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.

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.

Hybrid genome assemblies of four Shiga toxin-producing Escherichia coli strains containing a complete locus of enterocyte effacement and an O-Island 122.

This study describes the hybrid genome assemblies of four Shiga toxin-producing Escherichia coli strains isolated from the recto-anal junction of slaughter-age Irish sheep. In silico serotyping and genome analysis determined that each of the strains harbored a Shiga-toxin subtype, a complete locus of enterocyte effacement, and a rare O-island 122.

The efficiency of UV light-emitting diodes (UV-LED) in decontaminating Campylobacter and Salmonella and natural microbiota in chicken breast, compared to a UV pilot-plant scale device.

This study investigated the combined effect of Ultraviolet (UV) light-emitting diode (LED) technology treatment with refrigerated storage of chicken breast meat over 7 days on Campylobacter jejuni, Salmonella enterica serovar Typhimurium, total viable counts (TVC) and total Enterobacteriaceae counts (TEC). An optimised UV-LED treatment at 280 nm for 6 min decreased inoculated S. Typhimurium and C. jejuni populations by 0.6-0.64 log CFU/g, and TVC and TEC population by 1-1.2 log CFU/g in chicken samples. During a 7-day storage at 4 °C, a 0.73 log reduction in C. jejuni was achieved compared with non-treated samples. Moreover, the UV-LED effectiveness to reduce TVC and TEC during refrigerated storage was compared with a conventional UV lamp and a similar efficiency was observed. The impact of UV-LED and UV lamp devices on the microbial community composition of chicken meat during storage was further examined using 16 S rRNA gene amplicon sequencing. Although similar bacterial reductions were observed for both technologies, the microbial communities were impacted differently. Treatment with the UV conventional lamp increased the proportion of Brochothrix spp. In meat samples, whilst Photobacterium spp. Levels were reduced.

A Small Study on Clostridioides difficile in Spinach Field Soil and the Chemical and Microbial Factors that may Influence Prevalence.

Clostridioides difficile is a human pathogen that is ubiquitous in soil. Despite increasing infection rates and evidence of foodborne transmission, there is limited data on prevalence in soil or which factors influence persistence. The aim of this study was to investigate the prevalence of these bacteria in soil from three different spinach fields and to examine the chemical composition (carbon, organic carbon, nitrogen, organic matter, minerals and pH) and microbiota to gain insight into the factors that may promote/inhibit C. difficile. The overall C. difficile prevalence (10%) was lower than expected (based on international studies) and a significantly (P < 0.05) higher prevalence was obtained in Field 3 (20%) as compared to Fields 1 and 2 (5% each). Analysis of the soil suggested that the pH as well as organic matter, calcium and phosphorus content directly and indirectly (via the microbiota) influenced the prevalence of C. difficile in adjacent fields, where other factors (eg. climate) are similar. Although further studies are required to validate our findings, the data provides the first step in developing potential soil based control strategies.

Investigation of differences in susceptibility of Campylobacter jejuni strains to UV light-emitting diode (UV-LED) technology.

Campylobacter jejuni remains a high priority in public health worldwide. Ultraviolet light emitting-diode technology (UV-LED) is currently being explored to reduce Campylobacter levels in foods. However, challenges such as differences in species and strain susceptibilities, effects of repeated UV-treatments on the bacterial genome and the potential to promote antimicrobial cross-protection or induce biofilm formation have arisen. We investigated the susceptibility of eight C. jejuni clinical and farm isolates to UV-LED exposure. UV light at 280 nm induced different inactivation kinetics among strains, of which three showed reductions greater than 1.62 log CFU/mL, while one strain was particularly resistant to UV light with a maximum reduction of 0.39 log CFU/mL. However, inactivation was reduced by 0.46-1.03 log CFU/mL in these three strains and increased to 1.20 log CFU/mL in the resistant isolate after two repeated-UV cycles. Genomic changes related to UV light exposure were analysed using WGS. C. jejuni strains with altered phenotypic responses following UV exposure were also found to have changes in biofilm formation and susceptibility to ethanol and surface cleaners.

Differential impact of swine, bovine and poultry manure on the microbiome and resistome of agricultural grassland.

