Search results for: P. chrysogenum broth

Authors: Lucia Steenkamp, Chris V. D. Westhuyzen, Kgama Mathiba

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Ambergris, and more specifically its oxidation product (–)-ambrafuran, is a scarce, valuable, and sought-after perfumery ingredient. The material is used as a fixative agent to stabilise perfumes in formulations by reducing the evaporation rate of volatile substances. Ambergris is a metabolic product of the sperm whale (Physeter macrocephatus L.), resulting from intestinal irritation. Chemically, (–)-ambrafuran is produced from the natural product sclareol in eight synthetic steps – in the process using harsh and often toxic chemicals to do so. An overall yield of no more than 76% can be achieved in some routes, but generally, this is lower. A new 'green' route has been developed in our laboratory in which sclareol, extracted from the Clary sage plant, is converted to (–)-ambrafuran in two steps with an overall yield in excess of 80%. The first step uses a microorganism, Hyphozyma roseoniger, to bioconvert sclareol to an intermediate diol using substrate concentrations up to 50g/L. The yield varies between 90 and 67% depending on the substrate concentration used. The purity of the diol product is 95%, and the diol is used without further purification in the next step. The intermediate diol is then cyclodehydrated to the final product (–)-ambrafuran using a zeolite, which is not harmful to the environment and is readily recycled. The yield of the product is 96%, and following a single recrystallization, the purity of the product is > 99.5%. A preliminary LC-MS study of the bioconversion identified several intermediates produced in the fermentation broth under oxygen-restricted conditions. Initially, a short-lived ketone is produced in equilibrium with a more stable pyranol, a key intermediate in the process. The latter is oxidised under Norrish type I cleavage conditions to yield an acetate, which is hydrolysed either chemically or under lipase action to afford the primary fermentation product, an intermediate diol. All the intermediates identified point to the likely CYP450 action as the key enzyme(s) in the mechanism. This invention is an exceptional example of how the power of biocatalysis, combined with a mild, benign chemical step, can be deployed to replace a total chemical synthesis of a specific chiral antipode of a commercially relevant material.

Keywords: ambrafuran, biocatalysis, fragrance, microorganism

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Authors: Kanika Kalra, Harmeet Kaur, Dinesh Goyal

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Total phenolics were extracted from waste orange peels by solvent extraction and alkali hydrolysis method. The most efficient solvents for extracting phenolic compounds from waste biomass were methanol (60%) > dimethyl sulfoxide > ethanol (60%) > distilled water. The extraction yields were significantly impacted by solvents (ethanol, methanol, and dimethyl sulfoxide) due to varying polarity and concentrations. Extraction of phenolics using 60% methanol yielded the highest phenolics (in terms of gallic acid equivalent (GAE) per gram of biomass) in orange peels. Alkali hydrolyzed extract from orange peels contained 7.58±0.33 mg GAE g⁻¹. By using the solvent extraction technique, it was observed that 60% methanol is comparatively the best-suited solvent for extracting polyphenolic compounds and gave the maximum yield of 4.68 ± 0.47 mg GAE g⁻¹ in orange peel extracts. DPPH radical scavenging activity and reducing the power of orange peel extract were checked, where 60% methanolic extract showed the highest antioxidant activity, 85.50±0.009% for DPPH, and dimethyl sulfoxide (DMSO) extract gave the highest yield of 1.75±0.01% for reducing power ability of the orange peels extract. Characterization of the polyphenolic compounds was done by using Fourier transformation infrared (FTIR) spectroscopy. Solvent and alkali hydrolysed extracts were evaluated for antibacterial activity using the agar well diffusion method against Gram-positive Bacillus subtilis MTCC441 and Gram-negative Escherichia coli MTCC729. Methanolic extract at 300µl concentration showed an inhibition zone of around 16.33±0.47 mm against Bacillus subtilis, whereas, for Escherichia coli, it was comparatively less. Broth-based turbidimetric assay revealed the antibacterial effect of different volumes of orange peel extracts against both organisms.

Keywords: orange peels, total phenolic content, antioxidant, antibacterial

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Authors: Abdul Matin, Muazzama Akhtar, Shahid Nisar, Saddaf Mazzar, Umer Rashid

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Antibiotic resistance is an increasing concern worldwide now a day. Neisseria gonorrhea and Staphylococcus aureus are known to cause major human sexually transmitted and respiratory diseases respectively. Nanotechnology is an emerging discipline and its application in various fields especially in medical sciences is gigantic. In the present study, we synthesized multi-walled carbon nanotubes (MWNTs) using acid oxidation method and solubilized MWNTs were with length predominantly >500 nm and diameters ranging from 40 to 50 nm. The locally synthesized MWNTs were used against gram positive and negative bacteria to determine their impact on bacterial growth. Clinical isolates of Neisseria gonorrhea (isolate: 4C-11) and Staphylococcus aureus (isolate: 38541) were obtained from local hospital and normally cultured in LB broth at 37°C. Both clinical strains can be obtained on request from University of Gujarat. Spectophometric assay was performed to determine the impact of MWNTs on bacterial growth in vitro. To determine the effect of MWTNs on test organisms, various concentration of MWNTs were used and recorded observation on various time intervals to understand the growth inhibition pattern. Our results demonstrated that MWNTs exhibited toxic effects to Staphylococcus aureus while showed very limited growth inhibition to Neisseria gonorrhea, which suggests the resistant potential of Neisseria against nanoparticles. Our results clearly demonstrate the gradual decrease in bacterial numbers with passage of time when compared with control. Maximum bacterial inhibition was observed at maximum concentration (50 µg/ml). Our future work will include further characterization and mode of action of our locally synthesized MWNTs. In conclusion, we investigated and reported for the first time the inhibitory potential of locally synthesized MWNTs on local clinical isolates of Staphylococcus aureus and Neisseria gonorrhea.

Keywords: antibacterial activity, multi walled carbon nanotubes, Neisseria gonorrhea, spectrophotometer assay, Staphylococcus aureus

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Authors: Huyen T. Pham, Nhue P. Nguyen, Tien Q. Phi, Phuong T. Dang, Hy G. Le

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Since the marine environmental conditions are extremely different from the other ones, so that marine actinomycetes might produce novel bioactive compounds. Therefore, actinomycete strains were screened from marine water and sediment samples collected from the coastal areas of Northern Vietnam. Ninety-nine actinomycete strains were obtained on starch-casein agar media by dilution technique, only seven strains, named HP112, HP12, HP411, HPN11, HP 11, HPT13 and HPX12, showed significant antibacterial activity against both gram-positive and gram-negative bacteria (Bacillus subtilis ATCC 6633, Staphylococcus epidemidis ATCC 12228, Escherichia coli ATCC 11105). Further studies were carried out with the most active HP411strain against Candida albicans ATCC 10231. This strain could grow rapidly on starch casein agar and other media with high salt containing 7-10% NaCl at 28-30oC. Spore-chain of HP411 showed an elongated and circular shape with 10 to 30 spores/chain. Identification of the strain was carried out by employing the taxonomical studies including the 16S rRNA sequence. Based on phylogenetic and phenotypic evidence it is proposed that HP411 to be belongs to species Streptomyces variabilis. The potent of the crude extract of fermentation broth of HP411that are effective against wide range of pathogens: both gram-positive, gram-negative and fungi. Further studies revealed that the crude extract HP411 could obtain the anticancer activity for cancer cell lines: Hep-G2 (liver cancer cell line); RD (cardiac and skeletal muscle letters cell line); FL (membrane of the uterus cancer cell line). However, the actinomycetes from marine ecosystem will be useful for the discovery of new drugs in the furture.

Keywords: marine actinomycetes, antibacterial, anticancer, Streptomyces variabilis

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Authors: Kumudini Apsara Munasinghe, Devin Gregory Lissau, Ryan Thomas Wade

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Fresh fruits and vegetables are rich in vitamins, minerals, and fibers and help maintain a healthy weight over high-calorie food. Eating fruits and vegetables protects us from free radicals produced by metabolic reactions and safeguards us from cardiovascular disease and cancer. However, there has been an increased concern about foodborne diseases tied to contaminated farmers’ market produce. In addition, very little information is available about the contribution of eating raw fruits and vegetables to human exposure to antibiotic-resistant bacteria. This research aims to identify bacteria isolated from farmers’ market fruits and vegetables and understand their antibiotic resistance. Vegetables and fruits were collected from farmers’ markets around Frostburg and Cumberland areas in Maryland and transported to the microbiology lab at Frostburg State University for the isolation of bacteria. Bacteria were extracted from tomatoes, cucumber, strawberry, and lettuce using Tryptic soy broth overnight at 37°C, and Tryptic Soy agar was used for the streak plate technique to isolate bacteria. Pure cultures were used to identify bacteria using biochemical reactions after conducting Gram staining technique. The research used many biochemical reactions, including Mannitol Salt agar, MacConkey agar, and Eosin Methylene blue agar, for identification. Antibiotic sensitivity was tested for many different types of antibiotics, including amoxicillin, penicillin, tetracycline, ampicillin, and erythromycin. Most prevalent bacteria in the isolates were Staphylococcus, Bacillus, Micrococcus, Enterococcus, Enterobacter, Citrobacter, and other bacteria from the family Enterobacteriaceae. The data obtained from this research will be useful to educate and train farmers and individuals involved in post-harvest processes such as transportation and selling in farmers’ markets. Further results for bacterial antibiotic resistance will be obtained, and unculturable bacteria will be identified by next-generation DNA sequencing.

