Flagella-related gene mutations in Vibrio cholerae during extended cultivation in nutrient-limited media impair cell motility and prolong culturability.

IMPORTANCE: Vibrio cholerae undergoes a transition to a viable but non-culturable (VNC) state when subjected to various environmental stresses. We showed here that flagellar motility was involved in the development of the VNC state of V. cholerae. In this study, motility-defective isolates with mutations in various flagella-related genes, but not motile isolates, were predominantly obtained under the stress of long-term batch culture. Other genomic regions were highly conserved, suggesting that the mutations were selective. During the stationary phase of long-term culture, V. cholerae isolates with mutations in the acetate kinase and flagella-related genes were predominant. This study suggests that genes involved in specific functions in V. cholerae undergo mutations under certain environmental conditions.

Spatiotemporal variation in risk of Shigella infection in childhood: a global risk mapping and prediction model using individual participant data.

BACKGROUND: Diarrhoeal disease is a leading cause of childhood illness and death globally, and Shigella is a major aetiological contributor for which a vaccine might soon be available. The primary objective of this study was to model the spatiotemporal variation in paediatric Shigella infection and map its predicted prevalence across low-income and middle-income countries (LMICs). METHODS: Individual participant data for Shigella positivity in stool samples were sourced from multiple LMIC-based studies of children aged 59 months or younger. Covariates included household-level and participant-level factors ascertained by study investigators and environmental and hydrometeorological variables extracted from various data products at georeferenced child locations. Multivariate models were fitted and prevalence predictions obtained by syndrome and age stratum. FINDINGS: 20 studies from 23 countries (including locations in Central America and South America, sub-Saharan Africa, and south and southeast Asia) contributed 66 563 sample results. Age, symptom status, and study design contributed most to model performance followed by temperature, wind speed, relative humidity, and soil moisture. Probability of Shigella infection exceeded 20% when both precipitation and soil moisture were above average and had a 43% peak in uncomplicated diarrhoea cases at 33°C temperatures, above which it decreased. Compared with unimproved sanitation, improved sanitation decreased the odds of Shigella infection by 19% (odds ratio [OR]=0·81 [95% CI 0·76-0·86]) and open defecation decreased them by 18% (OR=0·82 [0·76-0·88]). INTERPRETATION: The distribution of Shigella is more sensitive to climatological factors, such as temperature, than previously recognised. Conditions in much of sub-Saharan Africa are particularly propitious for Shigella transmission, although hotspots also occur in South America and Central America, the Ganges-Brahmaputra Delta, and the island of New Guinea. These findings can inform prioritisation of populations for future vaccine trials and campaigns. FUNDING: NASA, National Institutes of Health-The National Institute of Allergy and Infectious Diseases, and Bill & Melinda Gates Foundation.

Emergence of Vibrio parahaemolyticus serotype O10:K4 in Thailand.

An emerging serotype O10:K4 of Vibrio parahaemolyticus has been predominantly isolated from outbreaks and sporadic cases in China. Herein, we report the first case of infection due to V. parahaemolyticus O10:K4 isolated from a hospitalized patient with acute diarrhea in Thailand. We sequenced the whole genome of the O10:K4 strain and compared it with those of the pandemic O3:K6 strain, O10:K4 strains in China, and other clinical and environmental strains. The results suggested that the O10:K4 strains are not a mere serotype variant diverged from the pandemic O3:K6 strain, confirming that the O10:K4 strain emergence has spread to Southeast Asia.

Associations Between Eight Earth Observation-Derived Climate Variables and Enteropathogen Infection: An Independent Participant Data Meta-Analysis of Surveillance Studies With Broad Spectrum Nucleic Acid Diagnostics.

