Understanding the molecular determinants driving the immunological specificity of the protective pilus 2a backbone protein of Group B Streptococcus

The pilus 2a backbone protein (BP-2a) is one of the most structurally and functionally characterized components of a potential vaccine formulation against Group B Streptococcus. It is characterized by six main immunologically distinct allelic variants, each inducing variant-specific protection. To investigate the molecular determinants driving the variant immunogenic specificity of BP-2a, in terms of single residue contributions, we generated six monoclonal antibodies against a specific protein variant based on their capability to recognize the polymerized pili structure on the bacterial surface. Three mAbs were also able to induce complement-dependent opsonophagocytosis killing of live GBS and target the same linear epitope present in the structurally defined and immunodominant domain D3 of the protein. Molecular docking between the modelled scFv antibody sequences and the BP-2a crystal structure revealed the potential role at the binding interface of some non-conserved antigen residues. Mutagenesis analysis confirmed the necessity of a perfect balance between charges, size and polarity at the binding interface to obtain specific binding of mAbs to the protein antigen for a neutralizing response

Molecular basis of the interactions between carbohydrate antigens and monoclonal antibodies

Carbohydrate-based vaccines induce an immune response against surface glycans found on pathogenic bacteria and are used to protect children from meningitis and other bacterial diseases. The definition of the epitope targeted by protective antibodies is a crucial information for the design, description and even registration of glycoconjugate vaccines. The aim of this project is the characterization at atomic level of the interaction between Group B Streptococcus (GBS) oligosaccharides with functional antibodies mediating bacterial phagocytic killing. The specificity of anti-GBS type III polysaccharide (GBS PSIII) antibodies has been ascribed to a conformational epitope with an extended helical structure which would be formed by multiple repeating units. Here fragments of GBS PSIII were obtained by chemical synthesis and by sizing of the native polysaccharide from bacterial source. The oligosaccharides (OSs) were purified by ion exchange chromatography and were characterized by mass spectrometry, HPLC and NMR analysis. First, the obtained OSs were used to characterize the interaction to protective anti PSIII rabbit monoclonal antibody (mAb) by competitive ELISA and surface plasmon resonance analysis. The dependence of binding affinity on oligosaccharide length was confirmed and the minimal structures showing comparable binding to full-length PS III were identified. Next, a combination of saturation transfer difference NMR (STD-NMR) and X-ray crystallography was used to map at atomic level the interactions in the rabbit mAb-OS complex. The challenging crystallization of the carbohydrate-protein complex was achieved and the molecular bases of the antigen-antibody interaction were unveiled. The obtained results were used to optimize a vaccine based on short GBS PSIII fragments

Streptococcus agalactiae causing human infections : genetic diversity and capsular switching

