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Typhoid fever is endemic in many parts of the world and remains a major public health concern in tropical and sub-tropical developing nations, including Fiji. To address high rates of typhoid fever, the Northern Division of Fiji implemented a mass vaccination with typhoid conjugate vaccine (Vi-polysaccharide conjugated to tetanus toxoid) as a public health control measure in 2023. In this study we define the genomic epidemiology of Salmonella Typhi in the Northern Division prior to island-wide vaccination, sequencing 85% ( n =419) of the total cases from the Northern and Central Divisions of Fiji that occurred in the period 2017-2019. We found elevated rates of nucleotide polymorphisms in the tviD and tviE genes (responsible for Vi-polysaccharide synthesis) relative to core genome levels within the Fiji endemic S . Typhi genotype 4.2. Expansion of these findings within a globally representative database of 12 382 S . Typhi (86 genotyphi clusters) showed evidence of convergent evolution of the same tviE mutations across the S . Typhi population, indicating that tvi selection has occurred both independently and globally. The functional impact of tvi mutations on the Vi-capsular structure and other phenotypic characteristics are not fully elucidated, yet commonly occurring tviE polymorphisms localize adjacent to predicted active site residues when overlayed against the predicted TviE protein structure. Given the central role of the Vi-polysaccharide in S . Typhi biology and vaccination, further integrated epidemiological, genomic and phenotypic surveillance is required to determine the spread and functional implications of these mutations.

Objectives: The aim of this study was to understand the status of extensively drug-resistance (XDR) genotype in Salmonella enterica serotype Typhi (S. Typhi) recovered during the pre to post COVID-19 pandemic period using Multiplex PCR., Methods: A longitudinal descriptive study was carried out during five years. Antibiotic susceptibility testing was performed according to the Clinical Laboratory Standards Institute antimicrobial susceptibility testing guidelines. The identification of S. Typhi, the detection of their high-risk lineages and XDR genotype was done using single nucleotide polymorphism-based multiplex PCR., Results: A total of four hundred nine (n = 409) S. Typhi isolates were recovered during pre to post COVID-19 pandemic period. Among them, 30.81% belonged to the pre COVID-19 period while 69.19% to the post COVID-19 period. Different trends in antibiotic resistance in S. Typhi isolates with high prevalence of XDR-S. Typhi were observed. However, there was comparatively different frequency of their occurrence among the S. Typhi isolates recovered during pre to post COVID-19 pandemic period. Multiplex PCR showed that the majority of S. Typhi isolates were the H58 haplotype or genotype 4.3.1 which contained XDR genotype., Conclusions: The increasing episodes of XDR-S. Typhi causing typhoid fever in endemic areas is alarming. The antibiotic resistance in food and water borne pathogens greatly contribute to the dissemination of the antimicrobial resistance in pathogenic bacteria, which has now been considered as a global concern., Competing Interests: Declaration of competing interest Authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The effectiveness of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas14a1, widely utilized for pathogenic microorganism detection, has been limited by the requirement of a protospacer adjacent motif (PAM) on the target DNA strands. To overcome this limitation, this study developed a Single Primer isothermal amplification integrated-Cas14a1 biosensor (SPCas) for detecting Salmonella typhi that does not rely on a PAM sequence. The SPCas biosensor utilizes a novel primer design featuring an RNA-DNA primer and a 3'-biotin-modified primer capable of binding to the same single-stranded DNA (ssDNA) in the presence of the target gene. The RNA-DNA primer undergoes amplification and is blocked at the biotin-modified end. Subsequently, strand replacement is initiated to generate ssDNA assisted by RNase H and Bst enzymes, which activate the trans-cleavage activity of Cas14a1 even in the absence of a PAM sequence. Leveraging both cyclic chain replacement reaction amplification and Cas14a1 trans-cleavage activity, the SPCas biosensor exhibits a remarkable diagnostic sensitivity of 5 CFU/mL. Additionally, in the assessment of 20 milk samples, the SPCas platform demonstrated 100% diagnostic accuracy, which is consistent with the gold standard qPCR. This platform introduces a novel approach for developing innovative CRISPR-Cas-dependent biosensors without a PAM sequence., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Bacterial relatedness measured using select chromosomal loci forms the basis of public health genomic surveillance. While approximating vertical evolution through this approach has proven exceptionally valuable for understanding pathogen dynamics, it excludes a fundamental dimension of bacterial evolution-horizontal gene transfer. Incorporating the accessory genome is the logical remediation and has recently shown promise in expanding epidemiological resolution for enteric pathogens. Employing k -mer-based Jaccard index analysis, and a novel genome length distance metric, we computed pangenome (i.e., core and accessory) relatedness for the globally important pathogen Salmonella enterica serotype Typhi (Typhi), and graphically express both vertical (homology-by-descent) and horizontal (homology-by-admixture) evolutionary relationships in a reticulate network of over 2,200 U.S. Typhi genomes. This analysis revealed non-random structure in the Typhi pangenome that is driven predominantly by the gain and loss of mobile genetic elements, confirming and expanding upon known epidemiological patterns, revealing novel plasmid dynamics, and identifying avenues for further genomic epidemiological exploration. With an eye to public health application, this work adds important biological context to the rapidly improving ways of analyzing bacterial genetic data and demonstrates the value of the accessory genome to infer pathogen epidemiology and evolution.IMPORTANCEGiven bacterial evolution occurs in both vertical and horizontal dimensions, inclusion of both core and accessory genetic material (i.e., the pangenome) is a logical step toward a more thorough understanding of pathogen dynamics. With an eye to public, and indeed, global health relevance, we couple contemporary tools for genomic analysis with decades of research on mobile genetic elements to demonstrate the value of the pangenome, known and unknown, annotated, and hypothetical, for stratification of Salmonella enterica serovar Typhi (Typhi) populations. We confirm and expand upon what is known about Typhi epidemiology, plasmids, and antimicrobial resistance dynamics, and offer new avenues of exploration to further deduce Typhi ecology and evolution, and ultimately to reduce the incidence of human disease., Competing Interests: The authors declare no conflict of interest.

Background: In Santiago, Chile, where typhoid had been hyperendemic (1977-1991), we investigated whether residual chronic carriers could be detected among household contacts of non-travel-related typhoid cases occurring during 2017-2019., Methods: Culture-confirmed cases were classified as autochthonous (domestically acquired) versus travel/immigration related. Household contacts of cases had stool cultures and serum Vi antibody measurements to detect chronic Salmonella Typhi carriers. Whole genome sequences of acute cases and their epidemiologically linked chronic carrier isolates were compared., Results: Five of 16 autochthonous typhoid cases (31.3%) were linked to 4 chronic carriers in case households; 2 cases (onsets 23 months apart) were linked to the same carrier. Carriers were women aged 69-79 years with gallbladder dysfunction and Typhi fecal excretion; 3 had highly elevated serum anti-Vi titers. Genomic analyses revealed close identity (≤11 core genome single-nucleotide polymorphism [SNP] differences) between case and epidemiologically linked carrier isolates; all were genotypes prevalent in 1980s Santiago. A cluster of 4 additional autochthonous cases unlinked to a carrier was identified based on genomic identity (0-1 SNPs). Travel/immigration isolate genotypes were typical for the countries of travel/immigration., Conclusions: Although autochthonous typhoid cases in Santiago are currently rare, 5 of 16 such cases (31.3%) were linked to elderly chronic carriers identified among household contacts of cases., Competing Interests: Potential conflicts of interest. M. M. L. and S. M. T. report grants from the Bill & Melinda Gates Foundation during the conduct of the study. M. M. L. and S. M. T. have patents issued for: “Broad spectrum vaccine against typhoidal and nontyphoidal Salmonella disease” (US Patent 9,011,871); and “Compositions and Methods for Producing Bacterial Conjugate Vaccines” (US Patent 10,716,839). M. M. L. also has a patent issued for: “Attenuated Salmonella Enterica Serovar Paratyphi and Uses Thereof” (US Patent 8,137,930). All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: Enteric fever is a serious public health concern. The causative agents, Salmonella enterica serovars Typhi and Paratyphi A, frequently have antimicrobial resistance (AMR), leading to limited treatment options and poorer clinical outcomes. We investigated the genomic epidemiology, resistance mechanisms, and transmission dynamics of these pathogens at three urban sites in Africa and Asia., Methods: S Typhi and S Paratyphi A bacteria isolated from blood cultures of febrile children and adults at study sites in Dhaka (Bangladesh), Kathmandu (Nepal), and Blantyre (Malawi) during STRATAA surveillance were sequenced. Isolates were charactered in terms of their serotypes, genotypes (according to GenoTyphi and Paratype), molecular determinants of AMR, and population structure. We used phylogenomic analyses incorporating globally representative genomic data from previously published surveillance studies and ancestral state reconstruction to differentiate locally circulating from imported pathogen AMR variants. Clusters of sequences without any single-nucleotide variants in their core genome were identified and used to explore spatiotemporal patterns and transmission dynamics., Findings: We sequenced 731 genomes from isolates obtained during surveillance across the three sites between Oct 1, 2016, and Aug 31, 2019 (24 months in Dhaka and Kathmandu and 34 months in Blantyre). S Paratyphi A was present in Dhaka and Kathmandu but not Blantyre. S Typhi genotype 4.3.1 (H58) was common in all sites, but with different dominant variants (4.3.1.1.EA1 in Blantyre, 4.3.1.1 in Dhaka, and 4.3.1.2 in Kathmandu). Multidrug resistance (ie, resistance to chloramphenicol, co-trimoxazole, and ampicillin) was common in Blantyre (138 [98%] of 141 cases) and Dhaka (143 [32%] of 452), but absent from Kathmandu. Quinolone-resistance mutations were common in Dhaka (451 [>99%] of 452) and Kathmandu (123 [89%] of 138), but not in Blantyre (three [2%] of 141). Azithromycin-resistance mutations in acrB were rare, appearing only in Dhaka (five [1%] of 452). Phylogenetic analyses showed that most cases derived from pre-existing, locally established pathogen variants; 702 (98%) of 713 drug-resistant infections resulted from local circulation of AMR variants, not imported variants or recent de novo emergence; and pathogen variants circulated across age groups. 479 (66%) of 731 cases clustered with others that were indistinguishable by point mutations; individual clusters included multiple age groups and persisted for up to 2·3 years, and AMR determinants were invariant within clusters., Interpretation: Enteric fever was associated with locally established pathogen variants that circulate across age groups. AMR infections resulted from local transmission of resistant strains. These results form a baseline against which to monitor the impacts of control measures., Funding: Wellcome Trust, Bill & Melinda Gates Foundation, EU Horizon 2020, and UK National Institute for Health and Care Research., Competing Interests: Declaration of interests AJP is Chair of the UK Government Department of Health and Social Care’s Joint Committee on Vaccination and Immunisation (unpaid) and was a member of WHO's SAGE until 2022 (unpaid). VEP has received travel reimbursement from Merck and Pfizer for attending scientific input engagements unrelated to the topic of the manuscript, and is a member of the WHO Immunization and Vaccine-related Implementation Research Advisory Committee. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

