The equine disease strangles, which is characterized by the formation of abscesses in the lymph nodes of the head and neck, is one of the most frequently diagnosed infectious diseases of horses around the world. The causal agent, Streptococcus equi subspecies equi , establishes a persistent infection in approximately 10 % of animals that recover from the acute disease. Such ‘carrier’ animals appear healthy and are rarely identified during routine veterinary examinations pre-purchase or transit, but can transmit S. equi to naïve animals initiating new episodes of disease. Here, we report the analysis and visualization of phylogenomic and epidemiological data for 670 isolates of S. equi recovered from 19 different countries using a new core-genome multilocus sequence typing (cgMLST) web bioresource. Genetic relationships among all 670 S. equi isolates were determined at high resolution, revealing national and international transmission events that drive this endemic disease in horse populations throughout the world. Our data argue for the recognition of the international importance of strangles by the Office International des Épizooties to highlight the health, welfare and economic cost of this disease. The Pathogenwatch cgMLST web bioresource described herein is available for tailored genomic analysis of populations of S. equi and its close relative S. equi subspecies zooepidemicus that are recovered from horses and other animals, including humans, throughout the world. This article contains data hosted by Microreact.

BackgroundUtilising next generation sequencing to interrogate saturated bacterial mutant libraries provides unprecedented information for the assignment of genome-wide gene essentiality. Exposure of saturated mutant libraries to specific conditions and subsequent sequencing can be exploited to uncover gene essentiality relevant to the condition. Here we present a barcoded transposon directed insertion-site sequencing (TraDIS) system to define an essential gene list for Streptococcus equi subsp. equi, the causative agent of strangles in horses, for the first time. The gene essentiality data for this group C Streptococcus was compared to that of group A and B streptococci.ResultsSix barcoded variants of pGh9:ISS1 were designed and used to generate mutant libraries containing between 33,000-66,000 unique mutants. TraDIS was performed on DNA extracted from each library and data were analysed separately and as a combined master pool. Gene essentiality determined that 19.5% of the S. equi genome was essential. Gene essentialities were compared to those of group A and group B streptococci, identifying concordances of 90.2% and 89.4%, respectively and an overall concordance of 83.7% between the three species.ConclusionsThe use of barcoded pGh9:ISS1 to generate mutant libraries provides a highly useful tool for the assignment of gene function in S. equi and other streptococci. The shared essential gene set of group A, B and C streptococci provides further evidence of the close genetic relationships between these important pathogenic bacteria. Therefore, the ABC of gene essentiality reported here provides a solid foundation towards reporting the functional genome of streptococci.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3794-3) contains supplementary material, which is available to authorized users.

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The human bacterial pathogen Streptococcus pyogenes (group A streptococcus [GAS]) causes more than 600 million cases of pharyngitis annually worldwide, 15 million of which occur in the United States. The human oropharynx is the primary anatomic site for GAS colonization and infection, and saliva is the first material encountered. Using a genome-wide transposon mutant screen, we identified 92 GAS genes required for wild-type fitness in human saliva. Many of the identified genes are involved in carbohydrate transport/metabolism, amino acid transport/metabolism, and inorganic ion transport/metabolism. The new information is potentially valuable for developing novel GAS therapeutics and vaccine research.

Streptococcus pyogenes (group A Streptococcus, or GAS) causes 23 600 million cases of pharyngitis each year. Despite this considerable disease 24

23[ABSTRACT] 24Necrotizing fasciitis and myositis are devastating infections characterized 25 by high mortality. Group A streptococcus (GAS) is a common cause of 26 these infections, but the molecular pathogenesis is poorly understood. We 27 report a genome-wide analysis using serotype M1 and M28 strains that 28 identified novel GAS genes contributing to necrotizing myositis in 29 nonhuman primates (NHP), a clinically relevant model. Using transposon 30 directed insertion-site sequencing (TraDIS) we identified 126 and 116 GAS 31 genes required for infection by serotype M1 and M28 organisms, 32 respectively. For both M1 and M28 strains, more than 25% of the GAS 33 genes required for necrotizing myositis encode known or putative 34 transporters. Thirteen GAS transporters contributed to both M1 and M28 35 strain fitness in NHP myositis, including putative importers for amino 36 acids, carbohydrates, and vitamins, and exporters for toxins, quorum 37 sensing peptides, and uncharacterized molecules. Targeted deletion of 38 genes encoding five transporters confirmed that each isogenic mutant 39 strain was significantly impaired in causing necrotizing myositis in NHPs. 40 qRT-PCR analysis showed that these five genes are expressed in infected 41 NHP and human skeletal muscle. Certain substrate-binding lipoproteins of 42 these transporters, such as Spy0271 and Spy1728, were previously 43 documented to be surface-exposed, suggesting that our findings have 44 translational research implications. 45 46 revealed some of the GAS molecules that contribute to the pathogenesis of 63 necrotizing fasciitis and myositis, including M protein (8, 9), extracellular cysteine 64 protease streptococcal pyrogenic exotoxin B (SpeB) (10-13), hyaluronic acid 65 capsule (14), and cytotoxins NADase and streptolysin O (15-21). However, 66although the genome of GAS is relatively small (~1,800 genes) (22, 23), current 67 understanding of the molecular pathogenesis of GAS necrotizing fasciitis and 68 myositis is limited. 69High-throughput genome-wide screens based on transposon mutagenesis 70 strategies are very useful in providing new information about the genetic basis of 71 bacterial virulence. Technologies such as signature-tagged mutagenesis (STM), 72 transposon site hybridization (TraSH), and Tn-seq have been applied 73 successfully to many bacterial pathogens to identify genes required for fitness 74 under diverse in vivo and ex vivo conditions (24-30). In GAS, genome-wide 75 transposon mutagenesis screens have been used to identify genes contributing 76 to fitness during growth in human blood ex vivo, human saliva ex vivo, and 77 mouse subcutaneous infections (24, 30-32). However, a genome-wide 78 investigation of the GAS genes contributing to fitness in necrotizing myositis has 79 not been undertaken. 80Analysis of the molecular pathogenesis of GAS necrotizing myositis 81 requires use of appropriate animal models. Toward this end, mouse and 82 nonhuman primate (NHP) necrotizing myositis models have been developed that 83 approximate this d...

Iceland is free of the major infectious diseases of horses. However, in 2010 an epidemic of respiratory disease of unknown cause spread through the country’s native horse population of 77,000. Microbiological investigations ruled out known viral agents but identified the opportunistic pathogen Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) in diseased animals. We sequenced the genomes of 257 isolates of S. zooepidemicus to differentiate epidemic from endemic strains. We found that although multiple endemic clones of S. zooepidemicus were present, one particular clone, sequence type 209 (ST209), was likely to have been responsible for the epidemic. Concurrent with the epidemic, ST209 was also recovered from a human case of septicemia, highlighting the pathogenic potential of this strain. Epidemiological investigation revealed that the incursion of this strain into one training yard during February 2010 provided a nidus for the infection of multiple horses that then transmitted the strain to farms throughout Iceland. This study represents the first time that whole-genome sequencing has been used to investigate an epidemic on a national scale to identify the likely causative agent and the link to an associated zoonotic infection. Our data highlight the importance of national biosecurity to protect vulnerable populations of animals and also demonstrate the potential impact of S. zooepidemicus transmission to other animals, including humans.

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