Land spreading of animal manure is an essential process in agriculture. Despite the importance of grassland in global food security the potential of the grass phyllosphere as a reservoir of antimicrobial resistance (AMR) is unknown. Additionally, the comparative risk associated with different manure sources is unclear. Due to the One Health nature of AMR there is an urgent need to fully understand the risk associated with AMR at the agriculture - environmental nexus. We performed a grassland field study to assess and compare the relative and temporal impact of bovine, swine and poultry manure application on the grass phyllosphere and soil microbiome and resistome over a period of four months, using 16S rRNA amplicon sequencing and high-throughput quantitative PCR (HT-qPCR). The soil and grass phyllosphere contained a diverse range of antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs). Manure treatment was found to introduce ARGs belonging to clinically important antimicrobial classes, such as aminoglycoside and sulphonamide into grass and soil. Temporal analysis of ARGs and MGEs associated with manure treatment indicated ARGs patterns were similar across the different manure types in the manure treated soil and grass phyllosphere. Manure treatment resulted in the enrichment in members of the indigenous microbiota and the introduction of manure associated bacteria, with this impact extending past the recommended six-week exclusion period. However, these bacteria were in low relative abundance and manure treatment was not found to significantly impact the overall composition of the microbiome or resistome. This provides evidence that the current guidelines facilitate reduction of biological risk to livestock. Additionally, in soil and grass samples MGEs correlated with ARGs from clinically important antimicrobial classes, indicating the key role MGEs play in horizontal gene transfer in agricultural grassland. These results demonstrate the role of the grass phyllosphere as an under-studied sink of AMR.

The Gut Microbiota of Broilers Reared with and without Antibiotic Treatment.

The aim of this study was to examine the microbiota in broilers reared with and without antibiotics and to investigate differences between the upper, middle and lower sections of the gastrointestinal tract (GIT). One of two commercial flocks was treated with an antibiotic (T) (20 mg trimethoprim and 100 mg sulfamethoxazole per ml in the drinking water for 3 days) and the other was left untreated (UT). The GIT contents of 51 treated and untreated birds were aseptically removed from the upper (U), middle (M) and lower (L) sections. These were pooled in triplicate (n = 17 per section per flock), the DNA extracted and purified, 16S amplicon metagenomic sequencing performed and the resultant data analysed using a range of bioinformatics software. There were significant differences in the microbiota of the upper, middle and lower GIT, and treatment with the antibiotic significantly affected the microbiota in each of these sections. This study provides new data on broiler GIT microbiota and suggests that GIT location is a more important determinant of the constituent bacterial flora rather than the use or otherwise of antimicrobial treatments, at least when applied early in the production cycle.

Characterization of cephalosporin and fluoroquinolone resistant Enterobacterales from Irish farm waste by whole genome sequencing.

Background: The Enterobacterales are a group of Gram-negative bacteria frequently exhibiting extended antimicrobial resistance (AMR) and involved in the transmission of resistance genes to other bacterial species present in the same environment. Due to their impact on human health and the paucity of new antibiotics, the World Health Organization (WHO) categorized carbapenem resistant and ESBL-producing as critical. Enterobacterales are ubiquitous and the role of the environment in the transmission of AMR organisms or antimicrobial resistance genes (ARGs) must be examined in tackling AMR in both humans and animals under the one health approach. Animal manure is recognized as an important source of AMR bacteria entering the environment, in which resistant genes can accumulate. Methods: To gain a better understanding of the dissemination of third generation cephalosporin and fluoroquinolone resistance genes between isolates in the environment, we applied whole genome sequencing (WGS) to Enterobacterales (79 E. coli, 1 Enterobacter cloacae, 1 Klebsiella pneumoniae, and 1 Citrobacter gillenii) isolated from farm effluents in Ireland before (n = 72) and after (n = 10) treatment by integrated constructed wetlands (ICWs). DNA was extracted using the MagNA Pure 96 system (Roche Diagnostics, Rotkreuz, Switzerland) followed by WGS on a MiSeq platform (Illumina, Eindhoven, Netherlands) using v3 chemistry as 300-cycle paired-end runs. AMR genes and point mutations were identified and compared to the phenotypic results for better understanding of the mechanisms of resistance and resistance transmission. Results: A wide variety of cephalosporin and fluoroquinolone resistance genes (mobile genetic elements (MGEs) and chromosomal mutations) were identified among isolates that mostly explained the phenotypic AMR patterns. A total of 31 plasmid replicon types were identified among the 82 isolates, with a subset of them (n = 24), identified in E. coli isolates. Five plasmid replicons were confined to the Enterobacter cloacae isolate and two were confined to the Klebsiella pneumoniae isolate. Virulence genes associated with functions including stress, survival, regulation, iron uptake secretion systems, invasion, adherence and toxin production were identified. Conclusion: Our study showed that antimicrobial resistant organisms (AROs) can persist even following wastewater treatment and could transmit AMR of clinical relevance to the environment and ultimately pose a risk to human or animal health.

