Vincent P. Richards

Evolutionary and population genomics of the cavity causing bacteria Streptococcus mutans

Streptococcus mutans is widely recognized as one of the key etiological agents of human dental caries. Despite its role in this important disease, our present knowledge of gene content variability across the species and its relationship to adaptation is minimal. Estimates of its demographic history are not available. In this study, we generated genome sequences of 57 S. mutans isolates, as well as representative strains of the most closely related species to S. mutans (S. ratti, S. macaccae, and S. criceti), to identify the overall structure and potential adaptive features of the dispensable and core components of the genome. We also performed population genetic analyses on the core genome of the species aimed at understanding the demographic history, and impact of selection shaping its genetic variation. The maximum gene content divergence among strains was approximately 23%, with the majority of strains diverging by 5-15%. The core genome consisted of 1,490 genes and the pan-genome approximately 3,296. Maximum likelihood analysis of the synonymous site frequency spectrum (SFS) suggested that the S. mutans population started expanding exponentially approximately 10,000 years ago (95% confidence interval [CI]: 3,268-14,344 years ago), coincidental with the onset of human agriculture. Analysis of the replacement SFS indicated that a majority of these substitutions are under strong negative selection, and the remainder evolved neutrally. A set of 14 genes was identified as being under positive selection, most of which were involved in either sugar metabolism or acid tolerance. Analysis of the core genome suggested that among 73 genes present in all isolates of S. mutans but absent in other species of the mutans taxonomic group, the majority can be associated with metabolic processes that could have contributed to the successful adaptation of S. mutans to its new niche, the human mouth, and with the dietary changes that accompanied the origin of agriculture.

Comparative genomics and the role of lateral gene transfer in the evolution of bovine adapted Streptococcus agalactiae.

In addition to causing severe invasive infections in humans, Streptococcus agalactiae, or group B Streptococcus (GBS), is also a major cause of bovine mastitis. Here we provide the first genome sequence for S. agalactiae isolated from a cow diagnosed with clinical mastitis (strain FSL S3-026). Comparison to eight S. agalactiae genomes obtained from human disease isolates revealed 183 genes specific to the bovine strain. Subsequent polymerase chain reaction (PCR) screening for the presence/absence of a subset of these loci in additional bovine and human strains revealed strong differentiation between the two groups (Fisher exact test: p<0.0001). The majority of the bovine strain-specific genes (∼ 85%) clustered tightly into eight genomic islands, suggesting these genes were acquired through lateral gene transfer (LGT). This bovine GBS also contained an unusually high proportion of insertion sequences (4.3% of the total genome), suggesting frequent genomic rearrangement. Comparison to other mastitis-causing species of bacteria provided strong evidence for two cases of interspecies LGT within the shared bovine environment: bovine S. agalactiae with Streptococcus uberis (nisin U operon) and Streptococcus dysgalactiae subsp. dysgalactiae (lactose operon). We also found evidence for LGT, involving the salivaricin operon, between the bovine S. agalactiae strain and either Streptococcus pyogenes or Streptococcus salivarius. Our findings provide insight into mechanisms facilitating environmental adaptation and acquisition of potential virulence factors, while highlighting both the key role LGT has played in the recent evolution of the bovine S. agalactiae strain, and the importance of LGT among pathogens within a shared environment.

Genetic connectivity in the Florida reef system: comparative phylogeography of commensal invertebrates with contrasting reproductive strategies.

