Characterization of pig tonsils as niches for the generation of Streptococcus suis diversity.

Streptococcus suis is a gram-positive bacterium that causes meningitis, septicemia, endocarditis, and other disorders in pigs and humans. We obtained 42 and 50 S. suis isolates from lesions of porcine endocarditis and palatine tonsils, respectively, of clinically healthy pigs in Japan; we then determined their sequence types (STs) by multilocus sequence typing (MLST), cps genotypes, serotypes, and presence of classical major virulence-associated marker genes (mrp, epf, and sly). The 42 isolates from endocarditis lesions were assigned to a limited number of STs and clonal complexes (CCs). On the other hand, the 50 isolates from tonsils were diverse in these traits and seemingly in the degree of virulence, suggesting that tonsils can accommodate a variety of S. suis isolates. The goeBURST full algorithm using tonsil isolates obtained in this study and those retrieved from the database showed that major CCs as well as many other clusters were composed of isolates originating from different countries, and some of the STs were very similar to each other despite the difference in country of origin. These findings indicate that S. suis with not only different but also similar mutations in the genome have survived in tonsils independently across different geographical locations. Therefore, unlike the lesions of endocarditis, the tonsils of pigs seemingly accommodate various S. suis lineages. The present study suggests that S. suis acquired its diversity by natural mutations during colonization and persistence in the tonsils of pigs.

Transition of microbiota in chicken cecal droppings from commercial broiler farms.

BACKGROUND: Chickens are major sources of human nutrition worldwide, but the chicken intestinal microbiota can be a source of bacterial infection. The microbiota has potential to regulate the colonization of pathogens by competitive exclusion, production of antimicrobial compounds, and stimulation of the mucosal immune system. But information on the microbiota in commercial broiler chickens is limited because of the difficulty of conducting studies at commercial farms. To obtain fundamental information that can be used to control pathogens in chickens, we determined the 6-week dynamics of microbiota in chicken cecal droppings from commercial broiler farms. RESULTS: Cecal droppings from four chickens were collected once a week from 1 to 6 weeks of age at three commercial broiler farms. A total of 168 samples were collected from 7 flocks and subjected to 16S rRNA amplicon sequencing. Despite the farms have distinctly different climate conditions, the microbiota in the same growth stages were similar among farms. Moreover, as the chickens grew and the feed types were switched, the richness and diversity of the microbiota gradually increased and convergence of the composition of the microbiota was apparent. Notably, minor bacterial taxa (i.e. OTUs with relative abundance < 0.05%) within the microbiota were changed by the chicken age, switching of feed types, and presence of Campylobacter. In particular, the effects of switching of feed types on the microbiota were larger than the effects of age and Campylobacter. CONCLUSIONS: Irrespective of the locations of the farms, the microbiota of chicken cecum, especially minor bacteria, was successively changed more affected by feed types than by ages. Switching of feed types inducing the alteration of the microbiota may be associated with the colonization of pathogens in the chicken gut. These results will also help with extrapolation of studies in experimental animals to those in the commercial farms.

Defining the taxonomic status of Streptococcus suis serotype 33: the proposal for Streptococcus ruminantium sp. nov.

To clarify the taxonomic classification of Streptococcus suis serotype 33, we performed biochemical and molecular genetic analyses using isolates (GUT-183, GUT-184, GUT-185, GUT-186, GUT-187T, GUT-188, GUT-189, GUT-190, GUT-191, GUT-192 and GUT-193) from bovine endocarditis. A comparative sequence analysis showed 99.2-100 % sequence similarity among the reference strain of S. suis serotype 33 and our isolates for the 16S rRNA gene. These similarities were higher than those between the isolate GUT-187T and S. suis and other streptococci. Comparison of sodA genes also showed high degrees of similarities among the reference strain of S. suis serotype 33 and our isolates (99.7-100 %), which were higher than those between the GUT-187T and S. suis and other streptococci. DNA-DNA relatedness among three isolates (GUT-186, GUT-187T, the reference strain of S. suis serotype 33) was over 76.7 %. In contrast, the relatedness between GUT-187T and the other streptococcal species (S. suis, Streptococcus parasuis, Streptococcus acidominimus and Streptococcus porci) was 8.4-24.9 %. Phylogenetic analyses showed that the isolates did not affiliate closely to any known species of the genus Streptococcus. Moreover, GUT-187T could be distinguished from S. suis and other closely related species of genus Streptococcus using biochemical tests. On the basis of the phenotypic and molecular genetic data, we propose that the isolates of S. suis serotype 33 should be classified into the genus Streptococcus, Streptococcus ruminantium sp. nov. with the type strain GUT-187T (=DSM 104980T=JCM 31869T).

