DODGE: Automated point source bacterial outbreak detection using cumulative long term genomic surveillance.

SUMMARY: The reliable and timely recognition of outbreaks is a key component of public health surveillance for foodborne diseases. Whole genome sequencing (WGS) offers high resolution typing of foodborne bacterial pathogens and facilitates the accurate detection of outbreaks. This detection relies on grouping WGS data into clusters at an appropriate genetic threshold, however, methods and tools for selecting and adjusting such thresholds according to the required resolution of surveillance and epidemiological context are lacking. Here we present DODGE (Dynamic Outbreak Detection for Genomic Epidemiology), an algorithm to dynamically select and compare these genetic thresholds. DODGE can analyse expanding datasets over time and clusters that are predicted to correspond to outbreaks (or 'investigation clusters') can be named with the established genomic nomenclature systems to facilitate integrated analysis across jurisdictions. DODGE was tested in two real-world genomic surveillance datasets of different duration, two months from Australia and nine years from the UK. In both cases only a minority of isolates were identified as investigation clusters. Two known outbreaks in the UK dataset were detected by DODGE and were recognised at an earlier timepoint than the outbreaks were reported. These findings demonstrated the potential of the DODGE approach to improve the effectiveness and timeliness of genomic surveillance for foodborne diseases and the effectiveness of the algorithm developed. AVAILABILITY: DODGE is freely available at https://github.com/LanLab/dodge and can easily be installed using Conda. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Emergence of Poultry-Associated Human Salmonella enterica Serovar Abortusovis Infections, New South Wales, Australia.

Salmonella enterica serovar Abortusovis is a ovine-adapted pathogen that causes spontaneous abortion. Salmonella Abortusovis was reported in poultry in 2009 and has since been reported in human infections in New South Wales, Australia. Phylogenomic analysis revealed a clade of 51 closely related isolates from Australia originating in 2004. That clade was genetically distinct from ovine-associated isolates. The clade was widespread in New South Wales poultry production facilities but was only responsible for sporadic human infections. Some known virulence factors associated with human infections were only found in the poultry-associated clade, some of which were acquired through prophages and plasmids. Furthermore, the ovine-associated clade showed signs of genome decay, but the poultry-associated clade did not. Those genomic changes most likely led to differences in host range and disease type. Surveillance using the newly identified genetic markers will be vital for tracking Salmonella Abortusovis transmission in animals and to humans and preventing future outbreaks.

A Multilocus Sequence Tying Scheme for Rapid Identification of Xanthomonas citri Based on Whole Genome Sequencing Data.

Xanthomonas citri is a plant-pathogenic bacterium associated with a diverse range of plant host species. It has undergone substantial reclassification and currently consists of fourteen different subspecies or pathovars that are responsible for a wide range of plant diseases. Whole genome sequencing (WGS) provides a cutting-edge advantage over other diagnostic techniques in epidemiological and evolutionary studies of X. citri because it has a higher discriminatory power and is replicable across laboratories. Also, WGS allows the improvement of multi-locus sequence typing (MLST) schemes. In this study, we used genome sequences of Xanthomonas isolates from the NCBI RefSeq database to develop a seven-gene MLST scheme that yielded 19 sequence types (STs) that correlated with phylogenetic clades of X. citri subspecies/pathovars. Using this MLST scheme, we examined 2,911 assemblies from NCBI GenBank and identified 15 novel STs from 37 isolates that were misclassified in the NCBI. In total, we identified 545 X. citri assemblies from GenBank with 95% average nucleotide identity to the X. citri type strain and all were classified as one of the 34 STs. All MLST classifications correlated with phylogenetic position inferred from alignments using 92 conserved genes. We observed several instances where strains from different pathovars formed closely related monophyletic clades and shared the same ST, indicating that further investigation of the validity of these pathovars is required. Our MLST scheme described here is a robust tool for rapid classification of X. citri pathovars using WGS and a powerful method for further comprehensive taxonomic revision of X. citri pathovars.

Early termination of the Shiga toxin transcript generates a regulatory small RNA.