A highly accurate, rapid, portable, and robust platform for detecting Salmonella enterica serovar Typhi ( S. Typhi) is crucial for early-stage diagnosis of typhoid to avert and control the outbreaks of this pathogen, which threaten global public health. This study presents a proof-of-concept for our developed label-free electrochemical DNA biosensor system for S. Typhi detection, which employs a printed circuit board gold electrode (PCBGE), integrated with a portable potentiostat reader. Initially, the functionalized DNA biosensor and target detection were characterized using cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) methods using a benchtop potentiostat. Interestingly, the newly developed DNA biosensor can identify target single-stranded DNA concentrations ranging from 10 nM to 20 μM, achieving a detection limit of 7.6 nM within a brief 5 minute timeframe. Under optimal detection conditions, the DNA biosensor exhibits remarkable selectivity, capable of distinguishing a single mismatch base pair from the target single-stranded DNA sequence. We then evaluated the feasibility of the developed DNA biosensor system as a diagnostic tool by detecting S. Typhi in 50 clinical samples using a portable potentiostat reader based on the DPV technique. Remarkably, the developed biosensor can distinctly distinguish between positive and negative samples, indicating that the miniaturised DNA biosensor system is practical for detecting S. Typhi in real biological samples. The developed DNA biosensor device in this work proves to be a promising point-of-care (POC) device for Salmonella detection due to its swift detection time, uncomplicated design, and streamlined workflow detection system.

Background: Understanding the source of typhoid infections and the genetic relatedness of Salmonella Typhi (S. Typhi) by cluster identification in endemic settings is critical for establishing coordinated public health responses for typhoid fever management. This study investigated the genotypic diversity, antibiotic resistance mechanisms, and clustering of 35 S.Typhi strains isolated from cases and carriers in the Mukuru Informal Settlement., Methods: We studied 35 S.Typhi isolates, including 32 from cases and 3 from carriers, from study participants in the informal settlement of Mukuru, Nairobi, Kenya. Genomic DNA was extracted, and whole-genome sequencing (WGS) was performed to determine the phylogenetic relatedness of strains and detect antimicrobial resistance determinants (AMR). WGS data were analyzed using bioinformatics tools available at the Center for Genomic Epidemiology and Pathogenwatch platforms., Results: Genotype 4.3.1.2 EA3 was found to be dominant at 46% (16/35), followed by 4.3.1.2 EA2 at 28% (10/35), and 4.3.1.1 EA1 at 27% (9/35). A comparison of the isolates with global strains from Pathogenwatch identified close clustering with strains from Uganda, Tanzania, Rwanda, and India. Three isolates (9%) distributed in each cluster were isolated from carriers. All genotype 4.3.1.2 EA3 isolates were genotypically multidrug-resistant to ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole. Single mutations in the quinolone resistance-determining region were identified in the gyrA (S83Y) and gyrB (S464F) genes. All isolates associated with multidrug resistance showed the presence of the IncQ1 plasmid with the following genes: blaTEM-1B, catA1, sul1, sul2, and dfrA7., Conclusion: The close phylogenetic relatedness between antimicrobial-resistant case isolates and carriage isolates indicates that typhoid carriage is a possible source of infection in the community. Comparative analysis with global isolates revealed that the Kenyan isolates share common lineages with strains from neighboring East African countries and India, suggesting regional dissemination of specific MDR clones. AMR was a major feature of the isolates. Surveillance and testing for antimicrobial susceptibility should inform options for the management of cases., (© 2024. The Author(s).)

Background: Salmonella typhi is a specific strain of the Salmonella bacterium, responsible for triggering typhoid fever; a significant public health concern in developing nations., Objective: The current study aimed to identify the bacteria from the gallbladder, taken during cholecystectomies of patients, by isolating Salmonella typhi and by using microscopic characteristics, biochemical and polymerase chain reaction (PCR) tests., Methods: A total of 120 specimens were collected from the Baghdad Teaching Hospital, Iraq. A cross-sectional descriptive study was carried out from October, 2021, to July, 2022. During that study, 26 (54.2%) male patient tested positive for Salmonella typhias well as 22 (45.8%) female patients. The age of the patients varied from < 30 to > 60 years. p-value > 0.05 was considered significant to confirm a relationship between age and Salmonella typhi effect for patients., Results: Out of the 120 blood samples taken for this study, 48 (40%) tested positive by use of PCR test, 40 (33.3%) tested positive by use of the Widal test, 35 (29.1%) were positive for biopsy culture, and 35 (29.1%) were positive for blood culture. All Salmonella typhi isolates were found to be sensitive to the imipenem, cefepime, and ceftriaxone, but were resistant to gentamycin, ciprofloxacin, amikacin, erythromycin, and tetracycline (72%, 29%, 43%, 100%, 100%, respectively)., Conclusions: The real time polymerase chain reaction (RT-PCR) tests and the Vitek 2 compact system showed a high level of accuracy in the detection of Salmonella typhi. Multidrug resistance was observed, which should be a signal to reduce antibiotic consumption.

Salmonella enterica serovar Typhi ( S . Typhi) causes typhoid fever, a systemic infection that affects millions of people worldwide. S . Typhi can invade and survive within host cells, such as intestinal epithelial cells and macrophages, by modulating their immune responses. However, the immunomodulatory capability of S . Typhi in relation to TolC-facilitated efflux pump function remains unclear. The role of TolC, an outer membrane protein that facilitates efflux pump function, in the invasion and immunomodulation of S . Typhi, was studied in human intestinal epithelial cells and macrophages. The tolC deletion mutant of S . Typhi was compared with the wild-type and its complemented strain in terms of their ability to invade epithelial cells, survive and induce cytotoxicity in macrophages, and elicit proinflammatory cytokine production in macrophages. The tolC mutant, which has a defective outer membrane, was impaired in invading epithelial cells compared to the wild-type strain, but the intracellular presence of the tolC mutant exhibited greater cytotoxicity and induced higher levels of proinflammatory cytokines ( IL-1β and IL-8 ) in macrophages compared to the wild-type strain. These effects were reversed by complementing the tolC mutant with a functional tolC gene. Our results suggest that TolC plays a role in S . Typhi to efficiently invade epithelial cells and suppress host immune responses during infection. TolC may be a potential target for the development of novel therapeutics against typhoid fever.

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., (© 2024. The Author(s).)

Many bacterial pathogens, including the human exclusive pathogen Salmonella Typhi, express capsular polysaccharides as a crucial virulence factor. Here, through S. Typhi whole genome sequence analyses and functional studies, we found a list of single point mutations that make S. Typhi hypervirulent. We discovered a single point mutation in the Vi biosynthesis enzymes that control Vi polymerization or acetylation is enough to result in different capsule variants of S. Typhi. All variant strains are pathogenic, but the hyper Vi capsule variants are particularly hypervirulent, as demonstrated by the high morbidity and mortality rates observed in infected mice. The hypo Vi capsule variants have primarily been identified in Africa, whereas the hyper Vi capsule variants are distributed worldwide. Collectively, these studies increase awareness about the existence of different capsule variants of S. Typhi, establish a solid foundation for numerous future studies on S. Typhi capsule variants, and offer valuable insights into strategies to combat capsulated bacteria., (© 2024. The Author(s).)

