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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: 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: Understanding the dynamics of infection and carriage of typhoid in endemic settings is critical to finding solutions to prevention and control., Methods: In a 3-year case-control study, we investigated typhoid among children aged <16 years (4670 febrile cases and 8549 age matched controls) living in an informal settlement, Nairobi, Kenya., Results: 148 S . Typhi isolates from cases and 95 from controls (stool culture) were identified; a carriage frequency of 1 %. Whole-genome sequencing showed 97% of cases and 88% of controls were genotype 4.3.1 (Haplotype 58), with the majority of each (76% and 88%) being multidrug-resistant strains in three sublineages of the H58 genotype (East Africa 1 (EA1), EA2, and EA3), with sequences from cases and carriers intermingled., Conclusions: The high rate of multidrug-resistant H58 S . Typhi, and the close phylogenetic relationships between cases and controls, provides evidence for the role of carriers as a reservoir for the community spread of typhoid in this setting., Funding: National Institutes of Health (R01AI099525); Wellcome Trust (106158/Z/14/Z); European Commission (TyphiNET No 845681); National Institute for Health Research (NIHR); Bill and Melinda Gates Foundation (OPP1175797)., Competing Interests: SK, ZD, CM, RN, SK, CW, SA, NG, RO, BO, SD, MA, JC, KH, GD No competing interests declared, (© 2021, Kariuki et al.)

The emergence of antimicrobial resistance (AMR) to first- and second-line treatment regimens of enteric fever is a global public-health problem, and routine genomic surveillance to inform clinical and public-health management guidance is essential. Here, we present the prospective analysis of genomic data to monitor trends in incidence, AMR and travel, and assess hierarchical clustering (HierCC) methodology of 1742 isolates of typhoidal salmonellae. Trend analysis of Salmonella Typhi and S . Paratyphi A cases per year increased 48 and 17.3%, respectively, between 2016 and 2019 in England, mainly associated with travel to South Asia. S . Paratyphi B cases have remained stable and are mainly associated with travel to the Middle East and South America. There has been an increase in the number of S . Typhi exhibiting a multidrug-resistant (MDR) profile and the emergence of extensively drug resistant (XDR) profiles. HierCC was a robust method to categorize clonal groups into clades and clusters associated with travel and AMR profiles. The majority of cases that had XDR S . Typhi reported recent travel to Pakistan (94 %) and belonged to a subpopulation of the 4.3.1 (H58) clone (HC5&#95;1452). The phenotypic and genotypic AMR results showed high concordance for S . Typhi and S . Paratyphi A, B and C, with 99.99 % concordance and only three (0.01 %) discordant results out of a possible 23 178 isolate/antibiotic combinations. Genomic surveillance of enteric fever has shown the recent emergence and increase of MDR and XDR S . Typhi strains, resulting in a review of clinical guidelines to improve management of imported infections.

The first imported case of XDR typhoid fever in Taiwan contracted with a bacterial strain, which was most closely related to the bla CTX-M-15 -carrying strains linked to Pakistan. Meropenem, in combination with an antimicrobial with intracellular activity against Salmonella, should be used for the treatment of XDR typhoid fever., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to be declared., (Copyright © 2020. Published by Elsevier B.V.)

In Korea, typhoid fever is a rare disease due to improved living standards. However, typhoid fever remains a major burden in developing countries and regions, such as India and Southeast Asia. In this study, we isolated Salmonella Typhi (S. Typhi) from eight patients with typhoid fever who were travelers returning from India. The strains isolated were characterized by antimicrobial susceptibility profiling and whole-genome sequencing (WGS) analysis. All strains were resistant to nalidixic acid and azithromycin. Among them, four isolates were highly resistant to ciprofloxacin (MIC ≥32 μg/ml); these strains have not been confirmed in Korea PulseNet DB. According to WGS, the ciprofloxacin-resistant strains belong to the global dominant multidrug-resistant (MDR) haplotype H58 (SNP glpA C1047T, SptP protein Q185* (premature stop codon)) and do not harbor the MDR plasmid. H58-associated SNPs in membrane and metabolism genes, including yhdA, yajI, hyaE, tryE, rlpB and metH, are present. Additionally, phylogenetic analysis assigned the H58 strains to sublineage II, whereas the non-H58 strains are closely related to haplotype H50. The presence of high-level ciprofloxacin-resistant S. Typhi haplotype H58 in Korea was first confirmed as due to influx from overseas via travelers. This study provides information about intercontinental drug-resistant transmission between countries and suggests that travelers need to be careful about personal hygiene., Competing Interests: The authors have declared that no competing interests exist.

The rising prevalence of antimicrobial resistance in Salmonella enterica serovars Typhi and Paratyphi A, causative agents of typhoid and paratyphoid, have led to fears of untreatable infections. Of specific concern is the emerging resistance against azithromycin, the only remaining oral drug to treat extensively drug resistant (XDR) typhoid. Since the first report of azithromycin resistance from Bangladesh in 2019, cases have been reported from Nepal, India, and Pakistan. The genetic basis of this resistance is a single point mutation in the efflux pump AcrB (R717Q/L). Here, we report 38 additional cases of azithromycin-resistant (AzmR) Salmonella Typhi and Paratyphi A isolated in Bangladesh between 2016 and 2018. Using genomic analysis of 56 AzmR isolates from South Asia with AcrB-R717Q/L, we confirm that this mutation has spontaneously emerged in different Salmonella Typhi and Paratyphi A genotypes. The largest cluster of AzmR Typhi belonged to genotype 4.3.1.1; Bayesian analysis predicts the mutation to have emerged sometime in 2010. A travel-related Typhi isolate with AcrB-R717Q belonging to 4.3.1.1 was isolated in the United Kingdom, increasing fears of global spread. For real-time detection of AcrB-R717Q/L, we developed an extraction-free, rapid, and low-cost mismatch amplification mutation assay (MAMA). Validation of MAMA using 113 AzmR and non-AzmR isolates yielded >98% specificity and sensitivity versus phenotypic and whole-genome sequencing assays currently used for azithromycin resistance detection. With increasing azithromycin use, AcrB-R717Q/L is likely to be acquired by XDR strains. The proposed tool for active detection and surveillance of this mutation may detect pan-oral drug resistance early, giving us a window to intervene. IMPORTANCE In the early 1900s, with mortality of ∼30%, typhoid and paratyphoid ravaged parts of the world; with improved water, sanitation, and hygiene in resource-rich countries and the advent of antimicrobials, mortality dwindled to <1%. Today, the burden rests disproportionately on South Asia, where the primary means for combatting the disease is antimicrobials. However, prevalence of antimicrobial resistance is rising and, in 2016, an extensively drug resistant Typhi strain triggered an ongoing outbreak in Pakistan, leaving only one oral drug, azithromycin, to treat it. Since the description of emergence of azithromycin resistance, conferred by a point mutation in acrB (AcrB-R717Q/L) in 2019, there have been increasing numbers of reports. Using genomics and Bayesian analysis, we illustrate that this mutation emerged in approximately 2010 and has spontaneously arisen multiple times. Emergence of pan-oral drug resistant Salmonella Typhi is imminent. We developed a low-cost, rapid PCR tool to facilitate real-time detection and prevention policies., (Copyright © 2021 Sajib et al.)

Salmonella Typhi is the primary causative agent of typhoid fever; an acute systemic infection that leads to chronic carriage in 3-5% of individuals. Chronic carriers are asymptomatic, difficult to treat and serve as reservoirs for typhoid outbreaks. Understanding the factors that contribute to chronic carriage is key to development of novel therapies to effectively resolve typhoid fever. Herein, although we observed no distinct clustering of chronic carriage isolates via phylogenetic analysis, we demonstrated that chronic isolates were phenotypically distinct from acute infection isolates. Chronic carriage isolates formed significantly thicker biofilms with greater biomass that correlated with significantly higher relative levels of extracellular DNA (eDNA) and DNABII proteins than biofilms formed by acute infection isolates. Importantly, extracellular DNABII proteins include integration host factor (IHF) and histone-like protein (HU) that are critical to the structural integrity of bacterial biofilms. In this study, we demonstrated that the biofilm formed by a chronic carriage isolate in vitro, was susceptible to disruption by a specific antibody against DNABII proteins, a successful first step in the development of a therapeutic to resolve chronic carriage., Competing Interests: The authors have declared that no competing interests exist.