Tese de doutoramento, Ciências e Tecnologias da Saúde (Microbiologia), Universidade de Lisboa, Faculdade de Medicina, 2011Streptococcus agalactiae (group B streptococci, GBS) is primarily a colonizing agent of the genitourinary and gastrointestinal tracts of a significant proportion of the human population. It is, however, well established as a leading cause of bacterial sepsis and meningitis in neonates and is increasingly associated with invasive infections in adults. While vertical transmission is commonly accepted to be the cause of early-onset disease, the source of bacterial strains causing infection in the late-onset period is less well understood. Administration of intrapartum antimicrobial prophylaxis to colonized women has resulted in a striking decline in early-onset and maternal GBS disease, but late-onset infections have mostly remained unchanged. Moreover, antimicrobial prophylaxis raised concerns as to selection and emergence of GBS resistant strains and alternative prevention strategies have focused on the development of vaccines that hold promising, although still preliminary results. The aim of the work presented in this thesis was to characterize the population structure of GBS in Portugal, and to assess the genetic diversity of isolates recovered from vaginal colonization and invasive disease in different age groups, to contribute to the global epidemiology of GBS and our understanding of GBS population biology. To this end a set of common techniques was chosen, including serotyping, antimicrobial susceptibility testing, pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and surface protein gene profiling. In combination, these methods allowed the identification of the main genetic lineages circulating in Portugal and Barcelona, providing the means for an appropriate comparison of both. These studies started with the comparison of 64 isolates recovered from invasive infections in newborns in the Lisbon area with 269 isolates colonizing women in the third trimester of pregnancy, from the same period. The genetic lineages defined by both PFGE and MLST identified very diverse populations with reported differences in the prevalence of serotypes and clones in carriage and invasive disease. A major finding concerned the identification of an unusually high proportion of ST24 isolates among serotype Ia, further strengthened by the independent study of another population (212 neonatal isolates) from the Barcelona area. Despite the geographic distance, both studies from Barcelona and Lisbon revealed extensive similarity in terms of clonal structure and genetic lineages. The high prevalence in both the studies of a particular lineage serotype Ia, defined by ST24 and the surface protein gene bca, highlighted the importance of local dynamics, indicating that genetic evolution of GBS presents with a geographic structure and may depend on local factors. The subsequent analysis of 225 isolates recovered from non-pregnant adults in Portugal revealed a GBS population dominated by a more diverse clonal composition when compared to that of neonates, consistent with the broader spectrum of disease presentation in these patients and consequent multiplicity of genetic lineages. Invasive disease in this population increased with age and was more frequent among men. The dominance of serotype Ia in this population, regardless of age, highlighted the importance of this serotype in GBS pathogenesis as a leading cause of invasive infections in adults, not reported elsewhere but already noted among neonatal infections in the Iberian Peninsula. Furthermore, the high prevalence of ST24 in all these studies, as opposed to rare descriptions elsewhere, suggested that this lineage had enhanced invasiveness and was probably expanding as a regionally successful clone that may disseminate more globally. Macrolide resistance rates in Portugal did not show significant trends, even if macrolides have been used in intrapartum prophylaxis increasing the selective pressure on GBS. Macrolide resistance is disseminated in Portugal by both a multiclonal mechanism resulting from the spread of resistance genes throughout most serotypes and genetic backgrounds, as well as by clonal expansion of particular lineages, such as the serotype V ST1/alp3. One of the main purposes of the analysis of a significant number of GBS isolates was their classification into lineages sharing the same genetic background, which would allow the inference of genetic relationships between strains and their contextualization in the global epidemiology of GBS. However, the associations of phenotype-genotype or between different genetic traits were never absolute, highlighting the role of horizontal genetic transfer in the evolution of GBS. Capsular switching was anticipated to occur frequently within GBS, even though this species is not recognized to be naturally competent for the acquisition of foreign DNA. Substantial evidence provided by the epidemiological studies performed on the Portuguese GBS collections drove the search for capsular transformants within these populations. The results obtained confirmed the existence of capsular switching in GBS, but questioned the high frequency of these events estimated from previous studies. Serotyping errors probably justified the overrepresentation of capsular switching in epidemiological studies. The mechanism for these genetic transfer events involved the replacement of the whole capsular locus instead of the previously proposed genetic transfer of only the serotypespecific genes. Globally, the results presented in this thesis suggest that GBS has an apparent remarkably stable, both temporally and geographically, clonal structure. Against this, background diversification is ongoing and can depend on local factors. Capsular switching is likely contributing to diversification, however not as frequently as initially thought and may impact on the vaccine formulations currently under development. Despite increasing information on maternal colonization and invasive disease, a better understanding of colonization in adults and natural reservoirs of GBS is required for the appropriate management of the GBS infections.Streptococcus agalactiae (estreptococos do grupo B, GBS) é maioritariamente um agente colonizador dos tratos genito-urinário e gastrointestinal de uma proporção significativa da população humana. É, no