Medium Agar Plates Research Articles

Role of HIF-1, Siah-1 and SKN-1 in Inducing Adiposity for Caenorhabditis elegans under Hypoxic Conditions

BACKGROUND: Hypoxia has been shown to be able to induce adiposity. However, the mechanism and factors involved in this effect still remains unclear. Hence, we sought to investigate the role of oxygensensitive factors regarding hypoxia-induced adiposity in nematode Caenorhabditis elegans.METHODS: The C. elegans were grown on nematode growth medium (NGM) agar plates seeded with Escherichia coli OP50 at 20°C. The ratio of width/body length was measured using the morphometry analysis. Fat accumulation was examined using Sudan Black methods. Protein levels of sterol binding protein (SBP)-1 were assessed by immunoblotting. Lifespan assay was performed at 20°C and was monitored every two days.RESULTS: The results showed that of all mutant used, only hif-1 mutant which did not experience an increase in the ratio of width/body length (p>0.05) and fat accumulation (p>0.05), indicating that hypoxia-inducible factors (HIF)-1 plays an important role in the pathogenesis of hypoxia-induced adiposity. Both siah-1 and skn-1 mutants experienced SBP-1 protein elevation (p<0.05), and increased fat-6 mRNA expression (p<0.05) which were not experienced by a hif-1 mutant (p>0.05) further supporting that HIF-1 acts as an upstream regulator fromSBP-1.CONCLUSION: In general, the results of this study provide evidences of the involvement of the transcription factor HIF-1 in inducing adiposity under the hypoxic conditions. However, we did not find the involvement of seven in absentia homolog-1 (Siah-1) and skinhead-1 (SKN-1).KEYWORDS: hypoxia, adiposity, fat, HIF-1, Siah-1, SKN-1, C. elegans

Rapid isolation of non-aflatoxigenicAspergillus flavus strains

In the present study, a method for screening non-aflatoxigenicAspergillus flavus in soil samples collected from major peanut-growing regions of China was developed. The single colonies were picked and cultured onAspergillus flavus andparasiticus agar (AFPA). If the reverse side of the colony on AFPA was orange-coloured, it was consideredA. flavus orAspergillus parasiticus. After the genomic DNA of each strain was extracted, 28S rRNA and calmodulin were amplified and sequenced to determine the species. The key gene,aflR, was amplified and digested via polymerase chain reaction-restriction fragment length polymorphism. The aflatoxigenicA. flavus and the non-aflatoxigenicA. flavus andA. parasiticus were distinguished by enzyme digestion ofaflR. 156 strains ofA. flavus were screened, which consisted of 135 aflatoxigenic and 21 non-aflatoxigenic strains. The aflatoxin producing ability of each strain was confirmed using solid-state fermentation experiments. Using the method developed in the present study, we confirmed that the non-aflatoxigenicA. flavus strains isolated lost their capacity to produce aflatoxins. Considering there could be some alterations in other functional genes, some non-aflatoxigenic strains could be identified inaccurately as aflatoxigenic strains, although that did not occur in the present study. The growth of non-aflatoxigenicA. flavus was observed, and the most rapidly growing non-aflatoxigenic strain was selected for plate confrontation assays and toxic mixed culture experiments. The inhibition rate of non-aflatoxigenicA. flavus against aflatoxigenicA. flavus was 55.4 and 72.6% in potato dextrose agar (PDA) plate and natural soybean medium, respectively. The screened non-aflatoxigenicA. flavus strains provide a microbial resource for biological control of aflatoxin contamination.

RNAi-Mediated Gene Silencing of Trcot1 Induces a Hyperbranching Phenotype in Trichoderma reesei.