Keywords: antibiotic resistance, farmers markets, fruits, bacteria, vegetables

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Authors: Constance Chivengwa, Tinashe Mandimutsira, Jephris Gere, Charles Magogo, Irene Chikanza, Jerneja Vidmar, Walter Chingwaru

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The use of traditional methods in the management of diarrhoea has remained a common practice among the indigenous African tribes of Southern Africa. Despite the widespread use of traditional medicines in Zimbabwe, very little research validating the activities of phytomedicines against diarrhoea, as claimed by the Shona people of Zimbabwe, has been reported. This study sought to determine the efficacies of the plants that are frequently used to treat stomach complaints, namely Dicoma anomala, Cassia abbreviata, Lannea edulis and Peltophorum africanum against Escherichia coli (an indicator of faecal contamination of water, and whose strains such as EHEC (O157), ETEC and EPEC, are responsible for a number of outbreaks of diarrhoea) and Salmonella spp. Ethanol and aqueous extracts from these plants were obtained, evaporated, dried and stored. The dried extracts were reconstituted and diluted 10-fold in nutrient broth (from 100 to 0.1 microgram/mL) and tested for inhibition against the bacteria. L. edulis exhibited the best antimicrobial effect (minimum inhibition concentration = 10 microgram/mL for both extracts and microorganisms). Runners up to L. edulis were C. abbreviata (20 microgram/mL for both microorganisms) and P. africanum (20 and 30 microgram/mL respectively). Interestingly, D. anomala, which is widely considered panacea in African medicinal practices, showed low antimicrobial activity (60 and 100 microgram/mL respectively). The high antimicrobial activity of L. edulis can be explained by its content of flavonoids, tannins, alkylphenols (cardonol 7 and cardonol 13) and dihydroalkylhexenones. The antimicrobial activities of C. abbreviata can be linked to its content of anthraquinones and triterpenoids. P. africanum is known to contain benzenoids, flavanols, flavonols, terpenes, xanthone and coumarins. This study therefore demonstrated that, among the plants that are used against diarrhoea in African traditional medicine, L. edulis is a clear winner against E. coli and Salmonella spp. Activity guided extraction is encouraged to establish the complement of compounds that have antimicrobial activities.

Keywords: diarrhoea, Escherichia coli, Salmonella, phytomedicine, MIC, Zimbabwe

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Authors: Kailas L. Wasewar

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Biobutanol has been considered as a potential and alternative biofuel relative to the most popular biodiesel and bioethanol. End product toxicity is the major problems in commercialization of fermentation based process which can be reduce to some possible extent by removing biobutanol simultaneously. Several techniques have been investigated for removing butanol from fermentation broth such as stripping, adsorption, liquid–liquid extraction, pervaporation, and membrane solvent extraction. Liquid–liquid extraction can be performed with high selectivity and is possible to carry out inside the fermenter. Conventional solvents have few drawbacks including toxicity, loss of solvent, high cost etc. Hence alternative solvents must be explored for the same. Room temperature ionic liquids (RTILs) composed entirely of ions are liquid at room temperature having negligible vapor pressure, non-flammability, and tunable physiochemical properties for a particular application which term them as “designer solvents”. Ionic liquids (ILs) have recently gained much attention as alternatives for organic solvents in many processes. In particular, ILs have been used as alternative solvents for liquid–liquid extraction. Their negligible vapor pressure allows the extracted products to be separated from ILs by conventional low pressure distillation with the potential for saving energy. Morpholinium, imidazolium, ammonium, phosphonium etc. based ionic liquids have been employed for the separation biobutanol. In present chapter, basic concepts of ionic liquids and application in separation have been presented. Further, type of ionic liquids including, conventional, functionalized, polymeric, supported membrane, and other ionic liquids have been explored. Also the effect of various performance parameters on separation of biobutanol by ionic liquids have been discussed and compared for different cation and anion based ionic liquids. The typical methodology for investigation have been adopted such as contacting the equal amount of biobutanol and ionic liquids for a specific time say, 30 minutes to confirm the equilibrium. Further, biobutanol phase were analyzed using GC to know the concentration of biobutanol and material balance were used to find the concentration in ionic liquid.

Keywords: biobutanol, separation, ionic liquids, sustainability, biorefinery, waste biomass

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Authors: Cengiz Demir, Recep Keşli, Gülşah Aşık

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Objective: This study was carried out with the purpose of defining by using the E test method and determining the antibiotic resistance profiles of Gram-negative anaerobic bacilli isolated from various clinical specimens obtained from patients with suspected anaerobic infections and referred to Medical Microbiology Laboratory of Afyon Kocatepe University, ANS Application and Research Hospital. Methods: Two hundred and seventy eight clinical specimens were examined for isolation of the anaerobic bacteria in Medical Microbiology Laboratory between the 1st November 2014 and 30th October 2015. Specimens were cultivated by using Scheadler agar that 5% defibrinated sheep blood added, and Scheadler broth. The isolated anaerobic Gram-negative bacilli were identified conventional methods and Vitek 2 (ANC ID Card, bioMerieux, France) cards. Antibiotic resistance rates against to penicillin G, clindamycin, cefoxitin, metronidazole, moxifloxacin, imipenem, meropenem, ertapenem and doripenem were determined with E-test method for each isolate. Results: Of the isolated twenty-eight anaerobic gram negative bacilli fourteen were identified as the B. fragilis group, 9 were Prevotella group, and 5 were Fusobacterium group. The highest resistance rate was found against penicillin (78.5%) and resistance rates against clindamycin and cefoxitin were found as 17.8% and 21.4%, respectively. Against to the; metronidazole, moxifloxacin, imipenem, meropenem, ertapenem and doripenem, no resistance was found. Conclusion: Since high rate resistance has been detected against to penicillin in the study penicillin should not be preferred in empirical treatment. Cefoxitin can be preferred in empirical treatment; however, carrying out the antibiotic sensitivity testing will be more proper and beneficial. No resistance was observed against carbapenem group antibiotics and metronidazole; so that reason, these antibiotics should be reserved for treatment of infectious caused by resistant strains in the future.

Keywords: anaerobic gram-negative bacilli, anaerobe, antibiotics and resistance profiles, e-test method

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Authors: Seyedeh Banafsheh Bagheri Marzouni, Sona Rostampour Yasouri

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Salmonella is one of the most important common pathogens between humans and animals worldwide. Globally, the prevalence of the disease in humans is due to the consumption of food contaminated with animal-derived Salmonella. These foods include eggs, red meat, chicken, and milk. Contamination of chicken and its products with Salmonella may occur at any stage of the chicken processing chain. Salmonella infection is usually not fatal. However, its occurrence is considered dangerous in some individuals, such as infants, children, the elderly, pregnant women, or individuals with weakened immune systems. If Salmonella infection enters the bloodstream, the possibility of contamination of tissues throughout the body will arise. Therefore, determining the potential risk of Salmonella at various stages is essential from the perspective of consumers and public health. The aim of this study is to isolate and identify Salmonella from chicken samples distributed in the Tehran market using the Gold standard culture method and PCR techniques based on specific genes, invA and ent. During the years 2022-2023, sampling was performed using swabs from the liver and intestinal contents of distributed chickens in the Tehran province, with a total of 120 samples taken under aseptic conditions. The samples were initially enriched in buffered peptone water (BPW) for pre-enrichment overnight. Then, the samples were incubated in selective enrichment media, including TT broth and RVS medium, at temperatures of 37°C and 42°C, respectively, for 18 to 24 hours. Organisms that grew in the liquid medium and produced turbidity were transferred to selective media (XLD and BGA) and incubated overnight at 37°C for isolation. Suspicious Salmonella colonies were selected for DNA extraction, and PCR technique was performed using specific primers that targeted the invA and ent genes in Salmonella. The results indicated that 94 samples were Salmonella using the PCR technique. Of these, 71 samples were positive based on the invA gene, and 23 samples were positive based on the ent gene. Although the culture technique is the Gold standard, PCR is a faster and more accurate method. Rapid detection through PCR can enable the identification of Salmonella contamination in food items and the implementation of necessary measures for disease control and prevention.

