In Silico Typing and Identification Confirmation of Isolates.

Streptococcus suis is an important zoonotic pathogen causing severe infections in pigs and humans. Serotyping of S. suis strains is crucial for epidemiological surveillance, outbreak investigations, and understanding the pathogenesis of this bacterium. Here, we describe a step-by-step approach that enhances a previously developed pipeline by utilizing a computational script for efficient and accurate typing of S. suis strains. The pipeline is implemented in Perl programming language and leverages the Short Read Sequence Typing for Bacterial Pathogens (SRST2) tool. It integrates various bioinformatics techniques and utilizes multiple databases, including a serotype database, cpsH confirmation database, multi-locus sequence typing (MLST) database, recN species-specific gene database, and virulence gene database. These databases contain comprehensive information on S. suis serotypes, genetic markers, and virulence factors. The script can utilize paired-end or single-end fastq files as input and first confirms the species by sequence read data aligning to the recN gene, ensuring the accurate identification of S. suis strains. The pipeline next performs MLST typing and virulence factor identification using SRST2 while in a parallel processes it performs in silico serotyping of the strains. The pipeline offers a streamlined and semiautomated approach to serotyping S. suis strains, facilitating large-scale studies and reducing the manual effort required for data analysis.

Streptococcus suis serotype 4: a population with the potential pathogenicity in humans and pigs.

Streptococcus suis is a major bacterial pathogen in pigs and an emerging zoonotic pathogen. Different S. suis serotypes exhibit diverse characteristics in population structure and pathogenicity. Surveillance data highlight the significance of S. suis serotype 4 (SS4) in swine streptococcusis, a pathotype causing human infections. However, except for a few epidemiologic studies, the information on SS4 remains limited. In this study, we investigated the population structure, pathogenicity, and antimicrobial characteristics of SS4 based on 126 isolates, including one from a patient with septicemia. We discovered significant diversities within this population, clustering into six minimum core genome (MCG) groups (1, 2, 3, 4, 7-2, and 7-3) and five lineages. Two main clonal complexes (CCs), CC17 and CC94, belong to MCG groups 1 and 3, respectively. Numerous important putative virulence-associated genes are present in these two MCG groups, and 35.00% (7/20) of pig isolates from CC17, CC94, and CC839 (also belonging to MCG group 3) were highly virulent (mortality rate ≥ 80%) in zebrafish and mice, similar to the human isolate ID36054. Cytotoxicity assays showed that the human and pig isolates of SS4 strains exhibit significant cytotoxicity to human cells. Antimicrobial susceptibility testing showed that 95.83% of strains isolated from our labs were classified as multidrug-resistant. Prophages were identified as the primary vehicle for antibiotic resistance genes. Our study demonstrates the public health threat posed by SS4, expanding the understanding of SS4 population structure and pathogenicity characteristics and providing valuable information for its surveillance and prevention.

Investigation of genomic and pathogenicity characteristics of Streptococcus suis ST1 human strains from Guangxi Zhuang Autonomous Region (GX) between 2005 and 2020 in China.

Streptococcus suis is a significant and emerging zoonotic pathogen. ST1 and ST7 strains are the primary agents responsible for S. suis human infections in China, including the Guangxi Zhuang Autonomous Region (GX). To enhance our understanding of S. suis ST1 population characteristics, we conducted an investigation into the phylogenetic structure, genomic features, and virulence levels of 73 S. suis ST1 human strains from GX between 2005 and 2020. The ST1 GX strains were categorized into three lineages in phylogenetic analysis. Sub-lineage 3-1a exhibited a closer phylogenetic relationship with the ST7 epidemic strain SC84. The strains from lineage 3 predominantly harboured 89K-like pathogenicity islands (PAIs) which were categorized into four clades based on sequence alignment. The acquirement of 89K-like PAIs increased the antibiotic resistance and pathogenicity of corresponding transconjugants. We observed significant diversity in virulence levels among the 37 representative ST1 GX strains, that were classified as follows: epidemic (E)/highly virulent (HV) (32.4%, 12/37), virulent plus (V+) (29.7%, 11/37), virulent (V) (18.9%, 7/37), and lowly virulent (LV) (18.9%, 7/37) strains based on survival curves and mortality rates at different time points in C57BL/6 mice following infection. The E/HV strains were characterized by the overproduction of tumour necrosis factor (TNF)-α in serum and promptly established infection at the early phase of infection. Our research offers novel insights into the population structure, evolution, genomic features, and pathogenicity of ST1 strains. Our data also indicates the importance of establishing a scheme for characterizing and subtyping the virulence levels of S. suis strains.