Trichoderma reesei is the major filamentous fungus used to produce cellulase and there is huge interest in promoting its ability to produce higher titers of cellulase. Among the many factors affecting cellulase production in T. reesei, the mycelial phenotype is important but seldom studied. Herein, a close homolog of the Neurospora crassa COT1 kinase was discovered in T. reesei and designated TrCOT1, which is of 83.3% amino acid sequence identity. Functional disruption of Trcot1 in T. reesei by RNAi-mediated gene silencing resulted in retarded sporulation on potato dextrose agar and dwarfed colonies on minimal medium agar plates containing glucose, xylan, lactose, xylose, or glycerol as the sole carbon source. The representative mutant strain, SUS2/Trcot1i, also displayed reduced mycelia accumulation but hyperbranching in the MM glucose liquid medium, with hyphal growth unit length values decreased to 73.0 µm/tip compared to 239.8 µm/tip for the parent strain SUS2. The hyperbranching phenotype led to slightly but significantly increased cellulase secretion from 24 to 72 h in a batch culture. However, the cellulase production per unit of mycelial biomass was much more profoundly improved from 24 to 96 h.

The Genome of Caenorhabditis bovis.

The free-living nematode Caenorhabditis elegans is akey laboratory model for metazoan biology. C.elegans has also become a model for parasitic nematodes despite being only distantly related to most parasitic species. All of the ∼65 Caenorhabditis species currently in culture are free-living, with most having been isolated from decaying plant or fungal matter. Caenorhabditis bovis is a particularly unusual species that has been isolated several times from the inflamed ears of Zebu cattle in Eastern Africa, where it is associated with thedisease bovine parasitic otitis. C.bovis is therefore of particular interest to researchers interested in the evolution of nematode parasitism. However, as C.bovis is not in laboratory culture, it remains little studied. Here, by sampling livestock markets and slaughterhouses in Western Kenya, we successfully reisolated C.bovis from the ear of adult female Zebu. We sequenced the genome of C.bovis using the Oxford Nanopore MinION platform in a nearby field laboratory and used the data to generate a chromosome-scaledraft genome sequence. We exploited this draft genome sequence to reconstruct the phylogenetic relationships of C.bovis to other Caenorhabditis species and reveal the changes in genome size and content that have occurred during its evolution. We also identified expansions in several gene families that have been implicated in parasitism in other nematode species. The high-quality draft genome and our analyses thereof represent a significant advancement in our understanding of this unusual Caenorhabditis species.

First Report of Plantain Soft Rot Caused by Klebsiella variicola in Haiti

Plantain and banana, both Musa spp. hybrids, constitute a major component of domestic food production in Haiti. In response to grower reports (Faculty of Agronomy and Veterinary Medicine, Port-au-Prince, personal communication) of up to 60% incidence of “toppling disease”, we conducted a preliminary study in July 2018 at four plantain farms in Arcahaie, Haiti. Symptoms observed on up to 30% of plants (n = ∼100) included a brown necrotic soft rot lesion on pseudostems extending up to 1 m in length and several centimeters deep. Toppling occurred at fruit onset, with plants folding over at the point of the soft rot. Six plants representing the four farms were sampled: 6-cm² sections were excised from the lesion edge, surface sterilized with 0.6% sodium hypochlorite, rinsed, and macerated with sterile tap water, and then the suspension was streaked onto nutrient agar (NA) and Miller-Schroth medium agar (MSM) plates, yielding six bacterial isolates. Colonies were round, glossy, convex, translucent gray-white on NA, produced a transient red-orange color change on MSM, and were aerobic, gram-negative, oxidase-negative and hypersensitive response negative on ‘Bonnie Best’ tomato and ‘Hicks’ tobacco (Schaad et al. 2001); all the above tests were repeated twice. All six isolates were identical in initial 16S rDNA sequencing, so pathogenicity of two randomly chosen isolates (G18-1365 and G18-1376) was tested on disease‐free ‘Dwarf Cavendish’ banana plantlets by drilling a 2.5-cm hole into the pseudostem, inserting sterile cotton-tipped swabs drawn through 48-h cultures on NA (negative control was dipped in sterile water) into the drilled incision, and sealing with Parafilm. Each plant was wounded once, with five plants per isolate or control. Rotting symptoms consistent with the field observations were observed 4 weeks after inoculation for all isolate inoculations; control plants remained symptomless. Reisolation and identification to fulfill Koch’s postulates were completed with results identical to the first isolations and tests. Genomic DNA was boil-extracted as previously described (Dashti et al. 2009) from the two isolates grown 24 h on NA. The amplification and sequencing of 16S rDNA, rpoB, phoE, and infB gene targets were carried out using primers and protocols previously described (Rosenblueth et al. 2004). One sequence for each isolate’s gene target (eight total) was deposited in NCBI GenBank (accessions MK217521 to MK217522; MK235993 to MK235998). In BLAST searches, sequence fragments of 16S rDNA, rpoB, phoE, and infB from isolates G18-1365 and G18-1376 resulted in 99% identity with Klebsiella variicola strains. Multilocus sequence analysis (MEGA X, Kumar et al. 2018) of those sequences yielded a concatenated phylogenetic tree that placed both isolates within the K. variicola clade. Isolates G18-1365 and G18-1376 have been deposited at the BCCM, Ghent University (accessions LMG 31122 and LMG 31123, respectively). K. variicola has been reported to cause similar bacterial rots in banana and carrot (Chandrashekar et al. 2018; Fan et al. 2016). Before management can be prescribed, further studies are necessary to determine disease incidence and if this species is the sole cause of toppling or is part of a larger disease complex, and to understand the mode of transmission within and among plantain farms. Additionally, similarity to human pathogenic strains should be studied to determine if there is a human health risk posed by K. variicola isolates causing soft rot in plantain.

