Search

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.

Despite the sporadic detection of fluoroquinolone-resistant Shigella in Asia in the early 2000s and the subsequent global spread of ciprofloxacin-resistant (cipR) Shigella sonnei from 2010, fluoroquinolones remain the recommended therapy for shigellosis1-7. The potential for cipR S. sonnei to develop resistance to alternative second-line drugs may further limit future treatment options8. Here, we aim to understand the evolution of novel antimicrobial resistant (AMR) S. sonnei variants after introduction into Vietnam. We found that cipR S. sonnei displaced the resident ciprofloxacin-susceptible (cipS) lineage while rapidly acquiring additional resistance to multiple alternative antimicrobial classes. We identified several independent acquisitions of extensively drug-resistant/multidrug-resistant-inducing plasmids, probably facilitated by horizontal transfer from commensals in the human gut. By characterizing commensal Escherichia coli from Shigella-infected and healthy children, we identified an extensive array of AMR genes and plasmids, including an identical multidrug-resistant plasmid isolated from both S. sonnei and E. coli in the gut of a single child. We additionally found that antimicrobial usage may impact plasmid transfer between commensal E. coli and S. sonnei. These results suggest that, in a setting with high antimicrobial use and a high prevalence of AMR commensals, cipR S. sonnei may be propelled towards pan-resistance by adherence to outdated international treatment guidelines.

The emergence of multi-drug (MDR) and extensive-drug resistance (XDR) in Salmonella Typhi and Paratyphi A hinder efficacious out-patient enteric fever treatment. We show that non-XDR and XDR S. Typhi and S. Paratyphi A are susceptible to the carbapenem tebipenem in vitro. Tebipenem demonstrated partial synergy with antimicrobials including azithromycin, signifying combination therapy may limit the emergence of resistance. Given recent evidence on tebipenem inhibitory activity against MDR Shigella, its broad-spectrum activity against MDR/XDR organisms renders it a good clinical trial candidate.

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. Here, aiming to identify alternative antimicrobial chemicals with activity against multi-drug resistant (MDR) Shigella, we initiated a collaborative academia-industry drug discovery project, applying high throughput phenotypic screening across broad chemical diversity. We identified several suitable compounds 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. 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. The orally bioavailable prodrug (tebipenem pivoxil) effectively cleared the gut of infecting organisms when administered in physiological doses to Shigella-infected mice and gnotobiotic piglets. Our data highlight the utility of broad compound screening for tackling the emerging antimicrobial resistance crisis and shows that tebipenem pivoxil (licenced for paediatric respiratory tract infections in Japan) could be repurposed as an effective treatment for severe diarrhoea caused by MDR Shigella and other enteric pathogens in LMICs.

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.

Non-typhoidal Salmonella (NTS) serovars, sequences types and antimicrobial susceptibility profiles have specific associations with animal and human infections in Vietnam. Antimicrobial resistance may have an effect on the manifestation of human NTS infections, with isolates from asymptomatic individuals being more susceptible to antimicrobials than those associated with animals and human diarrhoea.

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.

Non-typhoidal Salmonella (NTS) serovars, sequences types and antimicrobial susceptibility profiles have specific associations with animal and human infections in Vietnam. Antimicrobial resistance may have an effect on the manifestation of human NTS infections, with isolates from asymptomatic individuals being more susceptible to antimicrobials than those associated with animals and human diarrhoea.