Streptococcus suis Serotype 2 Capsule In Vivo.

Many Streptococcus suis isolates from porcine endocarditis in slaughterhouses have lost their capsule and are considered avirulent. However, we retrieved capsule- and virulence-recovered S. suis after in vivo passages of a nonencapsulated strain in mice, suggesting that nonencapsulated S. suis are still potentially hazardous for persons in the swine industry.

Estimating the burden of foodborne diseases in Japan.

OBJECTIVE: To assess the burden posed by foodborne diseases in Japan using methods developed by the World Health Organization's Foodborne Disease Burden Epidemiology Reference Group (FERG). METHODS: Expert consultation and statistics on food poisoning during 2011 were used to identify three common causes of foodborne disease in Japan: Campylobacter and Salmonella species and enterohaemorrhagic Escherichia coli (EHEC). We conducted systematic reviews of English and Japanese literature on the complications caused by these pathogens, by searching Embase, the Japan medical society abstract database and Medline. We estimated the annual incidence of acute gastroenteritis from reported surveillance data, based on estimated probabilities that an affected person would visit a physician and have gastroenteritis confirmed. We then calculated disability-adjusted life-years (DALYs) lost in 2011, using the incidence estimates along with disability weights derived from published studies. FINDINGS: In 2011, foodborne disease caused by Campylobacter species, Salmonella species and EHEC led to an estimated loss of 6099, 3145 and 463 DALYs in Japan, respectively. These estimated burdens are based on the pyramid reconstruction method; are largely due to morbidity rather than mortality; and are much higher than those indicated by routine surveillance data. CONCLUSION: Routine surveillance data may indicate foodborne disease burdens that are much lower than the true values. Most of the burden posed by foodborne disease in Japan comes from secondary complications. The tools developed by FERG appear useful in estimating disease burdens and setting priorities in the field of food safety.

Development of a two-step multiplex PCR assay for typing of capsular polysaccharide synthesis gene clusters of Streptococcus suis.

We developed a practical and easy two-step multiplex PCR assay to aid in serotyping of Streptococcus suis. The assay accurately typed almost all of the serotype reference strains and field isolates of various serotypes and also identified the genotypes of capsular polysaccharide synthesis gene clusters of some serologically nontypeable strains.

Genetic analysis of capsular polysaccharide synthesis gene clusters from all serotypes of Streptococcus suis: potential mechanisms for generation of capsular variation.

Streptococcus suis strains are classified into 35 serotypes on the basis of the antigenicity of their capsular polysaccharides (CPs). CP synthesis genes are known to be clustered on the chromosome (cps gene cluster). The entire cps gene clusters of S. suis have so far been sequenced in 15 serotypes and found to be located between orfZ and aroA. In this study, to provide comprehensive information about S. suis CPs, we sequenced the entire cps gene clusters of the remaining serotypes and analyzed the complete set of S. suis cps gene clusters. Among the 35 cps gene clusters, 22 were located between orfZ and aroA, whereas the other 13 were flanked by other gene(s) on the chromosomes, and the chromosomal locus was classified into five patterns. By clustering analysis, the predicted products of cps genes found in the 35 serotypes were assigned into 291 homology groups, and all serotypes possessed a serotype-specific gene, except for serotypes 1, 2, 1/2, and 14. Because of the presence of genes encoding flippase (wzx) and polymerase (wzy), CPs of all serotypes were thought to be synthesized by the Wzx/Wzy pathway. Our data also implied the possibility of the transfer of the entire or partial cps gene clusters among S. suis strains, as well as the influence of spontaneous mutations in a single gene or a few genes on the antigenicity of some serotypes. Accumulation of these gene transfers and small-scale mutations may have generated the antigenic diversity of S. suis CPs.