Genetic diversity and antimicrobial susceptibility of Streptococcus suis from diseased Swiss pigs collected between 2019 - 2022.

Streptococcus suis is an important pathogen causing severe disease in pigs and humans, giving rise to economic losses in the pig production industry. Out of 65 S. suis isolates collected from diseased pigs in Switzerland between 2019 and 2022, 57 isolates were thoroughly examined by phenotypic and whole genome sequence (WGS) based characterization. The isolates' genomes were sequenced allowing for a comprehensive analysis of their distribution in terms of serovar, sequence type (ST), clonal complex (CC), and classical virulence markers. Antimicrobial resistance (AMR) genes were screened, and phenotypic susceptibility to eight classes of antimicrobial agents was examined. Serovar 6, devoid of any resistance genes, was found to be most prevalent, followed by serovars 1, 3, 1/2, and 9. Thirty STs were identified, with ST1104 being the most prevalent. Serovar 2 and serovar 1/2 were associated with CC1, potentially containing the most virulent variants. Based on single nucleotide polymorphism (SNP) analyses, fifteen isolates belonged to one of seven putative transmission clusters each consisting of two or three isolates. High phenotypic AMR rates were detected for tetracyclines (80%) and macrolides (35%) and associated with the resistance genes tet(O) and erm(B), respectively. In contrast, susceptibility to ß-lactam antibiotics and phenicols was high. Determination of phenotypic AMR profiling, including the minimum inhibitory concentrations (MICs) of the tested antimicrobial agents, sets a baseline for future studies. The study provides valuable insights into the genetic diversity and antimicrobial susceptibility of Swiss S. suis isolates, facilitating the identification of emerging clones relevant to public health concerns.

Comparative genetic analyses provide clues about capsule switching in Streptococcus suis 2 strains with different virulence levels and genetic backgrounds.

Streptococcus suis (S. suis) is a major bacterial pathogen in the swine industry and an emerging zoonotic agent. S. suis produces an important extracellular component, capsular polysaccharide (CPS), based on which dozens of serotypes have been identified. Through virulence genotyping, we revealed the relatedness between subpopulations of S. suis serotype 2 (SS2), S. suis serotype 3 (SS3) and S. suis serotype 7 (SS7) strains despite their serotype differences. Multilocus sequence typing (MLST) was used to characterize the whole S. suis population and revealed capsule switching between S. suis strains. Importantly, capsule switching occurred in the SS2, SS3 and SS7 strains belonging to CC28 and CC29, which are phylogenetically distinct from the main CC1 SS2 lineage. To further explore capsule switching in S. suis, comparative genomic analyses were performed using available complete S. suis genomes. Phylogenetic analyses suggested that the SS2 strains could be divided into two clades (1 and 2), and those classified into clade 2 colocalized with SS3 and SS7 strains, in accordance with the above virulence genotyping and MLST analyses. Clade 2 SS2 strains presented high genetic similarity to SS3 and SS7 and shared common competence and defensive elements with them but were significantly different from Clade 1 SS2 strains. Notably, although the cps loci shared by Clade 1 and 2 SS2 strains were almost identical, a specific region of the cps locus of strain NSUI002 (Clade 2 SS2) could be found in the SS3 cps locus but not in the Clade 1 SS2 strain. These data indicated that the SS2 strains in CC28 and CC29 might have acquired the cps locus through capsule switching, which could explain the distinct genetic lineages within the SS2 population.