Conserved molecular signatures in multidrug-resistant Salmonella typhi can serve as novel therapeutic targets for mitigation of infection. In this regard, we present the S. typhi cell division activator protein (StCAP) as a conserved target across S. typhi variants. From in silico and fluorimetric assessments, we found that StCAP is a DNA-binding protein. Replacement of the identified DNA-interacting residue Arg 34 of StCAP with Ala 34 showed a dramatic (15-fold) increase in K d value compared to the wild type ( K d 546 nm) as well as a decrease in thermal stability (10 °C shift). Out of the two screened molecules against the DNA-binding pocket of StCAP, eltrombopag, and nilotinib, the former displayed better binding. Eltrombopag inhibited the stand-alone S. typhi culture with an IC 50 of 38 μM. The effect was much more pronounced on THP-1-derived macrophages (T1Mac) infected with S. typhi where colony formation was severely hindered with IC 50 reduced further to 10 μM. Apoptotic protease activating factor1 (Apaf1), a key molecule for intrinsic apoptosis, was identified as an StCAP-interacting partner by pull-down assay against T1Mac. Further, StCAP-transfected T1Mac showed a significant increase in LC3 II (autophagy marker) expression and downregulation of caspase 3 protein. From these experiments, we conclude that StCAP provides a crucial survival advantage to S. typhi during infection, thereby making it a potent alternative therapeutic target.

Background: Enteric fever is caused by Salmonella enterica serovars Typhi (S. Typhi) and Paratyphi A, B, and C. It continues to be a significant cause of morbidity and mortality worldwide. In highly endemic areas, children are disproportionately affected, and antimicrobial resistance reduces therapeutic options. It is estimated that 2-5% of enteric fever patients develop chronic asymptomatic infection. These carriers may act as reservoirs of infection; therefore, the prospective identification and treatment of carriers are critical for long-term disease control. We aimed to find the frequency of Salmonella Typhi carriers in patients undergoing cholecystectomy. We also compared the detection limit of culturing versus qPCR in detecting S. Typhi, performed a geospatial analysis of the carriers identified using this study, and evaluated the accuracy of anti-Vi and anti-YncE in identifying chronic typhoid carriage., Methods: We performed a cross-sectional study in two centers in Pakistan. Gallbladder specimens were subjected to quantitative PCR (qPCR) and serum samples were analyzed for IgG against YncE and Vi by ELISA. We also mapped the residential location of those with a positive qPCR result., Findings: Out of 988 participants, 3.4% had qPCR-positive gallbladder samples (23 S. Typhi and 11 S. Paratyphi). Gallstones were more likely to be qPCR positive than bile and gallbladder tissue. Anti-Vi and YncE were significantly correlated (r = 0.78 p<0.0001) and elevated among carriers as compared to qPCR negative controls, except for anti-Vi response in Paratyphi A. But the discriminatory values of these antigens in identifying carriers from qPCR negative controls were low., Conclusion: The high prevalence of typhoid carriers observed in this study suggests that further studies are required to gain information that will help in controlling future typhoid outbreaks in a superior manner than they are currently being managed., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Qureshi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Macrophages provide a crucial environment for Salmonella enterica serovar Typhi (S. Typhi) to multiply during typhoid fever, yet our understanding of how human macrophages and S. Typhi interact remains limited. In this study, we delve into the dynamics of S. Typhi replication within human macrophages and the resulting heterogeneous transcriptomic responses of macrophages during infection. Our study reveals key factors that influence macrophage diversity, uncovering distinct immune and metabolic pathways associated with different stages of S. Typhi intracellular replication in macrophages. Of note, we found that macrophages harboring replicating S. Typhi are skewed towards an M1 pro-inflammatory state, whereas macrophages containing non-replicating S. Typhi exhibit neither a distinct M1 pro-inflammatory nor M2 anti-inflammatory state. Additionally, macrophages with replicating S. Typhi were characterized by the increased expression of genes associated with STAT3 phosphorylation and the activation of the STAT3 transcription factor. Our results shed light on transcriptomic pathways involved in the susceptibility of human macrophages to intracellular S. Typhi replication, thereby providing crucial insight into host phenotypes that restrict and support S. Typhi infection., (© 2024. The Author(s).)

Enteric fever, a persistent public health challenge in developing regions, is exacerbated by suboptimal socioeconomic conditions, contaminated water and food sources, and insufficient sanitation. This study delves into the antimicrobial susceptibility of Salmonella Typhi, uncovering the genetic underpinnings of its resistance. Analyzing 897 suspected cases, we identified a significant prevalence of typhoid fever, predominantly in males (58.3 %) and younger demographics. Alarmingly, our data reveals an escalation in resistance to both primary and secondary antibiotics, with cases of multi-drug resistant (MDR) and extensively drug-resistant (XDR) S. Typhi reaching 14.7 % and 43.4 %, respectively, in 2021. The Multiple Antibiotic Resistance (MAR) index exceeded 0.2 in over half of the isolates, signaling widespread antibiotic misuse. The study discerned 47 unique antibiotic resistance patterns and pinpointed carbapenem and macrolide antibiotics as the remaining effective treatments against XDR strains, underlining the critical need to preserve these drugs for severe cases. Molecular examinations identified bla TEM , bla SHV , and bla CTX-M genes in ceftriaxone-resistant strains, while qnrS was specific to ciprofloxacin-resistant variants. Notably, all examined strains exhibited a singular mutation in the gyrA gene, maintaining wild-type gyrB and parC genes. The erm(B) gene emerged as the primary determinant of azithromycin resistance. Furthermore, a distressing increase in resistance genes was observed over three years, with erm(B), bla TEM and qnrS showing significant upward trends. These findings are a clarion call for robust antimicrobial stewardship programs to curtail inappropriate antibiotic use and forestall the burgeoning threat of antibiotic resistance in S. Typhi., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Salmonella enterica serovar Typhi and Paratyphi A are the cause of typhoid and paratyphoid fever in humans, which are systemic life-threatening illnesses. Both serovars are exclusively adapted to the human host, where they can cause life-long persistent infection. A distinct feature of these serovars is the presence of a relatively high number of degraded coding sequences coding for metabolic pathways, most likely a consequence of their adaptation to a single host. As a result of convergent evolution, these serovars shared many of the degraded coding sequences although often affecting different genes in the same metabolic pathway. However, there are several coding sequences that appear intact in one serovar while clearly degraded in the other, suggesting differences in their metabolic capabilities. Here, we examined the functionality of metabolic pathways that appear intact in S . Typhi but that show clear signs of degradation in S . Paratyphi A. We found that, in all cases, the existence of single amino acid substitutions in S . Typhi metabolic enzymes, transporters, or transcription regulators resulted in the inactivation of these metabolic pathways. Thus, the inability of S . Typhi to metabolize Glucose-6-Phosphate or 3-phosphoglyceric acid is due to the silencing of the expression of the genes encoding the transporters for these compounds due to point mutations in the transcriptional regulatory proteins. In contrast, its inability to utilize glucarate or galactarate is due to the presence of point mutations in the transporter and enzymes necessary for the metabolism of these sugars. These studies provide additional support for the concept of adaptive convergent evolution of these two human-adapted S. enterica serovars and highlight a limitation of bioinformatic approaches to predict metabolic capabilities., Importance: Salmonella enterica serovar Typhi and Paratyphi A are the cause of typhoid and paratyphoid fever in humans, which are systemic life-threatening illnesses. Both serovars can only infect the human host, where they can cause life-long persistent infection. Because of their adaptation to the human host, these bacterial pathogens have changed their metabolism, leading to the loss of their ability to utilize certain nutrients. In this study we examined the functionality of metabolic pathways that appear intact in S . Typhi but that show clear signs of degradation in S . Paratyphi A. We found that, in all cases, the existence of single amino acid substitutions in S . Typhi metabolic enzymes, transporters, or transcription regulators resulted in the inactivation of these metabolic pathways. These studies provide additional support for the concept of adaptive convergent evolution of these two human-adapted S. enterica serovars., Competing Interests: The authors declare no conflict of interest.

Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of Typhoid fever. Blood culture is the gold standard for clinical diagnosis, but this is often difficult to employ in resource limited settings. Environmental surveillance of waste-impacted waters is a promising supplement to clinical surveillance, however validating methods is challenging in regions where S. Typhi concentrations are low. To evaluate existing S. Typhi environmental surveillance methods, a novel process control organism (PCO) was created as a biosafe surrogate. Using a previous described qPCR assay, a modified PCR amplicon for the staG gene was cloned into E. coli. We developed a target region that was recognized by the Typhoid primers in addition to a non-coding internal probe sequence. A multiplex qPCR reaction was developed that differentiates between the typhoid and control targets, with no cross-reactivity or inhibition of the two probes. The PCO was shown to mimic S. Typhi in lab-based experiments with concentration methods using primary wastewater: filter cartridge, recirculating Moore swabs, membrane filtration, and differential centrifugation. Across all methods, the PCO seeded at 10 CFU/mL and 100 CFU/mL was detected in 100% of replicates. The PCO is detected at similar quantification cycle (Cq) values across all methods at 10 CFU/mL (Average = 32.4, STDEV = 1.62). The PCO was also seeded into wastewater at collection sites in Vellore (India) and Blantyre (Malawi) where S. Typhi is endemic. All methods tested in both countries were positive for the seeded PCO. The PCO is an effective way to validate performance of environmental surveillance methods targeting S. Typhi in surface water., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Philo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Aim: We investigated the molecular underpinnings of variation in immune responses to the live attenuated typhoid vaccine (Ty21a) by analyzing the baseline immunological profile. We utilized gene expression datasets obtained from the Gene Expression Omnibus (GEO) database (accession number: GSE100665) before and after immunization. We then employed two distinct computational approaches to identify potential baseline biomarkers associated with responsiveness to the Ty21a vaccine., Main Methods: The first pipeline (knowledge-based) involved the retrieval of differentially expressed genes (DEGs), functional enrichment analysis, protein-protein interaction network construction, and topological network analysis of post-immunization datasets before gauging their pre-vaccination expression levels. The second pipeline utilized an unsupervised machine learning algorithm for data-driven feature selection on pre-immunization datasets. Supervised machine-learning classifiers were employed to computationally validate the identified biomarkers., Key Findings: Baseline activation of NKIRAS2 (a negative regulator of NF-kB signalling) and SRC (an adaptor for immune receptor activation) was negatively associated with Ty21a vaccine responsiveness, whereas LOC100134365 exhibited a positive association. The Stochastic Gradient Descent (SGD) algorithm accurately distinguished vaccine responders and non-responders, with 88.8%, 70.3%, and 85.1% accuracy for the three identified genes, respectively., Significance: This dual-pronged novel analytical approach provides a comprehensive comparison between knowledge-based and data-driven methods for the prediction of baseline biomarkers associated with Ty21a vaccine responsiveness. The identified genes shed light on the intricate molecular mechanisms that influence vaccine efficacy from the host perspective while pushing the needle further towards the need for development of precise enteric vaccines and on the importance of pre-immunization screening., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Typhoid fever is a common cause of febrile illness in low- and middle-income countries. While multidrug-resistant (MDR) Salmonella Typhi (S. Typhi) has spread globally, fluoroquinolone resistance has mainly affected Asia., Methods: Consecutively, 1038 blood cultures were obtained from patients of all age groups with fever and/or suspicion of serious systemic infection admitted at Mnazi Mmoja Hospital, Zanzibar in 2015-2016. S. Typhi were analyzed with antimicrobial susceptibility testing and with short read (61 strains) and long read (9 strains) whole genome sequencing, including three S. Typhi strains isolated in a pilot study 2012-2013., Results: Sixty-three S. Typhi isolates (98%) were MDR carrying blaTEM-1B, sul1 and sul2, dfrA7 and catA1 genes. Low-level ciprofloxacin resistance was detected in 69% (43/62), with a single gyrase mutation gyrA-D87G in 41 strains, and a single gyrA-S83F mutation in the non-MDR strain. All isolates were susceptible to ceftriaxone and azithromycin. All MDR isolates belonged to genotype 4.3.1 lineage I (4.3.1.1), with the antimicrobial resistance determinants located on a composite transposon integrated into the chromosome. Phylogenetically, the MDR subgroup with ciprofloxacin resistance clusters together with two external isolates., Conclusions: We report a high rate of MDR and low-level ciprofloxacin resistant S. Typhi circulating in Zanzibar, belonging to genotype 4.3.1.1, which is widespread in Southeast Asia and African countries and associated with low-level ciprofloxacin resistance. Few therapeutic options are available for treatment of typhoid fever in the study setting. Surveillance of the prevalence, spread and antimicrobial susceptibility of S. Typhi can guide treatment and control efforts., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Onken et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Environmental surveillance (ES) for Salmonella Typhi potentially offers a low-cost tool to identify communities with a high burden of typhoid fever., Methods: We developed standardized protocols for typhoid ES, including sampling site selection, validation, characterization; grab or trap sample collection, concentration; and quantitative PCR targeting Salmonella genes (ttr, staG, and tviB) and a marker of human fecal contamination (HF183). ES was implemented over 12 months in a historically high typhoid fever incidence setting (Vellore, India) and a lower incidence setting (Blantyre, Malawi) during 2021-2022., Results: S. Typhi prevalence in ES samples was higher in Vellore compared with Blantyre; 39/520 (7.5%; 95% confidence interval [CI], 4.4%-12.4%) vs 11/533 (2.1%; 95% CI, 1.1%-4.0%) in grab and 79/517 (15.3%; 95% CI, 9.8%-23.0%) vs 23/594 (3.9%; 95% CI, 1.9%-7.9%) in trap samples. Detection was clustered by ES site and correlated with site catchment population in Vellore but not Blantyre. Incidence of culture-confirmed typhoid in local hospitals was low during the study and zero some months in Vellore despite S. Typhi detection in ES., Conclusions: ES describes the prevalence and distribution of S. Typhi even in the absence of typhoid cases and could inform vaccine introduction. Expanded implementation and comparison with clinical and serological surveillance will further establish its public health utility., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

AB 5 -type toxins are a diverse family of protein toxins composed of an enzymatic active (A) subunit and a pentameric delivery (B) subunit. Salmonella enterica serovar Typhi's typhoid toxin features two A subunits, CdtB and PltA, in complex with the B subunit PltB. Recently, it was shown that S . Typhi encodes a horizontally acquired B subunit, PltC, that also assembles with PltA/CdtB to produce a second form of typhoid toxin. S . Typhi therefore produces two AB 5 toxins with the same A subunits but distinct B subunits, an evolutionary twist that is unique to typhoid toxin. Here, we show that, remarkably, the Salmonella bongori species independently evolved an analogous capacity to produce two typhoid toxins with distinct B subunits. S. bongori 's alternate B subunit, PltD, is evolutionarily distant from both PltB and PltC and outcompetes PltB to form the predominant toxin. We show that, surprisingly, S. bongori elicits similar levels of CdtB-mediated intoxication as S . Typhi during infection of cultured human epithelial cells. This toxicity is exclusively due to the PltB toxin, and strains lacking pltD produce increased amounts of PltB toxin and exhibit increased toxicity compared to the wild type, suggesting that the acquisition of the PltD subunit potentially made S. bongori less virulent toward humans. Collectively, this study unveils a striking example of convergent evolution that highlights the importance of the poorly understood "two-toxin" paradigm for typhoid toxin biology and, more broadly, illustrates how the flexibility of A-B interactions has fueled the evolutionary diversification and expansion of AB 5 -type toxins., Importance: Typhoid toxin is an important Salmonella Typhi virulence factor and an attractive target for therapeutic interventions to combat typhoid fever. The recent discovery of a second version of this toxin has substantial implications for understanding S . Typhi pathogenesis and combating typhoid fever. In this study, we discover that a remarkably similar two-toxin paradigm evolved independently in Salmonella bongori , which strongly suggests that this is a critical aspect of typhoid toxin biology. We observe significant parallels between how the two toxins assemble and their capacity to intoxicate host cells during infection in S . Typhi and S. bongori , which provides clues to the biological significance of this unusual toxin arrangement. More broadly, AB5 toxins with diverse activities and mechanisms are essential virulence factors for numerous important bacterial pathogens. This study illustrates the capacity for novel A-B interactions to evolve and thus provides insight into how such a diverse arsenal of toxins might have emerged., Competing Interests: The authors declare no conflict of interest.

Salmonella serovars Typhi and Paratyphi cause a prolonged illness known as enteric fever, whereas other serovars cause acute gastroenteritis. Mechanisms responsible for the divergent clinical manifestations of nontyphoidal and enteric fever Salmonella infections have remained elusive. Here, we show that S . Typhi and S . Paratyphi A can persist within human macrophages, whereas S . Typhimurium rapidly induces apoptotic macrophage cell death that is dependent on Salmonella pathogenicity island 2 (SPI2). S . Typhi and S . Paratyphi A lack 12 specific SPI2 effectors with pro-apoptotic functions, including nine that target nuclear factor κB (NF-κB). Pharmacologic inhibition of NF-κB or heterologous expression of the SPI2 effectors GogA or GtgA restores apoptosis of S . Typhi-infected macrophages. In addition, the absence of the SPI2 effector SarA results in deficient signal transducer and activator of transcription 1 (STAT1) activation and interleukin 12 production, leading to impaired T H 1 responses in macrophages and humanized mice. The absence of specific nontyphoidal SPI2 effectors may allow S . Typhi and S . Paratyphi A to cause chronic infections., Importance: Salmonella enterica is a common cause of gastrointestinal infections worldwide. The serovars Salmonella Typhi and Salmonella Paratyphi A cause a distinctive systemic illness called enteric fever, whose pathogenesis is incompletely understood. Here, we show that enteric fever Salmonella serovars lack 12 specific virulence factors possessed by nontyphoidal Salmonella serovars, which allow the enteric fever serovars to persist within human macrophages. We propose that this fundamental difference in the interaction of Salmonella with human macrophages is responsible for the chronicity of typhoid and paratyphoid fever, suggesting that targeting the nuclear factor κB (NF-κB) complex responsible for macrophage survival could facilitate the clearance of persistent bacterial infections., Competing Interests: The authors declare no conflict of interest.