Keywords: culture, PCR, salmonella spp, salmonella enteritidis

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Authors: Sze Wing Ho, Nagendra P. Shah

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Lactic acid bacteria (LAB) have shown the beneficial effects on human gastrointestinal tract, such as protects diarrhea induced by lactose intolerance or enteric pathogens. Citrulline is a non-protein amino acid and also the precursors of arginine and nitric oxide, it has shown to enhance intestinal barrier function. Citrulline has shown to improve the growth of some strains of LAB, it is important for LAB to have a sufficient cell concentration to contribute the effects. Therefore, the aims of this study were to investigate the effect of combining citrulline with LAB on the anti-adhesion effect against pathogens and the effect on the cell integrity. The effect of citrulline on selected LAB was determined by incubating in 0%, 0.1% or 0.2% citrulline enriched MRS broth for 18 h. The adhesion ability of LAB and the anti-adhesion effect of LAB and citrulline against pathogens were performed on IPEC-J2 cell line. Transepithelial electrical resistance (TEER) assay was used to measure the tight junction (TJ) integrity. TJ proteins (claudin-1, occludin and zonula occluden-1 (ZO-1)) were determined by western blot analysis. It found that the growth of Lactobacillus helveticus ASCC 511 was significantly stimulated by 0.2% citrulline compared with control during 18 h fermentation. The adhesion of L. helveticus ASCC 511 and Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) ASCC 756 was increased when supplemented with citrulline. Citrulline has shown significant inhibitory effect on the adhesion of Escherichia coli PELI0480 (O157:H7), Shigella sonnei ATCC 25931, Staphyloccocus aureus CMCC26003 and Cronobacter sakazakii ATCC 29544. The anti-adhesion effect of L. helveticus ASCC 511, L. bulgaricus ASCC 756 and Lactobacillus paracasei ASCC 276 against Cronobacter sakazakii ATCC 29544 was significantly enhanced with citrulline supplementation. Treatments with citrulline and LAB were able to maintain the TEER of IPEC-J2 cell and shown the positive effect on the TJ proteins. In conclusion, citrulline had stimulating effect on some strains of LAB and determined to improve the adhesion of LAB on intestinal epithelial cell, to enhance the inhibitory effect on enteric pathogens adhesion as well as had beneficial effects on maintaining cell integrity. It implied LAB supplemented with citrulline might have advantageous effects on gastrointestinal tracts.

Keywords: citrulline, lactic acid bacteria, amino acid, anti-adhesion effect, cell integrity

Procedia PDF Downloads 215

Authors: Hyvee Gean Cabuso, Arvin Taruc, Danielle Villanueva, Channela Anais Hipolito, Jia Bianca Alfonso

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Introduction: Denture adhesive aids to provide additional retention, support and comfort for patients with loose dentures, as well as for patients who seek to achieve optimal denture adhesion. But due to its growing popularity, arising oral health issues should be considered, including its possible impact that may alter the microbiological condition of the denture. Changes as such may further resolve to denture-related oral diseases that can affect the day-to-day lives of patients. Purpose: The study aims to assess and compare the microbiological status of dentures without adhesives versus dentures when adhesives were applied. The study also intends to identify the presence of specific microorganisms, their colony concentration and their possible effects on the oral microflora. This study also aims to educate subjects by introducing an alternative denture cleaning method as well as denture and oral health care. Methodology: Edentulous subjects age 50-80 years old, both physically and medically fit, were selected to participate. Before obtaining samples for the study, the alternative cleaning method was introduced by demonstrating a step-by-step cleaning process. Samples were obtained by swabbing the intaglio surface of their upper and lower prosthesis. These swabs were placed in a thioglycollate broth, which served as a transport and enrichment medium. The swabs were then processed through bacterial culture. The colony-forming units (CFUs) were calculated on MacConkey Agar Plate (MAP) and Blood Agar Plate (BAP) in order to identify and assess the microbiological status, including species identification and microbial counting. Result: Upon evaluation and analysis of collected data, the microbiological assessment of the upper dentures with adhesives showed little to no difference compared to dentures without adhesives, but for the lower dentures, (P=0.005), which is less than α = 0.05; therefore, the researchers reject (Ho) and that there is a significant difference between the mean ranks of the lower denture without adhesive to those with, implying that there is a significant decrease in the bacterial count. Conclusion: These results findings may implicate the possibility that the addition of denture adhesives may contribute to the significant decrease of microbial colonization on the dentures.

Keywords: denture, denture adhesive, denture-related, microbiological assessment

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Authors: Naira Sahakyan, Margarit Petrosyan, Armen Trchounian

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One of the tasks in contemporary biotechnology, pharmacology and other fields of human activities is to obtain biologically active substances from plants. They are very essential in the treatment of many diseases due to their actually high therapeutic value without visible side effects. However, sometimes the possibility of obtaining the metabolites is limited due to the reduction of wild-growing plants. That is why the plant cell cultures are of great interest as alternative sources of biologically active substances. Besides, during the monitored cultivation, it is possible to obtain substances that are not synthesized by plants in nature. Isolated culture of Ajuga genevensis with high growth activity and ability of regeneration was obtained using MS nutrient medium. The agar-diffusion method showed that aqueous extracts of callus culture revealed high antimicrobial activity towards various gram-positive (Bacillus subtilis A1WT; B. mesentericus WDCM 1873; Staphylococcus aureus WDCM 5233; Staph. citreus WT) and gram-negative (Escherichia coli WKPM M-17; Salmonella typhimurium TA 100) microorganisms. The broth dilution method revealed that the minimal and half maximal inhibitory concentration values against E. coli corresponded to the 70 μg/mL and 140 μg/mL concentration of the extract respectively. According to the photochemiluminescent analysis, callus tissue extracts of leaf and root origin showed higher antioxidant activity than the same quantity of A. genevensis intact plant extract. A. genevensis intact plant and callus culture extracts showed no cytotoxic effect on K-562 suspension cell line of human chronic myeloid leukemia. The GC-MS analysis showed deep differences between the qualitative and quantitative composition of callus culture and intact plant extracts. Hexacosane (11.17%); n-hexadecanoic acid (9.33%); and 2-methoxy-4-vinylphenol (4.28%) were the main components of intact plant extracts. 10-Methylnonadecane (57.0%); methoxyacetic acid, 2-tetradecyl ester (17.75%) and 1-Bromopentadecane (14.55%) were the main components of A. genevensis callus culture extracts. Obtained data indicate that callus culture of A. genevensis can be used as an alternative source of biologically active substances.

Keywords: Ajuga genevensis, antibacterial activity, antioxidant activity, callus cultures

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Authors: William Kelly, Sorelle Veigne, Xianhua Li, Zuyi Huang, Shyamsundar Subramanian, Eugene Schaefer

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The ambr15™ bioreactor is a single-use microbioreactor for cell line development and process optimization. The ambr system offers fully automatic liquid handling with the possibility of fed-batch operation and automatic control of pH and oxygen delivery. With operating conditions for large scale biopharmaceutical production properly scaled down, micro bioreactors such as the ambr15™ can potentially be used to predict the effect of process changes such as modified media or different cell lines. In this study, gassing rates and dilution rates were varied for a semi-continuous cell culture system in the ambr15™ bioreactor. The corresponding changes to metabolite production and consumption, as well as cell growth rate and therapeutic protein production were measured. Conditions were identified in the ambr15™ bioreactor that produced metabolic shifts and specific metabolic and protein production rates also seen in the corresponding larger (5 liter) scale perfusion process. A Dynamic Flux Balance model was employed to understand and predict the metabolic changes observed. The DFB model-predicted trends observed experimentally, including lower specific glucose consumption when CO₂ was maintained at higher levels (i.e. 100 mm Hg) in the broth. A Computational Fluid Dynamic (CFD) model of the ambr15™ was also developed, to understand transfer of O₂ and CO₂ to the liquid. This CFD model predicted gas-liquid flow in the bioreactor using the ANSYS software. The two-phase flow equations were solved via an Eulerian method, with population balance equations tracking the size of the gas bubbles resulting from breakage and coalescence. Reasonable results were obtained in that the Carbon Dioxide mass transfer coefficient (kLa) and the air hold up increased with higher gas flow rate. Volume-averaged kLa values at 500 RPM increased as the gas flow rate was doubled and matched experimentally determined values. These results form a solid basis for optimizing the ambr15™, using both CFD and FBA modelling approaches together, for use in microscale simulations of larger scale cell culture processes.