Diarrheal disease, still a major cause of childhood illness, is caused by numerous, diverse infectious microorganisms, which are differentially sensitive to environmental conditions. Enteropathogen-specific impacts of climate remain underexplored. Results from 15 studies that diagnosed enteropathogens in 64,788 stool samples from 20,760 children in 19 countries were combined. Infection status for 10 common enteropathogens-adenovirus, astrovirus, norovirus, rotavirus, sapovirus, Campylobacter, ETEC, Shigella, Cryptosporidium and Giardia-was matched by date with hydrometeorological variables from a global Earth observation dataset-precipitation and runoff volume, humidity, soil moisture, solar radiation, air pressure, temperature, and wind speed. Models were fitted for each pathogen, accounting for lags, nonlinearity, confounders, and threshold effects. Different variables showed complex, non-linear associations with infection risk varying in magnitude and direction depending on pathogen species. Rotavirus infection decreased markedly following increasing 7-day average temperatures-a relative risk of 0.76 (95% confidence interval: 0.69-0.85) above 28°C-while ETEC risk increased by almost half, 1.43 (1.36-1.50), in the 20-35°C range. Risk for all pathogens was highest following soil moistures in the upper range. Humidity was associated with increases in bacterial infections and decreases in most viral infections. Several virus species' risk increased following lower-than-average rainfall, while rotavirus and ETEC increased with heavier runoff. Temperature, soil moisture, and humidity are particularly influential parameters across all enteropathogens, likely impacting pathogen survival outside the host. Precipitation and runoff have divergent associations with different enteric viruses. These effects may engender shifts in the relative burden of diarrhea-causing agents as the global climate changes.

Corrigendum to: Phylogenetic Analysis Revealed the Dissemination of Closely Related Epidemic Vibrio cholerae O1 Isolates in Laos, Thailand, and Vietnam.

[This corrects the article DOI: 10.1093/ofid/ofaa492.].

Phylogenetic Analysis Revealed the Dissemination of Closely Related Epidemic Vibrio cholerae O1 Isolates in Laos, Thailand, and Vietnam.

We performed whole-genome sequencing of Vibrio cholerae O1 isolates from Laos, Thailand, and Vietnam, where cholera outbreaks occurred, to determine their genetic lineages. Core genome phylogenetic analysis revealed that the isolates located in same lineage without regional clusters, which suggests that closely related strains circulated in Southeast Asia.

Etiologic features of diarrheagenic microbes in stool specimens from patients with acute diarrhea in Thailand.

Many microbial species have been recognized as enteropathogens for humans. Here, we predicted the causative agents of acute diarrhea using data from multiplex quantitative PCR (qPCR) assays targeting 19 enteropathogens. For this, a case-control study was conducted at eight hospitals in Thailand. Stool samples and clinical data were collected from 370 hospitalized patients with acute diarrhea and 370 non-diarrheal controls. Multiple enteropathogens were detected in 75.7% and 13.0% of diarrheal stool samples using multiplex qPCR and bacterial culture methods, respectively. Asymptomatic carriers of enteropathogens were found among 87.8% and 45.7% of individuals by qPCR and culture methods, respectively. These results suggested the complexity of identifying causative agents of diarrhea. An analysis using the quantification cut-off values for clinical relevance drastically reduced pathogen-positive stool samples in control subjects from 87.8% to 0.5%, whereas 48.9% of the diarrheal stool samples were positive for any of the 11 pathogens. Among others, rotavirus, norovirus GII, Shigella/EIEC, and Campylobacter were strongly associated with acute diarrhea (P-value < 0.001). Characteristic clinical symptoms, epidemic periods, and age-related susceptibility to infection were observed for some enteropathogens. Investigations based on qPCR approaches covering a broad array of enteropathogens might thus improve our understanding of diarrheal disease etiology and epidemiological trends.

Rapid screening and early precautions for carbapenem-resistant Acinetobacter baumannii carriers decreased nosocomial transmission in hospital settings: a quasi-experimental study.