Tomatoes are readily available and affordable vegetables that offer a range of health benefits due to their bioactive molecules, such as antioxidants and antimicrobials. In contrast to the widely recognized antioxidant properties of tomatoes, their antimicrobial properties remain largely unexplored. Here, we present our findings on the antimicrobial properties of tomato juice and peptides, namely, tomato-derived antimicrobial peptides (tdAMPs), in relation to their effectiveness against typhoidal Salmonella . Our research has revealed that tomato juice demonstrates significant antimicrobial properties against Salmonella Typhi, a pathogen that specifically affects humans and is responsible for causing typhoid fever. By employing computational analysis of the tomato genome sequence, conducting molecular dynamics simulation, and performing functional analyses, we have successfully identified two tdAMPs, namely, tdAMP-1 and tdAMP-2. These tdAMPs have demonstrated potent antimicrobial properties by effectively disrupting bacterial membranes. The efficacy of tdAMP-2 is shown to be more effective than tdAMP-1. The efficacy of tdAMP-1 and tdAMP-2 has been demonstrated against drug-resistant S . Typhi, as well as hyper-capsular S . Typhi variants that possess hypervirulent characteristics, which are presently circulating in countries with endemicity. Tomato juice, along with the two tdAMPs, has demonstrated effectiveness against uropathogenic Escherichia coli as well. This underscores their potential as viable agents in combating certain Gram-negative pathogens. This study provides valuable insights into the development of effective and sustainable public health strategies that utilize tomato and its derivatives as lifestyle interventions.IMPORTANCEIn this study, we investigate the antimicrobial properties of tomato juice, the most widely consumed affordable vegetables, as well as tomato-derived antimicrobial peptides, in relation to their effectiveness against foodborne pathogens with an emphasis on Salmonella Typhi, a deadly human-specific pathogen., Competing Interests: The authors declare no conflict of interest.

The intracellular bacteria, Salmonella Typhi adapts to acidic conditions in the host cell by resetting the chromosomal DNA topology majorly controlled by DNA Gyrase, a Type II topoisomerase. DNA Gyrase forms a heterodimer A 2 B 2 complex, which manages the DNA supercoiling and relaxation in the cell. DNA relaxation forms a part of the regulatory mechanism to activate the transcription of genes required to survive under hostile conditions. Acid-induced stress attenuates the supercoiling activity of the DNA Gyrase, resulting in DNA relaxation. Salmonella DNA becomes relaxed as the bacteria adapt to the acidified intracellular environment. Despite comprehensive studies on DNA Gyrase, the mechanism to control supercoiling activity needs to be better understood. A loss in supercoiling activity in E. coli was observed upon deletion of the non-conserved acidic C-tail of Gyrase A subunit. Salmonella Gyrase also contains an acidic tail at the C-terminus of Gyrase A, where its deletion resulted in reduced supercoiling activity compared to wild-type Gyrase. Interestingly, we also found that wild-type Gyrase compromises supercoiling activity at acidic pH 2-3, thereby causing DNA relaxation. The absence of a C-tail displayed DNA supercoiling to some extent between pH 2-9. Hence, the C-tail of Gyrase A might be one of the controlling factors that cause DNA relaxation in Salmonella at acidic pH conditions. We propose that the presence of the C-tail of GyraseA causes acid-mediated inhibition of the negative supercoiling activity of Gyrase, resulting in relaxed DNA that attracts DNA-binding proteins for controlling the transcriptional response., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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)., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Typhoid-conjugate vaccines (TCVs) provide an opportunity to reduce the burden of typhoid fever, caused by Salmonella Typhi, in endemic areas. As policymakers design vaccination strategies, accurate and high-resolution data on disease burden is crucial. However, traditional blood culture-based surveillance is resource-extensive, prohibiting its large-scale and sustainable implementation. Salmonella Typhi is a water-borne pathogen, and here, we tested the potential of Typhi-specific bacteriophage surveillance in surface water bodies as a low-cost tool to identify where Salmonella Typhi circulates in the environment. In 2021, water samples were collected and tested for the presence of Salmonella Typhi bacteriophages at two sites in Bangladesh: urban capital city, Dhaka, and a rural district, Mirzapur. Salmonella Typhi-specific bacteriophages were detected in 66 of 211 (31%) environmental samples in Dhaka, in comparison to 3 of 92 (3%) environmental samples from Mirzapur. In the same year, 4,620 blood cultures at the two largest pediatric hospitals of Dhaka yielded 215 (5%) culture-confirmed typhoid cases, and 3,788 blood cultures in the largest hospital of Mirzapur yielded 2 (0.05%) cases. 75% (52/69) of positive phage samples were collected from sewage. All isolated phages were tested against a panel of isolates from different Salmonella Typhi genotypes circulating in Bangladesh and were found to exhibit a diverse killing spectrum, indicating that diverse bacteriophages were isolated. These results suggest an association between the presence of Typhi-specific phages in the environment and the burden of typhoid fever, and the potential of utilizing environmental phage surveillance as a low-cost tool to assist policy decisions on typhoid control., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hooda et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Environmental surveillance, using detection of Salmonella Typhi DNA, has emerged as a potentially useful tool to identify typhoid-endemic settings; however, it is relatively costly and requires molecular diagnostic capacity. We sought to determine whether S. Typhi bacteriophages are abundant in water sources in a typhoid-endemic setting, using low-cost assays., Methodology: We collected drinking and surface water samples from urban, peri-urban and rural areas in 4 regions of Nepal. We performed a double agar overlay with S. Typhi to assess the presence of bacteriophages. We isolated and tested phages against multiple strains to assess their host range. We performed whole genome sequencing of isolated phages, and generated phylogenies using conserved genes., Findings: S. Typhi-specific bacteriophages were detected in 54.9% (198/361) of river and 6.3% (1/16) drinking water samples from the Kathmandu Valley and Kavrepalanchok. Water samples collected within or downstream of population-dense areas were more likely to be positive (72.6%, 193/266) than those collected upstream from population centers (5.3%, 5/95) (p=0.005). In urban Biratnagar and rural Dolakha, where typhoid incidence is low, only 6.7% (1/15, Biratnagar) and 0% (0/16, Dolakha) river water samples contained phages. All S. Typhi phages were unable to infect other Salmonella and non-Salmonella strains, nor a Vi-knockout S. Typhi strain. Representative strains from S. Typhi lineages were variably susceptible to the isolated phages. Phylogenetic analysis showed that S. Typhi phages belonged to the class Caudoviricetes and clustered in three distinct groups., Conclusions: S. Typhi bacteriophages were highly abundant in surface waters of typhoid-endemic communities but rarely detected in low typhoid burden communities. Bacteriophages recovered were specific for S. Typhi and required Vi polysaccharide for infection. Screening small volumes of water with simple, low-cost (~$2) plaque assays enables detection of S. Typhi phages and should be further evaluated as a scalable tool for typhoid environmental surveillance., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Shrestha et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Fluoroquinolone resistance in Salmonella has been reported worldwide and poses a serious public health threat in developing countries. Multiple factors contribute to fluoroquinolone resistance, including mutations in DNA gyrase and the acquisition of antimicrobial resistance genes. Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever in humans, which is highly prevalent in counties with poor sanitation and hygiene standards. Here, we reported S. Typhi clinical isolates that showed varying degrees of susceptibility to fluoroquinolones and were characterized by Analytical Profile Index 20E test kit and 16S rRNA sequencing. S. Typhi strain S27 was resistant to fluoroquinolones and had multiple mutations in the gyrA gene. The gyrA lies in the quinolone resistance determining region of S. Typhi and has mutations at codon 83 (Ser83Phe), codon 87 (Asp87Gly), codon 308 (Lys308Glu), and codon 328 (Val328Ile). S. Typhi strain S6 has no gyrA mutations and is sensitive to fluoroquinolones but forms a strong biofilm relative to S. Typhi S27. Transcriptional analysis of biofilm associated genes revealed that the waaG gene was significantly downregulated. The ΔwaaG mutant showed a significant decrease in persister cells and a strong biofilm formation relative to wild type and gyrA mutant. The gyrA tetra mutant persister assay revealed a significant increase in persister cells compared to wild type and ΔwaaG. Collectively, this is the first report of S. Typhi's two key genes and their roles in antibiotic tolerance, biofilm formation, and fluoroquinolone resistance that can help in understanding the mechanism of persister formation and eradication., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

