Insights on the effects of antimicrobial and heavy metal usage on the antimicrobial resistance profiles of pigs based on culture-independent studies.

Antimicrobial resistance is a global threat to human, animal, and environmental health. In pig production, antimicrobials and heavy metals such as zinc oxide are commonly used for treatment and prevention of disease. Nevertheless, the effects of antimicrobials and heavy metals on the porcine resistome composition and the factors influencing this resistance profile are not fully understood. Advances in technologies to determine the presence of antimicrobial resistance genes in diverse sample types have enabled a more complete understanding of the resistome and the factors which influence its composition. The aim of this review is to provide a greater understanding of the influence of antimicrobial and heavy metal usage on the development and transmission of antimicrobial resistance on pig farms. Furthermore, this review aims to identify additional factors that can affect the porcine resistome. Relevant literature that used high-throughput sequencing or quantitative PCR methods to examine links between antimicrobial resistance and antimicrobial and heavy metal use was identified using a systematic approach with PubMed (NCBI), Scopus (Elsevier), and Web of Science (Clarivate Analytics) databases. In total, 247 unique records were found and 28 publications were identified as eligible for inclusion in this review. Based on these, there is clear evidence that antimicrobial and heavy metal use are positively linked with antimicrobial resistance in pigs. Moreover, associations of genes conferring antimicrobial resistance with mobile genetic elements, the microbiome, and the virome were reported, which were further influenced by the host, the environment, or the treatment itself.

Salmonella Typhimurium Infection Along the Porcine Gastrointestinal Tract and Associated Lymphoid Tissues.

Salmonella is a major foodborne pathogen and pork is one of the main sources of human salmonellosis. Understanding the pathogenesis and progression of the infection within the host is of interest to establish potential approaches to control the disease in pigs. The present study evaluates factors such as intestinal colonization, fecal shedding, and pathogen persistence by 2 studies using experimental challenge with Salmonella Typhimurium in weaned pigs and euthanasia at different time points (1, 2, and 6 and 2, 14, and 30 days postinfection [dpi], respectively). Histopathology of intestine at early time points (1 dpi and 2 dpi) showed severe damage to the epithelium together with an increase in polymorphonuclear cells and macrophages (P < .001), particularly in jejunum and ileum. Large quantities of Salmonella were detected within the contents of the ileum, cecum, and colon in early infection. Salmonella could also be observed in the medulla of tonsils and mesenteric lymph nodes. From 6 dpi onward, signs of recovery were observed, with progressive restoration of the epithelium, reduction of the inflammatory infiltrate, and elimination of Salmonella from the mucosa. Concentration of Salmonella in feces and ileum content decreased, but shedding did not cease even at 4 weeks after infection. Persistence of the bacteria in mesenteric lymph nodes was identified within the connective tissue at 14 and 30 dpi. Our results demonstrate a recovery of the disease after an initial acute phase but also show persistence within the lumen and surrounding lymphoid tissue. These findings are relevant to developing effective control strategies.

Evaluation of an Alternative Experimental Infection Method, Which Closely Mimics the Natural Route of Transmission of Monophasic Salmonella Typhimurium in Pigs.

Salmonella carriage in pigs is a significant food safety issue. This study describes a new protocol of Salmonella infection based on exposure to an artificially contaminated environment that closely mimics natural exposure to the organism. The aim of the study was to develop and evaluate the effectiveness of this protocol, which could then be used as a tool in the investigation of control measures. In addition, Salmonella shedding pattern and growth performance of the pigs were examined. Trial pigs (n = 10) were placed in a pen that had been previously contaminated by housing two pigs experimentally challenged with a monophasic Salmonella Typhimurium (mST). A further 10 pigs were placed in a Salmonella-free pen. Pigs were weighed on days 0 and 28. Feces was collected on days 0, 2, 3, 5, 7, 14, 21, and 28 and examined for the presence and quantity of Salmonella. The trial was replicated once. All pigs in the contaminated pens shed Salmonella within the first 2 days of exposure with values ranging from 100 to 104 CFU/g. The noninfected pigs had significantly higher final body weights on day 28 than those exposed to the Salmonella contaminated environment in both replicates. The pigs in the Salmonella-free pen had significantly higher average daily weight gain over the 28-day period compared to the infected animals (p < 0.001). Although not significant, numerical improvements in average daily feed intake and feed conversion efficiency were observed in the Salmonella-free pigs when compared to the contaminated pigs. The approach used was successful in infecting pigs with Salmonella without the need for direct inoculation or exposure to seeder pigs. This "natural" method of infection in which pigs are exposed to low levels of environmental contamination with Salmonella may be an effective tool that could be utilized when investigating control measures.