The population structure, antimicrobial resistance, and pathogenicity of Streptococcus suis cps31.

Streptococcus suis is a zoonotic pathogen that can cause invasive infections in humans and pigs. The S. suis cps31 strains (SS31) were frequently isolated from healthy or diseased pigs and one human infection case caused by SS31 was reported in Thailand in 2015. However, except for a few epidemiologic studies, little information is available for SS31. To characterize SS31, a total of 75 SS31 strains were analyzed, including 52 strains that were isolated from healthy or diseased pigs and 23 strains whose information was accessed from NCBI. The MLST analysis showed that SS31 exhibited high heterogeneity. The phylogenetic analysis and minimum core-genome (MCG) classification revealed that 75 strains were clustered into 3 lineages. Strains from NCBI mainly at Lineage 2 belong to MCG7-3, and most of strains from China at Lineage 3 belong to MCG7-2. This finding indicated that their evolutionary path was different. All SS31 strains were resistant to more than three classes of antimicrobial agents, and major antimicrobial resistance genes for strains from Lineage 3 were carried by prophages. This observation is different from the previous observation that integrative conjugative elements and integrative and mobilizable elements are major vehicles of antimicrobial resistance genes for S. suis. In addition to strains isolated from diseased pigs, seven of 47 strains isolated from clinically healthy pigs were also pathogenic in a zebrafish infection model. These findings reveal unique characteristics of SS31 and contribute to establishing public health surveillance for SS31 and clarifying the diversity of S. suis.

The phosphorylation of phosphoglucosamine mutase GlmM by Ser/Thr kinase STK mediates cell wall synthesis and virulence in Streptococcus suis serotype 2.

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that causes serious economic losses in the pig industry. Phosphorylation is an important mechanism of protein modification. Recent studies have reported that the serine/threonine kinase (STK) gene contributes to the growth and virulence of SS2. However, the mechanism underlying the regulatory functions of STK in SS2 has not been thoroughly elucidated to date. In this study, phosphoproteomic analysis was performed to determine substrates of the STK protein. Twenty-two proteins with different cell functions were identified as potential substrates of STK. Phosphoglucosamine mutase (GlmM) was selected for further investigation among them. In vitro phosphorylation assay and immunoprecipitation assay indicated that GlmM was phosphorylated by STK at the Ser-101 site and the phosphorylation level of GlmM can be affected. We observed that compared to the wild-type strain ZY05719, the glmM-deficient strain (ΔglmM) and the glmM S101A point mutation strain (CΔglmM S101A) showed aberrant cell morphology and attenuated virulence, including enlarged cell volume, absent capsule, decreased resistance, lower survival caused by unusual peptidoglycan synthesis, and significantly attenuated pathogenicity in a mouse infection model. Additionally, compared to ZY05719 and CΔglmM, GlmM enzyme acivities and peptidoglycan concentrations of the stk-deficient strain (Δstk), CΔglmM S101A decreased significantly. These experiments revealed that STK phosphorylates GlmM at the Ser-101 site to impact GlmM enzyme activity and control cell wall peptidoglycan synthesis to affect SS2 pathogenicity.

