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Salmonella Paratyphi A, the primary etiology of paratyphoid, is estimated to cause 3.4 million infections annually, worldwide. With rising antimicrobial resistance and no licensed vaccines, genomic surveillance is key to track and monitor transmission, but there is currently no reliable genotyping framework for this pathogen. Here, we sequence 817 isolates from South Asia and add 562 publicly available genomes to build a global database representing 37 countries, covering 1917–2019. We develop a single nucleotide polymorphism-based genotyping scheme, Paratype, that segregates Salmonella Paratyphi A population into three primary and nine secondary clades, and 18 genotypes. Each genotype is assigned a unique allele definition located on an essential gene. Using Paratype, we identify spatiotemporal genomic variation and antimicrobial resistance markers. We release Paratype as an open-access tool that can use raw read files from both Illumina and Nanopore platforms, and thus can assist surveillance studies tracking Salmonella Paratyphi A across the globe.

Funder: Joint Programming Initiative on Antimicrobial Resistance; doi: http://dx.doi.org/10.13039/100013281, Funder: UK Medical Research Council, Streptococcus pneumoniae is a devastating global pathogen. Prevalent in sub-Saharan Africa, pneumococcal serotype 1 is atypical in that it is rarely found as a nasopharyngeal coloniser, yet is described as one of the most common causes of invasive pneumococcal disease. Clonal sequence type (ST)-306 and ST615 are representative of the two major serotype 1 lineages A and C, respectively. Here we investigated the virulence properties and haemolytic activities of these 2 clonal types using in vivo mouse models and in vitro assays. A lethal dose of ST615 administered intranasally to mice led to the rapid onset of disease symptoms and resulted in 90% mortality. In contrast, mice exposed to the same infection dose of ST306 or a pneumolysin (Ply)-deficient ST615 failed to develop any disease symptoms. Interestingly, the 2 strains did not differ in their ability to bind the immune complement or to undergo neutrophil-mediated phagocytosis. Upon comparative genomic analysis, we found higher within-ST sequence diversity in ST615 compared with ST306 and determined that ZmpA, ZmpD proteins, and IgA protease, were uniquely found in ST615. Using cell fractionation and cell contact-dependent assay, we made the unexpected finding that ST615 harbours the expression of two haemolytic variants of Ply: a cell-wall restricted fully haemolytic Ply, and a cytosolic pool of Ply void of any detectable haemolytic activity. This is the first time such a phenomenon has been described. We discuss the biological significance of our observation in relation to the aptitude of the pneumococcus for sustaining its human reservoir.

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.

Whole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Here, we describe a universal protocol for simultaneous DNA extraction and sequencing of numerous different bacterial species, allowing mixed species sequence runs to meet variable laboratory demand. We assembled test panels representing 20 clinically relevant bacterial species. The DNA extraction process used the QIAamp mini DNA kit, to which different combinations of reagents were added. Thereafter, a common protocol was used for library preparation and sequencing. The addition of lysostaphin, lysozyme or buffer ATL (a tissue lysis buffer) alone did not produce sufficient DNA for library preparation across the species tested. By contrast, lysozyme plus lysostaphin produced sufficient DNA across all 20 species. DNA from 15 of 20 species could be extracted from a 24-h culture plate, while the remainder required 48–72 h. The process demonstrated 100% reproducibility. Sequencing of the resulting DNA was used to recapitulate previous findings for species, outbreak detection, antimicrobial resistance gene detection and capsular type. This single protocol for simultaneous processing and sequencing of multiple bacterial species supports low volume and rapid turnaround time by local clinical microbiology laboratories.

Escherichia coli is the leading cause of urinary tract infection, one of the most common bacterial infections in humans. Despite this, a genomic perspective is lacking regarding the phylogenetic distribution of isolates associated with different clinical syndromes. Here, we present a large-scale phylogenomic analysis of a spatiotemporally and clinically diverse set of 907 E. coli isolates, including 722 uropathogenic E. coli (UPEC) isolates. A genome-wide association approach identifies the (P-fimbriae-encoding) papGII locus as the key feature distinguishing invasive UPEC, defined as isolates associated with severe UTI, i.e., kidney infection (pyelonephritis) or urinary-source bacteremia, from non-invasive UPEC, defined as isolates associated with asymptomatic bacteriuria or bladder infection (cystitis). Within the E. coli population, distinct invasive UPEC lineages emerged through repeated horizontal acquisition of diverse papGII-containing pathogenicity islands. Our findings elucidate the molecular determinants of severe UTI and have implications for the early detection of this pathogen.