Keywords: cell culture, computational fluid dynamics, dynamic flux balance analysis, microbioreactor

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Authors: Saifalarab A. Mohmmed, Morgana E. Vianna, Jonathan C. Knowles

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The issue of apical periodontitis has received considerable critical attention. Bacteria is integrated into communities, attached to surfaces and consequently form biofilm. The biofilm structure provides bacteria with a series protection skills against, antimicrobial agents and enhances pathogenicity (e.g. apical periodontitis). Sodium hypochlorite (NaOCl) has become the irrigant of choice for elimination of bacteria from the root canal system based on its antimicrobial findings. The aim of the study was to investigate the effect of different agitation techniques on the efficacy of 2.5% NaOCl to eliminate the biofilm from the surface of the lateral canal using the residual biofilm, and removal rate of biofilm as outcome measures. The effect of canal complexity (lateral canal) on the efficacy of the irrigation procedure was also assessed. Forty root canal models (n = 10 per group) were manufactured using 3D printing and resin materials. Each model consisted of two halves of an 18 mm length root canal with apical size 30 and taper 0.06, and a lateral canal of 3 mm length, 0.3 mm diameter located at 3 mm from the apical terminus. E. faecalis biofilms were grown on the apical 3 mm and lateral canal of the models for 10 days in Brain Heart Infusion broth. Biofilms were stained using crystal violet for visualisation. The model halves were reassembled, attached to an apparatus and tested under a fluorescence microscope. Syringe and needle irrigation protocol was performed using 9 mL of 2.5% NaOCl irrigant for 60 seconds. The irrigant was either left stagnant in the canal or activated for 30 seconds using manual (gutta-percha), sonic and ultrasonic methods. Images were then captured every second using an external camera. The percentages of residual biofilm were measured using image analysis software. The data were analysed using generalised linear mixed models. The greatest removal was associated with the ultrasonic group (66.76%) followed by sonic (45.49%), manual (43.97%), and passive irrigation group (control) (38.67%) respectively. No marked reduction in the efficiency of NaOCl to remove biofilm was found between the simple and complex anatomy models (p = 0.098). The removal efficacy of NaOCl on the biofilm was limited to the 1 mm level of the lateral canal. The agitation of NaOCl results in better penetration of the irrigant into the lateral canals. Ultrasonic agitation of NaOCl improved the removal of bacterial biofilm.

Keywords: 3D printing, biofilm, root canal irrigation, sodium hypochlorite

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Authors: Savitha Janakiraman, Shivakumar M. C

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Anidulafungin, an advanced class of antifungal agent used for the treatment of chronic fungal infections, is derived from Echinocandin B nucleus, an intermediate metabolite of Echinocandin B produced by Aspergillus nidulans. The enzyme acylase derived from the fermentation broth of Actinoplanes utahensis (NRRL 12052) plays a key role in the bioconversion of echinocandin B to echinocandin B nucleus. The membrane-bound nature of acylase and low levels of expression contributes to the rate-limiting process of enzymatic deacylation, hence low yields of ECB nucleus and anidulafungin. In the present study, this is addressed through novel genetic engineering approaches of overexpression and heterologous expression studies, immobilization of whole cells of Actinoplanes utahensis (NRRL 12052) and Co-cultivation studies. Overexpression of the acylase gene in Actinoplanes utahensis (NRRL 12052) was done by increasing the gene copy number to increase the echinocandin B nucleus production. Echinocandin B acylase gene, under the control of a PermE* promoter, was cloned in pSET152 vector and introduced into Actinoplanes utahensis (NRRL12052) by a ɸC31-directed site-specific recombination method. The resultant recombinant strain (C2-18) showed a 3-fold increase in acylase expression, which was confirmed by HPLC analysis. Pichia pastoris is one of the most effective and versatile host systems for the production of heterologous proteins. The ECB acylase gene was cloned into pPIC9K vector with AOX1 promoter and was transformed into Pichia pastoris (GS115). The acylase expression was confirmed by protein expression and bioconversion studies. The heterologous expression of acylase in Pichia pastoris, is a milestone in the development of antifungals. Actively growing cells of Actinoplanes utahensis (NRRL 12052) were immobilized and tested for bioconversion ability which showed >90% conversion in each cycle. The stability of immobilized cell beads retained the deacylation ability up to 60 days and reusability was confirmed up to 4 cycles. The significant findings from the study have revealed that immobilization of whole cells of Actinoplanes utahensis (NRRL 12052) could be an alternative option for bioconversion of echinocandin B to echinocandin B nucleus, which has not been reported to date. The concept of co-cultivation of Aspergillus nidulans and Actinoplanes utahensis strains for the production of the echinocandin B nucleus was also carried out in order to produce echinocandin B nucleus. The process completely reduced the ECB purification step and, therefore, could be recommended as an ingenious method to improve the yield of the ECB nucleus.

Keywords: acylase, anidulafungin, antifungals, Aspergillus nidulans

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Authors: Magdalena Diaka, Paweł Mazierski, Joanna Żebrowska, Michał Winiarski, Tomasz Klimczuk, Adriana Zaleska-Medynska

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During the last years, TiO2 nanotubes have been widely studied due to their unique highly ordered array structure, unidirectional charge transfer and higher specific surface area compared to conventional TiO2 powder. These photoactive materials, in the form of thin layer, can be activated by low powered and low cost irradiation sources (such as LEDs) to remove VOCs, microorganism and to deodorize air streams. This is possible due to their directly growth on a support material and high surface area, which guarantee enhanced photon absorption together with an extensive adsorption of reactant molecules on the photocatalyst surface. TiO2 nanotubes exhibit also lots of other attractive properties, such as potential enhancement of electron percolation pathways, light conversion, and ion diffusion at the semiconductor-electrolyte interface. Pure TiO2 nanotubes were previously used to remove organic compounds from the gas phase as well as in water splitting reaction. The major factors limiting the use of TiO2 nanotubes, which have not been fully overcome, are their relatively large band gap (3-3,2 eV) and high recombination rate of photogenerated electron–hole pairs. Many different strategies were proposed to solve this problem, however titania nanostructures containing incorporated metal oxides like Ag2O shows very promising, new optical and photocatalytic properties. Unfortunately, there is still very limited number of reports regarding application of TiO2/MxOy nanostructures. In the present work, we prepared TiO2/Ag2O nanotubes obtained by anodization of Ti-Ag alloys containing 5, 10 and 15 wt. % Ag. Photocatalysts prepared in this way were characterized by X-ray diffraction spectroscopy (XRD), scanning electron microscopy (SEM), luminescence spectroscopy and UV-Vis spectroscopy. The activities of new TiO2/Ag2O were examined by photocatalytic degradation of toluene in gas phase reaction and phenol in aqueous phase using 1000 W Xenon lamp (Oriel) and light emitting diodes (LED) as a irradiation sources. Additionally efficiency of bacteria (Pseudomonas aeruginosa) removal from the gas phase was estimated. The number of surviving bacteria was determined by the serial twofold dilution microtiter plate method, in Tryptic Soy Broth medium (TSB, GibcoBRL).

Keywords: photocatalysis, antibacterial properties, titania nanotubes, new TiO2/MxOy nanostructures

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Authors: Zorica Tomičić, Ružica Tomičić, Peter Raspor

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Growing resistance of pathogenic yeast Candida glabrata to many classes of antifungal drugs has stimulated efforts to discover new agents to combat a rising number of invasive C. glabrata infections, which deserves a great deal of concern due to the high mortality rate in immunocompromised populations. One promising strategy is the use of probiotic microorganisms, which, when administered in adequate amounts, confers a health benefit. A selected number of probiotic organisms, Saccharomyces boulardii among them, have been tested as potential biotherapeutic agents. The aim of this study was to investigate the effect of the probiotic yeast S. boulardii on the adhesion of clinical isolates of C. glabrata at different temperatures, pH values, and in the presence of three clinically important antifungal drugs, such as fluconazole, itraconazole and amphotericin B. The method used to assess adhesion was crystal violet staining. The selection of antimycotics concentrations used in the adhesion assay was based on minimum inhibitory concentrations (MICs) obtained by the preliminarily performed microdilution modification of the Reference method for broth dilution antifungal susceptibility testing of yeast (Clinical and Laboratory Standards Institute (CLSI), standard M27-A2). the results showed that despite the nonadhesiveness of S. boulardii cells, probiotic yeast significantly suppressed the adhesion of C. glabrata strains. Besides, at specific strain ratios, a slight stimulatory effect was observed in some C. glabrata strains, which highlights the importance of strain specificity and opens up further research interests. When environmental conditions are considered, temperature and pH significantly influenced co-culture adhesion of C. glabrata and S. boulardii. The adhesion of C. glabrata strains was relatively equally reduced over all tested temperature range (28°C, 37°C, 39°C and 42°C) in the presence of S. boulardii cells, while the adhesion of a few C. glabrata strains were significantly stimulated at 28°C and suppressed at 42°C. Further, the adhesion was highly dependent on pH, with the highest adherence at pH 4 and lowest at pH 8.5. It was observed that S. boulardii did not manage to suppress the adhesion of C. glabrata strains at high pH. Antimycotics on the other hand showed a greater impact, since S. boulardii failed to affect co-culture adhesion at higher antimycotics concentrations. As expected, exposure to various concentrations of amphotericin B significantly reduced the adherence ability of C.glabrata strains both in a single culture and co-culture with S. boulardii. Therefore, it can be speculated that S. boulardii could substitute the effect of antimycotics in a range concentrations and with specific type of strains. This would certainly change the view on the treatment of yeast infections in the future.