entanto, reconhecido como uma das principais causas de sépsis e meningite bacteriana em recém-nascidos, e crescentemente associado a infecções invasivas em adultos. Enquanto a transmissão vertical é normalmente aceite como causa do aparecimento precoce da doença, a origem das estirpes bacterianas que causam infecção no período tardio é menos bem compreendida. A administração de profilaxia antimicrobiana intraparto a mulheres colonizadas resultou num declínio acentuado das infecções por GBS de início precoce e materna, mas as infecções de início tardio, na sua maioria, mantiveram-se inalteradas. Mais ainda, a profilaxia antimicrobiana levantou preocupações quanto à selecção e ao aparecimento de estirpes de GBS resistentes e as estratégias alternativas de prevenção têm-se focado no desenvolvimento de vacinas com resultados promissores, embora ainda preliminares. O objectivo do trabalho apresentado nesta tese foi caracterizar a estrutura da população de GBS em Portugal e avaliar a diversidade genética das estirpes isoladas de colonização vaginal e doença invasiva em diferentes faixas etárias, de forma a contribuir para a epidemiologia global de GBS e para a compreensão da biologia populacional de GBS. Para atingir esse objectivo foi escolhido um conjunto de técnicas, incluindo a serotipagem, testes de susceptibilidade a antimicrobianos, electroforese em gel de campo pulsado (PFGE), “multilocus sequence typing” (MLST) e determinação de perfis de proteínas de superfície. Em combinação, estes métodos permitiram a identificação das principais linhagens genéticas que circulam em Portugal e Barcelona, fornecendo meios para uma comparação apropriada de ambas. Estes estudos começaram com a comparação de 64 estirpes isoladas de infecções invasivas em recém-nascidos na região de Lisboa com 269 estirpes de colonização em mulheres no terceiro trimestre de gravidez, no mesmo período. As linhagens genéticas definidas por PFGE e MLST identificaram populações muito diversificadas, com diferenças descritas na prevalência dos serótipos e clones, em colonização e doença invasiva. Uma constatação importante diz respeito à identificação de um número excepcionalmente elevado de estirpes ST24 no serótipo Ia, reforçado pelo estudo independente de outra população (212 estirpes neonatais) da área de Barcelona. Apesar da distância geográfica, ambos os estudos de Barcelona e Lisboa revelaram uma enorme semelhança em termos da estrutura clonal e linhagens genéticas. A elevada prevalência em ambos os estudos de uma linhagem particular do serótipo Ia, definida pelo ST24 e pelo gene que codifica a proteína de superfície bca, salientaram a importância de dinâmicas locais, indicando que a evolução genética de GBS apresenta uma estrutura geográfica e pode depender de factores locais A análise subsequente de 225 estirpes isoladas de adultos, exceptuando grávidas, em Portugal revelou uma população de GBS dominada por uma composição clonal mais diversificada quando comparada com a de recém-nascidos, consistente com o espectro mais alargado de manifestações de doença nestes indivíduos e consequente multiplicidade de linhagens genéticas. A doença invasiva nesta população aumentou com a idade, sendo mais frequente entre os homens. A predominância do serótipo Ia nessa população, independentemente da idade, destacou a importância deste serótipo na patogénese da GBS como uma das principais causas de infecções invasivas em adultos, não relatada noutros locais mas já identificada nas infecções neonatais na Península Ibérica. Além disso, a elevada prevalência de ST24 em todos estes estudos, por oposição a raras descrições noutros locais, sugerem que esta linhagem tem maior capacidade invasiva e está provavelmente em expansão como um clone bem-sucedido a nível regional que pode disseminar mais globalmente. As taxas de resistência aos macrólidos em Portugal não apresentaram tendências significativas, ainda que os macrólidos sejam usados na profilaxia intraparto, aumentando a pressão selectiva sobre GBS. A resistência aos macrólidos está disseminada em Portugal, tanto por um mecanismo multiclonal que resulta na dispersão de genes de resistência na maior parte serótipos e clones, como pela expansão clonal de linhagens específicas, tais como a do serótipo V ST1/alp3. Um dos principais objectivos da análise de um número significativo de estirpes de GBS era a sua classificação em linhagens com o mesmo património genético, o que permitiria a inferência de relações genéticas entre estirpes, e a sua contextualização na epidemiologia global de GBS. Contudo, as associações fenótipo-genótipo ou entre características genéticas diferentes nunca foram absolutas, salientando o papel da transferência genética horizontal na evolução de GBS. Foi proposto que a transformação capsular ocorre frequentemente em GBS, apesar de esta espécie não ser reconhecida como naturalmente competente para a aquisição de DNA exógeno. Evidências substanciais fornecidas pelos estudos epidemiológicos realizados nas colecções portuguesas de GBS levaram à pesquisa de transformantes capsular nessas populações. Os resultados obtidos confirmaram a existência de transformação capsular em GBS, mas questionaram a elevada frequência destes eventos, estimada em estudos anteriores. Erros de serotipagem provavelmente justificam a elevada representação de transformação capsular em estudos epidemiológicos. O mecanismo na base destes eventos de transferência genética envolveu a substituição de todo o locus capsular em vez da transferência genética de apenas os genes específicos do serótipo, proposta anteriormente. Globalmente, os resultados apresentados nesta tese sugerem que os GBS possuem uma estrutura clonal, extremamente estável, tanto geográfica como temporalmente. Por oposição, a diversificação do património genético é permanente e pode depender de factores locais. A transformação capsular provavelmente contribui para esta diversificação, porém não tão frequentemente quanto inicialmente se pensava e pode ter um impacto na formulação das vacinas actualmente em desenvolvimento. Apesar do aumento da informação sobre a colonização materna e a doença invasiva, uma melhor compreensão da colonização em adultos e dos reservatórios naturais de GBS é necessária para uma abordagem mais fundamentada das infecções por GBS.Fundação para a Ciência e a Tecnologia (FCT, SFRH/BD/41761/2007) e programa POPH/FS