Background: Active surveillance has the potential to prevent nosocomial transmission of carbapenem-resistant Acinetobacter baumannii (CRAB). We assessed whether rapid diagnosis using clinical specimen-direct loop-mediated isothermal amplification (LAMP), a rapid molecular diagnostic assay, and subsequent intervention, could reduce CRAB nosocomial transmission in intensive care units (ICUs). Methods: A before and after (quasi-experimental) study was conducted in two ICUs at the Mahidol University Faculty of Medicine Ramathibodi Hospital with 3 months of observational period followed by 9 months of interventional period. All patients were screened for CRAB using both the culture and LAMP method from rectal swab and/or bronchial aspirates (intubated patients only) upon admission, weekly thereafter, and upon discharge. During the pre-intervention period, we performed contact precautions based on culture results. In contrast, during the intervention period, we initiated contact precautions within a few hours after sample collection on the basis of LAMP results. Results: A total of 1335 patients were admitted to the ICUs, of which 866 patients (pre-intervention period: 187; intervention period: 679) were eligible for this study. Incidence rate of CRAB infection decreased to 20.9 per 1000 patient-days in the intervention period from 35.2 in the pre-intervention period (P < 0.02). The calculated hazard ratio of CRAB transmission was 0.65 (95% confidence interval [CI], 0.44-0.97). Risk factors for CRAB acquisition included exposure to carbapenem (hazard ratio, 2.54 [95% CI: 1.61-5.57]). Conclusions: LAMP screening for CRAB upon ICU admission proved feasible for routine clinical practice. Rapid screening using LAMP followed by early intervention may reduce CRAB transmission rates in ICUs when compared to conventional intervention.

Incidence of metachronous gastric cancer in patients whose primary gastric neoplasms were discovered after Helicobacter pylori eradication.

BACKGROUND AND AIMS: The incidence of metachronous gastric cancer (MGC) in patients whose primary gastric neoplasm is discovered after Helicobacter pylori eradication remains unclear. Here, we evaluated the long-term effect of previous H pylori eradication on development of MGC after endoscopic submucosal dissection (ESD). METHODS: We analyzed prospectively collected data of consecutive patients with successful H pylori eradication more than 1 year before (eradicated group, 180 patients) or after (control group, 602 patients) initial curative ESD. These patients were also followed by endoscopy for over 2 years. Propensity score matching and inverse probability of treatment weighting (IPTW) were used to adjust for confounding variables during data analysis. The main outcome was the incidence of MGC after initial ESD. RESULTS: In a propensity-matched analysis of 174 pairs, the incidence of MGC was similar in the 2 cohorts (33.9 per 1000 person-years vs 40.8 per 1000 person-years in the control group, P = .454) at a median follow-up of 4.1 years (interquartile range, 3.0-5.6). Incidences were also similar in the 2 groups when data were analyzed using IPTW, even after exclusion of 123 patients with successful H pylori eradication <5 years before initial ESD. Multiple Cox regression analysis revealed age, differentiated-type histology, and initial multiplicity were predictors of MGC in patients after initial curative ESD. CONCLUSIONS: The frequency of follow-up surveillance after initial curative ESD should be kept constant, irrespective of whether H pylori eradication is performed before or after initial curative ESD.

Draft genome sequence of a colistin-resistant Escherichia coli ST226: A clinical strain harbouring an mcr-1 variant.

