Development of whey protein beverage incorporating encapsulated probiotic strain Lactiplantibacillus rhamnosus NCDC 347 and its physico-chemical characteristics.

In the present study, encapsulated strain Lactiplantibacillus rhamnosus NCDC 347 was used to prepare a novel whey protein-based beverage. The encapsulation process utilized skimmed milk powder matrix and evaluated strain viability, physico-chemical properties, sensory assessment, and shelf-life stability. Encapsulated L. rhamnosus NCDC 347 within skim milk powder maintained viability at 8.0 log CFU/g, forming spherical microcapsules with 1-12 µm concavities. Probiotic addition to whey protein beverages maintained pH and acidity within desired ranges. Physico-chemical analysis showed protein content of 8.71 ± 0.21 % to 10.05 ± 0.42 %, fat content of 0.56 ± 0.24 % to 0.67 ± 0.13 %, viscosity of 5.14 pa/s, and total soluble solids (TSS) of 14.42 ± 0.31 to 16.16 ± 0.23° Brix. The shelf-life study revealed that the beverage remained stable for up to 90 days with no significant changes (p > 0.05) in sensory analysis. The sensory analysis scored the test sample's acceptability at 7.3 ± 0.41. The protein-rich probiotic drink exhibited favorable sensory qualities. Overall, incorporating encapsulated probiotic strain L. rhamnosus NCDC 347 into whey protein beverages could address daily protein requirements and enhance health.

Prevalence of antibiotic resistance in lactic acid bacteria isolated from traditional fermented Indian food products.

The emergence of antimicrobial resistance (AMR) in lactic acid bacteria (LAB) raises questions on qualified presumptive safety status and poses challenge of AMR transmission in food milieu. This study focuses on isolation, identification and characterization of AMR in LAB prevalent in traditional fermented Indian food products. The analysis of 16SrRNA based phylogenetic tree showed placements of isolates among four different genera Lactobacillus, Enterococcus, Weissella and Leuconostoc. In E-strip gradient test of susceptibility to 14 different antibiotics, over 50% of isolates showed resistance to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, kanamycin, linezolid, streptomycin, trimethoprim and vancomycin. A multivariate principal component analysis, an antibiogram and multiple antibiotic resistance index-values (> 0.2) indicated presence of multidrug-resistance among the isolates. This study reports prevalence of an alarmingly high rate of AMR LAB strains in traditional fermented foods and is important to regulators and public health authorities for developing strategies to control transmission in food systems. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01305-1.

Complete genome sequence of Lactiplantibacillus plantarum BBC32B isolated from human feces sample.

We report complete genome sequence of Lactiplantibacillus plantarum BBC32B, which was isolated from human feces sample and submitted to Microbial-Type Culture Collection (MTCC), India with deposition number MTCC 25432. The bacteria from Lactobacillaceae family contained 3,411,152 bp; 3,425 protein coding genes, sharing 69.67% average nucleotide identity with closest species of Lactobacillus brevis ATCC367.

Predictive Modeling of Riboflavin Production in Lactiplantibacillus plantarum MTCC 25432 Using Fuzzy Inference System.

Riboflavin (Vitamin B2) is an essential vitamin and a microbial metabolite produced by some lactic acid bacteria (LAB). This investigation aims to study the overproduction of riboflavin in selected Lactiplantibacillus plantarum strain by using the one factor at a time (OFAT) tool coupled with the Fuzzy Inference System (FIS) and its validation through fermentative production in semi-defined media. Out of three Lactiplantibacillus strains used in this study, the maximum riboflavin producing strain was selected based on its ability to grow and produce higher levels of riboflavin. In results, Lactiplantibacillus plantarum strain MTCC 25432 was able to produce 346 µg/L riboflavin in riboflavin deficient assay medium and was investigated further. By using the OFAT-fuzzy FIS system, casamino acid in the range of 5-20 g/L, GTP 0.01-0.04 g/L, sodium acetate 5-15 g/L, and glycine 5-15 g/L were used to predict their effect on riboflavin production. The conditions optimized with modeling showed a 24% increment in riboflavin production (429 µg/L) by Lactiplantibacillus plantarum MTCC 25432 vis-a-vis the unoptimized counterpart (346 µg/L). In conclusion, an FIS-based predictive model was effectively implemented to estimate the riboflavin within an acceptable limit of 3.4%. Riboflavin production enhancing effects observed with various levels of sodium acetate, casamino acid, and GTP could be useful to re-design matrices for riboflavin production.

