Systematic Evaluation of Parameters Important for Production of Native Toxin A and Toxin B from Clostridioides difficile

In the attempt to improve the purification yield of native toxin A (TcdA) and toxin B (TcdB) from Clostridioides difficile (C. difficile), we systematically evaluated culture parameters for their influence on toxin production. In this study, we showed that culturing C. difficile in a tryptone-yeast extract medium buffered in PBS (pH 7.5) that contained 5 mM ZnCl2 and 10 mM glucose supported the highest TcdB production, measured by the sandwich ELISA. These culture conditions were scalable into 5 L and 15 L dialysis tube cultures, and we were able to reach a TcdB concentration of 29.5 µg/mL of culture. Furthermore, we established a purification protocol for TcdA and TcdB using FPLC column chromatography, reaching purities of >99% for both toxins with a yield around 25% relative to the starting material. Finally, by screening the melting temperatures of TcdA and TcdB in various buffer conditions using differential scanning fluorimetry, we found optimal conditions for improving the protein stability during storage. The results of this study present a complete protocol for obtaining high amounts of highly purified native TcdA and TcdB from C. difficile.

Phenotypic Differentiation of Two Morphologically Similar Aflatoxin-Producing Fungi from West Africa

Aflatoxins (AF) are hepatocarcinogenic metabolites produced by several Aspergillus species. Crop infection by these species results in aflatoxin contamination of cereals, nuts, and spices. Etiology of aflatoxin contamination is complicated by mixed infections of multiple species with similar morphology and aflatoxin profiles. The current study investigates variation in aflatoxin production between two morphologically similar species that co-exist in West Africa, A. aflatoxiformans and A. minisclerotigenes. Consistent distinctions in aflatoxin production during liquid fermentation were discovered between these species. The two species produced similar concentrations of AFB1 in defined media with either urea or ammonium as the sole nitrogen source. However, production of both AFB1 and AFG1 were inhibited (p < 0.001) for A. aflatoxiformans in a yeast extract medium with sucrose. Although production of AFG1 by both species was similar in urea, A. minisclerotigenes produced greater concentrations of AFG1 in ammonium (p = 0.039). Based on these differences, a reliable and convenient assay for differentiating the two species was designed. This assay will be useful for identifying specific etiologic agents of aflatoxin contamination episodes in West Africa and other regions where the two species are sympatric, especially when phylogenetic analyses based on multiple gene segments are not practical.

Effect of peptones on the ability of plant pathogenic bacteria to grow on media supplemented with copper sulphate

The extensive use of copper compounds for control of bacterial plant pathogens could lead to selecting strains of the pathogens that are copper-resistant. The ability to grow on a medium supplemented with copper, and therefore the concentration of copper to which bacterial strains are resistant, depends on the composition of the medium used for the test. The effects of different peptones (casitone and proteose peptone No. 3) and different media on the ability of strains of Pseudomonas syringae to grow in presence of copper were determined. Similar results were obtained when casitone was substituted with proteose peptone No. 3 in casitone yeast extract medium, but not when those two peptones were interchanged in King’s B medium. In water, casitone allowed strains of P. syringae pv. actinidiae (Psa) to grow in the presence of higher amounts of copper than proteose peptone No. 3 did. In all cases, resistance to copper was increased with increased peptone concentration.

Biotransformation of major ginsenoside Rb1 to pharmacologically active ginsenoside Rg3 through fermentation by Weissella hellenica DC06 in newly developed medium

The study was conducted to develop an edible and low cost growth medium for cultivation of Weissella hellenica DC06, a lactic acid bacteria (LAB) and to study whether, the medium is suitable for bioconversion of major ginsenoside Rb1 into ginsenoside Rg3 through fermentation by W. hellenica DC06. Fourteen different media compositions were investigated to cultivate W. hellenica DC06. Among these, W. hellenica DC06 exhibited the highest growth in media containing 20 g/l radish, 20 g/l glucose, and 10 g/l yeast extract (Medium 3). The optical density of W. hellenica DC06 cultivated in medium 3 reached 1.8 (1.066 x 1010 CFU/ml) after 24 h of incubation. Importantly, the optimized medium was approximately four times cheaper compared to MRS medium. In addition to being economical, the new medium was also edible. Also W. hellenica DC06 showed strong fermentation ability in newly developed medium regarding on major ginsenoside Rb1 biotransformation. Ginsenoside Rb1 was converted into pharmacologically active ginsenoside Rg3 in new medium. In contrast,W. hellenica DC06 showed weak fermentation ability in MRS medium where ginsenoside Rb1 was converted intoginsenoside Rd. The transformation products were analyzed by TLC, and HPLC. Within seven days of fermentation, almost all ginsenoside Rb1 was decomposed and converted into Rg3 in optimized medium. W. hellenica DC06 hydrolyzed two glucose moieties attached to the C-20 position of the ginsenoside Rb1aglyconeand synthesized Rg3 in newly developed medium.Bangladesh J. Sci. Ind. Res. 51(4), 271-278, 2016

Biodegradation and decolorization of melanoidin solutions by manganese peroxidase yeasts

