Less effective selection leads to larger genomes.

The evolutionary origin of the striking genome size variations found in eukaryotes remains enigmatic. The effective size of populations, by controlling selection efficacy, is expected to be a key parameter underlying genome size evolution. However, this hypothesis has proved difficult to investigate using empirical data sets. Here, we tested this hypothesis using 22 de novo transcriptomes and low-coverage genomes of asellid isopods, which represent 11 independent habitat shifts from surface water to resource-poor groundwater. We show that these habitat shifts are associated with higher transcriptome-wide [Formula: see text] After ruling out the role of positive selection and pseudogenization, we show that these transcriptome-wide [Formula: see text] increases are the consequence of a reduction in selection efficacy imposed by the smaller effective population size of subterranean species. This reduction is paralleled by an important increase in genome size (25% increase on average), an increase also confirmed in subterranean decapods and mollusks. We also control for an adaptive impact of genome size on life history traits but find no correlation between body size, or growth rate, and genome size. We show instead that the independent increases in genome size measured in subterranean isopods are the direct consequence of increasing invasion rates by repeat elements, which are less efficiently purged out by purifying selection. Contrary to selection efficacy, polymorphism is not correlated to genome size. We propose that recent demographic fluctuations and the difficulty of observing polymorphism variation in polymorphism-poor species can obfuscate the link between effective population size and genome size when polymorphism data are used alone.

Life History Traits Impact the Nuclear Rate of Substitution but Not the Mitochondrial Rate in Isopods.

The rate of molecular evolution varies widely among species. Life history traits (LHTs) have been proposed as a major driver of these variations. However, the relative contribution of each trait is poorly understood. Here, we test the influence of metabolic rate (MR), longevity, and generation time (GT) on the nuclear and mitochondrial synonymous substitution rates using a group of isopod species that have made multiple independent transitions to subterranean environments. Subterranean species have repeatedly evolved a lower MR, a longer lifespan and a longer GT. We assembled the nuclear transcriptomes and the mitochondrial genomes of 13 pairs of closely related isopods, each pair composed of one surface and one subterranean species. We found that subterranean species have a lower rate of nuclear synonymous substitution than surface species whereas the mitochondrial rate remained unchanged. We propose that this decoupling between nuclear and mitochondrial rates comes from different DNA replication processes in these two compartments. In isopods, the nuclear rate is probably tightly controlled by GT alone. In contrast, mitochondrial genomes appear to replicate and mutate at a rate independent of LHTs. These results are incongruent with previous studies, which were mostly devoted to vertebrates. We suggest that this incongruence can be explained by developmental differences between animal clades, with a quiescent period during female gametogenesis in mammals and birds which imposes a nuclear and mitochondrial rate coupling, as opposed to the continuous gametogenesis observed in most arthropods.

No Evidence That Nitrogen Limitation Influences the Elemental Composition of Isopod Transcriptomes and Proteomes.

The field of stoichiogenomics aims at understanding the influence of nutrient limitations on the elemental composition of the genome, transcriptome, and proteome. The 20 amino acids and the 4 nt differ in the number of nutrients they contain, such as nitrogen (N). Thus, N limitation shall theoretically select for changes in the composition of proteins or RNAs through preferential use of N-poor amino acids or nucleotides, which will decrease the N-budget of an organism. While these N-saving mechanisms have been evidenced in microorganisms, they remain controversial in multicellular eukaryotes. In this study, we used 13 surface and subterranean isopod species pairs that face strongly contrasted N limitations, either in terms of quantity or quality. We combined in situ nutrient quantification and transcriptome sequencing to test if N limitation selected for N-savings through changes in the expression and composition of the transcriptome and proteome. No evidence of N-savings was found in the total N-budget of transcriptomes or proteomes or in the average protein N-cost. Nevertheless, subterranean species evolving in N-depleted habitats displayed lower N-usage at their third codon positions. To test if this convergent compositional change was driven by natural selection, we developed a method to detect the strand-asymmetric signature that stoichiogenomic selection should leave in the substitution pattern. No such signature was evidenced, indicating that the observed stoichiogenomic-like patterns were attributable to nonadaptive processes. The absence of stoichiogenomic signal despite strong N limitation within a powerful phylogenetic framework casts doubt on the existence of stoichiogenomic mechanisms in metazoans.

Genome Content and Phylogenomics Reveal both Ancestral and Lateral Evolutionary Pathways in Plant-Pathogenic Streptomyces Species.