Effective spatial management of coral reefs including design of marine protected areas requires an understanding of interpopulation genetic connectivity. We assessed gene flow along 355 km of the Florida reef system and between Florida and Belize in three commensal invertebrates occupying the same host sponge (Callyspongia vaginalis) but displaying contrasting reproductive dispersal strategies: the broadcast‐spawning brittle star Ophiothrix lineata and two brooding amphipods Leucothoe kensleyi and Leucothoe ashleyae. Multiple analytical approaches to sequence variation in the mitochondrial COI gene demonstrated a high degree of overall connectivity for all three species along the Florida reef system. Ophiothrix lineata showed significant genetic structuring between Florida and Belize, and a pattern of isolation by distance but no significant genetic structuring along the Florida coastline. Bayesian estimates of migration detected a strong southerly dispersal bias for O. lineata along the Florida reef system, contrary to the general assumption of northerly gene flow in this region based on the direction of the Florida Current. Both amphipods, despite direct development, also showed high gene flow along the Florida reef system. Multiple inferences of long‐distance dispersal from a nested clade analysis support the hypothesis that amphipod transport, possibly in detached sponge fragments, could generate the high levels of overall gene flow observed. However, this transport mechanism appears much less effective across deep water as connectivity between Florida and Belize (1072 km) is highly restricted.

Species Delineation and Evolutionary History of the Globally Distributed Spotted Eagle Ray (Aetobatus narinari)

The spotted eagle ray (Aetobatus narinari), a large coral reef-associated batoid of conservation concern, is currently described as a single, circumglobally distributed species. However, geographic differences in its morphology and parasite diversity have raised unconfirmed suspicions that A. narinari may constitute a species complex. We used 1570 bp of mitochondrial and nuclear sequence data (cytochrome b, cytochrome c oxidase subunit I, and internal transcribed spacer 2) to assess the validity of A. narinari as a single cosmopolitan species and infer its evolutionary history. Specimens from 4 major geographic regions were examined: the Central Atlantic, Eastern Pacific, Western Pacific, and Central Pacific. Phylogenies described 3 distinct, reciprocally monophyletic lineages with no genetic exchange among regions. Based on combined genealogical concordance and genetic distance criteria, we recommend that the Western/Central Pacific lineage be recognized as a distinct species from lineages in the Central Atlantic and Eastern Pacific. The latter 2 lineages, separated by the Isthmus of Panama, are proposed as subspecies. A basal position in phylogenetic analyses and statistical parsimony results support an Indo-West Pacific origin for the A. narinari species complex, with subsequent westerly dispersal around the southern tip of Africa into the Atlantic and then into the Eastern Pacific.

Phylogenomics and the Dynamic Genome Evolution of the Genus Streptococcus

The genus Streptococcus comprises important pathogens that have a severe impact on human health and are responsible for substantial economic losses to agriculture. Here, we utilize 46 Streptococcus genome sequences (44 species), including eight species sequenced here, to provide the first genomic level insight into the evolutionary history and genetic basis underlying the functional diversity of all major groups of this genus. Gene gain/loss analysis revealed a dynamic pattern of genome evolution characterized by an initial period of gene gain followed by a period of loss, as the major groups within the genus diversified. This was followed by a period of genome expansion associated with the origins of the present extant species. The pattern is concordant with an emerging view that genomes evolve through a dynamic process of expansion and streamlining. A large proportion of the pan-genome has experienced lateral gene transfer (LGT) with causative factors, such as relatedness and shared environment, operating over different evolutionary scales. Multiple gene ontology terms were significantly enriched for each group, and mapping terms onto the phylogeny showed that those corresponding to genes born on branches leading to the major groups represented approximately one-fifth of those enriched. Furthermore, despite the extensive LGT, several biochemical characteristics have been retained since group formation, suggesting genomic cohesiveness through time, and that these characteristics may be fundamental to each group. For example, proteolysis: mitis group; urea metabolism: salivarius group; carbohydrate metabolism: pyogenic group; and transcription regulation: bovis group.

Phenotypic heterogeneity of genomically-diverse isolates of Streptococcus mutans.

