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Antimicrobials are added to semen extenders to inhibit the growth of bacteria that are transferred to the semen during collection. However, this non-therapeutic use of antimicrobials could contribute to the development of antimicrobial resistance. The objective of this study was to determine changes in the antibiotic susceptibility of vaginal microbiota after artificial insemination. Swabs were taken from the vagina of 26 mares immediately before artificial insemination and again 3 days later. Bacteria isolated from the vagina at both time points were subjected to antibiotic susceptibility testing and whole-genome sequencing. In total, 32 bacterial species were identified. There were increases in the resistance of Escherichia coli to trimethoprim (p = 0.0006), chloramphenicol and (p = 0.012) tetracycline (p = 0.03) between day 0 and day 3. However, there was no significant effect of exposure to antibiotics in semen extenders with respect to the resistance of Staphylococcus simulans and Streptococcus equisimilis (p > 0.05). Whole-genome sequencing indicated that most phenotypic resistance was associated with genes for resistance. These results indicate that the resistance patterns of vaginal bacteria may be affected by exposure to antibiotics; therefore, it would be prudent to minimize, or preferably, avoid using antibiotics in semen extenders.

One major source of faecal bacteria and antibiotics to surface waters are effluents from sewage treatment plants (STPs) which highlights the importance of efficient sewage treatment for reduction of microorganisms as well as chemicals. In the present study a broad and exploratory screening survey was conducted on water and, fish and caged mussels samples from the sewage treatment plant and the recipient river Fyrisån in Uppsala, Sweden, during the years 2017-2019. The aim was to study the impact of STP effluent discharge on the river water microbial quality measured through the presence of Escherichia coli (E. coli) and Enterococcus spp. as fecal indicator bacteria, antibiotic resistance by extended-spectrum beta-lactamase producing E. coli (ESBL-E. coli), potentially pathogenic bacteria Salmonella spp., as well as presence and levels of antibiotics. In the STP, large reductions of faecal bacteria from influent to effluent sewage water (>90%) was measured, but less reduction was observed for the antibiotics (66%). Measurement of ESBL-E. coli in the STP sewage water revealed increased ratio of ESBL-E. coli isolates in the STP effluent compared with influent samples indicating potential selection for antibiotic resistance during the sewage process. Recipient river water samples revealed increased faecal bacteria and resistant ESBL-E. coli concentrations several kilometers downstream the STP outlet. Salmonella spp were isolated from all samples of STP influent and effluent water and at some sites in the river. Screening of wild fish and caged mussels in the river show that animals in the recipient environment to STPs can be carriers of faecal bacteria and ESBL-E. coli. This highlights the potential risk for transmissions of pathogens and antibiotic resistance from both the water and aquatic animals inhabiting microbial polluted environments. Large variations in concentrations of bacteria as well as antibiotics were observed between the years, which were probably due to annual differences in river discharge flow, indicating that prevailing weather conditions influence the levels of bacteria and antibiotics in the river. Generally, the resistance pattern to the 14 different tested antibiotics were similar in ESBL-E. coli isolated from the STP influent and effluent, river and biota samples. Apart from ampicillin and cefotaxime which were used for selection of ESBL-E. coli, high resistances were detected also for ceftazidime and ciprofloxacin. Large proportions