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Despite recent advances in typhoid fever control, asymptomatic carriage of Salmonella Typhi in the gallbladder remains poorly understood. Aiming to understand if S. Typhi becomes genetically adapted for long-term colonisation in the gallbladder, we performed whole genome sequencing on a collection of S. Typhi isolated from the gallbladders of typhoid carriers. These sequences were compared to contemporaneously sampled sequences from organisms isolated from the blood of acute patients within the same population. We found that S. Typhi carriage was not restricted to any particular genotype or conformation of antimicrobial resistance genes, but was largely reflective of S. Typhi circulating in the general population. However, gallbladder isolates showed a higher genetic variability than acute isolates, with median pairwise SNP distances of 21 and 13 SNPs (p = 2.8x10-9), respectively. Within gallbladder isolates of the predominant H58 genotype, variation was associated with a higher prevalence of nonsense mutations. Notably, gallbladder isolates displayed a higher frequency of non-synonymous mutations in genes encoding hypothetical proteins, membrane lipoproteins, transport/binding proteins, surface antigens, and carbohydrate degradation. Specifically, we identified several gallbladder-specific non-synonymous mutations involved in LPS synthesis and modification, with some isolates lacking the Vi capsular polysaccharide vaccine target due to the 134Kb deletion of SPI-7. S. Typhi is under strong selective pressure in the human gallbladder, which may be reflected phylogenetically by long terminal branches that may distinguish organisms from chronic and acute infections. Our work shows that selective pressures asserted by the hostile environment of the human gallbladder generate new antigenic variants and raises questions regarding the role of carriage in the epidemiology of typhoid fever.

Background Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa , Escherichia coli, Streptococcus pneumoniae and Staphylococcus aureus are major bacterial causes of lower respiratory tract infections (LRTIs) globally, leading to substantial morbidity and mortality. The rapid increase of antimicrobial resistance (AMR) in these pathogens poses significant challenges for their effective antibiotic therapy. In low-resourced settings, patients with LRTIs are prescribed antibiotics empirically while awaiting several days for culture results. Rapid pathogen and AMR gene detection could prompt optimal antibiotic use and improve outcomes. Methods Here, we developed multiplex quantitative real-time PCR using EvaGreen dye and melting curve analysis to rapidly identify six major pathogens and fourteen AMR genes directly from respiratory samples. The reproducibility, linearity, limit of detection (LOD) of real-time PCR assays for pathogen detection were evaluated using DNA control mixes and spiked tracheal aspirate. The performance of RT-PCR assays was subsequently compared with the gold standard, conventional culture on 50 tracheal aspirate and sputum specimens of ICU patients. Results The sensitivity of RT-PCR assays was 100% for K. pneumoniae , A. baumannii , P. aeruginosa , E. coli and 63.6% for S. aureus and the specificity ranged from 87.5% to 97.6%. The kappa correlation values of all pathogens between the two methods varied from 0.63 to 0.95. The limit of detection of target bacteria was 1600 CFU/ml. The quantitative results from the PCR assays demonstrated 100% concordance with quantitative culture of tracheal aspirates. Compared to culture, PCR assays exhibited higher sensitivity in detecting mixed infections and S. pneumoniae . There was a high level of concordance between the detection of AMR gene and AMR phenotype in single infections. Conclusions Our multiplex quantitative RT-PCR assays are fast and simple, but sensitive and specific in detecting six bacterial pathogens of LRTIs and their antimicrobial resistance genes and should be further evaluated for clinical utility.

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)(p<0.001). H58 is the dominant S. 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. Clinical trial registration: ISRCTN63006567. People who ingest a type of bacteria called Salmonella Typhi can develop the symptoms of typhoid fever. This disease is common in low-income settings in Asia and Africa, and causes a high rate of death in people who are not treated with antimicrobial drugs. During a study in Nepal, Thanh et al. tried to evaluate which of two antimicrobials was better for treating typhoid fever. One of the drugs – called gatifloxacin – did not work in some of the patients. To understand why this treatment failed, Thanh et al. decoded the entire DNA sequences of all the Salmonella Typhi bacteria isolated during the study. Comparing this genetic data to the clinical data of the patients identified a new variant of Salmonella Typhi. These bacteria have a specific combination of genetic mutations that render them resistant to the family of drugs that gatifloxacin belongs to – the fluoroquinolones. Patients infected with the variant bacteria and treated with gatifloxacin were highly likely to completely fail treatment and have longer-lasting fevers. On further investigation Thanh et al. found these organisms were likely recently introduced into Nepal from India. Fluoroquinolones are amongst the most effective and common antimicrobials used to treat typhoid fever and other bacterial infections. However, the presence of bacteria that are resistant to these compounds in South Asia means that they should no longer be the first choice of drug to treat typhoid fever in this location.

