin-resistant AFEC. Finally, exploration of the APEC plasmidome indicated that the small fraction of the APEC virulome carried by IncF plasmids is pivotal for the manifestation of the APEC pathotype; thus, plasmid exchange can promote pathogenicity in strains that are at the edge of the commensal and pathogenic states.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis, the disease with the highest economic loss for the broiler industry. However, studies focusing on the prevalence and population structure of APEC in the broiler production pyramid are scarce. Here, we used genotyping and serotyping data to elucidate the APEC population structure and its changes in different broiler production stages along with whole-genome sequencing (WGS) in a subset of APEC isolates to determine transmission patterns amongst dominant APEC sequence types (STs) and characterize them in detail. Comparison of genotypes encountered in both APEC and avian fecal E. coli (AFEC) provided further insights. Overall, APEC-related mortality, as the proportion of the total sampled mortality in the broiler production, was high (35%), while phylogroup C and serogroup O78 were predominant amongst APEC isolates. We found a low (34.0%) and high (53.3%) incidence of colibacillosis in chicks and end-cycle broilers, respectively, which may be related to a shift in APEC genotypes, suggesting a trend from commensalism to pathogenicity across different broiler production stages. Despite considerable APEC genotypic diversity, there was substantial genotype overlap (40.9%, overall) over the production stages and convergence of STs to the four clusters. Within these clusters, WGS data provided evidence of clonal transmission events and revealed an enriched virulence and resistance APEC repertoire. More specifically, sequenced APEC were assigned to defined pathotypes based on their virulence gene content while the majority (86%) was genotypically multi-drug resistant. Interestingly, WGS-based phylogeny showed that a subset of APEC, which are cephalosporin-resistant, may originate directly from cephalosporin-resistant AFEC. Finally, exploration of the APEC plasmidome indicated that the small fraction of the APEC virulome carried by IncF plasmids is pivotal for the manifestation of the APEC pathotype; thus, plasmid exchange can promote pathogenicity in strains that are at the edge of the commensal and pathogenic states.

Antimicrobials are commonly used in conventional livestock production and manure is widely applied to agricultural lands as fertilizer. This practice raises questions regarding the effects of fertilization on (i) soil microbiota composition and (ii) spread of antimicrobials and antimicrobial resistance (AMR) in the environment. This study was conducted in a high-density farming area of Northern Italy and aimed at assessing the impact of (dairy cattle, chickens and swine) manure application on soil microbiome, antimicrobial concentrations and antimicrobial resistance gene (ARG) abundance. We found the microbial community composition in manure to be different and less diverse than in soil, with manure application altering only marginally the soil microbiome. Exceptions were the phyla Firmicutes, Tenericutes and Cloacimonetes, which significantly enriched in fertilized soil. Of the antimicrobials investigated, only flumequine concentrations increased after manure application, albeit non-significantly. ARGs were more abundant in manure, with ermA, ermB, blaOXA-1 and oqxA being significantly enriched in fertilized soil. Positive correlations between oqxA and qnrS abundances and flumequine concentrations were observed, together with the co-occurrence of some ARGs and microbial taxa (e.g. oqxA correlated with Acidobacteria and Gemmatimonadetes). This study showed that manure application has little effect on soil microbiome but may contribute to the dissemination of specific ARGs into the environment. Moreover, flumequine residues seem to enhance the emergence of oqxA and qnrS in soil.

Presence of extended-spectrum β-lactamase (ESBL)- and AmpC β-lactamase (pAmpC)-producing Escherichia coli (ESBL/pAmpC-EC) in humans and animals is alarming due to the associated risks of antibiotic therapy failure. ESBL/pAmpC-EC transmission between the human and animal compartments remains controversial. Using cefotaxime-supplemented (selective) media, we recently showed high sample prevalence of ESBL/pAmpC-EC in an integrated broiler chain [i.e. Parent Stock (PS), offspring broilers and their carcasses]. Here, we used a different approach. In parallel with the selective isolation, samples were processed on non-selective media. E. coli isolates were tested for ESBL/pAmpC-production and those found positive were genotyped. For carcasses, total E. coli were enumerated. This approach enabled us to estimate prevalence at the isolate level, which mirrors ESBL/pAmpC-EC colonisation levels. We showed that although present in many animals, ESBL/pAmpC-EC were overall subdominant to intestinal E. coli, indicating that high sample prevalence is not associated with high levels of resistance in individual hosts. This is a relevant aspect for risk assessments, especially regarding the immediate exposure of farm personnel. An exception was a particularly dominant B2/blaCMY-2 lineage in the gut of imported PS chicks. This predominance obscured presence of latent genotypes, however bias towards particular ESBL/pAmpC-EC genotypes from the selective method or underestimation by the non-selective approach did not occur. At the slaughterhouse, we showed a link between total E. coli and ESBL/pAmpC-EC on carcasses. Mitigation strategies for reducing consumers’ exposure should aim at suppressing ESBL/pAmpC-EC in the broiler gut as well as controlling critical points in the processing line.

