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The expansion and intensification of livestock production is predicted to promote the emergence of pathogens. As pathogens sometimes jump between species this can affect the health of humans as well as livestock. Here we investigate how livestock microbiota can act as a source of these emerging pathogens through analysis ofStreptococcus suis, a ubiquitous component of the respiratory microbiota of pigs that is also a major cause of disease on pig farms and an important zoonotic pathogen. Combining molecular dating, phylogeography and comparative genomic analyses of a large collection of isolates, we find that several pathogenic lineages ofS. suisemerged in the 19th and 20th centuries, during an early period of growth in pig farming. These lineages have since spread between countries and continents, mirroring trade in live pigs. They are distinguished by the presence of three genomic islands with putative roles in metabolism and cell adhesion, and an ongoing reduction in genome size, which may reflect their recent shift to a more pathogenic ecology. Reconstructions of the evolutionary histories of these islands reveal constraints on pathogen emergence that could inform control strategies, with pathogenic lineages consistently emerging from one subpopulation ofS. suisand acquiring genes through horizontal transfer from other pathogenic lineages. These results shed light on the capacity of the microbiota to rapidly evolve to exploit changes in their host population and suggest that the impact of changes in farming on the pathogenicity and zoonotic potential ofS. suisis yet to be fully realised.

IntroductionStreptoccocus 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.ResultsWe 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.ConclusionsThe 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.

Viral respiratory infections are often associated with bacterial co-infections that often lead to increased severity and mortality of the disease. During the recent pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), hospitalized patients reported developing secondary bacterial infections ranging from 0 to 40% of the cases. In the previous influenza pandemics, Streptococcus pneumoniae was the most isolated bacterial pathogen causing increased mortality in patients affected by viral pneumonia. Due to the difficulty to detect pneumococcal infection in SARS-CoV-2 patients by a rapid clinical test, the real prevalence of S. pneumoniae might be underestimated, and only a few cases have been documented so far. It has been estimated that 90% of patients admitted to the Intensive Care Unit are empirically treated with antimicrobial. The application of more rapid and sensitive diagnostic methods could help with targeted antibiotic therapy. Additionally, pneumococcal vaccination of high-risk individuals could reduce bacterial pneumonia, hospital admissions, and comorbidities associated with serious illness.

Around weaning, piglets are susceptible to infection by bacterial pathobionts, leading to increased morbidity and mortality. We identified isolates of Rothia nasisuis in the upper respiratory tract of weaned healthy piglets that produce valinomycin in vitro and in vivo via its vlm-encoded non-ribosomal peptide synthase (NRPS) enzyme complex. Valinomycin is an antiviral and antibiotic ionophore that shuttles potassium ions across membranes and is capable of inflammasome activation and apoptosis in LPS-primed macrophages at concentrations of 1 uM. Polarized monolayers of epithelial cells were much less sensitive to valinomycin but concentrations ≥ 10 µM decreased trans-epithelial resistance. R. nasisuis inhibited growth of closely related species of Rothia. Deliberate inoculation of valinomycin-producing R. nasisuis into newborn piglets suggested this species can shape the microbiota post weaning. Our findings support the idea that valinomycin is a competitive niche factor potentially also compromising epithelial integrity to gain access to (micro)nutrients.

The acquisition of novel genetic traits by natural competence is a strategy used by bacteria in microbe-rich environments including animal or human hosts where microbial competition, antibiotics and host immune defences threaten their survival. We show here that several virulent strains of Streptococcus suis, an important porcine pathogen and zoonotic agent, become naturally competent for genetic transformation with plasmid or genomic DNA when cultured in active porcine and human serum, but not when it is pre-heated for 30 minutes at 56°C to inactivate complement. Competence is also not induced in active fetal bovine serum, which contains less complement factors and immunoglobulins than adult serum. Late competence genes, encoding the uptake machinery for environmental DNA, were highly upregulated in active serum. Competence development was independent of the early competence regulatory switch suggesting the presence of an alternative stress-induced pathway for regulation of the transformasome, a type 4-like pilus DNA binding and transport apparatus.

