Five Years of GenoTyphi: Updates to the Global Salmonella Typhi Genotyping Framework.

In 2016, a whole-genome sequence (WGS)-based genotyping framework (GenoTyphi) was developed and provided a phylogenetically informative nomenclature for lineages of Salmonella Typhi, the etiological agent of typhoid fever. Subsequent surveillance studies have revealed additional epidemiologically important subpopulations, which require the definition of new genotypes and extension of associated software to facilitate the detection of antimicrobial resistance (AMR) mutations. Analysis of 4632 WGS provide an updated overview of the global S Typhi population structure and genotyping framework, revealing the widespread nature of haplotype 58 ([H58] 4.3.1) genotypes and the diverse range of genotypes carrying AMR mutations.

Spontaneous Emergence of Azithromycin Resistance in Independent Lineages of Salmonella Typhi in Northern India.

BACKGROUND: The emergence and spread of antimicrobial resistance (AMR) pose a major threat to the effective treatment and control of typhoid fever. The ongoing outbreak of extensively drug-resistant Salmonella Typhi (S. Typhi) in Pakistan has left azithromycin as the only remaining broadly efficacious oral antimicrobial for typhoid in South Asia. Ominously, azithromycin-resistant S. Typhi organisms have been subsequently reported in Bangladesh, Pakistan, and Nepal. METHODS: Here, we aimed to understand the molecular basis of AMR in 66 S. Typhi organisms isolated in a cross-sectional study performed in a suburb of Chandigarh in Northern India using whole-genome sequencing and phylogenetic analysis. RESULTS: We identified 7 S. Typhi organisms with the R717Q mutation in the acrB gene that was recently found to confer resistance to azithromycin in Bangladesh. Six out of the seven azithromycin-resistant S. Typhi isolates also exhibited triple mutations in gyrA (S83F and D87N) and parC (S80I) genes and were resistant to ciprofloxacin. These contemporary ciprofloxacin/azithromycin-resistant isolates were phylogenetically distinct from each other and from those reported from Bangladesh, Pakistan, and Nepal. CONCLUSIONS: The independent emergence of azithromycin-resistant typhoid in Northern India reflects an emerging broader problem across South Asia and illustrates the urgent need for the introduction of typhoid conjugate vaccines in the region.

Multiple Introductions of Salmonella enterica Serovar Typhi H58 with Reduced Fluoroquinolone Susceptibility into Chile.

Salmonella enterica serovar Typhi H58, an antimicrobial-resistant lineage, is globally disseminated but has not been reported in Latin America. Genomic analysis revealed 3 independent introductions of Salmonella Typhi H58 with reduced fluoroquinolone susceptibility into Chile. Our findings highlight the utility of enhanced genomic surveillance for typhoid fever in this region.

Persistent circulation of a fluoroquinolone-resistant Salmonella enterica Typhi clone in the Indian subcontinent.

BACKGROUND: The molecular structure of circulating enteric fever pathogens was studied using hospital-based genomic surveillance in a tertiary care referral centre in South India as a first genomic surveillance study, to our knowledge, of blood culture-confirmed enteric fever in the region. METHODS: Blood culture surveillance was conducted at St John's Medical College Hospital, Bengaluru, between July 2016 and June 2017. The bacterial isolates collected were linked to demographic variables of patients and subjected to WGS. The resulting pathogen genomic data were also globally contextualized to gauge possible phylogeographical patterns. RESULTS: Hospital-based genomic surveillance for enteric fever in Bengaluru, India, identified 101 Salmonella enterica Typhi and 14 S. Paratyphi A in a 1 year period. Ninety-six percent of isolates displayed non-susceptibility to fluoroquinolones. WGS showed the dominant pathogen was S. Typhi genotype 4.3.1.2 (H58 lineage II). A fluoroquinolone-resistant triple-mutant clone of S. Typhi 4.3.1.2 previously associated with gatifloxacin treatment failure in Nepal was implicated in 18% of enteric fever cases, indicating ongoing inter-regional circulation. CONCLUSIONS: Enteric fever in South India continues to be a major public health issue and is strongly associated with antimicrobial resistance. Robust microbiological surveillance is necessary to direct appropriate treatment and preventive strategies. Of particular concern is the emergence and expansion of the highly fluoroquinolone-resistant triple-mutant S. Typhi clone and its ongoing inter- and intra-country transmission in South Asia, which highlights the need for regional coordination of intervention strategies, including vaccination and longer-term strategies such as improvements to support hygiene and sanitation.

