Search results for: antimicrobial

Authors: Amal Saafan, Kareem Al Sofy, Sameh AbdelGhani, Magdy Amin

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Background: Acinetobacter spp. resistance towards β-lactam antibiotics is mediated mainly by different classes of β-lactamases production; detection of some genes responsible for production of β-lactamases is the objective of the study. Methods: One hundred fifty bacterial isolates were recovered from blood, sputum, and urine specimens from different hospitals in Egypt. Sixty-nine isolate were identified as Acinetobacter baumannii using traditional biochemical tests, CHROM agar, MicroScan and PCR amplification of blaoxa-51like gene. Acinetobacterbaumannii isolates were grouped into carbapenem resistant group (GP1), cefotaxime, ceftazidime and cefoxitin resistant group (GP2) and carbapenem and cephalosporin non-resistant group (GP3). Carbapenemase activity was screened using modified Hodge test (MHT) for GP1.Metallo-β-lactamases screening was performed for MHT positive isolates using double disk synergy test (DDST) and combined disk test (CDT). Amp C activity was screened using Amp C disk test with Tris-EDTA, DDST, and CDT for GP2. Finally, PCR amplification of blaoxa-51like, blaoxa-23like, blaIMP-like, blaVIM-like, and blaADC-like genes was performed for isolates that showed, at least, two positive results of three for both AmpC and carbapenemases phenotypic screening tests (obvious activity), in addition to GP3 (for comparison). Detection of blaoxa-51like and blaADC-like genes preceded by ISAba1 was also performed. Results: Antibiogram of 69 pure Acinetobacter baumannii isolates resulted in 57, 64, and 2 isolates enrolled into GP1, GP2, and GP3, respectively. Carbapenemase activity was shown by 49(85.9%) isolate using MHT. Metallo-β-lactamases screening revealed 32(65.3%) and 35(71.4%) using DDST and CDT, respectively.AmpC activity was shown by 43(67.2%) and 50 (78.1%) isolates using AmpC disk test with Tris-EDTA, and both DDST and CDT, respectively. Twenty-seven isolates showed obvious activity, all of them (100%) were harboring blaoxa-51like and blaADC-like genes, while blaoxa-23like, blaIMP-like andblaVIM-like genes were harbored by 23(85.2%), 9 (33.%) and no isolate respectively. Only 12 (44.4%) isolates harbored blaoxa-51like and blaADC-like genes preceded by ISAba1. GP3 isolates showed only positive blaoxa-51like and blaADC-like genes. Conclusion: It is not possible to correlate resistance with presence of blaoxa-51like and blaADC-like genes and presence of ISAba1 was immediate as transcriptional promoter. A blaoxa-23like gene played an important role in carbapenem resistance when compared with blaIMP-like and blaVIM-like gene.

Keywords: acinetobacter, beta-lactams, resistance, antimicrobial agents

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Authors: Recep Keşli, Gülşah Aşık, Cengiz Demir, Onur Türkyılmaz

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Objective: Serratia species are identified as aerobic, motile Gram negative rods. The species Serratia marcescens (S. marcescens) causes both opportunistic and nosocomial infections. The genus Providencia is Gram-negative bacilli and includes urease-producing that is responsible for a wide range of human infections. Although most Providencia infections involve the urinary tract, they are also associated with gastroenteritis, wound infections, and bacteremia. The aim of this study was evaluate the antimicrobial resistance rates of S. marcescens and Providencia spp. strains which had been isolated from various clinical materials obtained from different patients who belongs to intensive care units (ICU) and inpatient clinics. Methods: A total of 35 S. marcescens and Providencia spp. strains isolated from various clinical samples admitted to Medical Microbiology Laboratory, ANS Research and Practice Hospital, Afyon Kocatepe University between October 2013 and September 2015 were included in the study. Identification of the bacteria was determined by conventional methods and VITEK 2 system (bio-Merieux, Marcy l’etoile, France) was used additionally. Antibacterial resistance tests were performed by using Kirby Bauer disc (Oxoid, Hampshire, England) diffusion method following the recommendations of CLSI. Results: The distribution of clinical samples were as follows: upper and lower respiratory tract samples 26, 74.2 % wound specimen 6, 17.1 % blood cultures 3, 8.5%. Of the 35 S. marcescens and Providencia spp. strains; 28, 80% were isolated from clinical samples sent from ICU. The resistance rates of S. marcescens strains against trimethoprim-sulfamethoxazole, piperacillin-tazobactam, imipenem, gentamicin, ciprofloxacin, ceftazidime, cefepime and amikacin were found to be 8.5 %, 22.8 %, 11.4 %, 2.8 %, 17.1 %, 40 %, 28.5 % and 5.7 % respectively. Resistance rates of Providencia spp. strains against trimethoprim-sulfamethoxazole, piperacillin-tazobactam, imipenem, gentamicin, ciprofloxacin, ceftazidime, cefepime and amikacin were found to be 10.2 %, 33,3 %, 18.7 %, 8.7 %, 13.2 %, 38.6 %, 26.7%, and 11.8 % respectively. Conclusion: S. marcescens is usually resistant to ampicillin, amoxicillin, amoxicillin/clavulanate, ampicillin/sulbactam, cefuroxime, cephamycins, nitrofurantoin, and colistin. The most effective antibiotic on the total of S. marcescens strains was found to be gentamicin 2.8 %, of the totally tested strains the highest resistance rate found against to ceftazidime 40 %. The lowest and highest resistance rates were found against gentamiycin and ceftazidime with the rates of 8.7 % and 38.6 % for Providencia spp.

Keywords: Serratia marcescens, Providencia spp., antibiotic resistance, intensive care unit

Procedia PDF Downloads 219

Authors: Christoph Hacker, Zeynep Karahaliloglu, Gunnar Seide, Emir Baki Denkbas, Thomas Gries

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A wound dressing material is designed to facilitate wound healing and minimize scarring. An ideal wound dressing material should protect the wound from any contaminations of exogeneous microorganism. In addition, the dressing material should provide a moist environment through extraction of body fluid from the wound area. Recently, wound dressing electrospun nanofibrous membranes are produced by electrospinning from a polymer solution or a polymer melt. These materials have a great potential as dressing materials for wound healing because of superior properties such as high surface-to-volume ratio, high porosity with excellent pore interconnectivity. Melt electrospinning is an attractive tissue engineering scaffold manufacturing process which eliminated the health risk posed by organic solvents used in electrospinning process and reduced the production costs. In this study, antibacterial wound dressing materials were prepared from TPU (Elastollan 1185A) by a melt-electrospinning technique. The electrospinning parameters for an efficient melt-electrospinning process of TPU were optimized. The surface of the fibers was modified with poly(ethylene glycol) (PEG) by radio-frequency glow discharge plasma deposition method and with silver nanoparticles (nAg) to improve their wettability and antimicrobial properties. TPU melt-electrospun mats were characterized using SEM, DSC, TGA and XPS. The cell viability and proliferation on modified melt-electrospun TPU mats were evaluated using a mouse fibroblast cell line (L929). Antibacterial effects of theirs against both Staphylococcus aureus strain and Escherichia coli were investigated by disk-diffusion method. TPU was successfully processed into a porous, fibrous network of beadless fibers in the micrometer range (4.896±0.94 µm) with a voltage of 50 kV, a working distance of 6 cm, a temperature of the thermocouple and hot coil of 225–230ºC, and a flow rate of 0.1 mL/h. The antibacterial test indicated that PEG-modified nAg-loaded TPU melt-electrospun structure had excellent antibacterial effects and cell study results demonstrated that nAg-loaded TPU mats had no cytotoxic effect on the fibroblast cells. In this work, the surface of a melt-electrospun TPU mats was modified via PEG monomer and then nAg. Results showed melt-electrospun TPU mats modified with PEG and nAg have a great potential for use as an antibacterial wound dressing material and thus, requires further investigation.