Use of Purple Non-Sulfur Photosynthetic Bacteria (Rhodobacter sphaeroides) in Promoting Ciliated Protozoa Growth.

Photosynthetic bacterium (PSB) was isolated from sediment samples of Yamagawa Bay, Kagoshima, Japan. Phylogenetic analysis results of PSB isolate were closely related to Rhodobacter sphaeroides, purple non-sulfur photosynthetic bacteria (PNSB). Pink-colored smooth edges of single bacterial colonies were observed after 3-5 days of incubation period on Basic I medium agar plates. Rhodobacter sphaeroides microscopic examination showed a short rod cell (1-2 µm length) with round ends. Sediment and water samples used for ciliates cultivation were collected from Kuwano-ura Bay, Koshiki Island, Japan. Ciliates were cultivated using fish meal with radish leaves medium (MI), with sediment into MI (MII) and algae media (MIII). The use of the algae media (MIII) in cultivation mixture produced the highest total number of ciliates. Big size ciliates were identified as Euplotes minuta and Cyclidium varibonneti, while small size was identified as Micrometopion nutans, based on PCR-DGGE. When ciliates were cultured with the PSB isolate, Rhodobacter sphaeroides as a feed, ciliates grow to 2,081 individual ml-1 72 hrs later. These findings indicate that PNSB can be used to promote ciliates growth.

Artificial small RNA-mediated growth inhibition in Escherichia coli and Salmonella enterica serovar Typhimurium

We developed a synthetic RNA approach to identify growth inhibition sequences by cloning random 24-nucleotide (nt) sequences into an arabinose-inducible expression vector. This vector expressed a small RNA (sRNA) of ∼140 nt containing a 24 nt random sequence insert. After transforming Escherichia coli with the vector, 10 out of 954 transformants showed strong growth defect phenotypes and two clones caused cell lysis. We then examined growth inhibition phenotypes in the Salmonella Typhimurium LT2 strain using the twelve sRNAs that exerted an inhibitory effect on E. coli growth. Three of these clones showed strong growth inhibition phenotypes in S. Typhimurium LT2. The most effective sRNA contained the same insert (N1) in both bacteria. The 24 nt random sequence insert of N1 was abundant in guanine residues (ten out of 24 nt), and other random sequences causing growth defects were also highly enriched for guanine (G) nucleotides. We, therefore, generated clones that express sRNAs containing a stretch of 16 to 24 continuous guanine sequences (poly-G16, -G18, -G20, -G22, and -G24). All of these clones induced growth inhibition in both liquid and agar plate media and the poly-G20 clone showed the strongest effect in E. coli. These results demonstrate that our sRNA expression system can be used to identify nucleotide sequences that are potential candidates for oligonucleotide antimicrobial drugs.

