Etiology of Acute Diarrheal Disease and Antimicrobial Susceptibility Pattern in Children Younger Than 5 Years Old in Nepal.

Diarrhea is a common cause of morbidity and mortality among children younger than 5 years in developing countries. Children from 3 to 60 months of age were recruited from two hospitals in Nepal- Bharatpur Hospital, Bharatpur, and Kanti Children's Hospital, Kathmandu-in 2006 to 2009. Stool specimens collected from 1,200 children with acute diarrhea (cases) and 1,200 children without diarrhea (control subjects) were examined for a broad range of enteropathogens by standard microbiology, including microscopy, enzyme immunoassay for viral pathogens (adenovirus, astrovirus, and rotavirus) and protozoa (Giardia, Cryptosporidium, and Entamoeba histolytica), as well as by using reverse transcription real-time polymerase for norovirus. Antimicrobial susceptibility testing was performed using the disk diffusion method. Overall, rotavirus (22% versus 2%), norovirus (13% versus 7%), adenovirus (3% versus 0%), Shigella (6% versus 1%), enterotoxigenic Escherichia coli (8% versus 4%), Vibrio (7% versus 0%), and Aeromonas (9% versus 3%) were identified significantly more frequently in cases than control subjects. Campylobacter, Plesiomonas, Salmonella, and diarrheagenic E. coli (enteropathogenic, enteroinvasive, enteroaggregative) were identified in similar proportions in diarrheal and non-diarrheal stools. Campylobacter was resistant to second-generation quinolone drugs (ciprofloxacin and norfloxacin), whereas Vibrio and Shigella were resistant to nalidixic acid and trimethoprim/sulfamethoxazole. This study documents the important role of rotavirus and norovirus in acute diarrhea in children younger than 5 years, followed by the bacteria Shigella, enterotoxigenic E. coli, Vibrio cholera, and Aeromonas. Data on the prevalence and epidemiology of enteropathogens identify potential pathogens for public health interventions, whereas pathogen antibiotic resistance pattern data may provide guidance on choice of therapy in clinical settings.

Design of a Bacteriophage Cocktail Active against Shigella Species and Testing of Its Therapeutic Potential in Galleria mellonella.

Shigellosis is a leading global cause of diarrheal disease and travelers' diarrhea now being complicated by the dissemination of antibiotic resistance, necessitating the development of alternative antibacterials such as therapeutic bacteriophages (phages). Phages with lytic activity against Shigella strains were isolated from sewage. The genomes of 32 phages were sequenced, and based on genomic comparisons belong to seven taxonomic genera: Teetrevirus, Teseptimavirus, Kayfunavirus, Tequatrovirus, Mooglevirus, Mosigvirus and Hanrivervirus. Phage host ranges were determined with a diverse panel of 95 clinical isolates of Shigella from Southeast Asia and other geographic regions, representing different species and serotypes. Three-phage mixtures were designed, with one possessing lytic activity against 89% of the strain panel. This cocktail exhibited lytic activity against 100% of S. sonnei isolates, 97.2% of S. flexneri (multiple serotypes) and 100% of S. dysenteriae serotypes 1 and 2. Another 3-phage cocktail composed of two myophages and one podophage showed both a broad host range and the ability to completely sterilize liquid culture of a model virulent strain S. flexneri 2457T. In a Galleria mellonella model of lethal infection with S. flexneri 2457T, this 3-phage cocktail provided a significant increase in survival.

Molecular characterization of multidrug-resistant ESKAPEE pathogens from clinical samples in Chonburi, Thailand (2017-2018).

