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In spite of its immutable susceptibility to penicillin, Treponema pallidum (T. pallidum) subsp. pallidum continues to cause millions of cases of syphilis each year worldwide, resulting in significant morbidity and mortality and underscoring the urgency of developing an effective vaccine to curtail the spread of the infection. Several technical challenges, including absence of an in vitro culture system until very recently, have hampered efforts to catalog the diversity of strains collected worldwide. Here, we provide near-complete genomes from 196 T. pallidum strains–including 191 T. pallidum subsp. pallidum–sequenced directly from patient samples collected from 8 countries and 6 continents. Maximum likelihood phylogeny revealed that samples from most sites were predominantly SS14 clade. However, 99% (84/85) of the samples from Madagascar formed two of the five distinct Nichols subclades. Although recombination was uncommon in the evolution of modern circulating strains, we found multiple putative recombination events between T. pallidum subsp. pallidum and subsp. endemicum, shaping the genomes of several subclades. Temporal analysis dated the most recent common ancestor of Nichols and SS14 clades to 1717 (95% HPD: 1543–1869), in agreement with other recent studies. Rates of SNP accumulation varied significantly among subclades, particularly among different Nichols subclades, and was associated in the Nichols A subclade with a C394F substitution in TP0380, a ERCC3-like DNA repair helicase. Our data highlight the role played by variation in genes encoding putative surface-exposed outer membrane proteins in defining separate lineages, and provide a critical resource for the design of broadly protective syphilis vaccines targeting surface antigens., Author summary Each year, millions of new cases of venereal and congenital syphilis, caused by the bacterium Treponema pallidum (T. pallidum) subsp. pallidum, are diagnosed worldwide, resulting in significant morbidity and mortality. Alongside endemic circulation of syphilis in low-income countries, disease resurgence in high-income nations has underscored the need for a vaccine. Due to prior technological limitations in culturing and sequencing the organism, the extent of the genetic diversity within modern strains of T. pallidum subsp. pallidum remains poorly understood, hampering development of a broadly protective vaccine. In this study, we obtained 196 near-complete T. pallidum genomes directly from clinical swabs from eight countries across six continents. Of these, 191 were identified as T. pallidum subsp. pallidum, including 90 Nichols clade genomes. Bayesian analysis revealed a high degree of variance in mutation rate among subclades. Interestingly, a Nichols subclade with a particularly high mutation rate harbors a non-synonymous mutation in a putative DNA repair helicase. Coupling sequencing data with protein structure prediction, we identified multiple novel amino acid variants in several proteins previously identified as potential vaccine candidates. Our data help inform current efforts to develop a broadly protective syphilis vaccine.

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.

Beside diagnostic uncertainties due to the lack of a perfect gold standard test for Helicobacter pylori infection, the diagnosis and the prevalence estimation for this infection encounter particular challenges in Africa including limited diagnostic tools and specific genetic background. We developed and evaluated the accuracy of an enzyme-linked immunosorbent assay (ELISA) system tailored for H. pylori genetics in Africa (HpAfr-ELISA). Strains belonging to main genetic populations infecting Africans were exploited as sources for whole-cell antigens to establish in-house the ELISA system. A phase II unmatched case-control study explored the diagnostic accuracy of the HpAfr-ELISA using a training set of samples collected from dyspeptic patients from Kinshasa, the Democratic Republic of Congo (DRC) who had been tested with invasive standard tests (i.e., histology, culture, and rapid urease test) in 2017. Then the assay was cross-validated through a community-based survey assessing the prevalence of H. pylori and associated factors in 425 adults from Mbujimayi, DRC in 2018. Bayesian inferences were used to deal with statistical uncertainties of estimates (true prevalence, sensitivity, and specificity) in the study population. At its optimal cut-off-value 20.2 U/mL, the assay achieved an estimated sensitivity of 97.6% (95% credible interval [95%CrI]: 89.2; 99.9%) and specificity of 90.5% (95%CrI: 78.6; 98.5). Consistent outcomes obtained at repeated tests attested the robustness of the assay (negative and positive agreements always > 70%). The true prevalence of H. pylori was estimated 53.8% [95%CrI: 42.8; 62.7%]. Increasing age (adjusted odds ratio [aOR] > 1.0 [95% confidence interval (CI): > 1.0; 1.1]; p, Author summary Enzyme-linked immunosorbent assay (ELISA) systems are useful tools for mass screening of Helicobacter pylori infection but their use encounters important challenges in Africa. This is mainly due to the specific genetic background of African H. pylori that may jeopardize the accuracy of currently available ELISA kits. In this study, we developed a new ELISA system, i.e. HpAfr-ELISA, based on pooled antigens of H. pylori that represent main genetic populations colonizing Africa. This new assay could be validated analytically using training samples collected in H. pylori positive and negative patients from the Democratic Republic of Congo (DRC). Then, the tool was successfully applied to a real-world community-based mass survey aiming at assessing the epidemiology of H. pylori in a rural area of the DRC. The characteristics of the HpAfr-ELISA assay indicated good robustness and high performances for detection of H. pylori. Statistical modeling enabled estimating the true latent prevalence of H. pylori infection and identifying factors predicting the exposition to the infection in the study population. This outcome will be useful for drawing efficient health policies and optimally allocating available resources to fight the H. pylori infection and related complications (e.g., gastric cancer) in the study area and globally in Africa.

