Genomic Surveillance of Salmonella Paratyphi A: Neglected No More?

Salmonella enterica serovar Paratyphi A, the causative agent of paratyphoid fever, is a neglected tropical disease with a high burden and mortality in low- and middle-income countries. Limited information is available regarding its genomic diversity, especially from South Asian countries that are collectively responsible for >80% of all paratyphoid cases. At the 2021 International Conference on Typhoid and Other Salmonelloses, researchers from the around the globe presented their work on Salmonella Paratyphi A genomics. Presentations described recent genomic data from South Asia and the development of Paratype, an open-access single-nucleotide polymorphism-based genotyping scheme, to segregate Salmonella Paratyphi A genomes in a systematic and sustainable manner. In this review, we attempt to summarize the progress made thus far on Salmonella Paratyphi A genomics and discuss the questions that remain to better understand the pathogen and develop interventions to fight it.

Salmonella enterica Serovar Typhi on an Island: No H58, No Multidrug Resistance, but for How Long?

Little genomic data is available for typhoid fever from island nations, though the disease has a moderately high burden there. Sikorski et al. (M. J. Sikorski, T. H. Hazen, S. N. Desai, S. Nimarota-Brown, et al., mBio 13:e01920-22, 2022, https://doi.org/10.1128/mbio.01920-22) studied 306 Salmonella enterica serovar Typhi genomes from the Samoan Islands collected during 1983 to 2020 and reported dominance of a rare genotype, 2.5.4, and no H58 (genotype 4.3.1). They found pansusceptibility of all isolates to three first lines of antimicrobial agents (ampicillin, chloramphenicol, and cotrimoxazole). This commentary evaluates the importance of these findings for the Samoan Islands and how they can help the global typhoid community. The microbial community in the environment and human gut could have played a role in the lack of antimicrobial resistance (AMR). However, drug-resistant strains may arrive soon at the island, as their international spread is common. Further investigation would help the global typhoid community to better understand the evolution of an isolated pathogen community and the effect of vaccination there.

The international and intercontinental spread and expansion of antimicrobial-resistant Salmonella Typhi: a genomic epidemiology study.

BACKGROUND: The emergence of increasingly antimicrobial-resistant Salmonella enterica serovar Typhi (S Typhi) threatens to undermine effective treatment and control. Understanding where antimicrobial resistance in S Typhi is emerging and spreading is crucial towards formulating effective control strategies. METHODS: In this genomic epidemiology study, we sequenced the genomes of 3489 S Typhi strains isolated from prospective enteric fever surveillance studies in Nepal, Bangladesh, Pakistan, and India (between 2014 and 2019), and combined these with a global collection of 4169 S Typhi genome sequences isolated between 1905 and 2018 to investigate the temporal and geographical patterns of emergence and spread of antimicrobial-resistant S Typhi. We performed non-parametric phylodynamic analyses to characterise changes in the effective population size of fluoroquinolone-resistant, extensively drug-resistant (XDR), and azithromycin-resistant S Typhi over time. We inferred timed phylogenies for the major S Typhi sublineages and used ancestral state reconstruction methods to estimate the frequency and timing of international and intercontinental transfers. FINDINGS: Our analysis revealed a declining trend of multidrug resistant typhoid in south Asia, except for Pakistan, where XDR S Typhi emerged in 2016 and rapidly replaced less-resistant strains. Mutations in the quinolone-resistance determining region (QRDR) of S Typhi have independently arisen and propagated on at least 94 occasions, nearly all occurring in south Asia. Strains with multiple QRDR mutations, including triple mutants with high-level fluoroquinolone resistance, have been increasing in frequency and displacing strains with fewer mutations. Strains containing acrB mutations, conferring azithromycin resistance, emerged in Bangladesh around 2013 and effective population size of these strains has been steadily increasing. We found evidence of frequent international (n=138) and intercontinental transfers (n=59) of antimicrobial-resistant S Typhi, followed by local expansion and replacement of drug-susceptible clades. INTERPRETATION: Independent acquisition of plasmids and homoplastic mutations conferring antimicrobial resistance have occurred repeatedly in multiple lineages of S Typhi, predominantly arising in south Asia before spreading to other regions. FUNDING: Bill & Melinda Gates Foundation.

