The Identification of Enteric Fever-Specific Antigens for Population-Based Serosurveillance.

BACKGROUND: Enteric fever, caused by Salmonella enterica serovars Typhi and Paratyphi A, is a major public health problem in low- and middle-income countries. Moderate sensitivity and scalability of current methods likely underestimate enteric fever burden. Determining the serological responses to organism-specific antigens may improve incidence measures. METHODS: Plasma samples were collected from blood culture-confirmed enteric fever patients, blood culture-negative febrile patients over the course of 3 months, and afebrile community controls. A panel of 17 Salmonella Typhi and Paratyphi A antigens was purified and used to determine antigen-specific antibody responses by indirect ELISAs. RESULTS: The antigen-specific longitudinal antibody responses were comparable between enteric fever patients, patients with blood culture-negative febrile controls, and afebrile community controls for most antigens. However, we found that IgG responses against STY1479 (YncE), STY1886 (CdtB), STY1498 (HlyE), and the serovar-specific O2 and O9 antigens were greatly elevated over a 3-month follow up period in S. Typhi/S. Paratyphi A patients compared to controls, suggesting seroconversion. CONCLUSIONS: We identified a set of antigens as good candidates to demonstrate enteric fever exposure. These targets can be used in combination to develop more sensitive and scalable approaches to enteric fever surveillance and generate invaluable epidemiological data for informing vaccine policies. CLINICAL TRIAL REGISTRATION: ISRCTN63006567.

The repurposing of Tebipenem pivoxil as alternative therapy for severe gastrointestinal infections caused by extensively drug-resistant Shigella spp.

Background: Diarrhoea remains one of the leading causes of childhood mortality globally. Recent epidemiological studies conducted in low-middle income countries (LMICs) identified Shigella spp. as the first and second most predominant agent of dysentery and moderate diarrhoea, respectively. Antimicrobial therapy is often necessary for Shigella infections; however, we are reaching a crisis point with efficacious antimicrobials. The rapid emergence of resistance against existing antimicrobials in Shigella spp. poses a serious global health problem. Methods: Aiming to identify alternative antimicrobial chemicals with activity against antimicrobial resistant Shigella, we initiated a collaborative academia-industry drug discovery project, applying high-throughput phenotypic screening across broad chemical diversity and followed a lead compound through in vitro and in vivo characterisation. Results: We identified several known antimicrobial compound classes with antibacterial activity against Shigella. These compounds included the oral carbapenem Tebipenem, which was found to be highly potent against broadly susceptible Shigella and contemporary MDR variants for which we perform detailed pre-clinical testing. Additional in vitro screening demonstrated that Tebipenem had activity against a wide range of other non-Shigella enteric bacteria. Cognisant of the risk for the development of resistance against monotherapy, we identified synergistic behaviour of two different drug combinations incorporating Tebipenem. We found the orally bioavailable prodrug (Tebipenem pivoxil) had ideal pharmacokinetic properties for treating enteric pathogens and was effective in clearing the gut of infecting organisms when administered to Shigella-infected mice and gnotobiotic piglets. Conclusions: Our data highlight the emerging antimicrobial resistance crisis and shows that Tebipenem pivoxil (licenced for paediatric respiratory tract infections in Japan) should be accelerated into human trials and could be repurposed as an effective treatment for severe diarrhoea caused by MDR Shigella and other enteric pathogens in LMICs. Funding: Tres Cantos Open Lab Foundation (projects TC239 and TC246), the Bill and Melinda Gates Foundation (grant OPP1172483) and Wellcome (215515/Z/19/Z).

The evolutionary history of Shigella flexneri serotype 6 in Asia.

