Development and application of a prophage integrase typing scheme for group B Streptococcus

Group B Streptococcus (GBS) is a gram-positive pathogen mainly affecting humans, cattle, and fishes. Mobile genetic elements play an important role in the evolution of GBS, its adaptation to host species and niches, and its pathogenicity. In particular, lysogenic prophages have been associated with a high virulence of certain strains and with their ability to cause invasive infections in humans. It is therefore important to be able to accurately detect and classify prophages in GBS genomes. Several bioinformatic tools for the identification of prophages in bacterial genomes are available on-line. However, genome searches for most of these programs are affected by the composition of their reference database. Lack of databases specific to GBS results in failure to recognize all prophages in the species. Additionally, performance of these programs is affected by genome fragmentation in the case of draft genomes, leading to underestimation of the number of phages. They also prove impractical when dealing with large genome datasets and they do not offer a quick way of classifying bacteriophages. We developed a GBS-specific method to screen genome assemblies for the presence of prophages and to classify them based on a reproducible typing scheme. This was achieved through an extensive search of a vast number of high-quality GBS sequences (n = 572) originating from different host species and countries in order to build a database of phage integrase types, on which the scheme is based. The proposed typing scheme comprises 12 integration sites and sixteen prophage integrase types, including multiple subtypes per integration site and integrase genes that were not site-specific. Two putative phage-inducible chromosomal islands (PICI) and their insertion sites were also identified during the course of these analyses. Phages were common and diverse in all major clonal complexes associated with human disease and detected in isolates from every animal species and continent included in the study. This database will facilitate further work on the prevalence and role of prophages in GBS evolution, and identifies the roles of PICIs in GBS and of prophage in hypervirulent ST283 as areas for further research

Bacterial genomics reveal the complex epidemiology of an emerging pathogen in Arctic and boreal ungulates

Northern ecosystems are currently experiencing unprecedented ecological change, largely driven by a rapidly changing climate. Pathogen range expansion, and emergence and altered patterns of infectious disease, are increasingly reported in wildlife at high latitudes. Understanding the causes and consequences of shifting pathogen diversity and host-pathogen interactions in these ecosystems is important for wildlife conservation, and for indigenous populations that depend on wildlife. Among the key questions are whether disease events are associated with endemic or recently introduced pathogens, and whether emerging strains are spreading throughout the region. In this study, we used a phylogenomic approach to address these questions of pathogen endemicity and spread for Erysipelothrix rhusiopathiae, an opportunistic multi-host bacterial pathogen associated with recent mortalities in arctic and boreal ungulate populations in North America. We isolated E. rhusiopathiae from carcasses associated with large-scale die-offs of muskoxen in the Canadian Arctic Archipelago, and from contemporaneous mortality events and/or population declines among muskoxen in northwestern Alaska and caribou and moose in western Canada. Bacterial genomic diversity differed markedly among these locations; minimal divergence was present among isolates from muskoxen in the Canadian Arctic, while in caribou and moose populations, strains from highly divergent clades were isolated from the same location, or even from within a single carcass. These results indicate that mortalities among northern ungulates are not associated with a single emerging strain of E. rhusiopathiae, and that alternate hypotheses need to be explored. Our study illustrates the value and limitations of bacterial genomic data for discriminating between ecological hypotheses of disease emergence, and highlights the importance of studying emerging pathogens within the broader context of environmental and host factors

Genomic and immunogenic protein diversity of Erysipelothrix rhusiopathiae isolated from pigs in Great Britain: implications for vaccine protection