Antibiotic Resistance and Typhoid.

Multiple drug (antibiotic) resistance (MDR) has become a major threat to the treatment of typhoid and other infectious diseases. Since the 1970s, this threat has increased in Salmonella enterica serovar Typhi, driven in part by the emergence of successful genetic clades, such as haplotype H58, associated with the MDR phenotype. H58 S. Typhi can express multiple antibiotic resistance determinants while retaining the ability to efficiently transmit and persist within the human population. The recent identification of extensively drug resistant S. Typhi only highlights the dangers of ignoring this threat. Here we discuss the evolution of the S. Typhi MDR phenotype and consider options for management.

Inferring demographic parameters in bacterial genomic data using Bayesian and hybrid phylogenetic methods.

BACKGROUND: Recent developments in sequencing technologies make it possible to obtain genome sequences from a large number of isolates in a very short time. Bayesian phylogenetic approaches can take advantage of these data by simultaneously inferring the phylogenetic tree, evolutionary timescale, and demographic parameters (such as population growth rates), while naturally integrating uncertainty in all parameters. Despite their desirable properties, Bayesian approaches can be computationally intensive, hindering their use for outbreak investigations involving genome data for a large numbers of pathogen isolates. An alternative to using full Bayesian inference is to use a hybrid approach, where the phylogenetic tree and evolutionary timescale are estimated first using maximum likelihood. Under this hybrid approach, demographic parameters are inferred from estimated trees instead of the sequence data, using maximum likelihood, Bayesian inference, or approximate Bayesian computation. This can vastly reduce the computational burden, but has the disadvantage of ignoring the uncertainty in the phylogenetic tree and evolutionary timescale. RESULTS: We compared the performance of a fully Bayesian and a hybrid method by analysing six whole-genome SNP data sets from a range of bacteria and simulations. The estimates from the two methods were very similar, suggesting that the hybrid method is a valid alternative for very large datasets. However, we also found that congruence between these methods is contingent on the presence of strong temporal structure in the data (i.e. clocklike behaviour), which is typically verified using a date-randomisation test in a Bayesian framework. To reduce the computational burden of this Bayesian test we implemented a date-randomisation test using a rapid maximum likelihood method, which has similar performance to its Bayesian counterpart. CONCLUSIONS: Hybrid approaches can produce reliable inferences of evolutionary timescales and phylodynamic parameters in a fraction of the time required for fully Bayesian analyses. As such, they are a valuable alternative in outbreak studies involving a large number of isolates.

Functional analysis of Salmonella Typhi adaptation to survival in water.

Contaminated water is a major risk factor associated with the transmission of Salmonella enterica serovar Typhi (S. Typhi), the aetiological agent of human typhoid. However, little is known about how this pathogen adapts to living in the aqueous environment. We used transcriptome analysis (RNA-seq) and transposon mutagenesis (TraDIS) to characterize these adaptive changes and identify multiple genes that contribute to survival. Over half of the genes in the S. Typhi genome altered expression level within the first 24 h following transfer from broth culture to water, although relatively few did so in the first 30 min. Genes linked to central metabolism, stress associated with arrested proton motive force and respiratory chain factors changed expression levels. Additionally, motility and chemotaxis genes increased expression, consistent with a scavenging lifestyle. The viaB-associated gene tviC encoding a glcNAc epimerase that is required for Vi polysaccharide biosynthesis was, along with several other genes, shown to contribute to survival in water. Thus, we define regulatory adaptation operating in S. Typhi that facilitates survival in water.