Characterization of the emerging Salmonella 4,[5],12:i:- in Danish animal production.

The monophasic Salmonella variant with the antigenic formula Salmonella 4,[5],12:i:- has emerged in the last decade as one of the main serotypes related to human salmonellosis. In the present study, a collection of 94 isolates of the S. 4,12:i:- and S. 4,5,12:i:- coming from Danish farm animals, swine (86), cattle (7), and poultry (1), with well-defined identification was further typed by polymerase chain reaction serotyping, phage typing, and molecular typing (polymerase chain reaction and multilocus variable-number tandem-repeat analysis [MLVA]). Moreover, the determination of antimicrobial resistance pattern of each isolate was tested. In 68 of the isolates the fljB gene was absent (i.e., they were true monophasic strains), whereas in 26 isolates, the gene was present despite the fact that the isolates did not express it. The results clustered the isolates in three main pulse-types. The predominant cluster was compatible with the previously described pattern STYMXB.0131. All the isolates included in this cluster lacked the fljB gene, and all the isolates except one belonged to phage type DT 193 with the AMP-STR-SMX-TET resistance pattern. MLVA analysis divided the clusters in several MLVA profiles previously reported by other studies. Finally, antimicrobial resistance and multiresistance was frequent, although no resistance was detected in critical compounds: fluoroquinolones and cephalosporins. The present study demonstrates the presence of monophasic Salmonella Typhimurium-like strains in Danish food animal production with well-characterized clones that are described by previous studies, demonstrating the emergence and spread of this serotype in Denmark.

Salmonella Control in Swine: A Thoughtful Discussion of the Pre- and Post-Harvest Control Approaches in Industrialized Countries.

Pork is among the major sources of human salmonellosis in developed countries. Since the 1990s, different surveys and cross-sectional studies, both national and international (i.e., the baseline studies performed in the European Union), have revealed and confirmed the widespread non-typhoidal Salmonella serotypes in pigs. A number of countries have implemented control programs with different approaches and degrees of success. The efforts could be implemented either at farms, in post-harvest stages, or both. The current review revises the current state of the art in Salmonella in swine, the control programs ongoing or conducted in the past, and their strengths and failures, with particular attention to the weight of pre- and post-harvest control and the implications that both have for the success of interventions or mitigation after outbreaks. This review provides a novel perspective on Salmonella control in swine, a matter that still includes uncertainties and room for improvement as a question of public health and One Health.

New insights into swine dysentery: faecal shedding, macro and microscopic lesions and biomarkers in early and acute stages of Brachyspira hyodysenteriae infection.

BACKGROUND: Swine dysentery (SD) is a severe mucohaemorrhagic colitis in pigs caused classically by Brachyspira hyodysenteriae. Although several aspects of B. hyodysenteriae infection dynamic are already described, further research in the early stage of this infection is required. In this study, 7-week-old pigs were orally challenged with B. hyodysenteriae to obtain information about faecal shedding, macro and microscopic intestinal lesions and serum acute phase proteins in pigs at the onset of B. hyodysenteriae shedding (early infection group, n = 8), in pigs with mucohaemorrhagic diarrhoea (acute infection group, n = 8) and in non-infected controls (n = 16). RESULTS: First B. hyodysenteriae detection by q-PCR and first loose stools with blood and mucus occurred both at 8 days post-inoculation. The lapse between a positive q-PCR and observation of mucohaemorrhagic diarrhoea ranged from 0 to 3 days, except in a single pig in which this period lasted 5 days. Macroscopic lesions were observed in the large intestine from both infected groups although more frequent and severe in acute infection group. Microscopic observation of the apex mucosa revealed that in early infection only higher ulceration values were observed compared to healthy controls. In contrast, the acute infection group exhibited higher ulceration, neutrophils infiltration and increased mucosal thickness compared to the other two groups. Among the serum biomarkers tested, only haptoglobin, C-reactive protein, and creatine kinase showed a significant increase in pigs in the acute infection period compared to controls, whereas haptoglobin was the only factor with a significant increase at the early infection compared to non-infected animals. CONCLUSIONS: This study provides new insights about SD and remarks the complex and limited options to perform an early detection of infected animals beyond PCR diagnosis.