Microencapsulation of riboflavin-producing Lactiplantibacillus Plantarum MTCC 25,432 and Evaluation of its Survival in Simulated Gastric and Intestinal Fluid.

Microencapsulation is an optimistic method for the delivery of live microbial cells through different food products. In this study, riboflavin-producing probiotic strain Lactiplantibacillus plantarum MTCC 25,432 was encapsulated using a spray drying technique with different wall materials including Inulin, maltodextrin (MD), and MD + Inulin (1:1). The obtained spray dried powder was investigated for probiotic viability, encapsulation efficiency, particle size, water activity, moisture content, hygroscopicity, bulk and tapped densities, storage stabilities, Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA). Besides this, the viability of the free and encapsulated probiotic cells was tested under simulated gastric and intestinal fluid conditions. In the results, microcapsules produced with the combination of MD + Inulin showed higher dry powder yield (36.5%) and viability of L. plantarum MTCC 25,432 (7.4 log CFU / g) as compared with individual coating materials. Further characterization revealed that MD + Inulin microcapsules are spherical (3.50 ± 1.61 µm in diameter) in shape with concavities, showed the highest encapsulation efficiency (82%), low water activity (0.307), moisture content (3.67%) and good survival ability at low pH (pH 2.0 and 3.0), high bile salt concentrations (1.0% and 2.0%), and long storage conditions. No differences in FTIR spectra were observed among the tested samples. However, TGA showed enhanced thermal stability of probiotic-loaded microcapsules when MD + Inulin was used together. In conclusion, MD + Inulin could be a potential encapsulation material for riboflavin-producing probiotic bacteria L. plantarum MTCC 25,432.

Techno-Functional Assessment of Riboflavin-Enriched Yogurt-Based Fermented Milk Prepared by Supplementing Riboflavin-Producing Probiotic Strains of Lactiplantibacillus plantarum.

Vitamin enrichment in fermented dairy products through the intervention of vitamin-producing probiotic strains during fermentation is a novel approach in the field of probioceuticals. In this study, riboflavin-enriched yogurt-based fermented milk was prepared by mixing 1% (v/v) riboflavin-producing strain [1.2 × 108 CFU/mL of Lactiplantibacillus plantarum MTCC 25432 or L. plantarum MTCC 25433 or L. plantarum MTCC 25434] with 2% (v/v) traditional yogurt cultures [Streptococcus thermophilus NCDC 295 and L. delbrueckii subsp. bulgaricus NCDC 293; each of 1.3 × 107 CFU/mL]. The yogurt-based fermented milk prepared with traditional yogurt cultures (2%, v/v) was served as a control. The prepared yogurt-based fermented milk samples were analyzed and compared for riboflavin content, antimicrobial activity, physicochemical, and functional properties. As a result, the yogurt-based fermented milk prepared with L. plantarum MTCC 25432 produced a significantly higher amount of riboflavin (2.49 mg/L) as compared with MTCC 25433 (2.33 mg/L), MTCC 25434 (2.14 mg/L), and control (1.70 mg/L). The probiotic supplementation to yogurt cultures maintained the pH and titratable acidity in the range of 4.1-4.4 and 1.0-1.05% (lactic acid/100 mL), as recommended by Indian yogurt standards. The rheological, texture, and antimicrobial properties of yogurt-based fermented milk were enhanced with the addition of riboflavin-producing probiotic strains. Moreover, all yogurt-based fermented milk samples prepared in this study were acceptable as per the sensory evolution scores. In conclusion, the use of riboflavin-producing L. plantarum strains along with standard yogurt cultures could be the best approach to enhancing riboflavin content in yogurt-based fermented milk and fulfilling the daily riboflavin requirement in humans.

Phenotypic and genotypic characterization of multi-drug resistant, biofilm forming, human invasive strain of Salmonella Typhimurium SMC25 isolated from poultry meat in India.