Salmonella Typhi (S. Typhi) is the causative agent of typhoid fever; a systemic disease affecting ~20 million people per year globally. There are little data regarding the contemporary epidemiology of typhoid in Latin America. Consequently, we aimed to describe some recent epidemiological aspects of typhoid in Colombia using cases reported to the National Public Health Surveillance System (Sivigila) between 2012 and 2015. Over the four-year reporting period there were 836 culture confirmed cases of typhoid in Colombia, with the majority (676/836; 80.1%) of reported cases originated from only seven departments. We further characterized 402 S. Typhi isolates with available corresponding data recovered from various departments of Colombia through antimicrobial susceptibility testing and molecular subtyping. The majority (235/402; 58.5%) of these typhoid cases occurred in males and were most commonly reported in those aged between 10 and 29 years (218/402; 54.2%); there were three (0.74%) reported fatalities. The overwhelming preponderance (339/402; 84.3%) of S. Typhi were susceptible to all tested antimicrobials. The most common antimicrobial to which the organisms exhibited non-susceptibility was ampicillin (30/402;7.5%), followed by nalidixic acid (23/402, 5.7%). Molecular subtyping identified substantial genetic diversity, which was well distributed across the country. Despite the diffuse pattern of S. Typhi genotypes, we identified various geographical hotspots of disease associated with local dominant genotypes. Notably, we found limited overlap of Colombian genotypes with organisms reported in other Latin American countries. Our work highlights a substantial burden of typhoid in Colombia, characterized by sustained transmission in some regions and limited epidemics in other departments. The disease is widely distributed across the country and associated with multiple antimicrobial susceptible genotypes that appear to be restricted to Colombia. This study provides a current perspective for typhoid in Latin America and highlights the importance of pathogen-specific surveillance to add insight into the limited epidemiology of typhoid in this region., Competing Interests: The authors have declared that no competing interests exist.

To conduct outbreak identification and transmission factor analysis of typhoid epidemic occurred in Xinqiao town, Jiangyin city from June to September 2016. A total of 14 strains of Salmonella typhi isolated from confirmed cases were collected, and 65 external environment samples and 13 food samples related to the outbreak were taken. Real-time PCR was used to detect specific gene of Salmonella typhi in the samples. Conventional method was used to isolate strains. The strains isolated from both the samples and patients in the epidemic were subjected to antimicrobial susceptibility testing and PFGE molecular characteristics. Salmonella typhi strain was isolated from one external sample (well water of a deli processing plant). The results of drug susceptibility showed that 15 strains were resistant to nalidixic acid. A total of 15 strains of Salmonella typhi were divided into 2 molecular patterns by pulsed field gel electrophoresis. The fingerprints of PFGE from the 13 patients and the environmental isolate were completely consistent, and there was one band difference from the other patient isolate. Combined with the epidemiological investigation and laboratory test results, it was determined that the outbreak was caused by genetic clone of the same Salmonella typhi. Food processing plant should be one of the key links.

Salmonella enterica infections remain a challenging health issue, causing significant morbidity and mortality worldwide. Current vaccines against typhoid fever display moderate efficacy whilst no licensed vaccines are available for paratyphoid fever or invasive non-typhoidal salmonellosis. Therefore, there is an urgent need to develop high efficacy broad-spectrum vaccines that can protect against typhoidal and non-typhoidal Salmonella . The Salmonella outer membrane porins OmpC and OmpF, have been shown to be highly immunogenic antigens, efficiently eliciting protective antibody, and cellular immunity. Furthermore, enterobacterial porins, particularly the OmpC, have a high degree of homology in terms of sequence and structure, thus making them a suitable vaccine candidate. However, the degree of the amino acid conservation of OmpC among typhoidal and non-typhoidal Salmonella serovars is currently unknown. Here we used a bioinformatical analysis to classify the typhoidal and non-typhoidal Salmonella OmpC amino acid sequences into different clades independently of their serological classification. Further, our analysis determined that the porin OmpC contains various amino acid sequences that are highly conserved among both typhoidal and non-typhoidal Salmonella serovars. Critically, some of these highly conserved sequences were located in the transmembrane β-sheet within the porin β-barrel and have immunogenic potential for binding to MHC-II molecules, making them suitable candidates for a broad-spectrum Salmonella vaccine. Collectively, these findings suggest that these highly conserved sequences may be used for the rational design of an effective broad-spectrum vaccine against Salmonella ., (Copyright © 2020 Valero-Pacheco, Blight, Aldapa-Vega, Kemlo, Pérez-Toledo, Wong-Baeza, Kurioka, Perez-Shibayama, Gil-Cruz, Sánchez-Torres, Pastelin-Palacios, Isibasi, Reyes-Sandoval, Klenerman and López-Macías.)

Background: Antimicrobial resistance (AMR) is an increasing threat to global health. There are > 14 million cases of enteric fever every year and > 135,000 deaths. The disease is primarily controlled by antimicrobial treatment, but this is becoming increasingly difficult due to AMR. Our objectives were to assess the prevalence and geographic distribution of AMR in Salmonella enterica serovars Typhi and Paratyphi A infections globally, to evaluate the extent of the problem, and to facilitate the creation of geospatial maps of AMR prevalence to help targeted public health intervention., Methods: We performed a systematic review of the literature by searching seven databases for studies published between 1990 and 2018. We recategorised isolates to allow the analysis of fluoroquinolone resistance trends over the study period. The prevalence of multidrug resistance (MDR) and fluoroquinolone non-susceptibility (FQNS) in individual studies was illustrated by forest plots, and a random effects meta-analysis was performed, stratified by Global Burden of Disease (GBD) region and 5-year time period. Heterogeneity was assessed using the I 2 statistics. We present a descriptive analysis of ceftriaxone and azithromycin resistance., Findings: We identified 4557 articles, of which 384, comprising 124,347 isolates (94,616 S. Typhi and 29,731 S. Paratyphi A) met the pre-specified inclusion criteria. The majority (276/384; 72%) of studies were from South Asia; 40 (10%) articles were identified from Sub-Saharan Africa. With the exception of MDR S. Typhi in South Asia, which declined between 1990 and 2018, and MDR S. Paratyphi A, which remained at low levels, resistance trends worsened for all antimicrobials in all regions. We identified several data gaps in Africa and the Middle East. Incomplete reporting of antimicrobial susceptibility testing (AST) and lack of quality assurance were identified., Interpretation: Drug-resistant enteric fever is widespread in low- and middle-income countries, and the situation is worsening. It is essential that public health and clinical measures, which include improvements in water quality and sanitation, the deployment of S. Typhi vaccination, and an informed choice of treatment are implemented. However, there is no licenced vaccine for S. Paratyphi A. The standardised reporting of AST data and rollout of external quality control assessment are urgently needed to facilitate evidence-based policy and practice., Trial Registration: PROSPERO CRD42018029432.

Due to the public health importance of flagellar genes for typing, it is important to understand mechanisms that could alter their expression or presence. Phenotypic novelty in flagellar genes arise predominately through accumulation of mutations but horizontal transfer is known to occur. A linear plasmid termed pBSSB1 previously identified in Salmonella Typhi, was found to encode a flagellar operon that can mediate phase variation, which results in the rare z66 flagella phenotype. The identification and tracking of homologs of pBSSB1 is limited because it falls outside the normal replicon typing schemes for plasmids. Here we report the generation of nine new pBSSB1-family sequences using Illumina and Nanopore sequence data. Homologs of pBSSB1 were identified in 154 genomes representing 25 distinct serotypes from 67,758 Salmonella public genomes. Pangenome analysis of pBSSB1-family contigs was performed using roary and we identified three core genes amenable to a minimal pMLST scheme. Population structure analysis based on the newly developed pMLST scheme identified three major lineages representing 35 sequence types, and the distribution of these sequence types was found to span multiple serovars across the globe. This in silico pMLST scheme has shown utility in tracking and subtyping pBSSB1-family plasmids and it has been incorporated into the plasmid MLST database under the name "pBSSB1-family"., Competing Interests: The authors have declared that no competing interests exist.