Streptococcus suis serotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Streptococcus suis, particularly S. suis serotype 2 (SS2), is an important zoonotic pathogen causing meningitis in humans worldwide. Although the proper classification of the causative and pathogenic serotype is salutary for the clinical diagnosis, cross-reactions leading to the indistinguishability of serotypes by the current serotyping methods are significant limitations. In the present study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of extracted peptides was developed to improve the classification of serotype of S. suis. The peptide mass fingerprint (PMFs) database of S. suis was generated from the whole-cell peptides of 32 reference strains of S. suis isolates obtained from pigs. Thirty-two human S. suis isolates from clinical cases in Thailand were used to validate this alternative serotyping method in direct comparison to the multiplex (m)PCR approach. All reference strains, representing 32 serotypes of S. suis, exhibited their individual PMFs patterns, thus allowing differentiation from one another. Highly pathogenic SS2 and SS14 were clearly differentiated from the otherwise serologically closely related SS1/2 and SS1, respectively. The developed MALDI-TOF-MS serotyping method correctly classified the serotype in 68.8% (22/32) of the same serotype isolates generated from the PMFs database; while the validity for the clinical human isolates was 62.5% (20/32). The agreement between the MALDI-TOF-MS and mPCR serotyping was moderate with a Kappa score of 0.522, considering that mPCR could correctly serotype up to 75%. The present study demonstrated that PMFs from the developed MALDI-TOF-MS-based method could successfully discriminate the previously indistinguishable highly pathogenic SS2 and SS14 from SS1/2 and SS1, respectively. Moreover, this serotyping method distinguished pathogenic SS6, and so is an alternative approach of choice to rapidly and reliably serotype clinically pathogenic S. suis isolates.

Study of the relationship between untypable and typable isolates of Streptococcus suis recovered from clinically ill and healthy nursery pigs.

Streptococcus suis naturally colonizes the upper respiratory tract of pigs and can lead to severe disease conditions. Although there are several serotypes associated with disease, untypable isolates have also been observed. The objective of this study was to investigate the relatedness of untypable S. suis isolates detected in clinical cases and healthy pigs in Ontario, Canada, and their relation to typing serotypes. One hundred fifty-six isolates obtained from 33 cases and 26 farm-and-pen-matched control pigs were sequenced using Illumina HiSeq sequencing. Protein sequences of the capsular polysaccharide genes (cps) were identified and analyzed using a maximum likelihood tree. Among the 27 untypable isolates, 3 were from systemic sites of cases and 13 and 11 were from upper respiratory sites of cases and controls, respectively. One hundred fifty-six isolates were grouped into 17 distinct groups based on the cps gene tree. Isolates from these 17 distinct individual cps groups were distributed among a minimum of one farm and maximum of eight farms. Untypable isolates were detected in 12 of those groups and each cps group had untypable isolates present amongst multiple farms. Interestingly, the three systemic untypable isolates not only coexisted with other serotypes found in the same location of the same pigs but were also found among different cps groups. These isolates are of interest and warrant further investigation. Overall, a wide diversity of S. suis among untypable isolates was observed in this study.

Caveolae/rafts protect human cerebral microvascular endothelial cells from Streptococcus suis serotype 2 α-enolase-mediated injury.

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that causes meningitis. The ubiquitously expressed 40S ribosome protein SA (RPSA) is a multifunctional protein involved in the pathogenesis of multiple pathogens, especially those causing meningitis. However, the role of RPSA in SS2-induced meningitis is not clear. In this study, immunofluorescence staining revealed that SS2 infection promoted the intracellular transfer of RPSA to the surface of human cerebral microvascular endothelial cells (HCMECs). Moreover, SS2 infection promoted the accumulation of caveolin 1 (CAV1) and the formation of membrane bulges where RPSA enveloped CAV1 on the cell surface. SS2 infection also caused dynamic changes in the localization of RPSA and CAV1 on the cell surface which could be eliminated by disruption of caveolae/rafts by addition of methyl-ß-cyclodextrin (MßCD). Co-immunoprecipitation analysis demonstrated that α-enolase (ENO), a key virulence factor of SS2, interacted with RPSA, and promoted the interaction between RPSA and CAV1. Immunofluorescence staining, western blotting and flow cytometry analyses showed that damaged caveolae/rafts significantly enhanced ENO adhesion to HCMECs, promoted the "destruction" of RPSA by ENO, and enhanced the toxic effect of ENO on HCMECs. Importantly, these effects could be relieved upon the addition of cholesterol. We conclude that caveolae/rafts weaken the toxic effect of SS2 ENO on RPSA-mediated events in HCMECs. Our study has led to better understanding of the roles of RPSA and caveolae/rafts upon SS2 infection, and a new pathological role for RPSA in infection.

Inactivation of the htpsA gene affects capsule development and pathogenicity of Streptococcus suis.