Keywords: melt electrospinning, nanofiber, silver nanoparticles, wound dressing

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Authors: Bunindro Nameirakpam, Sonia Sougrapakam, Shannon B. Olsson, Rajashekar Yallappa

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Natural sources for new pesticidal compounds hold promise in view of their eco-friendly nature, selectivity and mammalian safety. Despite a large number of plants that show insecticidal activity and diversity of natural chemistry with inherent eco-friendly nature, newer classes of insecticides have eluded discovery. Artemisia vulgaris, known as Mugwort, is a universal herb used for folk medicine and religious purposes throughout the ancient world. In India, the essential oils of Artemisia vulgaris are used for its insecticidal, anti parasiticidal and antimicrobial properties. Traditionally, the dried leaves of Artemisia vulgaris are used to repel insects as well as rats in and around the granaries in the North-East India. Artemisia vulgaris collected during November from different ecological sites were studied for the bio-pesticidal utility against the stored grain pests. The insecticidal activities were found in the crude extracts of n-hexane and methanol from the samples collected in Sikkim and Manipur respectively. Using silica gel column chromatography protocol, we have isolated one novel bioactive molecule from the aerial parts of Artemisia vulgaris L based on various physical-chemical and spectroscopic techniques (IR, 1H NMR, 13C NMR and mass). The novel bioactive molecule is highly toxic and very low concentration (4.35 µg/l) is needed to control the stored product insects. In additional experiment results clearly showed the involvement of sodium pumps inhibition in the insecticidal action of purified compound in the Sitophilus oryzae. The knockdown activity of the purified compound is concomitant with the in vivo inhibition of Na+/ K+- ATPase. Further, our study showed insignificant differences in the seed germination of control and the treated grains. The lack of adverse effect of the novel bioactive molecule on the seed germination is highly desirable for seed/grain protectant and showing the potential to be developed as possible natural fumigants for the control of stored grain pests. The novel bioactive molecule is selective insecticide with a high margin of safety to mammals and showed promise as novel biopesticide candidate for grain protection. It is believed that Bio-pesticides can serve as the most important pest management tools as far as global safety is concerned.

Keywords: Indian traditional plant, Artemisia vulgaris, bio-pesticides, Na+/ K+- ATPase, seed germination

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Authors: Astrid Tannert, Anuradha Ramoji, Christina Ebert, Frederike Gladigau, Lorena Tuchscherr, Jürgen Popp, Ute Neugebauer

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Staphylococcus aureus is a facultative intracellular pathogen, which by entering host cells may evade immunologic host response as well as antimicrobial treatment. In that way, S. aureus can cause persistent intracellular infections which are difficult to treat. Depending on the strain, S. aureus may persist at different intracellular locations like the phagolysosome. The first barrier invading pathogens from the blood stream that they have to cross are the endothelial cells lining the inner surface of blood and lymphatic vessels. Upon proceeding from an acute to a chronic infection, intracellular pathogens undergo certain biochemical and structural changes including a deceleration of metabolic processes to adopt for long-term intracellular survival and the development of a special phenotype designated as small colony variant. In this study, the endothelial cell line Ea.hy 926 was used as a model for acute and chronic S. aureus infection. To this end, Ea.hy 926 cells were cultured on QIAscout™ Microraft Arrays, a special graded cell culture substrate that contains around 12,000 microrafts of 200 µm edge length. After attachment to the substrate, the endothelial cells were infected with GFP-expressing S. aureus for 3 weeks. The acute infection and the development of persistent bacteria was followed by confocal laser scanning microscopy, scanning the whole Microraft Array for the presence and for detailed determination of the intracellular location of fluorescent intracellular bacteria every second day. After three weeks of infection representative microrafts containing infected cells, cells with protruded infections and cells that did never show any infection were isolated and fixed for Raman micro-spectroscopic investigation. For comparison, also microrafts with acute infection were isolated. The acquired Raman spectra are correlated with the fluorescence microscopic images to give hints about a) the molecular alterations in endothelial cells during acute and chronic infection compared to non-infected cells, and b) metabolic and structural changes within the pathogen when entering a mode of persistence within host cells. We thank Dr. Ruth Kläver from QIAGEN GmbH for her support regarding QIAscout technology. Financial support by the BMBF via the CSCC (FKZ 01EO1502) and from the DFG via the Jena Biophotonic and Imaging Laboratory (JBIL, FKZ PO 633/29-1, BA 1601/10-1) is highly acknowledged.

Keywords: correlative image analysis, intracellular infection, pathogen-host adaption, Raman micro-spectroscopy

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Authors: Raghuram Kandimalla, Sanjeeb Kalita, Bhaswati Choudhury, Jibon Kotoky

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The quest for developing an ideal suture material prompted our interest to develop a novel suture with advantageous characteristics to market available ones. We developed novel suture biomaterial from muga silk (Antheraea assama) and ramie (Boehmeria nivea) plant fiber. Field emission scanning electron microscopy (FE-SEM), energy-dispersive X-ray spectroscopy (EDX), attenuated total reflection fourier transform infrared spectroscopy (ATR-FTIR) and thermo gravimetric analysis (TGA) results revealed the physicochemical properties of the fibers which supports the suitability of fibers for suture fabrication. Tensile properties of the prepared sutures were comparable with market available sutures and it found to be biocompatible towards human erythrocytes and nontoxic to mammalian cells. The prepared sutures completely healed the superficial deep wound incisions within seven days in adult male wister rats leaving no rash and scar. Histopathology studies supports the wound healing ability of sutures, as rapid synthesis of collagen, connective tissue and other skin adnexal structures were observed within seven days of surgery. Further muga suture surface modified by exposing the suture to oxygen plasma which resulted in formation of nanotopography on suture surface. Broad spectrum antibiotic amoxicillin was functionalized on the suture surface to prepare an advanced antimicrobial muga suture. Surface hydrophilicity induced by oxygen plasma results in an increase in drug-impregnation efficiency of modified muga suture by 16.7%. In vitro drug release profiles showed continuous and prolonged release of amoxicillin from suture up to 336 hours. The advanced muga suture proves to be effective against growth inhibition of Staphylococcus aureus and Escherichia coli, whereas normal muga suture offers no antibacterial activity against both types of bacteria. In vivo histopathology studies and colony-forming unit count data revealed accelerated wound healing activity of advanced suture over normal one through rapid synthesis and proliferation of collagen, hair follicle and connective tissues.

Keywords: sutures, biomaterials, silk, Ramie

Procedia PDF Downloads 288

Authors: Ana Carolina Kogawa, Selma Gutierrez Antonio, Hérida Regina Nunes Salgado

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Rifaximin is an oral antimicrobial, gut - selective and not systemic with adverse effects compared to placebo. It is used for the treatment of hepatic encephalopathy, travelers diarrhea, irritable bowel syndrome, Clostridium difficile, ulcerative colitis and acute diarrhea. The crystalline form present in the rifaximin with minimal systemic absorption is α, being the amorphous form significantly different. Regulators are increasingly attention to polymorphisms. Polymorphs can change the form by altering the drug characteristics compromising the effectiveness and safety of the finished product. International Conference on Harmonization issued the ICH Guidance Q6A, which aim to improve the control of polymorphism in new and existing pharmaceuticals. The objective of this study was to obtain polymorphic forms of rifaximin employing recrystallization processes and characterize them by thermal analysis (thermogravimetry - TG and differential scanning calorimetry - DSC), X-ray diffraction, scanning electron microscopy and solubility test. Six polymorphic forms of rifaximin, designated I to VI were obtained by the crystallization process by evaporation of the solvent. The profiles of the TG curves obtained from polymorphic forms of rifaximin are similar to rifaximin and each other, however, the DTG are different, indicating different thermal behaviors. Melting temperature values of all the polymorphic forms were greater to that shown by the rifaximin, indicating the higher thermal stability of the obtained forms. The comparison of the diffractograms of the polymorphic forms of rifaximin with rifaximin α, β and γ constant in patent indicate that forms III, V and VI are formed by mixing polymorph β and α and form III is formed by polymorph β. The polymorphic form I is formed by polymorph β, but with a significant amount of amorphous material. Already, the polymorphic form II consists of polymorph γ, amorphous. In scanning electron microscope is possible to observe the heterogeneity of morphological characteristics of crystals of polymorphic forms among themselves and with rifaximin. The solubility of forms I and II was greater than the solubility of rifaximin, already, forms III, IV and V presented lower solubility than of rifaximin. Similarly, the bioavailability of the amorphous form of rifaximin is considered significantly higher than the form α, the polymorphic forms obtained in this work can not guarantee the excellent tolerability of the reference medicine. Therefore, studies like these are extremely important and they point to the need for greater requirements by the regulatory agencies competent about polymorphs analysis of the raw materials used in the manufacture of medicines marketed globally. These analyzes are not required in the majority of official compendia. Partnerships between industries, research centers and universities would be a viable way to consolidate researches in this area and contribute to improving the quality of solid drugs.