Microorganisms Isolated from Moroccan Olive-Mill Wastes: Screening of Their Enzymatic Activities for Biotechnological Use

In this study, three samples of olive oil wastes: Olive Rinse Water (OR), Olive Mill Wastewater (OM) and Olive Pomace (OP), which were collected from an olive oil mill located at Fez-Morocco, were analyzed for their microbiota. A total of 81 isolates were obtained and constituted a microbial bank formed of 35 bacteria, 41 yeasts and 5 fungi. The microbial communities have been compared in term of metabolic potential by testing hydrolytic enzymes activities of lipase, protease, amylase, cellulase, pectinase and tannase on agar plate media. The results reveal that among the examined microorganisms, 68 isolates were able to produce at least one of the screened enzymes. The pectinase activity was the predominant one (39.51%), followed by cellulase activity (34.57%) and by lipase activity (27.16%). However, the amylase activity was observed only for 11.11% of the studied isolates, followed by protease activity (9.88%) and by tannase activity 6.17%. Thereby, considering the isolates ability to produce enzymes, they can be considered as potential candidates for industrial and biotechnological applications. Lipases are versatile enzymes that are used widely, it’s why the mostly aim of this work was to isolate new bacterial strains producing high level of lipases with interesting biochemical criteria. Thus, two isolates OR34 and OM2 showed the maximum lipase production under the standard conditions, about 9 U/ml and 11 U/ml, respectively, using pH-stat technique. They were identified as Proteus vulgaris and Serratia marcescens, respectively, via partial 16S rDNA gene sequence analysis.

Bacteria affect Caenorhabditis elegans responses to MeHg toxicity

The organic form of mercurial complex, methylmercury (MeHg), is a neurotoxin that bioaccumulates in the food web. Studies in model organisms, such as Caenorhabditis elegans (C. elegans), provide powerful insights on the role of genetic factors in MeHg-induced toxicity. C. elegans is a free living worm that is commonly cultured in nematode growth medium (NGM) agar plates seeded with bacteria. The bacteria have broad impact on C. elegans biology, including development, reproduction and lifespan, as well as metabolism of experimental chemicals. We hypothesized that MeHg toxicity in C. elegans could be modified by the bacterial food. Using a C. elegans wild-type (WT) strain and transgenic reporter strains, we found that bacterial food reduced the chronic toxicity of MeHg in C. elegans in a dose- and live-status-dependent manner. The MeHg-induced death rate in C. elegans was highest in fasted worms, followed by dehydrated dead bacteria, dead bacteria and live bacteria fed worms. Among the different bacterial foods, dehydrated dead bacteria fed worms were most sensitive to the toxicity of MeHg. The distinct bacteria food (dehydrated dead bacteria food) attenuated oxidative stress and development delay in C. elegans exposed to MeHg. The FOXO transcriptional factor DAF16 was not changed by MeHg but modified by the distinct bacteria food. Independent of MeHg treatment, daf-16 expression in fed worms migrated from the intestine to muscle. We conclude that, in chronic exposure studies in C. elegans, the effects of bacteria on toxicological outcomes should be considered.

Assessment of Airborne Bacterial and Fungal Communities in Selected Areas of Teaching Hospital, Kandy, Sri Lanka

Nosocomial infections, in lay term known as hospital acquired infections, are caused mainly by airborne pathogens found in healthcare facilities and their surroundings. The aim of this study was to quantify and identify bacteria and fungi in a hospital, which is an understudied area of air quality in Sri Lanka. Air samples were collected in agar medium and petri plates containing sterile filter papers. The number of culturable and total airborne microorganisms was estimated by manual counting and fluorescent microscopy, respectively. The morphologically distant bacteria and fungi were identified by DNA sequencing. The statistical analysis revealed significant variances between studied sites (p < 0.05) where Outpatients Department and Respiratory Unit showed higher levels of airborne microbial load. Culturable microbial count was higher at noon (hospital visiting hours) compared to other sampling periods (after hospital visiting hours) within the hospital. Total count of airborne microbes was found to be the highest during the afternoon. The most sensitive zones such as Operating Theatre and Intensive Care Unit showed considerably higher counts of airborne microbes. Identification by molecular means revealed the presence of human pathogens in the hospital air including Bacillus sp, Micrococcus sp, Pseudomonas sp, Staphylococcu ssp, Exiguobacterium sp, Enterobacter sp, Escherichia sp, Sphingomonas sp, Massilia sp, Kocuria sp, Fusarium sp, and Aspergillus sp. In conclusion, the results from this study indicate that the hospital air was generally contaminated. Therefore, the implementation of proactive policies and strategies are needed to monitor hospital air quality in sensitive zones as well as other areas of the hospitals.