Three of a Kind: Genetically Similar Tsukamurella Phages TIN2, TIN3, and TIN4.

Three Tsukamurella phages, TIN2, TIN3, and TIN4, were isolated from activated sludge treatment plants located in Victoria, Australia, using conventional enrichment techniques. Illumina and 454 whole-genome sequencing of these Siphoviridae viruses revealed that they had similar genome sequences, ranging in size between 76,268 bp and 76,964 bp. All three phages shared 74% nucleotide sequence identity to the previously described Gordonia phage GTE7. Genome sequencing suggested that phage TIN3 had suffered a mutation in one of its lysis genes compared to the sequence of phage TIN4, to which it is genetically very similar. Mass spectroscopy data showed the unusual presence of a virion structural gene in the DNA replication module of phage TIN4, disrupting the characteristic modular genome architecture of Siphoviridae phages. All three phages appeared highly virulent on strains of Tsukamurella inchonensis and Tsukamurella paurometabola.

The origins of haplotype 58 (H58) Salmonella enterica serovar Typhi.

Antimicrobial resistance (AMR) poses a serious threat to the clinical management of typhoid fever. AMR in Salmonella Typhi (S. Typhi) is commonly associated with the H58 lineage, a lineage that arose comparatively recently before becoming globally disseminated. To better understand when and how H58 emerged and became dominant, we performed detailed phylogenetic analyses on contemporary genome sequences from S. Typhi isolated in the period spanning the emergence. Our dataset, which contains the earliest described H58 S. Typhi organism, indicates that ancestral H58 organisms were already multi-drug resistant (MDR). These organisms emerged spontaneously in India in 1987 and became radially distributed throughout South Asia and then globally in the ensuing years. These early organisms were associated with a single long branch, possessing mutations associated with increased bile tolerance, suggesting that the first H58 organism was generated during chronic carriage. The subsequent use of fluoroquinolones led to several independent mutations in gyrA. The ability of H58 to acquire and maintain AMR genes continues to pose a threat, as extensively drug-resistant (XDR; MDR plus resistance to ciprofloxacin and third generation cephalosporins) variants, have emerged recently in this lineage. Understanding where and how H58 S. Typhi originated and became successful is key to understand how AMR drives successful lineages of bacterial pathogens. Additionally, these data can inform optimal targeting of typhoid conjugate vaccines (TCVs) for reducing the potential for emergence and the impact of new drug-resistant variants. Emphasis should also be placed upon the prospective identification and treatment of chronic carriers to prevent the emergence of new drug resistant variants with the ability to spread efficiently.

Evaluating the relationship between ciprofloxacin prescription and non-susceptibility in Salmonella Typhi in Blantyre, Malawi: an observational study.

