Log Phase Of Growth Research Articles

Microbial Growth as Determinant of Antibiotic Production with Biotic Elicitors Stimulation

An antibiotic-resistant and multidrug-resistant (MDR) issue open the role of researchers to continue to search for natural potential as a source of new antimicrobials. One of the potential fungi isolates that can produce antimicrobial active compounds from Indonesian tropical peat soils is Penicillium sp. LBKURCC34. In this study, the production of antimicrobial compounds from local isolates was carried out by batch fermentation method in liquid media with the addition of biotic elicitors to increase the extraction activity and yield. This study aims to optimize the results based on the time the elicitor is added. Staphylococcus aureus was used as a biotic elicitor, which was added on days 2, 3, and 4 in the production of antibiotics by fermentation incubation of 6-14 days. The antibiotic production media was extracted with ethyl acetate and evaporated. The antimicrobial test was carried out by the disk diffusion method against pathogenic bacteria Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Staphylococcus epidermidis using three crude extract contents (19; 38; and 57 µg/disc). Amoxicillin® was used as a positive control (10 µL/disc). The results showed that the addition of S. aureus biotic elicitor extended the log phase growth of the fungus Penicillium sp. LBKURCC34. The optimum condition of production was obtained by adding initiator treatment on the 3rd day for 14 days incubation with the highest yield and could inhibit the growth of all pathogenic microbes.

Salinity, pH and temperature growth ranges of Halophytophthora isolates suggest their physiological adaptations to mangrove environments

ABSTRACT Species of Halophytophthora are early colonisers of fallen mangrove leaves in the tropics but recently found commonly in temperate areas. In mangrove habitats, temperature and salinity change rapidly daily (high/low tide) and seasonally (summer/winter, rainy/dry seasons). Mangrove organisms have to develop adaptive strategies to thrive in such a physiologically challenging environment. In this study, growth of three isolates of Halophytophthora avicenniae and two isolates of H. batemanensis was tested under combined effects of 3 temperatures (15°C, 25°C, 37°C), 3 pHs (6, 7, 8) and 4 salinities (4 ‰, 8 ‰, 16 ‰, 32 ‰). No/little growth was observed at 37°C and growth saturation occurred earlier at 25°C than at 15°C. The log phase of growth was steeper at pH 6 than pH 7 and 8. Temperature and pH were found to exert a greater effect on growth than salinity. Generally, a reduction of growth rate was observed at pH 8 and 15°C. Increase in salinity caused a slight decrease in growth, most noticeable at 32 ‰. The wide growth ranges of temperature, salinity and pH of Halophytophthora isolates suggest that they are well adapted to the physical and chemical conditions of mangrove habitats.

Psychrotolerant Antarctic bacteria biosynthesize gold nanoparticles active against sulphate reducing bacteria

This study evaluates the biosynthesis of gold nanoparticle (GNP) using Antarctic bacteria and assesses its potential antibacterial activity on sulfate-reducing bacteria (SRB). The GNPs were biosynthesized at distinct temperatures (4°, 10°, 25°, 30° and 37° C) using bacterial isolate GL1.3, obtained from Antarctic lake water. Biochemical and phylogenetic analysis concluded that the isolate GL1.3 belongs to Bacillus sp. The GNP biosynthesis was achieved at all the incubation temperatures (4°, 10°, 25°, 30° and 37° C) only during the log phase of growth. These formed nanoparticles were identified by UV–Visible spectroscopy, scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), transmission electron microscopy (TEM) and X-ray diffraction (XRD) to be of size 30–50 nm. These GNPs exhibited antibacterial activity against SRB (Desulfovibrio sp.) evaluated by broth micro-dilution method. At 200 µg mL−1 GNP concentrations, being the minimal inhibitory concentration (MIC), the growth rate and sulfate reducing activity of Desulfovibrio sp. were reduced by 12% and 7% respectively. Comet assay revealed that the genotoxic effect of GNP on SRB is responsible for the inhibition of its growth and sulfide production. This showed that the Antarctic microbes could be useful for GNP synthesis even under psychrophilic conditions for various biomedical applications.

Thrombin: A Driver or Attenuator of Bone Disease on Hemophilia?