Streptococcus suis (SS) is a zoonotic pathogen that can cause systemic infection in pigs and humans. The ingestion of contaminated pig meat is a well-established risk factor for zoonotic S. suis disease. In our studies, we provide experimental evidence that S. suis is capable to translocate across the host gastro-intestinal tract (GIT) using in vivo and in vitro models. Hence, S. suis should be considered an emerging foodborne pathogen. In this addendum, we give an overview of the complex interactions between S. suis and host-intestinal mucosa which depends on the host origin, the serotype and genotype of S. suis, as well as the presence and expression of virulence factors involved in host-pathogen interaction. Finally, we propose a hypothetical model of S. suis interaction with the host-GIT taking in account differences in conditions between the porcine and human host.

Streptococcus suis is an emerging zoonotic pathogen, which causes meningitis and septicaemia in humans and pigs [1, 2]. Ingestion of S. suis serotype 2 (SS2) contaminated food was identified as a risk factor for meningitis caused by SS2 in adult patients in Vietnam and Thailand [2], indicating that the gastrointestinal tract (GIT) might serve as an entry site of SS2 infection in humans [3, 4]. The upper respiratory tract (URT) is considered the main entry site of S. suis infection in pigs [5]. However, recent studies showed that the porcine GIT is rapidly colonized by S. suis after weaning [6] and that bacterial entry through the GIT occurs in piglets after mild stress induction [7], indicating that also the porcine GIT is an entry site of S. suis infection. The capsular polysaccharide (CPS), which determines the serotype, appears to play an essential role during the interaction with the URT epithelium [8]. Suilysin (Sly), a hemolytic pore-forming toxin, may also play a role facilitating translocation across the mucosal epithelium [9]. SS2 is responsible for most reported infections in humans and pigs worldwide [1, 10]. However, S. suis serotype 9 (SS9) is the most common serotype isolated from diseased pigs in Northern Europe [11] and is also prevalent in China [12] but has never been isolated from human patients [1, 10]. It is not known why isolates with serotypes that are highly prevalent among diseased pigs, such as SS9, have never been reported from human cases of SS infections. In addition to serotype, the genotype contributes to virulence of S. suis. Invasive S. suis strains are limited to certain sequence types (ST) and multi locus sequence typing (MLST) clonal complexes (CC) [13]. The majority of the SS2 strains isolated from human patients belong to CC1. However, we recently identified SS2 isolates from human patients in The Netherlands belonging to CC20, which is unrelated to CC1, indicating that multiple zoonotic clones are circulating worldwide [11]. In contrast, Dutch SS9 strains isolated from diseased pigs mainly belong to CC16, which is unrelated to both CC1 and CC20 [11, 14]. We aimed to provide experimental proof of concept that S. suis is capable of translocation in the GIT, in the absence of any invasive procedures. We then studied strain- and host-specific factors that may modulate S. suis infectivity at the intestinal mucosal surface, potentially providing clues to the zoonotic potential of SS2.

Histatins are multifunctional histidine-rich peptides secreted by the salivary glands and exclusively present in the saliva of higher primates, where they play a fundamental role in the protection of the oral cavity. Our previously published results demonstrated that histatin-1 (Hst1) promotes cell-substrate adhesion in various cell types and hinted that it could also be involved in cell-cell adhesion, a process of fundamental importance to epithelial and endothelial barriers. Here we explore the effects of Hst1 on cellular barrier function. We show that Hst1 improved endothelial barrier integrity, decreased its permeability for large molecules, and prevented translocation of bacteria across epithelial cell layers. These effects are mediated by the adherens junction protein E-cadherin (E-cad) and by the tight junction protein zonula occludens 1, as Hst1 increases the levels of zonula occludens 1 and of active E-cad. Hst1 may also promote epithelial differentiation as Hst1 induced transcription of the epithelial cell differentiation marker apolipoprotein A-IV (a downstream E-cad target). In addition, Hst1 counteracted the effects of epithelial-mesenchymal transition inducers on the outgrowth of oral cancer cell spheroids, suggesting that Hst1 affects processes that are implicated in cancer progression.-Van Dijk, I. A., Ferrando, M. L., van der Wijk, A.-E., Hoebe, R. A., Nazmi, K., de Jonge, W. J., Krawczyk, P. M., Bolscher, J. G. M., Veerman, E. C. I., Stap, J. Human salivary peptide histatin-1 stimulates epithelial and endothelial cell adhesion and barrier function.