Effects of removing in-feed antibiotics and zinc oxide on the taxonomy and functionality of the microbiota in post weaning pigs.

BACKGROUND: Post weaning diarrhoea (PWD) causes piglet morbidity and mortality at weaning and is a major driver for antimicrobial use worldwide. New regulations in the EU limit the use of in-feed antibiotics (Ab) and therapeutic zinc oxide (ZnO) to prevent PWD. New approaches to control PWD are needed, and understanding the role of the microbiota in this context is key. In this study, shotgun metagenome sequencing was used to describe the taxonomic and functional evolution of the faecal microbiota of the piglet during the first two weeks post weaning within three experimental groups, Ab, ZnO and no medication, on commercial farms using antimicrobials regularly in the post weaning period. RESULTS: Diversity was affected by day post weaning (dpw), treatment used and diarrhoea but not by the farm. Microbiota composition evolved towards the dominance of groups of species such as Prevotella spp. at day 14dpw. ZnO inhibited E. coli overgrowth, promoted higher abundance of the family Bacteroidaceae and decreased Megasphaera spp. Animals treated with Ab exhibited inconsistent taxonomic changes across time points, with an overall increase of Limosilactobacillus reuteri and Megasphaera elsdenii. Samples from non-medicated pigs showed virulence-related functions at 7dpw, and specific ETEC-related virulence factors were detected in all samples presenting diarrhoea. Differential microbiota functions of pigs treated with ZnO were related to sulphur and DNA metabolism, as well as mechanisms of antimicrobial and heavy metal resistance, whereas Ab treated animals exhibited functions related to antimicrobial resistance and virulence. CONCLUSION: Ab and particularly ZnO maintained a stable microbiota composition and functionality during the two weeks post weaning, by limiting E. coli overgrowth, and ultimately preventing microbiota dysbiosis. Future approaches to support piglet health should be able to reproduce this stable gut microbiota transition during the post weaning period, in order to maintain optimal gut physiological and productive conditions.

Fine-tuning of post-weaning pig microbiome structure and functionality by in-feed zinc oxide and antibiotics use.

Introduction: Post-weaning diarrhoea (PWD) is a multifactorial disease that affects piglets after weaning, contributing to productive and economic losses. Its control includes the use of in-feed prophylactic antibiotics and therapeutic zinc oxide (ZnO), treatments that, since 2022, are no longer permitted in the European Union due to spread of antimicrobial resistance genes and pollution of soil with heavy metals. A dysbiosis in the microbiota has been suggested as a potential risk factor of PWD onset. Understanding pig's microbiota development around weaning and its changes in response to ZnO and antibiotics is crucial to develop feasible alternatives to prophylactic and metaphylactic antimicrobial use. Methods: This study used shotgun metagenomic sequencing to investigate the environmental and faecal microbiota on 10 farms using (Treated) or not using (ZnO-free) in-feed antibiotics and ZnO during the first 14 days post-weaning (dpw). Environmental samples from clean pens were collected at weaning day (0dpw), and faecal samples at 0, 7 and 14dpw. Diarrhoeic faecal samples were collected at 7dpw when available. Results: The analysis of data revealed that the faecal microbiota composition and its functionality was impacted by the sampling time point (microbiota maturation after weaning) but not by the farm environment. Treatment with antibiotics and ZnO showed no effects on diversity indices while the analyses of microbiota taxonomic and functional profiles revealed increased abundance of taxa and metabolic functions associated with Phascolarctobacterium succinatutens or different species of Prevotella spp. on the Treated farms, and with Megasphaera elsdenii and Escherichia coli on the ZnO-free farms. The analysis of diarrhoea samples revealed that the treatment favoured the microbiota transition or maturation from 0dpw to 14dpw in Treated farms, resembling the composition of healthy animals, when compared to diarrhoea from ZnO-free farms, which were linked in composition to 0dpw samples. Discussion: The results provide a comprehensive overview of the beneficial effects of ZnO and antibiotics in PWD in the microbiota transition after weaning, preventing the overgrowth of pathogens such as pathogenic E. coli and revealing the key aspects in microbiota maturation that antibiotics or ZnO alternatives should fulfil.

Effect of the addition of organic acids in drinking water or feed during part of the finishing period on the prevalence of Salmonella in finishing pigs.

