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Salmonella entericaserotype 4,[5],12,i:- (Typhimurium monophasic variant) of sequence type (ST) 34 has emerged as the predominant pandemic genotype in recent decades. Despite increasing reports of resistance to antimicrobials in Southeast Asia,SalmonellaST34 population structure and evolution remained understudied in the region. Here we performed detailed genomic investigations on 454 ST34 genomes collected from Vietnam and diverse geographical sources to elucidate the pathogen’s epidemiology, evolution and antimicrobial resistance. We showed that ST34 has been introduced into Vietnam in at least nine occasions since 2000, forming five co-circulating major clones responsible for paediatric diarrhoea and bloodstream infection. Most expansion events were associated with acquisitions of large multidrug resistance plasmids of IncHI2 or IncA/C2. Particularly, the self-conjugative IncA/C2 pST34VN2 (co-transferringblaCTX-M-55,mcr-3.1, andqnrS1) underlies local expansion and intercontinental spread in two separate ST34 clones. At the global scale, Southeast Asia was identified as a hotspot for the emergence and dissemination of multidrug resistantSalmonellaST34, and mutation analysis suggests of selection in antimicrobial responses and key virulence factors. Our work enriches the understanding on epidemiology and evolution of this variant in Southeast Asia, and determines that multidrug resistance plasmids have driven its local and potentially global success.

Non-typhoidal Salmonella (NTS) is a major cause of bacterial enterocolitis globally but also causes invasive bloodstream infections. Antimicrobial resistance (AMR) hampers the treatment of these infections and understanding how AMR spreads between NTS may help in developing effective strategies. We investigated NTS isolates associated with invasive disease, diarrhoeal disease and asymptomatic carriage in animals and humans from Vietnam. Isolates included multiple serovars and both common and rare phenotypic AMR profiles; long- and short-read sequencing was used to investigate the genetic mechanisms and genomic backgrounds associated with phenotypic AMR profiles. We demonstrate concordance between most AMR genotypes and phenotypes but identified large genotypic diversity in clinically relevant phenotypes and the high mobility potential of AMR genes (ARGs) in this setting. We found that 84 % of ARGs identified were located on plasmids, most commonly those containing IncHI1A_1 and IncHI1B(R27)_1_R27 replicons (33%), and those containing IncHI2_1 and IncHI2A_1 replicons (31%). The vast majority (95%) of ARGS were found within 10 kbp of IS6/IS26 elements, which provide plasmids with a mechanism to exchange ARGs between plasmids and other parts of the genome. Whole genome sequencing with targeted long-read sequencing applied in a One Health context identified a comparatively limited number of insertion sequences and plasmid replicons associated with AMR. Therefore, in the context of NTS from Vietnam and likely for other settings as well, the mechanisms by which ARGs move contribute to a more successful AMR profile than the specific ARGs, facilitating the adaptation of bacteria to different environments or selection pressures.

Nontyphoidal Salmonella (NTS) are among the most common etiological agents of diarrheal diseases worldwide and have become the most commonly detected bacterial pathogen in children hospitalized with diarrhea in Vietnam. Aiming to better understand the epidemiology, serovar distribution, antimicrobial resistance (AMR), and clinical manifestation of NTS gastroenteritis in Vietnam, we conducted a clinical genomics investigation of NTS isolated from diarrheal children admitted to one of three tertiary hospitals in Ho Chi Minh City., Nontyphoidal Salmonella (NTS) are among the most common etiological agents of diarrheal diseases worldwide and have become the most commonly detected bacterial pathogen in children hospitalized with diarrhea in Vietnam. Aiming to better understand the epidemiology, serovar distribution, antimicrobial resistance (AMR), and clinical manifestation of NTS gastroenteritis in Vietnam, we conducted a clinical genomics investigation of NTS isolated from diarrheal children admitted to one of three tertiary hospitals in Ho Chi Minh City. Between May 2014 and April 2016, 3,166 children hospitalized with dysentery were recruited into the study; 478 (∼15%) children were found to be infected with NTS by stool culture. Molecular serotyping of the 450 generated genomes identified a diverse collection of serogroups (B, C1, C2 to C3, D1, E1, G, I, K, N, O, and Q); however, Salmonella enterica serovar Typhimurium was the most predominant serovar, accounting for 41.8% (188/450) of NTS isolates. We observed a high prevalence of AMR to first-line treatments recommended by WHO, and more than half (53.8%; 242/450) of NTS isolates were multidrug resistant (MDR; resistant to ≥3 antimicrobial classes). AMR gene detection positively correlated with phenotypic AMR testing, and resistance to empirical antimicrobials was associated with a significantly longer hospitalization (0.91 days; P = 0.04). Our work shows that genome sequencing is a powerful epidemiological tool to characterize the serovar diversity and AMR profiles in NTS. We propose a revaluation of empirical antimicrobials for dysenteric diarrhea and endorse the use of whole-genome sequencing for sustained surveillance of NTS internationally.