Background Nontyphoidal Salmonella (NTS) are associated with both diarrhea and bacteremia. Antimicrobial resistance (AMR) is common in NTS in low-middle income countries, but the major source(s) of AMR NTS in humans are not known. Here, we aimed to assess the role of animals as a source of AMR in human NTS infections in Vietnam. We retrospectively combined and analyzed 672 NTS human and animal isolates from four studies in southern Vietnam and compared serovars, sequence types (ST), and AMR profiles. We generated a population structure of circulating organisms and aimed to attribute sources of AMR in NTS causing invasive and noninvasive disease in humans using Bayesian multinomial mixture models. Results Among 672 NTS isolates, 148 (22%) originated from human blood, 211 (31%) from human stool, and 313 (47%) from animal stool. The distribution of serovars, STs, and AMR profiles differed among sources; serovars Enteritidis, Typhimurium, and Weltevreden were the most common in human blood, human stool, and animals, respectively. We identified an association between the source of NTS and AMR profile; the majority of AMR isolates were isolated from human blood (p, Highlights • The major source(s) of antimicrobial resistance (AMR) in nontyphoidal Salmonella (NTS) are unknown. • We analyzed 672 NTS isolates, 148 (41%) from human blood, 211 (59%) from human stool, and 136 (43%) from animal stool. • Enteritidis, Typhimurium, and Weltevreden were the common serovars in human blood, human stool, and animals, respectively. • The majority of AMR isolates were associated with human blood. • NTS from animals does explain the full extent of AMR in human NTS infections.

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.

The global dissemination of a ciprofloxacin-resistant (cipR)S. sonneiclone outlines the mobility of this important agent of diarrheal disease, and threatens the utility of ciprofloxacin as a first-line antimicrobial for shigellosis. Here, we aimed to track the emergence of cipRS. sonneiin Vietnam to understand how novel antimicrobial resistant (AMR)Shigellaclones become established in new locations. From 2014 to 2016, we isolated and genome sequenced 79S. sonneifrom children hospitalized with dysenteric diarrhea in southern Vietnam. The novel cipRS. sonneiclone displaced the resident ciprofloxacin-susceptible lineage while acquiring resistance against third-generation cephalosporins, macrolides, and aminoglycosides. This process was not the result of a single clonal expansion, as we identified at least thirteen independent acquisitions of ESBL-encoding plasmids. The frequency and diversity of the variable AMR repertoire in an expanding clonal background ofS. sonneiis unprecedented and we speculated that it was facilitated by horizontal gene transfer from commensal organisms in the human gut. Consequently, we characterized non-ShigellaEnterobacteriaceae fromShigella-infected and healthy children by shotgun metagenomics. We identified a wide array of AMR genes and plasmids in the commensal Enterobacteriaceae, including anE. coliisolated from aShigella-infected child with an identical ESBL plasmid to that characterized in the infectingS. sonnei. We confirmed that these AMR plasmids could be exchanged between commensalE. coliandS. sonneiand found that supplementation of ciprofloxacin into the conjugation media significantly increased the conjugation frequency of IncI/blaCTX-M-15, IncB/O/blaCTX-M-27and IncF/blaCTX-M-27plasmids. In a setting with high antimicrobial use and a high prevalence of AMR commensals, cipRS. sonneimay be propelled towards pan-resistance by adherence to outdated international treatment guidelines. Our work highlights the role of the gut microbiota in transferring resistance plasmids into enteric pathogens and provides essential data to restrict the use of ciprofloxacin globally.

OBJECTIVES: The objective of this study was to assess the presence of mcr-1 in Shigella sonnei isolated in Vietnam. METHODS: WGS data were analysed for the presence of the mcr-1 gene sequence. The association of mcr-1 with a plasmid was assessed by PCR and by conjugation. RESULTS: Through genome sequencing we identified a plasmid-associated inactive form of mcr-1 in a 2008 Vietnamese isolate of Shigella sonnei. The plasmid was conjugated into Escherichia coli and mcr-1 was activated upon exposure to colistin, resulting in highly colistin-resistant transconjugants. CONCLUSIONS: This is the first description of the mcr-1 gene in Shigella, which is atypical given that colistin is not ordinarily used to treat diarrhoea. Our data suggest the mcr-1 gene has been circulating in human-restricted pathogens for some time but likely carries a selective fitness cost.

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.