OBJECTIVES: Escherichia coli isolates carrying the mcr-1 gene are rarely reported in diarrhoeal patients. Here we report the draft genome sequence of a colistin-resistant E. coli isolated from a hospitalised patient with acute diarrhoea in Thailand. METHODS: Whole genomic DNA of the colistin-resistant E. coli isolate (MSF11) was extracted and was sequenced using an Ion Torrent sequencer with 400-bp read chemistry. The draft genome sequence of MSF11 was analysed with regard to multilocus sequence type (ST), serotype, acquired antimicrobial resistance genes, plasmid replicon types and virulence genes using tools from the Center for Genomic Epidemiology. RESULTS: E. coli strain MSF11 was serotype OUT:H10 and ST226. Acquired antimicrobial resistance genes [blaCTX-M-15, qnrS1, catA2, mdf(A) and mcr-1.1] and virulence-related genes (astA and gad) were identified. The mcr-1 gene contained a single nucleotide polymorphism at position 27 (C→T) of the prototype, and the variant gene was associated with an IncX4-type plasmid. This plasmid-borne colistin resistance mediated by the mcr-1 variant has been observed among colistin-resistant strains from humans, animals and the environment previously reported in Thailand, although the STs and serotypes of the E. coli strains were different. CONCLUSIONS: An mcr-1 variant was identified in an E. coli isolate harbouring the EAST1 (enteroaggregative E. coli heat-stable toxin 1) gene (astA) from a human diarrhoeal stool specimen. This study highlights the potential risk of dissemination of colistin-resistant E. coli in view of the prevalence of the variant gene on IncX4-type plasmids.

Simultaneous detection and quantification of 19 diarrhea-related pathogens with a quantitative real-time PCR panel assay.

Acute diarrheal diseases are causes of global public health concern, especially in developing countries. A variety of diarrhea-associated microbial species, including bacteria, viruses, and protozoa, have been recognized. Simplified methods for detecting a wide range of diarrheagenic enteric microbes can clarify the etiology and aid in the diagnosis of diarrheal diseases. Here, we report a quantitative real-time (q)PCR-based method for simultaneous detection of 24 targets from 19 microbes suspected of causing diarrhea in stool specimens. We first selected the 24 oligonucleotide primer sets and hydrolysis probes conjugated with the fluorescent reporter dyes FAM, NED, or ABY, along with an internal control, and the passive reference dye ROX to establish a single-plate panel assay. The 12-duplex qPCR panel showed high linearity, with R2 values of 0.981-1.0 and limits of detection ranging from 1 to 103 fg for bacterial DNA (1-200 cells), 10-102 copies for viral DNA/RNA, and 10 fg for parasitic DNA (equivalent to approximately 1 parasite) per reaction. The accuracy and robustness of the assay was demonstrated in experiments using clinical stool specimens. This platform is low cost and easily customizable, and can be applied to various types of qPCR instruments and experimental designs for surveillance of acute diarrhea.

Vibrio cholerae embraces two major evolutionary traits as revealed by targeted gene sequencing.

Vibrio cholerae inhabits aquatic environments worldwide and has over 200 recognized serogroups classified by O-polysaccharide specificity. Here, we report that V. cholerae selects either of two genetic traits during their evolution. Sequencing of the specific gene locus MS6_A0927 revealed that 339 of 341 strains of V. cholerae and closely related Vibrio species originating from 34 countries over a century carried either metY (M) (~1,269 bp) or luxR-hchA (LH) (~1,600 bp) genes, and consequently those vibrios were separated into two clusters, M (45.4%) and LH (54.6%). Only two strains contained both M and LH in the same locus. Moreover, extensive polymorphisms in those genes were detected in M and LH with 79 and 46 sequence variations, respectively. V. cholerae O1 strains isolated from cholera outbreaks worldwide, and some non-O1 strains evolving from O1 via exchange of genes encoding cell surface polysaccharides possessed LH alleles. Analysis of polymorphisms in the gene locus implicated a high degree of genetic diversity and identical subpopulations among the V. cholerae species.

Genetic characterization of blaNDM-harboring plasmids in carbapenem-resistant Escherichia coli from Myanmar.