Contribution of genomics and transcriptomics to the understanding of the biological role of DNases in Streptococcus agalactiae

Streptococcus agalactiae is the leading cause of neonatal pneumonia, sepsis, and meningitis. Strains III/ST17 emerged as a hypervirulent clone mostly associated with meningitis during late-onset disease. Production of extracellular DNases and biofilm formation have been proposed to explain the leading role of this clone in neonatal meningitis. The aim of this thesis was to shed some light on the genetic background of DNase production in S. agalactiae strains isolated in humans presenting diverse cell tropism (invasive vs carriage). Genomic approaches were used to decipher the molecular basis of the differential production of DNases, through the analysis of the “core-genome” and the “pan-genome” of ST17 and ST19 strains. All ST17 strains, except one, displayed DNase activity which was observed in only one ST19 strain. ST17 DNase(-) revealed an exclusive amino acid change in NucA, a TnGBS2.3 homolog and an intact phage without homology in S. agalactiae. ST19 DNase(+) revealed an exclusive amino acid change alteration in nuclease GBS0609. A transcriptomic approach allowed the analysis of the level of expression, by functional category, of genes of S. agalactiae reference strain NEM316 at the exponential growth phase; exposure to human DNA did not affect the transcriptome. The optimal conditions for S. agalactiae biofilm assembly were determined for ST17 and ST19 strains, contributing to the standardization of experimental procedures, thus allowing the comparison of results between different laboratories. In addition, the enzymatic digestion of mature biofilms for the three strongest biofilm producers, evidenced that proteins were the predominant component of the extracellular polymeric matrix. Overall, the findings presented in this Ph.D. thesis may contribute for the knowledge on the production of extracellular DNases, and provide new insights into biofilm formation, genomics and transcriptomics for ST17 and ST19 S. agalactiae lineages.Streptococcus agalactiae é a principal causa de pneumonia neonatal, sépsis e meningite. O clone hipervirulento III/ST17, tem sido associado à meningite neonatal de início tardio, tendo a atividade de DNases extracelulares e a formação de biofilmes sido apontados como fatores de virulência nessas estirpes. O objetivo da presente dissertação de doutoramento foi contribuir para a descodificação da base genómica de produção de DNases em estirpes de S. agalactiae, isoladas em humanos, com diferentes tropismos celulares (invasivo vs colonização). A produção diferencial de DNases de um conjunto de estirpes pertencentes às linhagens ST17 e ST19 foi investigada através da análise do “core-genome” e do “pan-genome”. As estirpes ST17, exceto uma, apresentaram atividade nucleásica contrariamente às estirpes ST19, em que apenas uma revelou um fenótipo DNase(+). A estirpe ST17 DNase(-) exibiu uma alteração aminoacídica exclusiva na proteína NucA e a presença de um homólogo do tranposão TnGBS2.3, para além de um fago intacto sem homologia em S. agalactiae. A estirpe ST19 DNase(+) revelou um aminoácido alterado na nuclease GBS0609. A análise transcriptómica permitiu avaliar os níveis de expressão génica, por categoria funcional, da estirpe de referência S. agalactiae NEM316, durante a fase exponencial de crescimento; a exposição ao DNA humano não induziu alterações no transcriptoma. Foram determinadas as condições ótimas para a formação de biofilmes em estirpes de S. agalactiae de ST17 e ST19, contribuindo para a padronização dos procedimentos experimentais do estudo de produção de biofilmes nesta espécie bacteriana. A digestão enzimática de biofilmes maduros, para as três estirpes mais produtoras de biofilmes, evidenciou que o componente predominante da matriz extracelular são as proteínas. Globalmente, a presente dissertação de doutoramento contribui para o conhecimento sobre a produção das DNases extracelulares e a capacidade de formação de biofilmes das linhagens ST17 e ST19 de S. agalactiae, providenciando novos dados genómicos e transcriptómicos

Towards ending maternal and infant preventable deaths : omics tools to support vaccine development against Plasmodium falciparum malaria and Streptococcus agalactiae disease