The ability of selected manganese peroxidase (MnP) yeast strains, isolated from the mixed liquor of an activated sludge bioreactor treating melanoidins wastewater, was investigated in this work, aiming to examine the degradation potential of melanoidins, in the presence or absence of nutrients. Ten yeast strains were initially isolated from the mixed liquor; four yeast strains (Y1, Y2, Y3 and Y4) were selected for further studies, based on their tolerance towards synthetic melanoidins (SMs) degradation and MnP activity onto solid agar medium. The Y1 strain exhibited almost 98% homology to Candida glabrata yeast, based on 28S rRNA identification studies. During experiments carried out using SM at 30 °C, the four isolated yeast cultures showed a noticeable organic matter reduction and decolorization capacity reaching up to 70% within 2–5 days. However, the corresponding yeast cultures grown in glucose peptone yeast extract medium using real melanoidin wastewater at 30°C showed lower organic matter and color removal capacity, reaching about 60% within 2–5 days. Nevertheless, it was found that the removal of real and synthetic melanoidins could be carried out by these strains under non-aseptic conditions, without requiring further addition of nutrients.

Gluconobacter cerevisiae sp. nov., isolated from the brewery environment

Three strains, LMG 27748T, LMG 27749 and LMG 27882 with identical MALDI-TOF mass spectra were isolated from samples taken from the brewery environment. Analysis of the 16S rRNA gene sequence of strain LMG 27748T revealed that the taxon it represents was closely related to type strains of the species Gluconobacter albidus (100 % sequence similarity), Gluconobacter kondonii (99.9 %), Gluconobacter sphaericus (99.9 %) and Gluconobacter kanchanaburiensis (99.5 %). DNA–DNA hybridization experiments on the type strains of these species revealed moderate DNA relatedness values (39–65 %). The three strains used d-fructose, d-sorbitol, meso-erythritol, glycerol, l-sorbose, ethanol (weakly), sucrose and raffinose as a sole carbon source for growth (weak growth on the latter two carbon sources was obtained for strains LMG 27748T and LMG 27882). The strains were unable to grow on glucose-yeast extract medium at 37 °C. They produced acid from meso-erythritol and sucrose, but not from raffinose. d-Gluconic acid, 2-keto-d-gluconic acid and 5-keto-d-gluconic acid were produced from d-glucose, but not 2,5-diketo-d-gluconic acid. These genotypic and phenotypic characteristics distinguish strains LMG 27748T, LMG 27749 and LMG 27882 from species of the genus Gluconobacter with validly published names and, therefore, we propose classifying them formally as representatives of a novel species, Gluconobacter cerevisiae sp. nov., with LMG 27748T ( = DSM 27644T) as the type strain.

Clostridium difficile glutamate dehydrogenase is a secreted enzyme that confers resistance to H2O2

Clostridium difficile produces an NAD-specific glutamate dehydrogenase (GDH), which converts l-glutamate into α-ketoglutarate through an irreversible reaction. The enzyme GDH is detected in the stool samples of patients with C. difficile‐associated disease and serves as one of the diagnostic tools to detect C. difficile infection (CDI). We demonstrate here that supernatant fluids of C. difficile cultures contain GDH. To understand the role of GDH in the physiology of C. difficile, an isogenic insertional mutant of gluD was created in strain JIR8094. The mutant failed to produce and secrete GDH as shown by Western blot analysis. Various phenotypic assays were performed to understand the importance of GDH in C. difficile physiology. In TY (tryptose yeast extract) medium, the gluD mutant grew slower than the parent strain. Complementation of the gluD mutant with the functional gluD gene reversed the growth defect in TY medium. The presence of extracellular GDH may have a functional role in the pathogenesis of CDI. In support of this assumption we found higher sensitivity to H2O2 in the gluD mutant as compared to the parent strain. Complementation of the gluD mutant with the functional gluD gene reversed the H2O2 sensitivity.

STRAIN IMPROVEMENT OF A FUNGUS PRODUCING CHITINASE BY A CHEMICAL MUTAGEN

Microbial chitinases are commercially exploited for their biocontrol properties and generation of useful products from chitinous waste. Availability of highly active chitinolytic enzymes is a major problem. The present study was carried out to improve chitinase production by Aspergillus terreus using a chemical mutagen, ethidium bromide. The organism was cultivated on lactose- yeast extract medium. The production medium consisting of chitin- yeast extract medium was seeded at 10% level. The wild strains were exposed to ethidium bromide in the concentration range 1.5- 6.0 µg/mL. Generally, all the mutated strains showed an improved chitinase yield compared to the control. Highest yield was observed with the strain exposed to 6 µg/mL of ethidium bromide. The yield was 25.03 % higher compared to the wild strain. The mutated strain was slimy in nature. Protein content of the mutated strain decreased by 11%. Ethidium bromide at a concentration of 1.5 µg/mL was considered optional, at which the strain was stable with increase of 21.80 % in enzyme activity and 4.41% increase in protein content. Increased enzyme yield with decreased non-specific protein could be useful in producing cost effective enzyme.