BACKGROUND: Ciprofloxacin is the first-line drug for treating typhoid fever in many countries in Africa with a high disease burden, but the emergence of non-susceptibility poses a challenge to public health programmes. Through enhanced surveillance as part of vaccine evaluation, we investigated the occurrence and potential determinants of ciprofloxacin non-susceptibility in Blantyre, Malawi. METHODS: We conducted systematic surveillance of typhoid fever cases and antibiotic prescription in two health centres in Blantyre, Malawi, between Oct 1, 2016, and Oct 31, 2019, as part of the STRATAA and TyVAC studies. In addition, blood cultures were taken from eligible patients presenting at Queen Elizabeth Central Hospital, Blantyre, as part of routine diagnosis. Inclusion criteria were measured or reported fever, or clinical suspicion of sepsis. Microbiologically, we identified Salmonella enterica serotype Typhi (S Typhi) isolates with a ciprofloxacin non-susceptible phenotype from blood cultures, and used whole-genome sequencing to identify drug-resistance mutations and phylogenetic relationships. We constructed generalised linear regression models to investigate associations between the number of ciprofloxacin prescriptions given per month to study participants and the proportion of S Typhi isolates with quinolone resistance-determining region (QRDR) mutations in the following month. FINDINGS: From 46 989 blood cultures from Queen Elizabeth Central Hospital, 502 S Typhi isolates were obtained, 30 (6%) of which had either decreased ciprofloxacin susceptibility, or ciprofloxacin resistance. From 11 295 blood cultures from STRATAA and TyVAC studies, 241 microbiologically confirmed cases of typhoid fever were identified, and 198 isolates from 195 participants sequenced (mean age 12·8 years [SD 10·2], 53% female, 47% male). Between Oct 1, 2016, and Aug 31, 2019, of 177 typhoid fever cases confirmed by whole-genome sequencing, four (2%) were caused by S Typhi with QRDR mutations, compared with six (33%) of 18 cases between Sept 1 and Oct 31, 2019. This increase was associated with a preceding spike in ciprofloxacin prescriptions. Every additional prescription of ciprofloxacin given to study participants in the preceding month was associated with a 4·2% increase (95% CI 1·8-7·0) in the relative risk of isolating S Typhi with a QRDR mutation (p=0·0008). Phylogenetic analysis showed that S Typhi isolates with QRDR mutations from September and October, 2019, belonged to two distinct subclades encoding two different QRDR mutations, and were closely related (4-10 single-nucleotide polymorphisms) to susceptible S Typhi endemic to Blantyre. INTERPRETATION: We postulate a causal relationship between increased ciprofloxacin prescriptions and an increase in fluoroquinolone non-susceptibility in S Typhi. Decreasing ciprofloxacin use by improving typhoid diagnostics, and reducing typhoid fever cases through the use of an efficacious vaccine, could help to limit the emergence of resistance. FUNDING: Wellcome Trust, Bill & Melinda Gates Foundation, and National Institute for Health and Care Research (UK).

Small but sufficient: the Rhodococcus phage RRH1 has the smallest known Siphoviridae genome at 14.2 kilobases.

Bacteriophages are considered to be the most abundant biological entities on the planet. The Siphoviridae are the most commonly encountered tailed phages and contain double-stranded DNA with an average genome size of ∼50 kb. This paper describes the isolation from four different activated sludge plants of the phage RRH1, which is polyvalent, lysing five Rhodococcus species. It has a capsid diameter of only ∼43 nm. Whole-genome sequencing of RRH1 revealed a novel circularly permuted DNA sequence (14,270 bp) carrying 20 putative open reading frames. The genome has a modular arrangement, as reported for those of most Siphoviridae phages, but appears to encode only structural proteins and carry a single lysis gene. All genes are transcribed in the same direction. RRH1 has the smallest genome yet of any described functional Siphoviridae phage. We demonstrate that lytic phage can be recovered from transforming naked DNA into its host bacterium, thus making it a potentially useful model for studying gene function in phages.

The genomic characterization of Salmonella Paratyphi A from an outbreak of enteric fever in Vadodara, India.

Salmonella enterica Typhi (S. Typhi) and Paratyphi A (S. Paratyphi A) are the causative agents of enteric fever, a systemic human disease with a burden of 300 000 cases per year in India. The majority of enteric fever cases are associated with S. Typhi, resulting in a paucity of data regarding S. Paratyphi A, specifically with respect to genomic surveillance and antimicrobial resistance (AMR). Here, we exploited whole-genome sequencing (WGS) to identify S. Paratyphi A genotypes and AMR determinants associated with an outbreak of S. Paratyphi A in Vadodara, India, from December 2018 to December 2019. In total 117 S. Paratyphi A were isolated and genome sequenced, most were genotype 2.4.2 (72.6 % of all cases), which is the globally dominant genotype. The remainder were genotype 2.3 (25.6 %), while only two isolates belonged to genotype 2.4.1. A single base-pair mutation in gyrA, associated with reduced susceptibility to fluoroquinolones, was present in all of the outbreak isolates; with 74.35 % of isolates having a S83F substitution and the remainder having an S83Y substitution. Our surveillance study suggests that S. Paratyphi A is an emergent pathogen in South Asia, which may become increasingly relevant with the introduction of Vi conjugate vaccines.