Enterohemorrhagic Escherichia coli is a significant human pathogen that causes disease ranging from hemorrhagic colitis to hemolytic uremic syndrome. The latter can lead to potentially fatal renal failure and is caused by the release of Shiga toxins that are encoded within lambdoid bacteriophages. The toxins are encoded within the late transcript of the phage and are regulated by antitermination of the PR' late promoter during lytic induction of the phage. During lysogeny, the late transcript is prematurely terminated at tR' immediately downstream of PR', generating a short RNA that is a byproduct of antitermination regulation. We demonstrate that this short transcript binds the small RNA chaperone Hfq, and is processed into a stable 74-nt regulatory small RNA that we have termed StxS. StxS represses expression of Shiga toxin 1 under lysogenic conditions through direct interactions with the stx1AB transcript. StxS acts in trans to activate expression of the general stress response sigma factor, RpoS, through direct interactions with an activating seed sequence within the 5' UTR. Activation of RpoS promotes high cell density growth under nutrient-limiting conditions. Many phages utilize antitermination to regulate the lytic/lysogenic switch and our results demonstrate that short RNAs generated as a byproduct of this regulation can acquire regulatory small RNA functions that modulate host fitness.

Genomic Epidemiology of Vibrio cholerae O139, Zhejiang Province, China, 1994-2018.

Cholera caused by Vibrio cholerae O139 was first reported in Bangladesh and India in 1992. To determine the genomic epidemiology and origins of O139 in China, we sequenced 104 O139 isolates collected from Zhejiang Province, China, during 1994-2018 and compared them with 57 O139 genomes from other countries in Asia. Most Zhejiang isolates fell into 3 clusters (C1-C3), which probably originated in India (C1) and Thailand (C2 and C3) during the early 1990s. Different clusters harbored different antimicrobial resistance genes and IncA/C plasmids. The integrative and conjugative elements carried by Zhejiang isolates were of a new type, differing from ICEVchInd4 and SXTMO10 by single-nucleotide polymorphisms and presence of genes. Quinolone resistance-conferring mutations S85L in parC and S83I in gyrA occurred in 71.2% of the Zhejiang isolates. The ctxB copy number differed among the 3 clusters. Our findings provided new insights for prevention and control of O139 cholera .

Citrobacter freundii Activation of NLRP3 Inflammasome via the Type VI Secretion System.

Citrobacter freundii is a significant cause of human infections, responsible for food poisoning, diarrhea, and urinary tract infections. We previously identified a highly cytotoxic and adhesive C. freundii strain CF74 expressing a type VI secretion system (T6SS). In this study, we showed that in mice-derived macrophages, C. freundii CF74 activated the Nucleotide Oligomerization Domain -Like Receptor Family, Pyrin Domain Containing 3(NLRP3) inflammasomes in a T6SS-dependent manner. The C. freundii T6SS activated the inflammasomes mainly through caspase 1 and mediated pyroptosis of macrophages by releasing the cleaved gasdermin-N domain. The CF74 T6SS was required for flagellin-induced interleukin 1ß release by macrophages. We further show that the T6SS tail component and effector, hemolysin co-regulation protein-2 (Hcp-2), was necessary and sufficient to trigger NLRP3 inflammasome activation. In vivo, the T6SS played a key role in mediating interleukin 1ß secretion and the survival of mice during C. freundii infection in mice. These findings provide novel insights into the role of T6SS in the pathogenesis of C. freundii.

Population Structure and Multidrug Resistance of Non-O1/Non-O139 Vibrio cholerae in Freshwater Rivers in Zhejiang, China.