On 6 April 2022, the Public Health Service of Kennemerland, the Netherlands, was notified about an outbreak of fever and abdominal complaints on a retired river cruise ship, used as shelter for asylum seekers. The diagnosis typhoid fever was confirmed on 7 April. An extensive outbreak investigation was performed. Within 47 days, 72 typhoid fever cases were identified among asylum seekers (n = 52) and staff (n = 20), of which 25 were hospitalised. All recovered after treatment. Consumption of food and tap water on the ship was associated with developing typhoid fever. The freshwater and wastewater tanks shared a common wall with severe corrosion and perforations, enabling wastewater to leak into the freshwater tank at high filling levels. Salmonella Typhi was cultured from the wastewater tank, matching the patient isolates. In the freshwater tank, Salmonella species DNA was detected by PCR, suggesting the presence of the bacterium and supporting the conclusion of contaminated freshwater as the probable source of the outbreak. Outbreaks of uncommon infections may occur if persons from endemic countries are accommodated in crowded conditions. Especially when accommodating migrants on ships, strict supervision on water quality and technical installations are indispensable to guarantee the health and safety of the residents.

Salmonella Typhi, the invasive serovar of S. enterica subspecies enterica, causes typhoid fever in healthy human hosts. The emergence of antibiotic-resistant strains has consistently challenged the successful treatment of typhoid fever with conventional antibiotics. Antimicrobial resistance (AMR) in Salmonella is acquired either by mutations in the genomic DNA or by acquiring extrachromosomal DNA via horizontal gene transfer. In addition, Salmonella can form a subpopulation of antibiotic persistent (AP) cells that can survive at high concentrations of antibiotics. These have reduced the effectiveness of the first and second lines of antibiotics used to treat Salmonella infection. The recurrent and chronic carriage of S. Typhi in human hosts further complicates the treatment process, as a remarkable shift in the immune response from pro-inflammatory Th1 to anti-inflammatory Th2 is observed. Recent studies have also highlighted the overlap between AP, persistent infection (PI) and AMR. These incidents have revealed several areas of research. In this review, we have put forward a timeline for the evolution of antibiotic resistance in Salmonella and discussed the different mechanisms of the same availed by the pathogen at the genotypic and phenotypic levels. Further, we have presented a detailed discussion on Salmonella antibiotic persistence (AP), PI, the host and bacterial virulence factors that can influence PI, and how both AP and PI can lead to AMR., (© 2023 John Wiley & Sons Ltd.)

Background: Typhoid fever, caused by Salmonella enterica serovar Typhi, is a significant public health concern due to the escalating of antimicrobial resistance (AMR), with limited treatment options for extensively drug-resistant (XDR) S. Typhi strains pose a serious threat to disease management and control. This study aimed to investigate the genomic characteristics, epidemiology and AMR genes of XDR S. Typhi strains from typhoid fever patients in Pakistan., Methodology: We assessed 200 patients with enteric fever symptoms, confirming 65 S. Typhi cases through culturing and biochemical tests. Subsequent antimicrobial susceptibility testing revealed 40 cases of extensively drug-resistant (XDR) and 25 cases of multi-drug resistance (MDR). Thirteen XDR strains were selected for whole-genome sequencing, to analyze their sequence type, phylogenetics, resistance genes, pathogenicity islands, and plasmid sequences using variety of data analysis resources. Pangenome analysis was conducted for 140 XDR strains, including thirteen in-house and 127 strains reported from other regions of Pakistan, to assess their genetic diversity and functional annotation., Results: MLST analysis classified all isolates as sequence type 1 (ST-1) with 4.3.1.1. P1 genotype characterization. Prophage and Salmonella Pathogenicity Island (SPI) analysis identified intact prophages and eight SPIs involved in Salmonella's invasion and replication within host cells. Genome data analysis revealed numerous AMR genes including dfrA7, sul1, qnrS1, TEM-1, Cat1, and CTX-M-15, and SNPs associated with antibiotics resistance. IncY, IncQ1, pMAC, and pAbTS2 plasmids, conferring antimicrobial resistance, were detected in a few XDR S. Typhi strains. Phylogenetic analysis inferred a close epidemiological linkage among XDR strains from different regions of Pakistan. Pangenome was noted closed among these strains and functional annotation highlighted genes related to metabolism and pathogenesis., Conclusion: This study revealed a uniform genotypic background among XDR S. Typhi strains in Pakistan, signifying a persistence transmission of a single, highly antibiotic-resistant clone. The closed pan-genome observed underscores limited genetic diversity and highlights the importance of genomic surveillance for combating drug-resistant typhoid infections., Competing Interests: Declaration of competing interest The authors declare that no competing financial interests/ personal relationships exist in relevancy to the above-subject submitted manuscript. Acknowledgement The authors acknowledge the support of the Research Center for Advanced Materials Science (RCAMS) at King Khalid University Abha, Saudi Arabia, for research grant RCAMS/KKU/024/23., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: To determine blaCTX-M-15(Cefotaxime-Munich) gene amongst the extensively drug resistant (XDR) Salmonella typhi (S. typhi) isolates by quantitative Polymerase Chain Reaction (qPCR)., Study Design: Observational, cross-sectional study. Place and Duration of the Study: PNS Shifa Hospital and Bahria University of Health Sciences (BUHS), from January to June 2022., Methodology: All the patients clinically suspected of enteric fever, whose blood culture specimens yielded growth of S. typhi were included in this study. These samples were confirmed by serotyping and biochemical reactions. The ceftriaxone resistance was evaluated by antibiotic susceptibility test according to CLSI 2020 guidelines, whereas blaCTX-M-15 gene was detected by (PCR) using gene-specific primers., Results: Out of 149 S. typhi isolates, 87.2% were confirmed XDR S. typhi resistant to ceftriaxone (CRO). Among these, 83.9% harboured blaCTX-M-15 gene., Conclusion: There was a very high frequency of XDR S. typhi harbouring blaCTX-M-15 in Karachi, Pakistan., Key Words: blaCTX-M-15, Salmonella typhi, Third generation cephalosporin, Typhoid fever, Extensively drug resistant.

Background: The continued emergence of Salmonella enterica serovar Typhi, with ever increasing antimicrobial resistance, necessitates the use of vaccines in endemic countries. A typhoid fever outbreak in Harare, Zimbabwe, in 2018 from a multidrug resistant S Typhi with additional resistance to ciprofloxacin was the catalyst for the introduction of a typhoid conjugate vaccine programme. We aimed to investigate the emergence and evolution of antimicrobial resistance of endemic S Typhi in Zimbabwe and to determine the population structure, gene flux, and sequence polymorphisms of strains isolated before a typhoid conjugate vaccine programme to provide a baseline for future evaluation of the effect of the vaccination programme., Methods: In this genomic epidemiology study, we used short-read whole-genome sequencing of S Typhi isolated from clinical cases of typhoid fever in Harare, Zimbabwe, between Jan 1, 2012, and Feb 9, 2019, to determine the S Typhi population structure, gene flux, and sequence polymorphisms and reconstructed the evolution of antimicrobial resistance. Maximum likelihood time-scaled phylogenetic trees of Zimbabwe isolates in the context of global isolates obtained from the National Center for Biotechnology Information were constructed to infer spread and emergence of antimicrobial resistance., Findings: The population structure of S Typhi in Harare, Zimbabwe, from 2012 to 2019 was dominated by multidrug resistant genotype 4.3.1.1.EA1 (H58) that spread to Zimbabwe from neighbouring countries in around 2009 (95% credible interval 2008·5-2010·0). Acquisition of an IncN plasmid carrying antimicrobial resistance genes including a qnrS gene and a mutation in the quinolone resistance determining region of gyrA gene contributed to non-susceptibility and resistance to quinolone antibiotics. A minority population of antimicrobial susceptible S Typhi genotype 3.3.1 strains were present throughout., Interpretation: The currently dominant S Typhi population is genotype 4.3.1.1 that spread to Zimbabwe and acquired additional antimicrobial resistance though acquisition of a plasmid and mutation in the gyrA gene. This study provides a baseline population structure for future evaluation of the effect of the typhoid conjugate vaccine programme in Harare., Funding: Bill & Melinda Gates Foundation and the Biotechnology and Biological Sciences Research Council Institute Strategic Programme., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Purpose: To investigate the antibiotic resistance and genetic profile of ceftriaxone-resistant Salmonella Typhi isolated from the blood culture of two paediatric cases of typhoid fever and one from the stool culture of their household contact, in North India., Methods: In this study, whole-genome sequencing was carried out with paired-end 2 ​× ​150 bp reads on Illumina MiSeq (Illumina, USA) employing v2 and v3 chemistry. To check data quality, adapters and low-quality sequences were removed through Trimmomatic-v0.36. High quality reads were then assembled de novo using A5-miseq pipeline. For further refinement, reference-guided contig ordering and orienting were performed on the scaffold assemblies using ABACAS (http://abacas.sourceforge.net/). The assembled genome was annotated using Prokka v1.12 to identify and annotate the gene content. Plasmid replicons in bacterial isolates were identified by PlasmidFinder, whereas mobile genetic elements were predicted using Mobile Element Finder. Referenced-based SNP tree with maximum likelihood method was built with CSI phylogeny v1.4., Results: All three isolates exhibited resistance to ceftriaxone, cefixime, ciprofloxacin, ampicillin, and co-trimoxazole, while demonstrating sensitivity to azithromycin and chloramphenicol. The whole-genome sequencing of these strains revealed the presence of blaCTX-M-15 gene for cephalosporin resistance in addition to gyrA, qnr and IncY plasmid replicon. A 5 ​kb IS91 Sbo1 gene cassette (IncY plasmid) was identified which carried extended spectrum β-lactamase blaCTX-M-15, blaTEM-1D (resistance to ampicillin and cephalosporin), sul2, dfrA14 (resistant to trimethoprim-sulfamethoxazole) and qnrS (resistant to ciprofloxacin). These isolates belong to H58 lineage and grouped as sequence type 1 (ST1) on multilocus sequence typing (MLST) analysis., Conclusion: In the present study we report the isolation of blaCTX-M-15 positive S. Typhi from two paediatric patients presenting with fever and one from stool culture of their contact from North India and highlight the need for further investigations to understand the different factors contributing to ceftriaxone resistance in Salmonella Typhi., (Copyright © 2023 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