Little is known about the genetic diversity of Salmonella enterica serovar Typhi (S. Typhi) circulating in Latin America. It has been observed that typhoid fever is still endemic in this part of the world; however, a lack of standardized blood culture surveillance across Latin American makes estimating the true disease burden problematic. The Colombian National Health Service established a surveillance system for tracking bacterial pathogens, including S. Typhi, in 2006. Here, we characterized 77 representative Colombian S. Typhi isolates collected between 1997 and 2018 using pulse field gel electrophoresis (PFGE; the accepted genotyping method in Latin America) and whole genome sequencing (WGS). We found that the main S. Typhi clades circulating in Colombia were clades 2.5 and 3.5. Notably, the sequenced S. Typhi isolates from Colombia were closely related in a global phylogeny. Consequently, these data suggest that these are endemic clades circulating in Colombia. We found that AMR in S. Typhi in Colombia was uncommon, with a small subset of organisms exhibiting mutations associated with reduced susceptibility to fluoroquinolones. This is the first time that S. Typhi isolated from Colombia have been characterized by WGS, and after comparing these data with those generated using PFGE, we conclude that PFGE is unsuitable for tracking S. Typhi clones and mapping transmission. The genetic diversity of pathogens such as S. Typhi is limited in Latin America and should be targeted for future surveillance studies incorporating WGS., Author summary Salmonella Typhi is the causative agent of typhoid fever, with between 9–13 million cases and 116,800 associated deaths annually. Typhoid fever is still a public health problem mainly in low and middle-income countries (LMICs), including in Latin America, which has a modelled incidence of up to 169 (32–642) cases per 100,000 person-years. Several international studies have aimed to fill data gaps regarding the global distribution and genetic landscape of typhoid; however, in spite of these efforts Latin America is still underrepresented. The globally dominant lineages of S. Typhi (e.g., H58), which often carry multi-drug resistance (MDR) plasmids, decreased fluoroquinolone susceptibility, and now azithromycin resistance, are not detectable by the accepted method (PFGE) used to track outbreaks of typhoid in Latin America. We compared PFGE with whole genome sequence (WGS) and found it correlated poorly, resulting in the over clustering of cases. We additionally found that unlike in most endemic countries, S. Typhi in Colombia are highly antimicrobial susceptible and restricted to a limited number of genotypes that are not as commonly identified in other S. Typhi endemic countries. Our study provides the first enhanced insights into the molecular epidemiology of S. Typhi in Colombia, using WGS data for the first time to investigate the population structure in Colombia and identifying predominant circulating genotypes. Our work demonstrates that routine surveillance with the integration of WGS is necessary not only to improve disease burden estimates, but also to track the national and regional transmission dynamics of S. Typhi.

Background Burkholderia sensu stricto is comprised mainly of opportunistic pathogens. This group is widely distributed in the environment but is especially important in clinical settings. In Mexico, few species have been correctly identified among patients, most often B. cepacia is described. Methodology/Principal findings In this study, approximately 90 strains identified as B. cepacia with the VITEK2 system were isolated from two medical centers in Mexico City and analyzed by MLSA, BOX-PCR and genome analysis. The initial identification of B. cepacia was confirmed for many strains, but B. contaminans, B. multivorans and B. vietnamiensis were also identified among clinical strains for the first time in hospitals in Mexico. Additionally, the presence of B. pseudomallei was confirmed, and a novel species within the B. cepacia complex was documented. Several strains misidentified as B. cepacia actually belong to the genera Pseudomonas, Stenotrophomonas and Providencia. Conclusions/Significance The presence of different Burkholderia species in Mexico was confirmed. Correct identification of Burkholderia species is important to provide accurate treatment for immunosuppressed patients., Author summary Burkholderia sensu stricto species are opportunistic pathogens in immunosuppressed patients. Both B. cepacia complex and B. pseudomallei are widely known around the world but not in Mexico. The identification of these bacteria can be difficult due to the similarity among the species. Tools like MLSA and genome analysis are currently used for identification. Approximately 90 strains identified as B. cepacia with the VITEK2 system were obtained from two hospitals in Mexico City. The strains were analyzed by MLSA, BOX-PCR and genome analysis. The analysis confirmed the presence of B. cepacia and identified for the first time B. multivorans, B. contaminans and B. vietnamiensis in Mexican clinical settings. The presence of B. pseudomallei was confirmed, and a novel species in the B. cepacia complex was also found. The correct identification of Burkholderia species in Mexico is important for appropriate treatment application.

Salmonella is a major cause of foodborne disease globally. Pigs can carry and shed non-typhoidal Salmonella (NTS) asymptomatically, representing a significant reservoir for these pathogens. To investigate Salmonella carriage by African domestic pigs, faecal and mesenteric lymph node samples were taken at slaughter in Nairobi, Busia (Kenya) and Chikwawa (Malawi) between October 2016 and May 2017. Selective culture, antisera testing and whole genome sequencing were performed on samples from 647 pigs; the prevalence of NTS carriage was 12.7% in Busia, 9.1% in Nairobi and 24.6% in Chikwawa. Two isolates of S. Typhimurium ST313 were isolated, but were more closely related to ST313 isolates associated with gastroenteritis in the UK than bloodstream infection in Africa. The discovery of porcine NTS carriage in Kenya and Malawi reveals potential for zoonotic transmission of diarrhoeal strains to humans in these countries, but not for transmission of clades specifically associated with invasive NTS disease in Africa., Author summary Healthy humans infected with non-typhoidal Salmonella (NTS) typically suffer from diarrhoeal disease which resolves without treatment. However, NTS infection of patients with an impaired immune system can lead to either bloodstream infection or infection in another part of the body; so-called ‘invasive’ NTS infection. Over the last twenty years, NTS have been the most prevalent bacteria to be isolated from human blood in sub-Saharan Africa. It is well known that pigs are able to carry a wide range of different NTS serovars without showing signs of disease themselves. Carrier pigs may be able to intermittently shed NTS in their faeces which may have the potential to cause disease in humans. If good hygiene protocols are not followed during the porcine slaughter process, pork intended for human consumption may become contaminated with NTS. In the next few years, pork consumption is projected to increase dramatically in sub-Saharan Africa. The degree of asymptomatic carriage of NTS by pigs in this region has never been investigated in detail. In this study, we report that pigs in sub-Saharan Africa are able to carry a wide variety of non-typhoidal Salmonella serovars, such as S. Typhimurium, that have the potential to cause diarrhoeal disease in humans. No clades of NTS which have previously been associated with invasive NTS disease in sub-Saharan Africa were detected.