of the isolated ESBL-E. coli were also multidrug-resistant, i.e. resistant to at least three different antibiotic classes. The present study show that the Uppsala STP serves as a major source for faecal bacteria, ESBL-E. coli and antibiotics to surface water of the river Fyrisån, which can remain at considerable levels for several kilometers downstream the outlet. Utsläppsvatten från avloppsreningsverk är en betydande källa för avföringsbakterier och antibiotika till recipienternas ytvatten. Kontaminering av vattendrag med fekala mikroorganismer är ett stort globalt problem då mikroorganismerna kan orsaka sjukdomar och sprida antibiotikaresistens. Antibiotikaresistens är ett av de största hoten mot folkhälsan och modern sjukvård. Ett av de allvarligaste resistensproblemen är så kallade ESBL (Extended Spectrum Beta-Lactamase)-bildande tarmbakterier, vilka påvisas allt oftare inom sjukvården och i samhället. ESBL är ett enzym som kan bildas av olika bakterier, bland annat av Escherichia coli (E. coli) som är en vanligt förekommande tarmbakterie. I Sverige är ESBL-E. coli den vanligaste rapporterade resistenstypen. Enzymet ESBL bryter ner de flesta antibiotika i betalaktamgruppen (penicilliner- och cefalosporiner) vilket gör att dessa antibiotika inte är effektiva vid behandling av infektioner orsakade av ESBL-producerande bakterier. I denna studie genomfördes en screeningundersökning av förekomsten av fekala bakterier och antibiotika i inkommande och utgående vatten från Uppsala avloppsreningsverk, samt i ytvatten, vild fisk och burutsatta musslor från recipienten Fyrisån under åren 2017-2019. Syftet var att studera påverkan av utsläpp av avloppsvatten på den mikrobiella kvalitet i recipienten, vilket mättes genom förekomst av de fekala indikatorbakterierna E. coli och Enterococcus spp., antibiotikaresistenta ESBL-E. coli, potentiellt sjukdomsframkallande bakterier av Salmonella spp., samt även haltbestämning av antibiotika. I reningsverket uppmättes relativt stora reduktioner av de fekala indikatorbakterierna från inkommande till utgående vatten (> 90%), men mindre reduktion observerades för antibiotika (66%). Förekomsten av ESBL-E. coli ökade från inkommande till utgående vatten i reningsverket, vilket kan indikera att selektionstryck för uppkomst av antibiotikaresistens förekommer under avloppsreningsprocessen. I Fyrisån uppmättes förhöjda halter av både indikatorbakterier, resistenta ESBL-E. coli, samt antibiotikahalter flera kilometer nedströms utloppet från reningsverket, jämfört med provlokaler uppströms. Salmonella spp kunde isoleras från alla prover av inkommande och utgående vatten i reningsverket, samt från vissa provlokaler i Fyrisån. Undersökning av vild fisk och burutsatta musslor i Fyrisån visar att akvatiska djur i mottagarmiljön till utsläpp från reningsverk kan vara bärare av fekala bakterier och resistenta ESBL-E. coli. Utvärderingen av resistens mot 14 olika antibiotika hos ESBL-E. coli som isolerats visade att resistensmönstret var liknande i både prover från reningsverket och prover från Fyrisån. Förutom ampicillin och cefotaxim som användes för selektion av ESBL-E. coli, påvisades resistens även för ceftazidim och ciprofloxacin. Många av de isolerade ESBL-E. coli var också multiresistenta (resistenta mot minst 3 olika antibiotikaklasser). Denna studie visar att Uppsala reningsverk fungerar som en källa för avföringsbakterier, resistenta ESBL-E. coli, Salmonella spp. och antibiotika till ytvattnet i Fyrisån, som kan förekomma i betydande nivåer flera kilometer nedströms utloppet. Utsläppen belyser vikten av effektiv avloppsrening för att minska utsläpp av mikroorganismer och kemikalier till omkringliggande miljö. Resultaten indikerar också den potentiella risken för överföring av sjukdomsframkallande- och antibiotikaresistenta bakterier från både ytvattnet och vattenlevande djur som bor i mikrobiellt förorenade miljöer.