Importance: Typhoid fever is a life-threatening disease caused by Salmonella enterica serovar Typhi, resulting in a significant disease burden across developing countries. Historically, China was very much close to the global epicenter of typhoid, but the role of typhoid transmission within China and among epicenter remains overlooked in previous investigations. By using newly produced genomics on a national scale, we clarify the complex local and global transmission history of such a notorious disease agent in China spanning the most recent five decades, which largely undermines the global public health network., Competing Interests: The authors declare no conflict of interest.

Enteric fever, caused by oral infection with typhoidal Salmonella serovars, presents as a non-specific febrile illness preceded by an incubation period of 5 days or more. The enteric fever human challenge model provides a unique opportunity to investigate the innate immune response during this incubation period, and how this response is altered by vaccination with the Vi polysaccharide or conjugate vaccine. We find that on the same day as ingestion of typhoidal Salmonella , there is already evidence of an immune response, with 199 genes upregulated in the peripheral blood transcriptome 12 hours post-challenge (false discovery rate <0.05). Gene sets relating to neutrophils, monocytes, and innate immunity were over-represented (false discovery rate <0.05). Estimating cell proportions from gene expression data suggested a possible increase in activated monocytes 12 hours post-challenge ( P = 0.036, paired Wilcoxon signed-rank test). Furthermore, plasma TNF-α rose following exposure ( P = 0.011, paired Wilcoxon signed-rank test). There were no significant differences in gene expression (false discovery rate <0.05) in the 12 hours response between those who did and did not subsequently develop clinical or blood culture confirmed enteric fever or between vaccination groups. Together, these results demonstrate early perturbation of the peripheral blood transcriptome after enteric fever challenge and provide initial insight into early mechanisms of protection., Competing Interests: A.J.P. is Chair of the UK Department of Health and Social Care's (DHSC) Joint Committee on Vaccination & Immunisation (JCVI) and was a member of the WHO's Strategic Advisory Group of Experts until 2022. C.J.B. is currently employed by GlaxoSmithKline.

Since its emergence in 2016, extensively drug resistant (XDR) Salmonella enterica serovar Typhi (S. Typhi) has become the dominant cause of typhoid fever in Pakistan. The establishment of sustained XDR S. Typhi transmission in other countries represents a major public health threat. We show that the annual volume of air travel from Pakistan strongly discriminates between countries that have and have not imported XDR S. Typhi in the past, and identify a significant association between air travel volume and the rate of between-country movement of the H58 haplotype of S. Typhi from fitted phylogeographic models. Applying these insights, we analyze flight itinerary data cross-referenced with model-based estimates of typhoid fever incidence to identify the countries at highest risk of importation and sustained onward transmission of XDR S. Typhi. Future outbreaks of XDR typhoid are most likely to occur in countries that can support efficient local S. Typhi transmission and have strong travel links to regions with ongoing XDR typhoid outbreaks (currently Pakistan). Public health activities to track and mitigate the spread of XDR S. Typhi should be prioritized in these countries., (© 2023. Springer Nature Limited.)

The Salmonella pathogenicity island 2 (SPI-2)-encoded type III secretion system (injectisome) is assembled following uptake of bacteria into vacuoles in mammalian cells. The injectisome translocates virulence proteins (effectors) into infected cells. Numerous studies have established the requirement for a functional SPI-2 injectisome for growth of Salmonella Typhimurium in mouse macrophages, but the results of similar studies involving Salmonella Typhi and human-derived macrophages are not consistent. It is important to clarify the functions of the S . Typhi SPI-2 injectisome, not least because an inactivated SPI-2 injectisome forms the basis for live attenuated S . Typhi vaccines that have undergone extensive trials in humans. Intracellular expression of injectisome genes and effector delivery take longer in the S . Typhi/human macrophage model than for S . Typhimurium and we propose that this could explain the conflicting results. Furthermore, strains of both S . Typhimurium and S . Typhi contain intact genes for several 'core' effectors. In S . Typhimurium these cooperate to regulate the vacuole membrane and contribute to intracellular bacterial replication; similar functions are therefore likely in S . Typhi.

Due to high mortality rates, typhoid fever still is one of the major health problems in the world, particularly in developing countries. The lack of highly specific and sensitive diagnostic tests and the great resemblance of typhoid fever symptoms to other diseases made the false-negative diagnosis a major challenge in typhoid fever management. Hence, we decided to design a Surface Plasmon Resonance (SPR) based biosensor for specific detection of Salmonella typhi through DNA hybridization. The results showed that the 10 nM of the synthetic target sequence, as well as 1 nM of PCR product, were the lowest feasible detected concentrations by the designed biosensor. This genosensor was also found to significantly distinguish the complementary sequence with the accuracy of one base mismatch sequence. The surface of the chip can be regenerated with NaOH solution and used for consecutive diagnosis. Therefore, the function of the designed biosensor indicates its high potential for Salmonella typhi detection practice., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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])., Competing Interests: MC, ZD, DI, AA, MA, MC, KC, JC, BH, KK, MM, CP, SV, HW, AA, AA, SA, JA, PA, BB, AB, JC, Kd, AD, Jd, PD, CD, SD, ME, LF, DG, GG, MG, AG, CG, MG, RH, RH, YH, JH, OI, JI, JJ, CJ, DJ, JJ, GK, AK, AK, AK, SK, RK, RK, AL, ML, RL, SL, GM, TM, CM, AM, GN, SN, SN, TN, SN, EN, IO, SP, AP, FQ, FQ, SR, SR, DR, PR, RR, TR, JR, SS, SS, KS, MS, JS, JS, VS, JS, RS, SS, MS, AS, AS, KT, DT, AT, MT, MT, RT, NT, ST, KV, MV, BV, FW, JW, GD, SA, JK, DA, SB, KH No competing interests declared, NF NAF chairs the Wellcome Surveillance and Epidemiology of Drug Resistant Infections (SEDRIC) group, which has a focus on antimicrobial resistance. This could be perceived as relevant although not a direct conflict, IB IB has consulted to BlueDot and the NHL Players' Association, AP AJP is chair of the UK Department of Health and Social Care's (DHSC) Joint Committee on Vaccination and Immunisation (JCVI) but does not take part in the JCVI COVID-19 committee. He was a member of WHO SAGE until 2022. AJPs employer, Oxford University has entered into a partnership with AstraZeneca for development of a COVID-19 vaccine. AJP has provided advice to Shionogi & Co., Ltd on development of a COVID19 vaccine, (© 2023, Carey et al.)

The human-specific Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a systemic disease with no known reservoir. Curli fimbriae are major components of biofilm produced by Salmonella and are encoded by the csg gene cluster (csgBAC and csgDEFG). The role of curli in S. Typhi is unknown, although detection of anti-curli antibodies suggests they are produced during host infection. In this study, we investigated curli gene expression in S. Typhi. We demonstrated that the CsgD regulatory protein binds weakly to the csgB promoter. Yet, replacing S. Typhi csgD with the csgD allele from S. Typhimurium did not modify the curli negative phenotype on Congo Red medium suggesting that differential regulation of curli gene expression in S. Typhi is not dependent on modification of the CsgD regulator. The entire csg gene cluster from S. Typhimurium was also cloned into S. Typhi, but again, despite introduction of a fully functional csg gene cluster from S. Typhimurium, curli were still not detected in S. Typhi. Thus, in addition to intrinsic genomic differences in the csg gene cluster that have resulted in production of a modified CsgD protein, S. Typhi has likely undergone other changes independent of the csg gene cluster that have led to distinctive regulation of csg genes compared to other Salmonella serovars., (© 2023. Springer Nature Limited.)

