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Cholera is a life-threatening infectious disease that remains an important public health issue in several low and middle-income countries. In 1992, a newly identified O139 Vibrio cholerae temporarily displaced the O1 serogroup. No study has been able to answer why the potential eighth cholera pandemic (8CP) causing V. cholerae O139 emerged so successfully and then died out. We conducted a genomic study, including 330 O139 isolates, covering emergence of the serogroup in 1992 through to 2015. We noted two key genomic evolutionary changes that may have been responsible for the disappearance of genetically distinct but temporally overlapping waves (A-C) of O139. Firstly, as the waves progressed, a switch from a homogenous toxin genotype in wave-A to heterogeneous genotypes. Secondly, a gradual loss of antimicrobial resistance (AMR) with the progression of waves. We hypothesize that these two changes contributed to the eventual epidemiological decline of O139.

The phylum Chlamydiae comprises a diverse group of obligate intracellular bacteria that share a unique biphasic developmental cycle. While many of these bacteria are considered pathogens of humans and animals, particularly in the well-described Chlamydiaceae family, other more distantly related members can be detected in the environment. The limiting factor to studying these diverse Chlamydia-related bacteria is their requirement to be cultured in a host cell, which for many novel and/or Candidatus species, is lacking. To gain insight into the biology and epidemiology of novel bacteria in the phylum Chlamydiae, my project aimed to develop a culture-independent genome sequencing (CIGS) work-flow to characterise the genomes of novel uncultivated chlamydiae in clinical samples. I addressed these aims by applying our CIGS method to two examples encompassing the taxonomic and biological diversity within the phylum, including (a) novel species in the genus Chlamydia from reptiles; and (b) deeply rooted chlamydiae that had previously been identified infecting fish.

Inclusion membrane prediction. (PDF 99 kb)

QUAST report. (PDF 127 kb)

Locations of â pmp islandsâ in the uncultured Ca. Chlamydia sanzinia genome and selected chlamydial genomes. Figure was constructed in Geneious. Figure S3. Schematic representation of pmp pseudogenes attributed to frameshift mutations. Open reading frames are coloured blue in frame and genes/pseudogenes are coloured white on the forward strand. Vertical black lines represent stop codons. Diagonal blue lines represent fragmentation due to premature stop codons. Figure constructed using Artemis. (PDF 140 kb)

Chlamydial contig BLAST analysis. Table S3. Metagenome coverage statistics. Table S4. Putative host contig analysis. Table S5. rRNA-containing contigs detected using Metaxa. Table S5. Phylogenetic marker gene BLAST analysis. (PDF 152 kb)

Funder: International Centre for Diarrhoeal Disease Research, Funder: NIHR BRC, Background: The proportion of enteric fever cases caused by Salmonella Paratyphi A is increasing and may increase further as we begin to introduce typhoid conjugate vaccines (TCVs). While numerous epidemiological and genomic studies have been conducted for S. Typhi, there are limited data describing the genomic epidemiology of S. Paratyphi A in especially in endemic settings, such as Bangladesh. Principal findings: We conducted whole genome sequencing (WGS) of 67 S. Paratyphi A isolated between 2008 and 2018 from eight enteric disease surveillance sites across Bangladesh. We performed a detailed phylogenetic analysis of these sequence data incorporating sequences from 242 previously sequenced S. Paratyphi A isolates from a global collection and provided evidence of lineage migration from neighboring countries in South Asia. The data revealed that the majority of the Bangladeshi S. Paratyphi A isolates belonged to the dominant global lineage A (67.2%), while the remainder were either lineage C (19.4%) or F (13.4%). The population structure was relatively homogenous across the country as we did not find any significant lineage distributions between study sites inside or outside Dhaka. Our genomic data showed presence of single point mutations in gyrA gene either at codon 83 or 87 associated with decreased fluoroquinolone susceptibility in all Bangladeshi S. Paratyphi A isolates. Notably, we identified the pHCM2- like cryptic plasmid which was highly similar to S. Typhi plasmids circulating in Bangladesh and has not been previously identified in S. Paratyphi A organisms. Significance: This study demonstrates the utility of WGS to monitor the ongoing evolution of this emerging enteric pathogen. Novel insights into the genetic structure of S. Paratyphi A will aid the understanding of both regional and global circulation patterns of this emerging pathogen and provide a framework for future genomic surveillance studies.

