A possible mechanism of macrolide resistance among multiple resistant Campylobacter jejuni and Campylobacter coli isolated from Thai children during 1991-2000.

A total of 495 Campylobacterjejuni and 122 C. coli isolated from Thai children were screened for macrolide (erythromycin and azithromycin) resistance by disk diffusion assay. Minimum inhibitory concentrations for erythromycin, azithromycin, nalidixic acid, ciprofloxacin, tetracycline, streptomycin, gentamicin and chloramphenicol were further determined for these macrolide-resistant Campylobacter isolates. Presence of known point mutations resulting in reduced susceptibility to macrolides was investigated by PCR and DNA sequencing. Seventeen percent (23/122) of C. coli and 2.4% (12/495) of C. jejuni isolates were resistant to macrolides. By sequencing domain V of the 23S ribosomal DNA from all 35 macrolide-resistant isolates, a known point mutation of 23S rRNA associated with reduced susceptibility to macrolides was detected in all isolates except one. Among the macrolide-resistant isolates, all were multiply resistant to nalidixic acid and ciprofloxacin, of which the latter is the preferred antimicrobial used for diarrheal treatment in Thailand. Furthermore, most macrolide-resistant isolates were also resistant to tetracycline and streptomycin. The spread of macrolide and quinolone resistant Campylobacter should be monitored closely in Thailand and elsewhere as these antimicrobials are preferred drugs for treatment of diarrhea.

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.

An observational clinic-based study of diarrheal illness in deployed United States military personnel in Thailand: presentation and outcome of Campylobacter infection.

Campylobacter is a leading cause of traveler's diarrhea in Thailand. Since resistance to quinolones is high among Campylobacter isolates, empiric therapy with quinolones for traveler's diarrhea may be ineffective in this region. We conducted an observational study among 169 U.S. military personnel with acute diarrhea and compared their microbiologic findings to those of 77 asymptomatic personnel deployed to Thailand in May 1998. Of 146 pathogenic bacterial isolates, the most common were nontyphoidal Salmonella (n = 31), enterotoxigenic Escherichia coli (n = 24), and C. jejuni/coli (n = 23). Campylobacter was strongly associated with disease (odds ratio = 5.9; 95% confidence interval = 1.3-37.3), with a more severe clinical presentation, and with a reduced functional ability at presentation (P = 0.02). In vitro resistance to ciprofloxacin was observed in 96% of the Campylobacter isolates. Sub-optimal treatment response to ciprofloxacin was observed in 17% of the cases of Campylobacter infection versus 6% due to other causes. These results highlight the importance of Campylobacter as a cause of severe traveler's diarrhea in Thailand and illustrates the ongoing problem with antibiotic-resistant strains and associated treatment problems.

Bacterial enteric pathogens in children with acute dysentery in Thailand: increasing importance of quinolone-resistant Campylobacter.

Current data on pathogen prevalence and drug resistance patterns are important for treatment and vaccine-development strategies. An etiologic study of acute bacterial dysentery was conducted in children up to 12 years of age in 2 major hospitals in and around Bangkok. Stool samples or rectal swabs and clinical data were collected. Standard microbiological methods were used to detect Salmonella, Shigella, Campylobacter, Vibrio, Aeromonas and Plesiomonas. Pathogenic E. coli (ETEC, EIEC, STEC) was identified by digoxigenin-labeled probes. A total of 623 cases were enrolled: median age 11.0 months (range 1 month-12 years). At least one bacterial pathogen was isolated in 55% of cases. Campylobacter was the most common pathogen found (28%), whereas Salmonella, Shigella and ETEC were isolated from 18%, 9% and 6% respectively. EIEC, Vibrio and Plesiomonas were isolated from <1% and no STEC was detected. C. jejuni serotypes 36, 4 and 11 were the most common. The mean age of cases with Campylobacter was significantly lower than with Shigella (17.9 vs 52.8 months, p<0.001). Clinical presentations of Campylobacter and Shigella infections were compared: fever (28% vs 37%), abdominal colic (62% vs 80%, p<0.05), vomiting (38% vs 70%, p<0.001) and bloody stools (52% vs 48%). The Campylobacter isolates (80% C. jejuni, 20% C. coli) were 90% resistant to ciprofloxacin but sensitive to macrolides. All the Shigella isolates (70% S. sonnei) were sensitive to quinolones. Our study illustrates the increasing importance of quinolone-resistant Campylobacter and the decline of Shigella in the etiology of dysentery in Thailand. The clinical presentation of campylobacteriosis is similar to that of shigellosis, except that the patients may be younger and there may be less association with colic and vomiting; having fecal leukocytes will be >10/HPF. The use of macrolide antibiotics rather than quinolones would be reasonable in children <24 months of age; fluoroquinolones will be ineffective in at least half of culture-positive cases.