Background Over the past recent years, Vibrio cholerae has been associated with outbreaks in sub-Saharan Africa, notably in Democratic Republic of the Congo (DRC). This study aimed to determine the genetic relatedness of isolates responsible for cholera outbreaks in eastern DRC between 2014 and 2017, and their potential spread to bordering countries. Methods/Principal findings Phenotypic analysis and whole genome sequencing (WGS) were carried out on 78 clinical isolates of V. cholerae associated with cholera in eastern provinces of DRC between 2014 and 2017. SNP-based phylogenomic data show that most isolates (73/78) were V. cholerae O1 biotype El Tor with CTX-3 type prophage. They fell within the third transmission wave of the current seventh pandemic El Tor (7PET) lineage and were contained in the introduction event (T)10 in East Africa. These isolates clustered in two sub-clades corresponding to Multiple Locus Sequence Types (MLST) profiles ST69 and the newly assigned ST515, the latter displaying a higher genetic diversity. Both sub-clades showed a distinct geographic clustering, with ST69 isolates mostly restricted to Lake Tanganyika basin and phylogenetically related to V. cholerae isolates associated with cholera outbreaks in western Tanzania, whereas ST515 isolates were disseminated along the Albertine Rift and closely related to isolates in South Sudan, Uganda, Tanzania and Zambia. Other V. cholerae isolates (5/78) were non-O1/non-O139 without any CTX prophage and no phylogenetic relationship with already characterized non-O1/non-O139 isolates. Conclusions/Significance Current data confirm the association of both DRC O1 7PET (T)10 sub-clades ST69 and ST515 with recurrent outbreaks in eastern DRC and at regional level over the past 10 years. Interestingly, while ST69 is predominantly a locally endemic sequence type, ST515 became adaptable enough to expand across DRC neighboring countries., Author summary Cholera is a severe diarrheal disease caused by the Gram-negative bacterium Vibrio cholerae. After originating in Asia, the disease spread across sub-Saharan Africa, notably Democratic Republic of the Congo. The aim of our study was to assess the transmission pattern of V. cholerae strains prevailing in eastern DRC, and determine their genetic relatedness to strains from other African countries and other parts of the world. Between 2014 and 2017, we isolated V. cholerae from fecal samples of patients with acute diarrhea in eastern DRC, and subsequently examined the DNA of the bacteria. The results show that they all clustered in two genetic groups (ST69 and ST515) falling within the third transmission wave of the current seventh pandemic El Tor (7PET) lineage and T10 introduction event in East Africa. The genetic signature of ST515 may be involved in its adaptation to environmental conditions found in eastern DRC, and contribute to its extended geographic distribution. Indeed, unlike the locally endemic ST69, ST515 is spreading extensively through DRC cross-border countries such as South Sudan, Tanzania, Uganda and Zambia. This plainly justifies a regional strategy to strengthen the fight against cholera in eastern Africa.

Non-toxigenic Vibrio cholerae isolates have been found associated with diarrheal disease globally, however, the global picture of non-toxigenic infections is largely unknown. Among non-toxigenic V. cholerae, ctxAB negative, tcpA positive (CNTP) isolates have the highest risk of disease. From 2001 to 2012, 71 infectious diarrhea cases were reported in Hangzhou, China, caused by CNTP serogroup O1 isolates. We sequenced 119 V. cholerae genomes isolated from patients, carriers and the environment in Hangzhou between 2001 and 2012, and compared them with 850 publicly available global isolates. We found that CNTP isolates from Hangzhou belonged to two distinctive lineages, named L3b and L9. Both lineages caused disease over a long time period with usually mild or moderate clinical symptoms. Within Hangzhou, the spread route of the L3b lineage was apparently from rural to urban areas, with aquatic food products being the most likely medium. Both lineages had been previously reported as causing local endemic disease in Latin America, but here we show that global spread of them has occurred, with the most likely origin of L3b lineage being in Central Asia. The L3b lineage has spread to China on at least three occasions. Other spread events, including from China to Thailand and to Latin America were also observed. We fill the missing links in the global spread of the two non-toxigenic serogroup O1 V. cholerae lineages that can cause human infection. The results are important for the design of future disease control strategies: surveillance of V. cholerae should not be limited to ctxAB positive strains., Author summary Non-toxigenic Vibrio cholerae isolates are associated with diarrheal disease globally. Among them, ctxAB negative, tcpA positive (CNTP) isolates have the highest risk of disease because they may be able to colonize the human intestine. By population genomic analysis of 850 previously published genomes and 119 newly sequenced genomes in this study, we found that most of CNTP isolates can be attributed into two distinctive lineages, L3b and L9. Both lineages are not only circulating in local regions causing endemic disease, but have been spreading worldwide over the past 100 years. Central Asia is the most likely origin of the L3b lineage, and the strains spread to China on at least three occasions. The L3b strains were also exported from China to other parts of the world, and one of the migrations are related to the groups that circulated in Latin America. Within Hangzhou, a modernized city in China, aquatic food products are the most likely medium of CNTP strains, and a spread pattern from rural aquafarms to the urban areas was observed. This work filled the missing links in the global spread of two non-toxigenic serogroup O1 V. cholerae lineages that can cause human infection. The results suggest that in future disease control efforts, the sampling of V. cholerae should not be limited to the apparently toxic isolates, and that a more thorough and unbiased sampling framework is needed.

