Introduction Streptoccocus suis is a Gram-positive opportunistic pathogen causing systemic disease in piglets around weaning age. Outbreaks of S. suis disease are controlled by metaphylactic use of antibiotics, leading to high levels of antimicrobial resistance in S. suis isolates. This is an issue for both animal and human health due to the zoonotic disease potential of S. suis. The mechanisms facilitating invasive disease are not known but may involve host and environmental factors. The palatine tonsils are considered a portal of entry for pathogenic strains to cause systemic disease. We hypothesised that tonsil colonization by pathogenic and commensal bacteria may impact on disease risk via colonization resistance and co-infections. We conducted a case-control study on 9 European farms, comparing the tonsil microbiome of piglets with S. suis systemic disease with asymptomatic controls. We also compared these to piglets on control farms and piglets reared naturally in a forest. Results We found a small but significant difference in the tonsil microbiota composition of case and control piglets. Case-control associations varied between amplicon sequence variants (ASVs) and metagenome assembled genomes (MAGs) within the same species. Variants of putatively commensal taxa including Rothia nasimurium were reduced in abundance in case piglets compared to asymptomatic controls. Case piglets had higher relative abundance of Fusobacterium gastrosuis, Bacteroides heparinolyticus, and uncultured Prevotella and Alloprevotella species. There was, however, no higher abundance of S. suis itself at the species-level or of clinical strain marker genes in case piglets. Piglets sampled prospectively weeks prior to developing clinical signs had reduced microbiota alpha diversity. Despite case-control pairs receiving equal antimicrobial treatment, case piglets had higher abundance of antimicrobial resistance genes (ARGs) conferring resistance to antimicrobial classes used to treat S. suis. Conclusions The tonsillar microbiota of S. suis case piglets had increased abundance of taxa not previously linked to S. suis disease. This coincided with increased ARG abundance in case piglets, possibly due to adaptation of the disease-associated microbiota to frequent antimicrobial treatment.

Background The palatine tonsils are part of the mucosal immune system and stimulate immune responses through M cell uptake sampling of antigens and bacteria in the tonsillar crypts. Little is known about the development of the tonsillar microbiota and the factors determining the establishment and proliferation of disease-associated bacteria such as Streptococcus suis. In this study, we assessed tonsillar microbiota development in piglets during the first 5 weeks of life and identified the relative importance of maternal and environmental farm parameters influencing the tonsillar microbiota at different ages. Additionally, we studied the effect sow vaccination with a bacterin against S. suis on microbiota development and S. suis colonisation in their offspring. Results Amplicon sequencing of the 16S rRNA gene V3-V4 region revealed that a diverse tonsillar microbiota is established shortly after birth, which then gradually changes during the first 5 weeks of life without a large impact of weaning on composition or diversity. We found a strong litter effect, with siblings sharing a more similar microbiota compared to non-sibling piglets. Co-housing in rooms, within which litters were housed in separate pens, also had a large impact on microbiota composition. Sow parity and prepartum S. suis bacterin vaccination of sows had weaker but significant associations with microbiota composition, impacting on the abundance of Streptococcus species before and after weaning. Sex and birthweight had limited impact on the tonsillar microbiota, and none of the measured factors had consistent associations with microbiota diversity. Conclusions The piglet tonsillar microbiota is established shortly after birth. While microbiota development is associated with both environmental and maternal parameters, weaning has limited impact on microbiota composition. Intramuscular vaccination of sows pre-partum had a significant effect on the tonsillar microbiota composition of their piglets. These findings provide new insights into the mechanisms shaping the tonsillar microbiota.

Streptococcus suis colonizes the upper respiratory tract of healthy pigs at high abundance but can also cause opportunistic respiratory and systemic disease. Disease-associated S. suis reference strains are well studied, but less is known about commensal lineages. It is not known what mechanisms enable some S. suis lineages to cause disease while others persist as commensal colonizers, or to what extent gene expression in disease-associated and commensal lineages diverge. In this study we compared the transcriptomes of 21  S . suis strains grown in active porcine serum and Todd–Hewitt yeast broth. These strains included both commensal and pathogenic strains, including several strains of sequence type (ST) 1, which is responsible for most cases of human disease and is considered to be the most pathogenic S. suis lineage. We sampled the strains during their exponential growth phase and mapped RNA sequencing reads to the corresponding strain genomes. We found that the transcriptomes of pathogenic and commensal strains with large genomic divergence were unexpectedly conserved when grown in active porcine serum, but that regulation and expression of key pathways varied. Notably, we observed strong variation of expression across media of genes involved in capsule production in pathogens, and of the agmatine deiminase system in commensals. ST1 strains displayed large differences in gene expression between the two media compared to strains from other clades. Their capacity to regulate gene expression across different environmental conditions may be key to their success as zoonotic pathogens.