Solubilization of inorganic phosphate by rhizospheric fungi isolated from soil cultivated with Sorghum bicolor L.

Three zinc-phosphate-solubilizing fungi (ZPSF) were isolated from rhizospheric soil cultivated with Sorghum bicolor L. The fungal isolates were identified as Aspergillus chevalieri, Fusarium moniliforme, and Trichoderma harzianum. The results showed that halo zone formation by ZPSF on Pikovskaya (PVK) agar medium plates was an indicator of zinc phosphate (ZP) solubilization. The lowest in pH of the inoculated medium containing ZP was observed with A. chevalieri, followed by T. harzianum, followed by F. moniliforme, compared with the initial pH (6.5) of the non-inoculated medium. ZP solubilization processes at different temperatures (10 °C, 20 °C, 30 °C, and 40 °C) were conducted using ZPSF at different doses of ZP (0.5 g/L, 1 g/L, and 2 g/L). The released P, to P2O5, was monitored during the solubilization process. The released phosphorus increased as the temperature increased, with the greatest values of phosphorus obtained with F. moniliforme, A. chevalieri, and T. harzianum being 11.54 mg/L, 24.40 mg/L, and 28.40 mg/L, at 30 °C and a dose of 2 g/L of ZP, respectively. In contrast, the smallest values of phosphorus were 11.89 mg/L, 8.2 mg/L, and 7.97 mg/L, at 10 °C and a dose of 0.5 g/L of ZP, with F. moniliforme, A. chevalieri, and T. harzianum, respectively.

Characterization and antifungal activity of chitosanase produced by Pedobacter sp. PR-M6

To investigate the temperature requirements of chitosanase activity, as well as the degradation patterns generated by enzyme-induced chitosan oligomer hydrolysis, Pedobacter sp. PR-M6 was inoculated onto 0.5% colloidal chitosan medium agar plates. Cell growth was higher at 30 °C than at 20 °C during the initial 2 days of incubation. The protein content rapidly increased on day 1 at both temperatures and then it slowly increased at 20 °C and slowly decreased at 30 °C during the following 5 days of incubation. In order to characterize the electrophoretic pattern, Pedobacter sp. PR-M6 was cultured in 1% powder chitosan medium at 20 °C and 30 °C for 5 days after incubation and analyzed by SDS-PAGE. Four bands were visible, corresponding to ct1 (25 kDa), ct2 (17 kDa), ct3 (15 kDa), and ct4 (14 kDa), at both 20 °C and 30 °C. The optimal conditions for the activity of chitosanase produced from Pedobacter sp. PR-M6 were 60 °C and 1.81 enzyme units/mg protein. Two major isozyme bands (ct3 and ct4) exhibited their strongest chitosanase activity at 50 °C in SDS-PAGE gel. The reaction products generated from (GlcN)2-(GlcN)5 substrates at 60 °C after a 1 h incubation were investigated by thin-layer chromatography. Low-molecular weight chitosan and oligochitosan (LCOC) and soluble chitosan showed antifungal activity against A. brassicicola, B. cinerea, F. solani, and R. solani. LCOC exhibited higher antifungal activity than soluble chitosan. Moreover, LCOC treatments (500 ppm and 1000 ppm) inhibited conidia germination in A. brassicicola.

Difference of colorectal cancer microbial community by metagenomics and culture-based methods.

