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Multidrug-resistant Klebsiella pneumoniae strains are one of the most prevalent causes of nosocomial infections and pose an increasingly dangerous public health threat. The lack of remaining treatment options has resulted in the utilization of older drug classes, including colistin. As a drug of last resort, the discovery of plasmid-mediated colistin resistance by mcr-1 denotes the potential development of pandrug-resistant bacterial pathogens. To address the emergence of the mcr-1 gene, 118 gram-negative Enterobacteriaceae isolated from clinical samples collected at Queen Sirikit Naval Hospital in Chonburi, Thailand were screened for colistin resistance using automated antimicrobial susceptibility testing and conventional PCR screening. Two K. pneumoniae strains, QS17-0029 and QS17-0161, were positive for mcr-1, and both isolates were sequenced to closure using short- and long-read whole-genome sequencing. QS17-0029 carried 16 antibiotic resistance genes in addition to mcr-1, including 2 carbapenemases, blaNDM-1 and blaOXA-232. QS17-0161 carried 13 antibiotic resistance genes in addition to mcr-1, including the extended-spectrum β-lactamase blaCTX-M-55. Both isolates carried multiple plasmids, but mcr-1 was located alone on highly similar 33.9 Kb IncX4 plasmids in both isolates. The IncX4 plasmid shared considerable homology to other mcr-1-containing IncX4 plasmids. This is the first report of a clinical K. pneumoniae strain from Thailand carrying mcr-1 as well as the first strain to simultaneously carry mcr-1 and multiple carbapenemase genes (QS17-0029). The identification and characterization of these isolates serves to highlight the urgent need for continued surveillance and intervention in Southeast Asia, where extensively drug-resistant pathogens are being increasingly identified in hospital-associated infections.

We observed multidrug resistance in 10 (91%) of 11 Shigella isolates from a diarrheal surveillance study in Cambodia. One isolate was resistant to fluoroquinolones and cephalosporins and showed decreased susceptibility to azithromycin. We found mutations in gyrA, parC, β-lactamase, and mphA genes. Multidrug resistance increases concern about shigellosis treatment options.

Background Traveler’s diarrhea (TD) is a common health problem among visitors from developed to developing countries. Although global and regional estimates of pathogen distribution are available, the etiology of diarrhea among non-military travelers to Thailand is largely unknown. Methods A prospective TD case-control study was conducted among adult travelers from developed countries at a prominent hospital in Bangkok, Thailand during 2001–2003. Stool samples were collected from acute TD cases and non-diarrheal controls and analyzed for bacterial, viral, and protozoan pathogens by microbiology, ELISA or PCR methods. Calculation of adjusted odd ratios for risk factors was performed by logistic regression using STATA statistical software. Results Stool samples were collected and analyzed from 389 TD cases and 400 non-diarrhea controls. At least one pathogen was detected in 227 (58%) cases and 124 (31%) controls. Plesiomonas (14%), Vibrio (14%), Campylobacter (14%), and norovirus (12%) were the most frequently isolated pathogens among cases and significantly associated with diarrhea at p = 0.006, p

Regression analyses for pathogen detection, bacterial isolation, norovirus detection, protozoa detection, and traveler origin. Supplementary tables of regression analyses results. (DOCX 44 kb)

Hawaii has one of the highest incidences of Campylobacteriosis in the United States, but there remains little published data on circulating strains or antimicrobial resistance. We characterized 110 clinical Campylobacter isolates (106 C. jejuni, 4 C. coli) processed at Tripler Army Medical Center in Honolulu, HI from 2012–2016. Twenty-five percent of C. jejuni isolates exhibited fluoroquinolone (FQ) resistance, compared with 16% for tetracycline (TET), and 0% for macrolides. Two of the four C. coli isolates were resistant to FQ, TET, and macrolides. C. jejuni isolates further underwent multilocus sequence typing, pulsed-field gel electrophoresis, and molecular capsular typing. Nineteen capsule types were observed, with two capsule types (HS2 and HS9) being associated with FQ resistance (p