Background Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. The global burden and distribution of melioidosis is poorly understood, including in the Caribbean. B. pseudomallei was previously isolated from humans and soil in eastern Puerto Rico but the abundance and distribution of B. pseudomallei in Puerto Rico as a whole has not been thoroughly investigated. Methodology/Principal findings We collected 600 environmental samples (500 soil and 100 water) from 60 sites around Puerto Rico. We identified B. pseudomallei by isolating it via culturing and/or using PCR to detect its DNA within complex DNA extracts. Only three adjacent soil samples from one site were positive for B. pseudomallei with PCR; we obtained 55 isolates from two of these samples. The 55 B. pseudomallei isolates exhibited fine-scale variation in the core genome and contained four novel genomic islands. Phylogenetic analyses grouped Puerto Rico B. pseudomallei isolates into a monophyletic clade containing other Caribbean isolates, which was nested inside a larger clade containing all isolates from Central/South America. Other Burkholderia species were commonly observed in Puerto Rico; we cultured 129 isolates from multiple soil and water samples collected at numerous sites around Puerto Rico, including representatives of B. anthina, B. cenocepacia, B. cepacia, B. contaminans, B. glumae, B. seminalis, B. stagnalis, B. ubonensis, and several unidentified novel Burkholderia spp. Conclusions/Significance B. pseudomallei was only detected in three soil samples collected at one site in north central Puerto Rico with only two of those samples yielding isolates. All previous human and environmental B. pseudomallei isolates were obtained from eastern Puerto Rico. These findings suggest B. pseudomallei is ecologically established and widely dispersed in the environment in Puerto Rico but rare. Phylogeographic patterns suggest the source of B. pseudomallei populations in Puerto Rico and elsewhere in the Caribbean may have been Central or South America., Author summary The objective of this study was to examine the distribution and abundance of Burkholderia pseudomallei in the environment in Puerto Rico. B. pseudomallei is a microbe that lives in soil and causes the disease melioidosis. We conducted sampling around Puerto Rico to survey for the presence of B. pseudomallei in the environment. Of the 600 environmental samples collected, we isolated live B. pseudomallei from just two soil samples collected from the same site, which was in a region of the island where B. pseudomallei had never been previously reported. These results suggest B. pseudomallei is widely dispersed but rare in the environment in Puerto Rico. B. pseudomallei isolates from Puerto Rico are most closely related to other strains from the Caribbean. Caribbean strains are inside a larger group that contained all analyzed isolates from Central/South America, suggesting that B. pseudomallei populations in the Caribbean may have been introduced from Central or South America.

Background Pathogenic treponemes related to Treponema pallidum are both human (causing syphilis, yaws, bejel) and animal pathogens (infections of primates, venereal spirochetosis in rabbits). A set of 11 treponemal genome sequences including those of five Treponema pallidum ssp. pallidum (TPA) strains (Nichols, DAL-1, Mexico A, SS14, Chicago), four T. p. ssp. pertenue (TPE) strains (CDC-2, Gauthier, Samoa D, Fribourg-Blanc), one T. p. ssp. endemicum (TEN) strain (Bosnia A) and one strain (Cuniculi A) of Treponema paraluisleporidarum ecovar Cuniculus (TPeC) were tested for the presence of positively selected genes. Methodology/Principal findings A total of 1068 orthologous genes annotated in all 11 genomes were tested for the presence of positively selected genes using both site and branch-site models with CODEML (PAML package). Subsequent analyses with sequences obtained from 62 treponemal draft genomes were used for the identification of positively selected amino acid positions. Synthetic biotinylated peptides were designed to cover positively selected protein regions and these peptides were tested for reactivity with the patient's syphilis sera. Altogether, 22 positively selected genes were identified in the TP genomes and TPA sets of positively selected genes differed from TPE genes. While genetic variability among TPA strains was predominantly present in a number of genetic loci, genetic variability within TPE and TEN strains was distributed more equally along the chromosome. Several syphilitic sera were shown to react with some peptides derived from the protein sequences evolving under positive selection. Conclusions/Significance The syphilis-, yaws-, and bejel-causing strains differed relative to sets of positively selected genes. Most of the positively selected chromosomal loci were identified among the TPA treponemes. The local accumulation of genetic variability suggests that the diversification of TPA strains took place predominantly in a limited number of genomic regions compared to the more dispersed genetic diversity differentiating TPE and TEN strains. The identification of positively selected sites in tpr genes and genes encoding outer membrane proteins suggests their role during infection of human and animal hosts. The driving force for adaptive evolution at these loci thus appears to be the host immune response as supported by observed reactivity of syphilitic sera with some peptides derived from protein sequences showing adaptive evolution., Author summary In the genus Treponema there are several human and animal pathogens that include the causative agent of syphilis (Treponema pallidum ssp. pallidum; TPA), the causative agent of yaws (T. p. ssp. pertenue; TPE), and the causative agent of endemic syphilis (T. p. ssp. endemicum; TEN). T. paraluisleporidarum causes venereal spirochetosis in rabbits. We used whole genome sequences of 11 treponemal strains together with additional 62 draft genomic data to identify genes evolving under positive selection. The identified genes evolving under positive selection partly overlapped with the genes previously reported as recombinant and were found to be different in treponemal subspecies. Since both genetic recombination and positive selection could allow a survival of pathogenic bacteria despite the human immune response, identification of such genes could predict the major antigens recognized by the human immune system and also identify the most suitable components for development of an anti-treponemal vaccine.

Background Invasive non-typhoidal Salmonella (iNTS) serovars S. Typhimurium and S. Enteritidis are major etiologic agents of invasive bacterial disease among infants and young children in sub-Saharan Africa, including in Mali. Early studies of iNTS serovars in several countries indicated that S. Typhimurium was more prevalent than S. Enteritidis, including in Mali before 2008. We investigated genomic and associated phenotypic changes associated with an increase in the relative proportion of iNTS caused by S. Enteritidis versus S. Typhimurium in Bamako, Mali, during the period 2002–2012. Methodology/Principal findings Comparative genomics studies identified homologs of tetracycline resistance and arsenic utilization genes that were associated with the temporal shift of serovars causing iNTS shift, along with several hypothetical proteins. These findings, validated through PCR screening and phenotypic assays, provide initial steps towards characterizing the genomic changes consequent to unknown evolutionary pressures associated with the shift in serovar prevalence. Conclusions/Significance This work identified a shift to S. Enteritidis from the more classic S. Typhimurium, associated with iNTS in Bamako, Mali, during the period 2002–2012. This type of shift in underlying iNTS pathogens are of great importance to pediatric public health in endemic regions of sub-Saharan Africa. Additionally, this work demonstrates the utility of combining epidemiologic data, whole genome sequencing, and functional characterization in the laboratory to identify and characterize genomic changes in the isolates that may be involved with the observed shift in circulating iNTS agents., Author summary Much remains unknown about the mode of transmission of iNTS or the reservoirs of infection. As such, insight into potential selective pressures on underlying serovars bears importance to public health. Longitudinal studies over the years 2002–2012 identified a shift in the proportion of invasive non-typhoidal Salmonella (iNTS) disease caused by S. Typhimurium or S. Enteritidis in Bamako, Mali. Since S. Enteritidis exhibits a higher rate of fatal cases among the pediatric population than S. Typhimurium, it is important to understand what led to the increased proportion of cases from this serovar. This study examined the genetic changes in S. Enteritidis associated with this serovar shift through comparative genomics and laboratory findings. Through these methods, genes related to tetracycline resistance and arsenic catabolism were associated with the shift in serovars. These findings represent preliminary steps in investigating this underlying shift and determining the long-term repercussions of these changes to the epidemiologic profile of iNTS disease.