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.

Prospective study of the incidence of diarrheal disease and Helicobacter pylori infection among children in an orphanage in Thailand.

To evaluate the hypothesis that gastric infection with Helicobacter pylori increases risk for diarrheal disease in children, we conducted a yearlong prospective study among 160 orphanage children < 5 years of age in Nonthaburi, Thailand. Serum samples collected at six-month intervals were examined by ELISA for antibodies to H. pylori, and children were followed daily for the development of diarrhea. Seven percent of children were seropositive on enrollment, 59% were seronegative, and 34% were indeterminate. Among the seronegative children, seroconversion occurred at a rate of 7% per six months. Forty-six percent of children developed 214 total episodes of diarrhea. By age group, children < 18 months, 18-24 months and > 24 months of age experienced 2.6, 1.1, and 0.2 mean diarrhea episodes per six months. The incidence of diarrhea was not significantly different between children by H. pylori serostatus. We conclude that H. pylori infection was not associated with an increased risk of diarrheal disease.

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.

Trends in antibiotic resistance among diarrheal pathogens isolated in Thailand over 15 years.

Antibiotic resistance trends were examined for Shigella species, nontyphoidal Salmonella species, enterotoxigenic Escherichia coli (ETEC), and Campylobacter species isolates from indigenous persons and travelers in Thailand for up to 15 years. Resistance to trimethoprim-sulfamethoxazole was found in >90% of Shigella and 40% of ETEC and nontyphoidal Salmonella isolates. Resistance to nalidixic acid was found in 97%-100% of Shigella dysenteriae 1 strains isolated between 1992 and 1995. Ciprofloxacin resistance was detected in 1% of ETEC isolates in 1994 and 1995 and in one of 349 nontyphoidal Salmonella isolates in 1995. Ciprofloxacin resistance among Campylobacter species increased from zero before 1991 to 84% in 1995 (P < .0001). Azithromycin resistance was found in 7%-15% of Campylobacter isolates in 1994 and 1995, as well as 15% of ETEC and 3% of Salmonella isolates in 1995. Enteric pathogens in Thailand have developed resistance to virtually all antibiotics routinely used in the treatment of diarrhea, as well as the newer fluoroquinolone and macrolide classes of drugs.

Diarrhea Among Expatriate Residents in Thailand: Correlation Between Reduced Campylobacter Prevalence and Longer Duration of Stay.

Background: The etiology of diarrhea among expatriates in Thailand was investigated. Methods: One hundred and five stool specimens were collected from Americans and Europeans who had developed diarrhea while residing in Thailand. These stools were analyzed for bacterial enteric pathogens. Results: Enterotoxigenic Escherichia coli was isolated from 18 (17%) persons and Campylobacter from 10 (10%). Shigella and Salmonella were each isolated from 8 (8%) persons. Vibrio sp., enteroinvasive Escherichia coli, and enteropathogenic Escherichia coli were found in rare cases. Conclusions: Bacterial pathogens, particularly Campylobacter, were isolated significantly less often in patients who had lived in Thailand for more than 1 year, compared with those who had lived there less than 1 year (relative risk: 0.62, 95% confidence interval: 0.40-0.97, p=.03).

Use of azithromycin for the treatment of Campylobacter enteritis in travelers to Thailand, an area where ciprofloxacin resistance is prevalent.

We evaluated the use of azithromycin (500 mg) or ciprofloxacin (500 mg) daily for 3 days for the treatment of acute diarrhea among United States military personnel in Thailand. Stool cultures were obtained and symptoms were recorded on study days 0, 1, 2, 3, and 10. Campylobacter species were the most common pathogen isolated (44 isolates from 42 patients). All Campylobacter isolates were susceptible to azithromycin; 22 were resistant to ciprofloxacin. Among the 42 patients with campylobacter infection, there were 2 clinical and 6 bacteriologic treatment failures in the ciprofloxacin group and no treatment failures in the azithromycin group (P = .021 for bacteriologic failures). Overall, azithromycin was as effective as ciprofloxacin in decreasing the duration of illness (36.9 hours vs. 38.2 hours, respectively) and the number of stools (6.4 vs. 7.8, respectively). Among those not infected with Campylobacter species (n = 30), the duration of illness was 32.9 hours vs. 20.7 hours (P = .03) for the azithromycin and ciprofloxacin groups, respectively. Azithromycin is superior to ciprofloxacin in decreasing the excretion of Campylobacter species and as effective as ciprofloxacin in shortening the duration of illness. Azithromycin therapy may be an effective alternative to ciprofloxacin therapy in areas where ciprofloxacin-resistant Campylobacter species are prevalent.