Background Multidrug-resistant (MDR) Gram-negative bacterial species are an increasingly dangerous public health threat, and are now endemic in many areas of South Asia. However, there are a lack of comprehensive data from many countries in this region determining historic and current MDR prevalence. Enterotoxigenic Escherichia coli (ETEC) is a leading cause of both acute infant diarrhea and traveler’s diarrhea in Nepal. The MDR prevalence and associated resistance mechanisms of ETEC isolates responsible for enteric infections in Nepal are largely unknown. Methods A total of 265 ETEC isolates were obtained from acute diarrheal samples (263/265) or patient control samples (2/265) at traveler’s clinics or regional hospitals in Nepal from 2001 to 2016. Isolates were screened for antibiotic resistance, to include extended spectrum beta-lactamase (ESBL) production, via the Microscan Automated Microbiology System. ETEC virulence factors, specifically enterotoxins and colonization factors (CFs), were detected using multiplex PCR, and prevalence in the total isolate population was compared to ESBL-positive isolates. ESBL-positive isolates were assessed using multiplex PCR for genetic markers potentially responsible for observed resistance. Results A total of 118/265 (44.5%) ETEC isolates demonstrated resistance to ≥2 antibiotics. ESBL-positive phenotypes were detected in 40/265 isolates, with isolates from 2008, 2013, 2014, and 2016 demonstrating ESBL prevalence rates of 1.5, 34.5, 31.2, and 35.0% respectively. No difference was observed in overall enterotoxin characterization between the total ETEC and ESBL-positive populations. The CFs CS2 (13.6%), CS3 (25.3%), CS6 (30.2%), and CS21 (62.6%) were the most prevalent in the total ETEC population. The ESBL-positive ETEC isolates exhibited a higher association trend with the CFs CS2 (37.5%), CS3 (35%), CS6 (42.5%), and CS21 (67.5%). The primary ESBL gene identified was bla CTX-M-15 (80%), followed by bla SHV-12 (20%) and bla CTX-M-14 (2.5%). The beta-lactamase genes bla TEM-1 (40%) and bla CMY-2 (2.5%) were also identified. It was determined that 42.5% of the ESBL-positive isolates carried multiple resistance genes. Conclusion Over 30% of ETEC isolates collected post-2013 and evaluated in this study demonstrated ESBL resistance. Persistent surveillance and characterization of enteric ETEC isolates are vital for tracking the community presence of MDR bacterial species in order to recommend effective treatment strategies and help mitigate the spread of resistant pathogens.

Helicobacter fennelliae (H. fennelliae) is associated with human gastroenteritis; however, H. fennelliae was isolated and confirmed by phenotypic and genotypic identification from a non-diarrheal child stool sample in Cambodia. Antimicrobial susceptibility testing demonstrated that this isolate had a high minimal inhibitory concentration against macrolides and quinolones, which are first-line antibiotic treatment choices for Campylobacter infections. Consequently, macrolides and quinolones were likewise expected to be ineffective against Campylobacter-like organisms such as H. fennelliae. This isolate warranted further genetic characterization to better understand associated antibiotic resistance mechanisms. Resistant pathogens from asymptomatic diarrheal cases are likely underestimated, and as such colonized individuals may spread resistant organisms to local community members and the environment.

Background Travelers' diarrhea (TD) is a common illness experienced by travelers from developed countries who visit developing countries. Recent questionnaire-based surveillance studies showed that approximately 6%-16% of travelers experienced TD while visiting Thailand; however, a majority of TD information was limited mainly to US military populations. Methods A TD surveillance study was conducted at Bumrungrad International Hospital in 2012-2014 in Bangkok, Thailand. Enteropathogens were identified using conventional methods and the TaqMan® array card (TAC), which uses real-time polymerase chain reaction for the simultaneous detection of multiple pathogens. Analyses to determine pathogen-disease and symptoms association were performed to elucidate the clinical relevance of each enteropathogen. Results TAC identified more pathogens per sample than conventional methods. Campylobacter spp. were the most prevalent, followed by the diarrheagenic Escherichia coli and norovirus GII. These agents had significant pathogen-disease associations as well as high attributable fractions among diarrheal cases. A wide range of pathogen loads for Campylobacter spp. was associated with TD, while heat-labile toxin enterotoxigenic Escherichia coli was associated with an increased pathogen load. Most cases were associated with inflammatory diarrhea, while Campylobacter spp. and Shigella spp. were associated with dysentery. Conclusions A pan-molecular diagnostic method such as TAC produces quantifiable and comparable results of all tested pathogens, thereby reducing the variability associated with multiple conventional methods. This allows better determination of the clinical relevance of each diarrhea etiologic agent, as well as their geographical relevance in Thailand.

Background: Enteroaggregative Escherichia coli (EAEC) is a major cause of acute diarrhea in children, adults, and travelers as well as an important cause of persistent diarrhea. Due to the limited information on EAEC virulence genes, EAEC isolates isolated from children with diarrhea and asymptomatic controls in Thailand were characterized for the presence of virulence factors. Method: A study of diarrhea etiology in children from Thailand was conducted during 2008-2009 and 301 EAEC isolates were identified by pCVD432 probe/hybridization assays from diarrhea cases and controls. A total of 200 EAEC isolates were further characterized by PCR for EAEC genotypes, aggregative adherence fimbria (AAF/I-AAF/ IV), serine protease autotransporters of Enterobacteriaceae (SPATEs) class I & II, and enterotoxin genes. Results: The prevalence of EAEC isolates in this study were 7.7% and 9.1% among cases (n=1803) and controls (n=1790). Of 200 EAEC isolates, 69% of case and 58% of control isolates were classified as typical EAEC genotype. AAF/I (aggA) was the major fimbria type for cases (37%) and controls (23%), followed by AAF/III (9%, 13%), AAF/II (11%, 17%) and AAF/IV (5%, 3%). In confirmed cases, class II SPATE-sepA (19%, 7%) and pic (75%, 60%) and enterotoxin-set (75%, 61%) genes were detected in significantly more strains when compared to the controls. Additionally, typical EAEC isolates containing genes for AAF/I, Class II SPATEs (sepA, pic), and enterotoxin (set) were detected more frequently in cases than controls (P value