Emergence of highly virulent and multi-drug resistant (MDR) strains of Salmonella in food products significantly impacts public health and demands continuous monitoring for their presence in the food chain. The ability of Salmonella to form biofilms under harsh environmental conditions accompanied by MDR serotypes underscores an important food safety threat. This study aimed to isolate, identify and characterize MDR, biofilm-forming Salmonella from local Indian dairy and meat products (n = 60). All of the 24 isolates of Salmonella produced biofilm and were categorized as strong (16.6%), moderate (58.3%), and weak (25%) biofilm producers. Multiple antimicrobial resistance (MAR) index of all the Salmonella isolates was ≥0.2. The strongest biofilm forming poultry meat isolate, Salmonella SMC25 demonstrated intermediate to complete resistance to 14 of 22 different antibiotics tested. Epifluorescence microscopy showed that biofilm formation initiated as early as 4 h, reaching zenith within 96 h and much denser and robust biofilm is formed on rough stainless steel (SS316) surface compared to smooth glass surface. The results corroborated with increased temporal production of exopolysaccharides (EPS), high cell surface hydrophobicity and upregulation of marker genes vital to biofilm-formation in Salmonella. Significantly, SMC25 was found to adhere and invade mammalian cell lines Caco2 and HepG2, thus posing a serious food safety threat. This study is important in comprehending the prevalence of multidrug resistant, biofilm-forming, invasive strains of foodborne Salmonella in Indian food products and is important for effective risk assessment besides ensuring better food safety and public health.

Amelioration for oxidative stability and bioavailability of N-3 PUFA enriched microalgae oil: an overview.

Technological improvements in dietary supplements and nutraceuticals have highlighted the significance of bioactive molecules in a healthy lifestyle. Eicosapentaenoic acid and Cervonic acid (DHA), omega-3 polyunsaturated fatty acids seem to be famed for their ability to prevent diverse physiological abnormalities. Selection of appropriate pretreatments and extraction techniques for extraction of lipids from robust microalgae cell wall are very important to retain their stability and bioactivity. Therefore, extraction techniques with optimized extraction parameters offer an excellent approach for obtaining quality oil with a high yield. Oils enriched in omega-3 are particularly imperiled to oxidation which ultimately affects customer acceptance. Bio active encapsulation could be one of the effective approaches to overcome this dilemma. This review paper aims to give insight into the cultivation methods, and downstream processes, various lipid extraction approaches, techniques for retaining oxidative stability, bioavailability and food applications based on extracted or encapsulated omega-3.

Nutritional, phytochemical composition and potential health benefits of taro (Colocasia esculenta L.) leaves: A review.

Colocasia esculenta(L) or taro is a tropical crop largely produced for its tubers (corms) while leaves and stems remain underutilized and untapped by-products with promising potential applications.Colocasialeaves are low in calories, rich in proteins, dietary fiber, and micronutrients. However, its utilization as food remains limited owing to the lack of awareness vis-à-vis its nutritional profile and the presence of antinutrients such as tannins, phytates and oxalates. The antinutritional factors can be overcome by cooking and varied processing techniques thereby unveiling the nutritional benefits. The high content of bioactive compounds and antioxidative potential of colocasia leaves renders several health benefits such as anticancer, antidiabetic, anti-inflammatory activity. The paper reviews the available literature on the nutritional, antinutritional, phytochemical profile of taro leaves and the advanced analytical techniques for their identification and quantification. Further, its health benefits and food applications have been discussed.

Anti-mycobacterial activity of heat and pH stable high molecular weight protein(s) secreted by a bacterial laboratory contaminant.