Building an International One Health Strain Level Database to Characterise the Epidemiology of AMR Threats: ESBL-AmpC Producing E. coli as An Example-Challenges and Perspectives.

Antimicrobial resistance (AMR) is one of the top public health threats nowadays. Among the most important AMR pathogens, Escherichia coli resistant to extended spectrum cephalosporins (ESC-EC) is a perfect example of the One Health problem due to its global distribution in animal, human, and environmental sources and its resistant phenotype, derived from the carriage of plasmid-borne extended-spectrum and AmpC ß-lactamases, which limits the choice of effective antimicrobial therapies. The epidemiology of ESC-EC infection is complex as a result of the multiple possible sources involved in its transmission, and its study would require databases ideally comprising information from animal (livestock, companion, wildlife), human, and environmental sources. Here, we present the steps taken to assemble a database with phenotypic and genetic information on 10,763 ESC-EC isolates retrieved from multiple sources provided by 13 partners located in eight European countries, in the frame of the DiSCoVeR Joint Research project funded by the One Health European Joint Programme (OH-EJP), along with its strengths and limitations. This database represents a first step to help in the assessment of different geographical and temporal trends and transmission dynamics in animals and humans. The work performed highlights aspects that should be considered in future international efforts, such as the one presented here.

Electrochemical nucleic acid-based sensors for detection of Escherichia coli and Shiga toxin-producing E. coli-Review of the recent developments.

Escherichia coli are a group of bacteria that are a natural part of the intestinal flora of warm-blooded animals, including humans. Most E. coli are nonpathogenic and essential for the normal function of a healthy intestine. However, certain types, such as Shiga toxin-producing E. coli (STEC), which is a foodborne pathogen, can cause a life-threatening illness. The development of point-of-care devices for the rapid detection of E. coli is of significant interest with regard to ensuring food safety. The most suitable way to distinguish between generic E. coli and STEC is by using nucleic acid-based detection, focusing on the virulence factors. Electrochemical sensors based on nucleic acid recognition have attracted much attention in recent years for use in pathogenic bacteria detection. This review has summarized nucleic acid-based sensors for the detection of generic E. coli and STEC since 2015. First, the sequences of the genes used as recognition probes are discussed and compared to the most recent research regarding the specific detection of general E. coli and STEC. Subsequently, the collected literature regarding nucleic acid-based sensors is described and discussed. The traditional sensors were divided into four categories such as gold, indium tin oxide, carbon-based electrodes, and those using magnetic particles. Finally, we summarized the future trends in nucleic acid-based sensor development for E. coli and STEC including some examples of fully integrated devices.

First reported detection of the mobile colistin resistance genes, mcr-8 and mcr-9, in the Irish environment.

The emergence and dissemination of mobile colistin resistance (mcr) genes across the globe poses a significant threat to public health, as colistin remains one of the last line treatment options for multi-drug resistant infections. Environmental samples (157 water and 157 wastewater) were collected in Ireland between 2018 and 2020. Samples collected were assessed for the presence of antimicrobial resistant bacteria using Brilliance ESBL, Brilliance CRE, mSuperCARBA and McConkey agar containing a ciprofloxacin disc. All water and integrated constructed wetland influent and effluent samples were filtered and enriched in buffered peptone water prior to culture, while wastewater samples were cultured directly. Isolates collected were identified via MALDI-TOF, were tested for susceptibility to 16 antimicrobials, including colistin, and subsequently underwent whole genome sequencing. Overall, eight mcr positive Enterobacterales (one mcr-8 and seven mcr-9) were recovered from six samples (freshwater (n = 2), healthcare facility wastewater (n = 2), wastewater treatment plant influent (n = 1) and integrated constructed wetland influent (piggery farm waste) (n = 1)). While the mcr-8 positive K. pneumoniae displayed resistance to colistin, all seven mcr-9 harbouring Enterobacterales remained susceptible. All isolates demonstrated multi-drug resistance and through whole genome sequencing analysis, were found to harbour a wide variety of antimicrobial resistance genes i.e., 30 ± 4.1 (10-61), including the carbapenemases, blaOXA-48 (n = 2) and blaNDM-1 (n = 1), which were harboured by three of the isolates. The mcr genes were located on IncHI2, IncFIIK and IncI1-like plasmids. The findings of this study highlight potential sources and reservoirs of mcr genes in the environment and illustrate the need for further research to gain a better understanding of the role the environment plays in the persistence and dissemination of antimicrobial resistance.