Keywords: adhesion, antimycotics, candida glabrata, saccharomyces boulardii

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Authors: Xiang Zheng, Zhaoping Zhong

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In this study, Aspen Plus was used to simulate the whole process of biomass conversion to liquid fuel in different ways, and the main results of material and energy flow were obtained. The process optimization and evaluation were carried out on the four routes of cellulosic biomass pyrolysis gasification low-carbon olefin synthesis olefin oligomerization, biomass water pyrolysis and polymerization to jet fuel, biomass fermentation to ethanol, and biomass pyrolysis to liquid fuel. The environmental impacts of three biomass species (poplar wood, corn stover, and rice husk) were compared by the gasification synthesis pathway. The global warming potential, acidification potential, and eutrophication potential of the three biomasses were the same as those of rice husk > poplar wood > corn stover. In terms of human health hazard potential and solid waste potential, the results were poplar > rice husk > corn stover. In the popular pathway, 100 kg of poplar biomass was input to obtain 11.9 kg of aviation coal fraction and 6.3 kg of gasoline fraction. The energy conversion rate of the system was 31.6% when the output product energy included only the aviation coal product. In the basic process of hydrothermal depolymerization process, 14.41 kg aviation kerosene was produced per 100 kg biomass. The energy conversion rate of the basic process was 33.09%, which can be increased to 38.47% after the optimal utilization of lignin gasification and steam reforming for hydrogen production. The total exergy efficiency of the system increased from 30.48% to 34.43% after optimization, and the exergy loss mainly came from the concentration of precursor dilute solution. Global warming potential in environmental impact is mostly affected by the production process. Poplar wood was used as raw material in the process of ethanol production from cellulosic biomass. The simulation results showed that 827.4 kg of pretreatment mixture, 450.6 kg of fermentation broth, and 24.8 kg of ethanol were produced per 100 kg of biomass. The power output of boiler combustion reached 94.1 MJ, the unit power consumption in the process was 174.9 MJ, and the energy conversion rate was 33.5%. The environmental impact was mainly concentrated in the production process and agricultural processes. On the basis of the original biomass pyrolysis to liquid fuel, the enzymatic hydrolysis lignin residue produced by cellulose fermentation to produce ethanol was used as the pyrolysis raw material, and the fermentation and pyrolysis processes were coupled. In the coupled process, 24.8 kg ethanol and 4.78 kg upgraded liquid fuel were produced per 100 kg biomass with an energy conversion rate of 35.13%.

Keywords: biomass conversion, biofuel, process optimization, life cycle assessment

Procedia PDF Downloads 46

Authors: Gail Louw, Helena Boshoff, Taeksun Song, Clifton Barry

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Drug resistance in Mycobacterium tuberculosis is primarily attributed to mutations in target genes. These mutations incur a fitness cost and result in bacterial generations that are less fit, which subsequently acquire compensatory mutations to restore fitness. We hypothesize that mutations in specific drug target genes influence bacterial metabolism and cellular function, which affects its ability to develop subsequent resistance to additional agents. We aim to determine whether the sequential acquisition of drug resistance and specific mutations in a well-defined clinical M. tuberculosis strain promotes or limits the development of additional resistance. In vitro mutants resistant to pretomanid, linezolid, moxifloxacin, rifampicin and kanamycin were generated from a pan-susceptible clinical strain from the Beijing lineage. The resistant phenotypes to the anti-TB agents were confirmed by the broth microdilution assay and genetic mutations were identified by targeted gene sequencing. Growth of mono-resistant mutants was done in enriched medium for 14 days to assess in vitro fitness. Double resistant mutants were generated against anti-TB drug combinations at concentrations 5x and 10x the minimum inhibitory concentration. Subsequently, mutation frequencies for these anti-TB drugs in the different mono-resistant backgrounds were determined. The initial level of resistance and the mutation frequencies observed for the mono-resistant mutants were comparable to those previously reported. Targeted gene sequencing revealed the presence of known and clinically relevant mutations in the mutants resistant to linezolid, rifampicin, kanamycin and moxifloxacin. Significant growth defects were observed for mutants grown under in vitro conditions compared to the sensitive progenitor. Mutation frequencies determination in the mono-resistant mutants revealed a significant increase in mutation frequency against rifampicin and kanamycin, but a significant decrease in mutation frequency against linezolid and sutezolid. This suggests that these mono-resistant mutants are more prone to develop resistance to rifampicin and kanamycin, but less prone to develop resistance against linezolid and sutezolid. Even though kanamycin and linezolid both inhibit protein synthesis, these compounds target different subunits of the ribosome, thereby leading to different outcomes in terms of fitness in the mutants with impaired cellular function. These observations showed that oxazolidinone treatment is instrumental in limiting the development of multi-drug resistance in M. tuberculosis in vitro.

Keywords: oxazolidinones, mutations, resistance, tuberculosis

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Authors: G. A. Oladosu1, P. O. Ogbodogbo, C. I. Makinde1, M. O. Tijani, O. A. Adegboyega

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Saprolegniasis is a major pathogenic infection that contributes significantly to poor hatching rates in incubated fish eggs in the Africa catfish hatchery in Nigeria. Malachite green known to be very effective against this condition has been banned because it is carcinogenic. There is, therefore, the need for other effective yet safer methods of controlling saprolegniasis in incubated fish eggs. A total of 50 ml crude, chloroform extract of pyocyanin from which solvent was removed to attain 30 ml, having a concentration of 12.16 ug/ml was produced from 700 ml broth culture of Pseudomonas aeruginosa isolated from a previous study. In-vitro susceptibility of the fungus was investigated by exposing fungal infected eggs to two different time-concentration ratios of pyocyanin; 0.275 ug/ml and 2.75 ug/ml for 1 and 24 hours, and 5 mg/L malachite green as positive control while normal saline was the control. The efficacy of pyocyanin was evaluated using the degree of mycelial growth inhibition in different treatments. Fertilized Clarias gariepinus eggs (between 45 to 64 eggs) were then incubated in 20 ml of medium containing similar concentrations of pyocyanin and malachite green, with freshwater as a control for 24 hours. Hatching rates of the incubated eggs were observed. Three samples of un-hatched eggs were taken from each medium and observed for the presence of fungal pathogens using microscopy. Another batch of three samples of un-hatched eggs from each treatment was also inoculated on Sabourand dextrose agar (SDA) using Egg-Agar Transfer Technique to observe for fungal growth. Mycelial growth was inhibited in fungal infected eggs treated with 2.75 ug/ml for 24 hrs and the 5 mg/L malachite green for both 1 hr and 24 hrs. The mortality rate was 100% in fertilized C. gariepinus eggs exposed for 24 hrs to 0.275 and 2.75 ug/ml of pyocyanin. The mortality rate was least in malachite green followed by the control treatment. Embryonic development was observed to be arrested in the eggs treated with the two pyocyanin concentrations as they maintain their colour but showed no development beyond the gastrula stage, whereas viable eggs in the control and malachite green treatments developed fully into healthy hatchlings. Furthermore, microscopy of the un-hatched eggs revealed the presence of a protozoan ciliate; Colpidium sp, (Tetrahymenidae), as well as a pathogenic fungus; Saprolegnia sp. in the control but not in the malachite green and pyocyanin treatments. Growth of Saprolegnia sp was also observed in SDA culture of un-hatched eggs from the control, but not from pyocyanin and malachite green treated eggs. Pyocyanin treatment of incubated eggs of Clarias gariepinus effectively prevented fungal infection in the eggs, but also arrested the development of the embryo. Therefore, crude chloroform extract of pyocyanin from Pseudomonas aeruginosa cannot be used in the control of Saprolegniasis in incubated Clarias gariepinus eggs at the concentration and duration tested in this study.

