Kiatichai Faksri

Serogroup, virulence, and molecular traits of Vibrio parahaemolyticus isolated from clinical and cockle sources in northeastern Thailand.

Vibrio parahaemolyticus is responsible for seafood-borne gastroenteritis worldwide. Isolates of V. parahaemolyticus from clinical samples (n=74) and cockles (Anadara granosa) (n=74) in Thailand were analyzed by serotyping, determination of virulence and related marker genes present, response to antimicrobial agents, and genetic relatedness. Serological analysis revealed 31 different serotypes, 10 of which occurred among both clinical and cockle samples. The clinical isolates commonly included the pandemic serogroup O3:K6, while a few of the cockle isolates exhibited likely pandemic serovariants such as O3:KUT and O4:KUT, but not O3:K6. The pandemic (orf8 gene-positive) strains were more frequently found among clinical isolates (78.4%) than cockle isolates (28.4%) (p<0.001). Likewise, the virulence and related marker genes were more commonly detected among clinical than cockle isolates; i.e., tdh gene (93.2% versus 29.7%), vcrD2 (97.3% versus 23.0%), vopB2 (89.2% versus 13.5%), vopT (98.6% versus 36.5%) (all p<0.001) and trh (10.8% versus 1.4%) (p<0.05). Pulsed-field gel electrophoresis of NotI-digested genomic DNA of 41 randomly selected V. parahaemolyticus isolates representing different serotypes produced 33 pulsotypes that formed 5 different clusters (clonal complexes) (A-E) in a dendrogram. Vibrio parahaemolyticus O3:K6 and likely related pandemic serotypes were especially common among the numerous clinical isolates in cluster C, suggesting a close clonal link among many of these isolates. Most clinical and cockle isolates were resistant to ampicillin. This study indicates that O3:K6 and its likely serovariants based on the PFGE clusters, are causative agents. Seafoods such as cockles potentially serve as a source of virulent V. parahaemolyticus, but further work is required to identify possible additional sources.

Bioinformatics tools and databases for whole genome sequence analysis of Mycobacterium tuberculosis.

Tuberculosis (TB) is an infectious disease of global public health importance caused by Mycobacterium tuberculosis complex (MTC) in which M. tuberculosis (Mtb) is the major causative agent. Recent advancements in genomic technologies such as next generation sequencing have enabled high throughput cost-effective generation of whole genome sequence information from Mtb clinical isolates, providing new insights into the evolution, genomic diversity and transmission of the Mtb bacteria, including molecular mechanisms of antibiotic resistance. The large volume of sequencing data generated however necessitated effective and efficient management, storage, analysis and visualization of the data and results through development of novel and customized bioinformatics software tools and databases. In this review, we aim to provide a comprehensive survey of the current freely available bioinformatics software tools and publicly accessible databases for genomic analysis of Mtb for identifying disease transmission in molecular epidemiology and in rapid determination of the antibiotic profiles of clinical isolates for prompt and optimal patient treatment.

Comparative Proteomics of Activated THP-1 Cells Infected with Mycobacterium tuberculosis Identifies Putative Clearance Biomarkers for Tuberculosis Treatment.