The purpose of this study was to compare the existence of virulence genes in hemolytic Escherichia coli (HEC) and nonhemolytic E. coli (NHEC) isolated from weaner pigs in Thailand, and to determine their susceptibility to 10 antimicrobial agents. A total of 304 E. coli isolates were obtained from 90 piglets with diarrhea and 110 healthy piglets. Of these, 74 HEC isolates were obtained from 70 pigs with diarrhea, and 4 were obtained from 4 healthy pigs, while 190 and 40 NHEC were recovered from 110 healthy and 20 pigs with diarrhea, respectively. A ten digoxigenin (DIG)-labeled probe system was utilized for detecting genes encoding virulence-associated toxins and proteins in these isolates, and the minimal inhibitory concentration values against 10 antimicrobials were determined by means of the agar dilution technique. In total, 70.3% of the HEC isolates contained an exotoxin gene, lth, estp or stx2e, whereas 2.6% of the NHEC isolates hybridized with a gene probe for estp or stx2e. Over 90% of the isolates were resistant to most agents other than colistin and halquinol. The MIC(90) values of the HEC isolates for halquinol and colistin were 4 and 8 times greater than those of the NHEC isolates, respectively. The results represent the first characterization of resistant pathogenic E. coli distributed in the Thai pig industry. Amongst the HEC isolates, there appeared to be an association between the presence of some exotoxin genes, including lth, estp and stx2e, and reduced antimicrobial susceptibility.

SUMMARYThis study investigated fluoroquinolone, macrolide resistances and serotype distributions amongCampylobacter jejuniandCampylobacter coliisolated from children in Bangkok and rural settings during 1991–2000. Phenotypic identification, serotyping, and susceptibility testing were performed by standard microbiological procedures. The predominant serotypes ofC. jejuniwere Lior 36, 2 and 4 and ofC. coliwere Lior 8, 29 and 55. Resistance to nalidixic acid increased significantly during 1991–2000 and the frequency of isolates resistant to both nalidixic acid and ciprofloxacin in Bangkok was significantly greater than in rural settings. In 1996–2000, a significant trend was observed inC. jejuniisolates resistant to ciprofloxacin from Bangkok but not for macrolide resistance from both settings. In summary, fluoroquinolone resistance amongC. jejuniandC. coliisolates became widespread in both Bangkok and rural settings in Thailand in the 1990s while widespread resistance to macrolides was undetected.

In the summer of 2002, a total of 5963 cases of typhoid fever were recorded in Bharatpur, Nepal (population, 92,214) during a 7-week period. A team from the Armed Forces Research Institute of Medical Sciences in Bangkok, Thailand, and the CIWEC Travel Medicine Clinic (Kathmandu, Nepal) assisted the Nepal National Public Health Laboratory (Kathmandu, Nepal) in the further investigation of this large, explosive febrile disease outbreak.Investigators conducted a thorough epidemiologic and laboratory investigation to assess the size and scope of the outbreak. In addition to subculturing of previously collected samples, blood samples were obtained from 100 febrile patients, and culture and susceptibility testing were done by standard laboratory methods. Pulsed field gel electrophoresis (PFGE) and plasmid analysis were done.The majority of the isolates, including 1 from the municipal water supply, were multidrug resistant. The minimum inhibitory concentrations (MICs) of ciprofloxacin ranged from 0.19 microg/mL to 0.125 microg/mL. With use of PFGE, all isolates, including isolates from the water supply, showed an analytical similarity of 96%-100%. Multidrug-resistant isolates had a plasmid encoding for resistance, and those with resistance to nalidixic acid had a single-point mutation.To the best of our knowledge, this outbreak is the largest single-point source outbreak of multidrug-resistant typhoid fever yet reported, and it was molecularly traced to the city's single municipal water supply. Isolates were uniformly resistant to nalidixic acid, there was a decrease in their susceptibility as measured by MIC of fluoroquinolones, and 90% of isolates obtained were resistant to1 antibiotic.

We sequenced and analyzed Shigella dysenteriae strain Sd1617 serotype 1 that is widely used as model strain for vaccine design, trials and research. A combination of next-generation sequencing platforms and assembly yielded two contigs representing a chromosome size of 4.34 Mb and the large virulence plasmid of 177 kb. This genome sequence is compared with other Shigella genomes in order to understand gene complexity and pathogenic factors., The Shigella dysenteriae strain Sd1617 serotype 1 has been sequenced and analyzed. It is widely used as model strain for vaccine design, trials and research. A combination of next-generation sequencing platforms and assembly yielded two contigs representing a chromosome size of 4.34 Mb and the large virulence plasmid of 177 kb.

In this study, 176 clinical and environmental Vibrio cholerae strains of different O serotypes isolated in Thailand from 1982 to 1995 were selected and studied for the presence of class 1 integrons, a new group of genetic elements which carry antibiotic resistance genes. Using PCR and DNA sequencing, we found that 44 isolates contained class 1 integrons harboring the aadB , aadA2 , blaP1 , dfrA1 , and dfrA15 gene cassettes, which encode resistance to gentamicin, kanamycin, and tobramycin; streptomycin and spectinomycin; β-lactams; and trimethoprim, respectively. Each cassette array contained only a single antibiotic resistance gene. Although resistance genes in class 1 integrons were found in strains from the same epidemic, as well as in unrelated non-O1, non-O139 strains isolated from children with diarrhea, they were found to encode only some of the antibiotic resistance expressed by the strains. Serotype O139 strains did not contain class 1 integrons. However, the appearance and disappearance of the O139 serotype in the coastal city Samutsakorn in 1992 and 1993 were associated with the emergence of a distinct V. cholerae O1 strain which contained the aadA2 resistance gene cassette. A 150-kb self-transmissible plasmid found in three O1 strains isolated in 1982 contained the aadB gene cassette. Surprisingly, several strains harbored two integrons containing different cassettes. Thus, class 1 integrons containing various resistance gene cassettes are distributed among different V. cholerae O serotypes of mainly clinical origin in Thailand.