The United Nations sustainable developmental goal 3 “good health and well-being” includes the aim to significantly reduce global maternal mortality and preventable deaths of newborns and children under 5 years of age until the year 2030. Two major contributors to global maternal and infant morbidity and mortality are Plasmodium falciparum severe malaria and Group B Streptococcus (GBS) invasive disease. The central aspect in the WHO strategy towards the elimination of these two diseases is the development of effective malaria and GBS vaccines. In the case of malaria, the immunization with radiation-attenuated P. falciparum sporozoites (PfSPZ) has been shown to convey protective immunity against controlled human malaria infection (CHMI), making this a promising vaccination approach. However, the molecular mechanisms underlying protective anti-malarial immune responses as well as the reasons for the poor immunogenicity of the PfSPZ vaccine in malaria-experienced individuals compared to malaria-naïve volunteers, remain poorly understood. Emerging system analysis approaches, including genome-wide accession of gene expression using RNA-Sequencing (RNA-Seq) provide valuable insight into post-vaccination systemic molecular dynamics and can help to identify immunological correlates of protection. In the case of GBS, multivalent glycoconjugate vaccines, targeting selected GBS capsular polysaccharide types, are currently under clinical trial evaluation. With demonstrated good safety and immunogenicity profiles, the licensure of such vaccines is foreseeable. Large-scale monitoring of vaccine recipients for GBS carriage and assessment of vaccine impact on vaginal colonization, potential serotype replacement and emergence of escape strains will be an important aspect of post-licensure epidemiological studies. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS), has emerged as the method of choice for high-throughput microbial species identification in clinical microbiology and has been suggested for strain level typing of bacteria. The overall aims of this thesis therefore included to (i) evaluate the safety and protective efficacy against CHMI of PfSPZ vaccination in Tanzanian volunteers and (ii) elucidate gene expression dynamics in unvaccinated Tanzanian volunteers following CHMI and (iii) to establish a MALDI-TOF MS typing method for GBS for rapid screening of circulating and emerging genotypes. Building on these objectives, the here presented thesis is structured around five manuscripts

The molecular basis for virulence in Streptococcus Agalactiae

Group B Streptococcus (GBS) is a leading cause of neonatal meningitis and septicaemia. During the progression of invasive disease, GBS must be able to detect and adapt to a diverse range of environments. One of the challenging environments the organisms will encounter is the antimicrobial phagosome of cells of the innate immune system. Combining microscopy with pharmaceutical approaches, I have been able to show that GBS is residing within a vacuole that acquires phagolysosomal markers and, that acidification of the phagosome is required for GBS to survive. In addition this work has demonstrated that GBS induces only a weak reactive oxygen burst in macrophages and consequently reactive oxygen species are of limited importance. Interestingly, however, the GBS acid response regulator CovS/R is crucial for the organism’s ability to survive within murine macrophages. This is most likely due to the regulation of genes required for adaption to the intracellular environment.Lastly, to facilitate investigations into the interaction of GBS with the phagosome it would be desirable to be able to visualise live organisms within cells. In the final part of this thesis, I describe an evaluation of different approaches to generate a suitable, fluorescently labelled, strain of GBS

Role of CD4+ T cells in the regulation of the immune response against encapsulated Group B Streptococcus