Salmonella Paratyphi A, one of the major etiologic agents of enteric fever, has increased in prevalence in recent decades in certain endemic regions in comparison to S . Typhi, the most prevalent cause of enteric fever. Despite this increase, data on the prevalence and molecular epidemiology of S . Paratyphi A remain generally scarce. Here, we analysed the whole genome sequences of 216 S . Paratyphi A isolates originating from Kathmandu, Nepal between 2005 and 2014, of which 200 were from patients with acute enteric fever and 16 from the gallbladder of people with suspected chronic carriage. By exploiting the recently developed genotyping framework for S . Paratyphi A (Paratype), we identified several genotypes circulating in Kathmandu. Notably, we observed an unusual clonal expansion of genotype 2.4.3 over a four-year period that spread geographically and systematically replaced other genotypes. This rapid genotype replacement is hypothesised to have been driven by both reduced susceptibility to fluoroquinolones and genetic changes to virulence factors, such as functional and structural genes encoding the type 3 secretion systems. Finally, we show that person-to-person is likely the most common mode of transmission and chronic carriers seem to play a limited role in maintaining disease circulation.

One of the UN sustainable development goals is to achieve universal access to safe and affordable drinking water by 2030. It is locations like Kathmandu, Nepal, a densely populated city in South Asia with endemic typhoid fever, where this goal is most pertinent. Aiming to understand the public health implications of water quality in Kathmandu we subjected weekly water samples from 10 sources for one year to a range of chemical and bacteriological analyses. We additionally aimed to detect the etiological agents of typhoid fever and longitudinally assess microbial diversity by 16S rRNA gene surveying. We found that the majority of water sources exhibited chemical and bacterial contamination exceeding WHO guidelines. Further analysis of the chemical and bacterial data indicated site-specific pollution, symptomatic of highly localized fecal contamination. Rainfall was found to be a key driver of this fecal contamination, correlating with nitrates and evidence of S. Typhi and S. Paratyphi A, for which DNA was detectable in 333 (77%) and 303 (70%) of 432 water samples, respectively. 16S rRNA gene surveying outlined a spectrum of fecal bacteria in the contaminated water, forming complex communities again displaying location-specific temporal signatures. Our data signify that the municipal water in Kathmandu is a predominant vehicle for the transmission of S. Typhi and S. Paratyphi A. This study represents the first extensive spatiotemporal investigation of water pollution in an endemic typhoid fever setting and implicates highly localized human waste as the major contributor to poor water quality in the Kathmandu Valley.

Multidrug‐resistant (MDR) Klebsiella pneumoniae has become a leading cause of nosocomial infections worldwide. Despite its prominence, little is known about the genetic diversity of K. pneumoniae in resource‐poor hospital settings. Through whole‐genome sequencing (WGS), we reconstructed an outbreak of MDR K. pneumoniae occurring on high‐dependency wards in a hospital in Kathmandu during 2012 with a case‐fatality rate of 75%. The WGS analysis permitted the identification of two MDR K. pneumoniae lineages causing distinct outbreaks within the complex endemic K. pneumoniae . Using phylogenetic reconstruction and lineage‐specific PCR, our data predicted a scenario in which K. pneumoniae , circulating for 6 months before the outbreak, underwent a series of ward‐specific clonal expansions after the acquisition of genes facilitating virulence and MDR. We suggest that the early detection of a specific NDM‐1 containing lineage in 2011 would have alerted the high‐dependency ward staff to intervene. We argue that some form of real‐time genetic characterisation, alongside clade‐specific PCR during an outbreak, should be factored into future healthcare infection control practices in both high‐ and low‐income settings. Synopsis Whole‐genome sequencing and further data analysis allowed for a comprehensive understanding of the emergence and spread of two highly virulent clones of drug‐resistant Klebsiella pneumonia bloodstream infections from one hospital in Kathmandu, Nepal. MDR Klebsiella pneumoniae (Kp) can cause serious hospital outbreaks and can be associated with high mortality in hospitals in developing countries. Whole‐genome sequencing (WGS) of Kp bloodstream infections was used to study localized transmission and genetic adaptation during a hospital outbreak in Nepal. Genetic material specific to outbreak lineages permitted a retrospective temporal reconstruction of two Kp outbreak lineages across the hospital. The added benefit of Kp WGS in this study was to dissect these outbreaks with extremely fine detail. Genetic characterization of Kp causing bloodstream infections in hospitals in developing countries should be performed routinely to identify and isolate outbreaks early. Graphical Abstract Whole‐genome sequencing and further data analysis allowed for a comprehensive understanding of the emergence and spread of two highly virulent clones of drug‐resistant Klebsiella pneumonia bloodstream infections from one hospital in Kathmandu, Nepal.