Keywords: African catfish, bioprophylaxis, catfish embryo, Saprolegniasis

Procedia PDF Downloads 88

Authors: Robin Anderson, Elizabeth Latham, David Nisbet

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Propagation and dissemination of antimicrobial resistant and pathogenic microbes from spoiled silages and composts represents a serious public health threat to humans and animals. In the present study, the antimicrobial activity of the short chain nitro-compound, 2-nitro-1-propanol (9 mM) as well as the medium chain fatty acid, lauric acid, and its glycerol monoester, monolaurin, (each at 25 and 17 µmol/mL, respectfully) were investigated against select pathogenic and multi-drug resistant antimicrobial resistant Gram-positive bacteria common to spoiled silages and composts. In an initial study, we found that growth rates of a multi-resistant Enterococcus faecalis (expressing resistance against erythromycin, quinupristin/dalfopristin and tetracycline) and Staphylococcus aureus strain 12600 (expressing resistance against erythromycin, linezolid, penicillin, quinupristin/dalfopristin and vancomycin) were more than 78% slower (P < 0.05) by 2-nitro-1-propanol treatment during culture (n = 3/treatment) in anaerobically prepared ½ strength Brain Heart Infusion broth at 37oC when compared to untreated controls (0.332 ± 0.04 and 0.108 ± 0.03 h-1, respectively). The growth rate of 2-nitro-1-propanol-treated Listeria monocytogenes was also decreased by 96% (P < 0.05) when compared to untreated controls cultured similarly (0.171 ± 0.01 h-1). Maximum optical densities measured at 600 nm were lower (P < 0.05) in 2-nitro-1-propanol-treated cultures (0.053 ± 0.01, 0.205 ± 0.02 and 0.041 ± 0.01, respectively) than in untreated controls (0.483 ± 0.02, 0.523 ± 0.01 and 0.427 ± 0.01, respectively) for E. faecalis, S. aureus and L. monocytogenes, respectively. When tested against mixed microbial populations during anaerobic 24 h incubation of spoiled silage, significant effects of treatment with 1 mg 2-nitro-1-propanol (approximately 9.5 µmol/g) or 5 mg lauric acid/g (approximately 25 µmol/g) on populations of wildtype Enterococcus and Listeria were not observed. Mixed populations treated with 5 mg monolaurin/g (approximately 17 µmol/g) had lower (P < 0.05) viable cell counts of wildtype enterococci than untreated controls after 6 h incubation (2.87 ± 1.03 versus 5.20 ± 0.25 log10 colony forming units/g, respectively) but otherwise significant effects of monolaurin were not observed. These results reveal differential susceptibility of multi-drug resistant enterococci and staphylococci as well as L. monocytogenes to the inhibitory activity of 2-nitro-1-propanol and the medium chain fatty acid, lauric acid and its glycerol monoester, monolaurin. Ultimately, these results may lead to improved treatment technologies to preserve the microbiological safety of silages and composts.

Keywords: 2-nitro-1-propanol, lauric acid, monolaurin, gram positive bacteria

Procedia PDF Downloads 74

Authors: Yik Sin Chan, Bee Ling Chuah, Wei Quan Chan, Ri Jin Cheng, Yan Hang Oon, Kong Soo Khoo, Nam Weng Sit

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Traditionally, medicinal plants have been used to treat different kinds of ailments including infectious diseases. They serve as a good source of lead compounds for the development of new and safer anti-infective agents. This study aimed to investigate the antimicrobial potential of the leaves of three medicinal plants, namely Catunaregam spinosa (Rubiaceae; Mountain pomegranate), Houttuynia cordata (Saururaceae; "fishy-smell herb") and Rhapis excelsa (Arecaceae; “broadleaf lady palm”). The leaves extracts were obtained by sequential extraction using hexane, chloroform, ethyl acetate, ethanol, methanol and water. The antibacterial and antifungal activities were assessed using a colorimetric broth microdilution method against a panel of human pathogenic bacteria (Gram-positive: Bacillus cereus and Staphylococcus aureus; Gram-negative: Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) and fungi (yeasts: Candida albicans, Candida parapsilosis and Cryptococcus neoformans; Moulds: Aspergillus fumigatus and Trichophyton mentagrophytes) respectively; while antiviral activity was evaluated against the Chikungunya virus on monkey kidney epithelial (Vero) cells by neutral red uptake assay. All the plant extracts showed bacteriostatic activity, however, only 72% of the extracts (13/18) were found to have bactericidal activity. The lowest minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were given by the hexane extract of C. spinosa against S. aureus with the values of 0.16 and 0.31 mg/mL respectively. All the extracts also possessed fungistatic activity. Only the hexane, chloroform and ethyl acetate extracts of H. cordata exerted inhibitory activity against A. fumigatus, giving the lowest fungal susceptibility index of 16.7%. In contrast, only 61% of the extracts (11/18) showed fungicidal activity. The ethanol extract of R. excelsa exhibited the strongest fungicidal activity against C. albicans, C. parapsilosis and T. mentagrophytes with minimum fungicidal concentration (MFC) values of 0.04–0.08 mg/mL, in addition to its methanol extract against T. mentagrophytes (MFC=0.02 mg/mL). For anti-Chikungunya virus activity, only chloroform and ethyl acetate extracts of R. excelsa showed significant antiviral activity with 50% effective concentrations (EC50) of 29.9 and 78.1 g/mL respectively. Extracts of R. excelsa warrant further investigations into their active principles responsible for antifungal and antiviral properties.

Keywords: bactericidal, Chikungunya virus, extraction, fungicidal

Procedia PDF Downloads 369

Authors: Priyanka Gupta, Nilanjana Bairagi, Deepti Gupta

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New pathogenic strains of microbes are continually emerging and resistance of bacteria to antibiotics is growing. Hospitals in India have a high burden of infections in their intensive care units and general wards. Rising incidence of hospital infections is a matter of great concern in India. This growth is often attributed to the absence of effective infection control strategies in healthcare facilities. Government, therefore, is looking for cost effective strategies that are effective against HAIs. One possible method is by application of an antimicrobial finish on the uniform. But there are limited studies to show the effect of antimicrobial activity of antimicrobial finish treated nurses’ uniforms in a real hospital set up. This paper proposes a prospective non-destructive sampling technique, based on the use of a detachable fabric patch, to assess the effectiveness of silver based antimicrobial agent across five wards in a tertiary care government hospital in Delhi, India. Fabrics like polyester and polyester cotton blend fabric which are more prevalent for making coats were selected for the study. Polyester and polyester cotton blend fabric was treated with silver based antimicrobial (AM) finish. At the beginning of shift, a composite patch of untreated and treated fabric respectively was stitched on the abdominal region on the left and right side of the washed white coat of participating nurse. At the end of the shift, the patch was removed and taken for bacterial sampling on Brain Heart Infusion (BHI) plates. Microbial contamination on polyester and blend fabrics after 6 hours shift was compared in Brain Heart Infusion broth (BHI). All patches treated with silver based antimicrobial agent showed decreased bacterial counts. Percent reduction in the bacterial colonies after the antimicrobial treatment in both fabrics was 81.0 %. Antimicrobial finish was equally effective in reducing microbial adhesion on both fabric types. White coats of nurses become progressively contaminated during clinical care. Type of fabric used to make the coat can affect the extent of contamination which is higher on polyester cotton blend as compared to 100% polyester. The study highlights the importance of silver based antimicrobial finish in the area of uniform hygiene. Bacterial load can be reduced by using antimicrobial finish on hospital uniforms. Hospital staff uniforms endowed with antimicrobial properties may be of great help in reducing the occurrence and spread of infections.

Keywords: antimicrobial finish, bacteria, infection control, silver, white coat

Procedia PDF Downloads 183

Authors: Hana Al-Baghaoi, Kumar Shiva Gubbiyappa, Mayuren Candasamy, Kiruthiga Perumal Vijayaraman

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Acne is a chronic inflammatory disease of the sebaceous glands characterized by areas of skin with seborrhea, comedones, papules, pustules, nodules, and possibly scarring. Propionibacterium acnes (P. acnes), plays a key role in the pathogenesis of acne. Their colonization and proliferation trigger the host’s inflammatory response leading to the production of pro-inflammatory cytokines such as interleukin-8 (IL-8) and tumour necrosis factor-α (TNF-α). The usage of honey and natural compounds to treat skin ailments has strong support in the current trend of drug discovery. The present study was carried out evaluate antimicrobial and anti-inflammatory potential of Doani Sidr honey and its fractions against P. acnes and to screen madecassoside alone and in combination with fractions of honey. The broth dilution method was used to assess the antibacterial activity. Also, ultra structural changes in cell morphology were studied before and after exposure to Sidr honey using transmission electron microscopy (TEM). The three non-toxic concentrations of the samples were investigated for suppression of cytokines IL 8 and TNF α by testing the cell supernatants in the co-culture of the human peripheral blood mononuclear cells (hPBMCs) heat killed P. acnes using enzyme immunoassay kits (ELISA). Results obtained was evaluated by statistical analysis using Graph Pad Prism 5 software. The Doani Sidr honey and polysaccharide fractions were able to inhibit the growth of P. acnes with a noteworthy minimum inhibitory concentration (MIC) value of 18% (w/v) and 29% (w/v), respectively. The proximity of MIC and MBC values indicates that Doani Sidr honey had bactericidal effect against P. acnes which is confirmed by TEM analysis. TEM images of P. acnes after treatment with Doani Sidr honey showed completely physical membrane damage and lysis of cells; whereas non honey treated cells (control) did not show any damage. In addition, Doani Sidr honey and its fractions significantly inhibited (> 90%) of secretion of pro-inflammatory cytokines like TNF α and IL 8 by hPBMCs pretreated with heat-killed P. acnes. However, no significant inhibition was detected for madecassoside at its highest concentration tested. Our results suggested that Doani Sidr honey possesses both antimicrobial and anti-inflammatory effects against P. acnes and can possibly be used as therapeutic agents for acne. Furthermore, polysaccharide fraction derived from Doani Sidr honey showed potent inhibitory effect toward P. acnes. Hence, we hypothesize that fraction prepared from Sidr honey might be contributing to the antimicrobial and anti-inflammatory activity. Therefore, this polysaccharide fraction of Doani Sidr honey needs to be further explored and characterized for various phytochemicals which are contributing to antimicrobial and anti-inflammatory properties.