Background Diarrhoeagenic Escherichia coli (DEC) infections are common in children in low-middle income countries (LMICs). However, detecting the various DEC pathotypes is complex as they cannot be differentiated by classical microbiology. We developed four multiplex real-time PCR assays were to detect virulence markers of six DEC pathotypes; specificity was tested using DEC controls and other enteric pathogens. PCR amplicons from the six E. coli pathotypes were purified and amplified to be used to optimize PCR reactions and to calculate reproducibility. After validation, these assays were applied to clinical samples from healthy and diarrhoeal Vietnamese children and associated with clinical data. Results The multiplex real-time PCRs were found to be reproducible, and specific. At least one DEC variant was detected in 34.7% (978/2815) of the faecal samples from diarrhoeal children; EAEC, EIEC and atypical EPEC were most frequent Notably, 41.2% (205/498) of samples from non-diarrhoeal children was positive with a DEC pathotype. In this population, only EIEC, which was detected in 34.3% (99/289) of diarrhoeal samples vs. 0.8% (4/498) non-diarrhoeal samples (p Conclusions This approach revealed high carriage rates of DEC pathotypes among Vietnamese children. We describe a novel diagnostic approach for DEC, which provides baseline data for future surveillance studies assessing DEC burden in LMICs.

Purpose. Antimicrobial-resistant bacterial infections in low- and middle-income countries (LMICs) are a well-established global health issue. We aimed to assess the prevalence of and epidemiological factors associated with the carriage of ciprofloxacin- and ceftriaxone-resistant Escherichia coli and associated resistance genes in a cohort of 498 healthy children residing in urban Vietnam. Methodology. We cultured rectal swabs onto MacConkey agar supplemented with resistant concentrations of ciprofloxacin and ceftriaxone. Additionally, we screened meta-E. coli populations by conventional PCR to detect plasmid-mediated quinolone resistance (PMQR)- and extended-spectrum β-lactamase (ESBL)-encoding genes. We measured the associations between phenotypic/genotypic resistance and demographic characteristics using logistic regression. Results/key findings. Ciprofloxacin- and ceftriaxone-resistant E. coli were cultured from the faecal samples of 67.7 % (337/498) and 80.3 % (400/498) of children, respectively. The prevalence of any associated resistance marker in the individual samples was 86.7 % (432/498) for PMQR genes and 90.6 % (451/498) for β-lactamase genes. Overweight children were significantly more likely to carry qnr genes than children with lower weight-for-height z-scores [odds ratios (OR): 1.24; 95 % confidence interval (CI): 10.5–1.48 for each unit increase in weight for height; P=0.01]. Additionally, younger children were significantly more likely to carry ESBL CTX-M genes than older children (OR: 0.97, 95 % CI: 0.94–0.99 for each additional year, P=0.01). Conclusion. The carriage of genotypic and phenotypic antimicrobial resistance is highly prevalent among E. coli in healthy children in the community in Vietnam. Future investigations on the carriage of antimicrobial resistant organisms in LMICs should focus on the progression of carriage from birth and structure of the microbiome in obesity.

Diarrheal disease is a complex syndrome that remains a leading cause of global childhood morbidity and mortality. The diagnosis of enteric pathogens in a timely and precise manner is important for making treatment decisions and informing public health policy, but accurate diagnosis is a major challenge in industrializing countries. Multiplex molecular diagnostic techniques may represent a significant improvement over classical approaches. We evaluated the Luminex xTAG gastrointestinal pathogen panel (GPP) assay for the detection of common enteric bacterial and viral pathogens in Vietnam. Microbiological culture and real-time PCR were used as gold standards. The tests were performed on 479 stool samples collected from people admitted to the hospital for diarrheal disease throughout Vietnam. Sensitivity and specificity were calculated for the xTAG GPP for the seven principal diarrheal etiologies. The sensitivity and specificity for the xTAG GPP were >88% for Shigella spp., Campylobacter spp., rotavirus, norovirus genotype 1/2 (GI/GII), and adenovirus compared to those of microbiological culture and/or real-time PCR. However, the specificity was low (∼60%) for Salmonella species. Additionally, a number of important pathogens that are not identified in routine hospital procedures in this setting, such as Cryptosporidium spp. and Clostridium difficile , were detected with the GPP. The use of the Luminex xTAG GPP for the detection of enteric pathogens in settings, like Vietnam, would dramatically improve the diagnostic accuracy and capacity of hospital laboratories, allowing for timely and appropriate therapy decisions and a wider understanding of the epidemiology of pathogens associated with severe diarrheal disease in low-resource settings.