Poultry are the main reservoir for thermophilic Campylobacter spp., which is the most common causative agent of human bacterial gastroenteritis. The epidemiology of Campylobacter in poultry, particularly in turkeys, is not completely understood. This study aimed at identifying potential sources and transmission routes of thermophilic Campylobacter spp. in commercial turkey farms. C. jejuni and C. coli isolates from breeders (n = 29, 20 C. jejuni and 9 C. coli) and their progeny (n = 51, 18 C. jejuni and 33 C. coli) reared in two different farms for three sequential production cycles were analysed by multilocus sequence typing (MLST). Strains (n = 88, 42 C. jejuni and 46 C. coli) isolated from environmental (i.e. anteroom and in-house overshoes), water (i.e. drinkers and water line), and pest (i.e. flies, Alphitobius diaperinus, and mice) sources were also examined. MLST of C. jejuni and C. coli isolates resulted in 13 and 12 different sequence types (STs) belonging to six and one previously-described clonal complexes (CCs), respectively. Three novel STs were identified. Genetic similarities were detected between isolates from fattening turkeys and the considered environmental, water, and pest sources, and with the breeders to a lesser extent. Source attribution analysis estimated that environmental and water sources accounted for most (∼75%) of fattening turkey isolates and were therefore identified as the most likely sources of flock colonization, followed by pests (∼20%) and breeders (∼5%). These sources may thus be targeted by control measures to mitigate the risk of Campylobacter colonization in commercial turkeys. RESEARCH HIGHLIGHTSHigh occurrence of C. jejuni and C. coli in commercial turkey flocks.High genetic diversity of C. jejuni and C. coli in commercial turkey flocks.Horizontal transmission responsible for Campylobacter colonization of commercial turkey flocks.Environmental and water sources involved in Campylobacter colonization of commercial turkey flocks.Strategies for prevention and control of Campylobacter colonization of commercial turkey flocks are needed. High occurrence of C. jejuni and C. coli in commercial turkey flocks. High genetic diversity of C. jejuni and C. coli in commercial turkey flocks. Horizontal transmission responsible for Campylobacter colonization of commercial turkey flocks. Environmental and water sources involved in Campylobacter colonization of commercial turkey flocks. Strategies for prevention and control of Campylobacter colonization of commercial turkey flocks are needed.

Extended-spectrum β-lactamase (ESBL)- and plasmid mediated AmpC-type cephalosporinase (pAmpC)-producing Escherichia coli (ESBL/pAmpC E. coli) in food-producing animals is a major public health concern. This study aimed at quantifying ESBL/pAmpC-E. coli occurrence and transfer in Italy's broiler production pyramid. Three production chains of an integrated broiler company were investigated. Cloacal swabs were taken from parent stock chickens and offspring broiler flocks in four fattening farms per chain. Carcasses from sampled broiler flocks were collected at slaughterhouse. Samples were processed on selective media, and E. coli colonies were screened for ESBL/pAmpC production. ESBL/pAmpC genes and E. coli phylogroups were determined by PCR and sequencing. Average pairwise overlap of ESBL/pAmpC E. coli gene and phylogroup occurrences between subsequent production stages was estimated using the proportional similarity index, modelling uncertainty in a Monte Carlo simulation setting. In total, 820 samples were processed, from which 513 ESBL/pAmpC E. coli isolates were obtained. We found a high prevalence (92.5%, 95% CI 72.1-98.3%) in day-old parent stock chicks, in which blaCMY-2 predominated; prevalence then dropped to 20% (12.9-29.6%) at laying phase. In fattening broilers, prevalence was 69.2% (53.6-81.3%) at the start of production, 54.2% (38.9-68.6%) at slaughter time, and 61.3% (48.1-72.9%) in carcasses. Significantly decreasing and increasing trends for respectively blaCMY-2 and blaCTX-M-1 gene occurrences were found across subsequent production stages. ESBL/pAmpC E. coli genetic background appeared complex and bla-gene/ phylogroup associations indicated clonal and horizontal transmission. Modelling revealed that the average transfer of ESBL/pAmpC E. coli genes between subsequent production stages was 47.7% (42.3-53.4%). We concluded that ESBL/pAmpC E. coli in the broiler production pyramid is prevalent, with substantial transfer between subsequent production levels.