Introduction: Hemophilic bone disease is a pathology inherent to factor VIII (FVIII) and factor IX (FIX) deficiency present in both adult and pediatric persons with hemophilia (PWH). This pathology is also present in mouse models. We have previously demonstrated that FVIII replacement restores lost mineralization activity in osteoblast-like cells from FVIII-deficient mice. We hypothesized that bone disease in PWH is secondary to decreased thrombin generation in PWH. Methods: Murine bone marrow stromal cells isolated from FVIII knockout (FVIII-/-) and wild-type (FVIII+/+) C57BL mice and human osteosarcoma (HOS) TE85 cells were cultured in α-MEM with 0-10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 25 µg/mL Amphotericin B. Ten millimolar ß-glycerophosphate and 50 µg/mL ascorbic acid were added to induce differentiation and mineralization. Mineralized deposits were stained with alizarin red. Alkaline phosphatase (ALP) activity and culture protein content were measured using colorimetric assays. Results: In , the addition of thrombin in serum-free conditions increases cell proliferation ~25% (p=5x10-4, ANCOVA) and decreases mineralization up to 84% (p=1x10-6) (Figure 1). Thrombin's effect on ALP activity in HOS TE85 cells is negative at low cell numbers and positive at high cell numbers (Figure 1). Osteoblast-like cells from FVIII-/- mice mineralize 70% less than those from FVIII+/+ cells (p=3x10-6). Administering FVIII to cells from FVIII-/- mice ameliorates the mineralization deficiency (Figure 1), while mineralization in cells from FVIII+/+ mice does not change (p=0.66). FVIII-/- cells express 30% lower levels of ALP activity than FVIII+/+ (p=3x10-4). FVIII+/+ cells proliferate 26% more rapidly than FVIII-/- cells (p=4x10-7, ANCOVA), but this is not affected by the addition of FVIII in vitro (p=0.45). The addition of FVIII causes a ~33% decrease in ALP activity in osteoblast-like cells from both FVIII-/- (p=2x10-4) and FVIII+/+ (p=5x10-5) mice. Although thrombin impacts osteoblast differentiation and function, it has paradoxical effects on their mineralization, suggesting thrombin deficiency is not the sole driver of reduced bone health in PWH. Deficient mineralization in cells from FVIII-/- mice compared to those from FVIII+/+ mice is corrected with FVIII replacement in vivo or in vitro, suggesting that circulating FVIII has a positive role in these cells' mineralization. More rapid proliferation in cells from FVIII+/+ mice likely contributes to the increase in mineralization, yet, as proliferation is unchanged by FVIII replacement, it appears unlikely that this effect occurs via the same mechanism. Thrombin causes increased proliferation of HOS TE85 cells, yet also causes a significant decline in mineralization activity (Figure 1). Whether this is a paradoxical effect present only in this transformed cell line is yet undetermined. ALP activity increases when HOS TE85 cells are given thrombin in vivo during the log phase of growth (Figure 1), indicating a positive effect on the osteoblast-like function of the cells. However, this notion is undermined by the decrease in mineralization. Identifying the role of FVIII, FIX, thrombin, and active protein C in bone mineralization in PWH is proving to be a challenging undertaking, yet it remains critical, especially as new drugs with the potential to supplant FVIII prophylaxis hit the market. Future experiments will include thrombin and a thrombin inhibitor, a PAR1 agonist and antagonist, a PAR2 antagonist, and an APC neutralizing antibody. Upcoming findings will be discussed. Disclosures Taylor: Baxalta: Research Funding; CTI BioPharma: Employment, Equity Ownership.

Methylsulfonylmethane exhibits bacteriostatic inhibition of Escherichia coli, and Salmonella enterica Kinshasa, in vitro.

To evaluate antibacterial properties of methylsulfonylmethane (MSM) on Escherichia coli (MDRE21) and Salmonella enterica serovar Kinshasa (SK132). Bacterial proliferation analysis was measured spectrophotometrically during log phase growth with 0, 3, 5, 7, 10, 12 and 16% MSM. To assess the mechanism of inhibition, cultures were grown overnight with 0-16% MSM and enumerated on unmedicated brain-heart infusion agar (BHIA) or BHIA with 0-16% MSM. The long-term viability studies were done to evaluate the impact of 10% MSM. Absorbance data indicated a dose-dependent inhibition from 0 to 16% MSM. There was no growth of MDRE21 or SK132 on BHIA in 10-16% MSM. Both strains enumerated on unmedicated BHIA from overnight cultures with 10-16% MSM were able to partially recover. Recovery after MSM removal may be indicative of a bacteriostatic mechanism of inhibition. The long-term viability studies illustrated that neither MDRE21 nor SK132 could be rescued from 10% MSM after 5 or 6days respectively. Methylsulfonylmethane antibacterial activity may prove useful during pre or postharvest food safety as a disinfectant. The primary benefit being, its clinical safety to humans. Comparisons to other disinfectants would also need to be done to determine if MSM was superior to those already on the market and would be cost effective.

