Immune response in diarrheal patients and asymptomatic carrier with CS6-producing enterotoxigenic Escherichia coli infection.

Enterotoxigenic Escherichia coli (ETEC) is one of the major causes of diarrhea in children and travelers in developing countries. ETEC colonization factors (CFs) are virulence determinants considered as protective antigens and major targets for vaccine development against ETEC infections. One of the most prevalent CFs, coli surface antigen 6 (CS6), a non-fimbrial polymeric protein consisting of two major subunits, CssA and CssB, is produced by approximately 25-35% of ETEC worldwide. We could isolate only CS6-producing ETEC strains from two diarrheal patients and one asymptomatic carrier, but we could not detect CssA- or CssB-specific antibodies in the feces and blood of two patients convalescing from natural ETEC infection and of an asymptomatic carrier using western blotting. Therefore, in order to protect against infection with CS6-producing ETEC, protective levels of CS6 immunity should be incorporated in any future vaccines against ETEC.

Occurrence of mcr-mediated colistin resistance in Salmonella clinical isolates in Thailand.

Nontyphoidal Salmonella, an important zoonotic pathogen and a major cause of foodborne illnesses, could be a potential reservoir of plasmids harbouring mobile colistin resistance gene (mcr). This study reported, for the first time, a high rate of mcr-carrying Salmonella clinical isolates (3.3%, 24/724) in Thailand, associated with mcr-3 gene (3.0%, 22/724) in S. 4,[5],12:i:-(15.4%, 4/26), S. Typhimurium (8.8%, 5/57), and S. Choleraesuis (5.6%, 13/231). Remarkably, the increasing trends of colistin and extended-spectrum cephalosporin resistances have displayed a high agreement over the years, with a dramatic rise in the mcr-carrying Salmonella from 1.1% (6/563) during 2005-2007 to 11.2% (18/161) during 2014-2018 when CTX-M-55 became abundant. Clonal and plasmid analysis revealed that the self-transferable IncA/C and a novel hybrid IncA/C-FIIs MDR plasmids were the major vehicles to disseminate both mcr-3 and blaCTX-M55 genes among diverse Salmonella strains, from as early as 2007. To our knowledge the occurrence of mcr-3 and the co-existence of it with blaCTX-M-55 in S. Choleraesuis are reported here for the first time, leading to clinical concern over the treatment of the invasive salmonellosis. This study provides evidence of the potential reservoirs and vectors in the dissemination of the mcr and highlights the co-selection by colistin and/or cephalosporins.

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.].

The eye fly Siphunculina funicola (Diptera: Chloropidae) as a carrier of pathogenic bacteria in Thailand.

The oriental eye fly Siphunculina funicola (1.0-1.6 mm) is extremely annoying to humans and domestic animals, feeding on mucous membranes, secretions, wounds, eyes, and other moist surfaces of the host body. In many rural areas of Thailand heavy populations of this fly prevail where they aggregate on a variety of hanging substrates, such as strings, nest trailings, electrical lines, decorations, ropes, cob webs, clothes hangers, automobile radio antennae and other items in open shade close to their hosts. Both males and females feed voraciously on wounds and moist skin. With this type of persistent feeding, the eye flies are suspected to carry and transfer germs to their hosts. In the present study, bacteria were isolated from S. funicola captured from wounds, host seeking flies and from their resting sites. Some enriched and bacterial culture media were more suitable for isolation than others. A diverse group of bacteria (64 species), both gram-posi-tive and gram-negative, most in risk category 2, were identified. Bacterial colony counts from Trypic soy broth ranged from 10 to > 3.0 x 10(3) cfu/ml. The most common bacteria isolated were Acinetobacter, Aeromonas, Bacillus, Corynebacterium, Enterobacter, Enterococcus, Escherichia, Kocuria, Pantoea, Pseudomonas, Staphylococcus and others. These bacteria may cause disease conditions in humans and animals. This is the first time bacteria from S. funicola have been reported.

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.

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.

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.

Genotypic and PFGE/MLVA analyses of Vibrio cholerae O1: geographical spread and temporal changes during the 2007-2010 cholera outbreaks in Thailand.

BACKGROUND: Vibrio cholerae O1 El Tor dominated the seventh cholera pandemic which occurred in the 1960s. For two decades, variants of V. cholerae O1 El Tor that produce classical cholera toxin have emerged and spread globally, replacing the prototypic El Tor biotype. This study aims to characterize V. cholerae O1 isolates from outbreaks in Thailand with special reference to genotypic variations over time. METHODS/FINDINGS: A total of 343 isolates of V. cholerae O1 from cholera outbreaks from 2007 to 2010 were investigated, and 99.4% were found to carry the classical cholera toxin B subunit (ctxB) and El Tor rstR genes. Pulsed-field gel electrophoresis (PFGE) differentiated the isolates into 10 distinct pulsotypes, clustered into two major groups, A and B, with an overall similarity of 88%. Ribotyping, multiple-locus variable-number tandem-repeat analysis (MLVA), and PCR to detect Vibrio seventh pandemic island II (VSP-II) related genes of randomly selected isolates from each pulsotype corresponded to the results obtained by PFGE. Epidemiological investigations revealed that MLVA type 2 was strongly associated with a cholera outbreak in northeastern Thailand in 2007, while MLVA type 7 dominated the outbreaks of the southern Gulf areas in 2009 and MLVA type 4 dominated the outbreaks of the central Gulf areas during 2009-2010. Only MLVA type 16 isolates were found in a Thai-Myanmar border area in 2010, whereas those of MLVA types 26, 39, and 41 predominated this border area in 2008. Type 39 then disappeared 1-2 years later as MLVA type 41 became prevalent. Type 41 was also found to infect an outbreak area. CONCLUSIONS: MLVA provided a high-throughput genetic typing tool for understanding the in-depth epidemiology of cholera outbreaks. Our epidemiological surveys suggest that some clones of V. cholerae O1 with similar but distinctive genetic traits circulate in outbreak sites, while others disappear over time.

A cholera outbreak of the Vibrio cholerae O1 El Tor variant carrying classical CtxB in northeastern Thailand in 2007.

Cholera outbreaks occurred in Thailand in 2007. Isolates from the northeastern regions were analyzed. Interestingly, the outbreak strain was identified as biotype El Tor; serotype Ogawa with cholera toxin B subunit gene (ctxB) of the classical type and CTX prophage repressor gene of the El Tor type. The clone was genetically closely related to pulsotype H, which is predominantly found in India. It was probably introduced into Thailand recently.

A rapid, simple, and sensitive loop-mediated isothermal amplification method to detect toxigenic Vibrio cholerae in rectal swab samples.

Loop-mediated isothermal amplification (LAMP) method was designed for clinical diagnosis of Vibrio cholerae carrying the ctxA gene. The detection limits of the method were 5 fg of purified genomic DNA/reaction and 0.54 CFU/reaction. The method was applied to rectal swab samples from cholera patients and healthy volunteers (19 subjects each) and yielded the same results as the "gold standard" culture method, while the polymerase chain reaction-based method failed to detect V. cholerae in 8 of the positive samples. Direct application of this LAMP method without precultivation enabled the rapid detection of 5 asymptomatic carriers from rectal swabs of 21 household contacts of cholera patients. This LAMP method could be a sensitive, specific, inexpensive, and rapid detection tool for V. cholerae carrying the ctxA gene in the clinical laboratory and in the field.