Erysipelas, caused by the bacterium Erysipelothrix rhusiopathiae, is re-emerging in swine and poultry production systems worldwide. While the global genomic diversity of this species has been characterized, how much of this genomic and functional diversity is maintained at smaller scales is unclear. Specifically, while several key immunogenic surface proteins have been identified for E. rhusiopathiae, little is known about their presence among field strains and their divergence from vaccines, which could result in vaccine failure. Here, a comparative genomics approach was taken to determine the diversity of E. rhusiopathiae strains in pigs in Great Britain over nearly three decades, as well as to assess the field strains’ divergence from the vaccine strain most commonly used in British pigs. In addition, the presence/absence and variability of 13 previously described immunogenic surface proteins was determined, including SpaA which is considered a key immunogen. We found a high diversity of E. rhusiopathiae strains in British pigs, similar to the situation described in European poultry but in contrast to swine production systems in Asia. Of the four clades of E. rhusiopathiae found globally, three were represented among British pig isolates, with Clade 2 being the most common. All British pig isolates had one amino acid difference in the immunoprotective domain of the SpaA protein compared to the vaccine strain. However, we were able to confirm using in silico structural protein analyses that this difference is unlikely to compromise vaccine protection. Of 12 other known immunogenic surface proteins of E. rhusiopathiae examined, 11 were found to be present in all British pig isolates and the vaccine strain, but with highly variable degrees of conservation at the amino acid sequence level, ranging from 0.3 to 27% variant positions. Moreover, the phylogenetic incongruence of these proteins suggests that horizontal transfer of genes encoding for antigens is commonplace for this bacterium. We hypothesize that the sequence variants in these proteins could be responsible for differences in the efficacy of the immune response. Our results provide the necessary basis for testing this hypothesis through in vitro and in vivo studies

The fall and rise of group B Streptococcus in dairy cattle: reintroduction due to human-to-cattle host jumps?

Group B Streptococcus (GBS; Streptococcus agalactiae ) is a major neonatal and opportunistic bacterial pathogen of humans and an important cause of mastitis in dairy cattle with significant impacts on food security. Following the introduction of mastitis control programmes in the 1950s, GBS was nearly eradicated from the dairy industry in northern Europe, followed by re-emergence in the 21st century. Here, we sought to explain this re-emergence based on short and long read sequencing of historical (1953–1978; n=44) and contemporary (1997–2012; n=76) bovine GBS isolates. Our data show that a globally distributed bovine-associated lineage of GBS was commonly detected among historical isolates but never among contemporary isolates. By contrast, tetracycline resistance, which is present in all major GBS clones adapted to humans, was commonly and uniquely detected in contemporary bovine isolates. These observations provide evidence for strain replacement and suggest a human origin of newly emerged strains. Three novel GBS plasmids were identified, including two showing >98 % sequence similarity with plasmids from Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis , which co-exist with GBS in the human oropharynx. Our findings support introduction of GBS into the dairy population due to human-to-cattle jumps on multiple occasions and demonstrate that reverse zoonotic transmission can erase successes of animal disease control campaigns

Genomic epidemiology of clinical ESBL-producing Enterobacteriaceae in a German hospital suggests infections are primarily community- and regionally-acquired

Clinical Enterobacteriaceae isolates that produce extended-spectrum β-lactamases (ESBLs) have been increasingly reported at a global scale. However, comprehensive data on the molecular epidemiology of ESBL-producing strains are limited and few studies have been conducted in non-outbreak situations. We used whole-genome sequencing to describe the population structure of 294 ESBL-producing Escherichia coli and Klebsiella pneumoniae isolates that were recovered from a German community hospital throughout a 1 year sampling period in a non-outbreak situation. We found a high proportion of E. coli isolates (61.5 %) belonged to the globally disseminated extraintestinal pathogenic ST131, whereas a wider diversity of STs was observed among K. pneumoniae isolates. The E. coli ST131 population in this study was shaped by multiple introductions of strains as demonstrated by contextual genomic analysis including ST131 strains from other geographical sources. While no recent common ancestor of the isolates of the current study and other international isolates was found, our clinical isolates clustered with those previously recovered in the region. Furthermore, we found that the isolation of ESBL-producing clinical strains in hospitalized patients could only rarely be associated with likely patient-to-patient transmission, indicating primarily a community and regional acquisition of strains. Further genomic analyses of clinical, carriage and environmental isolates is needed to uncover hidden transmissions and thus discover the most common sources of ESBL-producing pathogen infections in our hospitals

