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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. 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. 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.

In the early 1900s, with mortality of ∼30%, typhoid and paratyphoid (caused by Salmonella Typhi and Paratyphi A) 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. 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.

Surface lipoproteins (SLPs) are peripherally attached to the outer leaflet of the outer membrane in many Gram-negative bacteria, playing significant roles in nutrient acquisition and immune evasion in the host. While the factors that are involved in the synthesis and delivery of SLPs in the inner membrane are well characterized, the molecular machinery required for the movement of SLPs to the surface are still not fully elucidated. In this study, we investigated the translocation of a SLP TbpB through a Slam1-dependent pathway. Using purified components, we developed an in vitro translocation assay where unfolded TbpB is transported through Slam1-containing proteoliposomes, confirming Slam1 as an outer membrane translocon. While looking to identify factors to increase translocation efficiency, we discovered the periplasmic chaperone Skp interacted with TbpB in the periplasm of Escherichia coli . The presence of Skp was found to increase the translocation efficiency of TbpB in the reconstituted translocation assays. A knockout of Skp in Neisseria meningitidis revealed that Skp is essential for functional translocation of TbpB to the bacterial surface. Taken together, we propose a pathway for surface destined lipoproteins, where Skp acts as a holdase for Slam-mediated TbpB translocation across the outer membrane.

Salmonella Paratyphi A, the primary etiology of paratyphoid, is estimated to cause 3.4 million infections annually, worldwide. With rising antimicrobial resistance and no licensed vaccines, genomic surveillance is key to track and monitor transmission, but there is currently no reliable genotyping framework for this pathogen. Here, we sequence 817 isolates from South Asia and add 562 publicly available genomes to build a global database representing 37 countries, covering 1917–2019. We develop a single nucleotide polymorphism-based genotyping scheme, Paratype , that segregates Salmonella Paratyphi A population into three primary and nine secondary clades, and 18 genotypes. Each genotype is assigned a unique allele definition located on an essential gene. Using Paratype , we identify spatiotemporal genomic variation and antimicrobial resistance markers. We release Paratype as an open-access tool that can use raw read files from both Illumina and Nanopore platforms, and thus can assist surveillance studies tracking Salmonella Paratyphi A across the globe. The bacterium Salmonella Paratyphi A causes paratyphoid fever. Here, the authors sequence over 800 isolates from South Asia, build a global database representing 37 countries, and develop a genotyping tool that identifies genomic variation and antimicrobial resistance markers for surveillance studies.

We report an outbreak of ceftriaxone-resistant Salmonella enterica serovar Typhi in Bangladesh; 47 cases were identified during April-September 2024. Isolates belonged to genotype 4.3.1.2 and harbored the bla gene on the pCROB1 plasmid. This genotype-plasmid lineage represents a recent introduction, calling for strengthened surveillance, antimicrobial stewardship, and vaccination strategies.

Typhoid-conjugate vaccines (TCVs) provide an opportunity to reduce the burden of typhoid fever, caused by Salmonella Typhi, in endemic areas. As policymakers design vaccination strategies, accurate and high-resolution data on disease burden is crucial. However, traditional blood culture-based surveillance is resource-extensive, prohibiting its large-scale and sustainable implementation. Salmonella Typhi is a water-borne pathogen, and here, we tested the potential of Typhi-specific bacteriophage surveillance in surface water bodies as a low-cost tool to identify where Salmonella Typhi circulates in the environment. In 2021, water samples were collected and tested for the presence of Salmonella Typhi bacteriophages at two sites in Bangladesh: urban capital city, Dhaka, and a rural district, Mirzapur. Salmonella Typhi-specific bacteriophages were detected in 66 of 211 (31%) environmental samples in Dhaka, in comparison to 3 of 92 (3%) environmental samples from Mirzapur. In the same year, 4,620 blood cultures at the two largest pediatric hospitals of Dhaka yielded 215 (5%) culture-confirmed typhoid cases, and 3,788 blood cultures in the largest hospital of Mirzapur yielded 2 (0.05%) cases. 75% (52/69) of positive phage samples were collected from sewage. All isolated phages were tested against a panel of isolates from different Salmonella Typhi genotypes circulating in Bangladesh and were found to exhibit a diverse killing spectrum, indicating that diverse bacteriophages were isolated. These results suggest an association between the presence of Typhi-specific phages in the environment and the burden of typhoid fever, and the potential of utilizing environmental phage surveillance as a low-cost tool to assist policy decisions on typhoid control.