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.

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.

Evaluating the impact of heavy metals on antimicrobial resistance in the primary food production environment: A scoping review.

Heavy metals are naturally occurring environmental compounds, which can influence antimicrobial resistance (AMR) dissemination. However, there is limited information on how heavy metals may act as a selective pressure on AMR in the primary food production environment. This review aims to examine the literature on this topic in order to identify knowledge gaps. A total of 73 studies, which met pre-established criteria, were included. These investigations were undertaken between 2008 and 2021, with a significant increase in the last three years. The majority of studies included were undertaken in China. Soil, water and manure were the most common samples analysed, and the sampling locations varied from areas with a natural presence of heavy metals, areas intentionally amended with heavy metals or manure, to areas close to industrial activity or mines. Fifty-four per cent of the investigations focused on the analysis of four or more heavy metals, and copper and zinc were the metals most frequently analysed (n = 59, n = 49, respectively). The findings of this review highlight a link between heavy metals and AMR in the primary food production environment. Heavy metals impacted the abundance and dissemination of mobile genetic elements (MGEs) and antimicrobial resistance genes (ARGs), with MGEs also observed as playing a key role in the spread of ARGs and metal resistance genes (MRGs). Harmonization of methodologies used in future studies would increase the opportunity for comparison between studies. Further research is also required to broaden the availability of data at a global level.

Comparison of soil and grass microbiomes and resistomes reveals grass as a greater antimicrobial resistance reservoir than soil.

Grasslands cover a large proportion of global agricultural landmass used to feed herbivores and ruminants and link the environment to the food chain via animals onto humans. However, most scientific studies of antimicrobial resistance and microbiomes at the environmental - animal nexus have focused on soil or vegetables rather than grasslands. Based on previous microbiome phyllosphere-soil studies we hypothesised that the microbiome and resistomes across soil and grass would have a core of shared taxa and antimicrobial resistance genes (ARGs), but that in addition each would also have a minority of unique signatures. Our data indicated grass contained a wider variety and higher relative abundance of ARGs and mobile genetic elements (MGEs) than soil with or without slurry amendments. The microbiomes of soil and grass were similar in content but varied in the composition proportionality. While there were commonalities across many of the ARGs present in soil and on grass their correlations with MGEs and bacteria differed, suggesting a source other than soil is also relevant for the resistome of grass. The variations in the relative abundances of ARGs in soil and on grass also indicated that either the MGEs or the bacteria carrying the ARGs comprised a higher relative abundance on grass than in soil. We conclude that while soil may be a source of some of these genes it cannot be the source for all ARGs and MGEs. Our data identifies grass as a more diverse and abundant reservoir of ARGs and MGEs in the environment than soil, which is significant to human and animal health when viewed in the context of grazing food animals.

Examining the impact of zinc on horizontal gene transfer in Enterobacterales.

Antimicrobial resistance is one of the main international health concerns for humans, animals, and the environment, and substantial efforts have focused on reducing its development and spread. While there is evidence for correlations between antimicrobial usage and antimicrobial resistance development, specific information on the effect of heavy metal/antimicrobial usage on bacterial conjugation is more limited. The aim of this study was to investigate the effects of zinc and antimicrobials in different concentrations on horizontal gene transfer of an ampicillin resistance gene, using a multi-drug resistant Escherichia coli donor strain and three different Salmonella enterica serovars as recipient strains. Differences in conjugation frequencies for the different Salmonella recipients were observed, independent of the presence of zinc or the antimicrobials. Selective pressure on the recipient strains, in the form of ampicillin, resulted in a decrease in conjugation frequencies, while, the presence of rifampicin resulted in increases. Zinc exposure affected conjugation frequencies of only one of the three recipient strains, thus the effect of zinc on conjugation frequencies seemed to be concentration and strain dependent. Furthermore, differences in growth rates due to plasmid carriage were observed for one of the Salmonella strains.