Le Streptocoque de groupe B (GBS) est un important agent d’infection invasive pouvant mener à la mort et demeure la cause principale de septicémie néonatale à ce jour. Neuf sérotypes ont été officiellement décrits basés sur la composition de la capsule polysaccharidique (CPS). Parmi ces sérotypes, le type III est considéré le plus virulent et fréquemment associé aux maladies invasives graves, telle que la méningite. Malgré que plusieurs recherches aient été effectuées au niveau des interactions entre GBS type III et les cellules du système immunitaire innées, aucune information n’est disponible sur la régulation de la réponse immunitaire adaptative dirigée contre ce dernier. Notamment, le rôle de cellules T CD4+ dans l’immuno-pathogenèse de l’infection causée par GBS n’a jamais été étudié. Dans cet étude, trois différents modèles murins d’infection ont été développé pour évaluer l’activation et la modulation des cellules T CD4+ répondantes au GBS de type III : ex vivo, in vivo, et in vitro. Les résultats d’infections ex vivo démontrent que les splénocytes totaux répondent à l’infection en produisant des cytokines de type-1 pro-inflammatoires. Une forte production d’IL-10 accompagne cette cascade inflammatoire, probablement dans l’effort de l’hôte de maintenir l’homéostasie. Les résultats démontrent aussi que les cellules T sont activement recrutées par les cellules répondantes du système inné en produisant des facteurs chimiotactiques, tels que CXCL9, CXCL10, et CCL3. Plus spécifiquement, les résultats obtenus à partir des cellules isolées T CD4+ provenant des infections ex vivo ou in vivo démontrent que ces cellules participent à la production d’IFN-γ et de TNF-α ainsi que d’IL-2, suggérant un profil d’activation Th1. Les cellules isolées T CD4+ n’étaient pas des contributeurs majeurs d’IL-10. Ceci indique que cette cytokine immuno-régulatrice est principalement produite par les cellules de l’immunité innée de la rate de souris infectées. Le profil Th1 des cellules T CD4+ a été confirmé en utilisant un modèle in vitro. Nos résultats démontrent aussi que la CPS de GBS a une role immuno-modulateur dans le développement de la réponse Th1. En résumé, cette étude adresse pour la première fois, la contribution des cellules T CD4+ dans la production d’IFN-γ lors d’une infection à GBS et donc, dans le développement d’une réponse de type Th1. Ces résultats renforcent d’avantage le rôle central de cette cytokine pour un control efficace des infections causées par ce pathogène.Group B Streptococcus (GBS) is an important agent of life-threatening invasive infections and remains the leading cause of neonatal sepsis to this day. Nine serotypes have been officially described based on capsular polysaccharide (CPS) composition. Among them, capsular type III is considered one of the most virulent and frequently associated with severe invasive diseases, such as meningitis. Although extensive research has been done on the interactions between GBS type III and various cells of the innate immune system, no information is available on the regulation of the adaptive immune response against this pathogen. In particular, the role of CD4+ T cells in the immuno-pathogenesis of the infection caused by GBS has never been assessed. In this study, three different models of murine infection were developed to evaluate activation and modulation of responding CD4+ T cells against GBS type III: ex vivo, in vivo, and in vitro. Ex vivo analysis of total splenocytes showed that GBS induces the release of type-1 pro-inflammatory cytokines. A strong IL-10 production follows this inflammatory cascade, indicating the host effort to maintain homeostasis. Results also indicate that T cells were actively recruited by responding innate immune cells via the release of chemotactic factors such as CXCL9, CXCL10, and CCL3. More specifically, results obtained from isolated CD4+ T cells from ex vivo or in vivo infections showed that they actively participate in the production of IFN-γ and TNF-α, as well as IL-2, suggesting a Th1 profile of activation. On the other hand, isolated CD4+ T cells were not main sources of IL-10. This observation suggests that this immuno-regulatory cytokine is produced mainly by cells of the spleen innate immune system of infected animals. The CD4+ Th1 cell profile was confirmed using an in vitro model of infection. Our results also suggest that the GBS CPS plays an immuno-modulatory role in the development of a Th1 response. In summary, this study addresses for this first time the contribution of CD4+ T cells in IFN-γ production during GBS infection, and thus, in the development of a Th1 response. Our data further highlight the central role of this cytokine for effective control of GBS infections

Comparative and Functional Genomic Analysis of <i>Streptococcus Equi</i> and <i>Streptococcus Zooepidemicus</i> : Identifying Novel Vaccine Targets

Streptococcus equi subspecies equi (S. equi) is a host-restricted pathogen of horses and the aetiological agent of strangles. Available evidence suggests that S. equi evolved from Streptococcus equi subspecies zooepidemicus (S. zooepidemicus), a versatile bacterium that is often isolated from the equine respiratory tract, but can cause opportunistic disease in horses and other animals. A comparison of the genomes of S. equi 4047 and S. zooepidemicus H70 and the screening of diverse S. equi and S. zooepidemicus strains uncovered the genetic events that have shaped the evolution of S. equi, and led to its emergence as a niche-adapted pathogen. This analysis provides evidence of functional loss, changes in the organisation and sequence of genes, and pathogenic specialisation through the acquisition of prophage encoding a phospholipase A2 toxin, and 4 superantigens, and an integrative conjugative element carrying a novel siderophore-like nonribosomal peptide synthetase (NRPS) system. The NRPS shares similarity with the yersiniabactin system found in the high pathogenicity island of Yersinia pestis and is the first of its kind to be identified in streptococci. As this genetic feature is absent from the S. zooepidemicus population, its gain is considered to have been a key event in the emergence of S. equi. Further work determined a role for the NRPS in iron acquisition, and through its heterologous reconstitution in Escherichia coli and/or the analysis of allelic replacement mutants in S. equi, identified biosynthetic genes, transporters involved in efflux and import of the NRPS product(s), salicylate as a substrate for the NRPS and its regulation by a novel iron-dependent IdeR-like repressor, using various in vitro growth assays, including sensitivity to streptonigrin and 55Fe accumulation. Possible vaccine targets were identified in both subspecies and existing diagnostic tools were improved, which included the development of a quantitative PCR test for the detection of S. equi

Detailed genomic and antimicrobial resistance comparison of UK Streptococcus agalactiae isolates from adults to those of diverse global origins