Results of a surveillance on cholera conducted with patients seen at the Children Hospital in Bangkok, Thailand from August 1993 to July 1995 are presented. Annually, isolation rates for Vibrio cholerae varied between 1·7 and 4·4% of patients with diarrhoea. V. cholerae O1 serotype Ogawa accounted for between 31 and 47% of patients cultured positive for V. cholerae, whereas the O139 serotype dominated in early 1994 after which it disappeared. Non-O1, non-O139 strains were isolated at similar rates as serotype O1 in 1993 and 1994, but accounted for 69% of V. cholerae culture positive specimens in 1995. However, the annual proportion of the isolation of non-O1, non-O139 strains showed little variation and remained low between 1·0 and 1·3%. Serotyping of 69 epidemiological unrelated non-O1, non-O139 strains produced 37 different O-serotypes. BglI ribotyping of serotypes containing more than two strains demonstrated a high degree of heterogeneity within and between serotypes, except seven serotype O37 strains which showed an identical ribotype suggesting clonality. None of the 69 strains hybridized with a cholera toxin probe and only two strains hybridized with a heat-stable enterotoxin probe. Susceptibility testing to 12 antibiotics showed that 40 of 69 (58%) non-O1, non-O139 strains were resistant to colistin, streptomycin and sulphisoxazole and 28 of 69 (41%) were multiple antibiotic resistant (MAR; [ges ]4 antibiotics). Although 26 of 69 (38%) strains contained one or more plasmids, the plasmids were of low molecular weights and did not seem to encode antibiotic resistance. The results of the present study showed that a high proportion of heterogenous MAR V. cholerae non-O1, non-O139 strains were isolated from children at the hospital. With reference to the emergence of V. cholerae O139 in 1992, we suggest that non-O1, non-O139 strains should be monitored carefully to detect new serotypes with a possible epidemic potential, but also to determine the development and mechanism of antibiotic resistance.

Seventy clinical strains of Vibrio cholerae O1 isolated from 1982–96 in Samutsakorn, a port city 30 km southwest of Bangkok where cholera occurs at low levels with regular seasonality, were characterized to investigate if there were any differences among the O1 strains isolated before, during and after the O139 epidemic. Pulsed-field gel electrophoresis (PFGE) typing, ribotyping and southern blot hybridization with a cholera toxin probe (CT genotyping) demonstrated several genotypes among O1 strains isolated before the emergence of V. cholerae O139. However, O1 strains isolated during and after the advent of O139 showed identical ribotypes which were distinctly different from the types identified in strains isolated before the emergence of O139. Ribotypes identified in strains during and after the advent of O139 were also demonstrated by O1 strains isolated immediately before the emergence of O139. Considering the seasonality of cholera in Samutsakorn, the identical ribotype and CT genotype and the closely related PFGE types shown by all O1 strains isolated during and after the appearance of O139 is remarkable and suggest that the V. cholerae O1 strain may reemerge from an environmental source. A subgroup of V. cholerae O1 strains isolated before the emergence of the O139 epidemic had a ribotype identical to a type demonstrated by O139 strains isolated in Thailand. Our results support similar findings in Bangladesh and India that a distinct O1 strain appeared during the O139 epidemic. However, compared with the apparent identical strain which replaced O139 in Bangladesh and India, the emerged O1 strain in Samutsakorn showed a different ribotype and CT genotype.

Background Shigella species are an important cause of diarrhea in developing countries. These bacteria normally acquire their antibiotic resistance via several different mobile genetic elements including plasmids, transposons, and integrons involving gene cassettes. During a diarrhea surveillance study in Thimphu, Bhutan in June and July, 2011, Shigella sonnei were isolated more frequently than expected. This study describes the antibiotic resistance of these S. sonnei isolates. Methods A total of 29 S. sonnei isolates from Thimphu, Bhutan was characterized for antimicrobial susceptibility by disc diffusion assay and minimum inhibitory concentration (MIC) assay. All isolates were tested by pulsed-field gel electrophoresis (PFGE) with restriction enzyme XbaI and were tested for plasmid. The plasmid patterns and the PFGE patterns were analyzed by Bionumerics software. DNA sequencing was performed on amplified products for gyraseA gene and class 1 and class 2 integrons. S. sonnei isolates were classified for incompatibility of plasmids by PCR-based replicon typing (PBRT). Results These S. sonnei were resistant to multiple drugs like ciprofloxacin, nalidixic acid, trimethoprim-sulfamethoxazole, streptomycin, and tetracycline but susceptible to azithromycin. All isolates had class 2 integrons dfrA1, sat1 and aadA1 genes. Two point mutations in Gyrase A subunit at position Ser83Leu and Asp87Gly were detected in these quinolone resistant isolates. The plasmid and PFGE patterns of S. sonnei isolates suggested a clonal relationship of the isolates. All isolates carried common ColE plasmid. ColE plasmid co-resided with B/O plasmid (nine isolates) or I1 plasmid (one isolate). Conclusions The characteristics of 29 S. sonnei isolates from Thimphu, Bhutan in June and July, 2011 are identical in PFGE, plasmid and resistance pattern. This study suggests that these recent S. sonnei isolates are clonally related and multidrug-resistant.