Isolation and characterization of bacteriophage to control multidrug-resistant Pseudomonas aeruginosa planktonic cells and biofilm

Pseudomonas aeruginosa is Gram-negative bacterium, one of the leading cause of drug-resistant nosocomial infections in developing countries. This bacterium possesses chromosomally encoded efflux pumps, poor permeability of outer-membrane and high tendency for biofilm formation which are tools to confer resistance. Bacteriophages are regarded as feasible treatment option for control of resistant P. aeruginosa. The aim of the current study was isolate and characterized a bacteriophage against P. aeruginosa with MDR and biofilm ability.A bacteriophage MA-1 with moderate host range was isolated from waste water. The phage was considerable heat and pH stable. Electron microscopy revealed that phage MA-1 belongs to Myoviridae family. Its genome was dsDNA (≈50 kb), coding for eighteen different proteins (ranging from 12 to 250 KDa). P. aeruginosa-2949 log growth phase was significantly reduced by phage MA-1 (2.5 × 103 CFU/ml) as compared to control (without phage). Phage MA-1 also showed significant reductions of 2.0, 2.5 and 3.2 folds in 24, 48, and 74 h old biofilms after 6 h treatment with phage respectively as compared to control. It was concluded from this study that phage MA-1 has capability of killing P. aeruginosa planktonic cells and biofilm, but for complete eradication cocktail will more effective to avoid resistance.

A metabolic study to decipher amino acid catabolism-directed biofuel synthesis in Acetoanaerobium sticklandii DSM 519

Acetoanaerobium sticklandii DSM 519 is a hyper-ammonia-producing anaerobe. It has the ability to produce organic solvents and acids from protein catabolism through Stickland reactions and specialized pathways. Nevertheless, its protein catabolism-directed biofuel production has not yet been understood. The present study aimed to decipher such growth-associated metabolic potential of this organism at different growth phases using metabolic profiling. A seed culture of this organism was grown separately in metabolic assay media supplemented with gelatin and or a mixture of amino acids. The extracellular metabolites produced by this organism were qualitatively analyzed by gas chromatography-mass spectrometry platform. The residual amino acids after protein degradation and amino acids assimilation were identified and quantitatively measured by high-performance liquid chromatography (HPLC). Organic solvents and acids produced by this organism were detected and the quantity of them determined with HPLC. Metabolic profiling data confirmed the presence of amino acid catabolic products including tyramine, cadaverine, methylamine, and putrescine in fermented broth. It also found products including short-chain fatty acids and organic solvents of the Stickland reactions. It reported that amino acids were more appropriate for its growth yield compared to gelatin. Results of quantitative analysis of amino acids indicated that many amino acids either from gelatin or amino acid mixture were catabolised at a log-growth phase. Glycine and proline were poorly consumed in all growth phases. This study revealed that apart from Stickland reactions, a specialized system was established in A. sticklandii for protein catabolism-directed biofuel production. Acetone-butanol-ethanol (ABE), acetic acid, and butyric acid were the most important biofuel components produced by this organism. The production of these components was achieved much more on gelatin than amino acids. Thus, A. sticklandii is suggested herein as a potential organism to produce butyric acid along with ABE from protein-based wastes (gelatin) in bio-energy sectors.

Susceptibility testing of Leishmania spp. against amphotericin B and fluconazole using the Sensititre\u2122 YeastOne\u2122 YO9 platform

BackgroundCurrent drug regimens for cutaneous leishmaniasis (CL) include toxic systemic therapies such as amphotericin B (AB) and pentavalent antimonials. Fluconazole (FZ) is a well-tolerated potential oral alternative for the management CL. To date, few objective data exist to guide clinical decision-making when selecting a therapeutic agent a priori, and standardized, clinically-approved drug susceptibility testing platforms for Leishmania spp. have yet to be established. The Sensititre™ YeastOne™ YO9 plate is a commercialized drug susceptibility plate including AB and FZ used for routine testing of non-fastidious yeast. Our objective was to adapt the readily available Sensititre™ YeastOne™ YO9 plate, to determine drug susceptibility profiles of AB and FZ in cultured isolates of Old World and New World Leishmania spp. for the treatment of CL.MethodsPromastigotes were cultured in Tobie’s medium with Locke’s overlay until log phase growth was achieved, inoculated into the Sensititre™ system, and incubated over 96 H. minimum inhibitory concentrations (MICs) were determined colorimetrically, and promastigote death was assessed by conventional microscopy out to 96- h. Colour change correlated to MIC values.ResultsAll strains tested exhibited MIC values for FZ that were ≥ 256 μg/mL. New World strains demonstrated reduced susceptibility to AB (0.25 μg/mL – 0.50 μg/mL AB) compared to Old World strains at 0.12 μg/mL AB (p = 0.02). Seventeen (61%) of 28 Viannia isolates versus 82% (27/33) of non-Viannia isolates were resistant at 0.12 μg/mL AB (p = 0.09). For L. V. braziliensis isolates, mean MIC for AB was 0.375 ± 0.14 μg/mL (range 0.25–0.50 μg/mL), while for isolates of L. V. panamensis it was 0.314 ± 0.26 μg/mL (range 0.12–1.0 μg/mL).ConclusionsWe adapted the Sensititre™ YeastOne™ YO9 plate for testing of Leishmania spp. susceptibility profiles for commonly used antifungals in the treatment of CL, including AB and FZ. Given its current utility in mycology, optimization of the system for potential clinical implementation in parasitology should be pursued. However evaluation of clinically relevant amastigote-stage stages, and higher concentrations of FZ beyond the upper limit concentration of the Sensititre™ YeastOne™ Y09 plate would be required.