STREPTOCOCCUS SUIS: serotype 2 (S. suis 2) is an important swine pathogen and also an emerging zoonotic agent. HtpsA has been reported as an immunogenic cell surface protein on the bacterium. In the present study, we constructed an isogenic mutant strain of htpsA, namely ΔhtpsA, to study its role in the development and virulence of S. suis 2. Our results showed that the mutant strain lost its typical encapsulated structure with decreased concentrations of sialic acid. Furthermore, the survival rate in whole blood, the anti-phagocytosis by RAW264.7 murine macrophage, and the adherence ability to HEp-2 cells were all significantly affected in the ΔhtpsA. In addition, the deletion of htpsA sharply attenuated the virulence of S. suis 2 in an infection model of mouse. RNA-seq analysis revealed that 126 genes were differentially expressed between the ΔhtpsA and the wild-type strains, including 28 upregulated and 98 downregulated genes. Among the downregulated genes, many were involved in carbohydrate metabolism and synthesis of virulence-associated factors. Taken together, htpsA was demonstrated to play a role in the morphological development and pathogenesis of the highly virulent S. suis 2 05ZYH33 strain.

In vitro characterization of granulocyte-colony stimulating factor (G-CSF) production by dendritic cells and macrophages during Streptococcus suis infection.

Streptococcus suis serotype 2 is an important porcine bacterial pathogen and emerging zoonotic agent. Infections induce an exacerbated inflammation that can result in sudden death (septic shock) and meningitis. Though neutrophilic leukocytosis characterizes S. suis infection, the mediators involved are poorly understood. Among them, granulocyte-colony stimulating factor (G-CSF), a pro-inflammatory cytokine, triggers proliferation of neutrophil progenitors and neutrophil mobilization. However, the systemic production of G-CSF induced during S. suis infection, the cell types involved, and the underlying mechanisms remain unknown. In a S. suis serotype 2 mouse model of systemic infection, plasma levels of G-CSF rapidly increased after infection. S. suis activation of DCs and macrophages resulted in high (> 1000 pg/mL) and comparable production levels of G-CSF, as measured by ELISA. By using mutant strains deficient in capsular polysaccharide (CPS) or lipoprotein maturation in combination with purified lipoteichoic acid (LTA) from the latter mutant strain, it was showed that G-CSF production is mainly mediated by S. suis lipoproteins. The Toll-like receptor (TLR) pathway via myeloid differentiation primary response 88 (MyD88) is required for G-CSF production by DCs and macrophages following S. suis activation, with a partial involvement of TLR2. On the other hand, TLR2-independant G-CSF production induced by S. suis requires internalization and bacterial DNA might play a role in this pathway. Finally, these signals activated nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways leading to G-CSF production. In conclusion, this study demonstrated for the first time that S. suis induces G-CSF production in vivo and DCs and macrophages are key cellular sources of this cytokine mediator, mainly via the binding of lipoproteins to TLR2. The CPS significantly reduced this activation, confirming the powerful role of this component in S. suis virulence. As such, this study contributes to better understand how DCs and macrophages produce G-CSF in response to S. suis, and potentially to other streptococci.

Screening for phagocytosis resistance-related genes via a transposon mutant library of Streptococcus suis serotype 2.

