Oralak Serichantalergs

Distribution and Content of Class 1 Integrons in Different Vibrio cholerae O-Serotype Strains Isolated in Thailand

ABSTRACT 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, anddfrA15 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 aadA2resistance 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 differentV. cholerae O serotypes of mainly clinical origin in Thailand.

Typhoid Fever: A Massive, Single-Point Source, Multidrug-Resistant Outbreak in Nepal

BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSIONS 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 citys 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 to >1 antibiotic.

A high proportion of Vibrio cholerae strains isolated from children with diarrhoea in Bangkok, Thailand are multiple antibiotic resistant and belong to heterogenous non-O1, non-O139 O-serotypes

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-0139 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; > or = 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.

Diarrheagenic Escherichia coli and Shigella Strains Isolated from Children in a Hospital Case-Control Study in Hanoi, Vietnam

ABSTRACT 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.

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.

Development of a multiplex polymerase chain reaction assay for diarrheagenic Escherichia coli and Shigella spp. and its evaluation on colonies, culture broths, and stool

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.

Emerging fluoroquinolone and macrolide resistance of Campylobacter jejuni and Campylobacter coli isolates and their serotypes in Thai children from 1991 to 2000.

This study investigated fluoroquinolone, macrolide resistances and serotype distributions among Campylobacter jejuni and Campylobacter coli isolated 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 of C. jejuni were Lior 36, 2 and 4 and of C. coli were 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 in C. jejuni isolates resistant to ciprofloxacin from Bangkok but not for macrolide resistance from both settings. In summary, fluoroquinolone resistance among C. jejuni and C. coli isolates became widespread in both Bangkok and rural settings in Thailand in the 1990s while widespread resistance to macrolides was undetected.

Rotavirus disease in young children from Hanoi Vietnam.

Background: Pathogen prevalences and antimicrobial susceptibilities are essential for the rational development of preventive strategies for diarrheal diseases, but little recent information from Vietnam is available. We prospectively studied the prevalence of enteric pathogens in children less than 5 years of age with acute diarrhea and in nondiarrhea controls in a city hospital in Hanoi, Vietnam for 1 year. Methods: Enteric bacteria and viruses were detected by standard culture methods, and enzyme immunoassay in 291 cases and 291 controls. Results: Detection rates of viral pathogens among cases and controls were 31% and 3% for rotavirus, 12% and 1% for astrovirus and 4% and 1% for adenovirus. For bacterial pathogens, Aeromonas, Shigella, Salmonella, Campylobacter and enterotoxigenic E. coli were isolated from cases and controls in 15% and 8%, 9% and 1%, 7% and 1%, 4% and 0%, and 3% and 0%, respectively. The isolation of bacterial and viral pathogens except for adenovirus was significantly lower in controls than cases. Fluoroquinolones were effective against most bacterial enteropathogens, but resistance was observed in 27% of Campylobacter isolates. Conclusions: Viral etiologic agents especially rotavirus were the most important cause of acute diarrhea in children less than 5 years of age in Hanoi. The burden of rotavirus in young children in Hanoi warrants consideration of using the recently released rotavirus vaccine.

Phenotypic and molecular characterization of Vibrio cholerae O1 isolated in Samutsakorn, Thailand before, during and after the emergence of V. cholerae O139

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 0139 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 0139. However, O1 strains isolated during and after the advent of 0139 showed identical ribotypes which were distinctly different from the types identified in strains isolated before the emergence of 0139. Ribotypes identified in strains during and after the advent of 0139 were also demonstrated by O1 strains isolated immediately before the emergence of 0139. 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 0139 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 0139 epidemic had a ribotype identical to a type demonstrated by 0139 strains isolated in Thailand. Our results support similar findings in Bangladesh and India that a distinct O1 strain appeared during the 0139 epidemic. However, compared with the apparent identical strain which replaced 0139 in Bangladesh and India, the emerged O1 strain in Samutsakorn showed a different ribotype and CT genotype.

Molecular characterization and antibiotic susceptibility of Vibrio cholerae non-O1.

A collection of 64 clinical and environmental Vibrio cholerae non-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 (> or = 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. cholerae non-O1 strains were cholera toxin negative, whereas only strains recovered from patients were heat-stable enterotoxin positive. Forty-seven Bgl I 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 of V. cholerae non-O1 infections.