Ovine footrot is a contagious bacterial disease primarily caused by Dichelobacter nodosus. Footrot affects the feet of sheep and is characterised by two major clinical presentations. The milder form consists of inflammation confined to the interdigital space (interdigital dermatitis or benign footrot) and the more severe form includes underrunning of the hoof horn (underrunning or virulent footrot). Disease severity is dependent on several factors including the virulence of the D. nodosus strain, environmental conditions, farming practices, host susceptibility, and co-infecting bacteria. In Sweden, ovine footrot was first diagnosed in 2004, but fast and sensitive diagnostics for D. nodosus were not available. Likewise, knowledge was missing about the D. nodosus strains and co-infection with other lameness-associated bacteria. Hence the overall aim of this thesis project was to increase the knowledge about ovine footrot in Swedish sheep and to improve laboratory diagnostics for it. In this thesis project, sensitive and specific real-time PCR methods to detect and discriminate between virulent and benign strains of D. nodosus were developed and used to characterise D. nodosus from Swedish sheep. The results showed that most of the Swedish D. nodosus are benign and that the virulent type is uncommon. D. nodosus isolates from seven other countries included in the study showed that the D. nodosus genome is highly conserved and that it exists as a globally distributed bimodal population. Furthermore, D. nodosus is mainly associated with the early stages of footrot whereas Fusobacterium necrophorum is associated with the later ones. This confirms the suggested role of F. necrophorum as an opportunistic pathogen rather than the primary pathogen. Although previously proposed, there was no evidence of Treponema spp. in disease development. Finally, a sample pooling method was developed to meet the demands for cost-efficiency in control programs. The method allows samples to be analysed in groups of five with no loss of sensitivity compared to individual samples. It has been implemented in the Swedish Footrot Control Program as a result of this thesis project.

Funder: Department of Large Animal Clinical Sciences, Funder: USDA‐NIFA AgriLife and the Link Equine Research Endowment, Funder: Texas A&M University; Id: http://dx.doi.org/10.13039/100007904, Funder: Formas, Funder: Intervacc AB, Funder: The Horse Trust, Funder: SEIB, Funder: Paul Mellon Foundation, Funder: Alice Noakes Memorial Charitable Trust, Funder: Ivo Trust, Funder: Tattersalls, Funder: The Elise Pilkington Charitable Trust, Funder: The European Breeders Fund, Funder: The Serth and Gates Charity, Funder: Margaret Giffen Charitable Trust, Funder: The Payne Gallwey Charitable Trust, Funder: The Stafford Trust, Funder: Marjorie Coote Animal Charity Trust, Funder: Beryl Evetts and Robert Luff Animal Welfare Trust, Funder: The Anne Duchess of Westminster’s Charitable Trust, BACKGROUND: Streptococcus equi subspecies equi (S equi) is the cause of Strangles, one of the most prevalent diseases of horses worldwide. Variation within the immunodominant SeM protein has been documented, but a new eight-component fusion protein vaccine, Strangvac, does not contain live S equi or SeM and conservation of the antigens it contains have not been reported. OBJECTIVE: To define the diversity of the eight Strangvac antigens across a diverse S equi population. STUDY DESIGN: Genomic description. METHODS: Antigen sequences from the genomes of 759 S equi isolates from 19 countries, recovered between 1955 and 2018, were analysed. Predicted amino acid sequences in the antigen fragments of SEQ0256(Eq5), SEQ0402(Eq8), SEQ0721(EAG), SEQ0855(SclF), SEQ0935(CNE), SEQ0999(IdeE), SEQ1817(SclI) and SEQ2101(SclC) in Strangvac and SeM were extracted from the 759 assembled genomes and compared. RESULTS: The predicted amino acid sequences of SclC, SclI and IdeE were identical across all 759 genomes. CNE was truncated in the genome of five (0.7%) isolates. SclF was absent from one genome and another encoded a single amino acid substitution. EAG was truncated in two genomes. Eq5 was truncated in four genomes and 123 genomes encoded a single amino acid substitution. Eq8 was truncated in three genomes, one genome encoded four amino acid substitutions and 398 genomes encoded a single amino acid substitution at the final amino acid of the Eq8 antigen fragment. Therefore, at least 1579 (99.9%) of 1580 amino acids in Strangvac were identical in 743 (97.9%) genomes, and all genomes encoded identical amino acid sequences for at least six of the eight Strangvac antigens. MAIN LIMITATIONS: Three hundred and seven (40.4%) isolates in this study were recovered from horses in the UK. CONCLUSIONS: The predicted amino acid sequences of antigens in Strangvac were highly conserved across this collection of S equi.