Pork is a major cause of foodborne salmonellosis. Consequently, effective measures that could reduce the prevalence of Salmonella at the farm are of interest. In the present study, three field trials were performed to evaluate the effect of strategic administration of organic acids, at concentrations estimated by economic criteria, on the Salmonella prevalence in finishing pigs. Pigs received either a mixture of acids (lactic, formic, propionic, and acetic) added to their drinking water at a concentration of 0.035% (trial A), or a basal diet containing 0.5% potassium-diformate, KH(COOH)2, (trials B and C), during the last 6-7 weeks of the finishing period. Fecal Salmonella shedding and seroprevalence were monitored in the animals at four time points during the study. Mesenteric lymph nodes and cecal contents were collected from a subset of animals at the slaughterhouse and cultured for Salmonella. At the end of the finishing period in all three trials, the percentage of seropositive pigs was higher in the control group than in the experimental group, regardless of which cutoff value was used in the enzyme-linked immunosorbent assay (ELISA) assay. The frequency of fecal shedding was lower also in the treated pig groups from the trials A and B at the end of the finishing period. The results from the samples collected at the slaughterhouse did not yield differences between groups in trials A and B. However, Salmonella isolation was less frequent in the mesenteric lymph nodes from the experimental pigs in trial C. The seroprevalence reductions, together with some promising results in the reduction of shedding, support the idea that this intervention is a useful strategy to reduce Salmonella prevalence at the farm.

Detection and genetic characterization of enteric viruses in diarrhoea outbreaks from swine farms in Spain.

BACKGROUND: The aim of this work was to study the prevalence and distribution of Porcine astrovirus (PAstV), Porcine kobuvirus (PKoV), Porcine torovirus (PToV), Mammalian orthoreovirus (MRV) and Porcine mastadenovirus (PAdV) as well as their association with widely recognized virus that cause diarrhoea in swine such as coronavirus (CoVs) and rotavirus (RVs) in diarrhoea outbreaks from Spanish swine farms. Furthermore, a selection of the viral strains was genetically characterized. RESULTS: PAstV, PKoV, PToV, MRV and PAdV were frequently detected. Particularly, PAstV and PKoV were detected in almost 50% and 30% of the investigated farms, respectively, with an age-dependent distribution; PAstV was mainly detected in postweaning and fattening pigs, while PKoV was more frequent in sucking piglets. Viral co-infections were detected in almost half of the outbreaks, combining CoVs, RVs and the viruses studied, with a maximum of 5 different viral species reported in three investigated farms. Using a next generation sequencing approach, we obtained a total of 24 ARN viral genomes (> 90% genome sequence), characterizing for first time the full genome of circulating strains of PAstV2, PAstV4, PAstV5 and PToV on Spanish farms. Phylogenetic analyses showed that PAstV, PKoV and PToV from Spanish swine farms clustered together with isolates of the same viral species from neighboring pig producing countries. CONCLUSIONS: Although further studies to evaluate the role of these enteric viruses in diarrhoea outbreaks are required, their wide distribution and frequent association in co-infections cannot be disregard. Hence, their inclusion into routine diagnostic panels for diarrhoea in swine should be considered.

Comparison of antimicrobial resistant Escherichia coli isolated from Irish commercial pig farms with and without zinc oxide and antimicrobial usage.

BACKGROUND: The prophylactic use of antimicrobials and zinc oxide (ZnO) in pig production was prohibited by the European Union in 2022 due to potential associations between antimicrobial and heavy metal usage with antimicrobial resistance (AMR) and concerns regarding environmental pollution. However, the effects of their usage on the bacterial AMR profiles on commercial pig farms are still not fully understood and previous studies examining the effect of ZnO have reported contrasting findings. The objective of this study was to examine the effects of antimicrobial and ZnO usage on AMR on commercial pig farms. Faecal and environmental samples were taken on 10 Irish commercial farms, of which 5 farms regularly used ZnO and antimicrobials (amoxicillin or sulphadiazine-trimethoprim) for the prevention of disease. The other 5 farms did not use ZnO or any other form of prophylaxis. Escherichia coli numbers were quantified from all samples using non-supplemented and supplemented Tryptone Bile X-glucuronide agar. RESULTS: In total 351 isolates were phenotypically analysed, and the genomes of 44 AmpC/ESBL-producing E. coli isolates from 4 farms were characterised using whole-genome sequencing. Phenotypic analysis suggested higher numbers of multi-drug resistant (MDR) E. coli isolates on farms using prophylaxis. Furthermore, farms using prophylaxis were associated with higher numbers of isolates resistant to apramycin, trimethoprim, tetracycline, streptomycin, and chloramphenicol, while resistance to ciprofloxacin was more associated with farms not using any prophylaxis. Thirty-four of the 44 AmpC/ESBL-producing E. coli strains harboured the blaCTX-M-1 resistance gene and were multi drug resistant (MDR). Moreover, network analysis of plasmids and analysis of integrons showed that antimicrobial and biocide resistance genes were frequently co-located on mobile genetic elements, indicating the possibility for co-selection during antimicrobial or biocide usage as a contributor to AMR occurrence and persistence on farms. CONCLUSIONS: The results of this study showed evidence that antimicrobial and ZnO treatment of pigs post-weaning can favour the selection and development of AMR and MDR E. coli. Co-location of resistance genes on mobile genetic elements was observed. This study demonstrated the usefulness of phenotypic and genotypic detection of antimicrobial resistance by combining sequencing and microbiological methods.