Brucella abortus and B. melitensis have been reported in several studies in animals in Zimbabwe but the extent of the disease remains poorly known. Thus, characterizing the circulating strains is a critical first step in understanding brucellosis in the country. In this study we used an array of molecular assays including AMOS-PCR, Bruce-ladder, multiple locus variable number tandem repeats analysis (MLVA) and single nucleotide polymorphisms from whole genome sequencing (WGS-SNP) to characterize Brucella isolates to the species, biovar, and individual strain level. Sixteen Brucella strains isolated in Zimbabwe at the Central Veterinary laboratory from various hosts were characterized using all or some of these assays. The strains were identified as B. ovis, B. abortus, B. canis and B. suis, with B. canis being the first report of this species in Zimbabwe. Zimbabwean strains identified as B. suis and B. abortus were further characterized with whole genome sequencing and were closely related to reference strains 1330 and 86/8/59, respectively. We demonstrate the range of different tests that can be performed from simple assays that can be run in laboratories lacking sophisticated instrumentation to whole genome analyses that currently require substantial expertise and infrastructure often not available in the developing world., Author summary Brucellosis is endemic in Zimbabwe. This article describes the use of various assays such as AMOS, Bruce-ladder, MLVA, and whole genome sequencing to characterize Brucella species isolated from different animals in Zimbabwe. Choice of which assays to use in the laboratory is generally done considering reproducibility, robustness, expertise and affordability in a given setting. As evidenced in this study, most laboratories in Africa lack resources especially finances, equipments and expertise to perform necessary tests for diagnosis and identification of specific pathogens. The study shows that the differentiation of species can be correctly concluded from the analysis with AMOS, Bruce-ladder and MLVA16 assays. Furthermore, MLVA16 can be used as an epidemiological tool and traceback of outbreaks. These PCR assays can therefore add to the control and eradication of brucellosis, since the Brucella species (B. ovis, B. abortus, B. suis and B. canis) existing in Zimbabwe could be identified and characterized.

Background The Gram-negative soil dwelling bacterium Burkholderia pseudomallei is the etiological agent of melioidosis. The disease is endemic in most parts of Southeast Asia and northern Australia. Over last few years, there has been an increase in number of melioidosis cases from India; however the disease epidemiology is less clearly understood. Multi-locus sequence typing (MLST) is a powerful genotypic method used to characterize the genetic diversity of B. Pseudomallei both within and across the geographic regions. Methods In this study, MLST analysis was performed on 64 B. pseudomallei clinical isolates. These isolates were obtained between 2008–2014 from southwestern coastal region of India. Broad population patterns of Indian B. pseudomallei isolates in context with isolates of Southeast Asia or global collection was determined using in silico phylogenetic tools. Results A total of 32 Sequence types (STs) were reported among these isolates of which 17 STs (53%) were found to be novel. ST1368 was found as group founder and the most predominant genotype (n = 11, 17%). Most of the B. pseudomallei isolates reported in this study (or other Indian isolates available in MLST database) clustered in one major group suggesting clonality in Indian isolates; however, there were a few outliers. When analyzed by measure of genetic differentiation (FST) and other phylogenetic tools (e.g. PHYLOViZ), Indian STs were found closer to Southeast Asian isolates than Australian isolates. The phylogenetic analysis further revealed that within Asian clade, Indian isolates grouped more closely with isolates from Sri Lanka, Vietnam, Bangladesh and Thailand. Conclusions Overall, the results of this study suggest that the Indian B. pseudomallei isolates are closely related with lesser heterogeneity among them and cluster in one major group suggesting clonality of the isolates. However, it appears that there are a few outliers which are distantly related to the majority of Indian STs. Phylogenetic analysis suggest that Indian isolates are closely related to isolates from Southeast Asia, particularly from South Asia., Author summary Burkholderia pseudomallei, a gram negative bacterium, is the causative agent of melioidosis. B. pseudomallei is a soil saprophyte and causes infections in humans by percutaneous inoculation, inhalation or ingestion. Melioidosis is a life threatening disease, which requires prolonged antibiotic treatment and is classically characterized by pneumonia, septicemia and multiple abscesses. Melioidosis is widely prevalent in Southeast Asia and northern Australia. Of late it has been reported from tropical, subtropical and temperate regions. The predicted annual global burden of melioidosis is 165,000 cases. B. pseudomallei has been classified as a Category B threat agent by US Center for Disease Control. Melioidosis is an emerging disease in India that affects many regions. Over the past few years, there has been an increase in number of melioidosis cases, mainly from southwestern costal part of India. This study provides new insights into molecular epidemiology of melioidosis in India. By use of multi locus sequence typing (MLST), we show that Indian isolates are closely related and cluster in one major group suggesting clonality of the isolates. We further show that Indian isolates are more closely related to isolates from Asian countries particularly from South Asia.

