Draft genome sequence data of multidrug-resistant Pseudomonas aeruginosa WO7 from a hospital wastewater treatment plant in Thailand.

Multidrug-resistant Pseudomonas aeruginosa WO7 was isolated from an untreated water sample from a hospital wastewater treatment plant in Thailand. This report presents the draft genome sequence data of P. aeruginosa WO7. Genomic DNA was obtained from a pure culture of P. aeruginosa WO7, and paired-end reads were generated using an Illumina MiSeq sequencer. The draft genome consisted of 111 contigs with a total size of 6,784,206 base pairs, an N50 of 209,424 base pairs, and a GC content of 65.85%. The dDDH value between WO7 and Pseudomonas aeruginosa DSM 50071T was determined to be 90.7%, indicating that the strain is Pseudomonas aeruginosa. The data presented indicate the potential for bacterial classification, comparative genomics, comprehensive analysis of antimicrobial resistance, and assessment of bacterial virulence factors in P. aeruginosa. The draft genome sequence data have been deposited at the NCBI under Bioproject accession number PRJNA550309.

Molecular characterization of extended-spectrum cephalosporin and fluoroquinolone resistance genes in Salmonella and Shigella isolated from clinical specimens in Thailand.

Antimicrobial resistance of Salmonella and Shigella has become a major clinical and public health problem. The incident of co-resistance to third generation cephalosporins and fluoroquinolone is a serious therapeutic issue in Thailand. The present study aimed to investigate the antimicrobial resistance and molecular character of clinical Shigella and Salmonella isolates. A total of 33 Salmonella and 53 Shigella cefotaxime-resistant isolates were collected from human clinical cases in Thailand during the period from 2011-2018. The antimicrobial susceptibility of Salmonella and Shigella was determined by the disk diffusion method, and extended-spectrum beta-lactamase (ESBL) production was characterized by the double-disk synergy test. Genotype characterization was performed by PCR and DNA sequencing. Thirty-two (97.0%) and fifty-two (98.1%) isolates of cefotaxime-resistant Salmonella and Shigella, respectively, were identified as ESBL producers. Shigella sonnei (4 isolates), Salmonella serovar 4,5,12:i:- (6 isolates), Salmonella serovar Agona (2 isolates) and Salmonella serovar Rissen (2 isolates) showed co-resistance to ciprofloxacin and cefotaxime or ceftriaxone. The combination of bla CTX-M-15 plus other ESBL and/or AmpC ß-lactamase genes was the most dominant of the genotype patterns in ESBL-producing isolates. The plasmid harbouring the aac(6')-Ib-cr gene and mutations of gyrA (S83F, D87Y or D87G) and parC (T57S) genes was found in 2 ESBL-producing Salmonella isolates. Three Shigella sonnei isolates harboured mutations in gyrA (S83L, D87Y or D87G), and only one Shigella sonnei phase I isolate showed mutations in both gyrA (S83L and D87G) and parC (S80I) genes. Among these clinical Shigella sonnei isolates, qnrS determinants were identified. Production of ESBLs is an important mechanism for resistance to extended-spectrum cephalosporins in Salmonella and Shigella. The emergence of a decreased susceptibility to extended-spectrum cephalosporins and fluoroquinolone in ESBL-producing isolates has important clinical and therapeutic implications.

Molecular Characterization of Cephalosporin and Fluoroquinolone Resistant Salmonella Choleraesuis Isolated from Patients with Systemic Salmonellosis in Thailand.

The antimicrobial resistance of nontyphoidal Salmonella has become a major clinical and public health problem. Southeast Asia has a high level of multidrug-resistant Salmonella and isolates resistant to both fluoroquinolone and third-generation cephalosporins. The incidence of co-resistance to both drug classes is a serious therapeutic problem in Thailand. The aim of this study was to determine the antimicrobial resistance patterns, antimicrobial resistance genes and genotypic relatedness of third-generation cephalosporins and/or fluoroquinolone-resistant Salmonella Choleraesuis isolated from patients with systemic salmonellosis in Thailand. Antimicrobial susceptibility testing was performed using the agar disk diffusion method, and ESBL production was detected by the combination disc method. A molecular evaluation of S. Choleraesuis isolates was performed using PCR and DNA sequencing. Then, a genotypic relatedness study of S. Choleraesuis was performed by pulse field gel electrophoresis. All 62 cefotaxime-resistant S. Choleraesuis isolates obtained from 61 clinical specimens were multidrug resistant. Forty-four isolates (44/62, 71.0%) were positive for ESBL phenotypes. Based on the PCR sequencing, 21, 1, 13, 23, 20 and 6 ESBL-producing isolates harboured the ESBL genes blaCTX-M-14, blaCTX-M-15, blaCTX-M-55, blaCMY-2, blaACC-1 and blaTEM-1, respectively. This study also found that nine (9/62, 14.5%) isolates exhibited co-resistance to ciprofloxacin and cefotaxime. All of the co-resistant isolates harboured at least one PMQR gene. The qnr genes and the aac(6')-Ib-cr gene were the most prevalent genes detected. The QRDR mutation, including the gyrA (D87Y and D87G) and parC (T57S) genes, was also detected. PFGE patterns revealed a high degree of clonal diversity among the ESBL-producing isolates.

