Genetic characterization of coliform bacterial isolates from environmental water in Thailand.

INTRODUCTION: In contrast to the study in other part of the world, information about characteristics of plasmids carrying antimicrobial resistance genes (ARGs) in Enterobacteriaceae derived from environmental water in tropical Asian countries including Thailand is limited. This study, therefore, aimed to gain insight into genetic information of antimicrobial resistance in environmental water in Thailand. METHODS: Coliform bacteria were isolated from environmental water collected at 20 locations in Thailand and identified. Then, susceptibility profiles to ampicillin, cefazoline, cefotaxime, kanamycin, ciprofloxacin, sulfamethoxazole, tetracycline, and nalidixic acid were assessed. In addition, antimicrobial resistant genes integrons, and replicon types were analyzed. And furthermore, plasmids carrying blaTEM and tetM were identified by S1-PFGE analysis and confirmed transmissibility by transconjugation experiments. RESULTS: In 130 coliform bacteria isolated, 89 were resistant to cefazoline while 41 isolates were susceptible. Cefazoline-resistant coliform bacteria were found to be significantly resistant to cefotaxime and tetracycline as compared to susceptible isolates. Hence, blaTEM and tetM correlating with ß-lactam antibiotics and tetracycline, respectively, were analyzed found to co-localize on the IncFrepB plasmids in isolates from pig farms' wastewater by S1-PFGE analysis. And furthermore, transmissibility of the plasmids was confirmed. CONCLUSIONS: Results obtained in this study suggested that ARGs in coliform bacteria may have been spreading on the farm via IncFrepB plasmids. Hence, appropriate use of antimicrobials and good hygiene management on the farm are required to prevent the emergence and spread of resistant bacteria.

Co-harboring of cephalosporin (bla)/colistin (mcr) resistance genes among Enterobacteriaceae from flies in Thailand.

The spreading of antimicrobial-resistant Enterobacteriaceae, especially those co-harboring plasmid-mediated cephalosporin (bla) and colistin (mcr) resistance genes, is becoming increasingly problematic. As a vector, flies carry antimicrobial-resistant bacteria (ARB) into human and livestock habitats. To investigate ARB in flies, we collected 235 flies from 27 sites (18 urban areas, five pig farms and four chicken farms) in Thailand during 2013-2015. Cefotaxime-resistant Enterobacteriaceae (CtxRE) and bla-positive CtxRE were isolated from 70 (29.8%) and 48 (20.4%) flies, respectively. In 93 bla-positive CtxRE isolates that included Escherichia coli, Enterobacter spp., and Klebsiella pneumoniae from 48 flies, the most frequent bla gene was TEM (n = 62), followed by CTX-M-55 (n = 31), CTX-M-14 (n = 26), CMY-2 (n = 24) and SHV (n = 10), and 58 isolates harbored multiple types of these genes. In addition, we detected the mcr-1 (n = 1) and mcr-3 (n = 19) genes in bla-positive CtxRE isolates from 16 flies. In conjugation experiments, 10 mcr-3- and bla-positive isolates exhibited co-transfer of mcr-3 and blaTEM-1 genes. These results suggest that a relatively high proportion of flies in Thailand carries cephalosporin-resistant Enterobacteriaceae harboring co-transmissible cephalosporin and colistin resistance genes.

Use of Aeromonas spp. as General Indicators of Antimicrobial Susceptibility among Bacteria in Aquatic Environments in Thailand.

Antimicrobials are widely used, not only for treating human infections, but also for treatment of livestock and in fish farms. Human habitats in Southeastern Asian countries are located in close proximity to aquatic environments. As such, the human populations within these regions are at risk of exposure to antimicrobial resistant bacteria, and thereby disseminating antimicrobial resistance genes (ARGs). In this study, we collected water samples from 15 sites (5 sites in Chao Phraya River, 2 sites at the mouth of Chao Phraya River, 3 sites in Ta Chin River, and 5 sites at city canals) and 12 sites (6 sites at city canals; 2 sites at chicken farms; 2 sites at pig farms; and 2 samples from sites at pig farms, which were subsequently treated at a biogas plant) in Thailand in 2013 and 2014, respectively. In total, 117 Aeromonas spp. were isolated from the water samples, and these organisms exhibited various antimicrobial susceptibility profiles. Notably, there was a significant correlation between the environmental concentration of tetracyclines and the rates of tetracycline resistance in the isolated Aeromonas spp.; however, both the concentration and rates of tetracycline resistance in samples derived from pig farms were higher than those of samples harvested from other aquatic environments. These findings suggest that the high concentrations of antimicrobials observed in these aquatic environments likely select for ARGs. Furthermore, they indicate that Aeromonas spp. comprise an effective marker for monitoring antimicrobial resistance in aquatic environments.

Identification of a novel hedycaryol synthase gene isolated from Camellia brevistyla flowers and floral scent of Camellia cultivars.

MAIN CONCLUSION: A novel terpene synthase (Tps) gene isolated from Camellia brevistyla was identified as hedycaryol synthase, which was shown to be expressed specifically in flowers. Camellia plants are very popular because they bloom in winter when other plants seldom flower. Many ornamental cultivars of Camellia have been bred mainly in Japan, although the fragrance of their flowers has not been studied extensively. We analyzed floral scents of several Camellia cultivars by gas chromatography-mass spectrometry (GC-MS) and found that Camellia brevistyla produced various sesquiterpenes in addition to monoterpenes, whereas Camellia japonica and its cross-lines produced only monoterpenes, including linalool as the main product. From a flower of C. brevistyla, we isolated one cDNA encoding a terpene synthase (TPS) comprised of 554 amino acids, which was phylogenetically positioned to a sole gene clade. The cDNA, designated CbTps1, was expressed in mevalonate-pathway-engineered Escherichia coli, which carried the Streptomyces mevalonate-pathway gene cluster in addition to the acetoacetate-CoA ligase gene. A terpene product was purified from recombinant E. coli cultured with lithium acetoacetate, and analyzed by (1)H-nulcear magnetic resonance spectroscopy ((1)H-NMR) and GC-MS. It was shown that a sesquiterpene hedycaryol was produced, because (1)H-NMR signals of the purified product were very broad, and elemol, a thermal rearrangement product from hedycaryol, was identified by GC-MS analysis. Spectroscopic data of elemol were also determined. These results indicated that the CbTps1 gene encodes hedycaryol synthase. Expression analysis of CbTps1 showed that it was expressed specifically in flowers, and hedycaryol is likely to be one of the terpenes that attract insects for pollination of C. brevistyla. A linalool synthase gene, which was isolated from a flower of Camellia saluenensis, is also described.

Heterodyne Fourier transform spectrometer for the near- infrared region.

A Fourier transform spectrometer with heterodyne modulation achieved by a moving diffraction grating has been developed for the near-infrared (NIR) region. The grating simultaneously acts as a beam splitter and a modulator, which realizes the optical frequency shift of incident light for increasing the sensitivity of measurements by the heterodyne detection technique. The differences in diffraction angle among broad spectra are compensated by a collimating mirror and plane mirrors. The proposed spectrometer is used for the measurements of spectra in the NIR region. The signal-to-noise ratio of measurements is improved sevenfold with a heterodyne modulation of 410 Hz. As examples, this spectrometer is applied for quantitative calibration and discrimination of organic solutions. The measurement of transmission spectra of a grape is also demonstrated.