Advancing the development of new tuberculosis treatment regimens: The essential role of translational and clinical pharmacology and microbiology.

Application of clinical pharmacology best practices is essential to the efficient and rational development of drugs. In general, knowledge gained about exposure–response relationships in preclinical models aids drug and dose selection in human studies, and biomarkers and pharmacokinetic (PK) data one collects in early to middle drug development can be used to predict the dose and treatment response of promising therapeutics in definitive phase 3 trials. The essentiality of sound clinical pharmacology in tuberculosis (TB) drug and regimen development is heightened by unique challenges in assessing drugs for this disease—aspects of the organism’s biology, the variability in lung pathology, uncertainties about how to link treatment outcomes seen in preclinical models with those seen in humans (which thwarts preclinical–clinical translational work), the lack of predictive early clinical biomarkers, and the high variability in treatment response across patients and populations (Fig 1). In TB disease, Mycobacterium tuberculosis (M.tb) bacilli are detected in necrotic granulomas, large cavities with liquefied contents, and intracellularly within macrophages. We believe that drugs must access each of these compartments to achieve cure in patients [1]. We also believe that TB drugs and regimens must kill bacilli in different metabolic states, from actively multiplying to semidormant [2,3]. Both in vitro and in vivo preclinical models are leveraged to assess the clinical utility of new TB drugs and drug combinations. These models vary both in their ability to assess efficacy relative to the shifting metabolic states of M.tb infection and in their ability to recapitulate human disease. Still, two models are proving to be highly informative. The mouse model of infection has been invaluable in selecting rank-ordered drug combinations, whereas the now-validated in vitro pharmacodynamic (PD) system (IVPDS, or “hollow fiber model” for TB) has significantly improved our understanding of the PK drivers of treatment response in various growth and physiologic states [4,5]. In the IVPDS, an elaborate system of dialysis-like tubing allows the investigator to reproduce human-like concentration–time curves and see how different PK profiles affect killing of bacilli that are living in the system. Dose-fractionation studies, for example, can be carried out, and one can determine whether a drug’s activity is time dependent or, rather, concentration dependent. Or one can test a drug’s activity when the organism is nutrient starved, in log-phase growth, or intracellular. Whereas these models are informative, there remain gaps in our ability to bridge preclinical and clinical data using modern translational quantitative modeling [6]. There are also gaps in our ability to link surrogate end points in early-phase clinical trials (namely, longitudinally collected sputum cultures) and clinically relevant end points of treatment failure, relapse, and death in later-phase trials [7,8]. The identification of accurate tools that identify those patients who are unlikely to achieve cure with shortened regimens (specifically, patients with a disease phenotype that is “hard to treat”) would have immense value to both clinical trialists and TB clinicians [9]. The TB clinical pharmacology field has the opportunity to apply state-of-the-art quantitative pharmacology tools to bridge preclinical and clinical data more effectively and to enhance learning across the continuum of clinical development [9–13]. In this paper, based on discussions occurring at a WHO workshop held in March 2018, we describe our views on best practices for incorporating translational, PK-PD, and microbiologic assessments into drug development [14]. Open in a separate window Fig 1 Schema of preclinical and clinical pharmacology studies important for TB drug and regimen development. By phase of development, in green are the questions to be addressed, in blue are the tools to use to answer the questions, and in red are the outputs. ADME, absorption, distribution, metabolism, excretion; DDI, drug–drug interaction; Dz, disease; MIC, minimum inhibitory concentration; PBPK, physiologically based PK; PD, pharmacodynamic; Ph2A, phase 2A; Ph2B/C, phase 2B and C; Ph3, phase 3; PK, pharmacokinetic; TB, tuberculosis; y.o., year-olds.