Keywords: Doani sidr honey, Propionibacterium acnes, IL-8, TNF alpha

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Authors: S.Kauroo, J. Govinden Soulange, D. Marie

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Psiadia species from the Asteraceae are traditionally used in the folk medicine of Mauritius to treat cutaneous and bronchial infections. The present study aimed at validating the phytomedicinal properties of the selected species from the Asteraceae family, namely Psiadia arguta, Psiadia viscosa, Psiadia lithospermifolia, and Distephanus populifolius. Dried hexane, ethyl acetate, and methanol leaf extracts were studied for their antioxidant properties using the DPPH (1, 1-diphenyl-2-picryl-hydrazyl), FRAP (Ferric Reducing Ability of Plasma), and Deoxyribose assays. Antibacterial activity against human pathogenic bacteria namely Escherichia coli (ATCC 27853), Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212), Klebsiella pneumonia (ATCC27853), Pseudomonas aeruginosa (ATCC 27853), and Bacillus cereus (ATCC 11778) was measured using the broth microdilution assay. Qualitative phytochemical screening using standard methods revealed the presence of coumarins, tannins, leucoanthocyanins, and steroids in all the tested extracts. The measured phenolics level of the selected plant extracts varied from 24.0 to 231.6 mg GAE/g with the maximum level in methanol extracts in all four species. The highest flavonoids and proanthocyanidins content was noted in Psiadia arguta methanolic extracts with 65.7±1.8 mg QE/g and 5.1±0.0 mg CAT/g dry weight (DW) extract, respectively. The maximum free radical scavenging activity was measured in Psiadia arguta methanol and ethyl acetate extracts with IC50 11.3±0.2 and 11.6± 0.2 µg/mL, respectively and followed by Distephanus populifolius methanol extracts with an IC50 of 11.3± 0.8 µg/mL. The maximum ferric reducing antioxidant potential was noted in Psiadia lithospermifolia methanol extracts with a FRAP value of 18.8 ± 0.4 µmol Fe2+/L/g DW. The antioxidant capacity based on DPPH and Deoxyribose values were negatively related to total phenolics, flavonoid and proanthocyanidins content while the ferric reducing antioxidant potential were strongly correlated to total phenolics, flavonoid and proanthocyanidins content. All four species exhibited antimicrobial activity against the tested bacteria (both Gram-negative and Gram-positive). Interestingly, the hexane and ethyl acetate extracts of Psiadia viscosa and Psiadia lithospermifolia were more active than the control antibiotic Chloramphenicol. The Minimum inhibitory concentration (MIC) values for hexane and ethyl acetate extracts of Psiadia viscosa and Psiadia lithospermifolia against the tested bacteria ranged from (62.5 to 500 µg/ml). These findings validate the use of these tested Asteraceae in the traditional medicine of Mauritius and also highlight their pharmaceutical potential as prospective phytomedicines.

Keywords: antibacterial, antioxidant, DPPH, flavonoids, FRAP, Psiadia spp

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Authors: Danilo F. Rodrigues, Hérida Regina N. Salgado

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Introduction: The emergence of resistant microorganisms to a large number of clinically approved antimicrobials has been increasing, which restrict the options for the treatment of bacterial infections. As a strategy, drugs with high antimicrobial activities are in evidence. Stands out a class of antimicrobial, the cephalosporins, having as fourth generation cefepime (CEF) a semi-synthetic product which has activity against various Gram-positive bacteria (e.g. oxacillin resistant Staphylococcus aureus) and Gram-negative (e.g. Pseudomonas aeruginosa) aerobic. There are few studies in the literature regarding the development of microbiological methodologies for the analysis of this antimicrobial, so researches in this area are highly relevant to optimize the analysis of this drug in the industry and ensure the quality of the marketed product. The development of microbiological methods for the analysis of antimicrobials has gained strength in recent years and has been highlighted in relation to physicochemical methods, especially because they make possible to determine the bioactivity of the drug against a microorganism. In this context, the aim of this work was the development and validation of a microbiological method for quantitative analysis of CEF in powder lyophilized for injectable solution by turbidimetric assay. Method: For performing the method, Staphylococcus aureus ATCC 6538 IAL 2082 was used as the test microorganism and the culture medium chosen was the Casoy broth. The test was performed using temperature control (35.0 °C ± 2.0 °C) and incubated for 4 hours in shaker. The readings of the results were made at a wavelength of 530 nm through a spectrophotometer. The turbidimetric microbiological method was validated by determining the following parameters: linearity, precision (repeatability and intermediate precision), accuracy and robustness, according to ICH guidelines. Results and discussion: Among the parameters evaluated for method validation, the linearity showed results suitable for both statistical analyses as the correlation coefficients (r) that went 0.9990 for CEF reference standard and 0.9997 for CEF sample. The precision presented the following values 1.86% (intraday), 0.84% (interday) and 0.71% (between analyst). The accuracy of the method has been proven through the recovery test where the mean value obtained was 99.92%. The robustness was verified by the parameters changing volume of culture medium, brand of culture medium, incubation time in shaker and wavelength. The potency of CEF present in the samples of lyophilized powder for injectable solution was 102.46%. Conclusion: The turbidimetric microbiological method proposed for quantification of CEF in lyophilized powder for solution for injectable showed being fast, linear, precise, accurate and robust, being in accordance with all the requirements, which can be used in routine analysis of quality control in the pharmaceutical industry as an option for microbiological analysis.

Keywords: cefepime hydrochloride, quality control, turbidimetric assay, validation

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Authors: Minhas Alam, Muhammad Hidayat Rasool, Mohsin Khurshid, Bilal Aslam

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The genus Acinetobacter has emerged as a significant concern in hospital-acquired infections, particularly due to the versatility of Acinetobacter baumannii in causing nosocomial infections. The organism's remarkable metabolic adaptability allows it to thrive in various environments, including the environment, animals, and humans. However, the extent of antimicrobial resistance in Acinetobacter species from veterinary settings, especially in developing countries like Pakistan, remains unclear. This study aimed to isolate and characterize Acinetobacter spp. from veterinary settings in Punjab, Pakistan. A total of 2,230 specimens were collected, including 1,960 samples from veterinary settings (nasal and rectal swabs from dairy and beef cattle), 200 from the environment, and 70 from human clinical settings. Isolates were identified using routine microbiological procedures and confirmed by polymerase chain reaction (PCR). Antimicrobial susceptibility was determined by the disc diffusion method, and minimum inhibitory concentration (MIC) was measured by the micro broth dilution method. Molecular techniques, such as PCR and DNA sequencing, were used to screen for antimicrobial-resistant determinants. Genetic diversity was assessed using standard techniques. The results showed that the overall prevalence of A. baumannii in cattle was 6.63% (65/980). However, among cattle, a higher prevalence of A. baumannii was observed in dairy cattle, 7.38% (54/731), followed by beef cattle, 4.41% (11/249). Out of 65 A. baumannii isolates, the carbapenem resistance was found in 18 strains, i.e. 27.7%. The prevalence of A. baumannii in nasopharyngeal swabs was higher, i.e., 87.7% (57/65), as compared to rectal swabs, 12.3% (8/65). Class D β-lactamases genes blaOXA-23 and blaOXA-51 were present in all the CRAB from cattle. Among carbapenem-resistant isolates, 94.4% (17/18) were positive for class B β-lactamases gene blaIMP, whereas the blaNDM-1 gene was detected in only one isolate of A. baumannii. Among 70 clinical isolates of A. baumannii, 58/70 (82.9%) were positive for the blaOXA-23-like gene, and 87.1% (61/70) were CRAB isolates. Among all clinical isolates of A. baumannii, blaOXA-51-like gene was present. Hence, the co-existence of blaOXA-23 and blaOXA-51 was found in 82.85% of clinical isolates. From the environmental settings, a total of 18 A. baumannii isolates were recovered; among these, 38.88% (7/18) strains showed carbapenem resistance. All environmental isolates of A. baumannii harbored class D β-lactamases genes, i.e., blaOXA-51 and blaOXA-23 were detected in 38.9% (7/18) isolates. Hence, the co-existence of blaOXA-23 and blaOXA-51 was found in 38.88% of isolates. From environmental settings, 18 A. baumannii isolates were recovered, with 38.88% showing carbapenem resistance. All environmental isolates harbored blaOXA-51 and blaOXA-23 genes, with co-existence in 38.88% of isolates. MLST results showed ten different sequence types (ST) in clinical isolates, with ST 589 being the most common in carbapenem-resistant isolates. In veterinary isolates, ST2 was most common in CRAB isolates from cattle. Immediate control measures are needed to prevent the transmission of CRAB isolates among animals, the environment, and humans. Further studies are warranted to understand the mechanisms of antibiotic resistance spread and implement effective disease control programs.