We found a high transfer ratio of anti-nontyphoidal Salmonella (NTS) antibodies from mothers to infants. Furthermore, despite a high seroincidence of NTS in infants, maternally acquired antibodies provided protection from seroconversion. Therefore, we propose prenatal immunization against NTS as a possible strategy for protecting infants from NTS disease., Background Nontyphoidal Salmonella (NTS) organisms are a major cause of gastroenteritis and bacteremia, but little is known about maternally acquired immunity and natural exposure in infant populations residing in areas where NTS disease is highly endemic. Methods We recruited 503 pregnant mothers and their infants (following delivery) from urban areas in Vietnam and followed infants until they were 1 year old. Exposure to the dominant NTS serovars, Salmonella enterica serovars Typhimurium and Enteritidis, were assessed using lipopolysaccharide (LPS) O antigen–specific antibodies. Antibody dynamics, the role of maternally acquired antibodies, and NTS seroincidence rates were modeled using multivariate linear risk factor models and generalized additive mixed-effect models. Results Transplacental transfer of NTS LPS–specific maternal antibodies to infants was highly efficient. Waning of transplacentally acquired NTS LPS–specific antibodies at 4 months of age left infants susceptible to Salmonella organisms, after which they began to seroconvert. High seroincidences of S. Typhimurium and S. Enteritidis LPS were observed, and infants born with higher anti-LPS titers had greater plasma bactericidal activity and longer protection from seroconversion. Conclusions Although Vietnamese infants have extensive exposure to NTS, maternally acquired antibodies appear to play a protective role against NTS infections during early infancy. These findings suggest that prenatal immunization may be an appropriate strategy to protect vulnerable infants from NTS disease.

We identified high antimicrobial usage and high antimicrobial resistance in children hospitalized with bloody and/or mucoid bacteria-associated diarrhea. There was no clinical benefit of antimicrobial therapy in children with diarrhea; adequately powered randomized controlled trials are required., Background Pediatric diarrheal disease presents a major public health burden in low- to middle-income countries. The clinical benefits of empirical antimicrobial treatment for diarrhea are unclear in settings that lack reliable diagnostics and have high antimicrobial resistance (AMR). Methods We conducted a prospective multicenter cross-sectional study of pediatric patients hospitalized with diarrhea containing blood and/or mucus in Ho Chi Minh City, Vietnam. Clinical parameters, including disease outcome and treatment, were measured. Shigella, nontyphoidal Salmonella (NTS), and Campylobacter were isolated from fecal samples, and their antimicrobial susceptibility profiles were determined. Statistical analyses, comprising log-rank tests and accelerated failure time models, were performed to assess the effect of antimicrobials on disease outcome. Results Among 3166 recruited participants (median age 10 months; interquartile range, 6.5–16.7 months), one-third (1096 of 3166) had bloody diarrhea, and 25% (793 of 3166) were culture positive for Shigella, NTS, or Campylobacter. More than 85% of patients (2697 of 3166) were treated with antimicrobials; fluoroquinolones were the most commonly administered antimicrobials. AMR was highly prevalent among the isolated bacteria, including resistance against fluoroquinolones and third-generation cephalosporins. Antimicrobial treatment and multidrug resistance status of the infecting pathogens were found to have no significant effect on outcome. Antimicrobial treatment was significantly associated with an increase in the duration of hospitalization with particular groups of diarrheal diseases. Conclusions In a setting with high antimicrobial usage and high AMR, our results imply a lack of clinical benefit for treating diarrhea with antimicrobials; adequately powered randomized controlled trials are required to assess the role of antimicrobials for diarrhea.

BACKGROUND: Shigella spp. are one of the most common causes of paediatric dysentery globally, responsible for a substantial proportion of diarrhoeal disease morbidity and mortality, particularly in industrialising regions. Alarming levels of antimicrobial resistance are now reported in S. flexneri and S. sonnei, hampering treatment options. Little is known, however, about the burden of infection and disease due to Shigella spp. in the community. METHODS/DESIGN: In order to estimate the incidence of this bacterial infection in the community in Ho Chi Minh City, Vietnam we have designed a longitudinal cohort to follow up approximately 700 children aged 12-60 months for two years with active and passive surveillance for diarrhoeal disease. Children will be seen at 6 month intervals for health checks where blood and stool samples will be collected. Families will also be contacted every two weeks for information on presence of diarrhoea in the child. Upon report of a diarrhoeal disease episode, study nurses will either travel to the family home to perform an evaluation or the family will attend a study hospital at a reduced cost, where a stool sample will also be collected. Case report forms collected at this time will detail information regarding disease history, risk factors and presence of disease in the household.Outcomes will include (i) age-specific incidence of Shigella spp. and other agents of diarrhoeal disease in the community, (ii) risk factors for identified aetiologies, (iii) rates of seroconversion to a host of gastrointestinal pathogens in the first few years of life. Further work regarding the longitudinal immune response to a variety of Shigella antigens, host genetics and candidate vaccine/diagnostic proteins will also be conducted. DISCUSSION: This is the largest longitudinal cohort with active surveillance designed specifically to investigate Shigella infection and disease. The study is strengthened by the active surveillance component, which will likely capture a substantial proportion of episodes not normally identified through passive or hospital-based surveillance. It is hoped that information from this study will aid in the design and implementation of Shigella vaccine trials in the future.