Human COQ10A and COQ10B are distinct lipid-binding START domain proteins required for coenzyme Q function

Coenzyme Q (CoQ or ubiquinone) serves as an essential redox-active lipid in respiratory electron and proton transport during cellular energy metabolism. CoQ also functions as a membrane-localized antioxidant protecting cells against lipid peroxidation. CoQ deficiency is associated with multiple human diseases; CoQ10 supplementation in particular has noted cardioprotective benefits. In Saccharomyces cerevisiae, Coq10, a putative START domain protein, is believed to chaperone CoQ to sites where it functions. Yeast coq10 deletion mutants (coq10Δ) synthesize CoQ inefficiently during log phase growth and are respiratory defective and sensitive to oxidative stress. Humans have two orthologs of yeast COQ10, COQ10A and COQ10B Here, we tested the human co-orthologs for their ability to rescue the yeast mutant. We showed that expression of either human ortholog, COQ10A or COQ10B, rescues yeast coq10Δ mutant phenotypes, restoring the function of respiratory-dependent growth on a nonfermentable carbon source and sensitivity to oxidative stress induced by treatment with PUFAs. These effects indicate a strong functional conservation of Coq10 across different organisms. However, neither COQ10A nor COQ10B restored CoQ biosynthesis when expressed in the yeast coq10Δ mutant. The involvement of yeast Coq10 in CoQ biosynthesis may rely on its interactions with another protein, possibly Coq11, which is not found in humans. Coexpression analyses of yeast COQ10 and human COQ10A and COQ10B provide additional insights to functions of these START domain proteins and their potential roles in other biologic pathways.

Cellular therapy: Adoptive immunotherapy with expanded natural killer cells

Adoptive immunotherapy with natural killer cells was pioneered 30years ago in human clinical trials with the development of cytokine-induced killer cells-unfractionated peripheral blood mononuclear cell (PBMC) populations activated overnight with IL-2. Higher doses were subsequently made possible through the advent of steady-state apheresis, allowing the collection of PBMC numbers equivalent to an entire adult blood volume, and increased purity made feasible through magnetic CD3-depletion and/or CD56-selection methods. Still, these approaches rarely achieved clinical dosing above a single infusion of 108 NK cells/kg, except with substantial donor-recipient size mismatch (eg, parents donating cells to children). To address this shortcoming, leukemia cell lines with NK cell-like function or ex vivo expansion approaches centered on the homeostatic cytokine IL-15 were developed. Here, we describe the development of an ex vivo expansion system based on a feeder cell expressing membrane-bound IL-21 that enables log-phase growth of primary NK cells for many weeks without inducing senescence, and describe the biology, correlative science, and translation to clinical trials for patients with leukemia, brain tumors, and solid tumors.

Comparison of several non-specific skin mucus immune defences in three piscine species of aquaculture interest

Fish skin mucus is a viscous and semipermeable barrier made mainly of water, glycoproteins and soluble proteins. It represents an important defence against the environment and previous studies have reported the presence of different substances involved in immune defence responses in it. The aim of the present work was to characterize skin mucus protease activity by zymography and esterase activity of the subfamily of carboxylesterases in three species of interest for aquaculture: gilthead sea bream, sea bass and meagre. Mucus antioxidant power was also determined by adapting ferric reducing antioxidant power (FRAP) analysis. As a result of these non-specific immune defence parameters, we compared the antibacterial capacity of skin mucus in these species via in vitro dual bacteria strains–skin mucus co-culture growths. We used Pseudomonas anguilliseptica and Vibrio anguillarum as marine pathogenic bacteria and Escherichia coli as non-pathogenic. For each fish species, in the respective zymograms, we determined a pattern of proteolytic digestion bands. A high-molecular-weight band (around 200 kDa; H-band) was evident in sea bream and sea bass, and showed chymotrypsin activity. One or two intermediate-molecular-weight bands (around 75 kDa; I-bands) with non-trypsin and non-chymotrypsin activity, and putatively with metalloprotease activity, were evident in all species. Finally, low-molecular-weight bands (between 14 and 30 kDa; L-bands) showed distinct patterns for each species and matched trypsin activity. Despite the conservative pattern of digestion bands, the levels of total proteolytic activity (TPA) were 5 and 10 times higher in meagre than in sea bass and sea bream, respectively. In parallel, three carboxylesterase activities were detected in the mucus of the three fish species, using myristate (pNPM-CE activity), butyrate (pNPB-CE activity) and acetate (pNPA-CE activity) as substrates. Both pNPB-CE and pNPA-CE were the most abundant in fish mucus, and meagre was again the species with the highest levels. In contrast, the antioxidant power of meagre skin mucus was the lowest. We established the capacity of skin mucus to block or limit bacterial growth (lytic activity) using 24 h growth curves. The log-growth phase of V. anguillarum was strongly blocked by sea bream and meagre mucus for a few hours; but not by sea bass mucus. However, if mucus was not renewed, log-growth was at the end of 24 h studied period. For its part, P. anguilliseptica growth curve was delayed by the three mucus types during the entire growth period. Only meagre achieved lytic activity against E. coli growth. All parameters studied here will be of a great interest as non-invasive bioindicators of non-specific immune defences in fish skin mucus.