Unlocking the Potential of Genomic Data to Inform Typhoid Fever Control Policy: Supportive Resources for Genomic Data Generation, Analysis, and Visualization.

The global response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic demonstrated the value of timely and open sharing of genomic data with standardized metadata to facilitate monitoring of the emergence and spread of new variants. Here, we make the case for the value of Salmonella Typhi (S. Typhi) genomic data and demonstrate the utility of freely available platforms and services that support the generation, analysis, and visualization of S. Typhi genomic data on the African continent and more broadly by introducing the Africa Centres for Disease Control and Prevention's Pathogen Genomics Initiative, SEQAFRICA, Typhi Pathogenwatch, TyphiNET, and the Global Typhoid Genomics Consortium.

Acquisition of a large virulence plasmid (pINV) promoted temperature-dependent virulence and global dispersal of O96:H19 enteroinvasive Escherichia coli.

Enteroinvasive Escherichia coli (EIEC) and Shigella are closely related agents of bacillary dysentery. It is widely viewed that EIEC and Shigella species evolved from E. coli via independent acquisitions of a large virulence plasmid (pINV) encoding a type 3 secretion system (T3SS). Sequence Type (ST)99 O96:H19 E. coli is a novel clone of EIEC responsible for recent outbreaks in Europe and South America. Here, we use 92 whole genome sequences to reconstruct a dated phylogeny of ST99 E. coli, revealing distinct phylogenomic clusters of pINV-positive and -negative isolates. To study the impact of pINV acquisition on the virulence of this clone, we developed an EIEC-zebrafish infection model showing that virulence of ST99 EIEC is thermoregulated. Strikingly, zebrafish infection using a T3SS-deficient ST99 EIEC strain and the oldest available pINV-negative isolate reveals a separate, temperature-independent mechanism of virulence, indicating that ST99 non-EIEC strains were virulent before pINV acquisition. Taken together, these results suggest that an already pathogenic E. coli acquired pINV and that virulence of ST99 isolates became thermoregulated once pINV was acquired. IMPORTANCE Enteroinvasive Escherichia coli (EIEC) and Shigella are etiological agents of bacillary dysentery. Sequence Type (ST)99 is a clone of EIEC hypothesized to cause human disease by the recent acquisition of pINV, a large plasmid encoding a type 3 secretion system (T3SS) that confers the ability to invade human cells. Using Bayesian analysis and zebrafish larvae infection, we show that the virulence of ST99 EIEC isolates is highly dependent on temperature, while T3SS-deficient isolates encode a separate temperature-independent mechanism of virulence. These results indicate that ST99 non-EIEC isolates may have been virulent before pINV acquisition and highlight an important role of pINV acquisition in the dispersal of ST99 EIEC in humans, allowing wider dissemination across Europe and South America.

Global diversity and antimicrobial resistance of typhoid fever pathogens: Insights from a meta-analysis of 13,000 Salmonella Typhi genomes.