Streptococcus suis colonizes the upper respiratory tract of healthy pigs at high abundance but can also cause opportunistic respiratory and systemic disease. Disease-associated S. suis reference strains are well studied, but less is known about commensal lineages. It is not known what mechanisms enable some S. suis lineages to cause disease while others persist as commensal colonizers, or to what extent gene expression in disease-associated and commensal lineages diverge. In this study we compared the transcriptomes of 21 S. suis strains grown in active porcine serum and Todd-Hewitt yeast broth. These strains included both commensal and pathogenic strains, including several strains of sequence type (ST) 1, which is responsible for most cases of human disease and considered the most pathogenic S. suis lineage. We sampled the strains during their exponential growth phase and mapped RNA-sequencing reads to the corresponding strain genomes. We found that the transcriptomes of pathogenic and commensal strains with large genomic divergence were unexpectedly conserved when grown in active porcine serum, but that regulation and expression of key pathways varied. Notably, we observed strong variation of expression across media of genes involved in capsule production in pathogens, and of the agmatine deiminase system in commensals. ST1 strains displayed large differences in gene expression between the two media compared to strains from other clades. Their capacity to regulate gene expression across different environmental conditions may be key to their success as zoonotic pathogens.

Broadly pathogenic clades Broadly commensal clades General introduction | 17Chapter 1 targeting known pathogens. We can now gain information on both known and novel disease-associated microbes and assess how the occurrence and abundance of these correlate with disease symptoms, the environment, and the abundance of other species. Detection of health-associated bacteria is of great interest because they may have probiotic potential. Polymicrobial disease and co-infection can also be assessed, and disease-causing but non-invasive species can be discovered. A large effort has gone towards discovering the role of the microbiome in human diseases such as inflammatory bowel disease [94,95] and colorectal cancer [96,97] . Studies have also utilized metagenomics for infectious disease in joint fluid [98] , blood [99] , and respiratory samples [24] . Research on the respiratory microbiome is, however, lagging behind gut microbiome research despite the relevance to infectious disease [91] . Metagenomic studies on infectious disease in livestock are also lacking. Challenges and open questionsAlthough a large effort has been dedicated to understanding the ecology and evolution of S. suis, there are significant knowledge gaps. There is a lack of research on how commensal and pathogenic S. suis lineages colonize piglets and interact with biotic and abiotic factors to cause disease. The transcriptomic and phenotypic differences between commensal and pathogenic strains are not well described, and the exact (combination of ) mechanisms involved in S. suis virulence and transmission remain unknown. This hinders disease surveillance and the design of effective preventative measures and treatments. Recent amplicon-sequencing studies have shown high S. suis prevalence and abundance in the oral cavity of piglets, and conclusions from culture-based work need to be considered with caution because these likely found many false negatives of S. suis presence. It is not known whether S. suis colonizes the oral cavity of piglets at high abundances in all farms in different countries, or what proportion of the observed S. suis load consists of pathogenic and commensal strains.Knowledge is also lacking on the polymicrobial nature of S. suis disease. It is known that S. suis infection often co-occurs with infection by other bacteria and viruses, such as PRRSV, but little quantitative data exists on taxa co-occurring with S. suis disease. Associations between the commensal microbiota and S. suis are also of great interest. Activity of specific bacterial species may prevent S. suis invasive disease by interactions with the host immune system or by providing colonization resistance against pathogenic S. suis strains. Identifying taxa positively or negatively associated with S. suis disease may aid in development of prevention strategies, vaccines, and probiotics. These measures may contribute to reducing the global S. suis disease burden and antibiotic use in pig farming.