Treponema pallidum infections occur worldwide causing, among other diseases, syphilis and yaws. In particular sexually transmitted syphilis is regarded as a re-emerging infectious disease with millions of new infections annually. Here we present three historic T. pallidum genomes (two from T. pallidum ssp. pallidum and one from T. pallidum ssp. pertenue) that have been reconstructed from skeletons recovered from the Convent of Santa Isabel in Mexico City, operational between the 17th and 19th century. Our analyses indicate that different T. pallidum subspecies caused similar diagnostic presentations that are normally associated with syphilis in infants, and potential evidence of a congenital infection of T. pallidum ssp. pertenue, the causative agent of yaws. This first reconstruction of T. pallidum genomes from archaeological material opens the possibility of studying its evolutionary history at a resolution previously assumed to be out of reach., Author summary Among the worldwide prevalent treponemal diseases syphilis is a global threat that is currently re-emerging. The origins of syphilis and other treponemal diseases are as yet unresolved and are subject to an intensive scholarly debate. Until now, assumptions on its origins and evolutionary history could only be drawn from osteological analyses of past cases and genetic analysis of contemporary T. pallidum genomes; contributions from ancient DNA are very rare and have so far failed to provide genome-level data. The ancient T. pallidum genomes presented here allow us, for the first time, to perform genome-wide comparative analyses and to assess a connection between osteological manifestations of past treponemal cases and specific T. pallidum species. Our study demonstrates the possibility of retrieving ancient T. pallidum genomes from archeological material and thereby establishes a new method that could greatly contribute to uncover the mystery regarding the origins of treponemal diseases.

Background Plague, caused by the bacterium Yersinia pestis, is a highly infectious, zoonotic disease. Hundreds of human plague cases are reported across the world annually. Qinghai Plateau is one of the most severely affected plague regions in China, with more than 240 fatal cases of Y. pestis in the last 60 years. Conventional epidemiologic analysis has effectively guided the prevention and control of local plague transmission; however, molecular genetic analysis is more effective for investigating population diversity and transmission. In this report, we employed different genetic markers to analyze the population structure of Y. pestis in Qinghai Plateau. Methodology/Principal finding We employed a two-step hierarchical strategy to analyze the phylogeny of 102 Qinghai Plateau isolates of Y. pestis, collected between 1954 and 2011. First, we defined the genealogy of Y. pestis by constructed minimum spanning tree based on 25 key SNPs. Seven groups were identified, with group 1.IN2 being identified as the dominant population. Second, two methods, MLVA and CRISPR, were applied to examine the phylogenetic detail of group 1.IN2, which was further divided into three subgroups. Subgroups of 1.IN2 revealed a clear geographic cluster, possibly associated with interaction between bacteriophage and Y. pestis. More recently, Y. pestis populations appear to have shifted from the east toward the center and west of Qinghai Plateau. This shift could be related to destruction of the local niche of the original plague focus through human activities. Additionally, we found that the abundance and relative proportion of 1.IN2 subgroups varied by decade and might be responsible for the fluctuations of plague epidemics in Qinghai Plateau. Conclusion/Significance Molecular genotyping methods provided us with detailed information on population diversity and the spatial-temporal distribution of dominant populations of Y. pestis, which will facilitate future surveillance, prevention, and control of plague in Qinghai Plateau., Author summary Plague is a highly infectious disease caused by the Yersinia pestis bacterium. Since the first strain of Y. pestis was isolated in Qinghai in 1954, confirmed plague cases have occurred nearly every year, and more than 240 people have died from plague over the past 60 years. In this study, we analyzed 102 Y. pestis strains collected from Qinghai Plateau between 1954 and 2011. We determined their genetic diversity and inferred their spatial-temporal distribution, based on genetic markers including SNPs, VNTRs and CRISPRs. Our results indicate that 1.IN2 is the dominant group of Y. pestis in Qinghai Plateau, and its three subpopulations revealed clear geographic clustering that might be driven by interaction with bacteriophages. We observed that the Y. pestis population has moved from the east of Qinghai Plateau to central and western regions over the past 60 years. We also found that the abundance and relative proportion of 1.IN2 subgroups varied over time, leading to fluctuations in plague epidemics. These results extend our knowledge of the genetic diversity of Y. pestis, and its population dynamics in natural plague foci over a number of years. With ongoing risk of outbreaks, we recommend enhanced surveillance in this region.

Plague, caused by Yersinia pestis, was classified as a reemerging infectious disease by the World Health Organization. The five human pneumonic plague cases in Yulong County in 2005 gave rise to the discovery of a Yulong plague focus in Yunnan province, China. Thereafter, continuous wild rodent plague (sylvatic plague) was identified as the main plague reservoir of this focus. In this study, the epizootics in Yulong focus were described, and three molecular typing methods, including the different region (DFR) analysis, clustered regularly interspaced short palindromic repeats (CRISPRs), and the multiple-locus variable number of tandem repeats (VNTR) analysis (MLVA) (14+12), were used for the molecular typing and source tracing of Y. pestis isolates in the Yulong plague focus. Simultaneously, several isolates from the vicinity of Yunnan were used as controls. The results showed that during the 10-year period from 2006 to 2016, an animal plague epidemic occurred in 6 of those years, and 5 villages underwent an animal plague epidemic within a 30-km2 area of the Yulong plague focus. Searching for dead mice was the most effective monitoring method in this plague focus. No positive sample has been found in 6937 captured live rodents thus far, suggesting that the virulence of strains in the Yulong plague focus is stronger and the survival time of mice is shorter after infection. Strains from Lijiang, Sichuan and Tibet were of the same complex based on a typing analysis of DFR and CRISPR. The genetic relationship of Y. pestis illustrated by MLVA “14+12” demonstrates that Tibet and Sichuan strains evolved from the strains 1.IN2 (Qinghai, 1970 and Tibet, 1976), and Lijiang strains are closer to Batang strains (Batang County in Sichuan province, 2011, Himalaya marmot plague foci) in terms of genetic or phylogenic relationships. In conclusion, we have a deeper understanding of this new plague focus throughout this study, which provides a basis for effective prevention and control., Author summary Plague is a type of zoonosis that is highly lethal to humans. The surveillance of animal hosts is critical for the prevention and control of plague. The Yulong plague focus is a newly discovered plague focus in China in recent years. The plague outbreak had attracted widespread attention because 5 people were infected in 2005, 2 of whom died. We have monitored the plague focus for a decade, and isolated strains and DNAs of Yersinia pestis were studied. The structure, origin and evolutionary trend of the Yulong plague focus were clarified, which provides a scientific basis for the effective prevention and control of human plague. This article also provides a set of paradigms for the systematic study of new plague foci, which is a perfect combination of traditional monitoring methods and modern research methods.