BACKGROUND: The proportion of enteric fever cases caused by Salmonella Paratyphi A is increasing and may increase further as we begin to introduce typhoid conjugate vaccines (TCVs). While numerous epidemiological and genomic studies have been conducted for S. Typhi, there are limited data describing the genomic epidemiology of S. Paratyphi A in especially in endemic settings, such as Bangladesh. PRINCIPAL FINDINGS: We conducted whole genome sequencing (WGS) of 67 S. Paratyphi A isolated between 2008 and 2018 from eight enteric disease surveillance sites across Bangladesh. We performed a detailed phylogenetic analysis of these sequence data incorporating sequences from 242 previously sequenced S. Paratyphi A isolates from a global collection and provided evidence of lineage migration from neighboring countries in South Asia. The data revealed that the majority of the Bangladeshi S. Paratyphi A isolates belonged to the dominant global lineage A (67.2%), while the remainder were either lineage C (19.4%) or F (13.4%). The population structure was relatively homogenous across the country as we did not find any significant lineage distributions between study sites inside or outside Dhaka. Our genomic data showed presence of single point mutations in gyrA gene either at codon 83 or 87 associated with decreased fluoroquinolone susceptibility in all Bangladeshi S. Paratyphi A isolates. Notably, we identified the pHCM2- like cryptic plasmid which was highly similar to S. Typhi plasmids circulating in Bangladesh and has not been previously identified in S. Paratyphi A organisms. SIGNIFICANCE: This study demonstrates the utility of WGS to monitor the ongoing evolution of this emerging enteric pathogen. Novel insights into the genetic structure of S. Paratyphi A will aid the understanding of both regional and global circulation patterns of this emerging pathogen and provide a framework for future genomic surveillance studies.

Background The proportion of enteric fever cases caused by Salmonella Paratyphi A is increasing and may increase further as we begin to introduce typhoid conjugate vaccines (TCVs). While numerous epidemiological and genomic studies have been conducted for S. Typhi, there are limited data describing the genomic epidemiology of S. Paratyphi A in especially in endemic settings, such as Bangladesh. Principal findings We conducted whole genome sequencing (WGS) of 67 S. Paratyphi A isolated between 2008 and 2018 from eight enteric disease surveillance sites across Bangladesh. We performed a detailed phylogenetic analysis of these sequence data incorporating sequences from 242 previously sequenced S. Paratyphi A isolates from a global collection and provided evidence of lineage migration from neighboring countries in South Asia. The data revealed that the majority of the Bangladeshi S. Paratyphi A isolates belonged to the dominant global lineage A (67.2%), while the remainder were either lineage C (19.4%) or F (13.4%). The population structure was relatively homogenous across the country as we did not find any significant lineage distributions between study sites inside or outside Dhaka. Our genomic data showed presence of single point mutations in gyrA gene either at codon 83 or 87 associated with decreased fluoroquinolone susceptibility in all Bangladeshi S. Paratyphi A isolates. Notably, we identified the pHCM2- like cryptic plasmid which was highly similar to S. Typhi plasmids circulating in Bangladesh and has not been previously identified in S. Paratyphi A organisms. Significance This study demonstrates the utility of WGS to monitor the ongoing evolution of this emerging enteric pathogen. Novel insights into the genetic structure of S. Paratyphi A will aid the understanding of both regional and global circulation patterns of this emerging pathogen and provide a framework for future genomic surveillance studies., Author summary Salmonella enterica serovar Paratyphi A is an understudied cause of enteric fever which has started replacing Salmonella Typhi in some endemic regions. Currently, there are limited genomic epidemiology data to understand the true disease burden of disease caused by S. Paratyphi A in poor resource settings like Bangladesh. Our genomic data revealed that the population structure of S. Paratyphi A in Bangladesh comprised of only previously defined lineages A, C and F and carried single point mutation in gyrA gene associated with decreased fluoroquinolone susceptibility with no evidence of multi-drug resistance. Additionally, the global context revealed the clustering of organisms associated with neighboring countries in South Asia. We identified the pHCM2-like cryptic plasmid among our collection, which was not found in any S. Paratyphi A isolates previously. A comparative plasmid analysis showed that S. Paratyphi A pHCM2-like plasmid was highly similar to the plasmid found in S. Typhi but significantly different to a similar plasmid from non-invasive Salmonella Paratyphi B variant Java isolate from Bangladesh. This present study highlights the importance of sustained genomic surveillance to monitor the ongoing evolution of antimicrobial resistant determinants and plasmids in Bangladesh and transmission between different countries.