In order to control and eradicate epidemic cholera, we need to understand how epidemics begin, how they spread, and how they decline and eventually end. This requires extensive sampling of epidemic disease over time, alongside the background of endemic disease that may exist concurrently with the epidemic. The unique circumstances surrounding the Argentinian cholera epidemic of 1992–1998 presented an opportunity to do this. Here, we use 490 Argentinian V. cholerae genome sequences to characterise the variation within, and between, epidemic and endemic V. cholerae. We show that, during the 1992–1998 cholera epidemic, the invariant epidemic clone co-existed alongside highly diverse members of the Vibrio cholerae species in Argentina, and we contrast the clonality of epidemic V. cholerae with the background diversity of local endemic bacteria. Our findings refine and add nuance to our genomic definitions of epidemic and endemic cholera, and are of direct relevance to controlling current and future cholera epidemics., Pandemic cholera was reintroduced to Argentina in 1992, leading to epidemic spread. Here, the authors use whole genome sequencing to show how, over 6 years, epidemic cholera was caused by invariant 7PET lineage Vibrio cholerae, against a background of sporadic disease caused by diverse local strains.

Shigella sonnei increasingly dominates the international epidemiological landscape of shigellosis. Treatment options for S. sonnei are dwindling due to resistance to several key antimicrobials, including the fluoroquinolones. Here we analyse nearly 400 S. sonnei whole genome sequences from both endemic and non-endemic regions to delineate the evolutionary history of the recently emergent fluoroquinolone-resistant S. sonnei. We reaffirm that extant resistant organisms belong to a single clonal expansion event. Our results indicate that sequential accumulation of defining mutations (gyrA-S83L, parC-S80I, and gyrA-D87G) led to the emergence of the fluoroquinolone-resistant S. sonnei population around 2007 in South Asia. This clone was then transmitted globally, resulting in establishments in Southeast Asia and Europe. Mutation analysis suggests that the clone became dominant through enhanced adaptation to oxidative stress. Experimental evolution reveals that under fluoroquinolone exposure in vitro, resistant S. sonnei develops further intolerance to the antimicrobial while the susceptible counterpart fails to attain complete resistance., Shigella sonnei is one of the main species causing shigellosis worldwide. Here the authors analyse nearly 400 S. sonnei genome sequences and carry out experimental evolution experiments to shed light into the evolutionary processes underlying the recent emergence of fluoroquinolone resistance in this pathogen.

Bloodstream infections by Salmonella enterica serovar Typhimurium constitute a major health burden in sub-Saharan Africa (SSA). These invasive non-typhoidal (iNTS) infections are dominated by isolates of the antibiotic resistance-associated sequence type (ST) 313. Here, we report emergence of ST313 sublineage II.1 in the Democratic Republic of the Congo. Sublineage II.1 exhibits extensive drug resistance, involving a combination of multidrug resistance, extended spectrum β-lactamase production and azithromycin resistance. ST313 lineage II.1 isolates harbour an IncHI2 plasmid we name pSTm-ST313-II.1, with one isolate also exhibiting decreased ciprofloxacin susceptibility. Whole genome sequencing reveals that ST313 II.1 isolates have accumulated genetic signatures potentially associated with altered pathogenicity and host adaptation, related to changes observed in biofilm formation and metabolic capacity. Sublineage II.1 emerged at the beginning of the 21st century and is involved in on-going outbreaks. Our data provide evidence of further evolution within the ST313 clade associated with iNTS in SSA., Invasive non-typhoidal Salmonella (iNTS) infections are dominated by antibiotic resistant isolates of the sequence type (ST) 313. Here, the authors identify the ST313 sublineage II.1 in the Democratic Republic of the Congo exhibiting extensive drug resistance and genetic signatures potentially associated with host adaptation.

Bloodstream infections by Salmonella enterica serovar Typhimurium constitute a major health burden in sub-Saharan Africa (SSA). These invasive non-typhoidal (iNTS) infections are dominated by isolates of the antibiotic resistance-associated sequence type (ST) 313. Here, we report emergence of ST313 sublineage II.1 in the Democratic Republic of the Congo. Sublineage II.1 exhibits extensive drug resistance, involving a combination of multidrug resistance, extended spectrum β-lactamase production and azithromycin resistance. ST313 lineage II.1 isolates harbour an IncHI2 plasmid we name pSTm-ST313-II.1, with one isolate also exhibiting decreased ciprofloxacin susceptibility. Whole genome sequencing reveals that ST313 II.1 isolates have accumulated genetic signatures potentially associated with altered pathogenicity and host adaptation, related to changes observed in biofilm formation and metabolic capacity. Sublineage II.1 emerged at the beginning of the 21st century and is involved in on-going outbreaks. Our data provide evidence of further evolution within the ST313 clade associated with iNTS in SSA.