Keywords: electronic microscopy, polymorphism, rifaximin, solubility, X-ray diffraction

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Authors: Nadine khadraoui, Rym Essid, Olfa Tabbene, Imen Chniba, Safa Boujemaa, Selim Jallouli, Nadia Fares, Behija Mlik, Boutheina Ben Abdelmoumen Mardassi

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Background and aim: Mycoplasma hominis is an opportunistic pathogen that can cause various gynecological infections such cervicitis, infertility, and, less frequently, extra-genital infections. Previous studies on the antimicrobial susceptibility of Mycoplasma hominis Tunisian strains have highlighted a significant resistance, even multi-resistance, to the most used antibiotic in the therapy of consequential infections. To address this concern, the present study aimed for the alternative of phytotherapy. Peganum harmala seed extract was tested as an antibacterial agent against multidrug-resistant M.hominis clinical strains. Material and Methods: Peganum harmala plant was collected from Ain Sebaa, Tabarka, North West region of Tunisia in April 2018, air-dried, grounded and extracted by different solvents.The crude methanolic extract was further partitioned with n-HEX, DCM, EtOAC and n-BuOl. Antibacterial activity was evaluated against M. hominis ATCC 23114 and 20 M. hominis clinical strains.The antimycoplasmal activity was tested by the microdilution method, and MIC values were determined. Phytochemical analysis and hemolytic activity on human erythrocytes were also performed. The active fraction was then subjected to purification, and the chemical identification of the active compound was investigated. Results: Among the tested fractions, the n-BuOH extract was the most active fraction since it exhibited an inhibitory effect against M. hominis ATCC 23114 and 80% of the tested clinical strains with MIC between 125 and 1000 µg/ml. The phytochemical analysis of the n-BuOH revealed its metabolic abundance in polyphenols, flavonoids and condensed tannin with levels of 257.37 mg AGE/g, 172.27 mg EC/g and 58.27 mg EC/g, respectively. In addition, P. harmala n-BuOH extract exhibited potent bactericidal activity against all M. hominis isolates with CMB values ranging between 125 and 4000 µg/ml. Further, the active fraction exhibited weak cytotoxicity effect at active concentrations when tested on human erythrocytes. The active compound was identified by gas chromatography–mass spectrometry as an indole alkaloid harmaline. Conclusion: In summary, Peganum harmala extract demonstrated an interesting anti-mycoplasmal activity against M. hominis Tunisian strains. Therefore, it could be considered as a potential candidate for the treatment of consequential infections. However, further studies are necessary to evaluate its mechanism of action in mycoplasmas.

Keywords: mycoplasma hominis, peganum harmala, antibioresistance, phytotherapy, phytochemical analysis

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Authors: Arzu Karahan, Loriano Ballarin, Baruch Rinkevich

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Marine invertebrates, the highly diverse phyla of multicellular organisms, represent phenomena that are either not found or highly restricted in the vertebrates. These include phenomena like budding, fission, a fusion of ramets, and high regeneration power, such as the ability to create whole new organisms from either tiny parental fragment, many of which are controlled by totipotent, pluripotent, and multipotent stem cells. Thus, there is very much that can be learned from these organisms on the practical and evolutionary levels, further resembling Darwin's words, “It is not the strongest of the species that survives, nor the most intelligent, but the one most responsive to change”. The ‘stem cell’ notion highlights a cell that has the ability to continuously divide and differentiate into various progenitors and daughter cells. In vertebrates, adult stem cells are rare cells defined as lineage-restricted (multipotent at best) with tissue or organ-specific activities that are located in defined niches and further regulate the machinery of homeostasis, repair, and regeneration. They are usually categorized by their morphology, tissue of origin, plasticity, and potency. The above description not always holds when comparing the vertebrates with marine invertebrates’ stem cells that display wider ranges of plasticity and diversity at the taxonomic and the cellular levels. While marine/aquatic invertebrates stem cells (MISC) have recently raised more scientific interest, the know-how is still behind the attraction they deserve. MISC, not only are highly potent but, in many cases, are abundant (e.g., 1/3 of the entire animal cells), do not locate in permanent niches, participates in delayed-aging and whole-body regeneration phenomena, the knowledge of which can be clinically relevant. Moreover, they have massive hidden potential for the discovery of new bioactive molecules that can be used for human health (antitumor, antimicrobial) and biotechnology. The MARISTEM COST action (Stem Cells of Marine/Aquatic Invertebrates: From Basic Research to Innovative Applications) aims to connect the European fragmented MISC community. Under this scientific umbrella, the action conceptualizes the idea for adult stem cells that do not share many properties with the vertebrates’ stem cells, organizes meetings, summer schools, and workshops, stimulating young researchers, supplying technical and adviser support via short-term scientific studies, making new bridges between the MISC community and biomedical disciplines.

Keywords: aquatic/marine invertebrates, adult stem cell, regeneration, cell cultures, bioactive molecules

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Authors: S. Mamoucha, J. Rahul, N. Christodoulakis

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Introduction: Nature has been the source of medicinal agents since the dawn of the human existence on Earth. Currently, millions of people, in the developing world, rely on medicinal plants for primary health care, income generation and lifespan improvement. In Greece, more than 5500 plant taxa are reported while about 250 of them are considered to be of great pharmaceutical importance. Among the plants used for medical purposes, Vitex agnus-castus L. (Verbenaceae) is known since ancient times. It is a small tree or shrub, widely distributed in the Mediterranean basin up to the Central Asia. It is also known as chaste tree or monks pepper. Theophrastus mentioned the shrub several times, as ‘agnos’ in his ‘Enquiry into Plants’. Dioscorides mentioned the use of V. agnus-castus for the stimulation of lactation in nursing mothers and the treatment of several female disorders. The plant has important medicinal properties and a long tradition in folk medicine as an antimicrobial, diuretic, digestive and insecticidal agent. Materials and methods: Leaves were cleaned, detached, fixed, sectioned and investigated with light and Scanning Electron Microscopy (SEM). Histochemical tests were executed as well. Specific histochemical reagents (osmium tetroxide, H2SO4, vanillin/HCl, antimony trichloride, Wagner’ s reagent, Dittmar’ s reagent, potassium bichromate, nitroso reaction, ferric chloride and di methoxy benzaldehyde) were used for the sub cellular localization of secondary metabolites. Results: Light microscopical investigations of the elongated leaves of V. agnus-castus revealed three layers of palisade parenchyma, just below the single layered adaxial epidermis. The spongy parenchyma is rather loose. Adaxial epidermal cells are larger in magnitude, compared to those of the abaxial epidermis. Four different types of capitate, secreting trichomes, were localized among the abaxial epidermal cells. Stomata were observed at the abaxial epidermis as well. SEM revealed the interesting arrangement of trichomes. Histochemical treatment on fresh and plastic embedded tissue sections revealed the nature and the sites of secondary metabolites accumulation (flavonoids, steroids, terpenes). Acknowledgment: This work was supported by IKY - State Scholarship Foundation, Athens, Greece.

Keywords: Vitex agnus-castus, leaf anatomy, histochemical reagents, secondary metabolites

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Authors: Alaaeddeen M. Seufi

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Many have been published on therapeutic derivatives from living organisms including insects. In addition to traditional maggot therapy, more than 900 therapeutic products were isolated from insects. Most people look at insects as enemies and others believe that insects are friends. Many beneficial insects rather than Honey Bees, Silk Worms and Shellac insect could insure human-insect friendship. In addition, insects could be MicroFactories, Biosensors or Bioreactors. InsectFarm is an amazing example of the applied research that transfers insects from laboratory to market by Prof Mircea Ciuhrii and co-workers. They worked for 18 years to derive therapeutics from insects. Their research resulted in production of more than 30 commercial medications derived from insects (e.g. Imunomax, Noblesse, etc.). Two general approaches were followed to discover drugs from living organisms. Some laboratories preferred biochemical approach to purify components of the innate immune system of insects and insect metabolites as well. Then the purified components could be tested for many therapeutic trials. Other researchers preferred molecular approach based on proteomic studies. Components of the innate immune system of insects were then tested for their medical activities. Our Laboratory team preferred to induce insect immune system (using oral, topical and injection routes of administration), then a transcriptomic study was done to discover the induced genes and to identify specific biomarkers that can help in drug discovery. Biomarkers play an important role in medicine and in drug discovery and development as well. Optimum biomarker development and application will require a team approach because of the multifaceted nature of biomarker selection, validation, and application. This team uses several techniques such as pharmacoepidemiology, pharmacogenomics, and functional proteomics; bioanalytical development and validation; modeling and simulation to improve and refine drug development. Our Achievements included the discovery of four components of the innate immune system of Spodoptera littoralis and Musca domestica. These components were designated as SpliDef (defesin), SpliLec (lectin), SpliCec (cecropin) and MdAtt (attacin). SpliDef, SpliLec and MdAtt were confirmed as antimicrobial peptides, while SpliCec was additionally confirmed as anticancer peptide. Our current research is going on to achieve something in antioxidants and anticoagulants from insects. Our perspective is to achieve something in the mass production of prototypes of our products and to reach it to the commercial level. These achievements are the integrated contributions of everybody in our team staff.