Defining the Transcriptional and Post-transcriptional Landscapes of Mycobacterium smegmatis in Aerobic Growth and Hypoxia.

The ability of Mycobacterium tuberculosis to infect, proliferate, and survive during long periods in the human lungs largely depends on the rigorous control of gene expression. Transcriptome-wide analyses are key to understanding gene regulation on a global scale. Here, we combine 5′-end-directed libraries with RNAseq expression libraries to gain insight into the transcriptome organization and post-transcriptional mRNA cleavage landscape in mycobacteria during log phase growth and under hypoxia, a physiologically relevant stress condition. Using the model organism Mycobacterium smegmatis, we identified 6,090 transcription start sites (TSSs) with high confidence during log phase growth, of which 67% were categorized as primary TSSs for annotated genes, and the remaining were classified as internal, antisense, or orphan, according to their genomic context. Interestingly, over 25% of the RNA transcripts lack a leader sequence, and of the coding sequences that do have leaders, 53% lack a strong consensus Shine-Dalgarno site. This indicates that like M. tuberculosis, M. smegmatis can initiate translation through multiple mechanisms. Our approach also allowed us to identify over 3,000 RNA cleavage sites, which occur at a novel sequence motif. To our knowledge, this represents the first report of a transcriptome-wide RNA cleavage site map in mycobacteria. The cleavage sites show a positional bias toward mRNA regulatory regions, highlighting the importance of post-transcriptional regulation in gene expression. We show that in low oxygen, a condition associated with the host environment during infection, mycobacteria change their transcriptomic profiles and endonucleolytic RNA cleavage is markedly reduced, suggesting a mechanistic explanation for previous reports of increased mRNA half-lives in response to stress. In addition, a number of TSSs were triggered in hypoxia, 56 of which contain the binding motif for the sigma factor SigF in their promoter regions. This suggests that SigF makes direct contributions to transcriptomic remodeling in hypoxia-challenged mycobacteria. Taken together, our data provide a foundation for further study of both transcriptional and posttranscriptional regulation in mycobacteria.

Evaluation of Pertussis Toxin Expression in B2 and THIJS Media.

Whole-cell pertussis vaccine (wP) has been imperative and highly effective in preventing childhood deaths due to pertussis. Pertussis toxin is one of the virulence factors of Bordetella pertussis in all available pertussis vaccines. wP production in Razi Vaccine and Serum Research Institute is according to bioreactor culture of B. pertussis strains in B2 medium. The aim of this study was to evaluate B. pertussis strain 509 PT production in B2 and Thalen-IJssel (THIJS) media by Chinese Hamster Ovary (CHO) cell and enzyme-linked immunosorbent assay methods (ELISA). In the current study, B. pertussis strain 509 was cultured in B2 and THIJS media. Six samples were taken during the log growth phase within 2-3 h intervals (triplicate). The growth rate was calculated using opacity and the quantification of cell-associated and released PT measured by ELISA and CHO cell assays. THIJS medium was significantly showed an increase in the bacterial growth rate. During the first 29 h, bacterial concentrations in B2 and THIJS culture medium were 19 and 29 IOU, respectively. In THIJS medium, greater amount of pertussistoxin production was cell-associated. In B2 medium, maximum cell-associated toxin by ELISA and CHO cell assays were in the ODs of 1.1 and 0.9 and for THIJS medium in the ODs of 1.6 and 1.1, respectively. B. pertussis strain 509 in THIJS medium produced higher cell mass and cell-associated pertussis toxin than that of B2. It can be used for the production of whole-cell vaccine with higher pertussis toxin and accordingly using lower biomass per dose leading to the reduction of vaccine toxicity.

Once-a-week tigecycline for the treatment of drug-resistant TB.