High coverage, whole genome shotgun (WGS) sequencing of 57 geographically- and genetically-diverse isolates of Streptococcus mutans from individuals of known dental caries status was recently completed. Of the 57 sequenced strains, fifteen isolates, were selected based primarily on differences in gene content and phenotypic characteristics known to affect virulence and compared with the reference strain UA159. A high degree of variability in these properties was observed between strains, with a broad spectrum of sensitivities to low pH, oxidative stress (air and paraquat) and exposure to competence stimulating peptide (CSP). Significant differences in autolytic behavior and in biofilm development in glucose or sucrose were also observed. Natural genetic competence varied among isolates, and this was correlated to the presence or absence of competence genes, comCDE and comX, and to bacteriocins. In general strains that lacked the ability to become competent possessed fewer genes for bacteriocins and immunity proteins or contained polymorphic variants of these genes. WGS sequence analysis of the pan-genome revealed, for the first time, components of a Type VII secretion system in several S. mutans strains, as well as two putative ORFs that encode possible collagen binding proteins located upstream of the cnm gene, which is associated with host cell invasiveness. The virulence of these particular strains was assessed in a wax-worm model. This is the first study to combine a comprehensive analysis of key virulence-related phenotypes with extensive genomic analysis of a pathogen that evolved closely with humans. Our analysis highlights the phenotypic diversity of S. mutans isolates and indicates that the species has evolved a variety of adaptive strategies to persist in the human oral cavity and, when conditions are favorable, to initiate disease.

Comparative characterization of the virulence gene clusters (lipooligosaccharide [LOS] and capsular polysaccharide [CPS]) for Campylobacter coli, Campylobacter jejuni subsp. jejuni and related Campylobacter species.

Campylobacter jejuni subsp. jejuni and Campylobacter coli are leading causes of gastroenteritis, with virulence linked to cell surface carbohydrate diversity. Although the associated gene clusters are well studied for C. jejuni subsp. jejuni, C. coli has been largely neglected. Here we provide comparative analysis of the lipooligosaccharide (LOS) and capsular polysaccharide (CPS) gene clusters, using genome and cluster sequence data for 36 C. coli strains, 67 C. jejuni subsp. jejuni strains and ten additional Campylobacter species. Similar to C. jejuni subsp. jejuni, C. coli showed high LOS/CPS gene diversity, with each cluster delineated into eight gene content classes. This diversity was predominantly due to extensive gene gain/loss, with the lateral transfer of genes likely occurring both within and between species and also between the LOS and CPS. Additional mechanisms responsible for LOS/CPS diversity included phase-variable homopolymeric repeats, gene duplication/inactivation, and possibly host environment selection pressure. Analyses also showed that (i) strains of C. coli and Campylobacter upsaliensis possessed genes homologous to the sialic acid genes implicated in the neurological disorder Guillain-Barré syndrome (GBS), and (ii) C. coli LOS classes were differentiated between bovine and poultry hosts, potentially aiding post infection source tracking.

Genome characterization and population genetic structure of the zoonotic pathogen, Streptococcus canis

BackgroundStreptococcus canis is an important opportunistic pathogen of dogs and cats that can also infect a wide range of additional mammals including cows where it can cause mastitis. It is also an emerging human pathogen.ResultsHere we provide characterization of the first genome sequence for this species, strain FSL S3-227 (milk isolate from a cow with an intra-mammary infection). A diverse array of putative virulence factors was encoded by the S. canis FSL S3-227 genome. Approximately 75% of these gene sequences were homologous to known Streptococcal virulence factors involved in invasion, evasion, and colonization. Present in the genome are multiple potentially mobile genetic elements (MGEs) [plasmid, phage, integrative conjugative element (ICE)] and comparison to other species provided convincing evidence for lateral gene transfer (LGT) between S. canis and two additional bovine mastitis causing pathogens (Streptococcus agalactiae, and Streptococcus dysgalactiae subsp. dysgalactiae), with this transfer possibly contributing to host adaptation. Population structure among isolates obtained from Europe and USA [bovine = 56, canine = 26, and feline = 1] was explored. Ribotyping of all isolates and multi locus sequence typing (MLST) of a subset of the isolates (n = 45) detected significant differentiation between bovine and canine isolates (Fisher exact test: P = 0.0000 [ribotypes], P = 0.0030 [sequence types]), suggesting possible host adaptation of some genotypes. Concurrently, the ancestral clonal complex (54% of isolates) occurred in many tissue types, all hosts, and all geographic locations suggesting the possibility of a wide and diverse niche.ConclusionThis study provides evidence highlighting the importance of LGT in the evolution of the bacteria S. canis, specifically, its possible role in host adaptation and acquisition of virulence factors. Furthermore, recent LGT detected between S. canis and human bacteria (Streptococcus urinalis) is cause for concern, as it highlights the possibility for continued acquisition of human virulence factors for this emerging zoonotic pathogen.

