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Salmonella is a foodborne pathogen commonly associated with poultry products. The aims of this work were to (i) estimate the impact of critical steps of the slaughter process on Salmonella detection from broiler chicken carcasses in two commercial poultry slaughter plants in Quebec, Canada; (ii) investigate the presence of Salmonella in the slaughter plant environment; (iii) describe, using a high-resolution melting (HRM) approach, the HRM Salmonella profiles and serotypes present on carcasses and in the slaughter plant environment; and (iv) evaluate whether the HRM flock status after chilling could be predicted by the flock status at previous steps of the slaughter process, the status of previous flocks, or the status of the processing environment, for the same HRM profile. Eight visits were conducted in each slaughter plant over a 6-month period. In total, 379 carcass rinsates from 79 flocks were collected at five critical steps of the slaughter process. Environmental samples were also collected from seven critical sites in each slaughter plant. The bleeding step was the most contaminated, with >92% positive carcasses. A decrease of the contamination along the slaughtering process was noted, with carcasses sampled after dry-air chilling showing ≤2.5% Salmonella prevalence. The most frequently isolated serotypes were Salmonella Heidelberg, Kentucky, and Schwarzengrund. The detection of the Salmonella Heidelberg 1-1-1 HRM profile on carcasses after chilling was significantly associated with its detection at previous steps of the slaughter process and in previously slaughtered flocks from other farms during a same sampling day. Results highlight the importance of the chilling step in the control of Salmonella on broiler chicken carcasses and the need to further describe and compare the competitive advantage of Salmonella serotypes to survive processing. The current study also illustrates the usefulness of HRM typing in investigating Salmonella contamination along the slaughter process. HIGHLIGHTS

The intestinal microbiota plays several important roles in pig health and growth. The aim of the current study was to characterize the changes in the fecal microbiota diversity and composition of weaned piglets following an oral challenge with an ETEC: F4 strain and/or a treatment with colistin sulfate (CS). Twenty-eight piglets were used in this experiment and were divided into four groups: challenged untreated, challenged treated, unchallenged treated, and unchallenged untreated. Rectal swab samples were collected at five sampling times throughout the study. Total genomic DNA was used to assess the fecal microbiota diversity and composition using the V4 region of the 16S rRNA gene. The relative abundance, the composition, and the community structure of piglet fecal microbiota was highly affected by the ETEC: F4 challenge throughout the experiment, while the oral treatment with CS, a narrow spectrum antibiotic, resulted in a significant decrease of E. coli/Shigella populations during the treatment period only. This study was the first to identify some gut microbiota subgroups (e.g., Streptococcus, Lachnospiraceae) that are associated with healthy piglets as compared to ETEC: F4 challenged animals. These key findings might contribute to the development of alternative strategies to reduce the use of antimicrobials in the control of post-weaning diarrhea in pigs.

Recycled manure solids (RMS) bedding is an alternative bedding option that is growing in popularity on Canadian dairy farms. However, the microbiological characteristics and production of RMS bedding are poorly documented under on-farm conditions in eastern Canada. This bedding could support the presence of pathogens and could have an effect on cow and human health. The aim of this study was to describe the RMS microbiota when used under dairy cows and compare it with straw bedding. Unused and used bedding from 27 RMS and 61 straw-bedded dairy farms were collected and compared using 16S amplicon sequencing, bacterial counts, and Salmonella spp. and Listeria monocytogenes detection. Microbiota composition of unused RMS and unused straw were different. After use, both bedding microbiota were similar in their bacterial composition, structure, and diversity. Unused RMS generally contained higher bacterial counts than did unused straw, except for Klebsiella spp. counts. Salmonella spp. and L. monocytogenes were more frequently detected in unused RMS (Salmonella spp.: 11%; L. monocytogenes: 30%), than in unused straw (Salmonella spp.: 0%; L. monocytogenes: 11%). Finally, 2 RMS production systems (extraction of the liquid fraction followed by maturation in an opened or enclosed container vs. in a heap) did not influence the microbiota richness and bacteria distribution (α-diversity), but did influence the microbiota structure (β-diversity). In conclusion, animal and human pathogens were found in greater numbers and more frequently in unused RMS than unused straw, and this could eventually affect dairy cow or human health.