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.

Vibrio cholerae O139 in Thailand in 1994.

Vibrio 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.

Detection of Shigella and enteroinvasive Escherichia coli by PCR in the stools of patients with dysentery in Thailand.

The rate of detection of Shigella and enteroinvasive Escherichia coli (EIEC) using a PCR technique was compared with the rate detected by standard microbiological methods (bacteriology plus hybridization of E. coli colonies with a 17 kb EIEC probe) among patients with dysentery before and after antibiotic therapy. The PCR amplified DNA sequences encoding IpaH, a multiple copy sequence located on the chromosome and the invasion plasmid. Shigella or EIEC were detected using the IpaH PCR system among 72 (61%) of 119 patients with dysentery on the first day they were seen at hospital, compared to 50 (42%) using standard microbiological methods (p = 0.006). After three days of antibiotic therapy, IpaH sequences were detected in stools from 38 percent of patients, compared to 10 percent using standard microbiology (p < 0.001). After seven days of therapy, the rates were 26 percent vs. 8 percent respectively (p < 0.001). The IpaH PCR system appeared to be specific for Shigella or EIEC based on low rates of positive reactions among non-diarrhoea controls, and a strong correlation between persistently positive reactions and antibiotic resistance of bacterial isolates. IpaH sequences were detected in 10 (8%) of 119 drinking water samples from homes of patients with disease; none of these specimens were positive for Shigella or EIEC by standard microbiology. In conclusion, PCR amplification of IpaH sequences and detection of target DNA with a non-radioactive probe increased the rates of identification of Shigella and EIEC by 45% in initial clinical specimens and by nearly 300% in specimens obtained from patients receiving antibiotic therapy.

Isolation of group 2 aerotolerant Campylobacter species from Thai children with diarrhea.

Campylobacter species were isolated from 93 (15%) of 631 Thai children with diarrhea using the membrane filter technique on nonselective blood agar incubated at 37 degrees C. Campylobacter jejuni was isolated from 62 (10%), Campylobacter coli from 14 (2%), and atypical campylobacters from 17 (3%). The 17 atypical strains were first characterized biochemically and by dot blot DNA hybridization. Catalase-negative strains also were characterized by DNA hybridization and ribotype pattern. One strain was a catalase-negative "Campylobacter upsaliensis" and another was a nitrate-negative Campylobacter jejuni doylei. Fifteen isolates were aerotolerant strains most closely resembling Campylobacter cryaerophila or "C. upsaliensis" by dot hybridization. These aerotolerant strains, designated group 2 ("Campylobacter butzleri"), had ribotypes distinct from C. cryaerophila and have previously been shown to be related by DNA hybridization at the species level to the group 2 aerotolerant Campylobacter type strain (D2686). Group 2 aerotolerant Campylobacter were the atypical Campylobacter species most frequently isolated from Thai children with diarrhea.

Microbiology and diagnosis of infections with Shigella and enteroinvasive Escherichia coli.

The etiology of dysentery in Thailand and the existing methods of diagnosing infections with Shigella and enteroinvasive Escherichia coli (EIEC) are reviewed. The four Shigella species (S. dysenteriae, S. flexneri, S. boydii, and S. sonnei) are classically identified by culture of fecal specimens on selective media and testing of isolates for agglutination in species-specific antisera. DNA probes have been used to identify both lactose-fermenting and non-lactose-fermenting EIEC as well as Shigella isolates that do not agglutinate in antisera. These DNA probes are not necessary for the identification of Shigella if a competent bacteriology laboratory with shigella antisera is available. In Thailand Shigella and EIEC are isolated more often from children greater than 2 years of age than from younger children. The clinical illness associated with EIEC infections is similar to shigellosis. Fewer children with EIEC infections than with shigellosis, however, have occult blood in stool (36% vs. 82%) and more than 10 fecal leukocytes per high-power field (36% vs. 67%). Standard bacteriologic methods and testing of E. coli isolates for hybridization with the shigella/EIEC probe are currently the most sensitive means of diagnosing infections caused by these enteric pathogens. A more rapid method of identifying Shigella and EIEC infections in a situation where a bacteriology laboratory is not available will probably involve immunologic assays.