To understand the environmental reservoirs of Vibrio cholerae and their public health significance, we surveyed freshwater samples from rivers in two cities (Jiaxing [JX] and Jiande [JD]) in Zhejiang, China. A total of 26 sampling locations were selected, and river water was sampled 456 times from 2015 to 2016 yielding 200 V. cholerae isolates, all of which were non-O1/non-O139. The average isolation rate was 47.3% and 39.1% in JX and JD, respectively. Antibiotic resistance profiles of the V. cholerae isolates were examined with nonsusceptibility to cefazolin (68.70%, 79/115) being most common, followed by ampicillin (47.83%, 55/115) and imipenem (27.83%, 32/115). Forty-two isolates (36.52%, 42/115) were defined as multidrug resistant (MDR). The presence of virulence genes was also determined, and the majority of the isolates were positive for toxR (198/200, 99%) and hlyA (196/200, 98%) with few other virulence genes observed. The population structure of the V. cholerae non-O1/non-O139 sampled was examined using multilocus sequence typing (MLST) with 200 isolates assigned to 128 STs and 6 subpopulations. The non-O1/non-O139 V. cholerae population in JX was more varied than in JD. By clonal complexes (CCs), 31 CCs that contained isolates from this study were shared with other parts of China and/or other countries, suggesting widespread presence of some non-O1/non-O139 clones. Drug resistance profiles differed between subpopulations. The findings suggest that non-O1/non-O139 V. cholerae in the freshwater environment is a potential source of human infections. Routine surveillance of non-O1/non-O139 V. cholerae in freshwater rivers will be of importance to public health.

National Safety Survey of Animal-use Commercial Probiotics and Their Spillover Effects From Farm to Humans: An Emerging Threat to Public Health.

BACKGROUND: Human-use probiotics have recently been associated with clinical infections and antibiotic resistance transfer, raising public concern over their safety. However, despite their extensive application in aquaculture and animal husbandry, the safety of animal-use probiotics remains poorly described. METHODS: We evaluated the safety of 92 animal-use probiotics from China. The pattern of spread of pathogens from probiotics and the consequent public health implications were also examined by conducting in-field genomic surveillance at 2 farms. RESULTS: A total of 123 probiotic Bacillus species isolates were obtained from 92 brands of probiotics, of which 45 isolates were resistant to antibiotics. Notably, 33.7% of probiotic products were contaminated with life-threatening pathogens such as Klebsiella pneumoniae. Genomic surveillance at a chicken farm identified an anthrax toxin-positive Bacillus cereus strain in a probiotic product used as a feed supplement, which was transferred into the groundwater and to a nearby fish farm. Following up retrospective analysis of the surveillance data during 2015-2018 in 3 provinces retrieved 2 B. cereus strains from human with intestinal anthrax symptoms and confirmed the transmission of B. cereus from farm to human. Surveillance of anthrax toxin revealed that cya was detected in 8 of 31 farms. CONCLUSIONS: This study provides the first national safety survey of animal-use probiotics in China and confirms the spillover effects of probiotics from the farms to human. These results suggest that the large-scale application of pathogen-containing probiotics leads to the transfer of pathogens, with worrisome implications for public health. Good Manufacturing Practice should be implemented during the production of all probiotics.Animal-use probiotic products are frequently contaminated with viable pathogenic bacteria. This study revealed that virulent probiotic organisms and contaminating pathogens were colonized with farm animals and shed into the environment, which facilitated the transfer of pathogens to humans.

Nonsynonymous Polymorphism Counts in Bacterial Genomes: a Comparative Examination.

