Introduction: Knowledge of the prevalence and distribution of multidrug-resistant tuberculosis (MDR-TB) genotypes in northern Thailand is still limited. An accurate, rapid, and cost-effective diagnostic of MDR-TB is crucial to improve treatment and control of increased MDR-TB. Materials and Methods: The molecular diagnostic assays named "RIF-RD" and "INH-RD" were designed to detect rifampicin (RIF) and isoniazid (INH) resistance based on real-time PCR and high-resolution melting curve analysis. Applying the ∆T m cutoff values, the RIF-RD and INH-RD were evaluated against the standard drug susceptibility testing (DST) using 107 and 103 clinical Mycobacterium tuberculosis (Mtb) isolates from northern Thailand. DNA sequence analysis of partial rpoB, katG, and inhA promoter of 73 Mtb isolates, which included 30 MDR-TB, was performed to elucidate the mutations involved with RIF and INH resistance. Results: When compared with the phenotypic DST, RIF-RD targeting rpoB showed sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 83.9, 98.6, 96.9, and 92.0%, respectively. The multiplex reaction of the INH-RD targeted both katG and inhA promoter showed high sensitivity, specificity, PPV, and NPV of 97.1, 94.2, 89.2, and 98.5%, respectively. Six patterns of rpoB mutation, predominately at codons 531 (50%) and 526 (40%) along with a rare S522L (3.33%) and D516V (3.33%), were detected. A single pattern of katG mutation (S315T) (63.3%) and four patterns of inhA promoter mutation, predominately −15 (C>T), were found. Approximately, 17% of MDR-TB strains possessed double mutations within the katG and inhA promoter. Conclusion: Up to 86.7% and 96.7% of MDR-TB could be accurately detected by RIF-RD and INH-RD, emphasizing its usefulness as a low unit price assay for rapid screening of MDR-TB, with confirmation of INH resistance in low and middle-income countries. The MDR-TB genotypes provided will be beneficial for TB control and the development of drugresistant TB diagnostic technology in the future.
The onset of streptothricin (ST) biosynthesis in Streptomyces rochei F20 was studied by using reverse transcription-PCR (RT-PCR) to detect transcripts of ST genes during growth in liquid medium, soil, and the rhizosphere. In situ results correlated with those obtained in vitro, illustrating the growth phase-dependent manner of ST production by F20. Maximal transcription of ST resistance (sttR) and biosynthesis (sttA) genes occurred during the transition between the exponential and stationary phases of growth, when the specific growth rate () started to decline. A higher level of gene expression of sttR versus sttA was observed in all experiments. In liquid culture, maximal transcript accumulation of the sttA gene was only ca. 40% that of the sttR gene. sttA and sttR mRNAs were detected in soil containing approximately 10 6 CFU of growing cells g of soil ؊1 . sttR mRNA was detected in sterile and nonsterile rhizosphere colonized with growing mycelium of F20 at 1.2 ؋ 10 6 and 4.0 ؋ 10 5 CFU g of soil ؊1 , respectively. However, neither sttR nor sttA transcripts were detected by RT-PCR in the rhizoplane, which supported a lower population density of F20 than the rhizosphere.The involvement of antibiotics in biological control has been reported (3,14,24,27). However, antibiotic production in natural environments such as soil is difficult to demonstrate, because low levels of nutrients limit growth and production (32). Detection of antibiotic production in soil could be achieved only in the presence of nutrient sources, such as roots, seeds, or straw fragments. In addition, many antibiotics are adsorbed to clay particles and are difficult to detect by extraction (22,23). Only a few studies using conventional extraction methods have provided direct evidence supporting the crucial role of antibiotics in disease suppression by biological control agents in situ. By use of a bioassay technique, geldanamycin was detected at 88 g g of soil Ϫ1 in pea rhizosphere where Streptomyces hygroscopicus var. geldanus was used to control Rhizoctonia solani infection (24). Levels of thiostrepton production in sterile soil were reported by Wellington et al. (31) within the range of 30 to 50 ng g of soil Ϫ1 following extraction and bioassay via a specific thiostrepton-inducible promoter coupled to a resistance gene. A similar method using a highly sensitive inducible promoter driving gfp expression was used to assay oxytetracycline production by Streptomyces rimosus in soil microcosms (11). Fluorescence-activated cell sorter analysis was used to detect fluorescing cells in soil extracts. Extraction of phenazine from wheat rhizosphere inoculated with Pseudomonas fluorescens 2-79 and Pseudomonas aureofaciens 30-84 and the use of mutants with phenazine blocks unequivocally proved the significance of antibiotic production in control of take-all disease caused by Gaeumannomyces graminis var. tritici (27). Other studies have demonstrated the positive correlation between production of antibiotics using fluorescent pseudomonads and control of...
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