Streptomyces spp. are highly differentiated actinomycetes with large, linear chromosomes that encode an arsenal of biologically active molecules and catabolic enzymes. Members of this genus are well equipped for life in nutrient-limited environments and are common soil saprophytes. Out of the hundreds of species in the genus Streptomyces, a small group has evolved the ability to infect plants. The recent availability of Streptomyces genome sequences, including four genomes of pathogenic species, provided an opportunity to characterize the gene content specific to these pathogens and to study phylogenetic relationships among them. Genome sequencing, comparative genomics, and phylogenetic analysis enabled us to discriminate pathogenic from saprophytic Streptomyces strains; moreover, we calculated that the pathogen-specific genome contains 4,662 orthologs. Phylogenetic reconstruction suggested that Streptomyces scabies and S. ipomoeae share an ancestor but that their biosynthetic clusters encoding the required virulence factor thaxtomin have diverged. In contrast, S. turgidiscabies and S. acidiscabies, two relatively unrelated pathogens, possess highly similar thaxtomin biosynthesis clusters, which suggests that the acquisition of these genes was through lateral gene transfer.

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.

Timetree of Aselloidea reveals species diversification dynamics in groundwater.

A key challenge for biologists is to document and explain global patterns of diversification in a wide range of environments. Here, we explore patterns of continental-scale diversification in a groundwater species-rich clade, the superfamily Aselloidea (Pancrustacea: Isopoda). Our analyses supported a constant diversification rate during most of the course of Aselloidea evolution, until 4-15 Ma when diversification rates started to decrease. This constant accumulation of lineages challenges the view that groundwater species diversification in temperate regions might have been primarily driven by major changes in physical environment leading to the extinction of surface populations and subsequent synchronous isolation of multiple groundwater populations. Rather than acting synchronously over broad geographic regions, factors causing extinction of surface populations and subsequent reproductive isolation of groundwater populations may act in a local and asynchronous manner, thereby resulting in a constant speciation rate over time. Our phylogeny also revealed several cases of parapatric distributions among closely related surface-water and groundwater species suggesting that species diversification could also arise from a process of disruptive selection along the surface-subterranean environmental gradient. Our results call for re-evaluating the spatial scale and timing of factors causing diversification events in groundwater.

Both nuclear and cytoplasmic polymorphisms are involved in genetic conflicts over male fertility in the gynodioecious snail, Physa acuta.

Gynodioecy, the coexistence of hermaphrodites with females, often reflects conflicts between cytoplasmic male sterility (CMS) genes and nuclear genes restoring male fertility. CMS is frequent in plants and has been recently discovered in one animal: the freshwater snail, Physa acuta. In this system, CMS was linked to a single divergent mitochondrial genome (D), devoid of apparent nuclear restoration. Our study uncovers a second, novel CMS-associated mitogenome (K) in Physa acuta, demonstrating an extraordinary acceleration of molecular evolution throughout the entire K mitochondrial genome, akin to the previously observed pattern in D. This suggests a pervasive occurrence of accelerated evolution in both CMS-associated lineages. Through a 17-generation introgression experiment, we further show that nuclear polymorphisms in K-mitogenome individuals contribute to the restoration of male function in natural populations. Our results underscore shared characteristics in gynodioecy between plants and animals, emphasizing the presence of multiple CMS mitotypes and cytonuclear conflicts. This reaffirms the pivotal role of mitochondria in influencing male function and in generating genomic conflicts that impact reproductive processes in animals.

A collaborative backbone resource for comparative studies of subterranean evolution: The World Asellidae database.

Transition to novel environments, such as groundwater colonization by surface organisms, provides an excellent research ground to study phenotypic evolution. However, interspecific comparative studies on evolution to groundwater life are few because of the challenge in assembling large ecological and molecular resources for species-rich taxa comprised of surface and subterranean species. Here, we make available to the scientific community an operational set of working tools and resources for the Asellidae, a family of freshwater isopods containing hundreds of surface and subterranean species. First, we release the World Asellidae database (WAD) and its web application, a sustainable and FAIR solution to producing and sharing data and biological material. WAD provides access to thousands of species occurrences, specimens, DNA extracts and DNA sequences with rich metadata ensuring full scientific traceability. Second, we perform a large-scale dated phylogenetic reconstruction of Asellidae to support phylogenetic comparative analyses. Of 424 terminal branches, we identify 34 pairs of surface and subterranean species representing independent replicates of the transition from surface water to groundwater. Third, we exemplify the usefulness of WAD for documenting phenotypic shifts associated with colonization of subterranean habitats. We provide the first phylogenetically controlled evidence that body size of males decreases relative to that of females upon groundwater colonization, suggesting competition for rare receptive females selects for smaller, more agile males in groundwater. By making these tools and resources widely accessible, we open up new opportunities for exploring how phenotypic traits evolve in response to changes in selective pressures and trade-offs during groundwater colonization.