Background: With the rise in fluoroquinolone-resistant Salmonella Typhi and the recent emergence of ceftriaxone resistance, azithromycin is one of the last oral drugs available against typhoid for which resistance is uncommon. Its increasing use, specifically in light of the ongoing outbreak of extensively drug-resistant (XDR) Salmonella Typhi (resistant to chloramphenicol, ampicillin, cotrimoxazole, streptomycin, fluoroquinolones and third-generation cephalosporins) in Pakistan, places selective pressure for the emergence and spread of azithromycin-resistant isolates. However, little is known about azithromycin resistance in Salmonella, and no molecular data are available on its mechanism., Methods and Findings: We conducted typhoid surveillance in the two largest pediatric hospitals of Bangladesh from 2009-2016. All typhoidal Salmonella strains were screened for azithromycin resistance using disc diffusion and resistance was confirmed using E-tests. In total, we identified 1,082 Salmonella Typhi and Paratyphi A strains; among these, 13 strains (12 Typhi, 1 Paratyphi A) were azithromycin-resistant (MIC range: 32-64 μg/ml) with the first case observed in 2013. We sequenced the resistant strains, but no molecular basis of macrolide resistance was identified by the currently available antimicrobial resistance prediction tools. A whole genome SNP tree, made using RAxML, showed that the 12 Typhi resistant strains clustered together within the 4.3.1.1 sub-clade (H58 lineage 1). We found a non-synonymous single-point mutation exclusively in these 12 strains in the gene encoding AcrB, an efflux pump that removes small molecules from bacterial cells. The mutation changed the conserved amino acid arginine (R) at position 717 to a glutamine (Q). To test the role of R717Q present in azithromycin-resistant strains, we cloned acrB from azithromycin-resistant and sensitive strains, expressed them in E. coli, Typhi and Paratyphi A strains and tested their azithromycin susceptibility. Expression of AcrB-R717Q in E. coli and Typhi strains increased the minimum inhibitory concentration (MIC) for azithromycin by 11- and 3-fold respectively. The azithromycin-resistant Paratyphi A strain also contained a mutation at R717 (R717L), whose introduction in E. coli and Paratyphi A strains increased MIC by 7- and 3-fold respectively, confirming the role of R717 mutations in conferring azithromycin resistance., Conclusions: This report confirms 12 azithromycin-resistant Salmonella Typhi strains and one Paratyphi A strain. The molecular basis of this resistance is one mutation in the AcrB protein at position 717. This is the first report demonstrating the impact of this non-synonymous mutation in conferring macrolide resistance in a clinical setting. With increasing azithromycin use, strains with R717 mutations may spread and be acquired by XDR strains. An azithromycin-resistant XDR strain would shift enteric fever treatment from outpatient departments, where patients are currently treated with oral azithromycin, to inpatient departments to be treated with injectable antibiotics like carbapenems, thereby further burdening already struggling health systems in endemic regions. Moreover, with the dearth of novel antimicrobials in the horizon, we risk losing our primary defense against widespread mortality from typhoid. In addition to rolling out the WHO prequalified typhoid conjugate vaccine in endemic areas to decrease the risk of pan-resistant Salmonella Typhi strains, it is also imperative to implement antimicrobial stewardship and water sanitation and hygiene intervention to decrease the overall burden of enteric fever., Competing Interests: The authors have declared that no competing interests exist.

We combine methodology from history and genetics to reconstruct the biosocial history of antimicrobial resistance (AMR) in the bacterium Salmonella enterica serovar Typhi (S. Typhi). We show how evolutionary divergence in S. Typhi was driven by rising global antibiotic use and by the neglect of typhoid outside of high-income countries. Although high-income countries pioneered 1960s precautionary antibiotic regulations to prevent selection for multidrug resistance, new antibiotic classes, typhoid's cultural status as a supposedly ancient disease of "undeveloped" countries, limited international funding, and narrow biosecurity agendas helped fragment effective global collective action for typhoid control. Antibiotic-intensive compensation for weak water and healthcare systems subsequently fueled AMR selection in low- and middle-income countries but often remained invisible due to lacking surveillance capabilities. The recent rise of extensively drug-resistant typhoid bears the biosocial footprint of more than half a century of antibiotic-intensive international neglect., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.)

Rapid and accurate differentiation of Salmonella spp. causing enteric fever from nontyphoidal Salmonella is essential for clinical management of cases, laboratory risk management, and implementation of public health measures. Current methods used for confirmation of identification, including biochemistry and serotyping as well as whole-genome sequencing analyses, take several days. Here we report the development and evaluation of a real-time PCR assay that can be performed directly on crude DNA extracts from bacterial colonies for the rapid identification of typhoidal and nontyphoidal Salmonella ., (© Crown copyright 2019.)

Objectives: Plasmids harbour antibiotic resistance genes which contribute to the emergence of multidrug resistant pathogens. We detected the presence of plasmids in multidrug resistant Salmonella enterica serovar Typhi (S. Typhi) isolates from our previous study and consequently determined their incompatibility groups and possibility of conjugation transmission. Plasmids were extracted from 98 multidrug resistant S. Typhi isolates based on alkaline lysis technique. Plasmid incompatibility grouping was established by PCR replicon typing using 18 pairs of primers to amplify FIA, FIB, FIC, HI1, HI2, I1-Iγ, L/M, N, P, W, T, A/C, K, B/O, X, Y, F and FIIA replicons. Antibiotic resistance phenotypes were conjugally transferred from S. Typhi isolates with plasmids to Escherichia coli K12F strain devoid of plasmids., Results: Approximately 79.6% of the MDR S. Typhi isolates were related to the existence of plasmids. We detected 93.6% of plasmids belonging to incompatibility (Inc) group HI1. The other incompatibility groups identified included IncFIC (16.7%), IncP (1.3%), and IncI1 (1.3%) which appeared together with Inc HI1. MDR S. Typhi isolated carried a homologous plasmid of incompatibility group HI1 most of which transferred the resistance phenotypes of ampicillin, tetracycline and chloramphenicol to the transconjugants.

Background: Typhoid fever remains a major public health problem in Zimbabwe with recurrent outbreaks reported since 2009. To provide guidance on appropriate treatment choice in order to minimise the morbidity and mortality of typhoid fever and prevent large scale outbreaks, we investigated the antimicrobial susceptibility patterns, prevalence of Salmonella enterica serotype Typhi (S. Typhi) H58 haplotype and molecular subtypes of S. Typhi from outbreak strains isolated from 2009 to 2017 in Zimbabwe and compared these to isolates from neighbouring African countries., Methods: Antimicrobial susceptibility testing was performed on all isolates using the disk diffusion, and E-Test, and results were interpreted using Clinical and Laboratory Standards Institute (CLSI) guidelines (2017). S. Typhi H58 haplotype screening was performed on 161 (58.3%) isolates. Pulsed-field gel electrophoresis (PFGE) was performed on 91 selected isolates across timelines using antibiotic susceptibility results and geographical distribution (2009 to 2016)., Results: Between 2009 and 2017, 16,398 suspected cases and 550 confirmed cases of typhoid fever were notified in Zimbabwe. A total of 276 (44.6%) of the culture-confirmed S. Typhi isolates were analysed and 243 isolates (88.0%) were resistant to two or more first line drugs (ciprofloxacin, ampicillin and chloramphenicol) for typhoid. The most common resistance was to ampicillin-chloramphenicol (172 isolates; 62.3%). Increasing ciprofloxacin resistance was observed from 2012 to 2017 (4.2 to 22.0%). Out of 161 screened isolates, 150 (93.2%) were haplotype H58. Twelve PFGE patterns were observed among the 91 isolates analysed, suggesting some diversity exists among strains circulating in Zimbabwe. PFGE analysis of 2013, 2014 and 2016 isolates revealed a common strain with an indistinguishable PFGE pattern (100% similarity) and indistinguishable from PFGE patterns previously identified in strains isolated from South Africa, Zambia and Tanzania., Conclusions: Resistance to first line antimicrobials used for typhoid fever is emerging in Zimbabwe and the multidrug resistant S. Typhi H58 haplotype is widespread. A predominant PFGE clone circulating in Zimbabwe, South Africa, Zambia and Tanzania, argues for cross-border cooperation in the control of this disease.