BACKGROUND: ESKAPEE pathogens Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. and Escherichia coli are multi-drug resistant (MDR) bacteria that present increasing treatment challenges for healthcare institutions and public health worldwide. METHODS: 431 MDR ESKAPEE pathogens were collected from Queen Sirikit Naval Hospital, Chonburi, Thailand between 2017 and 2018. Species identification and antimicrobial resistance (AMR) phenotype were determined following CLSI and EUCAST guidelines on the BD Phoenix System. Molecular identification of antibiotic resistant genes was performed by polymerase chain reaction (PCR), real-time PCR assays, and whole genome sequencing (WGS). RESULTS: Of the 431 MDR isolates collected, 1.2% were E. faecium, 5.8% were S. aureus, 23.7% were K. pneumoniae, 22.5% were A. baumannii, 4.6% were P. aeruginosa, 0.9% were Enterobacter spp., and 41.3% were E. coli. Of the 401 Gram-negative MDR isolates, 51% were carbapenem resistant, 45% were ESBL producers only, 2% were colistin resistance and ESBLs producers (2%), and 2% were non-ESBLs producers. The most prevalent carbapenemase genes were blaOXA-23 (23%), which was only identified in A. baumannii, followed by blaNDM (17%), and blaOXA-48-like (13%). Beta-lactamase genes detected included blaTEM, blaSHV, blaOXA, blaCTX-M, blaDHA, blaCMY, blaPER and blaVEB. Seven E. coli and K. pneumoniae isolates showed resistance to colistin and carried mcr-1 or mcr-3, with 2 E. coli strains carrying both genes. Among 30 Gram-positive MDR ESKAPEE, all VRE isolates carried the vanA gene (100%) and 84% S. aureus isolates carried the mecA gene. CONCLUSIONS: This report highlights the prevalence of AMR among clinical ESKAPEE pathogens in eastern Thailand. E. coli was the most common MDR pathogen collected, followed by K. pneumoniae, and A. baumannii. Carbapenem-resistant Enterobacteriaceae (CRE) and extended spectrum beta-lactamases (ESBLs) producers were the most common resistance profiles. The co-occurrence of mcr-1 and mcr-3 in 2 E. coli strains, which did not affect the level of colistin resistance, is also reported. The participation of global stakeholders and surveillance of MDR remain essential for the control and management of MDR ESKAPEE pathogens.

Enteric etiological surveillance in acute diarrhea stool of United States Military Personnel on deployment in Thailand, 2013-2017.

BACKGROUND: Diarrhea remains a major public health problem for both civilian and military populations. This study describes the prevalence of acute diarrheal illness etiological agents, their antibiotic resistance distribution patterns, the resulting impact upon military force health protection, and potential prevention and treatment strategies. RESULTS: Forty-eight acute diarrhea stool samples from US military personnel deployed to Thailand from 2013-2017 were screened for enteric pathogens using ELISA, the TaqMan Array Card (TAC), and conventional microbiological methods. These isolates were also evaluated using antimicrobial susceptibility testing (AST) against ampicillin (AMP), azithromycin (AZM), ceftriaxone (CRO), ciprofloxacin (CIP), nalidixic acid (NA), erythromycin (ERY), and trimethoprim-sulfamethoxazole (SXT) using commercial methodology. Susceptibility results were interpreted following the CLSI and NARM guidelines. Questionnaire data obtained from 47/48 volunteers indicated that 89.4% (42/47) reported eating local food and the most common clinical symptoms were nausea and abdominal pain (51%; 24/47). Multiple bacterial species were identified from the 48 stool samples with diarrhea etiological agents being detected in 79% (38/48) of the samples distributed as follows: 43.8% (21/48) Campylobacter jejuni and Campylobacter species, 42% (20/48) diarrheagenic Escherichia coli, and 23% (11/48) Salmonella. Co-infections were detected in 46% (22/48) of the samples. All C. jejuni isolates were resistant to CIP and NA. One C. jejuni isolate exhibited resistance to both AZM and ERY. Lastly, an association between exposure to poultry and subsequent detection of the diarrhea-associated pathogens E. coli and P. shigelloides was significant (p < 0.05). CONCLUSION: The detection of Campylobacter isolates with CIP, AZM and ERY resistance has critical force health protection and public health implications, as these data should guide effective Campylobacteriosis treatment options for deployed military members and travelers to Southeast Asia. Additional research efforts are recommended to determine the association of pathogen co-infections and/or other contributing factors towards diarrheal disease in military and traveler populations. Ongoing surveillance and AST profiling of potential disease-causing bacteria is required for effective disease prevention efforts and treatment strategies.