BACKGROUND: Tuberculosis currently stands as the second leading cause of deaths worldwide due to single  infectious agent after Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The current challenges of drug resistance in tuberculosis highlight an urgent need to develop newer anti-mycobacterial compounds. In the present study, we report the serendipitous discovery of a bacterial laboratory contaminant (LC-1) exhibiting a zone of growth inhibition on an agar plate seeded with Mycobacterium tuberculosis. RESULTS: We utilized microbiological, biochemical and biophysical approaches to characterize LC-1 and anti-mycobacterial compound(s) in its secretome. Based on 16S rRNA sequencing and BIOLOG analysis, LC-1 was identified as Staphylococcus hominis, a human bacterial commensal. Anti-mycobacterial activity was initially found in 30 kDa retentate that was obtained by ultrafiltration of culture filtrate (CF). SDS-PAGE analysis of peak fractions obtained by size exclusion chromatography of 30 kDa retentate confirmed the presence of high molecular weight (≥ 30 kDa) proteins. Peak fraction-1 (F-1) exhibited inhibitory activity against M. bovis BCG, but not against M. smegmatis, E. coli and S. aureus. The active fraction F-1 was inactivated by treatment with Proteinase K and α-chymotrypsin. However, it retained its anti-mycobacterial activity over a wide range of heat and pH treatment. The anti-mycobacterial activity of F-1 was found to be maintained even after a long storage (~12 months) at - 20 °C. Mass spectrometry analysis revealed that the identified peptide masses do not match with any previously known bacteriocins. CONCLUSIONS: The present study highlights the anti-mycobacterial activity of high molecular weight protein(s) present in culture filtrate of LC-1, which may be tested further to target M. tuberculosis. The heat and pH stability of these proteins add to their characteristics as therapeutic proteins and may contribute to their long shelf life. LC-1 being a human commensal can be tested in future for its potential as a probiotic to treat tuberculosis.

Molecular docking, synthesis and anticancer activity of thiosemicarbazone derivatives against MCF-7 human breast cancer cell line.

BACKGROUND: The aim of this study was to synthesize and evaluate anticancer activity of 2-hydroxy benzaldehyde and 4-hydroxy benzaldehyde thiosemicarbazone (2-HBTSc and 4-HBTSc) against MCF-7 breast cancer cell line. MATERIALS AND METHODS: The ligands were prepared and characterized by UV vis, IR and NMR. MTT assay was used to assess viability of cells. RNA isolation, extraction and cDNA synthesis were done. Then all groups were subjected to RT-qPCR using Gene expression specific primers. Also, western blot protein expression and molecular docking were done. Two-way ANOVA with Tukey post-hoc test was employed to test the significance using GraphPad Prism. RESULTS: The IC50 values were 3.36µg/ml and 3.60µg/ml for 2-HBTSc and 4-HBTSc treated MCF-7 tumor cells respectively. Tumor cell growth inhibition ranged from 38 to 49.27% in 4-HBTSc treated cells, and 19 to 25% in 2-HBTSc treated cells with increase in doses 5 µg/ml to 20 µg/ml. The protein and gene expression result showed a significant upregulation in tumor suppressor and apoptosis inducing genes while, oncogene activity was significantly downregulated. Specifically, BRCA2 and pRB gene showed the highest expression in 4-HBTSc and 2-HBTSc treated cells respectively. Conversely, RAS oncogene was downregulated significantly. Docking result showed that both 2-HBTSc and 4-HBTSc have the potential to inhibit Estrogen Receptor Alpha Ligand Binding Domain, Human 17-Beta-hydroxysteroid dehydrogenase type 1 mutant protein and Human Topoisomerase II alpha that are expressed more during Breast Cancer. CONCLUSION: The findings of this study imply that the test compound has potential for further study.

Phenotypic and genotypic characterization of biofilm forming, antimicrobial resistant, pathogenic Escherichia coli isolated from Indian dairy and meat products.

Escherichia coli are commensal gastrointestinal microflora of humans, but few strains may cause food-borne diseases. Present study aimed to identify antimicrobial resistant (AMR), biofilm-forming E. coli from Indian dairy and meat products. A total of 32 E. coli isolates were identified and evaluated for biofilm-formation. EMC17, an E. coli isolate was established as a powerful biofilm-former that attained maximum biofilm-formation within 96 h on glass and stainless-steel surfaces. Presence and expression of virulence-associated genes (adhesins, invasins and polysaccharides) and ability to adhere and invade human liver carcinoma HepG2 cell lines implicates EMC17 to be pathotype belonging to Extra-intestinal Pathogenic E. coli (ExPEC). Antibiotic profiling of EMC17 identified it as multi-drug resistant (MDR) strain, possessing extended spectrum ß-lactamases (ESBL's) and biofilm phenotype. Early production of quorum sensing molecules (AHLs) alongside EPS production facilitated early onset of biofilm formation by EMC17. Furthermore, the biofilm-forming genes of EMC17 were significantly upregulated 3-27 folds in the biofilm-state. This study showed prevalence of MDR, biofilm-forming, pathogenic E. coli in Indian dairy and meat products that potentially serve as reservoirs for transmission of antimicrobial-resistant (AMR) genes of bacteria from food to humans and pose serious food safety threat.