The bacterial enzyme New Delhi metallo-ß-lactamase hydrolyzes almost all ß-lactam antibiotics, including carbapenems, which are drugs of last resort for severe bacterial infections. The spread of carbapenem-resistant Enterobacteriaceae that carry the New Delhi metallo-ß-lactamase gene, blaNDM, poses a serious threat to public health. In this study, we genetically characterized eight carbapenem-resistant Escherichia coli isolates from a tertiary care hospital in Yangon, Myanmar. The eight isolates belonged to five multilocus-sequence types and harbored multiple antimicrobial-resistance genes, resulting in resistance against nearly all of the antimicrobial agents tested, except colistin and fosfomycin. Nine plasmids harboring blaNDM genes were identified from these isolates. Multiple blaNDM genes were found in the distinct Inc-replicon types of the following plasmids: an IncA/C2 plasmid harboring blaNDM-1 (n = 1), IncX3 plasmids harboring blaNDM-4 (n = 2) or blaNDM-7 (n = 1), IncFII plasmids harboring blaNDM-4 (n = 1) or blaNDM-5 (n = 3), and a multireplicon F plasmid harboring blaNDM-5 (n = 1). Comparative analysis highlighted the diversity of the blaNDM-harboring plasmids and their distinct characteristics, which depended on plasmid replicon types. The results indicate circulation of phylogenetically distinct strains of carbapenem-resistant E. coli with various plasmids harboring blaNDM genes in the hospital.

Molecular Epidemiology of Cholera Outbreaks during the Rainy Season in Mandalay, Myanmar.

Cholera, caused by Vibrio cholerae, remains a global threat to public health. In Myanmar, the availability of published information on the occurrence of the disease is scarce. We report here that cholera incidence in Mandalay generally exhibited a single annual peak, with an annual average of 312 patients with severe dehydration over the past 5 years (since 2011) and was closely associated with the rainy season. We analyzed cholera outbreaks, characterized 67 isolates of V. cholerae serogroup O1 in 2015 from patients from Mandalay, and compared them with 22 V. cholerae O1 isolates (12 from Mandalay and 10 from Yangon) in 2014. The isolates carried the classical cholera toxin B subunit (ctxB), the toxin-coregulated pilus A (tcpA) of Haitian type, and repeat sequence transcriptional regulator (rstR) of El Tor type. Two molecular typing methods, pulsed-field gel electrophoresis and multiple-locus variable-number tandem repeat analysis (MLVA), differentiated the 89 isolates into seven pulsotypes and 15 MLVA profiles. Pulsotype Y15 and one MLVA profile (11, 7, 7, 16, 7) were predominantly found in the isolates from cholera outbreaks in Mandalay, 2015. Pulsotypes Y11, Y12, and Y15 with some MLVA profiles were detected in the isolates from two remote areas, Mandalay and Yangon, with temporal changes. These data suggested that cholera spread from the seaside to the inland area in Myanmar.

Comparative genomic characterization of a Thailand-Myanmar isolate, MS6, of Vibrio cholerae O1 El Tor, which is phylogenetically related to a "US Gulf Coast" clone.

BACKGROUND: The cholera outbreaks in Thailand during 2007-2010 were exclusively caused by the Vibrio cholerae O1 El Tor variant carrying the cholera toxin gene of the classical biotype. We previously isolated a V. cholerae O1 El Tor strain from a patient with diarrhea and designated it MS6. Multilocus sequence-typing analysis revealed that MS6 is most closely related to the U. S. Gulf Coast clone with the exception of two novel housekeeping genes. METHODOLOGY/PRINCIPAL FINDINGS: The nucleotide sequence of the genome of MS6 was determined and compared with those of 26 V. cholerae strains isolated from clinical and environmental sources worldwide. We show here that the MS6 isolate is distantly related to the ongoing seventh pandemic V. cholerae O1 El Tor strains. These strains differ with respect to polymorphisms in housekeeping genes, seventh pandemic group-specific markers, CTX phages, two genes encoding predicted transmembrane proteins, the presence of metY (MS6_A0927) or hchA/luxR in a highly conserved region of the V. cholerae O1 serogroup, and a superintegron (SI). We found that V. cholerae species carry either hchA/luxR or metY and that the V. cholerae O1 clade commonly possesses hchA/luxR, except for MS6 and U. S. Gulf Coast strains. These findings illuminate the evolutionary relationships among V. cholerae O1 strains. Moreover, the MS6 SI carries a quinolone-resistance gene cassette, which was closely related with those present in plasmid-borne integrons of other gram-negative bacteria. CONCLUSIONS/SIGNIFICANCE: Phylogenetic analysis reveals that MS6 is most closely related to a U. S. Gulf Coast clone, indicating their divergence before that of the El Tor biotype strains from a common V. cholerae O1 ancestor. We propose that MS6 serves as an environmental aquatic reservoir of V. cholerae O1.