489 Background: Dysbiosis of intestinal microbiota is promoting the development of colorectal cancer (CRC). We confirmed the intestinal microbiota composition from fecal sample of Korean CRC patients. Metagenomic analysis was performed and we isolated single microbes through culture-based method. Methods: CRC fecal samples were collected from 12 individuals. Metagenome Sequencing was based on the 16S rRNA gene amplicon on the Illumina MiSeq platform. The bacteria strains were subcultivated on the agar plate medium in aerobic and anaerobic and further identified by using the 16s rRNA gene sequencing. Results: Bacteria diversity by metagenome analysis was decreased in CRC group compared to control group. In CRC group, relative abundance of Firmicutes and Bacteroidetes were increased while the prevalence of Proteobacteria was decreased. The difference of microbial composition between control and CRC group was found at the genus level. Bacteroides, Parabacteroides of Bacteroidetes have increased and Acinetobacter, Pseudomonas of Proteobacteria have significantly decreased in CRC compared to control group. Using culture method, we isolated diverse bacteria of species level including five strains of Bacteroides; B. ovatus, B. uniformis, B. salyersiae, B. vulgatus and B. xylanisolvens and two strains of Fusobacterium; F. gonidiaformans and F. necrophorum from CRC patients. Conclusions: Metagenome analysis showed the genus Bacteroides, Parabacteroides of the phylum Bacteroidetes has increased and the genus Acinetobacter, Pseudomonas of Proteobacteria decreased in CRC group compared to control group. In addition to, we have isolated various strains associated with CRC by culture-based method.

Uptake and effects of lead and zinc on alfalfa (Medicago sativa L.) seed germination and seedling growth: Role of plant growth promoting bacteria

The present study was conducted to screen bacterial strains most effective in increasing alfalfa growth and metal accumulation in the presence of toxic levels of lead (Pb) and zinc (Zn). Results show that, compared to root and shoot growth, alfalfa seed germination is less sensitive to high levels of Pb and Zn. Pb and Zn concentrations of 4.4 and 7.9 mM, respectively, were required to give rise to a 50% reduction in in vitro seed germination. Root growth in alfalfa seedlings is, however, completely suppressed at a Pb concentration of 4 mM or a Zn concentration of 6 mM in the agar plate medium. Inoculation of the bacterial strains capable of producing indole-acetic acid and siderophore positively affected the plant growth parameter in the metal contaminated mediums. Root and/or shoot growths of alfalfa seedlings are significantly stimulated by the seven inoculated bacterial strains investigated, among which Bacillus filamentosus YSP110 is found to be the most effective. Inoculation of alfalfa plants with B. filamentosus strain YSP110 grown in a vermiculate medium is also seen to increase Pb accumulation in plant root and shoot by 18.0 and 72.4%, respectively. Bacillus cereus YSP4 is the bacterial strain most effective in stimulating root and shoot growth in alfalfa seedlings cultured in a Zn-contaminated medium. Compared to the non-inoculated plants, alfalfa seeds inoculated with B. cereus YSP4 also exhibit increased Zn accumulation in their roots and shoots by 43.2 and 48.7%, respectively. Results suggest that B. filamentosus YSP110 and B. cereus YSP4 offer a great potential for promoting plant growth and metal accumulation, which makes them good candidates for microbe-assisted phytoextraction of metal-contaminated soils.

2D Image Quantification of Microbial Iron Chelators (Siderophores) Using Diffusive Equilibrium in Thin Films Method.

Siderophores are natural metal chelating agents that strongly control the biogeochemical metal cycles such as Fe in the environment. This article describes a new methodology to detect and quantify at the micromolar concentration the spatial distribution at millimeter scale of siderophores within the root's system. The "universal" CAS assay originally designed for bacterial siderophores detection and later designed for fungus was adapted here for diffusive equilibrium in thin film gel techniques (DET). The method was calibrated against the marketed desferrioxamine mesylate (DFOM) siderophore and applied with experiments performed with sunflower ( Helianthus annuus) and wheat ( Triticum aestivum) cultivated on free iron agar medium plates. We present here the first results with 2D images of the siderophores distribution in the vicinity of the root system of plants. With this technique we detected (i) the production of siderophores on bacteria inoculated ( Pseudomonas fluorescens) environments and (ii) hotspots of natural iron binding ligands production up to 50 μM in the wheat rhizosphere. The lower detection limit in our experiment was 2.5 μmol/L. This new technique offers a unique opportunity to investigate the siderophore production in two dimensions in a wide range of applications from laboratory experiments to natural systems very likely using an in situ and nondestructive tool.