Invasive group B Streptococcus (GBS), a leading cause of illness and death among infants in the first week of life is also an important infectious agent able to cause invasive infections in adults. Serious life-threatening invasive GBS infections are increasingly recognized in the elderly and individuals compromised by underlying diseases such as diabetes, cirrhosis, and cancer. The significance of GBS as a cause of severe infections among adults is not widely appreciated. In adults, the modes of transmission and acquisition are less identified. Penicillin is the antibiotic of choice for treatment of GBS infection however, resistance to multiple antibiotics is increasing in Europe and worldwide in these organisms, making them increasingly difficult to treat, but also making them a potential danger as a silent resource for donating resistance genes to more aggressive pathogens. To date, there are no guidelines for the prevention of adult GBS disease; vaccines in development may hold promise. GBS emerged rapidly all over the world during the 1970s to become the leading cause of neonatal sepsis. However, several reports in recent years suggest that the incidence of GBS disease is also increasing among adults. The driving force behind this change has not been fully explained, and recent trends in disease incidence in adults have not been characterized in any systematic reviews due to inadequate data available on adult GBS disease.The aim of the work presented in this thesis was to characterize the population structure of human source isolated invasive and non-invasive GBS from adults in United Kingdom, and to evaluate the genetic diversity of isolates recovered from invasive disease in adult patients of Brazil and from vaginal and rectal colonization in Australian pregnant women with ≤22 weeks gestations to contribute to the global epidemiology of GBS and our understanding of GBS population biology. For this a set of conventional techniques were employed including antimicrobial susceptibility testing using disc diffusion test, serotyping using software Geneious (Biomatters ltd., New Zealand) and published primers for ten different GBS capsular types, multilocus sequence typing (MLST) and surface protein gene profiling using SRST2 v2.2. In combination, these methods allowed the identification of the main genetic lineages circulating in UK, Brazil, and Australia, providing the means for an appropriate comparison of Brazilian and Australian GBS to UK GBS population. In addition, analysis of WGS to determine GBS potential to switch capsule, antimicrobial resistance (AMR) genes associated with mobile genetic elements (MGEs), examining relatedness of the GBS strains using whole genome phylogeny and correlate serotyping, patient age group to AMR genes and pan genome wide association study (pan-GWAS) on UK, Brazil, and publicly available genomes of GBS isolates from Canada and the United States was performed. These analyses revealed a reasonable number of isolates with potential capsular switch including some cases switched from current vaccine (under trail) covered capsular type to non-vaccine covered serotypes. Further a diverse group of MGEs were identified with a capacity to disseminate the resistance phenotype, the association between strains clustered in a group based on patients age group they isolated from and the AMR genes they carry were studied. An intense pan-GWAS was performed to discover Clonal Complex (CC) specific genes that may play role in increased colonization, invasiveness, pathogenicity, and better survival of GBS in the host cell. The thesis work started with investigating 193 clinical GBS strains isolated from adults submitted to the UK national reference laboratory (179 invasive; 13 non-invasive; 1 with no information provided) for capsule type, MLST, presence of virulence factors, antimicrobial resistance genes, phylogeny, and genetic recombination. The genetic lineages defined by MLST identified very diverse populations but consistent in terms of serotypes prevalence and clonal structure identified previously in GBS invasive disease in United Kingdom. The prevalence of serotype III in this population, regardless of age, highlighted the importance of this serotype in GBS pathogenesis as a leading cause of invasive infections in adults. Macrolide resistance is disseminated in UK by both a multiclonal mechanism resulting from the spread of resistance genes throughout most serotypes and genetic backgrounds, as well as by clonal expansion of specific lineages, such as the serotype V ST1/alp3. Attachment and invasion of host cells are key steps in GBS pathogenesis, strong associations were identified between serotypes and virulence genes, such as serotype V/alp3, serotype II and III/bca+cba, serotype Ia/bibA predominantly clustered in CC1, CC8/CC10 and CC23, respectively, whereas serotype III/rib clustered in CC17 and CC19 demonstrating GBS strains belonging to a particular CC differ in their abilities to attach and invade to host cell types and express key virulence genes that are relevant to the disease process. A major finding includes a high number of capsular serotype-CC mismatches (14/179, 7.8%) iGBS identified with a concerning recombination of hypervirulent hvgA core genome expressing a non-vaccine covered serotype IV capsule. The mechanism for these genetic transfer events involved the replacement of the whole capsular locus instead of the previously proposed genetic transfer of only the serotype specific genes. The consequent analysis of MGEs carrying multidrug resistance genes in 41/193 GBS isolates