Epidemiology and etiology of Traveler's diarrhea in Bangkok, Thailand, a case-control study.

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 < 0.001, p < 0.001, p < 0.001, respectively. Shigella (3%) and ETEC (8%), detected in lower prevalence, also showed significant association with TD at p < 0.001 and p = 0.002, respectively. Travelers from East Asian countries had an increased risk of Vibrio infection (Crude odds ratio: 3.1, p-value = 0.001); travelers from the United States, Canada, and Europe had an increased risk of Campylobacter infection (Crude odds ratio: 2.6, p-value = 0.001); and travelers from Australia and New Zealand had an increased risk of Salmonella infection (Crude odds ratio: 3.2, p-value = 0.009). CONCLUSIONS: Etiology of TD in Thailand is mainly of bacterial origin. Plesiomonas, Vibrio, and norovirus are underappreciated diarrheagenic pathogens. In our study, the origin of the traveler plays an important role in diarrhea etiology. Understanding variations in TD severity and etiology among travelers from different regions warrants further study.

Antibiotic resistance, molecular characterizations, and clinical manifestations of Campylobacteriosis at a military medical center in Hawaii from 2012-2016: a retrospective analysis.

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 < 0.001 and p = 0.006, respectively). HS2 FQ-resistant isolates associated with clonal complex 21, possibly indicating clonal spread in FQ resistance. Macrolides should be considered for treatment of suspect cases due to lack of observed resistance.

Extended-spectrum ß-lactamase prevalence and virulence factor characterization of enterotoxigenic Escherichia coli responsible for acute diarrhea in Nepal from 2001 to 2016.

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 blaCTX-M-15 (80%), followed by blaSHV-12 (20%) and blaCTX-M-14 (2.5%). The beta-lactamase genes blaTEM-1 (40%) and blaCMY-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.

Genomic Characterization of Nonclonal mcr-1-Positive Multidrug-Resistant Klebsiella pneumoniae from Clinical Samples in Thailand.

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 ß-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.

Antibiotic resistance in Campylobacter and other diarrheal pathogens isolated from US military personnel deployed to Thailand in 2002-2004: a case-control study.

BACKGROUND: Campylobacter continues to be an important cause of diarrheal disease worldwide and a leading cause in Southeast Asia. Studies of US soldiers and marines deployed to Thailand for a 2 to 3 week field exercise provide a unique population in which to study traveler's diarrhea. METHODS: A case-control study of 217 deployed military personnel was conducted from 2002 through 2004. Of these, 155 subjects who presented to a field medical unit with acute diarrhea were enrolled as cases. These subjects referred an additional 62 diarrhea-free colleagues who served as controls. Frequencies of isolation of Campylobacter spp. and other enteric pathogens were compared in cases and controls, and antibiotic resistance of isolates was described. RESULTS: Of the 155 subjects with diarrhea, Campylobacter spp. was the most commonly identified pathogen, found in 54 (35%) of the subjects, followed by non-typhoidal Salmonella species found in 36 (23%) subjects. Of the 57 separate C. jejuni and C. coli isolates from these individuals, 51 (89%) were resistant to ciprofloxacin by the disc diffusion method. Nearly one-third of the Campylobacter species were resistant to ampicillin and trimethoprim-sulfamethoxazole. Resistance to azithromycin remained low at 2% (n = 1). CONCLUSIONS: The significant morbidity and marked fluoroquinolone resistance associated with Campylobacter infections in Thailand are important considerations for clinicians providing counseling on appropriate antibacterial regimens for civilian and military travelers.

Whole Genome Sequence Analysis of Salmonella Typhi Isolated in Thailand before and after the Introduction of a National Immunization Program.