Clinical and microbiologic features of Shigella and enteroinvasive Escherichia coli infections detected by DNA hybridization.

To determine the clinical and microbiologic features of Shigella and enteroinvasive Escherichia coli (EIEC) infections, we investigated 410 children with diarrhea and 410 control children without diarrhea who were seen at Children's Hospital, Bangkok, Thailand, from January to June 1985. Shigella spp. were isolated from 96 (23%) and EIEC were isolated from 17 (4%) of 410 children with diarrhea and from 12 (3%) and 6 (1%) of 410 control children, respectively. The isolation rates of both pathogens increased with age and peaked in children 3 to 5 years old from whom Shigella spp. were isolated from 38% and EIEC were isolated from 9%. Shigella spp. were isolated from 52% and EIEC were isolated from 7% of 91 children with bloody diarrhea and from 15 and 3% of 319 children with nonbloody diarrhea. Fifteen (65%) of 23 EIEC were lactose positive, and all belonged to recognized EIEC serotypes. Among children with diarrhea, the stool blots of 76% of 17 children infected with EIEC, 45% of 96 children infected with Shigella spp., and 1% of 297 culture-negative children hybridized with the 17-kilobase EcoRI digestion fragment of pRM17, a recombinant plasmid containing DNA derived from the 140-megadalton Shigella flexneri plasmid. Although EIEC colonies can be reliably detected by DNA hybridization, detection by stool blot is less sensitive. Shigella spp. and EIEC are important causes of endemic diarrhea among children greater than 1 year old in Thailand.

Influence of strain characteristics and immunity on the epidemiology of Campylobacter infections in Thailand.

To determine how strain differences and immunity affect the clinical expression of Campylobacter infections, we conducted a study of acute diarrheal disease in Thailand in which specimens from children with Campylobacter infections were cultured weekly for up to 12 weeks to determine the serotype-specific length of time of convalescent-phase excretion and rate of reinfection. Levels of immunoglobulin G to cell-surface antigens of C. jejuni were determined in another population of healthy children who were closely related by age and location to the children in the diarrheal disease study. Campylobacter species were initially isolated from 18% of 586 children under 5 years old with diarrhea; most isolates in Thailand belonged to serotypes commonly found in developed countries. C. coli was significantly less often associated with symptomatic infections and with bloody diarrhea than C. jejuni (P less than 0.001 and P = 0.045, respectively). The peak age of isolation and the peak level of immunoglobulin G to Campylobacter species occurred before 2 years of age. The mean duration of convalescent-phase excretion was 14 +/- 2 (standard error of the mean) days for children less than 1 year old and 8 +/- 2 days for children 1 to 5 years old (P = 0.02, t test). Infection with another Campylobacter serotype was found in 34% of 105 children during the 12-week follow-up period. The rate of reinfection in these children was 15% (range, 8 to 22%) each week. Hyperendemic exposure to Campylobacter species in Thailand confers immunity to infection that is associated with an early peak in specific serum antibodies and an age-related decrease in the case-to-infection ratio and duration of convalescent-phase excretion but does not prevent asymptomatic infections.

Application of DNA hybridization techniques in the assessment of diarrheal disease among refugees in Thailand.

The epidemiology and etiology of acute diarrheal disease were determined in a Hmong refugee camp on the Thai-Laotian border from April 11 to May 14, 1985. DNA hybridization techniques were used to detect Shigella species, enteroinvasive Escherichia coli, and enterotoxigenic E. coli. A monoclonal enzyme-linked immunosorbent assay was used to detect rotavirus, and standard microbiology was used to detect other enteropathogens. The age-specific diarrheal disease rates were 47 episodes per month per 1,000 children less than five years old and 113 episodes per month per 1,000 children less than one year old. Rotavirus, enterotoxigenic E. coli, Campylobacter, and Cryptosporidium were the predominant pathogens in children less than two years old. The DNA probe hybridized with 94% of 31 specimens identified as enterotoxigenic E. coli by the standard assays and with none of the specimens in which the standard assays were negative. The probe for Shigella and enteroinvasive E. coli hybridized in eight of 10 stools that contained Shigella and four of 314 stools from which Shigella and enteroinvasive E. coli were not isolated. The use of DNA probes allows specimens to be collected in remote areas with a minimum amount of equipment and technical expertise so that they can be easily transported to a central laboratory for further processing.