Funder: Queensland Government (State of Queensland); doi: https://doi.org/10.13039/501100003550, Syphilis, which is caused by the sexually transmitted bacterium Treponema pallidum subsp. pallidum, has an estimated 6.3 million cases worldwide per annum. In the past ten years, the incidence of syphilis has increased by more than 150% in some high-income countries, but the evolution and epidemiology of the epidemic are poorly understood. To characterize the global population structure of T. pallidum, we assembled a geographically and temporally diverse collection of 726 genomes from 626 clinical and 100 laboratory samples collected in 23 countries. We applied phylogenetic analyses and clustering, and found that the global syphilis population comprises just two deeply branching lineages, Nichols and SS14. Both lineages are currently circulating in 12 of the 23 countries sampled. We subdivided T. p. pallidum into 17 distinct sublineages to provide further phylodynamic resolution. Importantly, two Nichols sublineages have expanded clonally across 9 countries contemporaneously with SS14. Moreover, pairwise genome analyses revealed examples of isolates collected within the last 20 years from 14 different countries that had genetically identical core genomes, which might indicate frequent exchange through international transmission. It is striking that most samples collected before 1983 are phylogenetically distinct from more recently isolated sublineages. Using Bayesian temporal analysis, we detected a population bottleneck occurring during the late 1990s, followed by rapid population expansion in the 2000s that was driven by the dominant T. pallidum sublineages circulating today. This expansion may be linked to changing epidemiology, immune evasion or fitness under antimicrobial selection pressure, since many of the contemporary syphilis lineages we have characterized are resistant to macrolides.

Funder: U.S. Department of Health & Human Services | National Institutes of Health (NIH), In order to control and eradicate epidemic cholera, we need to understand how epidemics begin, how they spread, and how they decline and eventually end. This requires extensive sampling of epidemic disease over time, alongside the background of endemic disease that may exist concurrently with the epidemic. The unique circumstances surrounding the Argentinian cholera epidemic of 1992–1998 presented an opportunity to do this. Here, we use 490 Argentinian V. cholerae genome sequences to characterise the variation within, and between, epidemic and endemic V. cholerae. We show that, during the 1992–1998 cholera epidemic, the invariant epidemic clone co-existed alongside highly diverse members of the Vibrio cholerae species in Argentina, and we contrast the clonality of epidemic V. cholerae with the background diversity of local endemic bacteria. Our findings refine and add nuance to our genomic definitions of epidemic and endemic cholera, and are of direct relevance to controlling current and future cholera epidemics.

Funder: U.S. Department of Health & Human Services | National Institutes of Health (NIH), In order to control and eradicate epidemic cholera, we need to understand how epidemics begin, how they spread, and how they decline and eventually end. This requires extensive sampling of epidemic disease over time, alongside the background of endemic disease that may exist concurrently with the epidemic. The unique circumstances surrounding the Argentinian cholera epidemic of 1992–1998 presented an opportunity to do this. Here, we use 490 Argentinian V. cholerae genome sequences to characterise the variation within, and between, epidemic and endemic V. cholerae. We show that, during the 1992–1998 cholera epidemic, the invariant epidemic clone co-existed alongside highly diverse members of the Vibrio cholerae species in Argentina, and we contrast the clonality of epidemic V. cholerae with the background diversity of local endemic bacteria. Our findings refine and add nuance to our genomic definitions of epidemic and endemic cholera, and are of direct relevance to controlling current and future cholera epidemics.

Shigella sonnei increasingly dominates the international epidemiological landscape of shigellosis. Treatment options for S. sonnei are dwindling due to resistance to several key antimicrobials, including the fluoroquinolones. Here we analyse nearly 400 S. sonnei whole genome sequences from both endemic and non-endemic regions to delineate the evolutionary history of the recently emergent fluoroquinolone-resistant S. sonnei. We reaffirm that extant resistant organisms belong to a single clonal expansion event. Our results indicate that sequential accumulation of defining mutations (gyrA-S83L, parC-S80I, and gyrA-D87G) led to the emergence of the fluoroquinolone-resistant S. sonnei population around 2007 in South Asia. This clone was then transmitted globally, resulting in establishments in Southeast Asia and Europe. Mutation analysis suggests that the clone became dominant through enhanced adaptation to oxidative stress. Experimental evolution reveals that under fluoroquinolone exposure in vitro, resistant S. sonnei develops further intolerance to the antimicrobial while the susceptible counterpart fails to attain complete resistance.