Molecular analysis of population and De Novo transcriptome sequencing of Thai medaka, Oryzias minutillus (Teleostei: Adrianichthyidae).

Thai medaka (Oryzias minutillus) are alternatively known as Thai rice-fish or dwarf medaka, and they widely inhabit natural freshwater environments in all regions of Thailand. In this study, we aimed to investigate the molecular genetics of the Thai medaka population in Thailand inferred from the mitochondrial control region (D-loop) and the cytochrome c oxidase subunit 1 (coxI) sequences. Furthermore, we examined RNA sequencing (RNA-seq) of adult males and females was performed with next-generation sequencing. Together, the combination of the D-loop and coxI sequences clearly distinguished the Thai medaka populations into 2 groups, such as group 1, which generally included samples from the central, northern, western, and eastern regions of the northeastern region. In this group, the fish populations seem to be a little monophyly in which the first subpopulation comprised the main samples from the northern and central regions. The second subpopulation commonly contained fish from the eastern region and specimens from the southern part of the central region near the Gulf of Thailand. Although these subgroups related to geographical distribution, bootstrap values were low in branch considered significant for both subgroups. Group 2 consisted of almost all samples from the southern population and those from the central and southern part of the northeastern region. Group 2 was found that it was made of samples from the northeastern region and samples from the southern population. A total of 73551 unigenes were identified after gene annotation. Signal transduction was the predominant protein classification among the Thai medaka orthologous groups. A differentially expressed gene (DEG) analysis identified 6 subclusters between both sexes that were composed of 257, 131, 364, 386, 114 and 108 genes. Phototransduction was the most enriched pathway and was highly expressed in males, while viral carcinogenesis, oocyte genesis, and the complement and coagulation cascades were highly expressed in females. Further details of these DEGs are discussed below. These results suggest that Thai medaka may genetically exhibit independent populations in the geographic habitats of Thailand. Moreover, these fish also reveal the genes that are conserved in other organisms and those that may be specific to this species.

Contributions to the knowledge of Pseudolevinseniella (Trematoda: Digenea) and temnocephalans from alien crayfish in natural freshwaters of Thailand.

Redclaw crayfish (Cherax quadricarinatus) is a decapod species originating from Australian freshwater. For more than two decades, these crayfish have been re-distributing to environments in many countries, including Thailand. Moreover, they can carry endosymbionts and/or ectosymbionts into new environments. The aim of this study was to introduce a morphological description of Pseudolevinseniella anenteron as a metacercaria of the endoparasites of redclaw crayfish collected from natural water sources in Thailand. The occurrence of two ectosymbiotic temnocephalans (Diceratocephala boschmai and Temnosewellia sp.) in C. quadricarinatus was also reported. The internal morphology of P. anenteron, D. boschmai and Temnosewellia were described and discussed. The surface ultrastructure of the multidentate spines on the body and the metacercarial cyst wall of P. anenteron was investigated by scanning electron microscopy (SEM). By performing a search of the GenBank nucleotide database of partial sequences of 18S, 28S rDNA and cytochrome c oxidase subunit I (cox1), P. anenteron was found to be related to Maritrema, and Temnosewellia was found to be related to T. fasciata. However, according to the cox1 gene, Temnosewellia was found to be similar to T. minor. These results reveal that redclaw crayfish that inhabit natural freshwaters in Thailand may harbour endoparasites and ecto- and endosymbionts. Furthermore, these findings may be able to monitor invasive or non-invasive species in an ecosystem.

Rapid Colorimetric Assay for Detection of Listeria monocytogenes in Food Samples Using LAMP Formation of DNA Concatemers and Gold Nanoparticle-DNA Probe Complex.