Background: The Global Typhoid Genomics Consortium was established to bring together the typhoid research community to aggregate and analyse Salmonella enterica serovar Typhi (Typhi) genomic data to inform public health action. This analysis, which marks 22 years since the publication of the first Typhi genome, represents the largest Typhi genome sequence collection to date (n=13,000). Methods: This is a meta-analysis of global genotype and antimicrobial resistance (AMR) determinants extracted from previously sequenced genome data and analysed using consistent methods implemented in open analysis platforms GenoTyphi and Pathogenwatch. Results: Compared with previous global snapshots, the data highlight that genotype 4.3.1 (H58) has not spread beyond Asia and Eastern/Southern Africa; in other regions, distinct genotypes dominate and have independently evolved AMR. Data gaps remain in many parts of the world, and we show the potential of travel-associated sequences to provide informal 'sentinel' surveillance for such locations. The data indicate that ciprofloxacin non-susceptibility (>1 resistance determinant) is widespread across geographies and genotypes, with high-level ciprofloxacin resistance (≥3 determinants) reaching 20% prevalence in South Asia. Extensively drug-resistant (XDR) typhoid has become dominant in Pakistan (70% in 2020) but has not yet become established elsewhere. Ceftriaxone resistance has emerged in eight non-XDR genotypes, including a ciprofloxacin-resistant lineage (4.3.1.2.1) in India. Azithromycin resistance mutations were detected at low prevalence in South Asia, including in two common ciprofloxacin-resistant genotypes. Conclusions: The consortium's aim is to encourage continued data sharing and collaboration to monitor the emergence and global spread of AMR Typhi, and to inform decision-making around the introduction of typhoid conjugate vaccines (TCVs) and other prevention and control strategies. Funding: No specific funding was awarded for this meta-analysis. Coordinators were supported by fellowships from the European Union (ZAD received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 845681), the Wellcome Trust (SB, Wellcome Trust Senior Fellowship), and the National Health and Medical Research Council (DJI is supported by an NHMRC Investigator Grant [GNT1195210]).

Salmonella Typhi (Typhi) is a type of bacteria that causes typhoid fever. More than 110,000 people die from this disease each year, predominantly in areas of sub-Saharan Africa and South Asia with limited access to safe water and sanitation. Clinicians use antibiotics to treat typhoid fever, but scientists worry that the spread of antimicrobial-resistant Typhi could render the drugs ineffective, leading to increased typhoid fever mortality. The World Health Organization has prequalified two vaccines that are highly effective in preventing typhoid fever and may also help limit the emergence and spread of resistant Typhi. In low resource settings, public health officials must make difficult trade-off decisions about which new vaccines to introduce into already crowded immunization schedules. Understanding the local burden of antimicrobial-resistant Typhi and how it is spreading could help inform their actions. The Global Typhoid Genomics Consortium analyzed 13,000 Typhi genomes from 110 countries to provide a global overview of genetic diversity and antimicrobial-resistant patterns. The analysis showed great genetic diversity of the different strains between countries and regions. For example, the H58 Typhi variant, which is often drug-resistant, has spread rapidly through Asia and Eastern and Southern Africa, but is less common in other regions. However, distinct strains of other drug-resistant Typhi have emerged in other parts of the world. Resistance to the antibiotic ciprofloxacin was widespread and accounted for over 85% of cases in South Africa. Around 70% of Typhi from Pakistan were extensively drug-resistant in 2020, but these hard-to-treat variants have not yet become established elsewhere. Variants that are resistant to both ciprofloxacin and ceftriaxone have been identified, and azithromycin resistance has also appeared in several different variants across South Asia. The Consortium's analyses provide valuable insights into the global distribution and transmission patterns of drug-resistant Typhi. Limited genetic data were available fromseveral regions, but data from travel-associated cases helped fill some regional gaps. These findings may help serve as a starting point for collective sharing and analyses of genetic data to inform local public health action. Funders need to provide ongoing supportto help fill global surveillance data gaps.

Whole genome sequence analysis of Salmonella Typhi provides evidence of phylogenetic linkage between cases of typhoid fever in Santiago, Chile in the 1980s and 2010-2016.