Efficient establishment of pure cultures of yellow chanterelle Cantharellus anzutake from ectomycorrhizal root tips, and morphological characteristics of ectomycorrhizae and cultured mycelium

Abstract Species of fleshy yellow Cantharellus are known as chanterelles, which are among the most popular wild edible mycorrhizal mushrooms in the world. However, pure culture isolates of Cantharellus are rare. We report an efficient isolation technique of the Japanese golden chanterelle, Cantharellus anzutake, from its ectomycorrhizal root tips. Field-sampled fresh ectomycorrhizal root tips of C. anzutake on various hosts such as pines, spruce, and oaks were vortexed with 0.005% Tween 80 solution, surface sterilized with 1% calcium hypochlorite solution, rinsed with sterilized distilled water, and placed on modified Norkrans’ C (MNC) agar plate medium. Most ectomycorrhizal root tips of C. anzutake produced yellowish mycelial colonies within a few months. In contrast, tissue isolation from basidiomata provided limited cultures of C. anzutake but much contamination of bacteria and molds, even on media that contained antibiotics. The established C. anzutake cultures had clamp connections on the hyphae and contained intracellular oily droplets. These cultured isolates were identified as C. anzutake by sequence analysis of the rRNA internal transcribed spacer (ITS) region and translation elongation factor EF1-alpha (tef-1) genes.

Bioaerosol emissions associated with pit latrine emptying operations

Pit latrines are the most common sanitation option in the developing world. They are simple to build but require periodic emptying which results in widespread dispersion of fecal pathogens in the environment. While much is known about the health risks of fecal-oral exposure, little is known about those resulting from the aerosolization of pathogens from fecal material. Bioaerosols were sampled around seven pit latrines before, after, and during emptying in Blantyre, Malawi. Bioaerosols were collected directly onto nutrient and selective medium agar plates using an impact sampler. DNA was extracted from some plates and analyzed for selected enteric pathogens. Total heterotrophic bacteria in the air during active emptying ranged from 198 to >13,000 colony forming units (CFU) per m3, and generally increased above background levels during pit emptying. At about one meter from the pit latrine emptying, E. coli and total coliforms concentrations in air reached up to 350 and 790 CFU m−3, respectively. Additionally, at four out of the seven pit latrines sites sampled, enterotoxigenic E. coli (ETEC) LT/ST was confirmed to be present in bioaerosols. This work demonstrates the potential for airborne dispersion of enteric pathogens during pit latrine emptying operations.

Evaluation of antibacterial properties of hydroxyapatite/bioactive glass and fluorapatite/bioactive glass nanocomposite foams as a cellular scaffold of bone tissue.

AIMS AND OBJECTIVES:Infection is a serious problem for patients after implantation surgery, which is difficult to treat with antibiotic therapy. The present study was developed to evaluate and compare the antibacterial properties of hydroxyapatite/bioactive glass (HA/BG) and fluorapatite/bioactive glass (FA/BG) nanocomposite foams as a cellular scaffold for use in bone defects by two macrodilution and disk diffusion methods.MATERIALS AND METHODS:Staphylococcus aureus, Enterococcus faecalis, and Streptococcus mutans were cultured in brain heart infusion broth medium with nanocomposite powder for 5 days, and their bioactivity levels were evaluated by daily culturing on solid agar medium plates. To carry out the disk diffusion test, a disc form of nanocomposite foams was used on agar medium with 48 h incubation.RESULTS:None of two nanocomposites even at their highest concentration (200 mg/mL) did not prevent the growth of two Staphylococcus aureus and Enterococcus faecalis microorganisms. However, HA/BG nanocomposite on the 3rd day at a concentration of 200 mg/mL and on 4th and 5th day at a concentration of 100 mg/mL and FA/BG nanocomposite on the 4th day at a concentration of 100 mg/mL and on the 5th day at a concentration of 50 mg/mL could be able to kill Streptococcus mutans microorganism. In the disc diffusion test, none of the nanocomposites could create a nongrowth zone. Both tested biomaterials showed increased antibacterial properties over time and concentration increase.CONCLUSION:HA/BG and FA/BG nanocomposites, due to their biocompatibility and antimicrobial properties, are good choices for implantation instead of damaged bone tissue in tissue engineering.