STREPTOCOCCUS SUIS: serotype 2 (SS2) is a serious zoonotic pathogen which causes symptoms of streptococcal toxic shock syndrome (STSS) and septicemia; these symptoms suggest that SS2 may have evade innate immunity. Phagocytosis is an important innate immunity process where phagocytosed pathogens are killed by lysosome enzymes, reactive oxygen, and nitrogen species, and acidic environments in macrophages following engulfment. A previously constructed mutant SS2 library was screened, revealing 13 mutant strains with decreased phagocytic resistance. Through inverse PCR, the transposon insertion sites were determined. Through bioinformatic analysis, the 13 disrupted genes were identified as Cps2F, 3 genes belonging to ABC transporters, WalR, TehB, rpiA, S-transferase encoding gene, prs, HsdM, GNAT family N-acetyltransferase encoding gene, proB, and upstream region of DnaK. Except for the capsular polysaccharide biosynthesis associated Cps2F, the other genes had not been linked to a role in anti-phagocytosis. The survival ability in macrophages and whole blood of randomly picked mutant strains were significantly impaired compared with wild-type ZY05719. The virulence of the mutant strains was also attenuated in a mouse infection model. In the WalR mutant, the transcription of HP1065 decreased significantly compared with wild-type strain, indicating WalR might regulated HP1065 expression and contribute to the anti-phagocytosis of SS2. In conclusion, we identified 13 genes that influenced the phagocytosis resistant ability of SS2, and many of these genes have not been reported to be associated with resistance to phagocytosis. Our work provides novel insight into resistance to phagocytosis, and furthers our understanding of the pathogenesis mechanism of SS2.

Streptococcus suis in Swedish grower pigs: occurrence, serotypes, and antimicrobial susceptibility.

BACKGROUND: Streptococcus suis is a major cause of meningitis, arthritis, and pneumonia in pigs worldwide, and an emerging pathogen in humans. In Sweden, S. suis has previously received little attention but has in recent years become increasingly recognized as affecting the pig production. The aim of the present study was to investigate the occurrence, serotypes and antimicrobial susceptibility of S. suis in Swedish grower pigs from herds with and without reported S. suis associated disease, as well as possible associations between S. suis associated disease and selected environmental and production factors. Swab samples were taken from the tonsils of clinically healthy 8-13-week-old grower pigs from ten case herds and ten control herds. Isolates were cultured, identified using MALDI-TOF MS, and serotyped using latex agglutination. The antimicrobial susceptibility of 188 isolates was tested using broth microdilution. Production data was gathered and environmental parameters were measured on the farms. RESULTS: Streptococcus suis was isolated from 95% of the sampled pigs in both the case and the control herds. Serotypes 3, 4, 5, 7, 9, 10, 11, 15, 16, and 17-34 were detected, although a majority of the isolates (81.5%) were non-typeable. There was less diversity among the serotypes isolated from the case herds than among those from the control herds; four and nine different serotypes, respectively. Isolates resistant to penicillin (3.8%) were reported for the first time in Sweden. Tetracycline resistance was common (88.4%). No association was noted between the production and the environmental factors investigated, and the carriership of S. suis. CONCLUSIONS: The carriership of S. suis was found to be higher in clinically healthy Swedish pigs than previously estimated, and for the first time, the presence of Swedish isolates resistant to penicillin was reported. Many of the most commonly disease-associated serotypes, e.g. serotypes 2, 9, 3, and 7, were detected in healthy grower pigs although further studies are needed to investigate the virulence of these isolates.

Proteomics analysis of important molecules in serum from meningitic piglets caused by Streptococcus suis serotype 2.

INTRODUCTION: Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that causes meningitis in China. This study's aim was comparative analysis of serum proteomics from meningitis and non-meningitis piglets. METHODOLOGY: SS2 meningitis and non-meningitis piglet models were established. The serum samples were collected and analyzed by label-free LC-MS/MS proteomics technology. Differentially expressed proteins (DEPs) from serum were screened out by comparing the meningitis group and non-meningitis group to the healthy group (M/C; N/C), respectively. And then, globally and comparative analysis of DEPs in "M/C" and "N/C" in serum were performed using bioinformatics method. Finally, we comparatively analyzed the serum and cerebrospinal fluid proteomics in piglets that lived with meningitis. RESULTS: We obtained 316 and 191 DEPs from "M/C" and "N/C" which classification visualizations were established. 157 DEPs were common in both groups and 159 DEPs were unique to the "M/C". These DEPs and the signaling pathways which they participated in were visualized. Moreover, some DEPs which participated in multiple pathways were discovered and the interaction between 159 DEPs was also mapped. 39 common DEPs were also screened out in serum and cerebrospinal fluid during meningitis, and signaling pathways associated with these DEPs were further visualized. CONCLUSIONS: DEPs in "M/C" and "N/C" were comparatively analyzed and the similarities and differences of these DEPS which were involved in signal pathways were summarized. Moreover, several important molecules were screened out.

