Prevalence and antimicrobial susceptibility of Campylobacter isolated from retail chickens in Thailand.

Campylobacter is an important foodborne pathogen causing bacterial gastroenteritis worldwide; however, there has been a lack of information over the past decade on its occurrence, antibiotic susceptibility and genetic diversity in Thailand. Poultry meat is considered as a reservoir for transmission of Campylobacter to humans. This study determines the prevalence and antimicrobial resistance patterns of Campylobacter spp. on chicken samples purchased from 50 local wet markets and supermarkets in central Thailand. Of the 296 samples, 99 (33.5%) were contaminated with C. jejuni, 54 (18.2%) were C. coli and 15 (5.1%) were contaminated with both species. Antibiotic resistance rate is higher among C. coli isolates; 100%, 76.8%, 37.7%, 36.2% and 13.0% were resistant to quinolones, cyclines, macrolides, clindamycin and gentamicin, respectively. Most of the C. jejuni isolates were resistant to quinolones (79.8%) and cyclines (38.6%) whereas resistance to macrolides, clindamycin and gentamicin was found to be 1.8%. Multi-drug resistance (i.e. to three or more unrelated antimicrobials) was detected in 37.7% of C. coli and 1.8% of C. jejuni isolates. This study has revealed high contamination rates and alarming levels of antimicrobial resistance in Campylobacter spp. isolated from retail chicken samples in Thailand, suggesting the necessity of implementing interventions to reduce its prevalence from farm to table in the country.

Etiologic features of diarrheagenic microbes in stool specimens from patients with acute diarrhea in Thailand.

Many microbial species have been recognized as enteropathogens for humans. Here, we predicted the causative agents of acute diarrhea using data from multiplex quantitative PCR (qPCR) assays targeting 19 enteropathogens. For this, a case-control study was conducted at eight hospitals in Thailand. Stool samples and clinical data were collected from 370 hospitalized patients with acute diarrhea and 370 non-diarrheal controls. Multiple enteropathogens were detected in 75.7% and 13.0% of diarrheal stool samples using multiplex qPCR and bacterial culture methods, respectively. Asymptomatic carriers of enteropathogens were found among 87.8% and 45.7% of individuals by qPCR and culture methods, respectively. These results suggested the complexity of identifying causative agents of diarrhea. An analysis using the quantification cut-off values for clinical relevance drastically reduced pathogen-positive stool samples in control subjects from 87.8% to 0.5%, whereas 48.9% of the diarrheal stool samples were positive for any of the 11 pathogens. Among others, rotavirus, norovirus GII, Shigella/EIEC, and Campylobacter were strongly associated with acute diarrhea (P-value < 0.001). Characteristic clinical symptoms, epidemic periods, and age-related susceptibility to infection were observed for some enteropathogens. Investigations based on qPCR approaches covering a broad array of enteropathogens might thus improve our understanding of diarrheal disease etiology and epidemiological trends.

High prevalence and diversity of tcdA-negative and tcdB-positive, and non-toxigenic, Clostridium difficile in Thailand.

Studies on the prevalence and diversity of Clostridium difficile in Thailand have been limited to those derived from a few tertiary hospitals in Central Thailand. In this study, 145 C. difficile isolates collected in 13 provinces in Thailand during 2006-2018 were characterized by ribotyping and detection of toxin genes. Minimum inhibitory concentrations of eight antimicrobial agents were determined also for all 100 C. difficile strains collected from 2006 until 2015. Of the 145 strains of C. difficile, 71 (49%) were non-toxigenic, 46 (32%) were toxin A-negative, toxin B-positive (A-B+) and 28 (19%) were A+B+. No binary toxin-positive strain was found. The most common ribotype (RT) was RT 017 (A-B+CDT-, 19%, 28/145). Besides RT 017, 20 novel non-toxigenic and A-B+ ribotyping profiles, which may be related to RT 017 by the similarity of ribotyping profile, were identified. All C. difficile strains remained susceptible to metronidazole and vancomycin, however, a slight increase in MIC for metronidazole was seen in both toxigenic and non-toxigenic strains (overall MIC50/90 0.25/0.25 mg/L during 2006-2010 compared to overall MIC50/90 1.0/2.0 mg/L during 2011-2015). There was a high rate of fluoroquinolone resistance among RT 017 strains (77%), but there was little resistance among non-toxigenic strains. These results suggest that RT 017 is endemic in Thailand, and that the misuse of fluoroquinolones may lead to outbreaks of RT 017 infection in this country. Further studies on non-toxigenic C. difficile are needed to understand whether they have a role in the pathogenesis of C. difficile infection in Asia.