Vaccines against Salmonella Typhi, the causative agent of typhoid fever, are commonly used by travellers, however, there are few examples of national immunization programs in endemic areas. There is therefore a paucity of data on the impact of typhoid immunization programs on localised populations of S. Typhi. Here we have used whole genome sequencing (WGS) to characterise 44 historical bacterial isolates collected before and after a national typhoid immunization program that was implemented in Thailand in 1977 in response to a large outbreak; the program was highly effective in reducing typhoid case numbers. Thai isolates were highly diverse, including 10 distinct phylogenetic lineages or genotypes. Novel prophage and plasmids were also detected, including examples that were previously only reported in Shigella sonnei and Escherichia coli. The majority of S. Typhi genotypes observed prior to the immunization program were not observed following it. Post-vaccine era isolates were more closely related to S. Typhi isolated from neighbouring countries than to earlier Thai isolates, providing no evidence for the local persistence of endemic S. Typhi following the national immunization program. Rather, later cases of typhoid appeared to be caused by the occasional importation of common genotypes from neighbouring Vietnam, Laos, and Cambodia. These data show the value of WGS in understanding the impacts of vaccination on pathogen populations and provide support for the proposal that large-scale typhoid immunization programs in endemic areas could result in lasting local disease elimination, although larger prospective studies are needed to test this directly.

Immune responses to Campylobacter (C. jejuni or C. coli) infections: a two-year study of US forces deployed to Thailand.

Campylobacter spp. is a leading cause of diarrheal disease among US troops deployed to Thailand for exercise. We investigated the importance of immunological analysis and immune responses against Campylobacter infection in US troops deployed to Thailand. Blood and fecal samples were collected from volunteered soldiers with diarrhea and from healthy controls. Stool culture was performed to identify the pathogens. Campylobacter-specific antibodies, antibody secreting cells and cytokines were measured. Several bacterial protein fragments in the outer membrane extract of Campylobacter spp., were identified by an immunoblot analysis with plasma and fecal antibodies. Among all of the diarrheal cases, 35% were Campylobacter-positive. Based on antibody titers in plasma and in fecal extract and antibody secreting cells: 6% of healthy controls, 32% of the Campylobacter culture-negative diarrheal cases, and 85% of the Campylobacter culture-positive diarrheal cases were positive for Campylobacter. Our results indicate that the measurement of Campylobacter-specific antibodies in plasma and fecal extract samples is a good marker of exposure to Campylobacter, and this test may be a useful diagnostic tool for seroepidemiological studies. Elicited antibodies against several bacterial outer membrane protein fragments suggest that these protein fragments are vital in providing protective immunity against Campylobacter.

Evaluation of an intragastric challenge model for Shigella dysenteriae 1 in rhesus monkeys (Macaca mulatta) for the pre-clinical assessment of Shigella vaccine formulations.

Shigellosis is a worldwide disease, characterized by abdominal pain, fever, vomiting, and the passage of blood- and mucus-streaked stools. Rhesus monkeys and other primates are the only animals that are naturally susceptible to shigellosis. A suitable animal model is required for the pre-clinical evaluation of vaccines candidates. In this study, the minimal dose of Shigella dysenteriae1 1617 strain required to produce dysentery in four of five (80% attack rate) monkeys using an escalating dose range for three groups [2 × 10(8) , 2 × 10(9) and 2 × 10(10) colony forming unit (CFU)] was determined. In addition, the monkeys were re-infected. The identified optimal challenge dose was 2 × 10(9) CFU; this dose elicited 60% protection in monkeys when they were re-challenged with a one log higher dose (2 × 10(10) CFU). The challenge dose, 2 × 10(10) CFU, produced severe dysentery in all monkeys, with one monkey dying within 24 h, elicited 100% protection when re-challenged with the same dose. All monkeys exhibited immune responses. This study concludes that the rhesus monkey model closely mimics the disease and immune response seen in humans and is a suitable animal model for the pre-clinical evaluation of Shigella vaccine candidates. Prior infection with the 1617 strain can protect monkeys against subsequent re-challenges with homologous strains.

Bacterial pathogens isolated from raw meat and poultry compared with pathogens isolated from children in the same area of rural Thailand.

