The reduction of tetrazolium salts by NAD(P)H to formazan product has been widely used to determine the metabolic activity of cells, and as an indicator of cell viability. However, the application of a WST-8 based assay for the quantitative measurement of dehydrogenase enzyme activity has not been described before. In this study, we reported the application of an assay based on the tetrazolium salt WST-8 for the quantitative measurement of dehydrogenase activity. The assay is performed in a microplate format, where a single endpoint is measured at 450 nm. The optimized dehydrogenase-WST-8 assay conditions, the limit of detection (LOD), accuracy, and precision for measuring NAD(P)H, were demonstrated. The sensitivity of the WST-8 assay for detecting NAD(P)H was 5-fold greater than the spectrophotometric measurement of NAD(P)H absorption at 340 nm (LOD of 0.3 nmole vs 1.7 nmole, respectively). In the dehydrogenase assay, the colorimetric WST-8 method exhibits excellent assay reproducibility with a Z’ factor of 0.9. The WST-8 assay was also used to determine dehydrogenase activity in biological samples, and for screening the substrate of uncharacterized short-chain dehydrogenase/oxidoreductase from Burkholderia pseudomallei. The results suggest that the WST-8 assay is a sensitive and rapid method for determining NAD(P)H concentration and dehydrogenase enzyme activity, which can be further applied for the high-throughput screening of dehydrogenases.

Burkholderia pseudomallei is the causative agent of melioidosis, a frequently occurring disease in northeastern Thailand, where soil and water high in salt content are common. Using microarray analysis, we previously showed that B. pseudomallei up-regulated a short-chain dehydrogenase/oxidoreductase (SDO) under salt stress. However, the importance of SDO in B. pseudomallei infection is unknown. This study aimed to explore the function of B. pseudomallei SDO, and to investigate its role in interactions between B. pseudomallei and host cells.

Burkholderia pseudomalleiis a Gram-negative bacterium that causes melioidosis, which can be fatal in humans. Melioidosis is prevalent in the tropical regions of Southeast Asia and Northern Australia. Ecological data have shown that this bacterium can survive as a free-living organism in environmental niches, such as soil and water, as well as a parasite living in host organisms, such as ameba, plants, fungi, and animals. This review provides an overview of the survival and adaptation ofB. pseudomalleito stressful conditions induced by hostile environmental factors, such as salinity, oxidation, and iron levels. The adaptation ofB. pseudomalleiin host cells is also reviewed. The adaptive survival mechanisms of this pathogen mainly involve modulation of gene and protein expression, which could cause alterations in the bacteria’s cell membrane, metabolism, and virulence. Understanding the adaptations of this organism to environmental factors provides important insights into the survival and pathogenesis ofB. pseudomallei, which may lead to the development of novel strategies for the control, prevention, and treatment of melioidosis in the future.

Nontyphoidal-Salmonella bacteria cause foodborne gastroenteritis that may lead to fatal bacteremia, osteomyelitis, and meningitis if not treated properly. The emergence of multidrug-resistant Salmonella strains is a global public health threat. Regular monitoring of genotypes and phenotypes of Salmonella isolated from humans, animals, foods, and environments is mandatory for effective reduction and control of this food-borne pathogen. In this study, antimicrobial-resistant and virulent genotypes and phenotypes of Salmonella isolated from retail food samples in Bangkok, Thailand, were investigated. From 252 raw food samples, 58 Salmonella strains that belonged only to serotype Enteritidis were isolated. Disc diffusion method showed that all isolates were still sensitive to amikacin and carbapenems. More than 30% of the isolates were resistant to ampicillin, tetracycline, and ciprofloxacin. Twenty isolates resist at least three antibiotic classes. Minimum inhibitory concentration tests showed that 12.07% of the isolates produced extended-spectrum β-Lactamase. Polymerase chain reaction indicated that 32.76, 81.03, 39.66, and 5.17% of the isolates carried bla_TEM-1, tetA, sul2, and dfrA7, respectively. All isolates were positive for invasion-associated genes. Effective prevention and control of Salmonella (as well as other food-borne pathogens) is possible by increasing public awareness and applying food hygienic practices. Active and well harmonised “One Health” co-operation is required to effectively control food-borne zoonosis.

Antimicrobial resistance poses a global health threat, with Staphylococcus aureus emerging as a notorious pathogen capable of forming stubborn biofilms and regulating virulence through quorum sensing (QS). In the quest for novel therapeutic strategies, this groundbreaking study unveils the therapeutic potential of Paederia foetida Linn., an Asian medicinal plant containing various bioactive compounds, contributing to its antimicrobial activities, in the battle against S. aureus. Through a comprehensive approach, we investigated the effect of ethanolic P. foetida leaf extract on S. aureus biofilms, QS, and antimicrobial activity. The extract exhibited promising inhibitory effects against S. aureus including the biofilm-forming strain and MRSA. Real-time PCR analysis revealed significant downregulation of key virulence and biofilm genes, suggesting interference with QS. Biofilm assays quantified the extract’s ability to disrupt and prevent biofilm formation. LC-MS/MS analysis identified quercetin and kaempferol glycosides as potential bioactive constituents, while molecular docking studies explored their binding to the QS transcriptional regulator SarA. Computational ADMET predictions highlighted favorable intestinal absorption but potential P-glycoprotein interactions limiting oral bioavailability. While promising anti-virulence effects were demonstrated, the high molecular weights and excessive hydrogen bond donors/acceptors of the flavonoid glycosides raise concerns regarding drug-likeness and permeability. This integrated study offers valuable insights for developing novel anti-virulence strategies to combat antimicrobial resistance.

Serial passage is a problem among many bacterial species, especially those where strains have been stored (banked) for several decades. Prior to banking with an organization such as ATCC, many bacterial strains were passaged for many years, so the characteristics of each strain may be extremely different. This is in addition to any differences in the original host environment. For Burkholderia pseudomallei, the number of serial passages should be carefully defined for each experiment because it undergoes adaptation during the course of serial passages. In the present study, we found that passaged B. pseudomallei fresh clinical isolates and reference strain in Luria-Bertani broth exhibited increased plaque formation, invasion, intracellular replication, Galleria mellonella killing abilities, and cytokine production of host cells. These bacteria also modulated proteomic profiles during in vitro passage. We presume that the modulation of protein expression during in vitro passage caused changes in virulence and immunogenicity phenotypes. Therefore, we emphasize the need for caution regarding the use of data from passaged B. pseudomallei. These findings of phenotypic adaptation during in vitro serial passage can help researchers working on B. pseudomallei and on other species to better understand disparate findings among strains that have been reported for many years.