There is paucity of data regarding the geographical distribution, incidence, and phylogenetics of multi-drug resistant (MDR) Salmonella Typhi in sub-Saharan Africa. Here we present a phylogenetic reconstruction of whole genome sequenced 249 contemporaneous S. Typhi isolated between 2008-2015 in 11 sub-Saharan African countries, in context of the 2,057 global S. Typhi genomic framework. Despite the broad genetic diversity, the majority of organisms (225/249; 90%) belong to only three genotypes, 4.3.1 (H58) (99/249; 40%), 3.1.1 (97/249; 39%), and 2.3.2 (29/249; 12%). Genotypes 4.3.1 and 3.1.1 are confined within East and West Africa, respectively. MDR phenotype is found in over 50% of organisms restricted within these dominant genotypes. High incidences of MDR S. Typhi are calculated in locations with a high burden of typhoid, specifically in children aged <15 years. Antimicrobial stewardship, MDR surveillance, and the introduction of typhoid conjugate vaccines will be critical for the control of MDR typhoid in Africa.

The pathogenesis of Salmonella enterica serovar Typhi (S. Typhi), the cause of typhoid fever in humans, is mainly attributed to the acquisition of horizontally acquired DNA elements. Salmonella pathogenicity islands (SPIs) are indubitably the most important form of horizontally acquired DNA with respect to pathogenesis of this bacterium. The insertion or deletion of any of these transferrable SPIs may have impact on the virulence potential of S. Typhi. In this study, the virulence potential and genetic relatedness of 35 S. Typhi isolates, collected from 2004 to 2013 was determined by identification of SPI and non-SPI virulence factors through a combination of techniques including virulotyping, Whole Genome Sequencing (WGS), and Variable Number of Tandem Repeats (VNTR) profiling. In order to determine the virulence potential of local S. Typhi isolates, 56 virulence related genes were studied by PCR. These genes are located in the core as well as accessory genome (SPIs and plasmid). Major variations among studied virulence determinants were found in case of SPI-7 and SPI-10 associated genes. On the basis of presence of virulence related genes, the studied S. Typhi isolates from Pakistan were clustered into two virulotypes Vi-positive and Vi-negative. Interestingly, SPI-7 and SPI-10 were collectively absent or present in Vi-negative and Vi-positive strains, respectively. Two Vi-negative and 11 Vi-positive S. Typhi strains were also analyzed by whole genome sequencing (WGS) and their results supported the PCR results. Genetic diversity was tested by VNTR-based molecular typing. All 35 isolates were clustered into five groups. Overall, all Vi-negative isolates were placed in a single group (T5) whereas Vi-positive isolates were grouped into four types. Vi-negative and Vi-positive isolates were mutually exclusive. This is the first report on the comparative distribution of SPI and non-SPI related virulence genes in Vi-negative and Vi-positive S. Typhi isolates with an important finding that SPI-10 is absent in all Vi-negative isolates., Competing Interests: The authors have declared that no competing interests exist.

Background: Typhoid fever remains a public health concern in developing countries especially among the poor who live in informal settlements devoid of proper sanitation and clean water supply. In addition antimicrobial resistance poses a major challenge in management of the disease. This study assessed the antimicrobial susceptibility patterns of Salmonella enterica serotype Typhi (S. Typhi) isolated from typhoid fever cases (2004-2007)., Methods: A cross sectional study was conducted on 144 archived S. Typhi isolates (2004-2007) tested against 11 antimicrobial agents by quality controlled disk diffusion technique. Isolates resistant to ampicillin, chloramphenicol, and cotrimoxazole were considered Multidrug resistant (MDR). Thirty MDR isolates were selected randomly and further tested using minimum inhibitory concentration (MIC) E-test., Results: Sixteen percent (23/144) of the isolates were susceptible to all the antibiotics tested while 68% were resistant to three or more of the 11 antibiotics tested. The isolates showed a high susceptibility to ceftriaxone (94%) and gentamicin (97%). A high percentage of resistance was observed for the conventional first-line antibiotics; ampicillin (72%), chloramphenicol (72%), and cotrimoxazole (70%). Sixty-nine percent of the isolates (100/144) showed reduced susceptibility to ciprofloxacin. All the 30 (100%) isolates selected for MIC test were susceptible to amoxicillin-clavulanic acid. All except one of the 30 isolates were susceptible to ceftriaxone while majority 21 (70%) recorded an intermediate susceptibility to ciprofloxacin with MIC of 0.12-0.5 μg/mL., Conclusion: A large proportion of S. Typhi isolates were MDR and also showed reduced susceptibility to ciprofloxacin. Fluoroquinolone resistance is emerging and this may pose a challenge in treatment of typhoid in future. There is need for routine surveillance to monitor this phenotype in clinical settings.

Salmonella is one of major foodborne pathogens and the leading cause of foodborne illness-related hospitalizations and deaths. It is critical to develop a sensitive and rapid detection assay that can identify Salmonella to ensure food safety. In this study, a DNA sensor-based suspension array system of high multiplexing ability was developed to identify eight Salmonella serovars commonly associated with foodborne outbreaks to the serotype level. Each DNA sensor was prepared by activating pre-encoded microspheres with oligonucleotide probes that are targeting virulence genes and serovar-specific regions. The mixture of 12 different types of DNA sensors were loaded into a 96-well microplate and used as a 12-plex DNA sensor array platform. DNA isolated from Salmonella was amplified by multiplex polymerase chain reaction (mPCR), and the presence of Salmonella was determined by reading fluorescent signals from hybridization between probes on DNA sensors and fluorescently labeled target DNA using the Bio-Plex® system. The developed multiplex array was able to detect synthetic DNA at the concentration as low as 100 fM and various Salmonella serovars as low as 100 CFU/mL within 1 h post-PCR. Sensitivity of this assay was further improved to 1 CFU/mL with 6-h enrichment. The array system also correctly and specifically identified serotype of tested Salmonella strains without any cross-reactivity with other common foodborne pathogens. Our results indicate the developed DNA sensor suspension array can be a rapid and reliable high-throughput method for simultaneous detection and molecular identification of common Salmonella serotypes.

The aim of this study was to determine the prevalence and trends in the antimicrobial resistance of typhoidal salmonellae in children and adolescents at a pediatric tertiary care hospital in South India. Typhoidal salmonellae were isolated from 483 of the 77,713 blood cultures received during the ten-year study period (2007-2016). Isolates were speciated by conventional biochemical reactions and serotyping. Antimicrobial susceptibility testing was performed and interpreted according to the British Society for Antimicrobial Chemotherapy (BSAC)/European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The overall blood culture isolation rates of Salmonella enterica serovars Typhi and Paratyphi A in children were 0.5% (408 cases) and 0.1% (73 cases), respectively, with the highest isolation rates in school [299 (61.9%)] and preschool children [113 (23.4%)]. A decreasing prevalence of enteric fever was seen from 2012 to 2015, with a sudden surge in 2016. From 2011 onwards, a high fluoroquinolone resistance (90-100%) was observed. Multidrug resistance was observed in only four (0.9%) S. Typhi isolates. 100% susceptibility to third-generation cephalosporins and azithromycin was noted. Enteric fever as seen in a pediatric tertiary care hospital in India affects children and adolescents of all age groups, with greater isolation rates in school children, followed by those in preschool years, calling for targeted interventions against these age groups. The study findings support the use of third-generation cephalosporins and azithromycin as first-line therapy and ampicillin and co-trimoxazole as step-down therapy in pediatric enteric fever. However, continued local surveillance should be done to detect antimicrobial resistance trends to optimize treatment.

In 2015, a typhoid fever outbreak began in downtown Kampala, Uganda, and spread into adjacent districts. In response, an environmental survey of drinking water source types was conducted in areas of the city with high case numbers. A total of 122 samples was collected from 12 source types and tested for Escherichia coli , free chlorine, and conductivity. An additional 37 grab samples from seven source types and 16 paired large volume (20 liter) samples from wells and springs were also collected and tested for the presence of Salmonella enterica serovar Typhi. Escherichia coli was detected in 60% of kaveras (drinking water sold in plastic bags) and 80% of refilled water bottles; free chlorine was not detected in either source type. Most jerry cans (68%) contained E. coli and had free chlorine residuals below the WHO-recommended level of 0.5 mg/liter during outbreaks. Elevated conductivity readings for kaveras, refilled water bottles, and jerry cans (compared to treated surface water supplied by the water utility) suggested that they likely contained untreated groundwater. All unprotected springs and wells and more than 60% of protected springs contained E. coli Water samples collected from the water utility were found to have acceptable free chlorine levels and no detectable E. coli While S Typhi was not detected in water samples, Salmonella spp. were detected in samples from two unprotected springs, one protected spring, and one refilled water bottle. These data provided clear evidence that unregulated vended water and groundwater represented a risk for typhoid transmission. IMPORTANCE Despite the high incidence of typhoid fever globally, relatively few outbreak investigations incorporate drinking water testing. During waterborne disease outbreaks, measurement of physical-chemical parameters, such as free chlorine residual and electrical conductivity, and of microbiological parameters, such as the presence of E. coli or the implicated etiologic agent, in drinking water samples can identify contaminated sources. This investigation indicated that unregulated vended water and groundwater sources were contaminated and were therefore a risk to consumers during the 2015 typhoid fever outbreak in Kampala. Identification of contaminated drinking water sources and sources that do not contain adequate disinfectant levels can lead to rapid targeted interventions., (Copyright © 2017 American Society for Microbiology.)