Salmonella enterica serovar Typhi ( S . Typhi) is a human-restricted pathogen that replicates in macrophages. In this study, we investigated the roles of the S . Typhi type 3 secretion systems (T3SSs) encoded on Salmonella pathogenicity islands (SPI)-1 (T3SS-1) and SPI-2 (T3SS-2) during human macrophage infection. We found that mutants of S . Typhi deficient for both T3SSs were defective for intramacrophage replication as measured by flow cytometry, viable bacterial counts, and live time-lapse microscopy. T3SS-secreted proteins PipB2 and SifA contributed to S . Typhi replication and were translocated into the cytosol of human macrophages through both T3SS-1 and T3SS-2, demonstrating functional redundancy for these secretion systems. Importantly, an S . Typhi mutant strain that is deficient for both T3SS-1 and T3SS-2 was severely attenuated in the ability to colonize systemic tissues in a humanized mouse model of typhoid fever. Overall, this study establishes a critical role for S . Typhi T3SSs during its replication within human macrophages and during systemic infection of humanized mice. IMPORTANCE Salmonella enterica serovar Typhi is a human-restricted pathogen that causes typhoid fever. Understanding the key virulence mechanisms that facilitate S . Typhi replication in human phagocytes will enable rational vaccine and antibiotic development to limit the spread of this pathogen. While S . Typhimurium replication in murine models has been studied extensively, there is limited information available about S . Typhi replication in human macrophages, some of which directly conflict with findings from S . Typhimurium murine models. This study establishes that both of S . Typhi's two type 3 secretion systems (T3SS-1 and T3SS-2) contribute to intramacrophage replication and virulence., Competing Interests: The authors declare no conflict of interest.

Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever and, in some cases, chronic carriage after resolution of acute disease. This study examined sequential isolates of S. Typhi from a single host with persistent asymptomatic infection. These isolates, along with another S. Typhi isolate recovered from a household contact with typhoid fever, were subjected to whole genome sequencing and analysis. In addition, direct sequencing of the bile fluid from the host with persistent infection was also performed. Comparative analysis of isolates revealed three sub-populations of S. Typhi with distinct genetic patterns. Metagenomic sequencing recognised only two of the three sub-populations within the bile fluid. The detection and investigation of insertion sequences IS10R and associated deletions complemented analysis of single nucleotide polymorphisms. These findings improve our understanding of within-host dynamics of S. Typhi in cases of persistent infection and inform epidemiological investigations of transmission events associated with chronic carriers., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Sim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

BACKGROUNDTyphoid fever is caused by the Gram-negative bacterium Salmonella enterica serovar Typhi and poses a substantial public health burden worldwide. Vaccines have been developed based on the surface Vi-capsular polysaccharide of S. Typhi; these include a plain-polysaccharide-based vaccine, ViPS, and a glycoconjugate vaccine, ViTT. To understand immune responses to these vaccines and their vaccine-induced immunological protection, molecular signatures were analyzed using bioinformatic approaches.METHODSBulk RNA-Seq data were generated from blood samples obtained from adult human volunteers enrolled in a vaccine trial, who were then challenged with S. Typhi in a controlled human infection model (CHIM). These data were used to conduct differential gene expression analyses, gene set and modular analyses, B cell repertoire analyses, and time-course analyses at various post-vaccination and post-challenge time points between participants receiving ViTT, ViPS, or a control meningococcal vaccine.RESULTSTranscriptomic responses revealed strong differential molecular signatures between the 2 typhoid vaccines, mostly driven by the upregulation in humoral immune signatures, including selective usage of immunoglobulin heavy chain variable region (IGHV) genes and more polarized clonal expansions. We describe several molecular correlates of protection against S. Typhi infection, including clusters of B cell receptor (BCR) clonotypes associated with protection, with known binders of Vi-polysaccharide among these.CONCLUSIONThe study reports a series of contemporary analyses that reveal the transcriptomic signatures after vaccination and infectious challenge, while identifying molecular correlates of protection that may inform future vaccine design and assessment.TRIAL REGISTRATIONClinicalTrials.gov NCT02324751.

Salmonella enterica serovar Typhi (S. Typhi) is the cause of typhoid fever, presenting high rates of morbidity and mortality in low- and middle-income countries. The H58 haplotype shows high levels of antimicrobial resistance (AMR) and is the dominant S. Typhi haplotype in endemic areas of Asia and East sub-Saharan Africa. The situation in Rwanda is currently unknown and therefore to reveal the genetic diversity and AMR of S. Typhi in Rwanda, 25 historical (1984-1985) and 26 recent (2010-2018) isolates from Rwanda were analysed using whole genome sequencing (WGS). WGS was locally implemented using Illumina MiniSeq and web-based analysis tools, thereafter complemented with bioinformatic approaches for more in-depth analyses. Whereas historical S. Typhi isolates were found to be fully susceptible to antimicrobials and show a diversity of genotypes, i.e 2.2.2, 2.5, 3.3.1 and 4.1; the recent isolates showed high AMR rates and were predominantly associated with genotype 4.3.1.2 (H58, 22/26; 84,6%), possibly resulting from a single introduction in Rwanda from South Asia before 2010. We identified practical challenges for the use of WGS in endemic regions, including a high cost for shipment of molecular reagents and lack of high-end computational infrastructure for the analyses, but also identified WGS to be feasible in the studied setting and giving opportunity for synergy with other programs., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Rutanga et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The National Institute for Communicable Diseases in South Africa participates in national laboratory-based surveillance for human isolates of Salmonella species. Laboratory analysis includes whole-genome sequencing (WGS) of isolates. We report on WGS-based surveillance of Salmonella enterica serovar Typhi ( Salmonella Typhi) in South Africa from 2020 through 2021. We describe how WGS analysis identified clusters of enteric fever in the Western Cape Province of South Africa and describe the epidemiological investigations associated with these clusters. A total of 206 Salmonella Typhi isolates were received for analysis. Genomic DNA was isolated from bacteria and WGS was performed using Illumina NextSeq technology. WGS data were investigated using multiple bioinformatics tools, including those available at the Centre for Genomic Epidemiology, EnteroBase and Pathogenwatch. Core-genome multilocus sequence typing was used to investigate the phylogeny of isolates and identify clusters. Three major clusters of enteric fever were identified in the Western Cape Province; cluster one ( n =11 isolates), cluster two ( n =13 isolates), and cluster three ( n =14 isolates). To date, no likely source has been identified for any of the clusters. All isolates associated with the clusters, showed the same genotype (4.3.1.1.EA1) and resistome (antimicrobial resistance genes: bla TEM-1B , catA1 , sul1 , sul2 , dfrA7 ). The implementation of genomic surveillance of Salmonella Typhi in South Africa has enabled rapid detection of clusters indicative of possible outbreaks. Cluster identification allows for targeted epidemiological investigations and a timely, coordinated public health response.

Introduction: Salmonella enterica serovar Typhi ( S . Typhi) is a major cause of morbidity and mortality in developing countries, contributing significantly to the global disease burden., Methods: In this study, S . Typhi strains were isolated from 100 patients exhibiting symptoms of typhoid fever at a tertiary care hospital in Pakistan. Antimicrobial testing of all isolates was performed to determine the sensitivity and resistance pattern. Three MDR strains, namely QS194, QS430, and QS468, were subjected to whole genome sequencing for genomic characterization., Results and Discussion: MLST analysis showed that QS194, belonged to ST19, which is commonly associated with Salmonella enterica serovar typhimurium . In contrast, QS430 and QS468, belonged to ST1, a sequence type frequently associated with S . Typhi. PlasmidFinder identified the presence of IncFIB(S) and IncFII(S) plasmids in QS194, while IncQ1 was found in QS468. No plasmid was detected in QS430. CARD-based analysis showed that the strains were largely resistant to a variety of antibiotics and disinfecting agents/antiseptics, including fluoroquinolones, cephalosporins, monobactams, cephamycins, penams, phenicols, tetracyclines, rifamycins, aminoglycosides, etc. The S . Typhi strains possessed various virulence factors, such as Vi antigen, Agf/Csg, Bcf, Fim, Pef, etc. The sequencing data indicated that the strains had antibiotic resistance determinants and shared common virulence factors. Pangenome analysis of the selected S . Typhi strains identified 13,237 genes, with 3,611 being core genes, 2,093 shell genes, and 7,533 cloud genes. Genome-based typing and horizontal gene transfer analysis revealed that the strains had different evolutionary origins and may have adapted to distinct environments or host organisms. These findings provide important insights into the genetic characteristics of S . Typhi strains and their potential association with various ecological niches and host organisms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fatima, Ishaq, Irfan, AlAsmari, Achakzai, Zaheer, Ali and Akbar.)