Shigella flexneri serotype 6 is an understudied cause of diarrhoeal diseases in developing countries, and has been proposed as one of the major targets for vaccine development against shigellosis. Despite being named as S. flexneri, Shigella flexneri serotype 6 is phylogenetically distinct from other S. flexneri serotypes and more closely related to S. boydii. This unique phylogenetic relationship and its low sampling frequency have hampered genomic research on this pathogen. Herein, by utilizing whole genome sequencing (WGS) and analyses of Shigella flexneri serotype 6 collected from epidemiological studies (1987-2013) in four Asian countries, we revealed its population structure and evolutionary history in the region. Phylogenetic analyses supported the delineation of Asian Shigella flexneri serotype 6 into two phylogenetic groups (PG-1 and -2). Notably, temporal phylogenetic approaches showed that extant Asian S. flexneri serotype 6 could be traced back to an inferred common ancestor arising in the 18th century. The dominant lineage PG-1 likely emerged in the 1970s, which coincided with the times to most recent common ancestors (tMRCAs) inferred from other major Southeast Asian S. flexneri serotypes. Similar to other S. flexneri serotypes in the same period in Asia, genomic analyses showed that resistance to first-generation antimicrobials was widespread, while resistance to more recent first-line antimicrobials was rare. These data also showed a number of gene inactivation and gene loss events, particularly on genes related to metabolism and synthesis of cellular appendages, emphasizing the continuing role of reductive evolution in the adaptation of the pathogen to an intracellular lifestyle. Together, our findings reveal insights into the genomic evolution of the understudied Shigella flexneri serotype 6, providing a new piece in the puzzle of Shigella epidemiology and evolution.

Tebipenem as an oral alternative for the treatment of typhoid caused by XDR Salmonella Typhi.

BACKGROUND: Antimicrobial therapy is essential for the treatment of enteric fever, the infection caused by Salmonella serovars Typhi and Paratyphi A. However, an increase in resistance to key antimicrobials and the emergence of MDR and XDR in Salmonella Typhi poses a major threat for efficacious outpatient treatments. OBJECTIVES: We recently identified tebipenem, an oral carbapenem licensed for use for respiratory tract infections in Japan, as a potential alternative treatment for MDR/XDR Shigella spp. Here, we aimed to test the in vitro antibacterial efficacy of this drug against MDR and XDR typhoidal Salmonella. METHODS: We determined the in vitro activity of tebipenem in time-kill assays against a collection of non-XDR and XDR Salmonella Typhi and Salmonella Paratyphi A (non-XDR) isolated in Nepal and Bangladesh. We also tested the efficacy of tebipenem in combination with other antimicrobials. RESULTS: We found that both XDR and non-XDR Salmonella Typhi and Salmonella Paratyphi A are susceptible to tebipenem, exhibiting low MICs, and were killed within 8-24 h at 2-4×MIC. Additionally, tebipenem demonstrated synergy with two other antimicrobials and could efficiently induce bacterial killing. CONCLUSIONS: Salmonella Paratyphi A and XDR Salmonella Typhi display in vitro susceptibility to the oral carbapenem tebipenem, while synergistic activity with other antimicrobials may limit the emergence of resistance. The broad-spectrum activity of this drug against MDR/XDR organisms renders tebipenem a good candidate for clinical trials.

Evolutionary histories and antimicrobial resistance in Shigella flexneri and Shigella sonnei in Southeast Asia.

Conventional disease surveillance for shigellosis in developing country settings relies on serotyping and low-resolution molecular typing, which fails to contextualise the evolutionary history of the genus. Here, we interrogated a collection of 1,804 Shigella whole genome sequences from organisms isolated in four continental Southeast Asian countries (Thailand, Vietnam, Laos, and Cambodia) over three decades to characterise the evolution of both S. flexneri and S. sonnei. We show that S. sonnei and each major S. flexneri serotype are comprised of genetically diverse populations, the majority of which were likely introduced into Southeast Asia in the 1970s-1990s. Intranational and regional dissemination allowed widespread propagation of both species across the region. Our data indicate that the epidemiology of S. sonnei and the major S. flexneri serotypes were characterised by frequent clonal replacement events, coinciding with changing susceptibility patterns against contemporaneous antimicrobials. We conclude that adaptation to antimicrobial pressure was pivotal to the recent evolutionary trajectory of Shigella in Southeast Asia.