In vitro and in situ abrogation of biofilm formation in E. coli by vitamin C through ROS generation, disruption of quorum sensing and exopolysaccharide production.

Emergence of antimicrobial drug-resistance amongst food-borne pathogens has led to severe deficit of available therapeutics and requires novel interventions. This study determined the activity of vitamin C (VitC), a natural antioxidant as powerful antibacterial agent against multidrug-resistant (MDR), biofilm-forming E. coli. Our findings revealed that VitC wield antibacterial action in dose-time dependent manner with minimum inhibitory concentration (MIC) of 125 mM. At these concentrations VitC impaired quorum sensing (QS) and exopolysaccharide (EPS) production and induced sugar and protein leakage from the bacterial cells by virtue of reactive oxygen species (ROS) generation. Furthermore, VitC-treated bacteria showed downregulation of genes underpinning biofilm signaling (luxS) and regulation (bssR) by up to 27-folds. Finally, this study demonstrated the promising antimicrobial application of VitC, in situ, in Indian soft cheese (paneer) when applied as a coating. Therefore, VitC can be applied as natural and safe 'antimicrobial' against biofilm-forming bacteria in food systems vis-à-vis other conventional antimicrobials.

Zinc-Supported Multiwalled Carbon Nanotube Nanocomposite: A Synergism to Micronutrient Release and a Smart Distributor To Promote the Growth of Onion Seeds in Arid Conditions.

In the current scenario, nanotechnological applications in the agriculture sector showing potential impacts on the improvement of plant growth in terms of protection and safety are at a very nascent stage. The present study deals with the synergistic role of zinc (Zn) and multiwalled carbon nanotubes (MWCNTs) synthesized as a zinc oxide (ZnO)/MWCNT nanocomposite, a prospective applicant to modulate the micronutrient supply and enhance the growth of onion seeds, thereby replacing harmful, unsafe chemical fertilizers. To the best of our knowledge, this is the first report wherein MWCNTs have been envisaged as a micronutrient distributor and a nutrient stabilizer enhancing the growth of onion plant under arid conditions. The growth trend of onion seeds was evaluated in an aqueous medium with varied concentrations of (i) MWCNTs, (ii) zinc oxide nanoparticles, and (iii) ZnO/MWCNT nanocomposites. ZnO/MWCNT nanocomposites with 15 µg/mL concentration displayed the best seedling growth with the maximum number of cells in telophase. A significant growth trend with increased concentration of ZnO/MWCNTs displayed no negative impact on plant growth in contrast to that with the use of MWCNTs. The synergistic impact of Zn nanoparticles and MWCNTs in ZnO/MWCNT nanocomposites on the rate of germination was explained via a mechanism supported by scanning transmission electron microscopy.

The pleiotropic transcriptional response of Mycobacterium tuberculosis to vitamin C is robust and overlaps with the bacterial response to multiple intracellular stresses.