Keywords: AMPs, insect, innate immunitty, therappeutics

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Authors: Ira V. Stancheva, Ely G. Zayova, Maria P. Geneva, Marieta G. Hristozkova, Lyudmila I. Dimitrova, Maria I. Petrova

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The study describes a highly efficient and low-cost protocol for rapid and mass in vitro propagation of medicinal and aromatic plant species (Hyssopus officinalis L., Lamiaceae). Hyssop is an important aromatic herb used for its medicinal values because of its antioxidant, anti-inflammatory and antimicrobial properties. The protocol for large-scale multiplication of this aromatic plant was developed using young stem tips explants. The explants were sterilized with 0.04% mercuric chloride (HgCl₂) solution for 20 minutes and washing three times with sterile distilled water in 15 minutes. The cultural media was full and half strength Murashige and Skoog medium containing indole-3-butyric acid. Full and ½ Murashige and Skoog media without auxin were used as controls. For each variant 20 glass tubes with two plants were used. In each tube two tip and nodal explants were inoculated. Maximum shoot and root number were obtained on ½ Murashige and Skoog medium supplemented with 0.1 mg L-1 indole-3-butyric acid at the same time after four weeks of culture. The number of shoots per explant and shoot height were considered. The data on rooting percentage, the number of roots per plant and root length were collected after the same cultural period. The highest percentage of survival 85% for this medicinal plant was recorded in mixture of soil, sand and perlite (2:1:1 v/v/v). This mixture was most suitable for acclimatization of all propagated plants. Ex vitro acclimatization was carried out at 24±1 °C and 70% relative humidity under 16 h illuminations (50 μmol m⁻²s⁻¹). After adaptation period, the all plants were transferred to the field. The plants flowered within three months after transplantation. Phenotypic variations in the acclimatized plants were not observed. An average of 90% of the acclimatized plants survived after transferring into the field. All the in vitro propagated plants displayed normal development under the field conditions. Developed in vitro techniques could provide a promising alternative tool for large-scale propagation that increases the number of homologous plants for field cultivation. Acknowledgments: This study was conducted with financial support from National Science Fund at the Bulgarian Ministry of Education and Science, Project DN06/7 17.12.16.

Keywords: Hyssopus officinalis L., in vitro culture, micro propagation, acclimatization

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Authors: D. Berdous, H. Ferfera-Harrar

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Superabsorbent polymers (SAPs) or hydrogels with three-dimensional hydrophilic network structure are high-performance water absorbent and retention materials. The in situ synthesis of metal nanoparticles within polymeric network as antibacterial agents for bio-applications is an approach that takes advantage of the existing free-space into networks, which not only acts as a template for nucleation of nanoparticles, but also provides long term stability and reduces their toxicity by delaying their oxidation and release. In this work, SAP/nanosilver nanocomposites were successfully developed by a unique green process at room temperature, which involves in situ formation of silver nanoparticles (AgNPs) within hydrogels as a template. The aim of this study is to investigate whether these AgNPs-loaded hydrogels are potential candidates for antimicrobial applications. Firstly, the superabsorbents were prepared through radical copolymerization via grafting and crosslinking of acrylamide (AAm) onto chitosan backbone (Cs) using potassium persulfate as initiator and N,N’-methylenebisacrylamide as the crosslinker. Then, they were hydrolyzed to achieve superabsorbents with ampholytic properties and uppermost swelling capacity. Lastly, the AgNPs were biosynthesized and entrapped into hydrogels through a simple, eco-friendly and cost-effective method using aqueous silver nitrate as a silver precursor and curcuma longa tuber-powder extracts as both reducing and stabilizing agent. The formed superabsorbents nanocomposites (Cs-g-PAAm)/AgNPs were characterized by X-ray Diffraction (XRD), UV-visible Spectroscopy, Attenuated Total reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR), Inductively Coupled Plasma (ICP), and Thermogravimetric Analysis (TGA). Microscopic surface structure analyzed by Transmission Electron Microscopy (TEM) has showed spherical shapes of AgNPs with size in the range of 3-15 nm. The extent of nanosilver loading was decreased by increasing Cs content into network. The silver-loaded hydrogel was thermally more stable than the unloaded dry hydrogel counterpart. The swelling equilibrium degree (Q) and centrifuge retention capacity (CRC) in deionized water were affected by both contents of Cs and the entrapped AgNPs. The nanosilver-embedded hydrogels exhibited antibacterial activity against Escherichia coli and Staphylococcus aureus bacteria. These comprehensive results suggest that the elaborated AgNPs-loaded nanomaterials could be used to produce valuable wound dressing.

Keywords: antibacterial activity, nanocomposites, silver nanoparticles, superabsorbent Hydrogel

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Authors: Mohammad Abdollahi

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Tomato (Lycopersicon esculentum Mill.) is one of the most popular, widely grown and the second most important vegetable crop, after potatoes. Nematodes have been identified as one of the major pests affecting tomato production throughout the world. The most destructive nematodes are the genus Meloidogyne. Most widespread and devastating species of this genus are M. incognita, M. javanica, and M. arenaria. These species can cause complete crop loss under adverse growing conditions. There are several potential methods for management of the root knot nematodes. Although the chemicals are widely used against the phytonematodes, because of hazardous effects of these compounds on non-target organisms and on the environment, there is a need to develop other control strategies. Nowadays, non-chemical measures are widely used to control the plant parasitic nematodes. Biocontrol of phytonematodes is an important method among environment-friendly measures of nematode management. There are some soil-inhabiting fungi that have biocontrol potential on phytonematodes, which can be used in nematode management program. The fungus Metarhizium anisopliae, originally is an entomopathogenic bioagent. Biocontrol potential of this fungus on some phytonematodes has been reported earlier. Recently, use of organic soil amendments as well as the use of bioagents is under special attention in sustainable agriculture. This research aimed to reduce the pesticide use in control of root-knot nematode, Meloidogyne javanica in tomato. The effects of M. anisopliae IMI 330189 and different levels of oak tree debris on M. javanica were determined. The combination effect of the fungus as well as the different rates of soil amendments was determined. Pots were filled with steam pasteurized soil mixture and the six leaf tomato seedlings were inoculated with 3000 second stage larvae of M. javanica/kg of soil. After eight weeks, plant growth parameters and nematode reproduction factors were compared. Based on the results of our experiment, combination of M. anisopliae IMI 330189 and oak debris caused more than 90% reduction in reproduction factor of nematode, at the rates of 100 and 150 g/kg soil (P ≤ 0.05). As compared to control, the reduction in number of galls was 76%. It was 86% for nematode reproduction factor, showing the significance of combined effect of both tested agents. Our results showed that plant debris can increase the biological activity of the tested bioagent. It was also proved that there was no adverse effect of oak debris, which potentially has antimicrobial activity, on antagonistic power of applied bioagent.

Keywords: biological control, nematode management, organic soil, Quercus branti, root knot nematode, soil amendment

Procedia PDF Downloads 142

Authors: Judit Perjessy, Zsolt Zalan, Ferenc Hegyi, Eniko Horvath-Szanics, Krisztina Takacs, Andras Nagy, Adel Klupacs, Erika Koppany-Szabo, Zhirong Wang, Kaituo Wang, Muying Du, Jianquan Kan

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The post-harvest diseases cause great economic losses in the fruit and vegetables; the prevention of these deterioration has great importance. Against the fungi, which cause most of the diseases, are extensively used the fungicides. However, there are increasing consumer concerns over the presence of pesticide residues in food. An alternative and in recent years, increasingly studied method for the prevention of the diseases is biocontrol, where antagonistic microorganisms are used for the control of fungi. The genera of Lactobacillus is well known and extensively studied, but its applicability as biocontrol agents in post-harvest preservation of fruit and vegetables is poorly investigated. However these bacteria can be found on the surface of the plants and have great antimicrobial activity. In our study we have investigated the chitinase activity, the antifungal effect and the applicability of several Lactobacillus strains to select potential biocontrol agents. We investigated the determination of the environmental parameters of a gene (encoding chitinase) expression and we also investigated the relationship between actual antifungal activity and potential chitinase activity. Mixed cultures were also developed to enhance the antifungal activity and determined the optimal mold spore and bacteria concentration ratio for the appropriate efficacy. Five Lactobacillus strains (L. acidophilus N2, L. delbrueckii subsp. bulgaricus B397, L. sp. 2231, L. sake subsp. sake 2471, L. buchneri 1145) possess chitinase-coding gene from the 43 investigated Lactobacillus strains. Proteins with similar molecular weight and separation properties like bacterial chitinases were detected from these strains, which also possess chitin-binding property. Nevertheless, they were inactive, lacks the chitinolytic activity. In point of the cumulative activity of inhibition, our results showed that certain strains were statistically significant in a positive direction compared to other strains, e.g., L. rhamnosus VT1 and L. Casey 154 have shown great general antifungal effect against 11 molds from the genera Penicillium and Botrytis and isolated from spoiled fruit and vegetables. Also, some mixed cultures (L. rhamnosus VT1 - L. Plantarum 299v) showed significant antifungal effects against the indigenous molds on the surface of apple fruit during the industrial storage experiment. Thus, they could be promising for post-harvest biopreservation.