MDR-TB and XDR-TB have poor outcomes. To examine the efficacy of tigecycline monotherapy in the hollow fibre system model of TB. We performed pharmacokinetic/pharmacodynamic studies using tigecycline human-like concentration-time profiles in the hollow fibre system model of TB in five separate experiments using Mycobacterium tuberculosis in log-phase growth or as semi-dormant or intracellular bacilli, as monotherapy. We also compared efficacy with the isoniazid/rifampicin/pyrazinamide combination (standard therapy). We then applied extinction mathematics, morphisms and Latin hypercube sampling to identify duration of therapy with tigecycline monotherapy. The median tigecycline MIC for 30 M. tuberculosis clinical and laboratory isolates (67% MDR/XDR) was 2 mg/L. Tigecycline monotherapy was highly effective in killing M. tuberculosis in log-phase-growth and semi-dormant and intracellular M. tuberculosis. Once-a-week dosing had the same efficacy as daily therapy for the same cumulative dose; thus, tigecycline efficacy was linked to the AUC0-24/MIC ratio. Tigecycline replacement by daily minocycline after 4 weeks of therapy was effective in sterilizing bacilli. The AUC0-24/MIC ratio associated with optimal kill was 42.3. Tigecycline monotherapy had a maximum sterilizing effect (day 0 minus day 28) of 3.06 ± 0.20 log10 cfu/mL (r2 = 0.92) compared with 3.92 ± 0.45 log10 cfu/mL (r2 = 0.80) with optimized standard therapy. In our modelling, at a tigecycline monotherapy duration of 12 months, the proportion of patients with XDR-TB who reached bacterial population extinction was 64.51%. Tigecycline could cure patients with XDR-TB or MDR-TB who have failed recommended therapy. Once-a-week tigecycline could also replace second-line injectables in MDR-TB regimens.

Production of deuterated biomass by cultivation of Lemna minor (duckweed) in D2O.

Common duckweed Lemna minor was cultivated in 50% D2O to produce biomass with 50-60% deuterium incorporation containing cellulose with degree of polymerization close (85%) to that of H2O-grown controls. The small aquatic plant duckweed, particularly the genus Lemna, widely used for toxicity testing, has been proposed as a potential source of biomass for conversion into biofuels as well as a platform for production of pharmaceuticals and specialty chemicals. Ability to produce deuterium-substituted duckweed can potentially extend the range of useful products as well as assist process improvement. Cultivation of these plants under deuterating conditions was previously been reported to require addition of kinetin to induce growth and was hampered by anomalies in cellular morphology and protein metabolism. Here, we report the production of biomass with 50-60% deuterium incorporation by long-term photoheterotrophic growth of common duckweed Lemna minor in 50% D2O with 0.5% glucose. L. minor grown in 50% D2O without addition of kinetin exhibited a lag phase twice that of H2O-grown controls, before start of log phase growth at 40% of control rates. Compared to continuous white fluorescent light, growth rates increased fivefold for H2O and twofold for 50% D2O when plants were illuminated at higher intensity with a metal halide lamp and a diurnal cycle of 12-h light/12-h dark. Deuterium incorporation was determined by a combination of 1H and 2H nuclear magnetic resonance (NMR) to be 40-60%. The cellulose from the deuterated plants had an average-number degree of polymerization (DPn) and polydispersity index (PDI) close to that of H2O-grown controls, while Klason lignin content was reduced. The only major gross morphological change noted was root inhibition.

The effect of biotic and abiotic environmental factors on Pd(II) adsorption and reduction by Bacillus megaterium Y-4

In this study, we screened a new aerobic bacterium (Bacillus megaterium Y-4) that can efficiently reduce Pd(II) with different electron donors. The best electron donor was sodium formate and the best reduction of Pd(II) were by log growth phase cells. The high removal capacity of Pd(II) (1658.3 mg/g) was obtained with 30 mg/L dry cell weight and 50 mg/L Pd (II) in the presence of 5 mM sodium formate. The removal amount of Pd(II) increased with initial Pd(II) concentrations ranging from 50 to 200 mg/L with 100 mg/L Pd(II) being completely removed by 148 mg/L dry cell weight in 6 h. The cell wall, periplasmic space and intracellular contents of B. megaterium Y-4 contains different kinds of enzymes for reducing Pd(II). In addition, the activity of extracellular and periplasmic enzymes was more sensitive to temperature than intracellular enzymes. XRD and XPS analysis revealed that the enzyme for reducing Pd(II) in B. megaterium Y-4 can tolerate a broad range of temperatures (20–60 °C) and pH (2.0–7.0) but was sensitive to oxygen. TEM analysis showed that biogenic palladium nanoparticles (Pd-NPs) were mainly distributed evenly in the periplasmic space of the live cells and were released from cells into aqueous solution, which reduced the toxicity of Pd(II), allowing Pd-NP recovery without cell destruction. B. megaterium Y-4 is a potential bacterium for efficient treatment and reclamation of Pd(II) pollution and formation of Pd-NPs.

Chemogenic silver nanoparticles enhance lignans and neolignans in cell suspension cultures of Linum usitatissimum L.