1000 Ways to Die: Synthetic Lethality with an HDAC

Histones are proteins that can be post‐translationally modified and these modifications will determine the expression of genes. Many studies have shown that it is very common for cancers to have mutations in their histone genes. The concept of synthetic lethality states that if a cell has a single mutation it will be able to survive. But if a second mutation is applied, the cell will become synthetically sick and/or die. In our study, we are seeking to determine synthetically lethal interactions between the histone H3 gene and RPD3 histone deacetylase enzyme. In order to discover potential genetic targets to develop therapies against cancers. To achieve this we used a genetically engineered yeast strain that has the histone H3 gene and RPD3 gene deleted. The H3 gene deletion is covered by a wild type H3 plasmid. These yeast were then transformed with plasmids from the SHIMA library. The yeast were then grown on plates lacking uracil and tryptophan. The individual colonies were then patched and replica plated onto tryptophan deficient media. Finally, they were replica plated onto agar plate media containing 5‐ FOA. The DNA from yeast deemed synthetically lethal or sick were then put through “Library Prep” which is a high throughput method used to isolate the H3 gene within the plasmid with low error rate. The lethals were then put into Next Generation sequencing, aligned, and transferred in the IGV. Finally, we were able to estimate with high accuracy the frequency of H3 mutants in our lethal pool. The top three frequencies discovered were F67A, F84A, and Q93A. They represented 35%, 12% and 10% of our lethal pool respectively. In total we discovered ten new potential synthetically lethal interactions between histone H3 and RPD3. We also used this information in combination with previous knowledge to generate a genetic interaction map for RPD3 from our findings. Although still in the preliminary stages when verified with duplication studies our new potential synthetically lethal interactions can be targets to develop cancer therapies. Also, the knowledge gained from our study can be used to understand genetic interaction between histones and histone modification enzymes.Support or Funding InformationSummer Medical Research Fellowship at Rowan University School of Osteopathic MedicineThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inactivation Effect of Antibiotic-Resistant Gene Using Chlorine Disinfection

The aim of this study was to elucidate the inactivation effects on the antibiotic-resistance gene (vanA) of vancomycin-resistant enterococci (VRE) using chlorination, a disinfection method widely used in various water treatment facilities. Suspensions of VRE were prepared by adding VRE to phosphate-buffered saline, or the sterilized secondary effluent of a wastewater treatment plant. The inactivation experiments were carried out at several chlorine concentrations and stirring time. Enterococci concentration and presence of vanA were determined. The enterococci concentration decreased as chlorine concentrations and stirring times increased, with more than 7.0 log reduction occurring under the following conditions: 40 min stirring at 0.5 mg Cl2/L, 20 min stirring at 1.0 mg Cl2/L, and 3 min stirring at 3.0 mg Cl2/L. In the inactivation experiment using VRE suspended in secondary effluent, the culturable enterococci required much higher chlorine concentration and longer treatment time for complete disinfection than the cases of suspension of VRE. However, vanA was detected in all chlorinated suspensions of VRE, even in samples where no enterococcal colonies were present on the medium agar plate. The chlorine disinfection was not able to destroy antibiotic-resistance genes, though it can inactivate and decrease bacterial counts of antibiotic-resistant bacteria (ARB). Therefore, it was suggested that remaining ARB and/or antibiotic-resistance gene in inactivated bacterial cells after chlorine disinfection tank could be discharged into water environments.