Keywords: Acinetobacter baumannii, carbapenemases, drug resistance, MSLT

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Authors: Chee Wee Gan, Anthony Herr Cheun Ng, Yee Shan Wong, Subbu Venkatraman, Lynne Hsueh Yee Lim

Abstract:

Otitis media with effusion (OME) is the leading cause of hearing loss in children worldwide. Surgery to insert grommet tube into the eardrum is usually indicated for OME unresponsive to antimicrobial therapy. It is the most common surgery for children. However, current commercially available grommet tubes are non-bioresorbable, not drug-treated, with unpredictable duration of retention on the eardrum to ventilate middle ear. Their functionality is impaired when clogged or chronically infected, requiring additional surgery to remove/reinsert grommet tubes. We envisaged that a novel fully bioresorbable grommet tube with sustained antibiotic release technology could address these drawbacks. In this study, drug-loaded bioresorbable poly(L-lactide-co-ε-caprolactone)(PLC) copolymer grommet tubes were fabricated by microinjection moulding technique. In vitro drug release and degradation model of PLC tubes were studied. Antibacterial property was evaluated by incubating PLC tubes with P. aeruginosa broth. Surface morphology was analyzed using scanning electron microscopy. A preliminary animal study was conducted using guinea pigs as an in vivo model to evaluate PLC tubes with and without drug, with commercial Mini Shah grommet tube as comparison. Our in vitro data showed sustained drug release over 3 months. All PLC tubes revealed exponential degradation profiles over time. Modeling predicted loss of tube functionality in water to be approximately 14 weeks and 17 weeks for PLC with and without drug, respectively. Generally, PLC tubes had less bacteria adherence, which were attributed to the much smoother tube surfaces compared to Mini Shah. Antibiotic from PLC tube further made bacteria adherence on surface negligible. They showed neither inflammation nor otorrhea after 18 weeks post-insertion in the eardrums of guinea pigs, but had demonstrated severe degree of bioresorption. Histology confirmed the new PLC tubes were biocompatible. Analyses on the PLC tubes in the eardrums showed bioresorption profiles close to our in vitro degradation models. The bioresorbable antibiotic-loaded grommet tubes showed good predictability in functionality. The smooth surface and sustained release technology reduced the risk of tube infection. Tube functional duration of 18 weeks allowed sufficient ventilation period to treat OME. Our ongoing studies include modifying the surface properties with protein coating, optimizing the drug dosage in the tubes to enhance their performances, evaluating their functional outcome on hearing after full resoption of grommet tube and healing of eardrums, and developing animal model with OME to further validate our in vitro models.

Keywords: bioresorbable polymer, drug release, grommet tube, guinea pigs, otitis media with effusion

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Authors: Norihiro Kato, Yuriko Takayama

Abstract:

Some gram-negative bacteria enable the simultaneous activation of gene expression involved in N-acylhomoserine lactone (AHL) dependent cell-to-cell communication system. Such regulatory system for the bacterial group behavior is termed as quorum sensing (QS) because a diffusible AHL signal can accumulate around the cell during the increase of the cell density and trigger activation of the sequential QS process. By blocking the QS, the expression of diverse genes related to infection, antibiotic production, and biofilm formation is inhibited. Conditioning of QS by regulation of the DNA-receptor-AHL interaction is a potential target for enhancing host defenses against pathogenicity. We focused on engineered application of transcriptional regulator SpnR produced in opportunistic human pathogen Serratia marcescens. The SpnR can interact with AHL signals at an N-terminal domain and also with a promoter region of a QS target gene at a C-terminal domain. As the initial process of the QS activation, the SpnR forms a complex with the AHL to enhance the expression of pig cluster; the SpnR normally acts as an activator for the expression of the QS-dependent gene. In this research, we attempt to artificially control QS by changing the role of SpnR. The QS-dependent prodigiosin production is expected to inhibit by externally added SpnR in the culture broth of AS-1 strain because the AHL concentration was kept below the threshold by AHL-SpnR complex formation. Maltose-binding protein (MBP)-tagged SpnR (MBP-SpnR) was overexpressed in Escherichia coli and purified using an affinity chromatography equipped with an amylose resin column. The specific interaction between AHL and MBP-SpnR was demonstrated by quartz crystal microbalance (QCM) sensor. AHL with amino end-group was coupled with COOH-terminated self-assembled monolayer prepared on a gold electrode of 27-MHz quartz crystal sensor using water-soluble carbodiimide. After the injection of MBP-SpnR into a cup-type sensor cell filled with the buffer solution, time course of resonant frequency change (ΔFs) was determined. A decrease of ΔFs clearly showed the uptake of MBP-SpnR onto the AHL-immobilized electrode. Furthermore, no binding affinity was observed after the heat-inactivation of MBP-SpnR at 80ºC. These results suggest that MBP-SpnR possesses a specific affinity for AHL. MBP-SpnR was added to the culture medium as an AHL trap to study inhibitory effects on intracellularly accumulated prodigiosin. With approximately 2 µM MBP-SpnR, the amount of prodigiosin induced was half that of the control without any additives. In conclusion, the function of SpnR could be switched by adding it to the cell culture. Exogenously added MBP-SpnR possesses high affinity for AHL derived from cells and acts as an inhibitor of AHL-mediated QS.

Keywords: intracellular signaling, microbial biotechnology, quorum sensing, transcriptional regulator

Procedia PDF Downloads 240

Authors: Yuriko Takayama, Norihiro Kato

Abstract:

Quorum sensing (QS) is a cell-to-cell communication system in bacteria to regulate expression of target genes. In gram-negative bacteria, activation on QS is controlled by a concentration increase of N-acylhomoserine lactone (AHL), which can diffuse in and out of the cell. Effective control of QS is expected to avoid virulence factor production in infectious pathogens, biofilm formation, and antibiotic production because various cell functions in gram-negative bacteria are controlled by AHL-mediated QS. In this research, we applied cyclodextrins (CDs) as artificial hosts for the AHL signal to reduce the AHL concentration in the culture broth below its threshold for QS activation. The AHL-receptor complex induced under the high AHL concentration activates transcription of the QS-target gene. Accordingly, artificial reduction of the AHL concentration is one of the effective strategies to inhibit the QS. A hydrophobic cavity of the CD can interact with the acyl-chain of the AHL due to hydrophobic interaction in aqueous media. We studied N-hexanoylhomoserine lactone (C6HSL)-mediated QS in Serratia marcescens; accumulation of C6HSL is responsible for regulation of the expression of pig cluster. Inhibitory effects of added CDs on QS were demonstrated by determination of prodigiosin amount inside cells after reaching stationary phase, because production of prodigiosin depends on the C6HSL-mediated QS. By adding approximately 6 wt% hydroxypropyl-β-CD (HP-β-CD) in Luria-Bertani (LB) medium prior to inoculation of S. maecescens AS-1, the intracellularly accumulated prodigiosin was drastically reduced to 7-10%, which was determined after the extraction of prodigiosin in acidified ethanol. The AHL retention ability of HP-β-CD was also demonstrated by Chromobacterium violacuem CV026 bioassay. The CV026 strain is an AHL-synthase defective mutant that activates QS solely by adding AHLs from outside of cells. A purple pigment violacein is induced by activation of the AHL-mediated QS. We demonstrated that the violacein production was effectively suppressed when the C6HSL standard solution was spotted on a LB agar plate dispersing CV026 cells and HP-β-CD. Physico-chemical analysis was performed to study the affinity between the immobilized CD and added C6HSL using a quartz crystal microbalance (QCM) sensor. The COOH-terminated self-assembled monolayer was prepared on a gold electrode of 27-MHz AT-cut quartz crystal. Mono(6-deoxy-6-N, N-diethylamino)-β-CD was immobilized on the electrode using water-soluble carbodiimide. The C6HSL interaction with the β-CD cavity was studied by injecting the C6HSL solution to a cup-type sensor cell filled with buffer solution. A decrement of resonant frequency (ΔFs) clearly showed the effective C6HSL complexation with immobilized β-CD and its stability constant for MBP-SpnR-C6HSL complex was on the order of 102 M-1. The CD has high potential for engineered control of QS because it is safe for human use.

Keywords: acylhomoserine lactone, cyclodextrin, intracellular signaling, quorum sensing

Procedia PDF Downloads 208