Keywords: biocontrol, chitinase, Lactobacillus, post-harvest

Procedia PDF Downloads 121

Authors: L. D. Upegui, Isabel Alvarez-Solorza, Karina Garduno-Ulloa, Maren Boecker

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Introduction: The increasing prevalence rate of resistant and multiresistant bacterial strains to antibiotics is a threat to public health and requires a rapid multifunctional answer. Individuals that are affected by resistant strains present a higher morbidity and mortality than individuals that are infected with the same species of bacteria but with sensitive strains. There have been identified risk factors that are related to the misuse and overuse of antibiotics, like socio-demographic characteristics and psychological aspects of the individuals that have not been explored objectively due to a lack of valid and reliable instruments for their measurement. Objective: To validate a questionnaire for the evaluation of the levels of knowledge related to the use of antibiotics in a Mexican population. Materials and Methods: Analytical cross-sectional observational study. The questionnaire consists of 12 items to evaluated knowledge (1=no, 2=not sure, 3=yes) regarding the use of antibiotics, with higher scores corresponding to a higher level of knowledge. Data are collected in a sample of students. Data collection is still ongoing. In this abstract preliminary results of 30 respondents are reported which were collected during pilot-testing. The validation of the instrument was done using the Rasch model. Fit to the Rasch model was tested checking overall fit to the model, unidimensionality, local independence and evaluating the presence of Differential Item Functioning (DIF) by age and gender. The software Rumm2030 and the SPSS were used for the analyses. Results: The participants of the pilot-testing presented an average age of 32 years ± 12.6 and 53% were women. The preliminary results indicated that the items showed good fit to the Rasch model (chi-squared=12.8 p=0.3795). Unidimensionality (number of significant t-tests of 3%) could be proven, the items were locally independent, and no DIF was observed. Knowledge was the smallest regarding statements on the role of antibiotics in treating infections, e.g., most of the respondents did not know that antibiotics would not work against viral infections (70%) and that they could also cause side effects (87%). The knowledge score ranged from 0 to 100 points with a transformed measurement (mean of knowledge 27.1 ± 4.8). Conclusions: The instrument showed good psychometric proprieties. The low scores of knowledge about antibiotics suggest that misinterpretations on the use of these medicaments were prevalent, which could influence the production of antibiotic resistance. The application of this questionnaire will allow the objective identification of 'Hight risk groups', which will be the target population for future educational campaigns, to reduce the knowledge gaps on the general population as an effort against antibiotic resistance.

Keywords: antibiotics, knowledge, misuse, overuse, questionnaire, Rasch model, validation

Procedia PDF Downloads 122

Authors: Randa M. T. Mohamed

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Spices have long been used as traditional ingredients in the kitchen for seasoning, coloring, aromatic and food preservative properties. Besides, spices are equally used for therapeutic purposes. The objective of this study was to survey and document the medicinal properties of spices commonly used in the Sudanese kitchen for different food preparations. Also, extracts from reported spices were screened for the presence of secondary metabolites as well as their antioxidant and beta-lactamase inhibitory properties. This study was conducted in the Rekabbya Quartier in Omdurman, Khartoum State, Sudan. Information was collected by carrying out semi-structured interviews. All informants (30) in the present study were women. Spices were purchased from Attareen shop in Omdurman. Essential oils from spices were extracted by hydrodistillation and ethanolic extracts by maceration. Phytochemical screening was performed by thin layer chromatography (TLC). The antioxidant capacity of essential oils and ethanolic extracts was investigated through TLC bioautography. Beta lactamase inhibitory activity was performed by the acidimetric test. Ethnobotany study showed that a total of 16 spices were found to treat 36 ailments belonging to 10 categories. The most frequently claimed medicinal uses were for the digestive system diseases treated by 14 spices and respiratory system diseases treated by 8 spices. Gynaecological problems were treated by 4 spices. Dermatological diseases were cured by 5 spices while infections caused by tapeworms and other microbes causing dysentery were treated by 3 spices. 4 spices were used to treat bad breath, bleeding gum and toothache. Headache, eyes infection, cardiac stimulation and epilepsy were treated by one spice each. Other health problem like fatigue and loss of appetite and low breast milk production were treated by 1, 3 and 2 spices respectively. The majority (69%, 11/16) of spices were exported from different countries like India, China, Indonesia, Ethiopia, Egypt and Nigeria while 31% (5/16) was cultivated in Sudan. Essential oils of all spices were rich in terpenes while ethanolic extracts contained variable classes of secondary metabolites. Both essential oils and ethanolic extracts of all spices exerted considerable antioxidant activity. Only one extract, Syzygium aromaticum, possessed beta lactamase inhibitory activity. In conclusion, this study could contribute in conserving information on traditional medicinal uses of spices in Sudan. Also, the results demonstrated the potential of some of these spices to exert beneficial antimicrobial and antioxidant effect. Detailed phytochemical and biological assays of these spices are recommended.

Keywords: spices, ethnobotany, phytoconstituents, antioxidant, beta lactamase inhibition

Procedia PDF Downloads 45

Authors: Zachary Muthii Rukenya, James Mbaria, Peter Mbaabu, Kiama Stephen Gitahi, Ronald Onzago

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Aloe turkanensis is one of the widely used medicinal shrub and in Kenya the plant is mainly found in Baringo, Isiolo, Laikipia, Turkana and West Pokot Counties where it is used in ethno-medicine and ethno-veterinary medicine. The Turkana community uses the plant products to manage malaria, wounds, stomach ache, constipation, pain, skin infection, poultry diseases and retained afterbirth in cows. This evaluated the efficacy and safety of the plant obtained from Turkana County, Kenya. Preliminary data on the use of the plant in the county was collected through observation, photographing and interviews. A sample of the whole plant was harvested in Natira sublocation, in ex-Turkana west district in February 2012 after identification by Aloe-working group herbalists who voluntarily provided information on its medicinal uses. Botanical identification was done at Kenya Forest Research Centre in Karura where voucher specimen was deposited. Cold maceration using 70% methanol and distilled water was used for extraction. Bioassays were to determine the effects of the plant extracts on brine shrimp and selected bacterial and fungal cultures. The extracts were tested in-vitro activity against standard cultures of B. cereus (ATCC 11778), S. aureus (ATCC25923), P. aeroginosa (ATCC 27853), E. coli (ATCC 25922) and a human infections clinical isolate of C. albicans. The extracts of Aloe turkanensis inhibited the growth B. cereus (100-200 mg/ml), S. aureus (50-100 mg/ml), P. aeroginosa (200mg/ml), E. coli (400mg/ml) while C. albicans was not affected. The extracts also inhibited the growth of S. aureus and B. cereus with mean diameters of inhibition zones being 19.75±1 mm and 18.5±05 mm reapectively. Phytochemical screening showed the presence of alkaloids, tarpenoids, steroids, quinones, saponins and tannins in the plant extracts. The extract was found to be non-toxic at a concentration of 1000µg/ml with a 100% survival of Brine Shrimp larva. It was concluded that Aloe turkanensis growing the study area has metabolites that inhibit the growth of microorganisms and is however, there is need for further studies to validate the in-vivo bioactivity of the plant and more generate adequate toxicological data.to support conservation, value chain addition of its products and widespread use as a herbal remedy.