The Effect of Supplementation with Betaine and Zinc on In Vitro Large Intestinal Fermentation in Iberian Pigs under Heat Stress.

We investigated the effects of betaine and zinc on the in vitro fermentation of pigs under heat stress (HS). Twenty-four Iberian pigs (43.4 ± 1.2 kg) under HS (30 °C) were assigned to treatments for 4 weeks: control (unsupplemented), betaine (5 g/kg), and zinc (0.120 g/kg) supplemented diet. Rectal content was used as the inoculum in 24-hincubations with pure substrates (starch, pectin, inulin, cellulose). Total gas, short-chain fatty acid (SCFA), and methane production and ammonia concentration were measured. The abundance of total bacteria and several bacterial groups was assessed. Betaine increased the acetate production with pectin and inulin, butyrate production with starch and inulin, and ammonia concentration, and decreased propionate production with pectin and inulin. The abundance of Bifidobacterium and two groups of Clostridium decreased with betaine supplementation. Zinc decreased the production of SCFA and gas with starch and inulin, associated with diminished bacterial activity. Propionate production decreased with starch, pectin, and inulin while butyrate production increased with inulin, and isoacid production increased with cellulose and inulin in pigs supplemented with zinc. The ammonia concentration increased for all substrates. The Clostridium cluster XIV abundance decreased in pigs fed zinc supplemented diets. The results reported were dependent on the substrate fermented, but the augmented butyrate production with both betaine and zinc could be of benefit for the host.

Metagenomic comparison of the faecal and environmental resistome on Irish commercial pig farms with and without zinc oxide and antimicrobial usage.

BACKGROUND: Antimicrobials and heavy metals such as zinc oxide (ZnO) have been commonly used on Irish commercial pig farms for a 2-week period post-weaning to help prevent infection. In 2022, the prophylactic use of antimicrobials and ZnO was banned within the European Union due to concerns associated with the emergence of antimicrobial resistance (AMR) and contamination of the environment with heavy metals. In this study, faecal and environmental samples were taken from piglets during the weaning period from ten commercial farms, of which five farms used antimicrobial or ZnO prophylaxis (AB-ZnO farms) and five which had not used antimicrobials or ZnO for the previous 3 years (AB-ZnO free farms). A total of 50 samples were compared using a metagenomic approach. RESULTS: The results of this study showed some significant differences between AB-ZnO and AB-ZnO free farms and suggested positive selection for AMR under antimicrobial and ZnO treatment. Moreover, strong differences between environmental and faecal samples on farms were observed, suggesting that the microbiome and its associated mobile genetic elements may play a key role in the composition of the resistome. Additionally, the age of piglets affected the resistome composition, potentially associated with changes in the microbiome post-weaning. CONCLUSIONS: Overall, our study showed few differences in the resistome of the pig and its environment when comparing AB-ZnO farms with AB-ZnO free farms. These results suggest that although 3 years of removal of in-feed antimicrobial and ZnO may allow a reduction of AMR prevalence on AB-ZnO farms, more time, repeated sampling and a greater understanding of factors impacting AMR prevalence will be required to ensure significant and persistent change in on-farm AMR.

Genomic Insights into the Mobilome and Resistome of Sentinel Microorganisms Originating from Farms of Two Different Swine Production Systems.