Objective: To retrospectively analyze the antimicrobial resistance phenotype and molecular typing characteristics of Salmonella ( S .) typhi and S . paratyphi in Jiangsu province from 2012 to 2015. Methods: The samples were collected from typhoid and paratyphoid patients in Jiangsu province. The biochemical identification and serotyping were carried out after isolation and culture. Kirby-Bauer (K-B) testing was used to detect the drug susceptibility of the strains. The molecular typing characteristics of S . typhi and S . paratyphi were analyzed by pulsed field gel electrophoresis (PFGE). Results: The resistant rates of 134 S. typhi and S. paratyphi A strains to nalidixic acid were highest (61.2% and 86.7%), while the resistant rates to remaining antibiotics were less than 15.0%. Most of S. typhi and S. paratyphi A strains were resistant to only one antibiotic. Multidrug-resistant (MDR) strains of S. typhi and S. paratyphi A accounted for 2.6% and 13.3% respectively. The composition of the all-sensitive strains of S. typhi increased by 44.3% in 2015, at the same time, there were also MDR S. paratyphi A strains, which were resistant to 5 and 6 antibiotics. S. paratyphi A could be divided into eight molecular patterns by PFGE, showing that the similarity between the MDR strains and other strains was relatively low. The S. paratyphi A strains with same pattern were resistant to same antibiotics. S. typhi could be divided into 68 molecular patterns by PFGE, with large variability between different patterns. There was no corresponding relationship between the patterns and the drug resistance characteristics. Conclusions: The overall antibiotic resistance of S. typhi and S. paratyphi A strains showed a decreasing trend, but the number of antibiotics to which they were resistant increased. PFGE patterns of S. typhi showed diversity without correspondence to antibiotic characteristics. PFGE patterns of S. paratyphi A were less with correspondence to antibiotic characteristics. We should pay more attention to key patterns in key areas.

We aimed to describe the molecular epidemiological characteristics and clinical treatment outcome of typhoid fever in Ningbo, China during 2005-2014. Eighty-eight Salmonella Typhi isolates were obtained from 307 hospitalized patients. Three prevalent pulsed-field gel electrophoresis (PFGE) patterns of 54 isolates from 3 outbreaks were identified. Overall, there were 64 (72.7%) isolates from clustered cases and 24 (27.3%) isolates from sporadic cases. Resistance to nalidixic acid (NAL) (n = 47; 53.4%) and ampicillin (AMP) (n = 40; 45.4%) and rare resistance to tetracycline (TET) (n = 2; 2.3%) and gentamicin (GEN) (n = 2; 2.3%) were observed. No isolates resistant to cefotaxime (CTX), chloramphenicol (CL), ciprofloxacin (CIP), and trimethoprim-sulfamethoxazole (SXT) were found. The occurrence of reduced sensitivity to CIP was 52.3% (n = 46). The medians of fever clearance time in cases with and without complications were 7 (interquartile range (IQR): 4-10) and 5 (IQR: 3-7) days (P = 0.001), respectively, when patients were treated with CIP or levofloxacin (LEV) and/or third-generation cephalosporins (CEP). Rates of serious complications were at low levels: peritonitis (2.3%), intestinal hemorrhage (6.8%), and intestinal perforation (1.1%). The present study revealed a long-term clustering trend with respect to PFGE patterns, occasional outbreaks, and the rapid spread of AMP resistance and decreased CIP susceptibility among S. Typhi isolates in recent years.

Molecular subtyping and DNA sequencing-based methods, which are commonly used for discriminating Salmonella enterica serovar Typhi (S. Typhi) isolates, lead to improved molecular epidemiological investigations for prevention and control of typhoid fever. We obtained S. Typhi blood isolates (n = 66) from India during 2007-14 for molecular subtyping by pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) in association with antibiotic resistance profiles. Genotypic diversity was observed more by MLVA (Simpson's index of diversity, D value = 0.997) than PFGE (D value = 0.864). Two prevalent pulsotypes containing nalidixic acid-resistant (NAL R ) and NAL R -ciprofloxacin-resistant (CIP R ) S. Typhi isolates circulated in India. Multidrug-resistant (MDR), NAL R -CIP R , and most NAL R isolates were found to be clonal by PFGE. MLVA could differentiate the clonal isolates. Most of the MDR and NAL R- CIP R isolates showed variation in single or double VNTR loci, whereas NAL R isolates varied in more than 2 loci, reflecting higher genetic diversity among the NAL R isolates. Of the 6 VNTR loci, TR4,699 (D value = 0.838) and Sal02 (D value = 0.890) loci played important roles as MLVA cluster-supporting alleles. The rapid turnaround time and high-level discriminatory power of MLVA may be useful for tracking and controlling the transmission of S. Typhi isolates during epidemiological investigations.

Background: Enteric fever remains a major public health problem in low resource settings and antibiotic resistance is increasing. In Asia, an increasing proportion of infections is caused by Salmonella enterica serovar Paratyphi A, which for a long time was assumed to cause a milder clinical syndrome compared to Salmonella enterica serovar Typhi., Methodology: A retrospective chart review study was conducted of 254 unique cases of blood culture confirmed enteric fever who presented at a referral adult hospital in Phnom Penh, Cambodia between 2008 and 2015. Demographic, clinical and laboratory data were collected from clinical charts and antibiotic susceptibility testing was performed. Whole genome sequence analysis was performed on a subset of 121 isolates., Results: One-hundred-and-ninety unique patients were diagnosed with Salmonella Paratyphi A and 64 with Salmonella Typhi. In the period 2008-2012, Salmonella Paratyphi A comprised 25.5% of 47 enteric fever cases compared to 86.0% of 207 cases during 2013-2015. Presenting symptoms were identical for both serovars but higher median leukocyte counts (6.8 x 109/L vs. 6.3 x 109/L; p = 0.035) and C-reactive protein (CRP) values (47.0 mg/L vs. 36 mg/L; p = 0.034) were observed for Salmonella Typhi infections. All but one of the Salmonella Typhi isolates belonged to haplotype H58 associated with multidrug resistance (MDR) (i.e. resistance to ampicillin, chloramphenicol and co-trimoxazole).;42.9% actually displayed MDR compared to none of the Salmonella Paratyphi A isolates. Decreased ciprofloxacin susceptibility (DCS) was observed in 96.9% (62/64) of Salmonella Typhi isolates versus 11.5% (21/183) of Salmonella Paratyphi A isolates (all but one from 2015). All isolates were susceptible to azithromycin and ceftriaxone., Conclusions: In Phnom Penh, Cambodia, Salmonella Paratyphi A now causes the majority of enteric fever cases and decreased susceptibility against ciprofloxacin is increasing. Overall, Salmonella Typhi was significantly more associated with MDR and DCS compared to Salmonella Paratyphi A.

Purpose: Molecular epidemiological investigations of the highly clonal Salmonella enterica subspecies enterica serovar Typhi (S. Typhi) are important in outbreak detection and in tracking disease transmission. In this study, we developed and evaluated a multiple-locus variable-number tandem-repeats (VNTR) analysis (MLVA) assay for characterization of S. Typhi isolates from sub-Saharan Africa., Methodology: Twelve previously reported VNTR loci were evaluated and an MLVA assay consisting of five polymorphic loci was adopted. The MLVA assay was developed for use on capillary electrophoresis systems by testing a collection of 50 S. Typhi isolates. This S. Typhi strain panel consisted of six outbreak related isolates and 44 epidemiologically unlinked isolates. Amongst these were nine S.Typhi haplotype H58 isolates., Results: The MLVA assay characterized the 50 isolates into 47 MLVA profiles while PFGE analysis of the same isolates revealed 34 pulsotypes. MLVA displayed higher discriminatory power (Simpson's index of diversity (DI) 0.998 [95 % confidence interval (CI) 0.995-1.000)] as compared to pulsed-field gel electrophoresis [Simpson's DI 0.984 (95 % CI 0.974-0.994)]., Conclusion: The MLVA assay presented in this study is a simple, rapid and more accessible tool that serves as a good alternative to other molecular subtyping methods for S. Typhi.