CRISPRs in the human genome are differentially expressed between malignant and normal adjacent to tumor tissue.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) have been identified in bacteria, archaea and mitochondria of plants, but not in eukaryotes. Here, we report the discovery of 12,572 putative CRISPRs randomly distributed across the human chromosomes, which we termed hCRISPRs. By using available transcriptome datasets, we demonstrate that hCRISPRs are distinctively expressed as small non-coding RNAs (sncRNAs) in cell lines and human tissues. Moreover, expression patterns thereof enabled us to distinguish normal from malignant tissues. In prostate cancer, we confirmed the differential hCRISPR expression between normal adjacent and malignant primary prostate tissue by RT-qPCR and demonstrate that the SHERLOCK and DETECTR dipstick tools are suitable to detect these sncRNAs. We anticipate that the discovery of CRISPRs in the human genome can be further exploited for diagnostic purposes in cancer and other medical conditions, which certainly will lead to the development of point-of-care tests based on the differential expression of the hCRISPRs.

Reply to Fabre et al. Comment on "Tanmoy et al. CRISPR-Cas Diversity in Clinical Salmonella enterica Serovar Typhi Isolates from South Asian Countries. Genes 2020, 11, 1365".

We respectfully thank Fabre et al. [...].

Hospitalization of Pediatric Enteric Fever Cases, Dhaka, Bangladesh, 2017-2019: Incidence and Risk Factors.

BACKGROUND: Enteric fever causes substantial morbidity and mortality in low- and middle-income countries. Here, we analyzed Surveillance for Enteric Fever in Asia Project (SEAP) data to estimate the burden of enteric fever hospitalization among children aged <15 years and identify risk factors for hospitalization in Bangladesh. METHODS: SEAP used hospital surveillance paired with a community-based health-care utilization assessment. In SEAP hospital surveillance, blood was obtained for culture from children aged <15 years with ≥3 days of fever. In the hospital catchment area, a health-care utilization survey (HCUS) was conducted to estimate the proportion of febrile children hospitalized at the study hospitals. We analyzed hospital surveillance and HCUS data to estimate the health care-adjusted incidence of enteric fever hospitalization, and conducted univariable and multivariable logistic regressions. RESULTS: From July 2017 through June 2019, 2243 laboratory-confirmed enteric fever cases were detected in 2 study hospitals; 673 (30%) were hospitalized. The health care-adjusted incidence of enteric fever hospitalization among children <15 years old was 303/100 000 children/year (95% confidence interval [CI], 293-313). Salmonella Typhi contributed most to the enteric fever hospitalization incidence (277/100 000 children/year; 95% CI, 267-287). The incidence was highest among children aged 2 to <5 years (552/100 000 children/year; 95% CI, 522-583), followed by those aged <2 years (316/100 000 children/year; 95% CI, 288-344). Factors independently associated with enteric fever hospitalization included fever duration, diarrhea, vomiting, abdominal pain, and leukocytopenia. CONCLUSIONS: We estimated a high burden of hospitalization due to enteric fever among children aged <5 years in Bangladesh. The introduction of a typhoid conjugate vaccine would protect children from typhoid and avert typhoid hospitalizations.

CRISPR-Cas Diversity in Clinical Salmonella enterica Serovar Typhi Isolates from South Asian Countries.

Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), is a global health concern and its treatment is problematic due to the rise in antimicrobial resistance (AMR). Rapid detection of patients infected with AMR positive S. Typhi is, therefore, crucial to prevent further spreading. Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated genes (CRISPR-Cas), is an adaptive immune system that initially was used for typing purposes. Later, it was discovered to play a role in defense against phages and plasmids, including ones that carry AMR genes, and, at present, it is being explored for its usage in diagnostics. Despite the availability of whole-genome sequences (WGS), very few studied the CRISPR-Cas system of S. Typhi, let alone in typing purposes or relation to AMR. In the present study, we analyzed the CRISPR-Cas system of S. Typhi using WGS data of 1059 isolates obtained from Bangladesh, India, Nepal, and Pakistan in combination with demographic data and AMR status. Our results reveal that the S. Typhi CRISPR loci can be classified into two groups: A (evidence level >2) and B (evidence level ≤2), in which we identified a total of 47 unique spacers and 15 unique direct repeats. Further analysis of the identified spacers and repeats demonstrated specific patterns that harbored significant associations with genotype, demographic characteristics, and AMR status, thus raising the possibility of their usage as biomarkers. Potential spacer targets were identified and, interestingly, the phage-targeting spacers belonged to the group-A and plasmid-targeting spacers to the group-B CRISPR loci. Further analyses of the spacer targets led to the identification of an S. Typhi protospacer adjacent motif (PAM) sequence, TTTCA/T. New cas-genes known as DinG, DEDDh, and WYL were also discovered in the S. Typhi genome. However, a specific variant of the WYL gene was only identified in the extensively drug-resistant (XDR) lineage from Pakistan and ciprofloxacin-resistant lineage from Bangladesh. From this work, we conclude that there are strong correlations between variations identified in the S. Typhi CRISPR-Cas system and endemic AMR positive S. Typhi isolates.

Clinical Evaluation of a Multiplex PCR for the Detection of Salmonella enterica Serovars Typhi and Paratyphi A from Blood Specimens in a High-Endemic Setting.

Enteric fever is a major public health concern in endemic areas, particularly in infrastructure-limited countries where Salmonella Paratyphi A has emerged in increasing proportion of cases. We aimed to evaluate a method to detect Salmonella Typhi (S. Typhi) and Salmonella Paratyphi A (S. Paratyphi A) in febrile patients in Bangladesh. We conducted a prospective study enrolling patients with fever > 38°C admitted to two large urban hospitals and two outpatient clinics located in Dhaka, Bangladesh. We developed and evaluated a method combining short culture with a new molecular assay to simultaneously detect and differentiate S. Typhi and S. Paratyphi A from other Salmonella directly from 2 to 4 mL of whole blood in febrile patients (n = 680). A total of 680 cases were enrolled from the four participating sites. An increase in the detection rate (+38.8%) in S. Typhi and S. Paratyphi A was observed with a multiplex polymerase chain reaction (PCR) assay, and absence of non-typhoidal Salmonella detection was reported. All 45 healthy controls were culture and PCR negative, generating an estimated 92.9% of specificity on clinical samples. When clinical performance was assessed in the absence of blood volume prioritization for testing, a latent class model estimates clinical performance ≥ 95% in sensitivity and specificity with likelihood ratio (LR) LR+ > 10 and LR- < 0.1 for the multiplex PCR assay. The alternative method to blood culture we developed may be useful alone or in combination with culture or serological tests for epidemiological studies in high disease burden settings and should be considered as secondary endpoint test for future vaccine trials.

Ceftriaxone-resistant Salmonella Typhi carries an IncI1-ST31 plasmid encoding CTX-M-15.

PURPOSE: Ceftriaxone is the drug of choice for typhoid fever and the emergence of resistant Salmonella Typhi raises major concerns for treatment. There are an increasing number of sporadic reports of ceftriaxone-resistant S. Typhi and limiting the risk of treatment failure in the patient and outbreaks in the community must be prioritized. This study describes the use of whole genome sequencing to guide outbreak identification and case management. METHODOLOGY: An isolate of ceftriaxone-resistant S. Typhi from the blood of a child taken in 2000 at the Popular Diagnostic Center, Dhaka, Bangladesh was subjected to whole genome sequencing, using an Illumina NextSeq 500 and analysis using Geneious software.Results/Key findings. Comparison with other ceftriaxone-resistant S. Typhi revealed an isolate from the Democratic Republic of the Congo in 2015 as the closest relative but no evidence of an outbreak. A plasmid belonging to incompatibility group I1 (IncI1-ST31) which included blaCTX-M-15 (ceftriaxone resistance) associated with ISEcp-1 was identified. High similarity (90 %) was seen with pS115, an IncI1 plasmid from S. Enteritidis, and with pESBL-EA11, an incI1 plasmid from E. coli (99 %) showing that S. Typhi has access to ceftriaxone resistance through the acquisition of common plasmids. CONCLUSIONS: The transmission of ceftriaxone resistance from E. coli to S. Typhi is of concern because of clinical resistance to ceftriaxone, the main stay of typhoid treatment. Whole genome sequencing, albeit several years after the isolation, demonstrated the success of containment but clinical trials with alternative agents are urgently required.