Antimicrobial resistance (AMR) is a threat to public health due to long-term antimicrobial use (AMU), which promotes the bacterial acquisition of antimicrobial resistance determinants (ARDs). Within food-producing animals, organic and extensive Iberian swine production is based on sustainable and eco-friendly management systems, providing an excellent opportunity to evaluate how sustained differences in AMU impact the development and spread of AMR. Here, through a whole-genome sequencing approach, we provide an in-depth characterization of the resistome and mobilome and their interaction in 466 sentinel bacteria, namely, Escherichia coli, Enterococcus spp., Campylobacter coli, and Staphylococcus spp., recovered from 37 intensive and organic-extensive pig farms. Both ARDs and mobile genetic elements (MGEs) were primarily taxon-associated, with higher similarities among bacteria which were closely phylogenetically related. E. coli exhibited the most diverse resistome and mobilome, with 85.4% mobilizable ARDs, 50.3% of which were plasmid-associated. Staphylococcus spp. exhibited a broad repertoire of ARDs and MGEs, with 52.3% of its resistome being mobilizable. Although Enterococcus spp. carried the highest number of ARDs per isolate and its plasmidome was similar in size to that of E. coli, 43.7% of its resistome was mobilizable. A narrow spectrum of ARDs constituted the C. coli resistome, with point mutations as its main AMR driver. A constrained AMU, as observed in organic-extensive herds, determined a reduction in the quantitative composition of the resistome and the complexity of the resistome-mobilome interaction. These results demonstrate taxon-associated AMR-MGE interactions and evidence that responsible AMU can contribute to reducing AMR pressure in the food chain. IMPORTANCE This study provides the first integral genomic characterization of the resistome and mobilome of sentinel microorganisms for antimicrobial resistance (AMR) surveillance from two different swine production systems. Relevant differences were observed among taxa in the resistomes and mobilomes they harbored, revealing their distinctive risk in AMR dissemination and spread. Thus, Escherichia coli and, to a lesser extent, Staphylococcus spp. constituted the main reservoirs of mobilizable antimicrobial resistance genes, which were predominantly plasmid-associated; in contrast to Campylobacter coli, whose resistome was mainly determined by point mutations. The reduced complexity of mobilome-resistome interaction in Enterococcus spp. suggested its limited role in AMR dissemination from swine farms. The significant differences in antimicrobial use among the studied farms allowed us to assess the suitability of whole-genome sequencing as a rapid and efficient technique for the assessment of mid- to long-term on-farm interventions for the reduction of antimicrobial use and the evaluation of AMR status.

In vitro activity of essential oils against microbial isolates from otitis externa cases in dogs.

Despite the number of studies focused on the potential use of essential oils (EOs) as an alternative to conventional treatments of canine external otitis, there is controversy about their antimicrobial activity which could be explained by differences in technical or biological aspects. This study focuses on the antimicrobial activity of three single EO compounds (thymol, cinnamaldehyde and carvacrol) and two EOs (clove and oregano) against clinical isolates recovered from canine otitis externa cases (14 bacterial isolates belonging to five different genera and six Malassezia pachydermatis isolates). All compounds showed activity and cinnamaldehyde exhibited the highest bactericidal and fungicidal activity. The susceptibility was lower among bacterial isolates than fungal isolates, being this difference more evident for Gram-positive bacteria. No relationship between antibiotic multi-resistant profile and susceptibility to compounds was observed. To sum up, our results provide appropriate information about appropriate concentrations of promising candidates for the topic treatment of canine otitis.

Combined in-vitro and on-farm evaluation of commercial disinfectants used against Brachyspira hyodysenteriae.

BACKGROUND: Swine dysentery (SD) is a severe infectious disease with a relevant impact on pig production usually caused by Brachyspira hyodysenteriae, although B. hampsonii causes an identical clinical picture. SD control relies on antimicrobials, good management practices and strict biosecurity with cleaning and disinfection as crucial tools to avoid the pathogen transmission. This study evaluates the in-vitro efficacy of an array of commercial disinfectants against a collection of B. hyodysenteriae isolates using broth tests. The efficacy of cleaning and disinfection protocols was also evaluated on two farms with endemic SD using surface swabs collected in emptied pens before and after cleaning and disinfection procedures, using both real-time PCR and bacterial microbiological culture. RESULTS: Most of the commercial disinfectants evaluated were effective against all B. hyodysenteriae isolates tested, with a reduction of more than 5.00 log10 CFU/mL (bactericidal efficacy of 99.999%). However, some isolates exhibited reduced susceptibility to Virkon-S and Limoseptic disinfectants. The evaluation of cleaning and disinfection protocols on farms with SD outbreaks showed that approximately half the pens tested (n = 25) were positive by real-time PCR after pigs removal (mean B. hyodysenteriae counts 5.72 ± 1.04 log10 CFU/mL) while almost 20% of the pens remained positive after cleaning (n = 7) and disinfection (n = 5) procedures although with significantly lower, mean estimates (4.31 ± 0.43 log10 CFU/mL and 4.01 ± 0.55 log10 CFU/mL, respectively). CONCLUSIONS: These results show the efficacy of disinfectants against B. hyodysenteriae but also stress the need to implement adequately the cleaning and disinfection protocols on pig farms and review and revise their efficiency periodically.