Salmonella enterica serovar Typhi is a human-restricted Gram-negative bacterial pathogen responsible for causing an estimated 27 million cases of typhoid fever annually, leading to 217,000 deaths, and current vaccines do not offer full protection. The O-antigen side chain of the lipopolysaccharide is an immunodominant antigen, can define host-pathogen interactions, and is under consideration as a vaccine target for some Gram-negative species. The composition of the O-antigen can be modified by the activity of glycosyltransferase ( gtr ) operons acquired by horizontal gene transfer. Here we investigate the role of two gtr operons that we identified in the S Typhi genome. Strains were engineered to express specific gtr operons. Full chemical analysis of the O-antigens of these strains identified gtr -dependent glucosylation and acetylation. The glucosylated form of the O-antigen mediated enhanced survival in human serum and decreased complement binding. A single nucleotide deviation from an epigenetic phase variation signature sequence rendered the expression of this glucosylating gtr operon uniform in the population. In contrast, the expression of the acetylating gtrC gene is controlled by epigenetic phase variation. Acetylation did not affect serum survival, but phase variation can be an immune evasion mechanism, and thus, this modification may contribute to persistence in a host. In murine immunization studies, both O-antigen modifications were generally immunodominant. Our results emphasize that natural O-antigen modifications should be taken into consideration when assessing responses to vaccines, especially O-antigen-based vaccines, and that the Salmonella gtr repertoire may confound the protective efficacy of broad-ranging Salmonella lipopolysaccharide conjugate vaccines., (Copyright © 2017 Kintz et al.)

Objective: Establishment and application of Taq Man probe-based quadruple real-time PCR for detection of Salmonella paratyphi A/B/C and Salmonella typhi. Primers specific to Salmonella paratyphi A( SPAP), Salmonella paratyphi B( SPBP), Salmonella paratyphi C( SPCP), and Salmonella typhi( STP) were designed., Methods: A method of Taq Man probe-based quadruple real-time PCR was established according to the distinction of the 5'end of the probe mark of TET, ROX, FAM and HEX. 5 strains of SPA, 4 strains of SPB, 7 strains of SPC and 11 strains of ST were identified by amplification from SPAP, SPBP, SPCP and STP., Results: While other serotypes of salmonella and17 strains of non-salmonella got negative results of amplification. Amplification rate of SPAP, SPBP, SPCP, and STP were 84. 5%, 101. 8%, 92. 4% and 90. 9%, respectively. The linear correlation coefficient( R~2) were 0. 996, 0. 975, 0. 996 and 0. 984, respectively., Conclusion: The PCR system is specific and sensitive for the identification of SPA, SPB, SPC and ST.

Vaccines against Salmonella Typhi, the causative agent of typhoid fever, are commonly used by travellers, however, there are few examples of national immunization programs in endemic areas. There is therefore a paucity of data on the impact of typhoid immunization programs on localised populations of S. Typhi. Here we have used whole genome sequencing (WGS) to characterise 44 historical bacterial isolates collected before and after a national typhoid immunization program that was implemented in Thailand in 1977 in response to a large outbreak; the program was highly effective in reducing typhoid case numbers. Thai isolates were highly diverse, including 10 distinct phylogenetic lineages or genotypes. Novel prophage and plasmids were also detected, including examples that were previously only reported in Shigella sonnei and Escherichia coli. The majority of S. Typhi genotypes observed prior to the immunization program were not observed following it. Post-vaccine era isolates were more closely related to S. Typhi isolated from neighbouring countries than to earlier Thai isolates, providing no evidence for the local persistence of endemic S. Typhi following the national immunization program. Rather, later cases of typhoid appeared to be caused by the occasional importation of common genotypes from neighbouring Vietnam, Laos, and Cambodia. These data show the value of WGS in understanding the impacts of vaccination on pathogen populations and provide support for the proposal that large-scale typhoid immunization programs in endemic areas could result in lasting local disease elimination, although larger prospective studies are needed to test this directly., Competing Interests: The authors have declared that no competing interests exist.

Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), remains an unresolved public health problem in India. Emergence of antimicrobial resistant strains poses a great concern for typhoid treatment and influences reshaping of current S. Typhi population. We included representative S. Typhi strains (n=164) from retrospective studies, both community and hospital based, conducted at National Institute of Cholera and Enteric Diseases, Kolkata during 15 years period (1998-2012) to analyze their antimicrobial resistance (AMR) profiles, mechanism of AMR and molecular subtypes of the strains. More than 60% of the S. Typhi isolates were obtained from community based studies. During the study period, steady decline (46.4%-15.6%) in isolation of multidrug-resistant (MDR, resistant to ampicillin, chloramphenicol and co-trimoxazole) S. Typhi was noticed with parallel increase of nalidixic acid-resistant (NAL R ) strains (60.7%-93.8%) and ciprofloxacin resistant (CIP R ) strains (0%-25%). Of 53 MDR strains, 46 (86.8%) were NAL R showing decreased ciprofloxacin susceptible (DCS) (MIC for ciprofloxacin 0.12-0.5μg/ml) phenotype. Conjugative IncHI1 (230kb) and non-conjugative non-IncHI1 (180kb) plasmids were found in 23 (43.4%) and 14 (26.4%) MDR strains respectively, plasmid was absent in 16 (30.2%) MDR strains. MDR strains with or without plasmid shared the same set of resistance genes (bla TEM-1 , catA1, sul1, sul2, strA and strB) and class 1 integron possessing dfrA7 gene cassette. Two S. Typhi strains harbored 50kb transferrable plasmids carrying dfrA15 and aadA1 gene cassettes in class 1 integron. The majority of the strains (135/164, 82.3%) belonged to H58 haplotype. Among the MDR isolates, fluoroquinolone resistant or combined resistant isolates (n=147), 127 (86.4%) were H58 and 20 (13.6%) belonged to non-H58. NAL R S. Typhi strains with decreased susceptibility or resistance to ciprofloxacin had point mutation(s) in quinolone resistance-determining region of gyrA and parC genes. Pulsed-field gel electrophoresis showed more diversity among NAL R S. Typhi than MDR strains. Results of this study generated information useful for better understanding of the disease epidemiology and its control in endemic settings., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Enteric fever is an invasive infection predominantly caused by Salmonella enterica serovars Typhi and Paratyphi A. The pathogens have evolved from other nontyphoidal salmonellaeto become invasive and host restricted. Emergence of antimicrobial resistance in typhoidal salmonellae in some countries is a major therapeutic concern as the travelers returning from endemic countries carry resistant strains to non endemic areas. In order to understand the epidemiology and to design disease control strategies molecular typing of the pathogen is very important. We performed Multilocus Sequence Typing (MLST) of 251 S. Typhi and 18 S. Paratyphi strains isolated from enteric fever patients from seven centers across India during 2010-2013to determine the population structure and prevalence of MLST sequence types in India. MLST analysis revealed the presence of five sequence types (STs) of typhoidal salmonellae in India namely ST1, ST2 and ST3 for S. Typhi and ST85 and ST129 for S. Paratyphi A.S. Typhi strains showed monophyletic lineage and clustered in to 3 Sequence Types-ST1, ST2 and ST3 and S. Paratyphi A isolates segregated in two sequence types ST85 and ST129 respectively. No association was found between antimicrobial susceptibility and sequence types. This study found ST1 as the most prevalent sequence type of S. Typhi in India followed by ST2, which is in concordance with previous studies and MLST database. In addition a rare sequence type ST3 has been found which is reported for the first time from the Indian subcontinent. Amongst S. Paratyphi A, the most common sequence type is ST129 as also reported from other parts of world. This distribution and prevalence suggest the common spread of the sequence types across the globe and these findings can help in understanding the disease distribution., Competing Interests: The authors have declared that no competing interests exist.