Genomic data reveal single-nucleotide polymorphisms (SNPs) that may carry information about the evolutionary history of bacteria. However, it remains unclear what inferences about selection can be made from genomic SNP data. Bacterial species are often sampled during epidemic outbreaks or within hosts during the course of chronic infections. SNPs obtained from genomic analysis of these data are not necessarily fixed. Treating them as fixed during analysis by using measures such as the ratio of nonsynonymous to synonymous evolutionary changes (dN/dS) may lead to incorrect inferences about the strength and direction of selection. In this study, we consider data from a range of whole-genome sequencing studies of bacterial pathogens and explore patterns of nonsynonymous variation to assess whether evidence of selection can be identified by investigating SNP counts alone across multiple WGS studies. We visualize these SNP data in ways that highlight their relationship to neutral baseline expectations. These neutral expectations are based on a simple model of mutation, from which we simulate SNP accumulation to investigate how SNP counts are distributed under alternative assumptions about positive and negative selection. We compare these patterns with empirical SNP data and illustrate the general difficulty of detecting positive selection from SNP data. Finally, we consider whether SNP counts observed at the between-host population level differ from those observed at the within-host level and find some evidence that suggests that dynamics across these two scales are driven by different underlying processes.IMPORTANCE Identifying selection from SNP data obtained from whole-genome sequencing studies is challenging. Some current measures used to identify and quantify selection acting on genomes rely on fixed differences; thus, these are inappropriate for SNP data where variants are not fixed. With the increase in whole-genome sequencing studies, it is important to consider SNP data in the context of evolutionary processes. How SNPs are counted and analyzed can help in understanding mutation accumulation and trajectories of strains. We developed a tool for identifying possible evidence of selection and for comparative analysis with other SNP data. We propose a model that provides a rule-of-thumb guideline and two new visualization techniques that can be used to interpret and compare SNP data. We quantify the expected proportion of nonsynonymous SNPs in coding regions under neutrality and demonstrate its use in identifying evidence of positive and negative selection from simulations and empirical data.

Multilevel genome typing: genomics-guided scalable resolution typing of microbial pathogens.

BackgroundBoth long- and short-term epidemiology are fundamental to disease control and require accurate bacterial typing. Genomic data resulting from implementation of whole genome sequencing in many public health laboratories can potentially provide highly sensitive and accurate descriptions of strain relatedness. Previous typing efforts using these data have mainly focussed on outbreak detection.AimWe aimed to develop multilevel genome typing (MGT), using consecutive multilocus sequence typing (MLST) schemes of increasing sizes, stepping up from seven-gene MLST to core genome MLST, to allow examination of genetic relatedness at multiple resolution levels.MethodsThe system was applied to Salmonellaenterica serovar Typhimurium. The MLST scheme used at each step (MGT level), defined a given MGT-level specific sequence type (ST). The list of STs generated from all of these increasing MGT levels, was named a genome type (GT). Using MGT, we typed 9,096 previously characterised isolates with publicly available data.ResultsOur approach could identify previously described S. Typhimurium populations, such as the DT104 multidrug resistance lineage (GT 19-2-11) and two invasive lineages of African isolates (GT 313-2-3 and 313-2-752). Further, we showed that MGT-derived clusters can accurately distinguish five outbreaks from each other and five background isolates.ConclusionMGT provides a universal and stable nomenclature at multiple resolutions for S. Typhimurium strains and could be implemented as an internationally standardised strain identification system. While established so far only for S. Typhimurium, the results here suggest that MGT could form the basis for typing systems in other similar microorganisms.

Genomic dissection of the most prevalent Listeria monocytogenes clone, sequence type ST87, in China.

BACKGROUND: Listeria monocytogenes consists of four lineages that occupy a wide variety of ecological niches. Sequence type (ST) 87 (serotype 1/2b), belonging to lineage I, is one of the most common STs isolated from food products, food associated environments and sporadic listeriosis in China. Here, we performed a comparative genomic analysis of the L. monocytogenes ST87 clone by sequencing 71 strains representing a diverse range of sources, different geographical locations and isolation years. RESULTS: The core genome and pan genome of ST87 contained 2667 genes and 3687 genes respectively. Phylogenetic analysis based on core genome SNPs divided the 71 strains into 10 clades. The clinical strains were distributed among multiple clades. Four clades contained strains from multiple geographic regions and showed high genetic diversity. The major gene content variation of ST87 genomes was due to putative prophages, with eleven hotspots of the genome that harbor prophages. All strains carry an intact CRISRP/Cas system. Two major CRISPR spacer profiles were found which were not clustered phylogenetically. A large plasmid of about 90 Kb, which carried heavy metal resistance genes, was found in 32.4% (23/71) of the strains. All ST87 strains harbored the Listeria pathogenicity island (LIPI)-4 and a unique 10-open read frame (ORF) genomic island containing a novel restriction-modification system. CONCLUSION: Whole genome sequence analysis of L. monocytogenes ST87 enabled a clearer understanding of the population structure and the evolutionary history of ST87 L. monocytogenes in China. The novel genetic elements identified may contribute to its virulence and adaptation to different environmental niches. Our findings will be useful for the development of effective strategies for the prevention and treatment of listeriosis caused by this prevalent clone.