Using Shotgun Sequencing to Describe the Changes Induced by In-Feed Zinc Oxide and Apramycin in the Microbiomes of Pigs One Week Postweaning.

Postweaning diarrhea (PWD) is a relevant problem associated with early weaning on pig farms. For decades, in-feed antibiotics and therapeutic zinc oxide (ZnO) have been widely used to prevent PWD in piglets. The European Union is banning both strategies in 2022 due to antimicrobial resistance and environmental contamination concerns, respectively. Understanding the effects of these products on the pig microbiome is crucial for correcting potential microbial disbalances that would prompt PWD. Using shotgun sequencing, three trials were carried out to explore the impact of in-feed apramycin and ZnO, combined with different farm hygiene protocols, on the fecal microbiomes of piglets 7 days postweaning. In trial 1, 28-day-old piglets were allocated to one of three groups: control diet (Ct), Ct + ZnO (Zn), and Ct + apramycin (Ab). In trials 2 and 3, piglets were allocated to the same treatments, but the trials also included different cleaning protocols, achieving different hygiene levels. In-feed treatments impacted the richness, diversity, and relative abundance of the piglets' microbiome more than hygiene. Pigs in the Ct group showed higher species richness than pigs in the Ab and Zn groups. A clustering analysis evidenced a link between Enterobacteriaceae in the Ct group; Lactobacillaceae and Veillonellaceae mainly in the Ct group; and Bacteroidaceae, Ruminococcaceae, Oscillospiraceae, Acidaminococcaceae, and Lactobacillaceae in the Ab and Zn groups. Functional data analysis revealed a higher abundance of virulence genes in the Ct group microbiomes and heavy metal and antimicrobial resistance-related functions in the Zn treatment group. The results demonstrate that alternatives to Ab and ZnO should balance the microbial abundance and stimulate the growth of commensals to outcompete potential pathogens. IMPORTANCE Weaning is a critical period for piglets, during which potentially harmful bacteria such as Escherichia coli can increase in abundance in the intestine, creating digestive problems and diarrhea. In-feed antibiotics, the most frequent administration route for antibiotics in livestock, and therapeutic doses of zinc oxide (ZnO) help to control diarrhea but prompt secondary problems such as antimicrobial resistance and soil pollution from heavy metals. Understanding how these strategies impact the gut microbiota is crucial for establishing health biomarkers and designing successful replacement strategies. Using shotgun sequencing, this study compares the microbiota of pigs after early weaning when treated with in-feed antibiotics, ZnO, or treatment-free diets to describe differences that could define the susceptibility to infections, providing the basis for future research on improving intestinal resilience through microbiota-based strategies.

In vitro evaluation of gentamicin activity against Spanish field isolates of Brachyspira hyodysenteriae.