Salmonella Typhi (S. Typhi), the human-adapted agent of typhoid fever, is genetically monomorphic. SNPs accumulation divided the S. Typhi population in 85 haplotypes (H) of which one, H58, has undergone a clonal expansion. The surveillance of H58 S. Typhi is particularly important, especially in areas where typhoid fever is endemic. We developed a simple PCR and PCR-RFLP method to detect and subtype H58 S. Typhi based on the presence of genomic deletion and specific SNPs. The method was validated against 39 S. Typhi isolates of known haplotype, showing 100% of specificity and high sensitivity, and then used to screen a collection of 99 S. Typhi from Asia, demonstrating a high incidence of H58 S. Typhi in Jordan and India. Our method is designed to be applied in all laboratories with basic molecular biology equipment and few financial resources and allows the surveillance of H58 S. Typhi in resource poor settings., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Monophasic variant of Salmonella enterica subspecies enterica serovar Typhimurium (monophasic S. Typhimurium), with antigenic structure 1,4,[5],12:i:-, appears to be of increasing importance in Europe. In Italy, monophasic S. Typhimurium represented the third most frequent Salmonella serovar isolated from human cases between 2004 and 2008. From June 2013 to October 2014, a total of 206 human cases of salmonellosis were identified in Abruzzo region (Central Italy). Obtained clinical isolates characterised showed S. Typhimurium 1,4,[5],12:i:- with sole resistance to nalidixic acid, which had never been observed in Italy in monophasic S. Typhimurium, neither in humans nor in animals or foods. Epidemiological, microbiological and environmental investigations were conducted to try to identify the outbreak source. Cases were interviewed using a standardised questionnaire and microbiological tests were performed on human as well as environmental samples, including samples from fruit and vegetables, pigs, and surface water. Investigation results did not identify the final vehicle of human infection, although a link between the human cases and the contamination of irrigation water channels was suggested.

Bacterial antimicrobial resistance has been associated with the up regulation of genes encoding efflux pumps and the down regulation of genes encoding outer membrane proteins (OMPs). Gene expression in bacteria is primarily initiated by sigma factors (σ factors) such as RpoE, which plays an important role in responding to many environmental stresses. Here, we report the first observation that RpoE serves as an antibiotic resistance regulator in Salmonella enteric serovar Typhi (S. Typhi). In this study, we found that the rpoE mutant (ΔrpoE) of S. Typhi GIFU10007 has elevated resistance to several antimicrobial agents, including β-lactams, quinolones, and aminoglycosides. Genomic DNA microarray analysis was used to investigate the differential gene expression profiles between a wild type and rpoE mutant in response to ampicillin. The results showed that a total of 57 genes displayed differential expression (two-fold increase or decrease) in ΔrpoE versus the wild-type strain. The expressions of two outer membrane protein genes, ompF and ompC, were significantly down-regulated in ΔrpoE (six and seven-fold lower in comparison to wild-type strain) and RamA, a member of the efflux pump AraC/XylS family, was up-regulated about four-fold in the ΔrpoE. Our results suggest RpoE is a potential antimicrobial regulator in S. Typhi, controlling both the down regulation of the OMP genes and up-regulating the efflux system.

The interplay between bacterial antimicrobial susceptibility, phylogenetics and patient outcome is poorly understood. During a typhoid clinical treatment trial in Nepal, we observed several treatment failures and isolated highly fluoroquinolone-resistant Salmonella Typhi (S. Typhi). Seventy-eight S. Typhi isolates were genome sequenced and clinical observations, treatment failures and fever clearance times (FCTs) were stratified by lineage. Most fluoroquinolone-resistant S. Typhi belonged to a specific H58 subclade. Treatment failure with S. Typhi-H58 was significantly less frequent with ceftriaxone (3/31; 9.7%) than gatifloxacin (15/34; 44.1%)(Hazard Ratio 0.19, p=0.002). Further, for gatifloxacin-treated patients, those infected with fluoroquinolone-resistant organisms had significantly higher median FCTs (8.2 days) than those infected with susceptible (2.96) or intermediately resistant organisms (4.01)(pS. Typhi clade internationally, but there are no data regarding disease outcome with this organism. We report an emergent new subclade of S. Typhi-H58 that is associated with fluoroquinolone treatment failure.

Objective: To investigate the antibiotic resistance and pulsed field gel electrophoresis (PFGE) patterns of clinical isolates of Salmonella (S.) typhi and S. paratyphi in Henan province during 2009-2011., Methods: According to molecular typing and Salmonella K-B drug susceptibility test method published by international PulseNet bacterial infectious disease monitoring network and USA Clinical and Laboratory Standards Institute (CLSI), the drug susceptibility and PFGE molecule characteristics of 78 S. typhi and S. paratyphi strains isolated from sentinel hospitals in Henan were analyzed., Results: The 78 strains of S. typhi and S. paratyphi were resistant to 13 kinds of antibiotics, in which 62 were multidrug resistant (79.5%), 4 were resistant to 2-3 kinds of antibiotics (5.1%), 41 were resistant to 5-8 kinds of antibiotics (52.6%), 14 were resistant to 9-10 kinds of antibiotics (17.9%), 3 were resistant to 11-12 kinds of antibiotics (3.8%). The resistant rate to cephalosporins, quinolones and other 3 kinds of antibiotic showed an increase trends. Seventy two strains of S. typhi and S. paratyphi could be divided 14 molecular patterns by digestion with XbaⅠ and PFGE, each pattern contains 1-47 strains which shared the similarity of 66.03%-100.00%., Conclusions: The drug resistance of clinical isolates of S. typhi and S. paratyphi was serious in Henan. The PFGE patterns showed diversity, but the predominant patterns could be still found. The PFGE patterns of some strains were associated with their drug resistance.

Salmonella enterica serotype Typhi (S. Typhi) is the causative agent of typhoid fever and remains a major health threat in most of the developing countries. The prompt diagnosis of typhoid directly from the patient's blood requires high level of sensitivity and specificity. Some of us were the first to report PCR based diagnosis of typhoid. This approach has since then been reported by many scientists using different genomic targets. Since the number of bacteria circulating in the blood of a patient can be as low as 0.3 cfu ml(-1), there is always a room for improvement in diagnostic PCR. In the present study, the role of different types of nanoparticles was investigated to improve the existing PCR based methods for diagnosis and strain typing of S. Typhi (targeting Variable Number of Tandem Repeats [VNTR]) by using optimized PCR systems. Three different types of nanoparticles were used i.e., citrate stabilized gold nanoparticles, rhamnolipid stabilized gold and silver nanoparticles, and magnetic iron oxide nanoparticles. The non-specific amplification was significantly reduced in VNTR typing when gold and silver nanoparticles were used in an appropriate concentration. More importantly, the addition of nanoparticles decreased the non-specificity to a significant level in the case of multiplex PCR thus further validating the reliability of PCR for the diagnosis of typhoid.

Acute hepatic necrosis was diagnosed in a dog. Gram staining and fluorescence in situ hybridization identified Salmonella enterica in the liver, subsequently confirmed as S. enterica serotype I 4,5,12:-:1,2. This is the first report of acute hepatic necrosis with liver failure caused by Salmonella in a dog., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

In this work, IS200 and multi-locus sequence typing (MLST) were used to analyze 19 strains previously serotyped as Salmonella enterica serovar Typhi and isolated in Indonesia (16 strains), Mexico (2 strains), and Switzerland (1 strain). Most of the strains showed the most common Typhi sequence types, ST1 and ST2, and a new Typhi genotype (ST1856) was described. However, one isolate from Mexico and another from Indonesia were of the ST365 and ST426 sequence types, indicating that they belonged to serovars Weltevreden and Aberdeen, respectively. These results were supported by the amplification of IS200 fragments, which rapidly distinguish Typhi from other serovars. Our results demonstrate the utility of IS200 and MLST in the classification of Salmonella strains into serovars. These methods provide information on the clonal relatedness of strains isolated worldwide., (Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.)