Pertactin-Negative and Filamentous Hemagglutinin-Negative Bordetella pertussis, Australia, 2013-2017.

During the 2008-2012 pertussis epidemic in Australia, pertactin (Prn)-negative Bordetella pertussis emerged. We analyzed 78 isolates from the 2013-2017 epidemic and documented continued expansion of Prn-negative ptxP3 B. pertussis strains. We also detected a filamentous hemagglutinin-negative and Prn-negative B. pertussis isolate.

Whole-Genome Sequencing Reveals a Prolonged and Persistent Intrahospital Transmission of Corynebacterium striatum, an Emerging Multidrug-Resistant Pathogen.

Corynebacterium striatum is an emerging multidrug-resistant (MDR) pathogen that occurs primarily among immunocompromised and chronically ill patients. However, little is known about the genomic diversity of C. striatum, which contributes to its long-term persistence and transmission in hospitals. In this study, a total of 192 C. striatum isolates obtained from 14 September 2017 to 29 March 2018 in a hospital in Beijing, China, were analyzed by antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE). Whole-genome sequencing was conducted on 91 isolates. Nearly all isolates (96.3%, 183/190) were MDR. The highest resistance rate was observed for ciprofloxacin (99.0%, 190/192), followed by cefotaxime (90.6%, 174/192) and erythromycin (89.1%, 171/192). PFGE separated the 192 isolates into 79 pulsotypes, and differences in core genome single-nucleotide polymorphisms (SNPs) partitioned the 91 isolates sequenced into four clades. Isolates of the same pulsotype were identical or nearly identical at the genome level, with some exceptions. Two dominant subclones, clade 3a, and clade 4a, were responsible for the hospital-wide dissemination. Genomic analysis further revealed nine resistance genes mobilized by eight unique cassettes. PFGE and whole-genome sequencing revealed that the C. striatum isolates studied were the result mainly of predominant clones spreading in the hospital. C. striatum isolates in the hospital progressively acquired resistance to antimicrobial agents, demonstrating that isolates of C. striatum may adapt rapidly through the acquisition and accumulation of resistance genes and thus evolve into dominant and persistent clones. These insights will be useful for the prevention of C. striatum infection in hospitals.

Case report: whole genome sequencing based investigation of maternal-neonatal listeriosis in Sichuan, China.

BACKGROUND: Neonatal listeriosis is a rare but severe disease manifesting as septicemia and central nervous system (CNS) infections with a high fatality rate of around 20 to 30%. Whole genome sequencing (WGS) is a promising technique for pathogen identification and infection source tracing with its high resolution. CASE PRESENTATION: A case of neonatal sepsis with listeriosis was reported with positive blood culture for Listeria monocytogenes. The case was investigated to confirm the vertical transmission of the infection and identify the potential food source of the maternal L. monocytogenes infection using WGS. L. monocytogenes was isolated from the neonate's blood sample the day after caesarean delivery and from the mother's genital and pudenda swab samples 5 days and 13 days after caesarean delivery. WGS showed that the isolate from the neonate was identical to the genome type of the isolates from the mother, with only one of the 4 isolates from the mother differing by one single nucleotide polymorphism (SNP). By WGS, one L. monocytogenes isolate from a ready-to-eat (RTE) meat sample in the patients' community market shared the same sequence type but was ruled out as the cause of infection, with 57 SNP differences to the strain causing the maternal-neonatal infection. The food isolate also carried a novel plasmid pLM1686 that harbored heavy metal resistance genes. After caesarean section, the mother was treated with a third generation cephalosporin which L. monocytogenes is naturally resistant to, which may explain why genital and pudenda swabs were still culture-positive for L. monocytogenes 13 days after delivery. CONCLUSIONS: Genital swab culture for L. monocytogenes had been informative in the diagnosis of maternal listeriosis in this case. The high resolution of WGS confirmed the maternal-neonatal transmission of L. monocytogenes infection and ruled out the L. monocytogenes contaminated RTE meat from the local market as the direct source of the mother's infection.