Draft Genome Sequence of a Clostridium botulinum Isolate from Thailand Harboring the Subtype bont/B8 Gene.

In 2010, a Clostridium botulinum type B isolate was recovered from fermented soybeans during a foodborne botulism investigation. Molecular investigation of the botulinum neurotoxin (bont) gene operon determined that the sequence was a new subtype, denoted B8. Here, we describe the draft whole-genome sequence of the organism.

Characterization of Bacteriophages Infecting Clinical Isolates of Clostridium difficile.

Clostridium difficile is recognized as a problematic pathogen, causing severe enteric diseases including antibiotic-associated diarrhea and pseudomembranous colitis. The emergence of antibiotic resistant C. difficile has driven a search for alternative anti-infection modalities. A promising strategy for controlling bacterial infection includes the use of bacteriophages and their gene products. Currently, knowledge of phages active against C. difficile is still relatively limited by the fact that the isolation of phages for this organism is a technically demanding method since bacterial host themselves are difficult to culture. To isolate and characterize phages specific to C. difficile, a genotoxic agent, mitomycin C, was used to induce temperate phages from 12 clinical isolates of C. difficile. Five temperate phages consisting of ΦHR24, ΦHN10, ΦHN16-1, ΦHN16-2, and ΦHN50 were successfully induced and isolated. Spotting assays were performed against a panel of 92 C. difficile isolates to screen for susceptible bacterial hosts. The results revealed that all the C. difficile phages obtained in this work displayed a relatively narrow host range of 0-6.5% of the tested isolates. Electron microscopic characterization revealed that all isolated phages contained an icosahedral head connected to a long contractile tail, suggesting that they belonged to the Myoviridae family. Restriction enzyme analysis indicated that these phages possess unique double-stranded DNA genome. Further electron microscopic characterization revealed that the ΦHN10 absorbed to the bacterial surface via attachment to cell wall, potentially interacting with S-layer protein. Bacteriophages isolated from this study could lead to development of novel therapeutic agents and detection strategies for C. difficile.

Isolation and phylogenetic analysis of Bartonella species from Rusa deer (Rusa timorensis) in Thailand.

The aim of the present study is to investigate the prevalence of Bartonella infection in deer in Thailand and to characterize the isolates by biochemical, morphological and genetic analysis. A total of 247 blood samples were collected from Rusa deer (Rusa timorensis) in a livestock breeding facility in Thailand. Bartonella bacteria were isolated in 3.6% of the blood samples. Three out of 110 (2.7%) males and 6 of 137 (4.4%) females were positive for Bartonella. A higher prevalence of Bartonella was observed in young deer under 4 years of age compared to adults over 4 years of age, but no Bartonella was isolated from deer over 8 years of age. Phylogenetic analysis of concatenated sequences of seven loci of Bartonella indicated that all the isolates from Rusa deer in Thailand were identical and formed a distinct cluster from other known Bartonella species.

Molecular characterization of Clostridium botulinum isolates from foodborne outbreaks in Thailand, 2010.

BACKGROUND: Thailand has had several foodborne outbreaks of botulism, one of the biggest being in 2006 when laboratory investigations identified the etiologic agent as Clostridium botulinum type A. Identification of the etiologic agent from outbreak samples is laborious using conventional microbiological methods and the neurotoxin mouse bioassay. Advances in molecular techniques have added enormous information regarding the etiology of outbreaks and characterization of isolates. We applied these methods in three outbreaks of botulism in Thailand in 2010. METHODOLOGY/PRINCIPAL FINDINGS: A total of 19 cases were involved (seven each in Lampang and Saraburi and five in Maehongson provinces). The first outbreak in Lampang province in April 2010 was associated with C. botulinum type F, which was detected by conventional methods. Outbreaks in Saraburi and Maehongson provinces occurred in May and December were due to C. botulinum type A1(B) and B that were identified by conventional methods and molecular techniques, respectively. The result of phylogenetic sequence analysis showed that C. botulinum type A1(B) strain Saraburi 2010 was close to strain Iwate 2007. Molecular analysis of the third outbreak in Maehongson province showed C. botulinum type B8, which was different from B1-B7 subtype. The nontoxic component genes of strain Maehongson 2010 revealed that ha33, ha17 and botR genes were close to strain Okra (B1) while ha70 and ntnh genes were close to strain 111 (B2). CONCLUSION/SIGNIFICANCE: This study demonstrates the utility of molecular genotyping of C. botulinum and how it contributes to our understanding the epidemiology and variation of boNT gene. Thus, the recent botulism outbreaks in Thailand were induced by various C. botulinum types.