Background Yersinia pestis appears to be maintained in multiple, geographically separate, and phylogenetically distinct subpopulations within the highlands of Madagascar. However, the dynamics of these locally differentiated subpopulations through time are mostly unknown. To address that gap and further inform our understanding of plague epidemiology, we investigated the phylogeography of Y. pestis in Madagascar over an 18 year period. Methodology/Principal findings We generated whole genome sequences for 31 strains and discovered new SNPs that we used in conjunction with previously identified SNPs and variable-number tandem repeats (VNTRs) to genotype 773 Malagasy Y. pestis samples from 1995 to 2012. We mapped the locations where samples were obtained on a fine geographic scale to examine phylogeographic patterns through time. We identified 18 geographically separate and phylogenetically distinct subpopulations that display spatial and temporal stability, persisting in the same locations over a period of almost two decades. We found that geographic areas with higher levels of topographical relief are associated with greater levels of phylogenetic diversity and that sampling frequency can vary considerably among subpopulations and from year to year. We also found evidence of various Y. pestis dispersal events, including over long distances, but no evidence that any dispersal events resulted in successful establishment of a transferred genotype in a new location during the examined time period. Conclusions/Significance Our analysis suggests that persistent endemic cycles of Y. pestis transmission within local areas are responsible for the long term maintenance of plague in Madagascar, rather than repeated episodes of wide scale epidemic spread. Landscape likely plays a role in maintaining Y. pestis subpopulations in Madagascar, with increased topographical relief associated with increased levels of localized differentiation. Local ecological factors likely affect the dynamics of individual subpopulations and the associated likelihood of observing human plague cases in a given year in a particular location., Author summary Plague exists in several geographically separate phylogenetic groups within Madagascar, but little is known about the dynamics of these groups through time. We subtyped 773 Malagasy plague samples and identified 18 phylogenetic groups that showed persistence in the same locations over a period of almost two decades. Locations with more topographical relief contained more phylogenetic groups than flatter areas and different phylogenetic groups varied considerably in the number of samples collected each year. Transfers of plague from one location to another definitely occur, but appear to seldom result in the transplanted phylogenetic group becoming successfully established in a new location. Persistent, local transmission cycles are likely responsible for the long term maintenance of plague in Madagascar, rather than repeated wide scale disease transmission events. Landscape likely plays a role in maintaining different phylogenetic groups, with increased topographical relief associated with increased numbers of phylogenetic groups. Local ecological factors likely affect the dynamics of individual subpopulations and the associated likelihood of observing human plague cases in a given year in a particular location.

Background Depending on the epidemiological setting, a variable proportion of leprosy patients will suffer from excessive pro-inflammatory responses, termed type-1 reactions (T1R). The LRRK2 gene encodes a multi-functional protein that has been shown to modulate pro-inflammatory responses. Variants near the LRRK2 gene have been associated with leprosy in some but not in other studies. We hypothesized that LRRK2 was a T1R susceptibility gene and that inconsistent association results might reflect different proportions of patients with T1R in the different sample settings. Hence, we evaluated the association of LRRK2 variants with T1R susceptibility. Methodology An association scan of the LRRK2 locus was performed using 156 single-nucleotide polymorphisms (SNPs). Evidence of association was evaluated in two family-based samples: A set of T1R-affected and a second set of T1R-free families. Only SNPs significant for T1R-affected families with significant evidence of heterogeneity relative to T1R-free families were considered T1R-specific. An expression quantitative trait locus (eQTL) analysis was applied to evaluate the impact of T1R-specific SNPs on LRRK2 gene transcriptional levels. Principal Findings A total of 18 T1R-specific variants organized in four bins were detected. The core SNP capturing the T1R association was the LRRK2 missense variant M2397T (rs3761863) that affects LRRK2 protein turnover. Additionally, a bin of nine SNPs associated with T1R were eQTLs for LRRK2 in unstimulated whole blood cells but not after exposure to Mycobacterium leprae antigen. Significance The results support a preferential association of LRRK2 variants with T1R. LRRK2 involvement in T1R is likely due to a pathological pro-inflammatory loop modulated by LRRK2 availability. Interestingly, the M2397T variant was reported in association with Crohn’s disease with the same risk allele as in T1R suggesting common inflammatory mechanism in these two distinct diseases., Author Summary A major challenge of current leprosy control is the management of host pathological immune responses coined Type-1 Reactions (T1R). T1R are characterized by acute inflammatory episodes whereby cellular immune responses are directed against host peripheral nerve cells. T1R affects up half of all leprosy patients and are a major cause of leprosy-associated disabilities. Since there is evidence that host genetic factors predispose leprosy patients to T1R, we have conducted a candidate gene study to test if LRRK2 gene variants are T1R risk factors. The choice of LRRK2 was motivated by the fact that LRRK2 was associated with leprosy per se in some but not in other studies. We reasoned that this may reflect different proportions of leprosy patients with T1R in the different samples and that LRRK2 may in truth be a T1R susceptibility gene. Here, we show that variants overlapping the LRRK2 gene, reported as suggestive leprosy per se susceptibility factors in a previous genome-wide association study, are preferentially associated with T1R. The main SNP carrying most of the association signal is the amino-acid change M2397T (rs3761863) which is known to impact LRRK2 turnover. Interestingly, eQTL SNPs counterbalanced the effect of the M2397T variant but this compensatory mechanism was abrogated by Mycobacterium leprae antigen stimulation.