Keywords: Aloe turkanensis, bioactivity, cultivated, human infections

Procedia PDF Downloads 294

Authors: Koppula Prawan, Kehinde D. Oyeyemi, Kushal P. Singh

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As people become more conscious of the detrimental consequences of conventional agricultural practices on the environment and human health, organic, sustainable agriculture and plant protection employing microorganisms have grown in importance. Although the use of microorganisms in agriculture is a centuries-old tradition, it has recently attracted renewed interest as a sustainable alternative to chemical-based plant protection and fertilization. Healthy soil is the cornerstone of sustainable agriculture, and microbes are essential to this process. Synthetic fertilizers and pesticides can destroy the beneficial microorganisms in the soil, upsetting the ecosystem's equilibrium. By utilizing organic farming's natural practices, such as the usage of microbes, it aims to maintain and improve the health of the soil. Microbes have several functions in agriculture, including nitrogen fixation, phosphorus solubilization, and disease suppression. Nitrogen fixation is the process by which certain microbes, such as rhizobia and Azotobacter, convert atmospheric nitrogen into a form that plants can use. Phosphorus solubilization involves the conversion of insoluble phosphorus into a soluble form that plants can absorb. Disease suppression involves the use of microbes to control plant diseases by competing with pathogenic organisms for resources or by producing antimicrobial compounds. Microbes can be applied to plants through seed coatings, foliar sprays, or soil inoculants. Seed coatings involve applying a mixture of microbes and nutrients to the surface of seeds before planting. Foliar sprays involve applying microbes and nutrients to the leaves of plants during the growing season. Soil inoculants involve adding microbes to the soil before planting. The use of microbes in plant protection and fertilization has several advantages over conventional methods. Firstly, microbes are natural and non-toxic, making them safe for human health and the environment. Secondly, microbes have the ability to adapt to changing environmental conditions, making them more resilient to drought and other stressors. Finally, the use of microbes can reduce the need for synthetic fertilizers and pesticides, reducing costs and minimizing environmental impact. In conclusion, organic, sustainable agriculture and plant protection using microbes are an effective and sustainable alternatives to conventional farming practices. The use of microbes can help to preserve and enhance soil health, increase plant productivity, and reduce the need for synthetic fertilizers and pesticides. As the demand for organic and sustainable agriculture continues to grow, the use of microbes is likely to become more widespread, providing a more environmentally friendly and sustainable future for agriculture.

Keywords: microbes, inoculants, fertilization, soil health, conventional.

Procedia PDF Downloads 43

Authors: Piya Roychoudhury, Ruma Pal

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Cyanobacteria, commonly known as blue green algae were found to be an effective bioreagent for nanoparticle synthesis. Nowadays silver nanoparticles (AgNPs) are very popular due to their antimicrobial and anti-proliferative activity. To exploit these characters in different biotechnological fields, it is very essential to synthesize more stable, non-toxic nano-silver. For this reason silver-gold alloy (Ag-AuNPs) nanoparticles are of great interest as they are more stable, harder and more effective than single metal nanoparticles. In the present communication we described a simple technique for rapid synthesis of biocompatible AgNP and Ag-AuNP employing cyanobacteria, Leptolyngbya and Lyngbya respectively. For synthesis of AgNP the biomass of Leptolyngbya valderiana (200 mg Fresh weight) was exposed to 9 mM AgNO3 solution (pH 4). For synthesis of Ag-AuNP Lyngbya majuscula (200 mg Fresh weight) was exposed to equimolar solution of hydrogen tetra-auro chlorate and silver nitrate (1mM, pH 4). After 72 hrs of exposure thallus of Leptolyngyba turned brown in color and filaments of Lyngbya turned pink in color that indicated synthesis of nanoparticles. The produced particles were extracted from the cyanobacterial biomass using nano-capping agent, sodium citrate. Firstly, extracted brown and pink suspensions were taken for Energy Dispersive X-ray (EDAX) analysis to confirm the presence of silver in brown suspension and presence of both gold and silver in pink suspension. Extracted nanoparticles showed a distinct single plasmon band (AgNP at 411 nm; Ag-Au NP at 481 nm) in Uv-vis spectroscopy. It was revealed from Transmission electron microscopy (TEM) that all the synthesized particles were spherical in nature with a size range of ~2-25 nm. In X-ray powder diffraction (XRD) analysis four intense peaks appeared at 38.2°, 44.5°, 64.8°and 77.8° which confirmed the crystallographic nature of synthesized particles. Presence of different functional groups viz. N-H, C=C, C–O, C=O on the surface of nanoparticles were recorded by Fourier transform infrared spectroscopy (FTIR). Scanning Electron microscopy (SEM) images showed the surface topography of metal treated filaments of cyanobacteria. The stability of the particles was observed by Zeta potential study. Antibiotic property of synthesized particles was tested by Agar well diffusion method against gram negative bacteria Pseudomonas aeruginosa. Overall, this green-technique requires low energy, less manufacturing cost and produces rapidly eco-friendly metal nanoparticles.

Keywords: cyanobacteria, silver nanoparticles, silver-gold alloy nanoparticles, spectroscopy

Procedia PDF Downloads 289

Authors: Chrislaura Carmo, Luca Deiana, Mafalda Laranjo, Abilio Sobral, Armando Cordova

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Photodynamic therapy (PDT) is currently used for the treatment of malignancies and premalignant tumors. It is based on the capture of a photosensitizing molecule (PS) which, when excited by light at a certain wavelength, reacts with oxygen and generates oxidizing species (radicals, singlet oxygen, triplet species) in target tissues, leading to cell death. BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indaceno) derivatives are emerging as important candidates for photosensitizer in photodynamic therapy of cancer cells due to their high triplet quantum yield. Today these dyes are relevant molecules in photovoltaic materials and fluorescent sensors. In this study, it will be demonstrated the possibility that BODIPY can be covalently linked to thioglycolic acid through the click reaction. Thiol−ene click chemistry has become a powerful synthesis method in materials science and surface modification. The design of biobased allyl-terminated precursors with high renewable carbon content for the construction of the thiol-ene polymer networks is essential for sustainable development and green chemistry. The work aims to synthesize the BODIPY (10-(4-(allyloxy) phenyl)-2,8-diethyl-5,5-difluoro-1,3,7,9-tetramethyl-5H-dipyrrolo[1,2-c:2',1'-f] [1,3,2] diazaborinin-4-ium-5-uide) and to click reaction with Thioglycolic acid. BODIPY was synthesized by the condensation reaction between aldehyde and pyrrole in dichloromethane, followed by in situ complexation with BF3·OEt2 in the presence of the base. Then it was functionalized with allyl bromide to achieve the double bond and thus be able to carry out the click reaction. The thiol−ene click was performed using DMPA (2,2-Dimethoxy-2-phenylacetophenone) as a photo-initiator in the presence of UV light (320–500 nm) in DMF at room temperature for 24 hours. Compounds were characterized by standard analytical techniques, including UV-Vis Spectroscopy, 1H, 13C, 19F NMR and mass spectroscopy. The results of this study will be important to link BODIPY to polymers through the thiol group offering a diversity of applications and functionalization. This new molecule can be tested as third-generation photosensitizers, in which the dye is targeted by antibodies or nanocarriers by cells, mainly in cancer cells, PDT and Photodynamic Antimicrobial Chemotherapy (PACT). According to our studies, it was possible to visualize a click reaction between allyl BODIPY and thioglycolic acid. Our team will also test the reaction with other thiol groups for comparison. Further, we will do the click reaction of BODIPY with a natural polymer linked with a thiol group. The results of the above compounds will be tested in PDT assays on various lung cancer cell lines.

Keywords: bodipy, click reaction, thioglycolic acid, allyl, thiol-ene click

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Authors: Eliane G. Tótoli, Hérida Regina N. Salgado

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Daptomycin is an important antimicrobial agent used in clinical practice nowadays, since it is very active against some Gram-positive bacteria that are particularly challenges for the medicine, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). The importance of environmental preservation has receiving special attention since last years. Considering the evident need to protect the natural environment and the introduction of strict quality requirements regarding analytical procedures used in pharmaceutical analysis, the industries must seek environmentally friendly alternatives in relation to the analytical methods and other processes that they follow in their routine. In view of these factors, green analytical chemistry is prevalent and encouraged nowadays. In this context, infrared spectroscopy stands out. This is a method that does not use organic solvents and, although it is formally accepted for the identification of individual compounds, also allows the quantitation of substances. Considering that there are few green analytical methods described in literature for the analysis of daptomycin, the aim of this work was the development and validation of a green analytical method for the quantification of this drug in lyophilized powder for injectable solution, by Fourier-transform infrared spectroscopy (FT-IR). Method: Translucent potassium bromide pellets containing predetermined amounts of the drug were prepared and subjected to spectrophotometric analysis in the mid-infrared region. After obtaining the infrared spectrum and with the assistance of the IR Solution software, quantitative analysis was carried out in the spectral region between 1575 and 1700 cm-1, related to a carbonyl band of the daptomycin molecule, and this band had its height analyzed in terms of absorbance. The method was validated according to ICH guidelines regarding linearity, precision (repeatability and intermediate precision), accuracy and robustness. Results and discussion: The method showed to be linear (r = 0.9999), precise (RSD% < 2.0), accurate and robust, over a concentration range from 0.2 to 0.6 mg/pellet. In addition, this technique does not use organic solvents, which is one great advantage over the most common analytical methods. This fact contributes to minimize the generation of organic solvent waste by the industry and thereby reduces the impact of its activities on the environment. Conclusion: The validated method proved to be adequate to quantify daptomycin in lyophilized powder for injectable solution and can be used for its routine analysis in quality control. In addition, the proposed method is environmentally friendly, which is in line with the global trend.