Listeria monocytogenes is a major foodborne pathogen of global health concern. Herein, the rapid diagnosis of L. monocytogenes has been achieved using loop-mediated isothermal amplification (LAMP) based on the phosphatidylcholine-phospholipase C gene (plcB). Colorimetric detection was then performed through the formation of DNA concatemers and a gold nanoparticle/DNA probe complex (GNP/DNA probe). The overall detection process was accomplished within approximately 1 h with no need for complicated equipment. The limits of detection for L. monocytogenes in the forms of purified genomic DNA and pure culture were 800 fg and 2.82 CFU mL-1, respectively. No cross reactions were observed from closely related bacteria species. The LAMP-GNP/DNA probe assay was applied to the detection of 200 raw chicken meat samples and compared to routine standard methods. The data revealed that the specificity, sensitivity, and accuracy were 100, 90.20, and 97.50%, respectively. The present assay was 100% in conformity with LAMP-agarose gel electrophoresis assay. Five samples that were negative by both assays appeared to have the pathogen at below the level of detection. The assay can be applied as a rapid direct screening method for L. monocytogenes.

Cloning, Expression and Characterization of a Thermostable Esterase HydS14 from Actinomadura sp. Strain S14 in Pichia pastoris.

A thermostable esterase gene (hydS14) was cloned from an Actinomadura sp. S14 gene library. The gene is 777 bp in length and encodes a polypeptide of 258 amino acid residues with no signal peptide, no N-glycosylation site and a predicted molecular mass of 26,604 Da. The encoded protein contains the pentapeptide motif (GYSLG) and catalytic triad (Ser88-Asp208-His235) of the esterase/lipase superfamily. The HydS14 sequence shows 46%-64% identity to 23 sequences from actinomycetes (23 α/ß-hydrolases), has three conserved regions, and contains the novel motif (GY(F)SLG), which distinguishes it from other clusters in the α/ß-hydrolase structural superfamily. A plasmid containing the coding region (pPICZαA-hydS14) was used to express HydS14 in Pichia pastoris under the control of the AOXI promoter. The recombinant HydS14 collected from the supernatant had a molecular mass of ~30 kDa, which agrees with its predicted molecular mass without N-glycosylation. HydS14 had an optimum temperature of approximately 70 °C and an optimum pH of 8.0. HydS14 was stable at 50 and 60 °C for 120 min, with residual activities of above 80% and above 90%, respectively, as well as 50% activity at pH 6.0-8.0 and pH 9.0, respectively. The enzyme showed higher activity with p-nitrophenyl-C2 and C4. The Km and Vmax values for p-nitrophenyl-C4 were 0.21 ± 0.02 mM and 37.07 ± 1.04 µmol/min/mg, respectively. The enzyme was active toward short-chain p-nitrophenyl ester (C2-C6), displaying optimal activity with p-nitrophenyl-C4 (Kcat/Km = 11.74 mM(-1) · S(-1)). In summary, HydS14 is a thermostable esterase from Actinomadura sp. S14 that has been cloned and expressed for the first time in Pichia pastoris.

Cloning of a thermostable xylanase from Actinomadura sp. S14 and its expression in Escherichia coli and Pichia pastoris.

A thermophilic xylan-degrading Actinomadura sp. S14 was isolated from compost in Thailand. Hemicellulase activities such as endo-1,4-ß-xylanase, ß-xylosidase and α-arabinofuranosidase were induced with xylan-containing agriculture wastes and oat spelt xylan. The gene encoding xylanase consisting of 687bp was cloned from Actinomadura sp. S14. The deduced amino acid sequence contained a signal peptide of 41 amino acids and a probable mature xylanase of 188 amino acids. An open reading frame (xynS14) corresponding to a mature xylanase was expressed in Escherichia coli and Pichia pastoris. The specific activity of purified XynS14 (P. pastoris) was 2.4-fold higher than XynS14 (E. coli). Both XynS14s showed the same basic properties such as optimal pH and temperature (pH 6.0 and 80°C) and stability in a broad pH range (pH 5.0-11.0) and at high temperatures up to 80°C. Both XynS14s showed approximately the same substrate specificity and K(m) values toward various xylans, but XynS14 (P. pastoris) showed higher V(max) and K(cat) than XynS14 (E. coli). Higher specific activities of XynS14 (P. pastoris) may be due to protein-folding in the host. Purified XynS14 showed more endo-1,4-ß-xylanase activity on xylan and xylooligosaccharides than on xylotriose.