Background: Tail-biting (TB) is a serious behavioral disorder in pigs. It is defined as a pig chewing the tail of another pig. It is an important challenge in swine production as it impacts the animal welfare, its health and the economics and safety of the pork meat supply chain. Some treatments have been proposed but have not proven optimal. Nutrition, feed type and composition, appears to be an important factor in TB behavior, perhaps by modulating the intestinal microbiota (IM). In humans, IM is increasingly recognized as a modulator of behavior. Our aim was to assess an association between TB behavior and IM in pigs through comparisons of IM in groups of biter or bitten pigs to the IM in a non-biter and non-bitten negative control group, and through comparisons of IM between the biter and bitten groups. Each group, composed of 12 pigs, was formed at the beginning of the growing/finishing phase based on a target behavior analysis centered on TB behavior for the biter group and a score of damages caused to the tail for the bitten group. Fecal and blood samples were collected from each pig during a TB episode, at time 0, t0, and after a TB episode, four weeks later, at time 1, t1. Serum cortisol level was determined by ELISA and used as an indicator of stress. The pig’s fecal microbiota was analyzed from DNA extracted from freshly collected fecal matter using amplicon sequencing of the V4 hypervariable region of the 16S rRNA gene. Results: Serum cortisol levels were significantly higher in either the biter or bitten pig groups compared to the negative control group ( p =0.02 and p =0.01, respectively). Interestingly, no significant difference was revealed between the biter and bitten groups. The microbiota alpha-diversity was not significantly different between all groups, biter, bitten and negative control. Analyses of beta-diversity, however, revealed a significant difference between either the biter or the bitten group in comparison to the negative control group in terms of structure and composition of the microbiota. Lactobacillus were significantly more abundant in the negative control group compared to the two other groups ( p =0.001). No significant difference was revealed between the biter and bitten groups. Quantitative real-time PCR (qPCR) confirmed that lactobacilli were more abundant in the negative control group. Conclusions: This study demonstrates that TB behavior is associated to the IM composition in pigs.

The role of the accompanying microbiota in the presence of Listeria monocytogenes on meat processing surfaces is not yet understood, especially in industrial production conditions. In this study, 300 conveyor belt samples from the cutting room of a swine slaughterhouse were collected during production. The samples were subjected to the detection of L. monocytogenes. Recovered strains were characterized by serogrouping-PCR, InlA Sanger sequencing and for their ability to form biofilm. A selection of isolates was compared with core genome multi-locus sequence typing analysis (cgMLST). The sequencing of the V4 region of the 16S RNA gene of the microorganisms harvested from each sample was carried out in parallel using the Illumina MiSeq platform. Diversity analyses were performed and MaAsLin analysis was used to assess the link between L. monocytogenes detection and the surrounding bacteria. The 72 isolates collected showed a low genetic diversity and important persistence characteristics. L. monocytogenes isolates were not stochastically distributed on the surfaces: the isolates were detected on three out of six production lines, each associated with a specific meat cut: the half carcasses, the bostons and the picnics. MaAsLin biomarker analysis identified the taxa Veillonella (p ≤ 0.0397) as a bacterial determinant of the presence of L. monocytogenes on processing surfaces. The results of this study revealed a heterogenous contamination pattern of the processing surfaces by L. monocytogenes and targeted a bacterial indicator of the presence of the pathogen. These results could lead to a better risk assessment of the contamination of meat products.

The aim of the present study was to investigate the in vitro gastric stability of colistin sulfate (CS) and its antimicrobial activity against Escherichia coli and to study the impact of ETEC O149: F4 (K88) infection in pigs on CS intestinal absorption. The stability profile of CS was evaluated in a simulated gastric fluid (SGF). Antimicrobial activity of CS and its degradation products were examined in a 96-well polystyrene microplate model. The effect of experimental infection with ETEC O149: F4 on CS intestinal absorption was determined by quantification of CS systemic concentration using a validated LC–MS/MS method. A rapid degradation of CS accompanied by an increase in CS antimicrobial activity by comparison with non-degraded CS (P < 0.0001) was observed in SGF. Additionally, CS levels were not quantifiable in systemic circulation using a highly sensitive method and concurrent oral challenge did not affect CS absorption in an induction model of subclinical post-weaning diarrhea (PWD).

Background Enterotoxigenic Escherichia coli (ETEC) strains producing multiple enterotoxins are important causes of post-weaning diarrhea (PWD) in pigs. The aim of the present study was to investigate the fecal presence of ETEC enterotoxin as well as F4 and F18 genes as an indicator of colistin sulfate (CS) efficacy for treatment of PWD in pigs. Forty-eight piglets were weaned at the age of 21 days, and were divided into four groups: challenged treated, challenged untreated, unchallenged treated, and unchallenged untreated. Challenge was performed using 109 CFU of an ETEC: F4 strain, and treatment was conducted using oral CS at the dose of 50,000 IU/kg. The fecal presence of genes encoding for STa, STb, LT, F4 and F18 was detected using PCR. Results The PCR amplification of ETEC virulence genes showed that nearly 100% of pigs excreted genes encoding for STa and STb toxins in the feces before the challenge. These genes, in the absence of the gene encoding F4, were considered as a marker for F4-negative ETEC. One day after ETEC: F4 oral challenge pigs in the two challenged groups excreted the genes encoding LT and F4 in the feces. These genes were considered as a marker for F4-positive ETEC. However, the gene encoding F18 was not detected in any fecal samples of the 4 groups throughout the experiment. After only 3 days of successive oral treatment with CS, a significant reduction in both the F4-positive and negative ETEC populations was observed in the challenged treated group compared to the challenged untreated group (p

