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Background Streptococcus suis is divided into 29 serotypes based on a serological reaction against the capsular polysaccharide (CPS). Multiplex PCR tests targeting the cps locus are also used to determine S. suis serotypes, but they cannot differentiate between serotypes 1 and 14, and between serotypes 2 and 1/2. Here, we developed a pipeline permitting in silico serotype determination from whole-genome sequencing (WGS) short-read data that can readily identify all 29  S. suis serotypes. Results We sequenced the genomes of 121 strains representing all 29 known S. suis serotypes. We next combined available software into an automated pipeline permitting in silico serotyping of strains by differential alignment of short-read sequencing data to a custom S. suis cps loci database. Strains of serotype pairs 1 and 14, and 2 and 1/2 could be differentiated by a missense mutation in the cpsK gene . We report a 99 % match between coagglutination- and pipeline-determined serotypes for strains in our collection. We used 375 additional S. suis genomes downloaded from the NCBI’s Sequence Read Archive (SRA) to validate the pipeline. Validation with SRA WGS data resulted in a 92 % match. Included pipeline subroutines permitted us to assess strain virulence marker content and obtain multilocus sequence typing directly from WGS data. Conclusions Our pipeline permits rapid and accurate determination of S. suis serotype, and other lineage information, directly from WGS data. By discriminating between serotypes 1 and 14, and between serotypes 2 and 1/2, our approach solves a three-decade longstanding S. suis typing issue.

Streptococcussuis is an important zoonotic agent causing severe diseases in pigs and humans. To date, 33 serotypes of S. suis have been identified based on antigenic differences in the capsular polysaccharide. The capsular polysaccharide synthesis (cps) locus encodes proteins/enzymes that are responsible for capsular production and variation in the capsule structures are the basis of S. suis serotyping. Multiplex and/or simplex PCR assays have been developed for 15 serotypes based on serotype-specific genes in the cps gene cluster. In this study, we developed a set of multiplex PCR (mPCR) assays to identify the 33 currently known S. suis serotypes. To identify serotype-specific genes for mPCR, the entire genomes of reference strains for the 33 serotypes were sequenced using whole genome high-throughput sequencing, and the cps gene clusters from these strains were identified and compared. We developed a set of 4 mPCR assays based on the polysaccharide polymerase gene wzy, one of the serotype-specific genes. The assays can identify all serotypes except for two pairs of serotypes: 1 and 14, and 2 and 1/2, which have no serotype-specific genes between them. The first assay identifies 12 serotypes (serotypes 1 to 10, 1/2, and 14) that are the most frequently isolated from diseased pigs and patients; the second identifies 10 serotypes (serotypes 11 to 21 except 14); the third identifies the remaining 11 serotypes (serotypes 22 to 31, and 33); and the fourth identifies a new cps cluster of S. suis discovered in this study in 16 isolates that agglutinated with antisera for serotypes 29 and 21. The multiplex PCR assays developed in this study provide a rapid and specific method for molecular serotyping of S. suis.

Abstract O-antigen polysaccharide is a major immunogenic feature of the lipopolysaccharide of Gram-negative bacteria, and most species produce a large variety of forms that differ substantially from one another. There are 18 known O-antigen forms in the Yersinia pseudotuberculosis complex, which are typical in being composed of multiple copies of a short oligosaccharide called an O unit. The O-antigen gene clusters are located between the hemH and gsk genes, and are atypical as 15 of them are closely related, each having one of five downstream gene modules for alternative main-chain synthesis, and one of seven upstream modules for alternative side-branch sugar synthesis. As a result, many of the genes are in more than one gene cluster. The gene order in each module is such that, in general, the earlier a gene product functions in O-unit synthesis, the closer the gene is to the 5΄ end for side-branch modules or the 3΄ end for main-chain modules. We propose a model whereby natural selection could generate the observed pattern in gene order, a pattern that has also been observed in other species. O-antigen gene clusters in the Yersinia pseudotuberculosis complex.

Klebsiella pneumoniae is an important human pathogen associated with a variety of diseases and the prevalence of bla KPC carrying K. pneumoniae (KPC-Kp) is rapidly increasing. Capsule is an important virulence factor in K. pneumoniae . In this study, we determined to first systematically characterize capsular polysaccharide (CPS) and virulence traits in KPC-Kp strains. A total of 56 KPC-Kp isolates were recovered from clinical samples in a Chinese hospital, which were assigned to clonal lineages by multilocus sequence typing (MLST). Capsule typing ( wzi sequencing and wzc polymerase chain reaction [PCR]) and virulence genes were characterized by molecular approaches. The virulence of these strains was determined by biofilm formation, serum killing resistance, phagocytosis, and infection models. Six different STs were found among 56 KPC-Kp isolates: 76.8% (43 of 56 isolates) belonged to ST11, 6 isolates belonged to ST147, 4 isolates belonged to ST15, 1 isolate belonged to ST1456, 1 isolate belonged to ST65, and 1 isolate was ST23. Based on the wzi gene DNA sequences and wzc PCR, these 56 strains were classified as capsular type wzi 47-K47 ( n  = 37), wzi 64-K64 ( n  = 8), wzi 8-K8 ( n  = 4), wzi 37-K37 ( n  = 4), wzi 53-K53 ( n  = 1), wzi 125-K2 ( n  = 1), and wzi 1-K1 ( n  = 1). Heterogeneity was detected in biofilm formation and phagocytosis among different CPS types. ST11 strains were less virulent than other ST strains. KPC-Kp strains exhibit variability of virulence-associated traits. Differences were associated with the ST types and CPS.