Nontyphoidal Salmonella (NTS), particularly Salmonella enterica serovar Typhimurium, is among the leading etiologic agents of bacterial enterocolitis globally and a well-characterized cause of invasive disease (iNTS) in sub-Saharan Africa. In contrast, S. Typhimurium is poorly defined in Southeast Asia, a known hot spot for zoonotic disease with a recently described burden of iNTS disease. Here, we aimed to add insight into the epidemiology and potential impact of zoonotic transfer and antimicrobial resistance (AMR) in S. Typhimurium associated with iNTS and enterocolitis in Vietnam. We performed whole-genome sequencing and phylogenetic reconstruction on 85 human (enterocolitis, carriage, and iNTS) and 113 animal S. Typhimurium isolates isolated in Vietnam. We found limited evidence for the zoonotic transmission of S. Typhimurium. However, we describe a chain of events where a pandemic monophasic variant of S. Typhimurium (serovar I:4,[5],12:i:− sequence type 34 [ST34]) has been introduced into Vietnam, reacquired a phase 2 flagellum, and acquired an IncHI2 multidrug-resistant plasmid. Notably, these novel biphasic ST34 S. Typhimurium variants were significantly associated with iNTS in Vietnamese HIV-infected patients. Our study represents the first characterization of novel iNTS organisms isolated outside sub-Saharan Africa and outlines a new pathway for the emergence of alternative Salmonella variants into susceptible human populations. IMPORTANCE Salmonella Typhimurium is a major diarrheal pathogen and associated with invasive nontyphoid Salmonella (iNTS) disease in vulnerable populations. We present the first characterization of iNTS organisms in Southeast Asia and describe a different evolutionary trajectory from that of organisms causing iNTS in sub-Saharan Africa. In Vietnam, the globally distributed monophasic variant of Salmonella Typhimurium, the serovar I:4,[5],12:i:− ST34 clone, has reacquired a phase 2 flagellum and gained a multidrug-resistant plasmid to become associated with iNTS disease in HIV-infected patients. We document distinct communities of S. Typhimurium and I:4,[5],12:i:− in animals and humans in Vietnam, despite the greater mixing of these host populations here. These data highlight the importance of whole-genome sequencing surveillance in a One Health context in understanding the evolution and spread of resistant bacterial infections.

Background Broad-spectrum antimicrobials are commonly used as empirical therapy for infections of presumed bacterial origin. Increasing resistance to these antimicrobial agents has prompted the need for alternative therapies and more effective surveillance. Better surveillance leads to more informed and improved delivery of therapeutic interventions, potentially leading to better treatment outcomes. Methods We screened 1017 Gram negative bacteria (excluding Pseudomonas spp. and Acinetobacter spp.) isolated between 2011 and 2013 from positive blood cultures for susceptibility against third generation cephalosporins, ESBL and/or AmpC production, and associated ESBL/AmpC genes, at the Hospital for Tropical Diseases in Ho Chi Minh City. Results Phenotypic screening found that 304/1017 (30%) organisms were resistance to third generation cephalosporins; 172/1017 (16.9%) of isolates exhibited ESBL activity, 6.2% (63/1017) had AmpC activity, and 0.5% (5/1017) had both ESBL and AmpC activity. E. coli and Aeromonas spp. were the most common organisms associated with ESBL and AmpC phenotypes, respectively. Nearly half of the AmpC producers harboured an ESBL gene. There was no significant difference (p > 0.05) between the antimicrobial resistance phenotypes of the organisms associated with community and hospital-acquired infections. Conclusion AmpC and ESBL producing organisms were commonly associated with bloodstream infections in this setting, with antimicrobial resistant organisms being equally distributed between infections originating from the community and healthcare settings. Aeromonas spp., which was associated with bloodstream infections in cirrhotic/hepatitis patients, were the most abundant AmpC producing organism. We conclude that empirical monotherapy with third generation cephalosporins may not be optimum in this setting. Electronic supplementary material The online version of this article (10.1186/s13756-017-0265-1) contains supplementary material, which is available to authorized users.

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.