Salmonella enterica serovar Weltevreden (S. Weltevreden) is an emerging cause of diarrheal and invasive disease in humans residing in tropical regions. Despite the regional and international emergence of this Salmonella serovar, relatively little is known about its genetic diversity, genomics or virulence potential in model systems. Here we used whole genome sequencing and bioinformatics analyses to define the phylogenetic structure of a diverse global selection of S. Weltevreden. Phylogenetic analysis of more than 100 isolates demonstrated that the population of S. Weltevreden can be segregated into two main phylogenetic clusters, one associated predominantly with continental Southeast Asia and the other more internationally dispersed. Subcluster analysis suggested the local evolution of S. Weltevreden within specific geographical regions. Four of the isolates were sequenced using long read sequencing to produce high quality reference genomes. Phenotypic analysis in Hep-2 cells and in a murine infection model indicated that S. Weltevreden were significantly attenuated in these models compared to the classical S. Typhimurium reference strain SL1344. Our work outlines novel insights into this important emerging pathogen and provides a baseline understanding for future research studies., Author Summary Organisms belonging to the species Salmonella enterica are a major cause of infection globally. Such infections can be zoonotic in origin or transmitted between humans. One of the most notable features of the genus Salmonella is that the dominant serovars that cause human infections change over time, with new threats periodically emerging. These trends often go unnoticed and are underreported in low-income locations. There is good evidence that Salmonella Weltevreden is emerging (particularly in low-income countries in the tropics) as a significant cause of diarrhea and sometimes invasive bacterial disease in humans. However, little is known about the phylogenetic structure or virulence potential of this unstudied serovar. Here, we provide a detailed phylogenetic analysis of S. Weltevreden through whole genome sequencing and bioinformatics tools. We identify that specific phylogenetic clusters are associated with geographical regions, providing novel data regarding the global distribution of different clades and high quality reference genomes to facilitate future work. We additionally show that S. Weltevreden has a distinct virulence-associated phenotype in conventional laboratory Salmonella pathogenicity assays that will guide future investigations on this serovar.

Cholera is an acute diarrheal disease and a major public health problem in many developing countries in Asia, Africa, and Latin America. Since the Bay of Bengal is considered the epicenter for the seventh cholera pandemic, it is important to understand the genetic dynamism of Vibrio cholerae from Kolkata, as a representative of the Bengal region. We analyzed whole genome sequence data of V. cholerae O1 isolated from cholera patients in Kolkata, India, from 2007 to 2014 and identified the heterogeneous genomic region in these strains. In addition, we carried out a phylogenetic analysis based on the whole genome single nucleotide polymorphisms to determine the genetic lineage of strains in Kolkata. This analysis revealed the heterogeneity of the Vibrio seventh pandemic island (VSP)-II in Kolkata strains. The ctxB genotype was also heterogeneous and was highly related to VSP-II types. In addition, phylogenetic analysis revealed the shifts in predominant strains in Kolkata. Two distinct lineages, 1 and 2, were found between 2007 and 2010. However, the proportion changed markedly in 2010 and lineage 2 strains were predominant thereafter. Lineage 2 can be divided into four sublineages, I, II, III and IV. The results of this study indicate that lineages 1 and 2-I were concurrently prevalent between 2007 and 2009, and lineage 2-III observed in 2010, followed by the predominance of lineage 2-IV in 2011 and continued until 2014. Our findings demonstrate that the epidemic of cholera in Kolkata was caused by several distinct strains that have been constantly changing within the genetic lineages of V. cholerae O1 in recent years., Author summary Seven cholera pandemics have been recorded throughout history, and the sixth, and presumably earlier pandemics, emerged from the Bay of Bengal. The seventh pandemic strain also appeared and spread from this area to different area of the world. Thus, the Bay of Bengal has always been considered the epicenter of cholera pandemics. In this report, we characterized the V. cholerae strains isolated from patients with cholera in Kolkata as a representative area of the Bay of Bengal between 2007 and 2014. The analysis revealed that the cholera epidemics were caused by several distinct V. cholerae O1 strains and that the predominant strains have genetically changed several times in recent years.

Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), remains a serious global health concern. Since their emergence in the mid-1970s multi-drug resistant (MDR) S. Typhi now dominate drug sensitive equivalents in many regions. MDR in S. Typhi is almost exclusively conferred by self-transmissible IncHI1 plasmids carrying a suite of antimicrobial resistance genes. We identified over 300 single nucleotide polymorphisms (SNPs) within conserved regions of the IncHI1 plasmid, and genotyped both plasmid and chromosomal SNPs in over 450 S. Typhi dating back to 1958. Prior to 1995, a variety of IncHI1 plasmid types were detected in distinct S. Typhi haplotypes. Highly similar plasmids were detected in co-circulating S. Typhi haplotypes, indicative of plasmid transfer. In contrast, from 1995 onwards, 98% of MDR S. Typhi were plasmid sequence type 6 (PST6) and S. Typhi haplotype H58, indicating recent global spread of a dominant MDR clone. To investigate whether PST6 conferred a selective advantage compared to other IncHI1 plasmids, we used a phenotyping array to compare the impact of IncHI1 PST6 and PST1 plasmids in a common S. Typhi host. The PST6 plasmid conferred the ability to grow in high salt medium (4.7% NaCl), which we demonstrate is due to the presence in PST6 of the Tn6062 transposon encoding BetU., Author Summary Typhoid fever is caused by the bacterium Salmonella enterica serovar Typhi (S. Typhi). Treatment relies on antimicrobial drugs, however many S. Typhi are multi-drug resistant (MDR), severely compromising treatment options. MDR typhoid is associated with multiple drug resistance genes, which can be transferred between S. Typhi and other bacteria via self-transmissible plasmids. We used sequence analysis to identify single nucleotide polymorphisms (SNPs) within these plasmids, and used high-resolution SNP typing to trace the subtypes (termed haplotypes) of both the S. Typhi bacteria and their MDR plasmids isolated from more than 450 typhoid patients since 1958. Among isolates collected before 1995, a variety of plasmid haplotypes and S. Typhi haplotypes were detected, indicating that MDR typhoid was caused by a diverse range of S. Typhi and MDR plasmids. In contrast, 98% of MDR S. Typhi samples isolated from 1995 were of the same S. Typhi haplotype and plasmid haplotype, indicating that the recent increase in rates of MDR typhoid is due to the global spread of a dominant S. Typhi-plasmid combination. We demonstrate this particular plasmid type contains a transposon encoding two transporter genes, enabling its S. Typhi host to grow in the presence of high salt concentrations.