Detection and Genomic Characterisation of Clostridioides difficile from Spinach Fields.

Despite an increased incidence of Clostridioides difficile infections, data on the reservoirs and dissemination routes of this bacterium are limited. This study examined the prevalence and characteristics of C. difficile isolates in spinach fields. C. difficile was detected in 2/60 (3.3%) of spinach and 6/60 (10%) of soil samples using culture-based techniques. Whole genome sequencing (WGS) analysis identified the spinach isolates as belonging to the hypervirulent clade 5, sequence type (ST) 11, ribotypes (RT) 078 and 126 and carried the genes encoding toxins A, B and CDT. The soil isolates belonged to clade 1 with different toxigenic ST/RT (ST19/RT614, ST12/RT003, ST46/RT087, ST16/RT050, ST49/RT014/0) strains and one non-toxigenic ST79/RT511 strain. Antimicrobial resistance to erythromycin (one spinach isolate), rifampicin (two soil isolates), clindamycin (one soil isolate), both moxifloxacin and rifampicin (one soil isolate), and multi-drug resistance to erythromycin, vancomycin and rifampicin (two soil isolates) were observed using the E test, although a broader range of resistance genes were detected using WGS. Although the sample size was limited, our results demonstrate the presence of C. difficile in horticulture and provide further evidence that there are multiple sources and dissemination routes for these bacteria.

An Investigation of the Effect of Water Additives on Broiler Growth and the Caecal Microbiota at Harvest.

Campylobacter is the most common foodborne pathogen in developed countries and most cases are associated with poultry. This study investigated the effect of three anti-Campylobacter water additives on broiler growth and on the caecal microbiota at harvest using 16S rRNA amplicon sequencing. Mixtures of organic acids (OA) and essential oils (EO) were administered to broilers for the entirety of the production cycle (35 d) and medium-chain fatty acids (MCFA) for 5 d immediately before harvest, under commercial conditions. Bird weight gain was significantly (p < 0.001) reduced in broilers receiving the OA and EO treatments. While this was most likely due to reduced water intake and corresponding lower feed consumption, changes to the caecal microbiota may also have contributed. Firmicutes made up over 75% of the bacteria regardless of sample type, while the minor phyla included Bacteroidetes, Actinobacteria, Melainabacteria, and Proteobacteria. There were no significant (p > 0.05) differences in the alpha diversity as measured using ACE, Chao1, and Shannon indices, except for control (water) versus MCFA and OA versus MCFA, using the Wilcox test. In contrast, there was a significant (p < 0.05) difference in beta diversity when the treated were compared to the untreated control and main flock samples, while linear discriminant analysis effect size (LeFSe) identified three OTUs that were present in the control but absent in the treated birds. It was concluded that the water additives tested adversely affected broiler performance, which may, at least in part, be due to changes in the caecal microbiota, assuming that the altered microbiota at day 35 is indicative of a change throughout the production cycle.

Effect of Doxycycline Use in the Early Broiler Production Cycle on the Microbiome.

16S rRNA amplicon sequencing was used to investigate changes in the broiler gastrointestinal tract (GIT) microbiota throughout the rearing period and in combination with antibiotic treatment. Thirty birds (from a commercial flock) were removed at multiple points throughout the rearing period on days 13, 27, and 33, euthanised, and their GIT aseptically removed and divided into upper (the crop, proventriculus, and the gizzard), middle (the duodenum, jejunum, and ileum) and lower (the large intestine, the caeca, and the cloaca) sections. In a separate commercial flock, on the same farm with similar husbandry practices and feed, doxycycline (100 mg/ml per kg body weight) was administered in drinking water between day 8 and 12 (inclusive) of the production cycle. Birds were removed on days, 13, 27, and 33 and GIT samples prepared as above. The contents of three merged samples from each GIT section were pooled (n = 60), the DNA extracted and analysed by 16S rRNA amplicon metagenomic sequencing and analysed. Major changes in the broiler microbiota were observed as the birds aged particularly with the Firmicutes/Bacteroidetes ratio (F:B) of the lower GIT. Moreover, Chao1, ACE, and Shannon indices showed the antibiotic treatment significantly altered the microbiota, and this change persisted throughout the rearing period. Further research is required to investigate the effect of these changes on bird performance, susceptibility to infections and Campylobacter carriage.