Biomarkers for determining clearance of Mycobacterium tuberculosis (Mtb) infection during anti-tuberculosis therapy or following exposure could facilitate enhanced monitoring and treatment. We screened for biomarkers indicating clearance of Mtb infection in vitro. A comparative proteomic analysis was performed using GeLC MSI/MS. Intracellular and secreted proteomes from activated THP-1 cells infected with the Mtb H37Rv strain (MOI = 1) and treated with isoniazid and rifampicin for 1 day (infection stage) and 5 days (clearance stage) were analyzed. Host proteins associated with early infection (n = 82), clearance (n = 121), sustained in both conditions (n = 34) and suppressed by infection (n = 46) were elucidated. Of the potential clearance markers, SSFA2 and CAECAM18 showed the highest and lowest protein intensities, respectively. A western blot of CAECAM18 validated the LC MS/MS result. For three clearance markers (SSFA2, PARP14 and PSME4), in vivo clinical validation was concordantly reported in previous patient cohorts. A network analysis revealed that clearance markers were enriched amongst four protein interaction networks centered on: (i) CD44/CCND1, (ii) IFN-β1/NF-κB, (iii) TP53/TGF-β and (iv) IFN-γ/CCL2. After infection, proteins associated with proliferation, and recruitment of immune cells appeared to be enriched possibly reflecting recruitment of defense mechanisms. Counteracting proteins (CASP3 vs. Akt and NF-κB vs. TP53) associated with apoptosis regulation and its networks were enriched among the early and sustained infection biomarkers, indicating host-pathogen competition. The BRCA1/2 network was suppressed during infection, suggesting that cell proliferation suppression is a feature of Mtb survival. Our study provides insights into the mechanisms of host-Mtb interaction by comparing the stages of infection clearance. The identified clearance biomarkers may be useful in monitoring tuberculosis treatment.

Epidemiology and identification of potential fungal pathogens causing invasive fungal infections in a tertiary care hospital in northeast Thailand

Invasive fungal infections (IFIs) are life threatening and associated with a high mortality rate. Here, we describe the distribution of pathogens, host risk factors, and significance of fungi isolated from patients with IFIs. The study included 861 fungal isolates recovered between 2006 and 2011 from 802 patients at Srinagarind Hospital, Thailand. Based on the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group 2008 criteria, 28.5% (245/861 isolates) of the fungal isolates were considered to be causative agents of IFIs. The most common fungus was Candida albicans (46%, 396/861 isolates). However, the most common yeast causing IFIs was Cryptococcus neoformans (34.7%, 85/245 isolates), while the most common mould was Penicillium marneffei (10.6%, 26/245 isolates). Cryptococcosis was significantly associated with human immunodeficiency virus infections (P < 0.001). Trend analysis revealed that there was no significant increase in IFI cases (P = 0.34) from 2006 to 2011 or from 2007 to 2011 (P = 0.05), but there was a trend toward significant increases in candidiasis (P = 0.04). The fungal isolates were categorized according to the positive predictive value of their recovery in cultures as being true (>95%), moderate (5%-95%), and rare (<5%) pathogens. This classification system could facilitate the prediction of the likelihood of diseases caused by the isolated fungi.

Antimicrobial resistance and genetic diversity of the SXT element in Vibrio cholerae from clinical and environmental water samples in northeastern Thailand

Multidrug resistance in V. cholerae has been increasing around the world including northeastern Thailand. The aquatic environment is a reservoir of V. cholerae and might be an important source of resistant strains. The aims of this study were to investigate the phylogenetic relationships of intSXT gene sequences from 31 clinical and 14 environmental V. cholerae O1 and non-O1/non-O139 isolates and 11 sequences amplified directly from environmental water samples. We also amplified class 1 integrons, the SXT elements (targeting the intSXT gene) and antimicrobial resistance genes directly from water samples. Phylogenetic analysis displayed two major distinct clusters (clusters 1 and 2). Most V. cholerae O1 (19/20, 95%) and non-O1/non-O139 isolates (8/11, 72.7%) from clinical sources, and all sequences obtained directly from water samples, belonged to cluster 1. Cluster 2 mostly comprised environmental non-O1/non-O139 isolates (10/12, 83.3%). We successfully amplified the SXT elements directly from17.5% of water samples. Associated resistance genes were also amplified as follows: sul2 (41.3% of water samples), dfrA1 (60%), dfr18 (33.8%), strB (70%) and tetA (2.5%). Class 1 integrons were not found in water samples, indicating that the SXT element was the major contributor of multidrug resistance determinants in this region. The SXT element and antimicrobial resistance genes could be transferred from clinical V. cholerae O1 to environmental V. cholerae non-O1/non-O139 was demonstrated by conjugation experiment. These findings indicate that there may have been cross dissemination and horizontal gene transfer (HGT) of the SXT element harbored by V. cholerae O1 and non-O1/non-O139 strains isolated from clinical and environmental water sources. Environmental water might be an important source of antimicrobial resistance genes in V. cholerae in this region. Direct detection of antimicrobial resistance genes in water samples can be used for monitoring the spread of such genes in the ecosystem.