Prevalence of zoonotic Bartonella species among rodents and shrews in Thailand.

We investigated the prevalence of Bartonella species in 10 rodent and one shrew species in Thailand. From February 2008 to May 2010, a total of 375 small animals were captured in 9 provinces in Thailand. Bartonella strains were isolated from 57 rodents (54 from Rattus species and 3 from Bandicota indica) and one shrew (Suncus murinus) in 7 of the 9 provinces, and identified to the species level. Sequence analysis of the citrate synthase and RNA polymerase ß subunit genes identified the 58 isolates from each Bartonella-positive animal as B. tribocorum in 27 (46.6%) animals, B. rattimassiliensis in 17 (29.3%) animals, B. elizabethae in 10 (17.2%) animals and B. queenslandensis in 4 (6.9%) animals. R. norvegicus, R. rattus, and Suncus murinus carried B. elizabethae, which causes endocarditis in humans. The prevalence of Bartonella bacteremic animals by province was 42.9% of the animals collected in Phang Nga, 26.8% in Chiang Rai, 20.4% in Sa Kaeo, 16.7% in Nakhon Si Thammarat, 12.0% in Surat Thani, 9.1% in Mae Hong Son and Loei Provinces. These results indicate that Bartonella organisms are widely distributed in small mammals in Thailand and some animal species may serve as important reservoirs of zoonotic Bartonella species in the country.

Outbreak of norovirus gastroenteritis infection, Thailand.

Norovirus is a leading cause of gastrointestinal illness worldwide. We investigated an outbreak of gastrointestinal illness in Pattaya, Thailand, among participants of a course. We asked participants and family members to complete a questionnaire asking about symptoms, meals eaten, and foods consumed during the course. We collected stool samples from persons reporting illness and analyzed specimens for several viruses and enteropathogenic bacteria. We defined a case as a person having one or more episodes of diarrhea, with onset between 30 August and 1 September 2010, in a participant or family member who attended the course. Of 56 people who attended, 95% completed the questionnaire: nine met the case definition (attack rate, 17%). Common symptoms included abdominal cramps, nausea, fatigue, headache, and vomiting. Food items with elevated risk ratios included: crispy fish maw, dried squid, and cashew nut salad [risk ratio (RR) 5.1; 95% confidence interval (CI) 0.7-37]; assorted salad bar with dressing (RR 3.0; 95% CI 0.9-11); and seafood kebab (RR 5.8; 95% CI 0.8-43). Among ill persons, four (44%) provided stool samples and two (50%) were positive for norovirus. Our data suggest a foodborne outbreak of norovirus. Increased use of norovirus diagnostics as well as measures to prevent transmission may help identify additional outbreaks and improve control measures to limit the spread of outbreaks.

Development of a magnetic bead fluorescence microscopy immunoassay to detect and quantify Leptospira in environmental water samples.

Climate change, world population growth, and poverty have led to an increase in the incidence of leptospirosis. Leptospirosis is caused by pathogenic spirochaete bacteria that belong to the genus Leptospira. The bacteria are maintained in the renal tubules of the reservoir hosts (typically a rodent), then shed into the environment via the urine. Water is key for environmental survival and transmission, as leptospires can survive for several weeks in a moist environment. Therefore, environmental epidemiological studies are needed to study the contamination of environmental water sources. However, few such studies have been performed using cultivation of the isolates and PCR assays. But, leptospira cultivation can be easily contaminated by other organisms and takes usually several weeks. Moreover, PCR is a complex and costly analysis for the underdeveloped countries that have the highest incidence of leptospirosis. In this study, we describe two modifications of a fluorescence microscopy assay based on immuno-magnetic separation (IMS) to detect leptospires in environmental water samples that mainly differ in fluorescent dye staining. The first type uses acridine orange fluorescent dye staining combined with multiplexed IMS for sample screening. The detection limit ranged from 10(2) to 10(3) organisms per mL and largely depended on the capture efficiency (CE) of the immuno-magnetic particles. The second type uses serogroup-specific immuno-particles and direct fluorescence antibody staining (DFA) to detect leptospires; the detection limit of this second assay was approximately 10(1) cells per mL. Both assay types were applied to natural and experimentally infected water samples, which were also analysed with DFM and real-time PCR. Our data show that the fluorescent microscopy immunoassay successfully identified experimental leptospire contamination and was as sensitive as PCR. This modified immune-fluorescence assay may therefore enable epidemiological studies of leptospirosis.