Comparative genomic analysis of the genus Staphylococcus including Staphylococcus aureus and its newly described sister species Staphylococcus simiae.

BACKGROUND: Staphylococcus belongs to the Gram-positive low G + C content group of the Firmicutes division of bacteria. Staphylococcus aureus is an important human and veterinary pathogen that causes a broad spectrum of diseases, and has developed important multidrug resistant forms such as methicillin-resistant S. aureus (MRSA). Staphylococcus simiae was isolated from South American squirrel monkeys in 2000, and is a coagulase-negative bacterium, closely related, and possibly the sister group, to S. aureus. Comparative genomic analyses of closely related bacteria with different phenotypes can provide information relevant to understanding adaptation to host environment and mechanisms of pathogenicity. RESULTS: We determined a Roche/454 draft genome sequence for S. simiae and included it in comparative genomic analyses with 11 other Staphylococcus species including S. aureus. A genome based phylogeny of the genus confirms that S. simiae is the sister group to S. aureus and indicates that the most basal Staphylococcus lineage is Staphylococcus pseudintermedius, followed by Staphylococcus carnosus. Given the primary niche of these two latter taxa, compared to the other species in the genus, this phylogeny suggests that human adaptation evolved after the split of S. carnosus. The two coagulase-positive species (S. aureus and S. pseudintermedius) are not phylogenetically closest but share many virulence factors exclusively, suggesting that these genes were acquired by horizontal transfer. Enrichment in genes related to mobile elements such as prophage in S. aureus relative to S. simiae suggests that pathogenesis in the S. aureus group has developed by gene gain through horizontal transfer, after the split of S. aureus and S. simiae from their common ancestor. CONCLUSIONS: Comparative genomic analyses across 12 Staphylococcus species provide hypotheses about lineages in which human adaptation has taken place and contributions of horizontal transfer in pathogenesis.

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

BACKGROUND: Streptococcus 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. RESULTS: Here 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. CONCLUSION: This 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.

Passive sampling of environmental DNA in aquatic environments using 3D-printed hydroxyapatite samplers.

The study of environmental DNA (eDNA) released by aquatic organisms in their habitat offers a fast, noninvasive and sensitive approach to monitor their presence. Common eDNA sampling methods such as water filtration and DNA precipitation are time-consuming, require difficult-to-handle equipment and partially integrate eDNA signals. To overcome these limitations, we created the first proof of concept of a passive, 3D-printed and easy-to-use eDNA sampler. We designed the samplers from hydroxyapatite (HAp samplers), a natural mineral with a high DNA adsorption capacity. The porous structure and shape of the samplers were designed to optimize DNA adsorption and facilitate their handling in the laboratory and in the field. Here we show that HAp samplers can efficiently collect genomic DNA in controlled set-ups, but can also collect animal eDNA under controlled and natural conditions with yields similar to conventional methods. However, we also observed large variations in the amount of DNA collected even under controlled conditions. A better understanding of the DNA-hydroxyapatite interactions on the surface of the samplers is now necessary to optimize eDNA adsorption and to allow the development of a reliable, easy-to-use and reusable eDNA sampling tool.

Transcriptome-wide deregulation of gene expression by artificial light at night in tadpoles of common toads.

Artificial light at night (ALAN) affects numerous physiological and behavioural mechanisms in various species by potentially disturbing circadian timekeeping systems and modifying melatonin levels. However, given the multiple direct and indirect effects of ALAN on organisms, large-scale transcriptomic approaches are essential to assess the global effect of ALAN on biological processes. Moreover, although studies have focused mainly on variations in gene expression during the night in the presence of ALAN, it is necessary to investigate the effect of ALAN on gene expression during the day. In this study, we combined de novo transcriptome sequencing and assembly, and a controlled laboratory experiment to evaluate the transcriptome-wide gene expression response using high-throughput (RNA-seq) in Bufo bufo tadpoles exposed to ecologically relevant light levels. Here, we demonstrated for the first time that ALAN affected gene expression at night (3.5% and 11% of differentially expressed genes when exposed to 0.1 and 5 lx compared to controls, respectively), but also during the day (11.2% of differentially expressed genes when exposed to 5 lx compared to controls) with a dose-dependent effect. ALAN globally induced a downregulation of genes (during the night, 58% and 62% of the genes were downregulated when exposed to 0.1 and 5 lx compared to controls, respectively, and during the day, 61.2% of the genes were downregulated when exposed to 5 lx compared to controls). ALAN effects were detected at very low levels of illuminance (0.1 lx) and affected mainly genes related to the innate immune system and, to a lesser extend to lipid metabolism. These results provide new insights into understanding the effects of ALAN on organism. ALAN impacted the expression of genes linked to a broad range of physiological pathways at very low levels of ALAN during night-time and during daytime, potentially resulting in reduced immune capacity under environmental immune challenges.