Keywords: daptomycin, Fourier-transform infrared spectroscopy, green analytical chemistry, quality control, spectrometry in IR region

Procedia PDF Downloads 355

Authors: Valeria Velasco, Ana M. Bonilla, José L. Vergara, Alcides Lofa, Jorge Campos, Pedro Rojas-García

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Staphylococcus aureus is both commensal bacterium and opportunistic pathogen that can cause different diseases in humans and can rapidly develop antimicrobial resistance. Since this bacterium has the ability to colonize the nares and skin of humans and animals, there is a risk of contamination of food in different steps of the food chain supply. Emerging strains have been detected in food-producing animals and meat, such as methicillin-resistant S. aureus (MRSA). The aim of this study was to determine the prevalence and oxacillin susceptibility of S. aureus in the pork chain supply in Chile and to suggest some natural antimicrobials for control. A total of 487 samples were collected from pigs (n=332), carcasses (n=85), and retail pork meat (n=70). Presumptive S. aureus colonies were isolated by selective enrichment and culture media. The confirmation was carried out by biochemical testing (Api® Staph) and molecular technique PCR (detection of nuc and mecA genes, associated with S. aureus and methicillin resistance, respectively). The oxacillin (β-lactam antibiotic that replaced methicillin) susceptibility was assessed by minimum inhibitory concentration (MIC) using the Epsilometer test (Etest). A preliminary assay was carried out to test thymol, carvacrol, oregano essential oil (Origanum vulgare L.), Maqui or Chilean wineberry extract (Aristotelia chilensis (Mol.) Stuntz) as anti-staphylococcal agents using the disc diffusion method at different concentrations. The overall prevalence of S. aureus in the pork chain supply reached 33.9%. A higher prevalence of S. aureus was determined in carcasses (56.5%) than in pigs (28.3%) and pork meat (32.9%) (P ≤ 0.05). The prevalence of S. aureus in pigs sampled at farms (40.6%) was higher than in pigs sampled at slaughterhouses (23.3%) (P ≤ 0.05). The contamination of no packaged meat with S. aureus (43.1%) was higher than in packaged meat (5.3%) (P ≤ 0.05). The mecA gene was not detected in S. aureus strains isolated in this study. Two S. aureus strains exhibited oxacillin resistance (MIC ≥ 4µg/mL). Anti-staphylococcal activity was detected in solutions of thymol, carvacrol, and oregano essential oil at all concentrations tested. No anti-staphylococcal activity was detected in Maqui extract. Finally, S. aureus is present in the pork chain supply in Chile. Although the mecA gene was not detected, oxacillin resistance was found in S. aureus and could be attributed to another resistance mechanism. Thymol, carvacrol, and oregano essential oil could be used as anti-staphylococcal agents at low concentrations. Research project Fondecyt No. 11140379.

Keywords: antimicrobials, mecA gen, nuc gen, oxacillin susceptibility, pork meat

Procedia PDF Downloads 202

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 375

Authors: Kanti Sapkota, Do Hyun Lee, Sung Soo Han

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Nanocomposites have been widely used in various fields such as electronics, catalysis, and in chemical, biological, biomedical and optical fields. They display broad biomedical properties like antidiabetic, anticancer, antioxidant, antimicrobial and antibacterial activities. Moreover, nanomaterials have been used for wastewater treatment. Particularly, bimetallic hybrid nanocomposites exhibit unique features as compared to their monometallic components. Hybrid nanomaterials not only afford the multifunctionality endowed by their constituents but can also show synergistic properties. In addition, these hybrid nanomaterials have noteworthy catalytic and optical properties. Notably, Au−Ag based nanoparticles can be employed in sensor and catalysis due to their characteristic composition-tunable plasmonic properties. Due to their importance and usefulness, various efforts were developed for their preparation. Generally, chemical methods have been described to synthesize such bimetallic nanocomposites. In such chemical synthesis, harmful and hazardous chemicals cause environmental contamination and increase toxicity levels. Therefore, ecologically benevolent processes for the synthesis of nanomaterials are highly desirable to diminish such environmental and safety concerns. In this regard, here we disclose a simple, cost-effective, external additive free and eco-friendly method for the synthesis of Au-Ag@AgCl nanocomposites using Nephrolepis cordifolia root extract. Au-Ag@AgCl NCs were obtained by the simultaneous reduction of cationic Ag and Au into AgCl in the presence of plant extract. The particle size of 10 to 50 nm was observed with the average diameter of 30 nm. The synthesized nanocomposite was characterized by various modern characterization techniques. For example, UV−visible spectroscopy was used to determine the optical activity of the synthesized NCs, and Fourier transform infrared (FT-IR) spectroscopy was employed to investigate the functional groups present in the biomolecules that were responsible for both reducing and capping agents during the formation of nanocomposites. Similarly, powder X-ray diffraction (XRD), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), thermogravimetric analysis (TGA) and energy-dispersive X-ray (EDX) spectroscopy were used to determine crystallinity, size, oxidation states, thermal stability and weight loss of the synthesized nanocomposites. As a synthetic application, the synthesized nanocomposite exhibited excellent catalytic activity for the multicomponent synthesis of biologically interesting quinoline molecules via domino annulation-aromatization reaction of aniline, arylaldehyde, and phenyl acetylene derivatives. Interestingly, the nanocatalyst was efficiently recycled for five times without substantial loss of catalytic properties.

Keywords: nanoparticles, catalysis, multicomponent, quinoline

Procedia PDF Downloads 97

Authors: Sanjay Shukla, A. Nayak, R. K. Sharma, A. P. Singh, S. P. Tiwari

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Excessive use of antibiotics is a major problem in the treatment of wounds and other chronic infections, and antibiotic treatment is frequently non-curative, thus alternative treatment is necessary. Phage therapy is considered one of the most promising approaches to treat multi-drug resistant bacterial pathogens. Infections caused by Staphylococcus aureus are very efficiently controlled with phage cocktails, containing a different individual phages lysate infecting a majority of known pathogenic S. aureus strains. The aim of the present study was to evaluate the efficacy of a purified phage cocktail for prophylactic as well as therapeutic application in mouse model and in large animals with chronic septic infection of wounds. A total of 150 sewage samples were collected from various livestock farms. These samples were subjected for the isolation of bacteriophage by the double agar layer method. A total of 27 sewage samples showed plaque formation by producing lytic activity against S. aureus in the double agar overlay method out of 150 sewage samples. In TEM, recovered isolates of bacteriophages showed hexagonal structure with tail fiber. In the bacteriophage (ØVS) had an icosahedral symmetry with the head size 52.20 nm in diameter and long tail of 109 nm. Head and tail were held together by connector and can be classified as a member of the Myoviridae family under the order of Caudovirale. Recovered bacteriophage had shown the antibacterial activity against the S. aureus in vitro. Cocktail (ØVS1, ØVS5, ØVS9, and ØVS 27) of phage lysate were tested to know in vivo antibacterial activity as well as the safety profile. Result of mice experiment indicated that the bacteriophage lysate were very safe, did not show any appearance of abscess formation, which indicates its safety in living system. The mice were also prophylactically protected against S. aureus when administered with cocktail of bacteriophage lysate just before the administration of S. aureuswhich indicates that they are good prophylactic agent. The S. aureusinoculated mice were completely recovered by bacteriophage administration with 100% recovery, which was very good as compere to conventional therapy. In the present study, ten chronic cases of the wound were treated with phage lysate, and follow up of these cases was done regularly up to ten days (at 0, 5, and 10 d). The result indicated that the six cases out of ten showed complete recovery of wounds within 10 d. The efficacy of bacteriophage therapy was found to be 60% which was very good as compared to the conventional antibiotic therapy in chronic septic wounds infections. Thus, the application of lytic phage in single dose proved to be innovative and effective therapy for the treatment of septic chronic wounds.