Tuberculosis determination using SERS and chemometric methods

Nanostructures have been multiplying the advantages of Raman spectroscopy and further amplify the advantages of Raman spectroscopy is a continuous effort focused on the appropriate design of nanostructures. Herein, we designed different shapes of plasmonic nanostructures such as Vertical, Zig Zag, Slant nanorods and Spherical nanoparticles employing the DC magnetron sputtering system as SERS-active substrates for ultrasensitive detection of target molecules. The fabricated plasmonic nanostructures sensitivity and uniformity were exploited by reference dye analyte. These nanostructures were utilized in the label free detection of infectious disease, Tuberculosis (TB). For the first time, TB detection from serum samples using SERS has been demonstrated. Various multivariate statistical methods such as principal component analysis, support vector machine, decision tree and random forest were developed and tested their ability to discriminate the healthy and active TB samples. The results demonstrate the performance of the SERS spectra, chemometric methods and potential of the method in clinical diagnosis.

Comparative whole-genome sequence analysis of Mycobacterium tuberculosis isolated from tuberculous meningitis and pulmonary tuberculosis patients.

Tuberculous meningitis (TBM) is a severe form of tuberculosis with a high mortality rate. The factors associated with TBM pathogenesis are still unclear. Using comparative whole-genome sequence analysis we compared Mycobacterium tuberculosis (Mtb) isolates from cerebrospinal fluid of TBM cases (n = 73) with those from sputum of pulmonary tuberculosis (PulTB) patients (n = 220) from Thailand. The aim of this study was to seek genetic variants of Mtb associated with TBM. Regardless of Mtb lineage, we found 242 variants that were common to all TBM isolates. Among these variants, 28 were missense SNPs occurring mainly in the pks genes (involving polyketide synthesis) and the PE/PPE gene. Six lineage-independent SNPs were commonly found in TBM isolates, two of which were missense SNPs in Rv0532 (PE_PGRS6). Structural variant analysis revealed that PulTB isolates had 14 genomic regions containing 2–3-fold greater read depth, indicating higher copy number variants and half of these genes belonged to the PE/PPE gene family. Phylogenetic analysis revealed only two small clusters of TBM clonal isolates without support from epidemiological data. This study reported genetic variants of Mtb commonly found in TBM patients compared to PulTB patients. Variants associated with TBM disease warrant further investigation.

Detection and genotyping of Helicobacter pylori in saliva versus stool samples from asymptomatic individuals in Northeastern Thailand reveals intra-host tissue-specific H. pylori subtypes

BackgroundTwo-thirds of the world’s population is thought to be infected by Helicobacter pylori. Although most people infected with H. pylori are asymptomatic, this pathogen is associated with several gastric pathologies including cancer. The risk factors for colonization are still unclear and the genetic diversity within individual hosts has never been clearly investigated.ResultThis study determined the prevalence of, and explored risk factors for, H. pylori infection directly from paired saliva (n = 110) and stool (n = 110) samples from asymptomatic persons in Northeast Thailand. Samples were subjected to indirect immunofluorescence assay (IFA), 16S rRNA-based real-time PCR and vacA-based semi-nested PCR. Partial vacA gene sequences of H. pylori were compared between saliva and stool samples.The overall prevalence of H. pylori infection in our asymptomatic study population was 64%. Age, gender, occupation and frequency of brushing teeth were not found to be associated with H. pylori colonization. The vacA gene was successfully sequenced from both saliva and stool samples of 12 individuals. For seven of these individuals, saliva and stool sequences fell into different clusters on a phylogenetic tree, indicating intra-host genetic variation of H. pylori.ConclusionThis study reports a high prevalence of H. pylori infection in asymptomatic persons in this region of Thailand and demonstrates that genotypes (vacA gene sequences) of H. pylori may differ between the oral cavity and intestinal tract.