Extreme mitochondrial DNA divergence underlies genetic conflict over sex determination.

Cytoplasmic male sterility (CMS) is a form of genetic conflict over sex determination that results from differences in modes of inheritance between genomic compartments.1-3 Indeed, maternally transmitted (usually mitochondrial) genes sometimes enhance their transmission by suppressing the male function in a hermaphroditic organism to the detriment of biparentally inherited nuclear genes. Therefore, these hermaphrodites become functionally female and may coexist with regular hermaphrodites in so-called gynodioecious populations.3 CMS has been known in plants since Darwin's times4 but is previously unknown in the animal kingdom.5-8 We relate the first observation of CMS in animals. It occurs in a freshwater snail population, where some individuals appear unable to sire offspring in controlled crosses and show anatomical, physiological, and behavioral characters consistent with a suppression of the male function. Male sterility is associated with a mitochondrial lineage that underwent a spectacular acceleration of DNA substitution rates, affecting the entire mitochondrial genome-this acceleration concerns both synonymous and non-synonymous substitutions and therefore results from increased mitogenome mutation rates. Consequently, mitochondrial haplotype divergence within the population is exceptionally high, matching that observed between snail taxa that diverged 475 million years ago. This result is reminiscent of similar accelerations in mitogenome evolution observed in plant clades where gynodioecy is frequent,9,10 both being consistent with arms-race evolution of genome regions implicated in CMS.11,12 Our study shows that genomic conflicts can trigger independent evolution of similar sex-determination systems in plants and animals and dramatically accelerate molecular evolution.

Expanded multilocus sequence typing and comparative genomic hybridization of Campylobacter coli isolates from multiple hosts.

The purpose of this work was to evaluate the evolutionary history of Campylobacter coli isolates derived from multiple host sources and to use microarray comparative genomic hybridization to assess whether there are particular genes comprising the dispensable portion of the genome that are more commonly associated with certain host species. Genotyping and ClonalFrame analyses of an expanded 16-gene multilocus sequence typing (MLST) data set involving 85 isolates from 4 different hosts species tentatively supported the development of C. coli host-preferred groups and suggested that recombination has played various roles in their diversification; however, geography could not be excluded as a contributing factor underlying the history of some of the groups. Population genetic analyses of the C. coli pubMLST database by use of STRUCTURE suggested that isolates from swine form a relatively homogeneous genetic group, that chicken and human isolates show considerable genetic overlap, that isolates from ducks and wild birds have similarity with environmental water samples and that turkey isolates have a connection with human infection similar to that observed for chickens. Analysis of molecular variance (AMOVA) was performed on these same data and suggested that host species was a significant factor in explaining genetic variation and that macrogeography (North America, Europe, and the United Kingdom) was not. The microarray comparative genomic hybridization data suggested that there were combinations of genes more commonly associated with isolates derived from particular hosts and, combined with the results on evolutionary history, suggest that this is due to a combination of common ancestry in some cases and lateral gene transfer in others.

Enhancing DNA metabarcoding performance and applicability with bait capture enrichment and DNA from conservative ethanol.

Metabarcoding is often presented as an alternative identification tool to compensate for coarse taxonomic resolution and misidentification encountered with traditional morphological approaches. However, metabarcoding comes with two major impediments which slow down its adoption. First, the picking and destruction of organisms for DNA extraction are time and cost consuming and do not allow organism conservation for further evaluations. Second, current metabarcoding protocols include a PCR enrichment step which induces errors in the estimation of species diversity and relative biomasses. In this study, we first evaluated the capacity of capture enrichment to replace PCR enrichment using controlled freshwater macrozoobenthos mock communities. Then, we tested if DNA extracted from the fixative ethanol (etDNA) of the same mock communities can be used as an alternative to DNA extracted from pools of whole organisms (bulk DNA). We show that capture enrichment provides more reliable and accurate representation of species occurrences and relative biomasses in comparison with PCR enrichment for bulk DNA. While etDNA does not permit to estimate relative biomasses, etDNA and bulk DNA provide equivalent species detection rates. Thanks to its robustness to mismatches, capture enrichment is already an efficient alternative to PCR enrichment for metabarcoding and, if coupled to etDNA, is a time-saver option in studies where presence information only is sufficient.

Bedrock radioactivity influences the rate and spectrum of mutation.