Molecular genetics analysis for co-infection of Brugia malayi and Brugia pahangi in cat reservoirs based on internal transcribed spacer region 1.

This study described the diagnosis of a mixed infection of Brugia malayi and Brugia pahangi in a single domestic cat using the internal transcribed spacer 1 (ITS1) region. Following polymerase chain reaction amplification of the ITS1 region, the 580 bp amplicon was cloned, and 29 white colonies were randomly selected for DNA sequencing and phylogenetic tree construction. A DNA parsimony tree generated two groups of Brugia spp with one group containing 6 clones corresponding to B. pahangi and the other 23 clones corresponding to B. malayi. This indicated that mixed infection of the two Brugia spp, B. pahangi and B. malayi, had occurred in a single host.

Shigella species and serotypes among clinical isolates in Thailand from 2001 to 2005.

OBJECTIVE: To study the species and the serotypes of the clinical isolates of Shigella obtained from patients in Thailand. MATERIAL AND METHOD: The World Health Organization National Salmonella and Shigella Center Thailand, had confirmed the species and performed serotype identification of 1,913 clinical isolates of Shigella collected from the laboratory network of Department of Medical Sciences and the collaborated hospitals across Thailand from 2001 to 2005. RESULTS: Between the year 2001 and 2005, 728, 481, 160, 247, 297 clinical isolates were tested, respectively. There were 5 isolates of S. dysenteriae (group A), 416 isolates of S. flexneri (group B), 4 isolates of S. boydii (group C) and 1,488 isolates of S. sonnei (group D). A total of 21 Shigella serotypes were identified and there were 3 serotypes in group A, 11 serotypes in group B, 4 serotypes in group C, and 3 serotypes in group D. Throughout these five years, the five common serotypes were S. sonnei Phases I and II, 28.6% (548 isolates); S. sonnei Phase I, 24.6% (470 isolates); S. sonnei Phase II, 24.6% (470 isolates); S. flexneri Type 2a, 10.9% (208 isolates), and S. flexneri Type 3a, 6.3% (121 isolates), respectively. CONCLUSION: At the national scale in Thailand from 2001 to 2005, S. sonnei was the most frequent Shigella spp. isolated from patients in Thailand. In addition, S. dysenteriae and S. boydii were extremely uncommon. These findings are important in future vaccine development.

The epidemiological relationship between Salmonella enterica serovar typhimurium and Salmonella enterica serovar 4,[5],12:i:- isolates from humans and swine in Thailand.

A total of 138 isolates of S. Typhimurium and S. 4,[5],12:i:- from humans and swine in Thailand during 2003-2006, were evaluated for antimicrobial sensitivity by the disk diffusion method against 10 antimicrobial drugs and pulsed-field gel electrophoresis (PFGE) with endonuclease Xbal to investigate the epidemiological relationship among isolates. It was found that all isolates were classified into 27 antimicrobial resistance patterns, and 80% of S. Typhimurium and 95.4% of S. 4,[5],12:i:- isolates were resistant to three or more antimicrobial agents. By PFGE testing, the 84 PFGE patterns were categorized into A to Z patterns. Eighty percent of S. Typhimurium and 71.3% of S. 4,[5],12:i:- isolates in 7 major PFGE patterns had close clonal relationships (_85% similarity). Our studies indicate the spread of genetically identical clones of S. Typhimurium and S. 4,[5],12:i:- in humans and swine in Thailand.

Genetic diversity of Trypanosoma evansi in beef cattle based on internal transcribed spacer region.

This study was focused on genetic diversity of Trypanosoma evansi which is a widely distributed haemoflagellate of veterinary importance that infects a variety of larger mammals including horses, mules, camels, buffalo, cattle and deer. The genetic diversity of T. evansi of beef cattle LAM19 was accomplished by using phylogenetic analysis based on internal transcribed spacer region (ITS). Blood sample was collected from a naturally infected beef cattle LAM 19 and parasitemia was raised by mouse inoculation. The parasites were collected and isolated by using DE 52 DEAE cellulose anion exchange column prior to DNA extraction. Upon PCR amplification of ITS region, the product of 1300bp in size was obtained. The ITS nucleotide sequences were analyzed and revealed that it could demonstrate the genetic diversity of T. evansi of beef cattle LAM19. Based on the ITS tree, beef cattle LAM 19 T. evansi were categorized into two main groups where the genetic diversity occurred within Group 1. The data could be applicable for the survey of parasite dynamics, epidemiological studies as well as prevention and control of the disease.