Colonization with Staphylococcus aureus and Klebsiella pneumoniae causes infections in a Vietnamese intensive care unit.

Pre-existing colonization with Staphylococcus aureus or Klebsiella pneumoniae has been found to increase the risk of infection in intensive care patients. We previously conducted a longitudinal study to characterize colonization of these two organisms in patients admitted to intensive care in a hospital in southern Vietnam. Here, using genomic and phylogenetic analyses, we aimed to assess the contribution these colonizing organisms made to infections. We found that in the majority of patients infected with S. aureus or K. pneumoniae, the sequence type of the disease-causing (infecting) isolate was identical to that of corresponding colonizing organisms in the respective patient. Further in-depth analysis revealed that in patients infected by S. aureus ST188 and by K. pneumoniae ST17, ST23, ST25 and ST86, the infecting isolate was closely related to and exhibited limited genetic variation relative to pre-infection colonizing isolates. Multidrug-resistant S. aureus ST188 was identified as the predominant agent of colonization and infection. Colonization and infection by K. pneumoniae were characterized by organisms with limited antimicrobial resistance profiles but extensive repertoires of virulence genes. Our findings augment the understanding of the link between bacterial colonization and infection in a low-resource setting, and could facilitate the development of novel evidence-based approaches to prevent and treat infections in high-risk patients in intensive care.

Dissecting the molecular evolution of fluoroquinolone-resistant Shigella sonnei.

Shigella sonnei increasingly dominates the international epidemiological landscape of shigellosis. Treatment options for S. sonnei are dwindling due to resistance to several key antimicrobials, including the fluoroquinolones. Here we analyse nearly 400 S. sonnei whole genome sequences from both endemic and non-endemic regions to delineate the evolutionary history of the recently emergent fluoroquinolone-resistant S. sonnei. We reaffirm that extant resistant organisms belong to a single clonal expansion event. Our results indicate that sequential accumulation of defining mutations (gyrA-S83L, parC-S80I, and gyrA-D87G) led to the emergence of the fluoroquinolone-resistant S. sonnei population around 2007 in South Asia. This clone was then transmitted globally, resulting in establishments in Southeast Asia and Europe. Mutation analysis suggests that the clone became dominant through enhanced adaptation to oxidative stress. Experimental evolution reveals that under fluoroquinolone exposure in vitro, resistant S. sonnei develops further intolerance to the antimicrobial while the susceptible counterpart fails to attain complete resistance.

A Double-blind, Randomized, Placebo-controlled Trial of Lactobacillus acidophilus for the Treatment of Acute Watery Diarrhea in Vietnamese Children.

BACKGROUND: Probiotics are the most frequently prescribed treatment for children hospitalized with diarrhea in Vietnam. We were uncertain of the benefits of probiotics for the treatment of acute watery diarrhea in Vietnamese children. METHODS: We conducted a double-blind, placebo-controlled, randomized trial of children hospitalized with acute watery diarrhea in Vietnam. Children meeting the inclusion criteria (acute watery diarrhea) were randomized to receive either 2 daily oral doses of 2 × 10 CFUs of a local probiotic containing Lactobacillus acidophilus or placebo for 5 days as an adjunct to standard of care. The primary end point was time from the first dose of study medication to the start of the first 24-hour period without diarrhea. Secondary outcomes included the total duration of diarrhea and hospitalization, daily stool frequency, treatment failure, daily fecal concentrations of rotavirus and norovirus, and Lactobacillus colonization. RESULTS: One hundred and fifty children were randomized into each study group. The median time from the first dose of study medication to the start of the first 24-hour diarrhea-free period was 43 hours (interquartile range, 15-66 hours) in the placebo group and 35 hours (interquartile range, 20-68 hours) in the probiotic group (acceleration factor 1.09 [95% confidence interval, 0.78-1.51]; P = 0.62). There was also no evidence that probiotic treatment was efficacious in any of the predefined subgroups nor significantly associated with any secondary end point. CONCLUSIONS: This was a large double-blind, placebo-controlled trial in which the probiotic underwent longitudinal quality control. We found under these conditions that L. acidophilus was not beneficial in treating children with acute watery diarrhea.