Shigella flexneri is the most common cause of bacterial dysentery in low-income countries. Despite this, S. flexneri remains largely unexplored from a genomic standpoint and is still described using a vocabulary based on serotyping reactions developed over half-a-century ago. Here we combine whole genome sequencing with geographical and temporal data to examine the natural history of the species. Our analysis subdivides S. flexneri into seven phylogenetic groups (PGs); each containing two-or-more serotypes and characterised by distinct virulence gene complement and geographic range. Within the S. flexneri PGs we identify geographically restricted sub-lineages that appear to have persistently colonised regions for many decades to over 100 years. Although we found abundant evidence of antimicrobial resistance (AMR) determinant acquisition, our dataset shows no evidence of subsequent intercontinental spread of antimicrobial resistant strains. The pattern of colonisation and AMR gene acquisition suggest that S. flexneri has a distinct life-cycle involving local persistence. Dysentery is a disease in which the intestine becomes inflamed due to infection by bacteria, viruses or other microbes. Of the bacteria that can cause dysentery, bacteria called Shigella are most often responsible. Humans can acquire Shigella through contaminated food or water. Over the last century, improvements to sanitation combined with access to clean drinking water and better food hygiene have decreased the number of cases of dysentery in many countries. However, the disease continues to be common in low-income countries, especially in young children. One species of Shigella bacteria, called S. flexneri, causes far more cases of dysentry than other species of Shigella. Across the world, there are many different strains of S. flexneri, but it is not clear how these strains are related to each other, or how variable the genes that they carry are—known as genetic diversity. Here, Connor, Barker, Baker et al. used a technique called whole genome sequencing to map the evolutionary relationships of over 300 S. flexneri samples collected from around the globe over the past 100 years. This revealed that the bacterial strains can be split into seven groups that each have distinct geographic ranges and combinations of genes that enable the bacteria to infect humans. Many of the strains of bacteria within these groups seem to have colonized, and remained in, quite small geographic areas over long periods of time. This is different to other Shigella species, which appear to have spread between continents far more frequently over much shorter timescales. Connor, Barker, Baker et al.'s findings reveal that S. flexneri is more diverse than other Shigella bacteria, and suggest that the ability of strains to persist in local areas may have contributed to the species' long-term success. These results point towards the importance of the provision of clean water in the fight against S. flexneri, and underline the need for a greater understanding of how disease-causing bacteria colonize and interact with the local environment.

Non-orthogonal multiple access (NOMA) is considered a potential technique in fifth-generation (5G). Nevertheless, it is relatively complex when applying NOMA to a massive access scenario. Thus, in this paper, a hybrid NOMA/OMA scheme is considered for uplink wireless transmission systems where multiple cognitive users (CUs) can simultaneously transmit their data to a cognitive base station (CBS). We adopt a user-pairing algorithm in which the CUs are grouped into multiple pairs, and each group is assigned to an orthogonal sub-channel such that each user in a pair applies NOMA to transmit data to the CBS without causing interference with other groups. Subsequently, the signal transmitted by the CUs of each NOMA group can be independently retrieved by using successive interference cancellation (SIC). The CUs are assumed to harvest solar energy to maintain operations. Moreover, joint power and bandwidth allocation is taken into account at the CBS to optimize energy and spectrum efficiency in order to obtain the maximum long-term data rate for the system. To this end, we propose a deep actor-critic reinforcement learning (DACRL) algorithm to respectively model the policy function and value function for the actor and critic of the agent (i.e., the CBS), in which the actor can learn about system dynamics by interacting with the environment. Meanwhile, the critic can evaluate the action taken such that the CBS can optimally assign power and bandwidth to the CUs when the training phase finishes. Numerical results validate the superior performance of the proposed scheme, compared with other conventional schemes.

Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), remains a serious global health concern. Since their emergence in the mid-1970s multi-drug resistant (MDR) S. Typhi now dominate drug sensitive equivalents in many regions. MDR in S. Typhi is almost exclusively conferred by self-transmissible IncHI1 plasmids carrying a suite of antimicrobial resistance genes. We identified over 300 single nucleotide polymorphisms (SNPs) within conserved regions of the IncHI1 plasmid, and genotyped both plasmid and chromosomal SNPs in over 450 S. Typhi dating back to 1958. Prior to 1995, a variety of IncHI1 plasmid types were detected in distinct S. Typhi haplotypes. Highly similar plasmids were detected in co-circulating S. Typhi haplotypes, indicative of plasmid transfer. In contrast, from 1995 onwards, 98% of MDR S. Typhi were plasmid sequence type 6 (PST6) and S. Typhi haplotype H58, indicating recent global spread of a dominant MDR clone. To investigate whether PST6 conferred a selective advantage compared to other IncHI1 plasmids, we used a phenotyping array to compare the impact of IncHI1 PST6 and PST1 plasmids in a common S. Typhi host. The PST6 plasmid conferred the ability to grow in high salt medium (4.7% NaCl), which we demonstrate is due to the presence in PST6 of the Tn6062 transposon encoding BetU.

Fluoroquinolones (FQ) are the recommended antimicrobial treatment for typhoid, a severe systemic infection caused by the bacterium Salmonella enterica serovar Typhi. FQ-resistance mutations in S. Typhi have become common, hindering treatment and control efforts. Using in vitro competition experiments, we assayed the fitness of eleven isogenic S. Typhi strains with resistance mutations in the FQ target genes, gyrA and parC. In the absence of antimicrobial pressure, 6 out of 11 mutants carried a selective advantage over the antimicrobial-sensitive parent strain, indicating that FQ resistance in S. Typhi is not typically associated with fitness costs. Double-mutants exhibited higher than expected fitness as a result of synergistic epistasis, signifying that epistasis may be a critical factor in the evolution and molecular epidemiology of S. Typhi. Our findings have important implications for the management of drug-resistant S. Typhi, suggesting that FQ-resistant strains would be naturally maintained even if fluoroquinolone use were reduced. The fluoroquinolones are a group of antimicrobials that are used to treat a variety of life-threatening bacterial infections, including typhoid fever. Before the introduction of antimicrobials, the mortality rate from typhoid fever was 10–20%. Prompt treatment with fluoroquinolones has reduced this to less than 1%, and has also decreased the severity of symptoms suffered by people with the disease. Now, however, the usefulness of many antimicrobials, including the fluoroquinolones, is threatened by the evolution of antimicrobial resistance within the bacterial populations being treated. Drug resistance in bacteria typically arises through specific mutations, or following the acquisition of antimicrobial resistance genes from other bacteria. It is thought that the frequent use of antimicrobials in human and animal health puts selective pressure on bacterial populations, allowing bacterial strains with mutations or genes that confer antimicrobial resistance to survive, while bacterial strains that are sensitive to the antimicrobials die out. At first it was thought that specific mutations conferring antimicrobial resistance came at a fitness cost, which would mean that such mutations would be rare in the absence of antimicrobials. Now, based on research into typhoid fever, Baker et al. describe a system in which the majority of evolutionary routes to drug resistance are marked by significant fitness benefits, even in the absence of antimicrobial exposure. Typhoid is caused by a bacterial pathogen known as Salmonella Typhi, and mutations in two genes—gyrA and parC—result in resistance to fluoroquinolones. Baker et al. show that mutations in these genes confer a measurable fitness advantage over strains without these mutations, even in the absence of exposure to fluoroquinolones. Moreover, strains with two mutations in one of these genes exhibited a higher than predicted fitness, suggesting that there is a synergistic interaction between the two mutations. This work challenges the dogma that antimicrobial resistant organisms have a fitness disadvantage in the absence of antimicrobials, and suggests that increasing resistance to the fluoroquinolones is not solely driven by excessive use of this important group of drugs.