Retrospectively, we investigated the epidemiology of a massive Salmonella enterica serovar Typhi outbreak in Zambia during 2010 to 2012. Ninety-four isolates were susceptibility tested by MIC determinations. Whole-genome sequence typing (WGST) of 33 isolates and bioinformatic analysis identified the multilocus sequence type (MLST), haplotype, plasmid replicon, antimicrobial resistance genes, and genetic relatedness by single nucleotide polymorphism (SNP) analysis and genomic deletions. The outbreak affected 2,040 patients, with a fatality rate of 0.5%. Most (83.0%) isolates were multidrug resistant (MDR). The isolates belonged to MLST ST1 and a new variant of the haplotype, H58B. Most isolates contained a chromosomally translocated region containing seven antimicrobial resistance genes, catA1, blaTEM-1, dfrA7, sul1, sul2, strA, and strB, and fragments of the incompatibility group Q1 (IncQ1) plasmid replicon, the class 1 integron, and the mer operon. The genomic analysis revealed 415 SNP differences overall and 35 deletions among 33 of the isolates subjected to whole-genome sequencing. In comparison with other genomes of H58, the Zambian isolates separated from genomes from Central Africa and India by 34 and 52 SNPs, respectively. The phylogenetic analysis indicates that 32 of the 33 isolates sequenced belonged to a tight clonal group distinct from other H58 genomes included in the study. The small numbers of SNPs identified within this group are consistent with the short-term transmission that can be expected over a period of 2 years. The phylogenetic analysis and deletions suggest that a single MDR clone was responsible for the outbreak, during which occasional other S. Typhi lineages, including sensitive ones, continued to cocirculate. The common view is that the emerging global S. Typhi haplotype, H58B, containing the MDR IncHI1 plasmid is responsible for the majority of typhoid infections in Asia and sub-Saharan Africa; we found that a new variant of the haplotype harboring a chromosomally translocated region containing the MDR islands of IncHI1 plasmid has emerged in Zambia. This could change the perception of the term "classical MDR typhoid" currently being solely associated with the IncHI1 plasmid. It might be more common than presently thought that S. Typhi haplotype H58B harbors the IncHI1 plasmid or a chromosomally translocated MDR region or both., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The objective of the study was to conduct bacteriological analysis of water with special reference to Salmonella spp from natural sources of rural habitations of East Sikkim. A total of 28 Salmonella serovars isolated were biotyped, phage typed and tested for their anti-microbial susceptibility. All the isolates of Salmonella enterica serovar Typhi belonged to Biotype I. Four isolates of S. typhi belonged to phage type A. All S. paratyphi A isolates belong to phage 2. All the isolates were sensitive to chloramphenicol, cefixime and amikacin. Untreated natural water sources are unsafe for human consumption.

Background: Typhoid fever is an infectious disease of global importance that is caused by Salmonella enterica subsp. enterica serovar Typhi (S. Typhi). This disease causes an estimated 200,000 deaths per year and remains a serious global health threat. S. Typhi is strictly a human pathogen, and some recovered individuals become long-term carriers who continue to shed the bacteria in their faeces, thus becoming main reservoirs of infection., Results: A comparative genomics analysis combined with a phylogenomic analysis revealed that the strains from the outbreak and carrier were closely related with microvariations and possibly derived from a common ancestor. Additionally, the comparative genomics analysis with all of the other completely sequenced S. Typhi genomes revealed that strains BL196 and CR0044 exhibit unusual genomic variations despite S. Typhi being generally regarded as highly clonal. The two genomes shared distinct chromosomal architectures and uncommon genome features; notably, the presence of a ~10 kb novel genomic island containing uncharacterised virulence-related genes, and zot in particular. Variations were also detected in the T6SS system and genes that were related to SPI-10, insertion sequences, CRISPRs and nsSNPs among the studied genomes. Interestingly, the carrier strain CR0044 harboured far more genetic polymorphisms (83% mutant nsSNPs) compared with the closely related BL196 outbreak strain. Notably, the two highly related virulence-determinant genes, rpoS and tviE, were mutated in strains BL196 and CR0044, respectively, which revealed that the mutation in rpoS is stabilising, while that in tviE is destabilising. These microvariations provide novel insight into the optimisation of genes by the pathogens. However, the sporadic strain was found to be far more conserved compared with the others., Conclusions: The uncommon genomic variations in the two closely related BL196 and CR0044 strains suggests that S. Typhi is more diverse than previously thought. Our study has demonstrated that the pathogen is continually acquiring new genes through horizontal gene transfer in the process of host adaptation, providing novel insight into its unusual genomic dynamics. The understanding of these strains and virulence factors, and particularly the strain that is associated with the large outbreak and the less studied asymptomatic Typhi carrier in the population, will have important impact on disease control.

We report a case of typhoid fever in a traveler returning to Spain from Guatemala that was caused by Salmonella enterica serovar Typhi which produced an extended-spectrum β-lactamase (ESBL). This finding demonstrates the presence of ESBL-producing S. enterica ser. Typhi strains in the Americas. Enhanced surveillance is necessary to prevent further spread.

Background: The study aimed to compare the socio-demographic, host and clinical characteristics, seasonality and antimicrobial susceptibility of Typhoidal Salmonella (Salmonella enterica serovar Typhi and Paratyphi) (TS) with diarrhea between urban and rural Bangladesh., Methods: Relevant information of 77/25,767 (0.30%) and 290/17,622 (1.65%) patients positive with TS (in stool) were extracted from the data archive of Diarrheal Disease Surveillance System of icddr,b (urban Dhaka and rural Matlab Hospitals respectively) during 2000-2012. Comparison group (diarrhea patients negative for TS) was randomly selected from the database (1:3 ratio). Two poisson regression models were investigated for modelling seasonal effects on the number of cases., Results: Salmonella Typhi was more frequently isolated in Dhaka than Matlab (57% vs. 5%, p < 0.001); while Salmonella Paratyphi was more frequent in Matlab than Dhaka (96% vs. 43%; p < 0.001). Fever [adj. OR-5.86 (95% CI: 2.16, 15.94)], antimicrobial use at home [5.08 (2.60, 9.90)], and fecal red blood cells [2.53 (1.38, 4.64)] were significantly associated with detection of TS in stool of patient from Dhaka. For Matlab, the correlates were, vomiting [1.88 (1.35, 2.64)], fecal macrophage [1.89 (1.29, 2.74)] in addition to fever and duration of diarrhea and antimicrobial use. At Dhaka, all Salmonella Typhi isolates were susceptible to ceftriaxone; while in Dhaka and Matlab however, for ciprofloxacin it was 45% and 91%, respectively. Susceptibility to chloramphenicol, ampicillin, trimethoprim-sulphamethoxazole and nalidixic acid ranged from 12%-58%. Salmonella Paratyphi were susceptible to ceftriaxone (99%). A significant seasonal trend and year difference (before and after 2007) for Matlab was observed (p < 0.001 for all effects). Dhaka does not show significant year or seasonal effects (p = 0.07 for years and p = 0.81 and p = 0.18 for the cos and sin components, respectively). While not significant, two seasonal peaks were observed in Dhaka (January-February and September-November); while a single peak (August-November) was observed in Matlab., Conclusions: Proportion of serovar distribution of TS and their clinical characteristics, antimicrobial susceptibility and seasonal pattern were different among diarrhea patients in urban Dhaka and rural Matlab of Bangladesh.

Background: The genus Salmonella contains more than 2600 serovars. While most cause a self-limiting gastroenteritis, four serovars, S. Typhi, S. Paratyphi A, B and C, elicit typhoid, a potentially fatal systemic infection. Because of the prevalence in certain regions, such as South Asia, and the disease severity of typhoidal Salmonella infections, comprehensive studies are needed to elucidate the pathogenesis of diseases caused by these typhoidal serovars., Results: We performed comparative genomic analyses on eight human typhoidal strains and 27 non-human typhoidal Salmonella strains to elucidate their evolutionary relationships and identify the genes specific to the four typhoidal serovars. Our results indicate that Salmonella may have an open pan-genome. A core-genome based phylogeny demonstrated that divergence between S. Paratyphi A and S. Typhi took place not long ago and S. Paratyphi B shared a recent common ancestor with S. Paratyphi C. Of great interest, the divergence between S. Paratyphi B and S. Paratyphi C was shown to be more recent than that between S. Paratyphi A and S. Typhi. Alignment and comparisons of the genomes identified unique complements of protein families to each of the typhoidal serovars. Most of these protein families are phage related and some are candidate virulence factors. Importantly, we found 88 protein families specific to two to three of the four typhoidal serovars. All but two of the 88 genes are present in S. Typhi, with a few in the three paratyphoidal serovars but none in the non-human typhoidal serovars. Most of these genes are predicted to encode hypothetical proteins and some are known to code for virulence factors such as Vi polysaccharide related proteins., Conclusions: By comprehensive genomic comparisons, we identified protein families specific to the human typhoidal serovars, which will greatly facilitate investigations on typhoid pathogenesis., (Copyright © 2014 Elsevier B.V. All rights reserved.)