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

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

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

Alterations in Genes rib, scpB and Pilus Island Decrease the Prevalence of Predominant Serotype V, Not III and VI, of Streptococcus agalactiae from 2008 to 2012

Streptococcus agalactiae (GBS) can infect newborns, pregnant women and immunocompromised or elderly people. This study aimed to investigate differences in three pilus genes and virulence genes pavA, cfb, rib and scpB and changes in predominant serotypes III, V and VI from 2008 to 2012. The susceptibilities to penicillin, ceftriaxone, azithromycin, erythromycin, clindamycin, levofloxacin and moxifloxacin of 145 GBS strains of serotype III, V and VI strains from 2008 and 2012 were determined using disc diffusion method. PCR identification of ST-17, the pilus genes and virulence genes; multilocus sequence typing (MLST); and conserved domain and phylogenetic analysis of scpB-1 and scpB-2 proteins were performed. A dramatic number reduction was observed in serotype V, not III and V, from 2008 to 2012. The rate of resistance to azithromycin, clindamycin and erythromycin was the highest in serotype V. ST-17 was only found in serotype III with pilus genes PI-1+PI-2b. The major pilus genotype was PI-1+PI-2a. Serotype V without the rib gene was reduced in number between two studied years. Compared to scpB-1, scpB-2 had a 128-bp deletion in a PA C5a-like peptidase domain and putative integrin-binding motif RGD. In conclusion, reduction in serotype V may be due to presence of scpB-2 or lack of genes scpB and rib