A Highly Arginolytic Streptococcus Species That Potently Antagonizes Streptococcus mutans

ABSTRACT The ability of certain oral biofilm bacteria to moderate pH through arginine metabolism by the arginine deiminase system (ADS) is a deterrent to the development of dental caries. Here, we characterize a novel Streptococcus strain, designated strain A12, isolated from supragingival dental plaque of a caries-free individual. A12 not only expressed the ADS pathway at high levels under a variety of conditions but also effectively inhibited growth and two intercellular signaling pathways of the dental caries pathogen Streptococcus mutans. A12 produced copious amounts of H2O2 via the pyruvate oxidase enzyme that were sufficient to arrest the growth of S. mutans. A12 also produced a protease similar to challisin (Sgc) of Streptococcus gordonii that was able to block the competence-stimulating peptide (CSP)–ComDE signaling system, which is essential for bacteriocin production by S. mutans. Wild-type A12, but not an sgc mutant derivative, could protect the sensitive indicator strain Streptococcus sanguinis SK150 from killing by the bacteriocins of S. mutans. A12, but not S. gordonii, could also block the XIP (comX -inducing peptide) signaling pathway, which is the proximal regulator of genetic competence in S. mutans, but Sgc was not required for this activity. The complete genome sequence of A12 was determined, and phylogenomic analyses compared A12 to streptococcal reference genomes. A12 was most similar to Streptococcus australis and Streptococcus parasanguinis but sufficiently different that it may represent a new species. A12-like organisms may play crucial roles in the promotion of stable, health-associated oral biofilm communities by moderating plaque pH and interfering with the growth and virulence of caries pathogens.

Genome Based Phylogeny and Comparative Genomic Analysis of Intra-Mammary Pathogenic Escherichia coli

Escherichia coli is an important cause of bovine mastitis and can cause both severe inflammation with a short-term transient infection, as well as less severe, but more chronic inflammation and infection persistence. E. coli is a highly diverse organism that has been classified into a number of different pathotypes or pathovars, and mammary pathogenic E. coli (MPEC) has been proposed as a new such pathotype. The purpose of this study was to use genome sequence data derived from both transient and persistent MPEC isolates (two isolates of each phenotype) to construct a genome-based phylogeny that places MPEC in its phylogenetic context with other E. coli pathovars. A subsidiary goal was to conduct comparative genomic analyses of these MPEC isolates with other E. coli pathovars to provide a preliminary perspective on loci that might be correlated with the MPEC phenotype. Both concatenated and consensus tree phylogenies did not support MPEC monophyly or the monophyly of either transient or persistent phenotypes. Three of the MPEC isolates (ECA-727, ECC-Z, and ECA-O157) originated from within the predominately commensal clade of E. coli, referred to as phylogroup A. The fourth MPEC isolate, of the persistent phenotype (ECC-1470), was sister group to an isolate of ETEC, falling within the E. coli B1 clade. This suggests that the MPEC phenotype has arisen on numerous independent occasions and that this has often, although not invariably, occurred from commensal ancestry. Examination of the genes present in the MPEC strains relative to the commensal strains identified a consistent presence of the type VI secretion system (T6SS) in the MPEC strains, with only occasional representation in commensal strains, suggesting that T6SS may be associated with MPEC pathogenesis and/or as an inter-bacterial competitive attribute and therefore could represent a useful target to explore for the development of MPEC specific inhibitors.