All organisms on Earth are exposed to low doses of natural radioactivity but some habitats are more radioactive than others. Yet, documenting the influence of natural radioactivity on the evolution of biodiversity is challenging. Here, we addressed whether organisms living in naturally more radioactive habitats accumulate more mutations across generations using 14 species of waterlice living in subterranean habitats with contrasted levels of radioactivity. We found that the mitochondrial and nuclear mutation rates across a waterlouse species' genome increased on average by 60% and 30%, respectively, when radioactivity increased by a factor of three. We also found a positive correlation between the level of radioactivity and the probability of G to T (and complementary C to A) mutations, a hallmark of oxidative stress. We conclude that even low doses of natural bedrock radioactivity influence the mutation rate possibly through the accumulation of oxidative damage, in particular in the mitochondrial genome.

Positive selection in penicillin-binding proteins 1a, 2b, and 2x from Streptococcus pneumoniae and its correlation with amoxicillin resistance development.

The efficacy of beta-lactam antibiotics in Streptococcus pneumoniae has been compromised because of the development of altered penicillin-binding proteins (PBPs), however, this has been less so for amoxicillin than for penicillin. Recently, there have been a number of important methods developed to detect molecular adaptation in protein coding genes. The purpose of this study is to employ modern molecular selection approaches to predict sites under positive selection pressure in PBPs, derived from a large international S. pneumoniae collection of amoxicillin resistant and susceptible isolates, and encompassing a comparative data set of 354 pbp1a, 335 pbp2b, and 389 pbp2x gene sequences. A correspondence discriminant analysis (CDA) of positively selected pbp sites and amoxicillin MIC (minimum inhibitory concentration) values is then used to detect sites under positive selection pressure that are important in discriminating different amoxicillin MICs. Molecular adaptation was evident throughout PBP2X, with numerous positively selected sites in both the transpeptidase (TP) and C-terminal domains, strongly correlated with discriminating amoxicillin MICs. In the case of PBP1A positive selection was present in the glycosyltransfer (GT), TP and C-terminal domains. Sites within the TP domain tended to be correlated with the discrimination of low from intermediate MICs, whereas sites within the C-terminal tail, with a discrimination of intermediate from fully resistant. Most of the positively selected sites within PBP2B were in the N-terminal domain and were not correlated with amoxicillin MICs, however, several sites taken from the literature for the TP domain were strongly associated with discriminating high from intermediate level amoxicillin resistance. Many of the positively selected sites could be directly associated with functional inferences based on the crystal structures of these proteins. Our results suggest that clinical emphasis on TP domain sequences of these proteins may result in missing information relevant to antibiotic resistance development.

The relative frequency of intraspecific lateral gene transfer of penicillin binding proteins 1a, 2b, and 2x, in amoxicillin resistant Streptococcus pneumoniae.

Evidence exists for both interspecific and intraspecific recombination (lateral gene transfer; LGT) involving Streptococcus pneumoniae pbp (penicillin binding protein) loci. LGT of capsular genes, or serotype switching, is also know to occur between S. pneumoniae of different serotype. It is not clear whether intraspecific pbp LGT is relatively common, whether there is a difference in the relative frequency of intraspecific LGT of different pbps, and whether serotype switching is more or less frequent than pbp LGT. The purpose of this study was to use comparative evolutionary biology analysis of 216 international clinical S. pneumoniae isolates, from the Alexander Project collection, to gain insight on these issues, as well as the possible role they might be playing in spreading amoxicillin resistance. All 216 isolates were genotyped using MLST and complete or nearly complete sequences for pbp1a, pbp2b, and pbp2x were determined. Amoxicillin MICs were available for each isolate. pbps were genotyped using phylogenetics and two or more pbp types within a MLST sequence type (ST) or clonal complex were taken as putative cases of pbp LGT; these hypotheses were statistically evaluated using the approximately unbiased (AU) test. Serotypes were determined for 171 of these isolates and the minimum number of switching events necessary to explain the serotype phenotypes for each of the STs and clonal complexes were evaluated. The majority (78%) of the amoxicillin resistant isolates were comprised in 5 clonal complexes. The relative frequency of pbp LGT was greatest for pbp2b and 2x (minimum of 10.2 and 7.8%, respectively, of the isolates consistent with the LGT hypothesis), followed by 1a (3.9%). Serotype switching was more frequent than intraspecific pbp LGT (33% of isolates consistent with serotype switching hypothesis). Although intraspecific LGT of pbps is occurring and has played a role in the spread of amoxicillin resistance in S. pneumoniae, clonal dissemination appears to be more significant.

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.