Following the emergence of cholera in Lima, Peru, in 1991, isolates of Vibrio cholerae O1 biotype El Tor recovered from patients in various parts of Lima were selected and characterized. Ribotyping and pulsed-field gel electrophoresis (PFGE) revealed four BglI ribotypes and eight NotI PFGE types among 50 V. cholerae O1 strains recovered from patients with cholera in Lima from 1991 to 1995, with certain genotypes appearing to cluster geographically. While differences in ribotype and PFGE type patterns suggest that genetic changes are occurring in the strain responsible for the Latin American cholera epidemic, more frequently than previously reported, 40 (80%) O1 strains showed an identical ribotype pattern and 41 (82%) strains showed closely related PFGE types, types 1, 2, or 3, that differed by less than three restriction fragments. All strains were susceptible to nine antibacterial agents studied. In 1991, more than 95% of the clinical V. cholerae O1 strains were serotype Inaba, whereas from 1992, serotype Ogawa began to predominate, with more than 90% of the isolates being of the Ogawa serotype in 1995. The small differences in genotypes of V. cholerae O1 is remarkable because cholera is highly seasonal in coastal areas of Peru and support the hypothesis that the epidemic strain reemerges from an environmental source. However, the relative high rate of genetic changes within V. cholerae O1 as shown by ribotyping and PFGE should be taken into consideration when typing patterns of V. cholerae O1 associated with cholera in Latin America are evaluated.

Samples (1,318) of enterotoxigenic Escherichia coli (ETEC) isolated in 1994-1995 from children with diarrhea from Nepal, Indonesia, Peru, and Thailand were examined for colonization factor antigen (CFA) and coli surface (CS) antigens. Fifty-five percent of 361 heat-labile and heat-stable (LT-ST), 14% of 620 LT-only, and 48% of 337 ST-only ETEC had CFA/CS antigens. LT-ST ETEC strains were predominantly in the CFA II group, and ST only strains were in the CFA IV group. Additional studies are needed to identify ETEC strains that do not have CFA/CS antigens.

SummaryPulsed-field gel electrophoresis (PFGE) ofCpoI-digested genomic DNA and ribotyping (BglI) were applied to 60Vibrio choleraestrains including 48V. choleraeO139 from Thailand to compare their value in differentiating strains of the presentV. choleraeO139 epidemic. PFGE patters were divided into groups A and B representing five and four subtypes, respectively, while ribotyping showed four different patterns. PFGE group B subtypes were only presented among O139 isolates from Thailand, whereas four O139 strains from Bangladesh and India showed identical PFGE group A subtypes observed in O139 isolates from Thailand. Two nontoxigenic O139 isolates from Thailand showed different and unique PFGE types as did fiveV. choleraenon-Ol non-O139 isolates containing a gene virulence complex found inV. choleraeO139. These results indicate that PFGE (CpoI) can resolve recent evolutionary divergence withinV. choleraeO139 and offers a useful supplementary tool for following the progressingV. choleraeO139 epidemic.

In the present study, 19 strains of Vibrio cholerae O1 biotype El Tor isolated during outbreaks of cholera in Guinea-Bissau in 1987, 1994, and 1995 were characterized to investigate a possible epidemiological relationship among the isolates. On the basis of ribotyping with the restriction enzyme BglI, 5 strains isolated in 1987 showed two closely related ribotypes, while 14 strains isolated in 1994 and 1995 showed the same ribotype that was distinct from the ribotypes of strains isolated in 1987. Southern blot hybridization of BglI-digested genomic DNA with a cholera toxin probe demonstrated that the strains isolated in 1987 showed an identical cholera toxin genotype, whereas O1 strains isolated in 1994 and 1995 showed the same genotype that was distinct from the genotype of strains isolated in 1987. These results were supported by the results of antibiotic susceptibility testing, in which strains isolated in 1987 showed resistance to polymyxin B only, while each of the strains from 1994 and 1995 showed resistance to polymyxin B, trimethoprim-sulfamethoxazole, and the vibriostatic agent O/129. Although our results are based on a limited number of V. cholerae O1 strains, they suggest that the epidemic in Guinea-Bissau in 1994 and 1995 was due to the introduction of a new strain to the country.

Detection of diarrheagenic Escherichia coli (DEC) typically depends on identification of virulence genes from stool cultures, not on stool itself. We developed a multiplex polymerase chain reaction (PCR) assay that detects key DEC virulence genes (stx1, stx2, eae, bfpA, ipaH, LT, STh, aaiC, aatA). The assay involved a multiplex PCR reaction followed by detection of amplicon(s) using Luminex beads. The assay was evaluated on over 100 colony and broth specimens. We then evaluated the assay using DNA extracted from stool, colony pools, and Gram-negative broths, using stool spiked with known quantities of DEC. Performance of the assay on stool DNA was most quantitative, while stool broth DNA offered the lowest limit of detection. The assay was prospectively evaluated on clinical specimens in Tanzania. Stool DNA yielded higher sensitivity than colony pools compared with broth DNA as the standard. We propose using this assay to screen for DEC directly in stool or stool broths.