Background: Enteroaggregative (EAEC) and Shiga-toxin producing Escherichia coli (STEC) are a major cause of diarrhea worldwide. E. coli carrying both virulence factors characteristic for EAEC and STEC and producing extended-spectrum beta-lactamase caused severe and protracted disease during an outbreak of E. coli O104:H4 in Europe in 2011. We assessed the opportunities for E. coli carrying the aggR and stx genes to emerge in ‘backyard’ farms in south-east Asia.Results: Faecal samples collected from 204 chicken farms; 204 farmers and 306 age- and gender-matched individuals not exposed to poultry farming were plated on MacConkey agar plates with and without antimicrobials being supplemented. Sweep samples obtained from MacConkey agar plates without supplemented antimicrobials were screened by multiplex PCR for the detection of the stx1, stx2 and aggR genes. One chicken farm sample each (0.5 %) contained the stx1 and the aggR gene. Eleven (2.4 %) human faecal samples contained the stx1 gene, 2 samples (0.4 %) contained stx2 gene, and 31 (6.8 %) contained the aggR gene. From 46 PCR-positive samples, 205 E. coli isolates were tested for the presence of stx1, stx2, aggR, wzx O104 and fliC H4 genes. None of the isolates simultaneously contained the four genetic markers associated with E. coli O104:H4 epidemic strain (aggR, stx2, wzx O104 and fliC H4 ). Of 34 EAEC, 64.7 % were resistant to 3rd-generation cephalosporins.Conclusion: These results indicate that in southern Vietnam, the human population is a more likely reservoir of aggR and stx gene carrying E. coli than the chicken population. However, conditions for transmission of isolates and/or genes between human and animal reservoirs resulting in the emergence of highly virulent E. coli strains are still favorable, given the nature of‘backyard’ farms in Vietnam.

The toxigenic bacterium Vibrio cholerae belonging to the O1 and O139 serogroups is commonly associated with epidemic diarrhea in tropical settings; other diseases caused by this environmental pathogen are seldom identified. Here we report two unassociated cases of nonfatal, nontoxigenic V. cholerae non-O1, non-O139 bacteremia in patients with comorbidities in Ho Chi Minh City, Vietnam, that occurred within a 4-week period.

Acinetobacter baumannii has become one of the major infection threats in intensive care units (ICUs) globally. Since 2008, A. baumannii has been the leading cause of ventilator-associated pneumonia (VAP) in our ICU at an infectious disease hospital in southern Vietnam. The emergence of this pathogen in our setting is consistent with the persistence of a specific clone exhibiting resistance to carbapenems. Antimicrobial combinations may be a strategy to treat infections caused by these carbapenem-resistant A. baumannii. Therefore, we assessed potential antimicrobial combinations against local carbapenem-resistant A. baumannii by measuring in vitro interactions of colistin with four antimicrobials that are locally certified for treating VAP. We first performed antimicrobial susceptibility testing and multilocus variable number tandem repeat analysis (MLVA) genotyping on 74 A. baumannii isolated from quantitative tracheal aspirates from patients with VAP over an 18-month period. These 74 isolates could be subdivided into 21 main clusters by MLVA and >80 % were resistant to carbapenems. We selected 56 representative isolates for in vitro combination synergy testing. Synergy was observed in four (7 %), seven (13 %), 20 (36 %) and 38 (68 %) isolates with combinations of colistin with ceftazidime, ceftriaxone, imipenem and meropenem, respectively. Notably, more carbapenem-resistant A. baumannii isolates (36/43; 84 %) exhibited synergistic activity with a combination of colistin and meropenem than carbapenem-susceptible A. baumannii isolates (2/13; 15 %) (P = 0.023; Fisher's exact test). Our findings suggest that combinations of colistin and meropenem should be considered when treating carbapenem-resistant A. baumannii infections in Vietnam, and we advocate clinical trials investigating combination therapy for VAP.

