Comparative analysis of Streptococcus suis genomes identifies novel candidate virulence-associated genes in North American isolates.

Streptococcus suis is a significant economic and welfare concern in the swine industry. Pan-genome analysis provides an in-silico approach for the discovery of genes involved in pathogenesis in bacterial pathogens. In this study, we performed pan-genome analysis of 208 S. suis isolates classified into the pathogenic, possibly opportunistic, and commensal pathotypes to identify novel candidate virulence-associated genes (VAGs) of S. suis. Using chi-square tests and LASSO regression models, three accessory pan-genes corresponding to S. suis strain P1/7 markers SSU_RS09525, SSU_RS09155, and SSU_RS03100 (>95% identity) were identified as having a significant association with the pathogenic pathotype. The proposed novel SSU_RS09525 + /SSU_RS09155 + /SSU_RS03100 + genotype identified 96% of the pathogenic pathotype strains, suggesting a novel genotyping scheme for predicting the pathogenicity of S. suis isolates in North America. In addition, mobile genetic elements carrying antimicrobial resistance genes (ARGs) and VAGs were identified but did not appear to play a major role in the spread of ARGs and VAGs.

Genomic and pathogenic investigations of Streptococcus suis serotype 7 population derived from a human patient and pigs.

Streptococcus suis is one of the important emerging zoonotic pathogens. Serotype 2 is most prevalent in patients worldwide. In the present study, we first isolated one S. suis serotype 7 strain GX69 from the blood culture of a patient with septicemia complicated with pneumonia in China. In order to deepen the understanding of S. suis serotype 7 population characteristics, we investigated the phylogenetic structure, genomic features, and virulence of S. suis serotype 7 population, including 35 strains and 79 genomes. Significant diversities were revealed in S. suis serotype 7 population, which were clustered into 22 sequence types (STs), five minimum core genome (MCG) groups, and six lineages. Lineages 1, 3a, and 6 were mainly constituted by genomes from Asia. Genomes of Lineages 2, 3b, and 5a were mainly from Northern America. Most of genomes from Europe (41/48) were clustered into Lineage 5b. In addition to strain GX69, 13 of 21 S. suis serotype 7 representative strains were classified as virulent strains using the C57BL/6 mouse model. Virulence-associated genes preferentially present in highly pathogenic S. suis serotype 2 strains were not suitable as virulence indicators for S. suis serotype 7 strains. Integrative mobilizable elements were widespread and may play a critical role in disseminating antibiotic resistance genes of S. suis serotype 7 strains. Our study confirmed S. suis serotype 7 is a non-negligible pathotype and deepened the understanding of the population structure of S. suis serotype 7, which provided valuable information for the improved surveillance of this serotype.

Proposed virulence-associated genes of Streptococcus suis isolates from the United States serve as predictors of pathogenicity.

BACKGROUND: There is limited information on the distribution of virulence-associated genes (VAGs) in U.S. Streptococcus suis isolates, resulting in little understanding of the pathogenic potential of these isolates. This lack also reduces our understanding of the epidemiology associated with S. suis in the United States and thus affects the efficiency of control and prevention strategies. In this study we applied whole genome sequencing (WGS)-based approaches for the characterization of S. suis and identification of VAGs. RESULTS: Of 208 S. suis isolates classified as pathogenic, possibly opportunistic, and commensal pathotypes, the genotype based on the classical VAGs (epf, mrp, and sly encoding the extracellular protein factor, muramidase-release protein, and suilysin, respectively) was identified in 9% (epf+/mrp+/sly+) of the pathogenic pathotype. Using the chi-square test and LASSO regression model, the VAGs ofs (encoding the serum opacity factor) and srtF (encoding sortase F) were selected out of 71 published VAGs as having a significant association with pathotype, and both genes were found in 95% of the pathogenic pathotype. The ofs+/srtF+ genotype was also present in 74% of 'pathogenic' isolates from a separate validation set of isolates. Pan-genome clustering resulted in the differentiation of a group of isolates from five swine production companies into clusters corresponding to clonal complex (CC) and virulence-associated (VA) genotypes. The same CC-VA genotype patterns were identified in multiple production companies, suggesting a lack of association between production company, CC, or VA genotype. CONCLUSIONS: The proposed ofs and srtF genes were stronger predictors for differentiating pathogenic and commensal S. suis isolates compared to the classical VAGs in two sets of U.S. isolates. Pan-genome analysis in combination with metadata (serotype, ST/CC, VA genotype) was illustrated to be a valuable subtyping tool to describe the genetic diversity of S. suis.

MinION sequencing of Streptococcus suis allows for functional characterization of bacteria by multilocus sequence typing and antimicrobial resistance profiling.

In recent years, high-throughput sequencing has revolutionized disease diagnosis by its powerful ability to provide high resolution genomic information. The Oxford Nanopore MinION sequencer has unparalleled potential as a rapid disease diagnostic tool due to its high mobility, accessibility, and short turnaround time. However, there is a lack of rigorous quality assessment and control processes standardizing the testing on the MinION, which is necessary for incorporation into a diagnostic workflow. Thus, our study examined the use of the MinION sequencer for bacterial whole genome generation and characterization. Using Streptococcus suis as a model, we optimized DNA isolation and treatments to be used for MinION sequencing and standardized de novo assembly to quickly generate a full-length consensus sequence achieving a 99.4% average accuracy. The consensus genomes from MinION sequencing were able to accurately predict the multilocus sequence type in 8 out of 10 samples and identified antimicrobial resistance profiles for 100% of the samples, despite the concern of a high error rate. The inability to unequivocally predict sequence types was due to difficulty in differentiating high identity alleles, which was overcome by applying additional error correction methods to increase consensus accuracy. This manuscript provides methods for the use of MinION sequencing for identification of S. suis genome sequence, sequence type, and antibiotic resistance profile that can be used as a framework for identification and classification of other pathogens.

Serotype and Genotype (Multilocus Sequence Type) of Streptococcus suis Isolates from the United States Serve as Predictors of Pathotype.

Streptococcus suis is a significant cause of mortality in piglets and growing pigs worldwide. The species contains pathogenic and commensal strains, with pathogenic strains causing meningitis, arthritis, endocarditis, polyserositis, and septicemia. Serotyping and multilocus sequence typing (MLST) are primary methods to differentiate strains, but the information is limited for strains found in the United States. The objective of this study was to characterize the diversity of 208 S. suis isolates collected between 2014 and 2017 across North America (mainly the United States) by serotyping and MLST and to investigate associations between subtype and pathotype classifications (pathogenic, possibly opportunistic, and commensal), based on clinical information and site of isolation. Twenty serotypes were identified, and the predominant serotypes were 1/2 and 7. Fifty-eight sequence types (STs) were identified, and the predominant ST was ST28. Associations among serotypes, STs, and pathotypes were investigated using odds ratio and clustering analyses. Evaluation of serotype and ST with pathotype identified a majority of isolates of serotypes 1, 1/2, 2, 7, 14, and 23 and ST1, ST13, ST25, ST28, ST29, ST94, ST108, ST117, ST225, ST373, ST961, and ST977 as associated with the pathogenic pathotype. Serotypes 21 and 31, ST750, and ST821 were associated with the commensal pathotype, which is composed of isolates from farms with no known history of S. suis-associated disease. Our study demonstrates the use of serotyping and MLST to differentiate pathogenic from commensal isolates and establish links between pathotype and subtype, thus increasing the knowledge about S. suis strains circulating in the United States.