Safety, efficacy, and long-term outcomes for endoscopic submucosal dissection of early esophagogastric junction cancer.

BACKGROUND: Early esophagogastric junction (EGJ) cancer is currently being treated in the same way as early gastric cancer, by endoscopic submucosal dissection (ESD), but long-term outcomes are still unknown. Our aim was to retrospectively evaluate the safety and efficacy of ESD in treating early EGJ cancer and compare risk factors in curative and non-curative resection cases. METHODS: Forty-four cases of early EGJ cancer, defined as a Siewert's type II tumor, in 44 patients with a mean age of 70.0 years and a male/female ratio of 90.9:9.1 % were treated by ESD between January 2004 and June 2010. There were 30 standard indication cases; the remaining 14 cases were expanded indication cases. RESULTS: Mean resected specimen and tumor sizes were 35 and 17 mm, respectively, and median procedure time was 121 min, with no bleeding or perforation complications. All cases were resected en bloc with an 84.1 % curative resection rate (37/44). The curative resection rates in the standard and expanded indication cases were 90.0 % (27/30) and 71.4 % (10/14), respectively. There were no significant differences in tumor location, tumor morphology, tumor size, histology of biopsy specimens, or standard versus expanded indication cases with regard to risk factors for curative and non-curative resections. However, submucosal invasion, positive tumor margins, lymphovascular invasion, and some components of poorly differentiated adenocarcinomas in just the submucosal layer were significantly more common in the non-curative resection cases. CONCLUSIONS: ESD was a safe, effective, and minimally invasive treatment for early EGJ cancer. For tumors without any submucosal invasion findings, therefore, ESD is an acceptable treatment option, in addition to also being suitable for diagnostic purposes in evaluating the need for surgery.

Simultaneous endoscopic submucosal dissection for synchronous double early gastric cancer.

BACKGROUND: This study aims to evaluate the safety and efficacy of simultaneous endoscopic submucosal dissection (ESD) for synchronous double early gastric cancers. METHODS: We retrospectively evaluated 832 single gastric cancers from 789 patients treated by single ESD (single group) and 124 synchronous double cancers from 62 patients treated by simultaneous ESD (simultaneous group). RESULTS: The overall rate of en bloc resection and curative resection was comparable between the two groups. Procedure time was significantly longer in the simultaneous group than in the single group (131.0 ± 66.5 and 94.8 ± 64.1 min, respectively, P < 0.001). White blood cell count on the day after ESD was significantly higher in the simultaneous group (9310 ± 2774/µl) than in the single group (8633 ± 2341/µl) (P = 0.032). Length of fasting period after ESD was 1.1 ± 0.5 days in the single group and 1.4 ± 1.1 days in the simultaneous group (P = 0.082). Complications were more frequent in the simultaneous group than in the single group (11.3 vs. 5.4 %, respectively), but the difference was not significant (P = 0.082). Complication rate per one lesion did not differ between the two groups (5.6 vs. 5.4 %, respectively, P = 0.914). Multivariate analysis showed procedure time longer than 150 min was independently predictive for complications of simultaneous ESD (P < 0.042, odds ratio = 6.094). Large tumors, upper portion location and tumors not in the standard guideline criteria were significantly associated with long procedure time. CONCLUSIONS: Simultaneous ESD for synchronous early gastric cancer can be a feasible and safe option, and it can reduce hospital stay. These results need to be validated by further studies.