Proteomic Adaptation of Australian Epidemic Bordetella pertussis.

Bordetella pertussis causes whooping cough. The predominant strains in Australia changed to single nucleotide polymorphism (SNP) cluster I (pertussis toxin promoter allele ptxP3/pertactin gene allele prn2) from cluster II (non-ptxP3/non-prn2). Cluster I was mostly responsible for the 2008-2012 Australian epidemic and was found to have higher fitness compared to cluster II using an in vivo mouse competition assay, regardless of host's immunization status. This study aimed to identify proteomic differences that explain higher fitness in cluster I using isobaric tags for relative and absolute quantification (iTRAQ), and high-resolution multiple reaction monitoring (MRM-hr). A few key differences in the whole cell and secretome were identified between the cluster I and II strains tested. In the whole cell, nine proteins were upregulated (>1.2 fold change, q < 0.05) and three were downregulated (<0.8 fold change, q < 0.05) in cluster I. One downregulated protein was BP1569, a TLR2 agonist for Th1 immunity. In the secretome, 12 proteins were upregulated and 1 was downregulated which was Bsp22, a type III secretion system (T3SS) protein. Furthermore, there was a trend of downregulation in three T3SS effectors and other virulence factors. Three proteins were upregulated in both whole cell and supernatant: BP0200, molybdate ABC transporter (ModB), and tracheal colonization factor A (TcfA). Important expression differences in lipoprotein, T3SS, and transport proteins between the cluster I and II strains were identified. These differences may affect immune evasion, virulence and metabolism, and play a role in increased fitness of cluster I.

Novel Salmonella enterica Serovar Typhimurium Genotype Levels as Herald of Seasonal Salmonellosis Epidemics.

We examined the population dynamics of Salmonella enterica serovar Typhimurium during seasonal salmonellosis epidemics in New South Wales, Australia, during 2009-2016. Of 15,626 isolates, 5%-20% consisted of novel genotypes. Seasons with salmonellosis epidemics were associated with a reduction in novel genotypes in the preceding winter and spring.

Detection of Multiple Parallel Transmission Outbreak of Streptococcus suis Human Infection by Use of Genome Epidemiology, China, 2005.

Streptococcus suis sequence type 7 emerged and caused 2 of the largest human infection outbreaks in China in 1998 and 2005. To determine the major risk factors and source of the infections, we analyzed whole genomes of 95 outbreak-associated isolates, identified 160 single nucleotide polymorphisms, and classified them into 6 clades. Molecular clock analysis revealed that clade 1 (responsible for the 1998 outbreak) emerged in October 1997. Clades 2-6 (responsible for the 2005 outbreak) emerged separately during February 2002-August 2004. A total of 41 lineages of S. suis emerged by the end of 2004 and rapidly expanded to 68 genome types through single base mutations when the outbreak occurred in June 2005. We identified 32 identical isolates and classified them into 8 groups, which were distributed in a large geographic area with no transmission link. These findings suggest that persons were infected in parallel in respective geographic sites.

Population and evolutionary dynamics of Shiga-toxin producing Escherichia coli O157 in a beef herd: A longitudinal study.