Mycobacterium tuberculosis (Mtb) owes its success as a pathogen in large measure to its ability to exist in a persistent state of 'dormancy' resulting in a lifelong latent tuberculosis (TB) infection. An understanding of bacterial adaptation during dormancy will help in devising approaches to counter latent TB infection. In vitro models have provided valuable insights into bacterial adaptation; however, they have limitations because they do not disclose the bacterial response to the intracellular environment wherein the bacteria are simultaneously exposed to multiple stresses. We describe the pleiotropic response of Mtb in the vitamin C (vit C) model of dormancy developed in our laboratory. Vit C mediates a rapid regulation of genes representing ~14 % of the genome in Mtb cultures. The upregulated genes were better represented in lipid, intermediary metabolism and regulatory protein categories. The downregulated genes mainly related to virulence, detoxification, information pathways and cell wall processes. A comparison of this response to that in other models indicates that vit C generates a multiple-stress environment for axenic Mtb cultures that resembles a macrophage-like environment. The bacterial response to vit C resembles responses to gaseous stresses such as hypoxia and nitric oxide, oxidative and nitrosative stresses, nutrient starvation and, notably, the activated macrophage environment itself. These responses demonstrate that the influence of vit C on Mtb gene expression extends well beyond the DevR dormancy regulon. A detailed characterization of the response to vit C is expected to disclose useful strategies to counter the adaptive mechanisms essential to Mtb dormancy.

DevR (DosR) mimetic peptides impair transcriptional regulation and survival of Mycobacterium tuberculosis under hypoxia by inhibiting the autokinase activity of DevS sensor kinase.

BACKGROUND: Two-component systems have emerged as compelling targets for antibacterial drug design for a number of reasons including the distinct histidine phosphorylation property of their constituent sensor kinases. The DevR-DevS/DosT two component system of Mycobacterium tuberculosis (M. tb) is essential for survival under hypoxia, a stress associated with dormancy development in vivo. In the present study a combinatorial peptide phage display library was screened for DevS histidine kinase interacting peptides with the aim of isolating inhibitors of DevR-DevS signaling. RESULTS: DevS binding peptides were identified from a phage display library after three rounds of panning using DevS as bait. The peptides showed sequence similarity with conserved residues in the N-terminal domain of DevR and suggested that they may represent interacting surfaces between DevS and DevR. Two DevR mimetic peptides were found to specifically inhibit DevR-dependent transcriptional activity and restrict the hypoxic survival of M. tb. The mechanism of peptide action is majorly attributed to an inhibition of DevS autokinase activity. CONCLUSIONS: These findings demonstrate that DevR mimetic peptides impede DevS activation and that intercepting DevS activation at an early step in the signaling cascade impairs M. tb survival in a hypoxia persistence model.

D-alanine modification of a protease-susceptible outer membrane component by the Bordetella pertussis dra locus promotes resistance to antimicrobial peptides and polymorphonuclear leukocyte-mediated killing.

Bordetella pertussis is the causative agent of pertussis, a highly contagious disease of the human respiratory tract. Despite very high vaccine coverage, pertussis has reemerged as a serious threat in the United States and many developing countries. Thus, it is important to pursue research to discover unknown pathogenic mechanisms of B. pertussis. We have investigated a previously uncharacterized locus in B. pertussis, the dra locus, which is homologous to the dlt operons of Gram-positive bacteria. The absence of the dra locus resulted in increased sensitivity to the killing action of antimicrobial peptides (AMPs) and human phagocytes. Compared to the wild-type cells, the mutant cells bound higher levels of cationic proteins and peptides, suggesting that dra contributes to AMP resistance by decreasing the electronegativity of the cell surface. The presence of dra led to the incorporation of d-alanine into an outer membrane component that is susceptible to proteinase K cleavage. We conclude that dra encodes a virulence-associated determinant and contributes to the immune resistance of B. pertussis. With these findings, we have identified a new mechanism of surface modification in B. pertussis which may also be relevant in other Gram-negative pathogens.

Mycobacterium tuberculosis transcriptional adaptation, growth arrest and dormancy phenotype development is triggered by vitamin C.