One health research in Northern Tanzania – challenges and progress

East Africa has one of the world’s fastest growing human populations—many of whom are dependent on livestock—as well as some of the world’s largest wildlife populations. Humans, livestock, and wildlife often interact closely, intimately linking human, animal, and environmental health. The concept of One Health captures this interconnectedness, including the social structures and beliefs driving interactions between species and their environments. East African policymakers and researchers are recognising and encouraging One Health research, with both groups increasingly playing a leading role in this subject area. One Health research requires interaction between scientists from different disciplines, such as the biological and social sciences and human and veterinary medicine. Different disciplines draw on norms, methodologies, and terminologies that have evolved within their respective institutions and that may be distinct from or in conflict with one another. These differences impact interdisciplinary research, both around theoretical and methodological approaches and during project operationalisation. We present experiential knowledge gained from numerous ongoing projects in northern Tanzania, including those dealing with bacterial zoonoses associated with febrile illness, foodborne disease, and anthrax. We use the examples to illustrate differences between and within social and biological sciences and between industrialised and traditional societies, for example, with regard to consenting procedures or the ethical treatment of animals. We describe challenges encountered in ethical approval processes, consenting procedures, and field and laboratory logistics and offer suggestions for improvement. While considerable investment of time in sensitisation, communication, and collaboration is needed to overcome interdisciplinary challenges inherent in One Health research, this can yield great rewards in paving the way for successful implementation of One Health projects. Furthermore, continued investment in African institutions and scientists will strengthen the role of East Africa as a world leader in One Health research

Practical and effective diagnosis of animal anthrax in endemic low-resource settings

Anthrax threatens human and animal health, and people’s livelihoods in many rural communities in Africa and Asia. In these areas, anthrax surveillance is challenged by a lack of tools for on-site detection. Furthermore, cultural practices and infrastructure may affect sample availability and quality. Practical yet accurate diagnostic solutions are greatly needed to quantify anthrax impacts. We validated microscopic and molecular methods for the detection of Bacillus anthracis in field-collected blood smears and identified alternative samples suitable for anthrax confirmation in the absence of blood smears. We investigated livestock mortalities suspected to be caused by anthrax in northern Tanzania. Field-prepared blood smears (n = 152) were tested by microscopy using four staining techniques as well as polymerase chain reaction (PCR) followed by Bayesian latent class analysis. Median sensitivity (91%, CI 95% [84–96%]) and specificity (99%, CI 95% [96–100%]) of microscopy using azure B were comparable to those of the recommended standard, polychrome methylene blue, PMB (92%, CI 95% [84–97%] and 98%, CI 95% [95–100%], respectively), but azure B is more available and convenient. Other commonly-used stains performed poorly. Blood smears could be obtained for <50% of suspected anthrax cases due to local customs and conditions. However, PCR on DNA extracts from skin, which was almost always available, had high sensitivity and specificity (95%, CI 95% [90–98%] and 95%, CI 95% [87–99%], respectively), even after extended storage at ambient temperature. Azure B microscopy represents an accurate diagnostic test for animal anthrax that can be performed with basic laboratory infrastructure and in the field. When blood smears are unavailable, PCR using skin tissues provides a valuable alternative for confirmation. Our findings lead to a practical diagnostic approach for anthrax in low-resource settings that can support surveillance and control efforts for anthrax-endemic countries globally

Genomic analysis of group B Streptococcus from milk demonstrates the need for improved biosecurity: a cross-sectional study of pastoralist camels in Kenya

Background Streptococcus agalactiae (Group B Streptococcus, (GBS)) is the leading cause of mastitis (inflammation of the mammary gland) among dairy camels in Sub-Saharan Africa, with negative implications for milk production and quality and animal welfare. Camel milk is often consumed raw and presence of GBS in milk may pose a public health threat. Little is known about the population structure or virulence factors of camel GBS. We investigated the molecular epidemiology of camel GBS and its implications for mastitis control and public health. Results Using whole genome sequencing, we analysed 65 camel milk GBS isolates from 19 herds in Isiolo, Kenya. Six sequence types (STs) were identified, mostly belonging to previously described camel-specific STs. One isolate belonged to ST1, a predominantly human-associated lineage, possibly as a result of interspecies transmission. Most (54/65) isolates belonged to ST616, indicative of contagious transmission. Phylogenetic analysis of GBS core genomes showed similar levels of heterogeneity within- and between herds, suggesting ongoing between-herd transmission. The lactose operon, a marker of GBS adaptation to the mammary niche, was found in 75 % of the isolates, and tetracycline resistance gene tet(M) in all but two isolates. Only the ST1 isolate harboured virulence genes scpB and lmb, which are associated with human host adaptation. Conclusions GBS in milk from Kenyan camel herds largely belongs to ST616 and shows signatures of adaptation to the udder. The finding of similar levels of within- and between herd heterogeneity of GBS in camel herds, as well as potential human-camel transmission highlights the need for improved internal as well as external biosecurity to curb disease transmission and increase milk production