The development of vaccines against Campylobacter jejuni would be facilitated by the ability to perform phase II challenge studies. However, molecular mimicry of the lipooligosaccharide (LOS) of most C. jejuni strains with human gangliosides presents safety concerns about the development of Guillain-Barré syndrome. Clinical isolates of C. jejuni that appeared to lack genes for the synthesis of ganglioside mimics were identified by DNA probe analyses. Two clinical isolates from Southeast Asia (strains BH-01-0142 and CG8421) were determined to express the LOS type containing N -acetyl quinovosamine. No ganglioside structures were observed to be present in the LOSs of these strains, and pyrosequence analyses of the genomes of both strains confirmed the absence of genes involved in ganglioside mimicry. The capsule polysaccharide (CPS) of BH-01-0142 was determined to be composed of galactose (Gal), 6-deoxy- ido -heptose, and, in smaller amounts, d - glycero - d - ido -heptose, and the CPS of CG8421 was observed to contain Gal, 6-deoxy- altro -heptose, N -acetyl-glucosamine, and minor amounts of 6-deoxy-3- O -Me- altro -heptose. Both CPSs were shown to carry O -methyl-phosphoramidate. The two genomes contained strain-specific zones, some of which could be traced to a plasmid origin, and both contained a large chromosomal insertion related to the CJEI3 element of C. jejuni RM1221. The genomes of both strains shared a high degree of similarity to each other and, with the exception of the capsule locus of CG8421, to the type strain of the HS3 serotype, TGH9011.

This case-control study detected and characterized Shigella and diarrheagenic Escherichia coli (DEC) types among Vietnamese children less than 5 years old. In 249 children with diarrhea and 124 controls, Shigella spp. was an important cause of diarrhea ( P < 0.05). We used multiplex PCR and DNA probes to detect enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAggEC), enteropathogenic E. coli (EPEC), attaching and effacing E. coli (A/EEC), verocytotoxin-producing E. coli (VTEC), and enterotoxigenic E. coli (ETEC). The prevalences of DEC in the diarrhea and control groups were 25.7 and 10.5%, respectively. In 62 children with diarrhea, 64 DEC strains included 22 EAggEC (8.8%), 2 EIEC (0.8%), 23 A/EEC (9.2%), 7 EPEC (2.8%), and 10 ETEC strains (4.0%). Among controls, 13 DEC strains included 5 EAggEC strains (4.0%), 7 A/EEC strains (5.6%), and 1 EPEC strain. The characterization of DEC by serotypes, antimicrobial susceptibility patterns, virulence genes, and pulsed-field gel electrophoresis showed the occurrence of many different and highly heterogenic DEC subtypes, but common serotypes were found among ETEC, EIEC and EPEC, respectively. Serotyping was used to distinguish between A/EEC and EPEC. However, A/EEC, EPEC, and EAggEC were isolated at high frequency from both cases and controls. Further in-depth studies are needed to better understand important virulence factors of DEC, especially A/EEC, EPEC, and EAggEC.

Vibrio parahaemolyticus is a major cause of gastroenteritis worldwide. A total of 95 V. parahaemolyticus isolates belonging to 23 different serovars were identified in a case–control study of expatriates and Thai adults from 2001 to 2002 in Thailand. Fifty-two per cent of isolates (49/95) were resistant to ampicillin and sulfisoxazole, but all isolates were susceptible to ciprofloxacin and trimethoprim–sulfamethoxazole, two antibiotics commonly used to treat traveller’s diarrhoea. All isolates were positive for the species-specific toxR gene, and 91 and 5 were positive for the thermostable direct haemolysin (tdh) gene and the tdh-related (trh) gene, respectively. Sixty-five isolates were assigned to the pandemic group of V. parahaemolyticus by a group-specific PCR and the presence of the orf8 gene. The pandemic isolates belonged to three recognized serovars (O3 : K6, O1 : K25, O1 : KUT) and a new serovar, O3 : K46. This new serovar harboured pandemic traits. PFGE analysis revealed that all pandemic isolates including serovar O3 : K46 were closely related and clearly distinct from the non-pandemic isolates. In summary, three well-known serovars of pandemic V. parahaemolyticus isolates were identified as a major cause of diarrhoea in Thailand and a new V. parahaemolyticus isolate, serovar O3 : K46, with pandemic traits was detected.