Colistin (Polymyxin E) is one of the few cationic antimicrobial peptides commercialized in both human and veterinary medicine. For several years now, colistin has been considered the last line of defense against infections caused by multidrug-resistant (MDR) Gram-negative such as Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Colistin has been extensively used orally since the 1960s in food animals and particularly in swine for the control of Enterobacteriaceae infections. However, with the recent discovery of plasmid-mediated colistin resistance encoded by the mcr-1 gene and the higher prevalence of samples harboring this gene in animal isolates compared to other origins, livestock has been singled out as the principal reservoir for colistin resistance amplification and spread. Co-localization of the mcr-1 gene and Extended-Spectrum- β-lactamase (ESBL) genes on a unique plasmid has been also identified in many isolates from animal origin. The use of colistin in pigs as a growth promoter and for prophylaxis purposes should be banned, and the implantation of sustainable measures in pig farms for microbial infection prevention should be actively encouraged and financed. The scientific research should be encouraged in swine medicine to generate data helping to reduce the exacerbation of colistin resistance in pigs and in manure. The establishment of guidelines ensuring a judicious therapeutic use of colistin in pigs, in countries where this drug is approved, is of crucial importance. The implementation of a microbiological withdrawal period that could reduce the potential contamination of consumers with colistin resistant bacteria of porcine origin should be encouraged. Moreover, the management of colistin resistance at the human-pig-environment interface requires the urgent use of the One Health approach for effective control and prevention. This approach needs the collaborative effort of multiple disciplines and close cooperation between physicians, veterinarians, and other scientific health and environmental professionals. This review is an update on the chemistry of colistin, its applications and antibacterial mechanism of action, and on Enterobacteriaceae resistance to colistin in pigs. We also detail and discuss the One Health approach and propose guidelines for colistin resistance management.

Colistin sulfate (CS) is used in Canada for the treatment of post weaning diarrhea (PWD), to overcome conventional therapeutic antibiotics failures. The aim of the present study was to determine the effect of a conventional oral regimen of CS for the treatment of PWD, on the development of E. coli CS resistance and to evaluate the effect of ETEC: F4 infection on CS intestinal absorption. A total of 48 pigs were used, challenge was carried out by oral administration of 10 9 CFU of a hemolytic ETEC: F4 strain resistant to nalidixic acid. CS was administered at a dose of 50.000 UI/kg twice a day for 5 days. Feces were examined clinically and bacteriologically before and after challenge to evaluate presence of diarrhea and E. coli fecal excretion. ETEC: F4 virulence factors were monitored and CS plasma concentrations were quantified by an HPLC-MS/MS. From one until six days after CS administration, a significant reduction in the fecal excretion of ETEC: F4, total E. coli, ETEC: F4 virulence factors and in diarrhea scores was observed in the challenged treated group compared to the challenged untreated group (p 0.71). A significant selection pressure on E. coli total population was observed following CS treatment (p

Neomycin, an aminoglycoside, is use at farm level for Escherichia coli treatment of piglets postweaning diarrhea, with 40% of unsuccessfully treatment due to the antimicrobial resistance. To overcome this situation, veterinarians use colistin also call polymyxin E, but this antibiotic is not homologated in Canada, even if it seems to be effective against E.coli. The described resistance to polymyxin is associated to a modification of the LPS core and the lipid A regions in the bacteria. For Salmo nella, those modifications are associated to the two components system PmrA-PmrB. This system is known in E.coli but not yet reported to be implicated in colistin resistance. The neomycin resistance is linked to enzymatic modifications associated to genes located on plasmids. This study has for objectives to investigate the acquisition of colistin resistance and study the transfer of neomycin resistance between E.coli strains and other enterobacteria. E.coli O149 strains isolated from clinical cases (2008 to 2011) were used and susceptibility testing of strains was performed by the disk diffusion method. E-test and a micro-dilution method were used to determine the minimal inhibitory concentration (MIC) of colistin and neomycin respectively. All tested strains had a MIC higher than 128 ppm for neomycin whereas MICs were between 0.064 to 0.128 ppm for colistin, indicating that all strains were resistance to neomycin but all susceptible to colistin. In these isolates, neomycin antimicrobial resistance genes aac(3)-IV, apkA1 and aphA2 have been detected using PCR. Conjugation experiments are presently being performed. Colistin natural mutants (n=22) were created through serial passages on LB agar with 25xMIC. Sequencing of the PmrA-PmrB region of these mutants was performed to identify mutations. Three PmrA and seven PmrB mutations were for the first time reported in E.coli O149. Others mutants are still under investigation for other possible resistance mechanism.