Shiga toxin producing Escherichia coli O157:H7 (STEC O157) is naturally found in the gastrointestinal tract of cattle and can cause severe disease in humans. There is limited understanding of the population dynamics and microevolution of STEC O157 at herd level. In this study, isolates from a closed beef herd of 23 cows were used to examine the population turnover in the herd. Of the nine STEC O157 clades previously described, clade 7 was found in 162 of the 169 isolates typed. Multiple locus variable number tandem repeat analysis (MLVA) differentiated 169 isolates into 33 unique MLVA types. Five predominant MLVA types were evident with most of the remaining types containing only a single isolate. MLVA data suggest that over time clonal replacement occurred within the herd. Genome sequencing of 18 selected isolates found that the isolates were divided into four lineages, representing four different 'clones' in the herd. Genome data confirmed clonal replacement over time and provided evidence of cross transmission of strains between cows. The findings enhanced our understanding of the population dynamics of STEC O157 in its natural host that will help developing effective control measures to prevent the spread of the pathogen to the human population.

Streptococcus himalayensis sp. nov., isolated from the respiratory tract of Marmota himalayana.

Five strains of Gram-positive-staining, catalase-negative, coccus-shaped, chain-forming organisms isolated separately from the respiratory tracts of five Marmota himalayana animals in the Qinghai-Tibet Plateau of China were subjected to phenotypic and molecular taxonomic analyses. Comparative analysis of the 16S rRNA gene indicated that these singular organisms represent a new member of the genus Streptococcus, being phylogenetically closest to Streptococcus marmotae DSM 101995T (98.4 % similarity). The groEL, sodA and rpoB sequence analysis showed interspecies similarity values between HTS2T and Streptococcus. marmotae DSM 101995T, its closest phylogenetic relative based on 16S rRNA gene sequences, of 98.2, 78.8 and 93.7 %, respectively. A whole-genome phylogenetic tree built from 82 core genes of genomes from 16 species of the genus Streptococcus validated that HTS2T forms a distinct subline and exhibits specific phylogenetic affinity with S. marmotae. In silico DNA-DNA hybridization of HTS2T showed an estimated DNA reassociation value of 40.5 % with Streptococcus. marmotae DSM 101995T. On the basis of their phenotypic characteristics and phylogenetic findings, it is proposed that the five isolates be classified as representatives of a novel species of the genus Streptococcus, Streptococcus himalayensis sp. nov. The type strain is HTS2T (=DSM 101997T=CGMCC 1.15533T). The genome of Streptococcus himalayensis sp. nov. strain HTS2T contains 2195 genes with a size of 2 275 471 bp and a mean DNA G+C content of 41.3 mol%.

Novel Capsular Polysaccharide Loci and New Diagnostic Tools for High-Throughput Capsular Gene Typing in Streptococcus suis.

Streptococcus suis is an important pathogen of pigs and may cause serious disease in humans. Serotyping is an important tool for detection and epidemiological studies of S. suis Thirty-three reference serotypes and nine novel cps loci (NCLs) are recognized in S. suis To gain a better understanding of the prevalence and genetic characteristics of NCLs, we investigated the serotype identity of 486 isolates isolated between 2013 and 2015 in China by capsular gene typing methods. Two hundred seventy-six isolates carried NCLs belonging to 16 groups, 8 of which appear to have not been reported previously. These isolates showed autoagglutination, polyagglutination, or nonagglutination with reference antisera and thus were nonserotypeable. Almost all isolates carrying the unknown NCLs were encapsulated, with various capsular thicknesses, indicating that they are most likely novel serotypes. To simultaneously identify the currently recognized 17 NCLs, an 18-plex detection system using the Luminex xTAG universal array technology was developed. Our data also provide valuable genetic information for monitoring the variations within NCLs by investigating the genetic characteristics of different subtypes within NCLs. IMPORTANCE: Nonserotypeable Streptococcus suis isolates have been reported in many studies, and 9 novel cps loci (NCLs) have already been identified in nonserotypeable isolates. Moreover, novel cps loci are continually being found. The main purpose of this study was to investigate the prevalence and characteristics of NCLs in S. suis isolates recovered between 2013 and 2015 in China. This study provides valuable genetic information for monitoring the variations within NCLs. Meanwhile, a fast and cost-effective 18-plex detection system that can simultaneously identify the currently recognized 17 NCLs was developed in this study. This system will serve as a valuable tool for detecting known and identifying additional novel cps loci among nonserotypeable S. suis isolates.