Many proteins involved in signal transduction contain peptide recognition modules (PRMs) that recognize short linear motifs (SLiMs) within their interaction partners. Here, we used large‐scale peptide‐phage display methods to derive optimal ligands for 163 unique PRMs representing 79 distinct structural families. We combined the new data with previous data that we collected for the large SH3, PDZ, and WW domain families to assemble a database containing 7,984 unique peptide ligands for 500 PRMs representing 82 structural families. For 74 PRMs, we acquired enough new data to map the specificity profiles in detail and derived position weight matrices and binding specificity logos based on multiple peptide ligands. These analyses showed that optimal peptide ligands resembled peptides observed in existing structures of PRM‐ligand complexes, indicating that a large majority of the phage‐derived peptides are likely to target natural peptide‐binding sites and could thus act as inhibitors of natural protein–protein interactions. The complete dataset has been assembled in an online database ( http://www.prm‐db.org ) that will enable many structural, functional, and biological studies of PRMs and SLiMs. SYNOPSIS A database of thousands of peptides targeting hundreds of diverse peptide recognition modules is presented ( www.prm‐db.org ). Applications of this large dataset include exploring natural protein‐protein interactions and inhibitor design. The database contains 7,984 unique peptides, each binding to one of 500 peptide recognition modules (PRMs) representing 82 distinct structural families. Phage‐derived peptides reflect the hydrophobic character of natural peptides at specific positions critical for PRM recognition. Comparative structural analysis highlights the functional importance of specific positions in peptide binding profiles. Phage‐derived peptides resemble natural short linear motifs (SLiMs) and likely bind to the same sites. Graphical Abstract A database of thousands of peptides targeting hundreds of diverse peptide recognition modules is presented ( www.prm‐db.org ). Applications of this large dataset include exploring natural protein‐protein interactions and inhibitor design.

Rising antimicrobial resistance (AMR) in Salmonella Typhi restricts typhoid treatment options, heightening concerns for pan-oral drug-resistant outbreaks. However, lack of long-term temporal surveillance data on AMR in countries with high burden like Bangladesh is scarce. Our study explores the AMR trends of Salmonella Typhi isolates from Bangladesh, drawing comparisons with antibiotic consumption to optimize antibiotic stewardship strategies for the country. The typhoid fever surveillance from 1999 to 2022 included two pediatric hospitals and three private clinics in Dhaka, Bangladesh. Blood cultures were performed at treating physicians' discretion; cases were confirmed by microbiological, serological, and biochemical tests. Antibiotic susceptibility was determined following CLSI guidelines. National antibiotic consumption data for cotrimoxazole, ciprofloxacin, and azithromycin was obtained from IQVIA-MIDAS database for comparison. Over the 24 years of surveillance, we recorded 12,435 culture-confirmed typhoid cases and observed declining resistance to first-line drugs (amoxicillin, chloramphenicol, and cotrimoxazole); multidrug resistance (MDR) decreased from 38% in 1999 to 17% in 2022. Cotrimoxazole consumption dropped from 0.8 to 0.1 Daily defined doses (DDD)/1000/day (1999-2020). Ciprofloxacin non-susceptibility persisted at >90% with unchanged consumption (1.1-1.3 DDD/1000/day, 2002-2020). Low ceftriaxone resistance (<1%) was observed, with slightly rising MIC (0.03 to 0.12 mg/L, 1999-2019). Azithromycin consumption increased (0.1 to 3.8 DDD/1000/day, 1999-2020), but resistance remained ≤4%. Our study highlights declining MDR amongst Salmonella Typhi in Bangladesh; first-line antimicrobials could be reintroduced as empirical treatment options for typhoid fever if MDR rates further drops below 5%. The analysis also provides baseline data for monitoring the impact of future interventions like typhoid conjugate vaccines on typhoid burden and associated AMR.

Background Invasive pneumococcal disease (IPD), caused by Streptococcus pneumoniae , remains a major global health concern, particularly for children. Among more than 100 pneumococcal serotypes, 19A is known for its multidrug resistance (MDR) and increased incidence following PCV7/PCV10 introduction in many countries. Bangladesh introduced PCV10 in 2015 considering the low burden of 19A in the country. Methods Utilizing our IPD surveillance from 2004 to 2023, we investigated the hospital incidence of serotype 19A before and after PCV10 introduction among < 5 years old children in Bangladesh. Whole-genome sequencing was done for 153 serotype 19A isolates from IPD, otitis media, carriage, and urine samples. We used phylogenetic and BEAST analyses to investigate population structure, circulating subtypes, global pneumococcal sequence clusters (GPSCs), sequence types (STs), and antimicrobial resistance genes, and compared them with global 19A genomes. Results Our findings indicate no increase in hospital IPD incidence due to serotype 19A following PCV10 introduction. The MDR 19A-ST320 lineage (GPSC1) remains absent in Bangladesh. ST12888 (GPSC84) became dominant in the post-PCV10 introduction (from 15% to 70%). GPSC84 carries the capsular locus of 19A subtype-I (19A-I), which emerged independently from the standard 19A locus. Macrolide resistance is increasing within the 19A-I/GPSC84 lineage and is estimated to have originated between 2007 and 2011. Conclusions This study presents the first comprehensive genomic analysis of the serotype 19A population in Bangladesh, supporting the decision to introduce PCV10 in Bangladesh based on local pneumococcal serotype burden data. However, the rapid evolution within the local 19A population highlights the need for continuous epidemiological and genomic surveillance to support effective vaccination programs.