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.
\nThe 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 v

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