The Burkholderia cepacia Complex assembles at least eighteen closely related species that are ubiquitous in nature. Some isolates show beneficial potential for biocontrol, bioremediation and plant growth promotion. On the contrary, other strains are pathogens for plants and immunocompromised individuals, like cystic fibrosis patients. In these subjects, they can cause respiratory tract infections sometimes characterised by fatal outcome. Most of the Burkholderia cepacia Complex species are mucoid when grown on a mannitol rich medium and they also form biofilms, two related characteristics, since polysaccharides are important component of biofilm matrices. Moreover, polysaccharides contribute to bacterial survival in a hostile environment by inhibiting both neutrophils chemotaxis and antimicrobial peptides activity, and by scavenging reactive oxygen species. The ability of these microorganisms to produce exopolysaccharides with different structures is testified by numerous articles in the literature. However, little is known about the type of polysaccharides produced in biofilms and their relationship with those obtained in non-biofilm conditions. The aim of this study was to define the type of exopolysaccharides produced by nine species of the Burkholderia cepacia Complex. Two isolates were then selected to compare the polysaccharides produced on agar plates with those formed in biofilms developed on cellulose membranes. The investigation was conducted using NMR spectroscopy, high performance size exclusion chromatography, and gas chromatography coupled to mass spectrometry. The results showed that the Complex is capable of producing a variety of exopolysaccharides, most often in mixture, and that the most common exopolysaccharide is always cepacian. In addition, two novel polysaccharide structures were determined: one composed of mannose and rhamnose and another containing galactose and glucuronic acid. Comparison of exopolysaccharides obtained from cultures on agar plates with those extracted from biofilms on cellulose membranes showed important differences, thus suggesting that extrapolating data from non-biofilm conditions might not always be applicable.

SIGN-R1, a recently discovered C-type lectin expressed at high levels on macrophages within the marginal zone of the spleen, mediates the uptake of dextran polysaccharides by these phagocytes. We now find that encapsulated Streptococcus pneumoniae are rapidly cleared by these macrophages from the bloodstream, and that capture also takes place when different cell lines express SIGN-R1 after transfection. To assess the role of the capsular polysaccharide of S. pneumoniae (CPS) in the interaction of SIGN-R1 with pneumococci, we first studied binding and uptake of serotype 14 CPS in transfected cells. Binding was observed and was of a much higher avidity (3,000-fold) for CPS 14 than dextran. The CPSs from four different serotypes were also cleared by marginal zone macrophages in vivo. To establish a role for SIGN-R1 in this uptake, we selectively down-regulated expression of the lectin by pretreatment of the mice with SIGN-R1 antibodies, including a newly generated hamster monoclonal called 22D1. For several days after this transient knockout, the marginal zone macrophages were unable to take up either CPSs or dextrans. Therefore, marginal zone macrophages in mice have a receptor that interacts with capsular pneumococcal polysaccharides, setting the stage for further studies of the functional consequences of this interaction.

Aggregatibacter actinomycetemcomitans is divided into 6 serotypes. Occurrence of non-serotypeable strains is known, but background reasons are unclear. We hypothesized that non-serotypeable strains represent new serotypes or have altered expression of serotype-specific polysaccharide antigen (S-PA). We first characterized 311 strains from 189 individuals using both immunoassay- and PCR-based serotyping. Next, using natural human infection and rabbit immunization approaches, we clarified whether the phenotypically non-serotypeable strains expressed S-PA. Immunoassay identified serotypes a–f among 216 strains from 159 individuals. The remaining 95 strains from 30 individuals were phenotypically non-serotypeable. Yet, all these strains were identified by PCR-typing as serotype a-, b-, c-, or f. Non-serotypeability was confirmed by Western immunoblot with respective rabbit antisera. Patient sera remained non-reactive with autologous non-serotypeable strains at the serotype-specific region. Rabbit immunization with a phenotypically non-serotypeable strain induced no antibody production against S-PA. Thus, phenotypically non-serotypeable strains did not include novel serotypes, but lacked S-PA expression.

Genome sequence analysis of Xanthomonas oryzae pv. oryzae KACC10331 provides insight into the X. oryzae gum gene cluster that is composed of 14 open-reading frames (ORFs), designated gumB, -C, -D, -E, -F, -G, -H, -I, -J, -K, -L, -M, XOO3167, and -N. We analyzed the transcriptional linkage of the X. oryzae gum gene cluster by using RT-PCR. Analyses of the gum gene cluster by RT-PCR with the wild-type and mutant strains, which carried a deletion of the promoter-like region upstream of gumB or an insertion of the rrnB transcriptional terminator into the gumF gene, revealed that the ORFs of this gene cluster were transcribed as polycistronic mRNA, from gumB to gumN, and the secondary promoter was located upstream of gumG. Taken together, these results suggest that the genes of this cluster constitute an operon expressed from overlapping transcripts.

Knowledge about the type distribution of Streptococcus pneumoniae is fundamental to ensure an effective formulation of pneumococcal vaccine, especially with the possibility of producing a polysaccharide-protein-conjugated vaccine for the prevention of invasive disease in children. During the 6-year period 1982–1987, we received and typed 10,298 isolates from patients with invasive pneumococcal disease: 7,812 (76%) from blood and 2,486 (24%) from CSF. Of all isolates, 81% were recovered from individuals in Europe and 23% were from children. In order of frequency, S. pneumoniae types 6A + 6B, 14, 18C, 19F, 1, 7F, 23F, 19A, 4, and 5 were most commonly isolated from children, and types 3, 1, 14, 7F, 4, 6A + 6B, 8, 23F, 9V, and 19F, from adults. The pneumococcal types in the currently available 23-valent vaccine represented 87% of all isolates in this study, but the proportion of vaccine types varied somewhat with age and source. In all pneumococcal groups included in the vaccine, the vaccine types represented >80% of the isolates, except in groups 6, 15, and 18.