Short review: Geographical distribution of equine-associated pVAPA plasmids in Rhodococcus equi in the world.

Virulent Rhodococcus equi strains expressing virulence-associated 15-17 kDa protein (VapA) and having a large virulence plasmid (pVAPA) of 85-90 kb containing vapA gene are pathogenic for horses. In the last two decades, following pVAPA, two host-associated virulence plasmid types of R. equi have been discovered: a circular plasmid, pVAPB, associated with porcine isolates in 1995, and a recently detected linear plasmid, pVAPN, related to bovine and caprine isolates. Molecular epidemiological studies of R. equi infection in foals on horse-breeding farms in Japan and many countries around the world have been conducted in the last three decades, and the epidemiological studies using restriction enzyme digestion patterns of plasmid DNAs from virulent isolates have shown 14 distinct pVAPA subtypes and their geographical preference. This short review summarizes previous reports regarding equine-associated pVAPA subtypes in the world and discusses their geographic distribution from the standpoint of horse movements.

Complete Genome Sequences of Four Streptococcus parasuis Strains Obtained from Saliva of Domestic Pigs in Japan.

Streptococcus parasuis is a close relative of Streptococcus suis, an important zoonotic pathogen that causes various diseases in pigs and humans. Here, we report the complete genome sequences of four strains, including the type strain of S. parasuis, isolated from the saliva of healthy pigs in Japan.

Complete Genome Sequences of Three Streptococcus ruminantium Strains Obtained from Endocarditis Lesions of Cattle in Japan.

Streptococcus ruminantium is a close relative of Streptococcus suis, an important zoonotic pathogen that causes various diseases in pigs and humans. Here, we report the complete genome sequences of three S. ruminantium strains isolated from bovine endocarditis in Japan.

The minor pilin subunit Sgp2 is necessary for assembly of the pilus encoded by the srtG cluster of Streptococcus suis.

Gram-positive pili are composed of covalently bound pilin subunits whose assembly is mediated via a pilus-specific sortase(s). Major subunits constitute the pilus backbone and are therefore essential for pilus formation. Minor subunits are also incorporated into the pilus, but they are considered to be dispensable for backbone formation. The srtG cluster is one of the putative pilus gene clusters identified in the major swine pathogen Streptococcus suis. It consists of one sortase gene (srtG) and two putative pilin subunit genes (sgp1 and sgp2). In this study, by constructing mutants for each of the genes in the cluster and by both immunoblotting and immunogold electron microscopic analysis with antibodies against Sgp1 and Sgp2, we found that the srtG cluster mediates the expression of pilus-like structures in S. suis strain 89/1591. In this pilus, Sgp1 forms the backbone, whereas Sgp2 is incorporated as the minor subunit. In accordance with the current model of pilus assembly by Gram-positive organisms, the major subunit Sgp1 was indispensable for backbone formation and the cognate sortase SrtG mediated the polymerization of both subunits. However, unlike other well-characterized Gram-positive bacterial pili, the minor subunit Sgp2 was required for polymerization of the major subunit Sgp1. Because Sgp2 homologues are encoded in several other Gram-positive bacterial pilus gene clusters, in some types of pili, minor pilin subunits may contribute to backbone formation by a novel mechanism.

Lineage and virulence of Streptococcus suis serotype 2 isolates from North America.