To better understand the epidemiology of bacterial food borne pathogens in children, in relation to pathogens in meats from a market in rural Thailand, we collected 73 cultures samples from raw chicken, pork and fish at a local market where diarrheal disease surveillance was conducted. Standard methods were employed to isolate, identify and serotype enteric pathogens from children and food samples. Antibiotic susceptibility testing was performed. Ninety-seven percent of food samples were contaminated with at least one enteric pathogen. The pathogens most commonly isolated from food were Salmonella spp (84%), Arcobacter butzleri (74%) and Campylobacter spp (51%). The most common serovars of Salmonella obtained from humans with diarrhea were S. Risen, S. Stanley and S. Anatum. Most common serovars of Salmonella isolated from food were S. Anatum, S. Stanley, and S. Corvallis. Fifty-one percent and 25% of children infected with Salmonella and Campylobacter, respectively, infected with the same serotypes isolated from food samples, suggesting these pathogens are widespread in food and humans. Pulsed-field gel analysis of Salmonella spp revealed 65 pulsotypes, but no point-sources of salmonellosis were identified. Joint epidemiologic/laboratory studies are useful to describe the epidemiology of enteric pathogens in rural populations.

Prevalence and antimicrobial susceptibilities of Vibrio, salmonella, and Aeromonas isolates from various uncooked seafoods in Thailand.

Uncooked seafood samples were collected from open markets and supermarkets in Bangkok, Thailand, and were examined for the presence of Vibrio, Salmonella, and Aeromonas species from January to February 2008. From 120 samples, 272 bacterial isolates were identified through biochemical testing. Of all sea bass, shrimp, oyster, and blood cockle samples (30 of each) that were processed for culture, 114 (95%) samples had at least one detectable isolate of Vibrio, Salmonella, or Aeromonas, leaving only 6 (5%) samples free of them. All oyster sample (100%) had at least one pathogen, followed by sea bass (97%), blood cockles (97%), and shrimp (90%). Overall, 111 (92%) of all samples had detectable Vibrio spp., 32 (27%) had detectable Aeromonas spp., and 25 (21%) had detectable Salmonella enterica. There was no overall difference between positive samples collected from fresh markets versus supermarkets (relative risk, 0.97; 95% CI, 0.89 to 1.05). Resistance to ampicillin among isolated pathogens was relatively high (56%), while resistance to 12 other antibiotics, including azithromycin, ciprofloxacin, and trimethoprim-sulfamethoxazole, was relatively low (0, 0, and 3%, respectively). Study results indicate that uncooked seafood in Bangkok, Thailand, commonly harbors enteric pathogens and that consumption of uncooked seafood should be avoided to reduce foodborne illnesses.

Etiology of diarrhea in young children and patterns of antibiotic resistance in Cambodia.

BACKGROUND: Little is known about diarrhea etiology and antibiotic resistance in developing countries where diarrhea is a major public health problem. METHODS: To describe diarrhea etiology and antibiotic resistance patterns in Cambodia, 600 children aged 3 months to 5 years with acute diarrhea (cases) and 578 children without diarrhea (controls) were enrolled from a hospital in Phnom Penh. Stool samples were collected, and pathogens and antibiotic resistance patterns were described. RESULTS: The most frequently isolated pathogens in these cases were enteroaggregative Escherichia coli (20%) and rotavirus (26%). Enterotoxigenic E. coli, enteroaggregative E. coli, Shigella, Aeromonas, rotavirus, and adenovirus were statistically significantly associated with diarrhea. Among cases, vomiting was associated with viral infections, whereas bloody stool was associated with Shigella. Enterotoxigenic E. coli isolates were highly resistant to ampicillin, sulfonamides, and tetracycline. Approximately 50% of Campylobacter coli and 30% of Campylobacter jejuni isolates were resistant to nalidixic acid and ciprofloxacin. Over 33% of Salmonella isolates were resistant to ampicillin and tetracycline, and almost 100% of Shigella isolates were resistant to trimethoprim/sulfamethoxazole. CONCLUSIONS: These data on the etiology of diarrhea and antibiotic resistance patterns in Cambodia will have significant effect on local public health policies and on local resource prioritization practices.

Case-control study of diarrheal disease etiology in a remote rural area in Western Thailand.