Development of a mismatch amplification mutation assay to correctly serotype isolates of Streptococcus suis serotypes 1, 2, 1/2, and 14.

Streptococcus suis is one of the most important bacterial swine pathogens worldwide and is an emerging pathogen in humans. There are 29 serotypes, and serotyping, which is based on the antigenicity of the capsular polysaccharide (CPS) or on its coding genes, is often part of routine identification and provides further information regarding S. suis virulence and zoonotic potential. Serotypes 2 and 14 possess high zoonotic potential, and serotype 1/2 is the serotype most frequently isolated from diseased pigs in North America. PCR has replaced antibody-based techniques to perform serotyping. However, traditional PCR is not able to differentiate serotype 2 from 1/2 and serotype 1 from 14, given that the only difference in the cps loci of those serotype pairs is a nonsynonymous single-nucleotide polymorphism. We developed a mismatch amplification mutation assay (MAMA)-PCR that was able to correctly serotype 148 isolates previously known to be serotypes 1, 2, 1/2, or 14. This technique will be highly useful in animal and human health laboratories performing PCR serotyping of S. suis isolates.

Diversity of serotypes and new cps loci variants among Streptococcus suis isolates from pigs in Poland and Belarus.

Streptococcus suis plays an important role in infections in pigs but information about the epidemiology of this pathogen in Poland and Belarus remains scarce. Ninety-six isolates from brain and lungs were studied by PCR-based serotyping, analysis of virulence-associated determinants and multilocus sequence typing (MLST). Selected six isolates were further analyzed by genomic sequencing and transmission electron microscopy (TEM). Serotype 2 was most prevalent, followed by serotypes 3, 4, 8 and 7. All isolates carried fbpS; 30, 74 and 79 isolates were positive for epf, mrp and sao, respectively. MLST revealed that while widely distributed clonal complexes, such as 1, 16, 25 and 28 circulate in both countries, a significant part of the population is composed of novel singletons. Six isolates, all positive for the capsule in TEM, harbored cps loci differing to a various degree from these previously described, including one with a novel cps locus (putative NCL21). In conclusion, our study provides first molecular data on S. suis from pigs in the Central/Eastern Europe and contributes to a better characterization of diversity of loci responsible for capsule production in this pathogen.

Rapid high resolution melting assay to differentiate Streptococcus suis serotypes 2, 1/2, 1, and 14.

This rapid high resolution melting (HRM) assay allows distinguishing between Streptococcus suis serotype pairs 2 and 1/2 as well as 1 and 14, respectively, based on a single-nucleotide polymorphism within capsular polysaccharide synthesis gene cluster K. This assay is easy to implement and identifies potential zoonotic serotypes.

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.

Isolation and identification of Streptococcus suis from sick pigs in Bali, Indonesia.

OBJECTIVE: Streptococcus suis (S. suis) is a causative agent for various syndromes in pigs. It can be transmitted to humans with typical symptoms of meningitis and death. Although human infections have been confirmed at Bali Referral Hospital, Indonesia, since 2014, the bacteria have not been isolated from pigs. Here, we provide confirmation of the presence of the bacteria in sick pigs in the province. RESULTS: Streptococcus suis was confirmed in 8 of 30 cases. The final confirmation was made using PCR and sequencing of the glutamate dehydrogenase (GDH) and recombination/repair protein (recN) gene fragments. Upon PCR serotyping, two were confirmed to be serotype 2 or 1/2. Prominent histopathological lesions of confirmed cases were meningitis, endocarditis, pericarditis, bronchopneumonia, enteritis and glomerulonephritis. The dominant inflammatory cells were neutrophils and macrophages. Further research is needed to understand the risk factors for human infection. Community awareness on the risk of contracting S. suis and vaccine development are needed to prevent human infections.