Cell suspension culture of Linum usitatissimum is a great source of the novel and multipurpose medicinal compounds lignans and neolignans. Conventional culturing practices usually result in low yield of plant secondary metabolites; therefore, we conceived a successful mechanism to elicit production of lignans and neolignans in cell suspension cultures, simply, by addition of chemogenic Ag-NPs into the culture medium. A three stage feeding strategy (day 10, 10 and 15, and 10 and 20, respectively, after inoculation) spanning the log growth phase (day 10–20), was implemented to elicit cell suspension cultures of Linum usitatissimum. Though enhancing effects of Ag-NPs were observed at each stage, feeding Ag-NPs at day 10 resulted in comparatively, highest production of lignans (secoisolariciresinol diglucoside, 252.75 mg/l; lariciresinol diglucoside, 70.70 mg/l), neolignans (dehydrodiconiferyl alcohol glucoside, 248.20 mg/l; guaiacylglycerol-β-coniferyl alcohol ether glucoside, 34.76 mg/l), total phenolic content (23.45 mg GAE/g DW), total flavonoid content (11.85 mg QUE/g DW) and biomass (dry weight: 14.5 g/l), respectively. Furthermore, a linear trend in accumulation of lignans and neolignans was observed throughout log phase as compared to control, wherein growth non-associated trend in biosynthesis of these metabolites was observed. Optimum production of both lignans and neolignans occurred on day 20 of culture; a ten fold increase in secoisolariciresinol diglucoside, 2.8 fold increase in lariciresinol diglucoside, five fold increase in dehydrodiconiferyl alcohol glucoside and 1.75 fold increase in guaiacylglycerol-β-coniferyl alcohol ether glucoside was observed in production levels compared to control treatments, respectively.

Activity of Moxifloxacin against Mycobacterium tuberculosis in Acid Phase and Nonreplicative-Persister Phenotype Phase in a Hollow-Fiber Infection Model.

A major goal for improving tuberculosis therapy is to identify drug regimens with improved efficacy and shorter treatment durations. Shorter therapies improve patient adherence to the antibiotic regimens, which, in turn, decreases resistance emergence. Mycobacterium tuberculosis exists in multiple metabolic states. At the initiation of therapy, the bulk of the population is in log-phase growth. Consequently, it is logical to focus initial therapy on those organisms. Moxifloxacin has good early bactericidal activity against log-phase bacteria and is a logical component of initial therapy. It would be optimal if this agent also possessed activity against acid-phase and nonreplicative-persister (NRP) phenotype organisms. In our hollow-fiber infection model, we studied multiple exposures to moxifloxacin (equivalent to 200 mg to 800 mg daily) against strain H37Rv in the acid phase and against strain 18b in streptomycin starvation, which is a model for NRP-phase organisms. Moxifloxacin possesses good activity against acid-phase organisms, generating cell killing of 3.75 log10(CFU/ml) (200 mg daily) to 5.16 log10(CFU/ml) (800 mg daily) over the 28 days of the experiment. Moxifloxacin also has activity against streptomycin-starved strain 18b. The 400- to 800-mg daily regimens achieved extinction at day 28, while the no-treatment control still had 1.96 log10(CFU/ml) culturable. The lowest dose (200 mg daily) still had 0.7 log10(CFU/ml) measurable at day 28, a net kill of 1.26 log10(CFU/ml). Moxifloxacin is an attractive agent for early therapy, because it possesses activity against three metabolic states of M. tuberculosis.

Characterization of the squalene-rich Botryococcus braunii Abt02 strain

Botryococcus braunii is widely studied due to its high hydrocarbon content. In this study, B. braunii Abt02 was subjected to several analyses, including cytological observation, hydrocarbon composition analysis by gas chromatography mass spectrometry (GC-MS), phylogenetic identification using known races (A, B and L) of B. braunii strains based on their 18S rDNA sequences, and qPCR-based investigation of transcript accumulation levels of hydrocarbon biosynthesis-related enzymes (DXS, MCS, DLS, SQS) during different growth phases (lag phase, log phase, early stationary growth phase, late stationary growth phase) under nitrogen-replete and nitrogen-depleted growth conditions, respectively. Based on cytological observation and on the 18S rDNA phylogenetic analysis, strain Abt02 was assigned to race B. Analysis of the strain’s chemical composition showed that the B. braunii Abt02 contained high levels of hydrocarbons, which accounted for 43.75% of the cell’s dry weight. Of these hydrocarbons, squalene and its derivatives accounted for up to 87.54%. In addition, all four enzymes investigated were expressed at higher levels during the log growth phase under nitrogen depleted conditions than under nitrogen replete conditions.