An evaluation of purified Salmonella Typhi protein antigens for the serological diagnosis of acute typhoid fever.

OBJECTIVES: The diagnosis of typhoid fever is a challenge. Aiming to develop a typhoid diagnostic we measured antibody responses against Salmonella Typhi (S. Typhi) protein antigens and the Vi polysaccharide in a cohort of Bangladeshi febrile patients. METHODS: IgM against 12 purified antigens and the Vi polysaccharide was measured by ELISA in plasma from patients with confirmed typhoid fever (n = 32), other confirmed infections (n = 17), and healthy controls (n = 40). ELISAs with the most specific antigens were performed on plasma from 243 patients with undiagnosed febrile disease. RESULTS: IgM against the S. Typhi protein antigens correlated with each other (rho > 0.8), but not against Vi (rho < 0.6). Typhoid patients exhibited higher IgM against 11/12 protein antigens and Vi than healthy controls and those with other infections. Vi, PilL, and CdtB exhibited the greatest sensitivity and specificity. Specificity and sensitivity was improved when Vi was combined with a protein antigen, generating sensitivities and specificities of 0.80 and >0.85, respectively. Applying a dynamic cut-off to patients with undiagnosed febrile disease suggested that 34-58% had an IgM response indicative of typhoid. CONCLUSIONS: We evaluated the diagnostic potential of several S. Typhi antigens; our assays give good sensitivity and specificity, but require further assessment in differing patient populations.

South Asia as a Reservoir for the Global Spread of Ciprofloxacin-Resistant Shigella sonnei: A Cross-Sectional Study.

BACKGROUND: Antimicrobial resistance is a major issue in the Shigellae, particularly as a specific multidrug-resistant (MDR) lineage of Shigella sonnei (lineage III) is becoming globally dominant. Ciprofloxacin is a recommended treatment for Shigella infections. However, ciprofloxacin-resistant S. sonnei are being increasingly isolated in Asia and sporadically reported on other continents. We hypothesized that Asia is a primary hub for the recent international spread of ciprofloxacin-resistant S. sonnei. METHODS AND FINDINGS: We performed whole-genome sequencing on a collection of 60 contemporaneous ciprofloxacin-resistant S. sonnei isolated in four countries within Asia (Vietnam, n = 11; Bhutan, n = 12; Thailand, n = 1; Cambodia, n = 1) and two outside of Asia (Australia, n = 19; Ireland, n = 16). We reconstructed the recent evolutionary history of these organisms and combined these data with their geographical location of isolation. Placing these sequences into a global phylogeny, we found that all ciprofloxacin-resistant S. sonnei formed a single clade within a Central Asian expansion of lineage III. Furthermore, our data show that resistance to ciprofloxacin within S. sonnei may be globally attributed to a single clonal emergence event, encompassing sequential gyrA-S83L, parC-S80I, and gyrA-D87G mutations. Geographical data predict that South Asia is the likely primary source of these organisms, which are being regularly exported across Asia and intercontinentally into Australia, the United States and Europe. Our analysis was limited by the number of S. sonnei sequences available from diverse geographical areas and time periods, and we cannot discount the potential existence of other unsampled reservoir populations of antimicrobial-resistant S. sonnei. CONCLUSIONS: This study suggests that a single clone, which is widespread in South Asia, is likely driving the current intercontinental surge of ciprofloxacin-resistant S. sonnei and is capable of establishing endemic transmission in new locations. Despite being limited in geographical scope, our work has major implications for understanding the international transfer of antimicrobial-resistant pathogens, with S. sonnei acting as a tractable model for studying how antimicrobial-resistant Gram-negative bacteria spread globally.

A novel ciprofloxacin-resistant subclade of H58 Salmonella Typhi is associated with fluoroquinolone treatment failure.