This study was conducted to investigate the presence of intestinal parasites among pre-school children (aged 3 months to 5 years) in Sangkhlaburi, a rural district in the west of Thailand along the Thai-Myanmar border. Stool specimens were collected from October 2001 through October 2002. A total of 472 pre-school children, 233 males and 239 females, 236 children with diarrhea and 236 asymptomatic children were recruited for the study. Each specimen was processed and examined by direct wet smear, modified acid fast stain, formalin-ethylacetate sedimentation concentration technique, and trichrome stain. In detecting Giardia lamblia and Cryptosporidium species ProSpecT Microplate assays (Alexon-Trend, Lenexa, KS) were performed. There were 107 individuals (22.7%), 41 diarrheal and 66 asymptomatic children, infected with intestinal parasites. The most frequent parasites identified in cases and controls were G. lamblia and Cryptosporidium spp. Eighteen specimens (3.8%) showed mixed parasite infections. Highest proportion of intestinal parasites occurred during the rainy season (June-October).

SUMMARYVibrio cholerae O139 first appeared in India and Bangladesh in 1992. Surveillance for O139 was started at three hospitals in Thailand in 1993. By 1994 all three hospitals surveyed in Thailand had experienced an increase in Vibrio cholerae O139 infections.

We report sporadic cases of a severe gastroenteritis associated with Vibrio cholerae serogroup O141. Like O1 and O139 serogroup strains of V. cholerae isolated from cholera cases, the O141 clinical isolates carry DNA sequences that hybridize to cholera toxin (CT) gene probes. The CT genes of O1 and O139 strains are carried by a filamentous bacteriophage (termed CTX phage) which is known to use toxin-coregulated pili (TCP) as its receptor. In an effort to understand the mechanism of emergence of toxigenic O141 V. cholerae , we probed a collection of O141 clinical and environmental isolates for genes involved in TCP production, toxigenicity, virulence regulation, and other phylogenetic markers. The collection included strains isolated between 1964 and 1995 from diverse geographical locations, including eight countries and five U.S. states. Information collected about the clinical and environmental sources of these isolates suggests that they had no epidemiological association. All clinical O141 isolates hybridized to probes specific for genes encoding CT ( ctx ), zonula occludens toxin ( zot ), repetitive sequence 1 (RS1), RTX toxin ( rtxA ), the major subunit of TCP ( tcpA ), and the essential regulatory gene that controls expression of both CT and TCP ( toxR ). In contrast, all but one of the nonclinical O141 isolates were negative for ctx , zot , RS1, and tcpA , although these strains were positive for rtxA and toxR . The one toxigenic environmental O141 isolate was also positive for tcpA . Ribotyping and CT typing showed that the O141 clinical isolates were indistinguishable or closely related, while a toxigenic water isolate from Louisiana showed a distantly related ribotype. Nonclinical O141 isolates displayed a variety of unrelated ribotypes. These data support a model for emergence of toxigenic O141 that involves acquisition of the CTX phage sometime after these strains had acquired the pathogenicity island encoding TCP. The clonal nature of toxigenic O141 strains isolated from diverse geographical locations suggests that the emergence is a rare event but that once it occurs, toxigenic O141 strains are capable of regional and perhaps even global dissemination. This study stresses the importance of monitoring V. cholerae non-O1, non-O139 serogroup strains for their virulence gene content as a means of assessing their epidemic potential.

In February 1994, an outbreak of diarrhea caused by non-O1 Vibrio cholerae occurred among volunteers in a vaccine trial study area in Lima, Peru. Clinically, 95% of the patients presented with liquid diarrhea with either no or mild dehydration. Serogrouping of 58 isolates recovered from diarrheal patients affected in the outbreak revealed seven different serogroups, with serogroups O10 (21%) and O12 (65%) being predominant. Most of these isolates were susceptible to a variety of antimicrobial agents. None of the 58 isolates hybridized with a DNA probe previously used to detect the gene encoding the heat-stable enterotoxin NAG-ST or produced cholera toxin as assessed by GM1 ganglioside enzyme-linked immunosorbent assay. Ribotyping exhibited 10 different BglI ribotype patterns among the 58 V. cholera non-O1 strains studied. However, ribotyping showed that all isolates belonging to serogroup O12 exhibited identical ribotypes and that 83% of the serogroup O10 isolates belonged to another identical ribotype, thus showing excellent correlation between ribotypes and serogroups. Among a group of O10 and O12 isolates selected for virulence studies, none produced enterotoxin whereas the majority produced a cytotoxin, as assessed in Y1 and HeLa cells. These isolates were also negative for the gene encoding zonula occludens toxin (Zot) as assessed by a PCR assay. The isolates tested showed strong adherence and some degree of invasion in the HEp-2 cell assay, whereas none of the isolates was positive in the PCR assay for the gene encoding the toxin coregulated pilus subunit A antigen (tcpA). In the removable intestinal tie adult rabbit diarrhea model, O10 and O12 serogroup isolates produced severe diarrhea and occasionally death when rabbits were challenged with 10(10) bacterial cells. Fluid accumulation was shown in the rabbit intestinal loop test when whole cultures were injected. No significant difference in virulence was shown between serogroup O10 and O12 isolates. This study provides further evidence that V. chlorae non-O1 non-O139 strains have diarrhegenic potential for humans through a yet-undefined mechanism(s) and that such strains can cause outbreaks.