Typhoid fever epidemiology was investigated rigorously in Santiago, Chile during the 1980s, when Salmonella enterica serovar Typhi (S. Typhi) caused seasonal, hyperendemic disease. Targeted interventions reduced the annual typhoid incidence rates from 128-220 cases/105 population occurring between 1977-1984 to <8 cases/105 from 1992 onwards. As such, Santiago represents a contemporary example of the epidemiologic transition of an industrialized city from amplified hyperendemic typhoid fever to a period when typhoid is no longer endemic. We used whole genome sequencing (WGS) and phylogenetic analysis to compare the genotypes of S. Typhi cultured from acute cases of typhoid fever occurring in Santiago during the hyperendemic period of the 1980s (n = 74) versus the nonendemic 2010s (n = 80) when typhoid fever was rare. The genotype distribution between "historical" (1980s) isolates and "modern" (2011-2016) isolates was similar, with genotypes 3.5 and 2 comprising the majority of isolations, and 73/80 (91.3%) of modern isolates matching a genotype detected in the 1980s. Additionally, phylogenomically 'ancient' genotypes 1.1 and 1.2.1, uncommon in the global collections, were also detected in both eras, with a notable rise amongst the modern isolates. Thus, genotypes of S. Typhi causing acute illness in the modern nonendemic era match the genotypes circulating during the hyperendemic 1980s. The persistence of historical genotypes may be explained by chronic typhoid carriers originally infected during or before the 1980s.

Paratype: a genotyping tool for Salmonella Paratyphi A reveals its global genomic diversity.

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.

Genetic diversity of Salmonella Paratyphi A isolated from enteric fever patients in Bangladesh from 2008 to 2018.

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.

Genome-wide analysis provides a deeper understanding of the population structure of the Salmonella enterica serotype Paratyphi B complex in Bangladesh.

The Salmonella enterica serotype Paratyphi B complex causes a wide range of diseases, from gastroenteritis to paratyphoid fever, depending on the biotypes Java and sensu stricto. The burden of Paratyphi B biotypes in Bangladesh is still unknown, as these are indistinguishable by Salmonella serotyping. Here, we conducted the first whole-genome sequencing (WGS) study on 79 Salmonella isolates serotyped as Paratyphi B that were collected from 10 nationwide enteric disease surveillance sites in Bangladesh. Placing these in a global genetic context revealed that these are biotype Java, and the addition of these genomes expanded the previously described PG4 clade containing Bangladeshi and UK isolates. Importantly, antimicrobial resistance (AMR) genes were scarce amongst Bangladeshi S. Java isolates, somewhat surprisingly given the widespread availability of antibiotics without prescription. This genomic information provides important insights into the significance of S. Paratyphi B biotypes in enteric disease and their implications for public health.

Multiple introductions of multidrug-resistant typhoid associated with acute infection and asymptomatic carriage, Kenya.

Background: Understanding the dynamics of infection and carriage of typhoid in endemic settings is critical to finding solutions to prevention and control. Methods: In a 3-year case-control study, we investigated typhoid among children aged <16 years (4670 febrile cases and 8549 age matched controls) living in an informal settlement, Nairobi, Kenya. Results: 148 S. Typhi isolates from cases and 95 from controls (stool culture) were identified; a carriage frequency of 1 %. Whole-genome sequencing showed 97% of cases and 88% of controls were genotype 4.3.1 (Haplotype 58), with the majority of each (76% and 88%) being multidrug-resistant strains in three sublineages of the H58 genotype (East Africa 1 (EA1), EA2, and EA3), with sequences from cases and carriers intermingled. Conclusions: The high rate of multidrug-resistant H58 S. Typhi, and the close phylogenetic relationships between cases and controls, provides evidence for the role of carriers as a reservoir for the community spread of typhoid in this setting. Funding: National Institutes of Health (R01AI099525); Wellcome Trust (106158/Z/14/Z); European Commission (TyphiNET No 845681); National Institute for Health Research (NIHR); Bill and Melinda Gates Foundation (OPP1175797).