The objective was to assess the association of enteric pathogens in diarrheal disease in a remote rural area in Thailand. Stool specimens were collected from 236 children aged 3 months to 5 years with acute diarrhea (cases) and from 236 asymptomatic controls. Standard microbiologic methods, and enzyme immunoassay for viral pathogens, Giardia, and Cryptosporidium, were used to identify enteric pathogens with susceptibility testing by disk diffusion. Campylobacter, Plesiomonas, Salmonella, and enterotoxigenic Escherichia coli were commonly isolated from cases and controls (22% versus 25%, 10% versus 11%, 6% versus 9%, and 10% versus 6%, respectively). Only Shigella, rotavirus, and adenovirus were identified significantly more frequently in cases than controls (9% versus 0%, 18% versus 3%, and 16% versus 2%, respectively), whereas Giardia lamblia was detected less often in cases than controls. Most pre-school children were infested with enteric pathogens; laboratory-based studies are important to understand the epidemiology of enteric pathogens in remote areas among marginal populations.

Enteric pathogen sampling of tourist restaurants in Bangkok, Thailand.

BACKGROUND: Travelers' diarrhea (TD) is the most prevalent disorder affecting travelers to developing countries. Thailand is considered "moderately risky" for TD acquisition, but the risk by city visited or behavior of the visitor has yet to be definitely defined. Restaurant eating is consistently associated with the acquisition of diarrhea while traveling, and pathogen-free meals serve as a marker of public health success. This study seeks to ascertain a traveler's risk of exposure to certain bacterial gastric pathogens while eating at Bangkok restaurants recommended in popular tourist guide books. METHODS: A cross-sectional tourist restaurant survey was conducted. Thirty-five restaurants recommended in the two top selling Bangkok guidebooks on Amazon.com were sampled for bacterial pathogens known to cause diarrhea in Thailand, namely Salmonella, Campylobacter, and Arcobacter (a Campylobacter-like organism). A total of 70 samples from two meals at each restaurant were obtained. Suspected bacterial pathogens were isolated by differential culture and tested for antibiotic resistance. RESULTS: Salmonella group E was isolated from one meal (2%), and Arcobacter butzleri from nine meals (13%). Campylobacter spp. were not found. The large majority of A butzleri isolates were resistant to azithromycin but susceptible to ciprofloxacin and an aminoglycoside. CONCLUSIONS: A traveler's risk of exposure to established bacterial pathogens, Salmonella and Campylobacter, by eating in recommended restaurants is small. Arcobacter butzleri exposure risk is 13% per meal eaten, and rises to 75% when 10 meals are eaten. All restaurants, regardless of price, appear to be equally "risky." Current evidence points to Arcobacter being pathogenic in humans; however, further research is needed to conclusively define pathogenicity. Routine prophylaxis for diarrhea is not recommended; however, travelers should be aware of the risk and come prepared with adequate and appropriate self-treatment medications.

Antibacterial activity of Thai medicinal plants Pikutbenjakul.

BACKGROUND: Bacterial infections caused by resistant strains have been increased dramatically. Pikutbenjakul, a Thai medicinal plant formula containing Piper longum, Piper sarmentosum, Piper interruptum, Plumbago indica and Zingiber officinale have been widely used in Thai traditional medicine. OBJECTIVE: To determine antimicrobial activity of Pikutbenjakul formula and its components in order to develop the medicinal plants for alternative treatment of bacteria causing diarrhea. MATERIAL AND METHOD: Activity of Pikutbenjakul formula and its components was tested using disc diffusion and broth dilution methods against bacteria associated a set of bacteria associated with diarrheal disease including Vibrio cholerae, Vibrio vulnificus, Salmonella, Shigella, Escherichia coli (EIEC, ETEC, EPEC, EAEC and EHEC) and Staphylococcus aureus. The extraction was performed by maceration in 95% ethanol. RESULTS: The results showed all tested strains were susceptible to P. indica while other components were able to inhibit some strains. P. sarmentosum showed antimicrobial activity against Vibrios with the MIC values between 0.625 to > or =5 mg/ml. P. sarmentosum, P. indica and Pikutbenjakul formulas inhibited the growth of all Vibrios. P. interruptum inhibited V. cholerae serogroups O1 and non-O1/non-O139. P. longum was able to inhibit only two isolates of V. cholerae serogroup O139 (MIC = 1.25 mg/ml) and V. vulnificus (MIC > or =5 mg/ml). The activity of Pikutbenjakul containing Zingiber spp. and Pikutbenjakul containing Z. officinal against Vibrios, Shigella spp. and S. aureus was not significantly different. P. indica could inhibit Salmonella (MIC > or =5 mg/ml), E. coli (MIC > or =5 mg/ml) and S. aureus (MIC = 1.25 mg/ml). CONCLUSION: The results support the Thai medicinal plants for treatment of diarrhea caused by these bacteria. This study also provides an insightful knowledge on antimicrobial activity which would lead to further development of an effective formula of Pikutbenjakul for diarrheal disease and other infectious diseases in future.

