Sumalee Boonmar

Identification of Bartonella Infections in Febrile Human Patients from Thailand and Their Potential Animal Reservoirs

To determine the role of Bartonella species as causes of acute febrile illness in humans from Thailand, we used a novel strategy of co-cultivation of blood with eukaryotic cells and subsequent phylogenetic analysis of Bartonella-specific DNA products. Bartonella species were identified in 14 blood clots from febrile patients. Sequence analysis showed that more than one-half of the genotypes identified in human patients were similar or identical to homologous sequences identified in rodents from Asia and were closely related to B. elizabethae, B. rattimassiliensis, and B. tribocorum. The remaining genotypes belonged to B. henselae, B. vinsonii, and B. tamiae. Among the positive febrile patients, animal exposure was common: 36% reported owning either dogs or cats and 71% reported rat exposure during the 2 weeks before illness onset. The findings suggest that rodents are likely reservoirs for a substantial portion of cases of human Bartonella infections in Thailand.

Significant increase in antibiotic resistance of Salmonella isolates from human beings and chicken meat in Thailand

A total of 1715 Salmonella strains, including 600 S. enteritidis, 290 S.derby, 257 S. Weltevreden, 122 S. 1,4,5,12:i:-, 235 S. anatum, and 211 S. typhimurium, originating from 1308 human beings and 407 frozen chicken meat specimens collected in 1993 and 1994 were tested for antibiotic resistance. The disk diffusion method was used with nine disks of chloramphenicol, ceftriaxone, amikacin, kanamycin, ampicillin, sulfamethoxazole plus trimethoprim, nalidixic acid, gentamicin and ofloxacin. The resistance rates of human beings isolates in 1994 to ceftriaxone, amikacin and kanamycin were, respectively, 10.7%, 8.6%, 17.8% in S. enteritidis; 23.1%, 17.3% 33.0% in S. derby; 30.9%, 40.2%, 60.4% in S. weltevreden; 16.1%, 17.7%, 70.9% in S.1, 4, 5, 12:i:-; 25.7%, 21.6%, 24.7% in S. anatum; 18.9%, 15.7%, 37.8% in S. typhimurium, while those isolates in 1993 to the same three antibiotics were, respectively, 1.8%, 0.6%, 3.7% in S. enteritidis; 0.8%, 0%, 9.1% in S.derby; 1.8%, 2.7%, 10.8% in S. weltevreden; 3.8%, 0%, 23.0% in S.1, 4, 5, 12:i:-; 2.2%, 2.2%, 6.7% in S. anatum; 4.5%, 1.5%, 10.6% in S. typhimurium. It was shown that the resistance rates in 1994 were significantly higher than those in 1993. All isolates were susceptible to ofloxacin with the exception of one isolate.

Isolation and phylogenetic analysis of Arcobacter spp. in ground chicken meat and environmental water in Japan and Thailand

Prevalence of Arcobacter spp. in chicken meat samples and environmental water samples in Japan and Thailand was investigated. Arcobacter was isolated from 48% of chicken meat samples (20/41) and 23% of river water samples (4/17) from Japan, and 100% of chicken meat samples (10/10) and 100% of canal water samples (7/7) from Thailand. A. butzleri was among the species isolated from all positive samples. About 10% genetic diversity was seen in the rpoB‐rpoC in Arcobacters, and phylogenetic trees were divided into two clusters. In both countries, the results suggested that chicken and environmental water were highly contaminated with a genetically diverse population of Arcobacter.

Enrichment culture and molecular identification of diverse Bartonella species in stray dogs

Using pre-enrichment culture in Bartonella alpha-Proteobacteria growth medium (BAPGM) followed by PCR amplification and DNA sequence identification that targeted a fragment of the citrate synthase gene (gltA), we provide evidence of common bartonella infections and diverse Bartonella species in the blood of stray dogs from Bangkok and Khon Kaen, Thailand. The overall prevalence of all Bartonella species was 31.3% (60/192), with 27.9% (31/111) and 35.8% (29/81) in the stray dogs from Bangkok and Khon Kaen, respectively. Phylogenetic analyzes of gltA identified eight species/genotypes of Bartonella in the blood of stray dogs, including B. vinsonii subsp. arupensis, B. elizabethae, B. grahamii, B. quintana, B. taylorii, and three novel genotypes (BK1, KK1 and KK2) possibly representing unique species with ≤ 90.2% similarities to any of the known Bartonella species B. vinsonii subsp. arupensis was the only species detected in dogs from both sites, B. quintana and BK1 were found in the dogs from Bangkok, B. elizabethae, B. taylorii, KK1 and KK2 were found in the dogs from Khon Kaen. We conclude that stray dogs in Thailand are frequently infected with Bartonella species that vary with geographic region. As some Bartonella species detected in the present study are considered pathogenic for humans, stray dogs in Thailand may serve as possible reservoirs for Bartonella causing human illnesses. Further work is needed to determine the role of those newly discovered Bartonella genotypes/species in human and veterinary medicine.