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.

The Ecological Dynamics of Fecal Contamination and Salmonella Typhi and Salmonella Paratyphi A in Municipal Kathmandu Drinking Water.

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.

Salmonella Typhi and Salmonella Paratyphi A elaborate distinct systemic metabolite signatures during enteric fever.

The host-pathogen interactions induced by Salmonella Typhi and Salmonella Paratyphi A during enteric fever are poorly understood. This knowledge gap, and the human restricted nature of these bacteria, limit our understanding of the disease and impede the development of new diagnostic approaches. To investigate metabolite signals associated with enteric fever we performed two dimensional gas chromatography with time-of-flight mass spectrometry (GCxGC/TOFMS) on plasma from patients with S. Typhi and S. Paratyphi A infections and asymptomatic controls, identifying 695 individual metabolite peaks. Applying supervised pattern recognition, we found highly significant and reproducible metabolite profiles separating S. Typhi cases, S. Paratyphi A cases, and controls, calculating that a combination of six metabolites could accurately define the etiological agent. For the first time we show that reproducible and serovar specific systemic biomarkers can be detected during enteric fever. Our work defines several biologically plausible metabolites that can be used to detect enteric fever, and unlocks the potential of this method in diagnosing other systemic bacterial infections.

Differential epidemiology of Salmonella Typhi and Paratyphi A in Kathmandu, Nepal: a matched case control investigation in a highly endemic enteric fever setting.

BACKGROUND: Enteric fever, a systemic infection caused by the bacteria Salmonella Typhi and Salmonella Paratyphi A, is endemic in Kathmandu, Nepal. Previous work identified proximity to poor quality water sources as a community-level risk for infection. Here, we sought to examine individual-level risk factors related to hygiene and sanitation to improve our understanding of the epidemiology of enteric fever in this setting. METHODOLOGY AND PRINCIPAL FINDINGS: A matched case-control analysis was performed through enrollment of 103 blood culture positive enteric fever patients and 294 afebrile community-based age and gender-matched controls. A detailed questionnaire was administered to both cases and controls and the association between enteric fever infection and potential exposures were examined through conditional logistic regression. Several behavioral practices were identified as protective against infection with enteric fever, including water storage and hygienic habits. Additionally, we found that exposures related to poor water and socioeconomic status are more influential in the risk of infection with S. Typhi, whereas food consumption habits and migration play more of a role in risk of S. Paratyphi A infection. CONCLUSIONS AND SIGNIFICANCE: Our work suggests that S. Typhi and S. Paratyphi A follow different routes of infection in this highly endemic setting and that sustained exposure to both serovars probably leads to the development of passive immunity. In the absence of a polyvalent vaccine against S. Typhi and S. Paratyphi A, we advocate better systems for water treatment and storage, improvements in the quality of street food, and vaccination with currently available S. Typhi vaccines.

Identification of Salmonella enterica serovar Typhi genotypes by use of rapid multiplex ligation-dependent probe amplification.

Salmonella enterica serovar Typhi, the causative agent of typhoid fever, is highly clonal and genetically conserved, making isolate subtyping difficult. We describe a standardized multiplex ligation-dependent probe amplification (MLPA) genotyping scheme targeting 11 key phylogenetic markers of the S. Typhi genome. The MLPA method demonstrated 90% concordance with single nucleotide polymorphism (SNP) typing, the gold standard for S. Typhi genotyping, and had the ability to identify isolates of the H58 haplotype, which is associated with resistance to multiple antimicrobials. Additionally, the assay permitted the detection of fluoroquinolone resistance-associated mutations in the DNA gyrase-encoding gene gyrA and the topoisomerase gene parC with a sensitivity of 100%. The MLPA methodology is simple and reliable, providing phylogenetically and phenotypically relevant genotyping information. This MLPA scheme offers a more-sensitive and interpretable alternative to the nonphylogenetic subgrouping methodologies that are currently used in reference and research laboratories in areas where typhoid is endemic.