We performed multilocus sequence typing of 64 North American Streptococcus suis serotype 2 porcine isolates. Strains were sequence type (ST) 28 (51%), ST25 (44%), and ST1 (5%). We identified nonrandom associations between STs and expression of the virulence markers suilysin (SLY), muramidase-relased protein (MRP), and extracellular factor (EF). Expression of pili encoded by the srtF and srtG pilus clusters was also nonrandomly associated with STs. ST1 strains were SLY+ EF+ MRP+ srtF pilus+ srtG pilus-. ST25 strains were SLY- EF- MRP- srtF pilus- srtG pilus+, and most ST28 strains were SLY- MRP+ EF- srtF pilus+ srtG pilus+. ST28 isolates proved essentially nonvirulent in a mouse infection model; ST25 strains showed moderate virulence and ST1 isolates were highly virulent. ST1 is responsible for a high proportion of S. suis disease in humans worldwide. Its presence in North America indicates that potential zoonotic S. suis outbreaks in this continent cannot be disregarded.

Basis of the persistence of capsule-negative Streptococcus suis in porcine endocarditis inferred from comparative genomics.

The capsule (cap) of Streptococcus suis is an anti-phagocytic element and is one of the major virulence factors. However, we have found cap-positive and cap-negative isolates in porcine endocarditis. Here, we compared genome sequences of multiple cap-negative isolates with those of a cap-positive isolate from a single endocarditis. Cap-positive and cap-negative isolates from the same pig were phylogenetically closest compared with those from other pigs. Some of cap-negative isolates from the same pig showed different mutations in capsular polysaccharide synthesis (cps) genes, suggesting that these isolates arisen in pigs after infection. Different mutations in whole-genomes were also found among isolates with identical mutations in cps genes, indicating that mutations in cps genes and the whole-genome occurred independently. Since cap-negative isolates are rarely found in lesions of other diseases, these results suggest that endocarditis lesions may simply favored cap-negative mutants to survive the niches, leading to their persistence in the lesions.

16S rRNA Gene Amplicon Sequence Data from Feces of Wild Deer (Cervus nippon) in Japan.

We report 16S rRNA amplicon sequence data from feces of 109 wild deer in Japan. The dominant bacterial taxa in fecal microbiota of wild deer hunted between village and mountainous areas and those living on Miyajima Island and in Nara Park were similar but differed in abundance.

16S rRNA Gene Amplicon Sequence Data from Feces of Five Species of Wild Animals in Japan.

We report 16S rRNA amplicon sequence data from feces from 58 wild boars, 60 feral raccoons, 9 wild Japanese badgers, 21 wild masked palm civets, and 8 wild raccoon dogs in Japan. The predominant bacterial taxa in the fecal microbiota were similar in part but varied among the animal species.

Complete Genome Sequences of Two Streptococcus suis Strains Isolated from Asymptomatic Pigs.

Streptococcus suis is an important zoonotic pathogen that causes major economic problems in the pig industry worldwide and serious infections in humans, including meningitis and septicemia. Here, we report the complete genome sequences of two strains isolated from asymptomatic pigs.

Significant contribution of the pgdA gene to the virulence of Streptococcus suis.

Streptococcus suis is a major swine pathogen and emerging zoonotic agent. In this study we have determined the muropeptide composition of S. suis peptidoglycan (PG) and found, among other modifications, N-deacetylated compounds. Comparison with an isogenic mutant showed that the product of the pgdA gene is responsible for this specific modification which occurred in very low amounts. Low level of PG N-deacetylation correlated with absence of significant lysozyme resistance when wild-type S. suis was grown in vitro. On the other hand, expression of the pgdA gene was increased upon interaction of the bacterium with neutrophils in vitro as well as in vivo in experimentally inoculated mice, suggesting that S. suis may enhance PG N-deacetylation under these conditions. Evaluation of the DeltapgdA mutant in both the CD1 murine and the porcine models of infection revealed a significant contribution of the pgdA gene to the virulence traits of S. suis. Reflecting a severe impairment in its ability to persist in blood and decreased ability to escape immune clearance mechanisms mediated by neutrophils, the DeltapgdA mutant was highly attenuated in both models. The results of this study suggest that modification of PG by N-deacetylation is an important factor in S. suis virulence.

