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.

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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...

Resistance to common drugs by microorganisms and cancers has become a major issue in modern healthcare, increasing the number of deaths worldwide. Novel therapeutic agents with a higher efficiency and less side effects for the treatment of certain diseases are urgently needed. Plant defensins have an integral role in a hosts’ immune system and are attractive candidates for combatting drug-resistant microorganisms. Interestingly, some of these defensins also showed great potential due to their cytotoxic activity toward cancer cells. In this study, a defensin encoding gene was isolated from five legume seeds using 3′ rapid amplification of cDNA ends (3′ RACE) with degenerate primers and cDNA cloning strategies. Bioinformatic tools were used for in silico identification and the characterization of new sequences. To study the functional characteristics of these unique defensins, the gene encoded for Sesbania javanica defensin, designated as javanicin, was cloned into pTXB-1 plasmid and expressed in the Escherichia coli Origami 2 (DE3) strain. Under optimized conditions, a 34-kDa javanicin-intein fusion protein was expressed and approximately 2.5–3.5 mg/L of soluble recombinant javanicin was successfully extracted with over 90% purity. Recombinant javanicin displayed antifungal properties against human pathogenic fungi, including resistant strains, as well as cytotoxic activities toward the human breast cancer cell lines, MCF-7 & MDA-MB-231. Recombinant javanicin holds great promise as a novel therapeutic agent for further medical applications.

Streptococcus suis is an emerging zoonotic bacterium causing septicemia and meningitis in humans. Due to rapid disease progression, high mortality rate, and many underdiagnosed cases by time-consuming routine identification methods, alternative diagnostic testing is essential. Among 29 broadly accepted S. suis serotypes, serotypes 2 and 14 are high prevalent; however, many PCR assays showed an inability to differentiate serotype 2 from 1/2, and 1 from 14. In this study, we developed and validated a new multiplex PCR assay that facilitates the identification of only the 29 true serotypes of S. suis and simultaneously differentiates serotypes 1, 1/2, 2, and 14 within a single reaction. Importantly, the multiplex PCR could detect S. suis directly from positive hemocultures and CSF. The results revealed high sensitivity, specificity, and 100% accuracy with almost perfect agreement (κ = 1.0) compared to culture and serotyping methods. Direct detection enables a decrease in overall diagnosis time, rapid and efficient treatment, reduced fatality rates, and proficient disease control. This multiplex PCR offers a rapid, easy, and cost-effective method that can be applied in a routine laboratory. Furthermore, it is promising for developing point-of-care testing (POCT) for S. suis detection in the future.

Cryptococcus neoformans, a life-threatening human yeast pathogen, has the ability to produce melanin, which is one of the common virulence factors contributing to cryptococcal pathogenesis. This virulence factor is closely associated with the cryptococcal cell wall, specifically chitin and chitosan polysaccharides, a complex structure that is essential for maintaining cellular structure and integrity. In this study, we aim to investigate the effects of two stingless bee (SLB) propolis from Tetragonula laeviceps and Tetrigona melanoleuca against cell wall-associated melanin in C. neoformans, and its immune response in RAW 264.7 macrophage. The ethanolic extract of SLB propolis (EEP) has strongly exhibited anti-cryptococcal activity. Moreover, EEP from both sources reduced chitin/chitosan and melanin production against C. neoformans in a dose-dependent manner. Likewise, the mRNA expression level of CDA1, IPC1-PKC1 and LAC1 genes involved in the cryptococcal melanization pathway was significantly decreased at 2 mg/mL in EEP treatment. Additionally, pretreatment with EEP prior to yeast infection dramatically reduced intracellular replication of C. neoformans in RAW 264.7 macrophages in a dose-dependent manner. This study might be a new insight to use a natural powerful source, not only acting to target cell wall-associated molecules, but also being capable to explore a novel strategy by which dysregulation of these molecules leads to promote immunomodulatory activity.

This study characterizes clinical methicillin-resistant staphylococcal (MRS) isolates obtained from superficial pyoderma infections in dogs. Our interest was to determine the staphylococcal cassette chromosome mec (SCCmec) type and the antimicrobial susceptibility among MRS isolates from clinical cases. Skin swabs were collected and cultured. Staphylococcus species were identified and characterized with biochemical tests and MALDI-TOF-MS and antimicrobial susceptibility testing by disk diffusion. mecA detection and staphylococcal cassette chromosome mec (SCCmec) typing were achieved by PCR. Of the 65 clinical samples, 56 (86.2%) staphylococcal infections were identified. Twelve (21%) of 56 isolates were MRS infections. All MRS isolates were multidrug resistant. The ccrC and class-C2 mec, which were SCCmec type V, were the most prevalent (66.7%) among the 12 MRS isolates. The predominant SCCmec type V was found in S. aureus, S. intermedius group, S. lentus, S. xylosus, and S. arlettae. Treatment failure is a concern with the emergence of highly resistant MRS in dogs associated with superficial pyoderma. The detection of type V SCCmec MRS has previously been reported among veterinarians and dog owners but not in Northern Thailand. These infections serve as a reminder to improve infection prevention and control measures including reducing environmental contamination and potential zoonotic exposures to MRS. In addition, educational awareness of these risks in small animal hospitals needs to be increased among veterinary hospital staff, clients, and patients.