Emergence and properties of fluoroquinolone resistant Salmonella enterica serovar Typhi strains isolated from Nepal in 2002 and 2003.

A total of 171 Salmonella enterica serovar Typhi strains isolated from Nepal, mostly from patients with typhoid fever in 2002-2003, were tested for antimicrobial susceptibility by disk diffusion assay. Selected S. enterica serovar Typhi isolates were tested for MICs by E-test for ceftriaxone, ciprofloxacin and ofloxacin. Mutations of DNA gyrase gyrA and gyrB and topoisomerase IV parC and parE were identified by sequencing of PCR amplicons. By disk diffusion assay, 75/171 S. enterica serovar Typhi isolates were resistant to nalidixic acid, ampicillin, choramphenicol, streptomycin, tetracycline, sulfisoxazole, and trimethroprim/sulfamethoxazoles. Multiple drug resistance to the 7 antimicrobials was most predominant among S. enterica serovar Typhi isolates in this study. Resistance to nalidixic acid was detected in 76/111 and 56/60 of total isolates collected in 2002 and 2003, respectively. Nalidixic acid-resistant isolates in 2002 and 2003 showed MIC range for ciprofloxacin of 0.125-0.250 mg/l. Nalidixic acid-resistant isolates contained point mutations in gyrA and parC but not gyrB and parE. The gyrA mutation of nalidixic acid-resistant isolates obtained in 2002 and 2003 had amino acid substitution at position 83 of Serine-->Tyrosine and Serine-->Phenylalanine, respectively. Two different mutations of gyrA were detected among nalidixic acid-resistant isolates. Thus it is necessary to monitor mutation in DNA topoisomerase associated with increases in quinolones resistance.

First multi-locus sequence typing scheme for Arcobacter spp.

BACKGROUND: Arcobacter spp. are a common contaminant of food and water, and some species, primarily A. butzleri and A. cryaerophilus, have been isolated increasingly from human diarrheal stool samples. Here, we describe the first Arcobacter multilocus sequence typing (MLST) method for A. butzleri, A. cryaerophilus, A. skirrowii, A. cibarius and A. thereius. RESULTS: A sample set of 374 arcobacters, including 275 A. butzleri, 72 A. cryaerophilus, 15 A. skirrowii and 8 A. cibarius isolates from a wide variety of geographic locations and sources, was typed in this study. Additionally, this sample set contained four strains representing a new Arcobacter species, A. thereius. The seven loci used in the four-species Arcobacter MLST method are the same as those employed previously in C. jejuni, C. coli, C. helveticus and C. fetus (i.e. aspA, atpA(uncA), glnA, gltA, glyA, pgm and tkt). A large number of alleles were identified at each locus with the majority of isolates containing a unique sequence type. All Arcobacter isolates typed in this study contain two glyA genes, one linked to lysS (glyA1) and the other linked to ada (glyA2). glyA1 was incorporated into the Arcobacter MLST method while glyA2 was not because it did not increase substantially the level of discrimination. CONCLUSION: No association of MLST alleles or sequence types with host or geographical source was observed with this sample set. Nevertheless, the large number of identified alleles and sequence types indicate that this MLST method will prove useful in both Arcobacter strain discrimination and in epidemiological studies of sporadic Arcobacter-related gastroenteritis. A new Arcobacter MLST database was created http://pubmlst.org/arcobacter/; allele and ST data generated in this study were deposited in this database and are available online.