revealed a diverse group of MGES used three different insertion sites (rumA, rplL and rpsI) to disseminate phenotypic resistance in GBS isolated from adult patients of United Kingdom. Out of 41 isolates, only one isolate carried the macrolide resistance (ermT) gene was on a plasmid, while for 4 isolates fluoroquinolone resistance was mediated by double point somatic mutation in parC and gyrA; for all other isolates ARGs were acquired by MGEs including five novel MGEs identified in this study, including ICESag84 and ICESag100414 carrying ermA alone, ICESag662 containing ermB, tetS, ant(6-Ia) and aph(3'-III), ICESag71 carrying ermB and tetO, and ICESag139 containing ermA and the high gentamicin level resistance gene aac(6')-aph(2"). The Tn916 and Tn5801 belonging to Tn916/Tn1545 family harboured majority of tetM genes (88%, 154/175) found in UK tetracycline resistant GBS isolates and were significantly associated to CC1, CC8/10, CC19 and CC17 and CC23 isolates, respectively suggesting these ICEs are clonally related, acquired through limited and rare insertion events and led to expansion of these lineages, also supporting the earlier interpretations [1]. In addition, the Tn916/Tn1545 family ICE identified in this study were mostly integrated at two insertion sites, adjacent to two target genes (TGs) – rumA for Tn916 and guaA for Tn5801 demonstrating their integration preferences at hot spot regions. The high prevalence of MGEs carrying ARGs in UK adults GBS isolates implied that in GBS, these MGEs probably act like a reservoir of ARGs, and play a central role in the dissemination of resistance genes via horizontal gene transfer. Antibiotic susceptibility testing (AST) of GBS isolates recovered from UK (n=193) and Brazil (n=26) adults, and from vaginal and rectal sites of Australian pregnant women (n=171) revealed that all tested GBS populations were sensitive to ampicillin, vancomycin, and gentamicin, except a single UK strain that conferred high level gentamicin resistance through aminoglycoside modifying enzyme encoding gene aac(6')-aph(2"). Further all UK and Brazil GBS isolates carried five penicillin binding protein (PBPs) types with amino acid substitutions that did not appear to be associated with decreased β-lactam susceptibility suggesting penicillin as the first choice and vancomycin as the second choice of drug for GBS disease treatment as currently recommended by Centers for Disease Control and Prevention (CDC) and Royal College of Obstetricians and Gynaecologists (RCOG) guidelines [2, 3]. Accelerating resistance rates to erythromycin and clindamycin were observed in UK, which in comparison found less in Australian pregnant women colonized GBS and Brazilian GBS populations suggesting routine susceptibility testing of erythromycin and clindamycin for penicillin allergic patients to ensure effective treatment. A very few UK and Australian GBS strains were found resistant to chloramphenicol and/or levofloxacin while all Brazilian GBS were susceptible to these two antibiotics. High resistance rate to tetracycline was detected in UK, Brazilian and Australian GBS isolates and the tetM gene was found widespread and carried predominantly by Tn916/Tn1545 family elements. Three distinctive variants of Tn916 were observed in UK GBS strains, respectively including two Tn916 variants carrying tetM as a single resistance gene with additional 9 orfs in conjugation module, while the third Tn916 variant carried tetracycline efflux MFS transporter (tetL) gene near the tetM gene. The pangenome wide association study (pan-GWAS) was conducted on 447 GBS genomes, including this study sequenced UK (n=193) and Brazil (n=26) GBS strains, in addition to deliberately selected serotype V and III publicly available genomes from Canada (n=134) and the United States (n=94) to study genes specific to CC and their role in pathogenicity and invasiveness, since these two serotypes are universally found highly associated with adults and neonatal diseases, respectively. Each CC was characterized by specific genes that provides selective advantage to GBS for improved colonization, invasion, virulence, and survival within host. This analysis identified 97 CC-specific genes associated (excluding hypothetical proteins) with virulence, metabolism, and regulation of cellular mechanisms that may explain the differential virulence potential of the CCs. Among CC17 and CC23 GBS isolates, micronutrient uptake proteins (iron and manganese), two component systems, accessory secondary proteins, pilus and quorum-sensing genes were identified which were absent in less invasive lineages (CC1, CC8 and CC19). Metal resistance genes (arsenic, cadmium, and copper) and CRISPR associated genes (cas1/cas2) were confined to CC8 whereas the type IV secretory protein (VirD4) was significantly associated to CC19. Collectively this analysis underlines the lineage-specific basis of GBS niche adaptation and virulence. In summary, in this thesis GBS shown to have an evident stable clonal structure both temporally and geographically. Interestingly, capsular switching occurred across multiple serotypes and among strains with dissimilar genomic backgrounds in high numbers demonstrated ongoing GBS diversification due to recombination and highlights the importance of ongoing surveillance of GBS and may have implications for vaccine development strategies. Regardless of increasing information on invasive disease and maternal colonization, a thorough understanding of colonization in adults and natural reservoirs of GBS is required for the appropriate management of the GBS infections