High rate misidentification of biochemically determined Streptococcus isolates from swine clinical specimens.

In this study, 22 bacterial isolates from swine necropsy specimens, which were biochemically identified as Streptococcus suis and other Streptococcus species, were re-examined using species-specific PCR for authentic S. suis and 16S rRNA gene sequencing for the verification of the former judge. Identification of S. suis on the basis of biochemical characteristics showed high false-positive (70.6%) and false-negative (60%) rates. The authentic S. suis showed various capsular polysaccharide synthesis gene types, including type 2 that often isolated from human cases. Five of 22 isolates did not even belong to the genus Streptococcus. These results suggested that the misidentification of the causative pathogen in routine veterinary diagnosis could be a substantial obstacle for the control of emerging infectious diseases.

Functional dysbiosis within dental plaque microbiota in cleft lip and palate patients.

BACKGROUND: Dental caries is a polymicrobial disease and prevalent among cleft lip and palate (CLP) patients, although their oral hygiene is well maintained. Dysbiosis, the state of imbalance within the dental plaque microbiota, may cause caries prevalence among these patients. However, little is known about how dysbiosis occurs and affects cariogenicity. To find dysbiotic signs, here we conducted a metatranscriptomic analysis for the plaque microbiota in six CLP patients and four controls. METHODS: Total bacterial RNA was extracted from each sample and sequenced. Bacterial composition and functional profiles were estimated from 16S rRNA and mRNA reads, respectively. The mRNA reads were further used for estimating bacterial composition. Species listed in both rRNA-based and mRNA-based bacterial composition were identified as viable taxa with in situ function (VTiF), and the VTiF with a high mRNA-to-rRNA ratio were considered to be transcriptionally active. A network was constructed for each group by connecting two VTiF if their mRNA abundances were positively correlated. RESULTS: The bacterial composition and functional profiles themselves did not provide remarkable signs of dysbiosis in the CLP group. However, the group-specific active taxa were identified, including streptococcal and Prevotella species in the CLP group. Moreover, the network structure was different between groups; Actinomyces johnsonii and several species in the CLP group were the active taxa, which were connected based on positive correlations with statistical significance. CONCLUSIONS: Functional dysbiosis within the plaque microbiota was observed such as difference of the network structure between groups, and may be associated with cariogenicity. The observed functional dysbiosis was an invisible change within the microbiota in the oral cavity of CLP patients. This may emphasize the importance of maintaining good oral hygiene of the patients with cleft anomalies.

Characterization of pig saliva as the major natural habitat of Streptococcus suis by analyzing oral, fecal, vaginal, and environmental microbiota.

It is generally difficult to specify the sources of infection by which domestic animals may acquire pathogens. Through 16S rRNA gene amplicon sequencing, we compared the composition of microbiota in the saliva, vaginal mucus, and feces of pigs, and in swabs of feeder troughs and water dispensers collected from pig farms in Vietnam. The composition of the microbiota differed between samples in each sample group. Streptococcus, Actinobacillus, Moraxella, and Rothia were the most abundant genera and significantly discriminative in saliva samples, regardless of the plasticity and changeability of the composition of microbiota in saliva. Moreover, species assignment of the genus Streptococcus revealed that Streptococcus suis was exceptional in the salivary microbiota, due to being most abundant among the streptococcal species and sharing estimated proportions of 5.7%-9.4% of the total bacteria in saliva. Thus, pig oral microbiota showed unique characteristics in which the major species was the pig pathogen. On the other hand, ß-diversity analysis showed that the microbiota in saliva was distinct from those in the others. From the above results, pig saliva was shown to be the major natural habitat of S. suis, and is suggested to be the most probable source of S. suis infection.