Infection of less virulent Helicobacter pylori strains in asymptomatic healthy individuals in Thailand as a potential contributing factor to the Asian enigma.

In Thailand, gastric cancer incidence is considerably low despite the high prevalence of Helicobacter pylori infection. We investigated the prevalence of H. pylori infection and the genotypes of cagA by using 179 stool specimens obtained from asymptomatic Thai individuals. In this study, the prevalence of H. pylori infection was 43.6%, and the detection rate of cagA-positive strains was 43.5%. In addition, the proportion of the highly virulent East-Asian type of cagA was 7.2%. These results indicate that the low prevalence of cagA-positive H. pylori strain as well as the low prevalence of East-Asian genotype cagA-positive strains may contribute to the low gastric cancer incidence.

An application of duplex PCR for detection of Leptospira spp. and Orientia tsutsugamushi from wild rodents.

Duplex PCR is useful for detecting two different agents from the same specimen. Kidney specimens are the most suitable for detection of Leptospira spp. For Orientia tsutsugamushi, blood clots, spleen, and liver specimens are considered the most suitable. For this study, kidney tissues were the only specimens obtainable for the PCR. Blood clots, spleen, and liver specimens were not available. However, by using the PCR for scrub typhus, O. tsutsugamushi was detected in the kidney of one rodent. This result shows that kidney specimens can be used to detect O. tsutsugamushi using PCR. Further studies will be necessary in order to be able to compare the detection ratio of O. tsutsugamushi using kidney specimens and blood clots, spleen, and liver specimens.

Identification of clinical isolates of Leptospira spp by pulsed field gel-electrophoresis and microscopic agglutination test.

Twenty-five clinical isolates of Leptospira spp were characterized by microscopic agglutination test (MAT) and pulsed field gel-electrophoresis (PFGE) in comparison with 23 reference Leptospira serovars and with the saprophytic L. biflexa serovar Patoc. PFGE DNA profiling was more specific and reliable than MAT.

Molecular typing of Leptospira spp. based on putative O-antigen polymerase gene (wzy), the benefit over 16S rRNA gene sequence.

Molecular typing of leptospiral strains based on variation within putative O-antigen polymerase gene (wzy) was determined among reference strains and those isolated from patients. Using the PCR primers designed from the flanking gene of wzy derived from Leptospira interrogans serovar Copenhageni, all L. interrogans serovars as well as human and rodent leptospiral isolates from Thailand could be amplified. The size of PCR product ranged from 1 to 1.5 kb. The limitation of these primer pairs was the inability to amplify those strains whose sequences differ in the region of the primers, these included Leptospira biflexa (serovar Patoc), Leptospira borgpetersenii (serovar Tarassovi) and Leptospira kirschneri (serovar Bim, Bulgarica, Butembo). Notably, amplification was not limited to L. interrogans as demonstrated by the amplification of some strains from L. kirschneri, Leptospira meyeri, Leptospira noguchii, Leptospira santarosai, L. borgpetersenii and Leptospira weilii. The phylogenetic tree of wzy sequence, inferred by posterior probability of the Bayesian, enabled the categorization of leptospiral serovars into seven genetically related group, of which its differentiation power was better than that of the more highly conserved 16S rRNA gene, which is used extensively for genotyping.

Epidemiology of severe pneumonia caused by Legionella longbeachae, Mycoplasma pneumoniae, and Chlamydia pneumoniae: 1-year, population-based surveillance for severe pneumonia in Thailand.

BACKGROUND: Legionella species, Mycoplasma pneumoniae, and Chlamydia pneumoniae are recognized as important causes of pneumonia in high-income countries, but their significance in middle-income countries, such as Thailand, is unknown. METHODS: Population-based surveillance identified inpatient 3489 cases of clinically-defined pneumonia in a rural Thai province for 1 year. Patients who had a chest radiograph performed (for 2059 cases of pneumonia) were enrolled in an etiology study (which included 755 cases of pneumonia among 738 patients). Paired serum, nasopharyngeal swab, and urine specimens were obtained for diagnostic immunologic and molecular tests. Patients aged <18 years were not systematically tested for Legionella species. We report a lower limit of incidence (observed incidence) and an upper limit extrapolated to persons not tested or not enrolled in the study. RESULTS: The incidence of pneumonia due to Legionella longbeachae requiring hospitalization was 5-29 cases per 100,000 population. No case of Legionella pneumophila pneumonia was observed. The definite C. pneumoniae pneumonia incidence was 3-23 cases per 100,000 population; rates were highest among patients aged <1 year (18-166 cases per 100,000 population) and those aged >or=70 years (23-201 cases per 100,000 population). M. pneumoniae pneumonia had a similar age distribution, with an overall incidence of 6-44 cases per 100,000 population. These pathogens were associated with 15% of all cases of pneumonia. A nonsignificantly higher proportion of patients with pneumonia associated with L. longbeachae, compared with patients with pneumonia associated with M. pneumoniae or C. pneumoniae, required supplemental oxygen or mechanical ventilation (45% vs. 18%; P<.1). Among patients with atypical pneumonia, only 15% received antibiotics with activity against the associated pathogen. CONCLUSION: M. pneumoniae, C. pneumoniae, and L. longbeachae, but not L. pneumophila, are frequently associated with severe pneumonia in rural Thailand. Few patients receive antibiotics that cover atypical pathogens.