Whole-Genome Sequencing Analysis of Serially Isolated Multi-Drug and Extensively Drug Resistant Mycobacterium tuberculosis from Thai Patients.

Multi-drug and extensively drug-resistant tuberculosis (MDR and XDR-TB) are problems that threaten public health worldwide. Only some genetic markers associated with drug-resistant TB are known. Whole-genome sequencing (WGS) is a promising tool for distinguishing between re-infection and persistent infection in isolates taken at different times from a single patient, but has not yet been applied in MDR and XDR-TB. We aim to detect genetic markers associated with drug resistance and distinguish between reinfection and persistent infection from MDR and XDR-TB patients based on WGS analysis. Samples of Mycobacterium tuberculosis (n = 7), serially isolated from 2 MDR cases and 1 XDR-TB case, were retrieved from Siriraj Hospital, Bangkok. The WGS analysis used an Illumina Miseq sequencer. In cases of persistent infection, MDR-TB isolates differed at an average of 2 SNPs across the span of 2–9 months whereas in the case of reinfection, isolates differed at 61 SNPs across 2 years. Known genetic markers associated with resistance were detected from strains susceptible to streptomycin (2/7 isolates), p-aminosalicylic acid (3/7 isolates) and fluoroquinolone drugs. Among fluoroquinolone drugs, ofloxacin had the highest phenotype-genotype concordance (6/7 isolates), whereas gatifloxcain had the lowest (3/7 isolates). A putative candidate SNP in Rv2477c associated with kanamycin and amikacin resistance was suggested for further validation. WGS provided comprehensive results regarding molecular epidemiology, distinguishing between persistent infection and reinfection in M/XDR-TB and potentially can be used for detection of novel mutations associated with drug resistance.

Epidemiology of and risk factors for extrapulmonary nontuberculous mycobacterial infections in Northeast Thailand

Background Nontuberculous mycobacterial (NTM) infection is increasing worldwide. Current epidemiological data and knowledge of risk factors for this disease are limited. We investigated the trends in and risk of NTM infection in Northeast Thailand during 2012–2016. Methods Patient demographics, infection site(s), and underlying disease or conditions from 530 suspected cases of NTM infections were retrieved from medical records, reviewed and analyzed. A diagnosis of true NTM infection was accepted in 150 cases. Risk factor analyses were done for extrapulmonary NTM infections compared to pulmonary NTM infections and for Mycobacterium abscessus compared to members of the Mycobacterium avium complex (MAC). Trend analysis among NTM species causing NTM infections was performed. Results The most common species of NTMs causing extrapulmonary (n = 114) and pulmonary (n = 36) NTM infections in Northeast Thailand were M. abscessus (25.4% of extrapulmonary infected cases and 27.8% of pulmonary cases) followed by MAC (14.9% of extrapulmonary and 13.9% of pulmonary cases). Presence of anti-IFN-γ autoantibodies was the major risk factor for extrapulmonary (odds ratio (OR) = 20.75, 95%CI [2.70–159.24]) compared to pulmonary NTM infection. M. abscessus infection was less likely (OR = 0.17; 95%CI [0.04–0.80]) to be found in patients with HIV infection than was MAC infection. The prevalence of NTM infection, especially M. abscessus, in Northeast Thailand has recently increased. Extrapulmonary NTM and complicated NTM infections have increased in concordance with the recent trend of increasing frequency of anti-IFN-γ autoantibodies in the population. Conclusions M. abscessus was the commonest NTM pathogen followed by MAC. The prevalence of NTM infections and anti-IFN-γ are showing an upward trend. Autoimmune disease due to anti-IFN-γ is the major risk factor for extrapulmonary NTM infection in Northeast Thailand.