Global Distribution of Bartonella Infections in Domestic Bovine and Characterization of Bartonella bovis Strains Using Multi-Locus Sequence Typing

Bartonella bovis is commonly detected in cattle. One B. bovis strain was recently isolated from a cow with endocarditis in the USA, suggesting its role as an animal pathogen. In the present study, we investigated bartonella infections in 893 cattle from five countries (Kenya, Thailand, Japan, Georgia, and Guatemala) and 103 water buffaloes from Thailand to compare the prevalence of the infection among different regions and different bovid hosts. We developed a multi-locus sequence typing (MLST) scheme based on nine loci (16S rRNA, gltA, ftsZ, groEL, nuoG, ribC, rpoB, ssrA, and ITS) to compare genetic divergence of B. bovis strains, including 26 representatives from the present study and two previously described reference strains (one from French cows and another from a cow with endocarditis in the USA). Bartonella bacteria were cultured in 6.8% (7/103) of water buffaloes from Thailand; all were B. bovis. The prevalence of bartonella infections in cattle varied tremendously across the investigated regions. In Japan, Kenya, and the Mestia district of Georgia, cattle were free from the infection; in Thailand, Guatemala, and the Dusheti and Marneuli districts of Georgia, cattle were infected with prevalences of 10–90%. The Bartonella isolates from cattle belonged to three species: B. bovis (n=165), B. chomelii (n=9), and B. schoenbuchensis (n=1), with the latter two species found in Georgia only. MLST analysis suggested genetic variations among the 28 analyzed B. bovis strains, which fall into 3 lineages (I, II, and III). Lineages I and II were found in cattle while lineage III was restricted to water buffaloes. The majority of strains (17/28), together with the strain causing endocarditis in a cow in the USA, belonged to lineage I. Further investigations are needed to determine whether B. bovis causes disease in bovids.

Serotypes, Antimicrobial Susceptibility, and gyr A Gene Mutation of Campylobacter jejuni Isolates from Humans and Chickens in Thailand

In Thailand, 51% (36/70) Campylobacter jejuni isolates from humans and 68% (47/69) isolates from poultry were classified into 10 Penner serotypes (serotype B, C, R, E, G, A, K, D, I, and L) and 9 serotypes (serotype A, C, I, K, B, E, S, D, and L), respectively. The rate of antimicrobial drug resistance to nalidixic acid, ciprofloxacin, ampicillin, tetracycline, and erythromycin shown by human isolates were 96%, 96%, 29%, 57%, and 14%, while that shown by poultry isolates were 77%, 77%, 22%, 26%, and 17%, respectively. All quinolone‐resistant strains contained a mutation in the gyrA gene (T86→I86), suggesting that the strains were already widespread in Thailand.

Bartonella vinsonii subsp. arupensis in Humans, Thailand

We identified Bartonella vinsonii subsp. arupensis in pre-enriched blood of 4 patients from Thailand. Nucleotide sequences for transfer-messenger RNA gene, citrate synthase gene, and the 16S–23S rRNA internal transcribed spacer were identical or closely related to those for the strain that has been considered pathogenic since initially isolated from a human in Wyoming, USA.

Salmonella prevalence in meat at retail markets in Pakse, Champasak Province, Laos, and antimicrobial susceptibility of isolates

Non-typhoidal Salmonella is one of the most common causes of human gastroenteritis worldwide and most human outbreaks are associated with the consumption of contaminated food. However, there are no reports on Salmonella contamination in market meat in Laos. The objective of this study was to determine the prevalence of Salmonella in meat samples in Pakse, Champasak Province, Laos, as well as the antimicrobial susceptibility of isolates. The prevalence of Salmonella was 82% in beef, 93% in pork and 80% in buffalo meat. In total, 80 isolates and 11 serovars were found, including serovars Stanley (n=15), Anatum (n=14), Derby (n=11), Rissen (n=9) and Amsterdam (n=7). The drug susceptibility of 60 strains against 10 antimicrobial agents was tested. The 60 isolates examined were sensitive to ciprofloxacin (100% susceptible), norfloxacin (100%), cefotaxime (95%), nalidixic acid (90%) and chloramphenicol (88%), but were resistant to streptomycin (67% resistant), tetracycline (67%) and ampicillin (63%). Of the isolates, 73% were multidrug-resistant. These findings indicate a high Salmonella prevalence in market meat in Pakse. Therefore, programmes to control Salmonella contamination are needed.

Diversity and phylogenetic relationships among Bartonella strains from Thai bats

Bartonellae are phylogenetically diverse, intracellular bacteria commonly found in mammals. Previous studies have demonstrated that bats have a high prevalence and diversity of Bartonella infections globally. Isolates (n = 42) were obtained from five bat species in four provinces of Thailand and analyzed using sequences of the citrate synthase gene (gltA). Sequences clustered into seven distinct genogroups; four of these genogroups displayed similarity with Bartonella spp. sequences from other bats in Southeast Asia, Africa, and Eastern Europe. Thirty of the isolates representing these seven genogroups were further characterized by sequencing four additional loci (ftsZ, nuoG, rpoB, and ITS) to clarify their evolutionary relationships with other Bartonella species and to assess patterns of diversity among strains. Among the seven genogroups, there were differences in the number of sequence variants, ranging from 1–5, and the amount of nucleotide divergence, ranging from 0.035–3.9%. Overall, these seven genogroups meet the criteria for distinction as novel Bartonella species, with sequence divergence among genogroups ranging from 6.4–15.8%. Evidence of intra- and intercontinental phylogenetic relationships and instances of homologous recombination among Bartonella genogroups in related bat species were found in Thai bats.

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 β 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.