Background Streptococcus suis is a zoonotic pathogen, causing meningitis and septicemia. We previously demonstrated that the gastrointestinal tract (GIT) is an entry site for zoonotic S. suis infection. Here we studied the contribution of (S) under bar treptococcal (a) under bar(d) under bar hesin (P) under bar rotein (SadP) to host-pathogen interaction at GIT level. Methods SadP expression in presence of Intestinal Epithelial Cells (IEC) was compared with expression of other virulence factors by measuring transcript levels using quantitative Real Time PCR (qRT-PCR). SadP variants were identified by phylogenetic analysis of complete DNA sequences. The interaction of SadP knockout and complementation mutants with IEC was tested in vitro. Results Expression of sadP was significantly increased in presence of IEC. Sequence analysis of 116 invasive strains revealed five SadP sequence variants, correlating with genotype. SadP1, present in zoonotic isolates of clonal complex 1, contributed to binding to both human and porcine IEC and translocation across human IEC. Antibodies against the globotriaosylceramide Gb3/CD77 receptor significantly inhibited adhesion to human IEC. Conclusion SadP is involved in the host-pathogen interaction in the GIT. Differences between SadP variants may determine different affinities to the Gb3/CD77 host-receptor, contributing to variation in adhesion capacity to host IEC and thus to S. suis zoonotic potential

We have identified apuA in Streptococcus suis, which encodes a bifunctional amylopullulanase with conserved α-amylase and pullulanase substrate-binding domains and catalytic motifs. ApuA exhibited properties typical of a Gram-positive surface protein, with a putative signal sequence and LPKTGE cell-wall-anchoring motif. A recombinant protein containing the predicted N-terminal α-amylase domain of ApuA was shown to have α-(1,4) glycosidic activity. Additionally, an apuA mutant of S. suis lacked the pullulanase α-(1,6) glycosidic activity detected in a cell-surface protein extract of wild-type S. suis. ApuA was required for normal growth in complex medium containing pullulan as the major carbon source, suggesting that this enzyme plays a role in nutrient acquisition in vivo via the degradation of glycogen and food-derived starch in the nasopharyngeal and oral cavities. ApuA was shown to promote adhesion to porcine epithelium and mucus in vitro, highlighting a link between carbohydrate utilization and the ability of S. suis to colonize and infect the host.

Bluetongue virus (BTV) is the causative agent of Bluetongue (BT) disease in ruminant livestock and occurs almost worldwide between latitudes 35 degrees S and 50 degrees N; 24 serotypes of BTV are known of which 8 circulate periodically within parts of the Mediterranean Region. A fast (about 3.5 h) and versatile diagnostic procedure able to detect and quantify BTV-RNA, has been developed using a Molecular Beacon (MB) fluorescent probe; PCR primers were designed to target 91 bp within the NS3 conserved region of the viral RNA segment 10 (S10) and bracketed the MB fluorescence probe hybridisation site. The MB fluorescent probe was used to develop two Bluetongue serogroup-specific assays: a quantitative real time reverse transcriptase polymerase chain reaction (RT-PCR) and a traditional RT-PCR. These were tested using BTV-RNAs extracted from the blood and organs of BT-affected animals, and from virus isolate suspensions. The samples included ten serotypes (BTV-1-BTV-9 and BTV-16); of these, BTV serotypes -1, -2, -4, -9 and -16 have since 1998 been involved in the extensive outbreaks of BT across the Mediterranean Region. To evaluate the specificity and sensitivity of the MB probe, all positive samples (and negative controls) were tested using the developed quantitative real time RT-PCR and traditional RT-PCR assays. The former test had a detection limit of 10(3) cDNA molecules per reaction with a log-linear quantification range of up to 10(11) (R2 = 0.98), while the latter test was able to detect 500 cDNA-BTV molecules/PCR. The results show that the MB fluorescent probe is both rapid and versatile for the laboratory diagnosis of Bluetongue and for quantifying levels of viraemia in BTV-affected animals. An "in silico" comparison of the primers and MB fluorescent probe used in this study showed that it is possible to detect all 24 serotypes of BTV.