Keywords: phage therapy, S aureus, antimicrobial resistance, lytic phage, and bacteriophage

Procedia PDF Downloads 92

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

Abstract:

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: N. Selvi Gunel, L. M. Oktay, H. Memmedov, B. Durmaz, H. Kalkan Yildirim, E. Yildirim Sozmen

Abstract:

Object: Propolis which consists of compounds that are accepted as antioxidant, antimicrobial, antiseptic, antibacterial, anti-inflammatory, anti-mutagenic, immune-modulator and cytotoxic, is frequently used in current therapeutic applications. However, some of them result in allergic side effects, causing consumption to be restricted. Previously our group has succeeded in producing a new biotechnological product which was less allergenic. In this study, we purpose to optimize production conditions of this biologically-transformed propolis and determine the cytotoxic effects of obtained new products on colon cancer cell line (HCT-116). Method: Firstly, solid propolis samples were dissolved in water after weighing, grinding and sizing (sieve-35mesh) and applied 40 kHz/10 min ultrasonication. Samples were prepared according to inoculation with Lactobacillus plantarum in two different proportions (2.5% and 3.5%). Chromatographic analyzes of propolis were performed by UPLC-MS/MS (Waters, Milford, MA) system. Results were analysed by UPLC-MS/MS system MassLynx™ 4.1 software. HCT-116 cells were treated with propolis examples at 25-1000 µg/ml concentrations and cytotoxicity were measured by using WST-8 assay at 24, 48, and 72 hours. Samples with biological transformation were compared with the non-transformed control group samples. Our experiment groups were formed as follows: untreated (group 1), propolis dissolved in water ultrasonicated at 40 kHz/10 min (group 2), propolis dissolved in water ultrasonicated at 40 kHz/10 min and inoculated 2.5% L. plantarum L1 strain (group 3), propolis dissolved in water ultrasonicated at 40 kHz/10 min and inoculated 3.5% L. plantarum L3 strain (group 4). Obtained data were calculated with Graphpad Software V5 and analyzed by two-way ANOVA test followed by Bonferroni test. Result: As a result of our study, the cytotoxic effect of propolis samples on HCT-116 cells was evaluated. There was a 7.21 fold increase in group 3 compared to group 2 in the concentration of 1000 µg/ml, and it was a 6.66 fold increase in group 3 compared to group 1 at the end of 24 hours. At the end of 48 hours, in the concentration of 500 µg/ml, it was determined 4.7 fold increase in group 4 compared to group 3. At the same time, in the concentration of 750 µg/ml it was determined 2.01 fold increase in group 4 compared to group 3 and in the same concentration, it was determined 3.1 fold increase in group 4 compared to group 2. Also, at the 72 hours, in the concentration of 750 µg/ml, it was determined 2.42 fold increase in group 3 according to group 2 and in the same time, in the concentration of 1000 µg/ml, it was determined 2.13 fold increase in group 4 according to group 2. According to cytotoxicity results, the group which were ultrasonicated at 40 kHz/10min and inoculated 3.5% L. plantarum L3-strain had a higher cytotoxic effect. Conclusion: It is known that bioavailability of propolis is halved in six months. The data obtained from our results indicated that biologically-transformed propolis had more cytotoxic effect than non-transformed group on colon cancer cells. Consequently, we suggested that L. plantarum-transformation provides both reduction of allergenicity and extension of bioavailability period by enhancing healthful polyphenols.

Keywords: bio-transformation, propolis, colon cancer, cytotoxicity

Procedia PDF Downloads 113

Authors: Vivek Bhat, Rohini Kelkar, Sanjay Biswas

Abstract:

Introduction: Cancer patients are at increased risk of bacterial infections. This may due to the disease process itself, the effect of chemotherapeutic drugs or invasive procedures such as catheterization. A wide variety of bacteria including some emerging pathogens are increasingly being reported from these patients. The incidence of multidrug-resistant organisms particularly in the Gram negative group is also increasing, with higher resistance rates seen to cephalosporins, β-lactam/β-lactam inhibitor combinations, and the carbapenems. This study documents the bacteriological spectrum of infections and their resistance patterns in cancer patients. Methods: This study includes all bacterial isolates recovered from infections cancer patients over a period of 18 months. Samples included Blood cultures, Pus/wound swabs, urine, tissue biopsies, body fluids, catheter tips and respiratory specimens such as sputum and bronchoalveolar lavage (BAL). All samples were processed in the microbiology laboratory as per standard laboratory protocols. Organisms were identified to species level and antimicrobial susceptibility testing was performed manually by the disc diffusion technique or in the Vitek-2 (Biomereux, France) instrument. Interpretations were as per Clinical laboratory Standards Institute (CLSI) guidelines. Results: A total of 1150 bacterial isolates were cultured from 884 test samples during the study period. Of these 227 were Gram-positive and 923 were Gram-negative organisms. Staphylococcus aureus (99 isolates) was the commonest Gram-positive isolate followed by Enterococcus (79) and Gr A Streptococcus (30). Among the Gram negatives, E. coli (304), Pseudomonas aeruginosa (201) and Klebsiella pneumoniae (190) were the most common. Of the Staphylococcus aureus isolates 27.2% were methicillin resistant. Only 5.06% enterococci were vancomycin resistant. High rates of resistance to cefotaxime and ciprofloxacin were seen amongst E. coli (84.8% & 83.55%) and Klebsiella pneumoniae (71 & 62.1%) respectively. Resistance to carbapenems (meropenem) was high at 70% in Acinetobacter spp.; however all isolates were sensitive to colistin. Among the aminoglycosides, amikacin retained good efficacy against Escherichia coli (82.9%) and Pseudomonas aeruginosa (78.1%). Occasional isolates of emerging pathogens such as Chryseobacterium indologens, Roseomonas, and Achromobacter xyloxidans were also recovered. Conclusion: The common infections in cancer patients include respiratory, wound, tract infections and sepsis. The commonest isolates include Staphylococcus aureus, Enterococci, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. There is a high level of resistance to the commonly used antibiotics among Gram-negative organisms.

Keywords: bacteria, resistance, infection, cancer

Procedia PDF Downloads 274

Authors: Mihai Palade, Gina Cecilia Pistol, Mariana Stancu, Veronica Chedea, Ionelia Taranu

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Nutrition is an important determinant of general health status, with especially focus on prevention and/or attenuation of the inflammatory-associated pathologies. People with chronic kidney disease can experience chronic inflammation that can lead to cardiovascular disease and even an increased rate of death. There are important links between chronic kidney diseases, inflammation and nutritional strategies that may prevent or protect against undesirable inflammation and oxidative stress. The grape by-products either seeds or pomace are rich in polyphenols which may be beneficial in prevention of inflammatory, antioxidant and antimicrobial processes. As a model for studying the impact of grape seeds on renal inflammation and oxidative stress, we used in this study weaned piglets. After a feeding trial of 30 days with a control diet and an experimental diet containing 5% grape seed (GS), kidney samples were collected. In renal tissues were determined the expression and activity of important markers of immune respose and oxidative stress: pro-inflammatory cytokines (TNF-alpha, IL-1 beta, IL-6, IL-8, IFN-gamma), anti-inflammatory cytokines (IL-4, IL-10), anti-oxidant enzymes (catalase CAT, superoxide dismutase SOD, glutathione peroxidise GPx) and important mediators belonging to nuclear receptors (NF-kB1, Nrf-2 and PPAR-gamma). Gene expression was evaluated by qPCR, whereas protein concentration was determined using proteomic techniques (ELISA). The activity of anti-oxidant enzymes was determined using specific kits. Our results showed that GS enriched in polyphenols does not have effect on TNF-alpha, IL-6 and IL-1 beta gene expression and protein concentration in kidney. By contrast, the gene expression and protein level of IL-8 and IFN-gamma were decreased in GS kidney. Anti-inflammatory cytokines IL-4 and IL-10 gene levels were increased in kidneys collected from GS piglets in comparison with controls, with no modification of protein levels between the two groups. The activities of anti-oxidant enzymes CAT and GPx were increased in kidney by GS, whereas SOD activity was unmodified in comparison with control samples. Also, the GS diet was associated with no modulation of mRNAs for nuclear receptors NF-kB1, Nrf-2 and PPAR-gamma gene expressions in kidneys. In conclusion, our results demonstrated that GS enriched in bioactive compounds such polyphenols could modulate inflammation and oxidative stress markers in kidney tissues. Further studies are necessary to elucidate the mechanism of action of GS compounds in case kidney inflammation associated with oxidative stress, and signalling molecules involved in these mechanisms.

Keywords: animal model, kidney inflammation, oxidative stress, grape seed

Procedia PDF Downloads 271