Plasmid-mediated quinolone resistance (PMQR) refers to a family of closely related genes that confer decreased susceptibility to fluoroquinolones. PMQR genes are generally associated with integrons and/or plasmids that carry additional antimicrobial resistance genes active against a range of antimicrobials. In Ho Chi Minh City (HCMC), Vietnam, we have previously shown a high frequency of PMQR genes within commensal Enterobacteriaceae. However, there are limited available sequence data detailing the genetic context in which the PMQR genes reside, and a lack of understanding of how these genes spread across the Enterobacteriaceae. Here, we aimed to determine the genetic background facilitating the spread and maintenance of qnrS1, the dominant PMQR gene circulating in HCMC. We sequenced three qnrS1-carrying plasmids in their entirety to understand the genetic context of these qnrS1-embedded plasmids and also the association of qnrS1-mediated quinolone resistance with other antimicrobial resistance phenotypes. Annotation of the three qnrS1-containing plasmids revealed a qnrS1-containing transposon with a closely related structure. We screened 112 qnrS1-positive commensal Enterobacteriaceae isolated in the community and in a hospital in HCMC to detect the common transposon structure. We found the same transposon structure to be present in 71.4 % (45/63) of qnrS1-positive hospital isolates and in 36.7 % (18/49) of qnrS1-positive isolates from the community. The resulting sequence analysis of the qnrS1 environment suggested that qnrS1 genes are widely distributed and are mobilized on elements with a common genetic background. Our data add additional insight into mechanisms that facilitate resistance to multiple antimicrobials in Gram-negative bacteria in Vietnam.

We performed a prospective multicenter study to address the lack of data on the etiology, clinical and demographic features of hospitalized pediatric diarrhea in Ho Chi Minh City (HCMC), Vietnam. Over 2,000 (1,419 symptomatic and 609 non-diarrheal control) children were enrolled in three hospitals over a 1-year period in 2009–2010. Aiming to detect a panel of pathogens, we identified a known diarrheal pathogen in stool samples from 1,067/1,419 (75.2%) children with diarrhea and from 81/609 (13.3%) children without diarrhea. Rotavirus predominated in the symptomatic children (664/1,419; 46.8%), followed by norovirus (293/1,419; 20.6%). The bacterial pathogens Salmonella, Campylobacter, and Shigella were cumulatively isolated from 204/1,419 (14.4%) diarrheal children and exhibited extensive antimicrobial resistance, most notably to fluoroquinolones and third-generation cephalosporins. We suggest renewed efforts in generation and implementation of policies to control the sale and prescription of antimicrobials to curb bacterial resistance and advise consideration of a subsidized rotavirus vaccination policy to limit the morbidity due to diarrheal disease in Vietnam.

Ventilator-associated pneumonia (VAP) is a serious healthcare-associated infection that affects up to 30 % of intubated and mechanically ventilated patients in intensive care units (ICUs) worldwide. The bacterial aetiology and corresponding antimicrobial susceptibility of VAP is highly variable, and can differ between countries, national provinces and even between different wards in the same hospital. We aimed to understand and document changes in the causative agents of VAP and their antimicrobial susceptibility profiles retrospectively over an 11 year period in a major infectious disease hospital in southern Vietnam. Our analysis outlined a significant shift from Pseudomonas aeruginosa to Acinetobacter spp. as the most prevalent bacteria isolated from quantitative tracheal aspirates in patients with VAP in this setting. Antimicrobial resistance was common across all bacterial species and we found a marked proportional annual increase in carbapenem-resistant Acinetobacter spp. over a 3 year period from 2008 (annual trend; odds ratio 1.656, P = 0.010). We further investigated the possible emergence of a carbapenem-resistant Acinetobacter baumannii clone by multiple-locus variable number tandem repeat analysis, finding a bla OXA-23-positive strain that was associated with an upsurge in the isolation of this pathogen. We additionally identified a single bla NDM-1-positive A. baumannii isolate. This work highlights the emergence of a carbapenem-resistant clone of A. baumannii and a worrying trend of antimicrobial resistance in the ICU of the Hospital for Tropical Diseases in Ho Chi Minh City, Vietnam.