Distribution of Chlamydia trachomatis serovars among female prostitutes and non-prostitutes in Thailand, and non-prostitutes in Japan during the mid-90s.

The distribution of Chlamydia trachomatis serovars in Thailand and Japan during the same period of the mid-90s was determined. Seventy-one C . trachomatis specimens isolated from female patients who visited the Venereal Diseases Center at Bangkok, Thailand in 1994 were used in this study. Of these, 56 patients were prostitutes. Forty-seven specimens obtained from female non-prostitutes who attended the Department of Obstetrics and Gynecology, Saitama Medical School, Japan during the period from 1993 to 1995 were also used in this study. DNA was extracted from these specimens and typing of C. trachomatis serovars was performed by the polymerase chain reaction-restriction fragment length polymorphism method. The identified serovars among prostitutes of Thailand (n = 56)/non-prostitutes of Thailand (n = 15)/non-prostitutes of Japan (n = 47) were as follows: Ba 1/0/2, D 8/1/15, E 11/2/8, F 16/9/8, G 4/0/7, H 3/2/3, I 1/0/1, J 3/0/0, and K 10/1/4. Serovar F was most prevalent (35.2%) in both prostitutes and non-prostitutes from Thailand, followed by serovar E (18.3%). On the other hand, serovar D was the most frequent serovar in non-prostitutes in Japan (31.9%) followed by serovars F (17.0%) and E (17.0%). A difference in the distribution of C. trachomatis serovars of Thailand and Japan was noted.

Chlamydophila pneumoniae infection among young children with respiratory diseases in Thailand.

A total of 136 children aged 5 years and under with respiratory tract diseases were examined for Chlamydophila pneumoniae infection. By means of the micro-immunofluorescence test, an acute infection was suggested in 37 (27.2%) of them. Infection was found in 23 (43.4%) of 53 children with bronchitis, seven (70.0%) of 10 with pharyngitis, and two (22.2%) of nine with pneumonia. C. pneumoniae DNA was detected in seven of 55 children by means of nasopharyngeal swabs, and serological evidence was present in all of seven. Five of them were suggested the acute infection and four of the five showed IgG titers increasing four times and over. By age distribution, five of the seven DNA-positive children were 1 year old, and the remaining two were 2 and 4 years old, respectively. The clinical findings of the seven DNA-positive children were characterized as indicative of bronchitis (n = 4), pharyngitis (n = 2), and pneumonia (n = 1). In Thailand, C. pneumoniae infection occurs frequently among children aged 5 years and under, and may cause pharyngitis, bronchitis, and sometimes pneumonia. However, it is suggested that C. pneumoniae infection is not a major cause of severe pneumonia among children in that age group.

Chlamydophila pneumoniae specific antibodies in Thai patients with myocardial infarction.

To investigate the correlation between Chlamydia pneumoniae infection and acute myocardial infarction (AMI), a total of 101 serum specimens collected from patients with AMI admitted to the coronary care unit, Bhumibol Adulyadej Hospital, and serum specimens collected from healthy blood donors (control group) were examined by using the micro-immunofluorescence test. C. pneumoniae antibody-positive cases were found in 52 (52%) patients, consisting of 30 males and 22 females, though no significant difference of prevalence rate was observed when compared with the rate in the control group. However, the level of IgG titers in patients was higher than that in the controls, and this finding may support an association between C. pneumoniae infection and AMI. Among patients with AMI, several cases were suspected to have current infections because of a fourfold or higher titer increase in IgG or titers in IgM antibody of 1:32 or 1:64. There is no significant correlation between serologic test results and diabetes mellitus, hypertension, hyper cholesterol, a smoking habit, or the location of myocardial infarction among patients with AMI.