A total of 148 Vibrio cholerae isolates from a major shrimp production area in Southern Thailand were examined by colony hybridisation for genes encoding heat-stable enterotoxin (NAG-ST) and cholera toxin (CT). Only non-O1 V. cholerae strains were found to harbour NAG-ST (14 of 146) whereas no strains hybridised with the CT probe. NAG-ST-positive V. cholerae non-O1 strains were isolated from shrimp farms situated close to urban areas. Five different O serogroups were found among NAG-ST positive non-O1 strains. Southern blot and restriction endonuclease analysis of NAG-ST-positive strains revealed a high degree of genetic divergence. A total of seven classes of enterotoxin gene patterns were found with HindIII and EcoRI restriction endonucleases. Enterotoxin gene patterns correlated with O-antigen expression in 84% of isolates tested. In combination with other molecular techniques Southern blot analysis with an NAG-ST oligonucleotide probe could be useful for studying the molecular epidemiology of V. cholerae non-O1 strains.

SUMMARYA collection of 64 clinical and environmentalVibrio choleraenon-O1 strains isolated in Asia and Peru were characterized by molecular methods and antibiotic susceptibility testing. All strains were resistant to at least 1 and 80% were resistant to two or more antibiotics. Several strains showed multiple antibiotic resistance (≥ three antibiotics). Plasmids most often of low molecular weight were found in 21/64 (33%) strains. The presence of plasmids did not correlate with antibiotic resistance or influence ribotype patterns. In colony hybridization studies 63/64 (98%)V. choleraenon-O1 strains were cholera toxin negative, whereas only strains recovered from patients were heat-stable enterotoxin positive. Forty-sevenBglI ribotypes were observed. No correlation was shown between ribotype and toxin gene status. Ribotype similarity was compared by cluster analysis and two main groups of 13 and 34 ribotypes was found. Ribotyping is apparently a useful epidemiological tool in investigations ofV. choleraenon-O1 infections.

In 1992, a serologically novel clone of Vibrio cholerae, designated O139, caused large epidemics of diarrhea in India and Bangladesh. To determine the extent of the spread of V. cholerae O139 worldwide, 484 V. cholerae non-O1 strains isolated from different patients with diarrhea in Thailand, Indonesia, the Philippines, and Peru in 1993 were tested for agglutination in O139 antisera. One hundred fifty-one of these 484 isolates were examined for genes encoding cholera toxin, zonula occlulans toxin, the repetitive sequence 1, and the toxin coregulated pilin A (the V. cholerae virulence gene complex). Thirty-three percent (122 of 364) of V. cholerae non-O1 strains isolated from different patients with diarrhea in Thailand agglutinated in O139 antisera. Ninety-eight percent (120 of 122) of V. cholerae O139 contained the V. cholerae virulence gene complex. None of the 104 V. cholerae non-O1 strains isolated from patients with diarrhea in Indonesia or the 14 strains from patients with diarrhea in the Philippines were serotype O139. Four different ribotypes were found in V. cholerae O139 isolated in Asia. Twenty-three (47%) of 49 Thai O139 strains examined were of different ribotypes than isolates from India and Bangladesh; V. cholerae strains that were not O1 or O139 that were isolated from flies and water in Thailand 11 years previously in 1981 contained the same V. cholerae virulence gene complex found in V. cholerae O1 and O139. This suggests that other unidentified virulence determinants are involved in V. cholerae O139 pathogenesis.

An epidemic of a cholera-like disease occurred among Khmers in a camp in Aranyaprathet, Thailand, in May 1990. Of 215 patients with diarrhea, Vibrio cholerae O1 was isolated from 25 (12%) and V. cholerae non-O1 was isolated from 15 (7%). Five of 15 (33%) non-O1 V. cholerae isolates hybridized with two different oligonucleotide probes previously used to detect V. cholerae non-O1 that produces a heat-stable toxin. This is the first description of an epidemic of diarrhea caused by V. cholerae non-O1 that produces heat-stable toxin.

Background Campylobacter jejuni is a major cause of gastroenteritis worldwide. In Thailand, several strains of C. jejuni have been isolated and identified as major diarrheal pathogens among adult travelers. To study the epidemiology of C. jejuni in adult travelers and U.S. military personnel with acute diarrhea in Thailand from 1998-2003, strains of C. jejuni were isolated and phenotypically identified, serotyped, tested for antimicrobial susceptibility, and characterized using pulsed-field gel electrophoresis (PFGE). Results A total of 312 C. jejuni isolates were obtained from travelers (n = 46) and U.S. military personnel (n = 266) in Thailand who were experiencing acute diarrhea. Nalidixic acid and ciprofloxacin resistance was observed in 94.9% and 93.0% of the isolates, respectively. From 2001-2003, resistance to tetracycline (81.9%), trimethoprim-sulfamethoxazole (57.9%), ampicillin (28.9%), kanamycin (5.9%), sulfisoxazole (3.9%), neomycin (2.0%), and streptomycin (0.7%) was observed. Combined PFGE analysis showed considerable genetic diversity among the C. jejuni isolates; however, four PFGE clusters included isolates from the major Lior serotypes (HL: 36, HL: 11, HL: 5, and HL: 28). The PFGE analysis linked individual C. jejuni clones that were obtained at U.S. military exercises with specific antimicrobial resistance patterns. Conclusions In summary, most human C. jejuni isolates from Thailand were multi-resistant to quinolones and tetracycline. PFGE detected spatial and temporal C. jejuni clonality responsible for the common sources of Campylobacter gastroenteritis.