Rotavirus Surveillance at a WHO-Coordinated Invasive Bacterial Disease Surveillance Site in Bangladesh: A Feasibility Study to Integrate Two Surveillance Systems.

The World Health Organization (WHO) currently coordinates rotavirus diarrhea and invasive bacterial disease (IBD) surveillance at 178 sentinel sites in 60 countries. However, only 78 sites participate in both surveillance systems using a common sentinel site. Here, we explored the feasibility of extending a WHO-IBD surveillance platform to generate data on the burden of rotaviral diarrhea and its epidemiological characteristics to prepare the countries to measure the impact of rotaviral vaccine. A six-month (July to December, 2012) surveillance, managed by IBD team, collected stool samples and clinical data from under-five children with acute watery diarrhea at an IBD sentinel site. Samples were tested for rotavirus antigen by ELISA and genotyped by PCR at the regional reference laboratory (RRL). Specimens were collected from 79% (n=297) of eligible cases (n=375); 100% of which were tested for rotavirus by ELISA and 54% (159/297) of them were positive. At RRL, all the cases were confirmed by PCR and genotyped (99%; 158/159). The typing results revealed the predominance of G12 (40%; 64/159) genotype, followed by G1 (31%; 50/159) and G9 (19%; 31/159). All in all, this exploratory surveillance collected the desired demographic and epidemiological data and achieved almost all the benchmark indicators of WHO, starting from enrollment number to quality assurance through a number of case detection, collection, and testing of specimens and genotyping of strains at RRL. The success of this WHO-IBD site in achieving these benchmark indicators of WHO can be used by WHO as a proof-of-concept for considering integration of rotavirus surveillance with WHO-IBD platforms, specifically in countries with well performing IBD site and no ongoing rotavirus surveillance.

Application of a subtractive genomics approach for in silico identification and characterization of novel drug targets in Mycobacterium tuberculosis F11.

Extensive dead ends or host toxicity of the conventional approaches of drug development can be avoided by applying the in silico subtractive genomics approach in the designing of potential drug target against bacterial diseases. This study utilizes the advanced in silico genome subtraction methodology to design potential and pathogen specific drug targets against Mycobacterium tuberculosis, causal agent of deadly tuberculosis. The whole proteome of Mycobacterium tuberculosis F11 containing 3941 proteins have been analyzed through a series of subtraction methodologies to remove paralogous proteins and proteins that show extensive homology with human. The subsequent exclusion of these proteins ensured the absence of host cytotoxicity and cross reaction in the identified drug targets. The high stringency (expectation value 10(-100)) analysis of the remaining 2935 proteins against database of essential genes resulted in 274 proteins to be essential for Mycobacterium tuberculosis F11. Comparative analysis of the metabolic pathways of human and Mycobacterium tuberculosis F11 by KAAS at the KEGG server sorted out 20 unique metabolic pathways in Mycobacterium tuberculosis F11 that involve the participation of 30 essential proteins. Subcellular localization analysis of these 30 essential proteins revealed 7 proteins with outer membrane potentialities. All these proteins can be used as a potential therapeutic target against Mycobacterium tuberculosis F11 infection. 66 of the 274 essential proteins were uncharacterized (described as hypothetical) and functional classification of these proteins showed that they belonged to a wide variety of protein classes including zinc binding proteins, transferases, transmembrane proteins, other metal ion binding proteins, oxidoreductase, and primary active transporters etc. 2D and 3D structures of these 15 membrane associated proteins were predicted using PRED-TMBB and homology modeling by Swiss model workspace respectively. The identified drug targets are expected to be of great potential for designing novel anti-tuberculosis drugs and further screening of the compounds against these newly targets may result in discovery of novel therapeutic compounds that can be effective against Mycobacterium tuberculosis.