BACKGROUND: The treatment of swine dysentery (SD) has become constrained in recent years due to the limited availability of effective drugs combined with a rise in antimicrobial resistance. Gentamicin, an aminoglycoside antibiotic, is authorised for the control of this disease in several European countries but has not been extensively used so far. In this study, the in vitro susceptibility of 56 Brachyspira hyodysenteriae field isolates was evaluated against gentamicin using a broth microdilution test. The molecular basis of decreased susceptibility to gentamicin was also investigated by sequencing the 16S rRNA gene and phylogenetic relatedness by multiple-locus variable number tandem-repeat analysis (MLVA). RESULTS: Most B. hyodysenteriae isolates presented low minimum inhibitory concentration (MIC) values to gentamicin, with a mode of 2 µg/mL, a median or MIC50 of 4 µg/mL and percentile 90 or MIC90 of 16 µg/mL. The distribution of these values over the period studied (2011-2019) did not show a tendency towards the development of resistance to gentamicin. Differences in susceptibility among isolates could be explained by two point-mutations in the 16S rRNA gene, C990T and A1185G, which were only present in isolates with high MICs. These isolates were typed in three different MLVA clusters. Analyses of co-resistance between gentamicin and antimicrobials commonly used for the treatment of SD revealed that resistance to tiamulin and valnemulin was associated with low MICs for gentamicin. CONCLUSIONS: The results provide an accurate characterisation of antimicrobial sensitivity to gentamicin and possible mechanisms of resistance in Spanish B. hyodysenteriae isolates. These findings allow us to propose gentamicin as an alternative in the antibiotic management of SD, particularly in outbreaks caused by pleuromutilin resistant isolates.

Microbial community in resistant and susceptible Churra sheep infected by Teladorsagia circumcincta.

Gastrointestinal nematodes (GIN) are a major threat to health and welfare in small ruminants worldwide. Teladorsagia circumcincta is a nematode that inhabits the abomasum of sheep, especially in temperate regions, causing important economic losses. Given that T. circumcincta and microbiome share the same niche, interactions between them and the host are expected. Although it is known that within a sheep breed there are animals that are more resistant than others to infection by GIN, it is not known if the microbiome influences the phenotype of these animals. Under this condition, 12 sheep were classified according to their cumulative faecal egg count (cFEC) at the end of a first experimental infection, 6 as resistant group (RG) and 6 as susceptible group (SG) to T. circumcincta infection. Then, all sheep were experimentally infected with 70,000 L3 of T. circumcincta and at day 7 days post-infection were euthanized. At necropsy, gastric mucosa and gastric content from abomasum were collected to extract bacterial DNA and sequence V3-V4 region from 16S rRNA gene using Ilumina technology. After bioanalysis performed, results showed that α-diversity and ß-diversity remained similar in both groups. However, resistant phenotype sheep showed a higher number of bacteria butyrate-fermenting species as Clostridium sensu stricto 1 (abundance in RG: 1.29% and in SG: 0.069%; p = 0.05), and Turicibacter (abundance in RG: 0.31% and in SG: 0.027%; p = 0.07) in gastric content but also Serratia spp in gastric mucosa (abundance in RG: 0.12% and in SG: 0.041%; p = 0.07). A trend towards a significant negative correlation between cFEC and Clostridium sensu stricto 1 abundance in gastric content was detected (r = - 0.537; p = 0.08). These data suggest that microbiome composition could be another factor associated with the development of the resistant phenotype modifying the interaction with the host and the in last instance affecting the individual risk of infection.

Porcine reproductive and respiratory syndrome virus impacts on gut microbiome in a strain virulence-dependent fashion.

Porcine reproductive and respiratory syndrome (PRRS) is a viral disease defined by reproductive problems, respiratory distress and a negative impact on growth rate and general condition. Virulent PRRS virus (PRRSV) strains have emerged in the last years with evident knowledge gaps in their impact on the host immune response. Thus, the present study examines the impact of acute PRRS virus (PRRSV) infection, with two strains of different virulence, on selected immune parameters and on the gut microbiota composition of infected pigs using 16S rRNA compositional sequencing. Pigs were infected with a low virulent (PRRS_3249) or a virulent (Lena) PRRSV-1 strain and euthanized at 1, 3, 6, 8 or 13 days post-inoculation (dpi). Faeces were collected from each animal at the necropsy time-point. Alpha and beta diversity analyses demonstrated that infection, particularly with the Lena strain, impacted the microbiome composition from 6 dpi onwards. Taxonomic differences revealed that infected pigs had higher abundance of Treponema and Methanobrevibacter (FDR < 0.05). Differences were more considerable for Lena- than for PRRS_3249-infected pigs, showing the impact of strain virulence in the intestinal changes. Lena-infected pigs had reduced abundancies of anaerobic commensals such as Roseburia, Anaerostipes, Butyricicoccus and Prevotella (P < 0.05). The depletion of these desirable commensals was significantly correlated to infection severity measured by viraemia, clinical signs, lung lesions and immune parameters (IL-6, IFN-γ and Hp serum levels). Altogether, the results from this study demonstrate the indirect impact of PRRSV infection on gut microbiome composition in a strain virulence-dependent fashion and its association with selected immune markers.