Colistin sulfate (CS), a polymyxin antibiotic, is used in Canada for the treatment of post-weaning diarrhea in pigs as an alternative to neomycin. The aim of the present study was to evaluate some pharmacokinetics parameters of CS and its effect on the evolution of the intestinal Escherichia coli population in pigs challenged with enterotoxigenic E. coli (ETEC): F4. A total of 14 weaned piglets were divided into two groups, a non-challenged, treated group (n=7) and a challenged, treated group (n=7). Both groups received a single oral dose of CS at 50,000 IU/Kg. Challenge was carried out by oral administration of 10 9 CFU of a hemolytic ETEC: F4 strain resistant to nalidixic acid. Blood samples were taken at 30 min and 1, 2, 4, 6, 8, 12, 24, 36 and 48 hours post treatment from each pig and CS quantification was performed by LC-MS/MS. In the challenged group, severity of diarrhea was monitored and the presence of the ETEC:F4 strain in the feces was enumerated using 5% bovine blood agar plates containing nalidixic acid. In both groups, total E. coli counts were carried out using Petrifilm E. coli/Coliform count plates. In both groups, the plasma concentration of CS was below the lower limit of quantification (20 ng/ml). Following CS treatment, a decrease in the total E. coli and ETEC: F4 fecal counts were observed at 24 h post treatment. The fecal consistency was not affected by CS treatment. For the first time, a study of some CS pharmacokinetics parameters with a highly sensitive method showed that CS levels are not detectable in systemic circulation following oral administration, and concurrent oral challenge with an ETEC strain did not affect CS absorption. A single dose of CS resulted in reduced bacterial counts of the total E. coli and ETEC: F4 populations in the feces.

Enterotoxigenic Escherichia coli (ETEC: F4) associated with post-weaning diarrhea (PWD) in pigs has developed resistance against several antimicrobial families, leading to increased use of colistin sulfate (CS) for the treatment of this disease. The objective of this study was to determine the efficacy of oral CS treatment in experimental PWD due to ETEC: F4 challenge and determine the effect of this challenge on CS intestinal absorption. In this study, 96 pigs were divided into two trials based on CS dose (100 000 or 50 000 IU/kg). Fecal shedding of ETEC: F4, total E. coli, and CS-resistant E. coli, diarrhea scores, and weight changes were evaluated. Colistin sulfate plasma concentrations were determined by HPLC–MS/MS. Regardless of the dose, CS treatment resulted in a reduction of fecal ETEC: F4 and total E. coli shedding, and in diarrhea scores but only during the treatment period. However, CS treatment resulted in a slight increase in fecal shedding of CS resistant E. coli and did not prevent weight loss in challenged pigs. In addition, challenge with ETEC: F4 resulted in an increase of CS intestinal absorption. Our study is among the first to demonstrate that under controlled conditions, CS was effective in reducing fecal shedding of ETEC: F4 and total E. coli in experimental PWD. However, CS treatment was associated with a slight selection pressure on E. coli and did not prevent pig weight loss. Further studies are needed in field conditions, to better characterize CS therapeutic regimen efficacy and bacterial resistance dissemination. Electronic supplementary material The online version of this article (doi:10.1186/s13567-016-0344-y) contains supplementary material, which is available to authorized users.

International audience; Faced with ever-increasing demand, the industrial production of food animals is under pressure to increase its production. In order to keep productivity, quality, and safety standards up while reducing the use of antibiotics, farmers are seeking new feed additives. In chicken production, one of these additives is selenium. This element is expected to confer some advantages in terms of animal health and productivity, but its impact on chicken intestinal microbiota as well as on the carriage of foodborne pathogens is unknown. In this study, chickens raised in a level 2 animal facility were fed or not 0.3 ppm of in-feed selenium-yeast until 35 days of age and were inoculated or not with the foodborne pathogen Campylobacter jejuni at the age of 14 days. At the end of the study, body weight, seric IgY, intestinal IgA, seric gluthatione peroxydase activity, the caecal microbiota (analyzed by MiSeq 16S rRNA gene sequencing), and C. jejuni caecal levels were analyzed. The experiment was completely replicated twice, with two independent batches of chickens. This study revealed that, for healthy chickens raised in very good hygienic conditions, selenium-yeast does not influence the bird's body weight and lowers their seric gluthatione peroxidase activity as well as their intestinal IgA concentrations. Furthermore, selenium-yeast did not modify the caecal microbiota or the colonization of C. jejuni. The results also showed that C. jejuni colonization does not impact any of the measured chicken health parameters and only slightly impacts the caecal microbiota. This study also clearly illustrated the need for true biological replication (independent animal trials) when assessing the microbiota shifts associated with treatments as the chickens microbiotas clearly clustered according to study replicate.