How GBS got its hump: genomic analysis of Group B Streptococcus from camels identifies host restriction as well as mobile genetic elements shared across hosts and pathogens

Group B Streptococcus (GBS) literature largely focuses on humans and neonatal disease, but GBS also affects numerous animals, with significant impacts on health and productivity. Spill-over events occur between humans and animals and may be followed by amplification and evolutionary adaptation in the new niche, including changes in the core or accessory genome content. Here, we describe GBS from one-humped camels (Camelus dromedarius), a relatively poorly studied GBS host of increasing importance for food security in arid regions. Genomic analysis shows that virtually all GBS from camels in East Africa belong to a monophyletic clade, sublineage (SL)609. Capsular types IV and VI, including a new variant of type IV, were over-represented compared to other host species. Two genomic islands with signatures of mobile elements contained most camel-associated genes, including genes for metal and carbohydrate utilisation. Lactose fermentation genes were associated with milk isolates, albeit at lower prevalence in camel than bovine GBS. The presence of a phage with high identity to Streptococcus pneumoniae and Streptococcus suis suggests lateral gene transfer between GBS and bacterial species that have not been described in camels. The evolution of camel GBS appears to combine host restriction with the sharing of accessory genome content across pathogen and host species

The modification and evaluation of an ELISA test for the surveillance of <it>Mycobacterium avium</it> subsp. <it>paratuberculosis</it> infection in wild ruminants

<p>Abstract</p> <p>Background</p> <p>Enzyme-linked immunosorbent assay (ELISA) is often used to test wildlife samples for <it>Mycobacterium avium</it> subsp. <it>paratuberculosis</it> (MAP) infection. However, commercially available kits are only validated for use with domestic ruminant species. A literature review was performed to document the current use of MAP serum ELISA in wild and semi-domestic ruminants. We then modified and evaluated a commercial ELISA kit (IDEXX <it>Mycobacterium paratuberculosis</it> Antibody Test Kit) for use with species for which it was not originally developed: elk (<it>Cervus elaphus</it>), bison (<it>Bison bison</it>) and caribou (<it>Rangifer tarandus</it>). We tested the affinity of different conjugates for immunoglobulin G (IgG) isolated from these species, performed checkerboard tests to determine the optimal dilutions of samples and conjugates, and established cut-off values using two different methods: a Receiver Operational Curve on a panel of known samples for elk, and an alternate method involving a panel of unknown serum samples for the three species.</p> <p>Results</p> <p>We found that the anti-bovine conjugate included in the IDEXX ELISA kit has limited affinity for elk, bison, and caribou IgG. Protein G showed good affinity for IgG of all three species, while anti-deer conjugate also bound elk and caribou IgG. Using Protein G with elk serum, a cut-off sample-to-positive (S/P) value of 0.22 was selected, resulting in a sensitivity and specificity of 73% and 90%, respectively, whereas, using an anti-deer conjugate with elk serum, an S/P cut-off value of 0.29 gave a sensitivity of 68%, with 100% specificity. Cut-off values for bison and caribou using the Protein G conjugate were 0.17 and 0.25 respectively.</p> <p>Conclusions</p> <p>Due to incomplete reporting and a lack of test validation, it is difficult to critically appraise results of many sero-surveys that have previously been done for MAP in wildlife. Commercial ELISA kits may have limited or no capacity to detect antibodies from species other than for which they were developed. In order to generate reliable test results, it is essential to evaluate the test and perform modifications if deemed necessary. Despite the challenges inherent to wildlife diagnostics, we have shown that several methods can be used to improve confidence in test results.</p