BACKGROUND: Tubercle bacilli are thought to persist in a dormant state during latent tuberculosis (TB) infection. Although little is known about the host factors that induce and maintain Mycobacterium tuberculosis (M. tb) within latent lesions, O(2) depletion, nutrient limitation and acidification are some of the stresses implicated in bacterial dormancy development/growth arrest. Adaptation to hypoxia and exposure to NO/CO is implemented through the DevRS/DosT two-component system which induces the dormancy regulon. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that vitamin C (ascorbic acid/AA) can serve as an additional signal to induce the DevR regulon. Physiological levels of AA scavenge O(2) and rapidly induce the DevR regulon at an estimated O(2) saturation of <30%. The kinetics and magnitude of the response suggests an initial involvement of DosT and a sustained DevS-mediated response during bacterial adaptation to increasing hypoxia. In addition to inducing DevR regulon mechanisms, vitamin C induces the expression of selected genes previously shown to be responsive to low pH and oxidative stress, triggers bacterial growth arrest and promotes dormancy phenotype development in M. tb grown in axenic culture and intracellularly in THP-1 cells. CONCLUSIONS/SIGNIFICANCE: Vitamin C mimics multiple intracellular stresses and has wide-ranging regulatory effects on gene expression and physiology of M. tb which leads to growth arrest and a 'dormant' drug-tolerant phenotype, but in a manner independent of the DevRS/DosT system. The 'AA-dormancy infection model' offers a potential alternative to other models of non-replicating persistence of M. tb and may be useful for investigating host-'dormant' M. tb interactions. Our findings offer a new perspective on the role of nutritional factors in TB and suggest a possible role for vitamin C in TB.

Co-expression of DevR and DevR(N)-Aph proteins is associated with hypoxic adaptation defect and virulence attenuation of Mycobacterium tuberculosis.

BACKGROUND: The DevR response regulator is implicated in both hypoxic adaptation and virulence of Mycobacterium tuberculosis (M. tb). DevR regulon genes are powerfully induced in vivo implicating them in bacterial adaptation to host control strategies. A better understanding of DevR function will illumine the way for new strategies to control and treat tuberculosis. METHODOLOGY/PRINCIPAL FINDINGS: Towards this objective, we used a combination of genetic, microbiological, biochemical, cell biological tools and a guinea pig virulence assay to compare the hypoxic adaptation and virulence properties of two novel M. tb strains, namely, a devR disruption mutant, Mut1, that expresses C-terminal truncated N-terminal domain of DevR (DevR(NTD)) as a fusion protein with AphI (DevR(N)-Kan), and its complemented strain, Comp1, that expresses intact DevR along with DevR(N)-Kan. Comp1 bacteria exhibit a defect in DevR-mediated phosphosignalling, hypoxic induction of HspX and also hypoxic survival. In addition, we find that Comp1 is attenuated in virulence in guinea pigs and shows decreased infectivity of THP-1 cells. While Mut1 bacilli are also defective in hypoxic adaptation and early growth in spleen, they exhibit an overall virulence comparable to that of wild-type bacteria. CONCLUSIONS/SIGNIFICANCE: The hypoxic defect of Comp1 is associated to a defect in DevR expression level. The demonstrated repression of DevR function by DevR(N)-Kan suggests that such a knockdown approach could be useful for evaluating the activity of DevRS and other two-component signaling pathways. Further investigation is necessary to elucidate the mechanism underlying Comp1 attenuation.

Resazurin reduction assays for screening of anti-tubercular compounds against dormant and actively growing Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis.

OBJECTIVES: To develop simple, rapid, low-cost and robust assays for screening drugs against dormant and actively growing mycobacteria. METHODS: Actively growing aerobic and hypoxia-adapted dormant cultures of Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis were tested for susceptibility to standard antimicrobial drugs by resazurin reduction assay. The visual and fluorimetric MICs were compared with those obtained by the standard cfu assay. RESULTS: Drug MICs for M. tuberculosis and M. bovis BCG were determined by the aerobic resazurin microplate assay (REMA) and correlated well with those obtained by the cfu assay. Metronidazole and nitrofurans showed comparable bactericidal activity in the hypoxic resazurin reduction assay (HyRRA). The HyRRA assay was noted to be superior to the cfu assay in that it distinguished between metabolically active dormant bacteria and non-viable organisms, unlike the cfu assay that could not differentiate between these two populations. The HyRRA assay performed with good concordance in both fluorimetric and visual formats to distinguish between bactericidal and bacteriostatic effects of a drug. CONCLUSIONS: The REMA and HyRRA assays will be useful for anti-tubercular anti-dormancy compound screening and drug susceptibility testing in a safe, reliable, easy and cost-effective manner particularly in low resource countries. The application of the assays in M. smegmatis or M. bovis BCG offers the distinct advantage of rapidly and safely screening anti-tubercular compounds in a high-throughput format.