Search results for: immobilized bacteria

Authors: Danny Barash

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Riboswitches are RNA genetic control elements that were originally discovered in bacteria and provide a unique mechanism of gene regulation. They work without the participation of proteins and are believed to represent ancient regulatory systems in the evolutionary timescale. One of the biggest challenges in riboswitch research is that many are found in prokaryotes but only a small percentage of known riboswitches have been found in certain eukaryotic organisms. The few examples of eukaryotic riboswitches were identified using sequence-based bioinformatics search methods that include some slight structural considerations. These pattern-matching methods were the first ones to be applied for the purpose of riboswitch detection and they can also be programmed very efficiently using a data structure called affix arrays, making them suitable for genome-wide searches of riboswitch patterns. However, they are limited by their ability to detect harder to find riboswitches that deviate from the known patterns. Several methods have been developed since then to tackle this problem. The most commonly used by practitioners is Infernal that relies on Hidden Markov Models (HMMs) and Covariance Models (CMs). Profile Hidden Markov Models were also carried out in the pHMM Riboswitch Scanner web application, independently from Infernal. Other computational approaches that have been developed include RMDetect by the use of 3D structural modules and RNAbor that utilizes Boltzmann probability of structural neighbors. We have tried to incorporate more sophisticated secondary structure considerations based on RNA folding prediction using several strategies. The first idea was to utilize window-based methods in conjunction with folding predictions by energy minimization. The moving window approach is heavily geared towards secondary structure consideration relative to sequence that is treated as a constraint. However, the method cannot be used genome-wide due to its high cost because each folding prediction by energy minimization in the moving window is computationally expensive, enabling to scan only at the vicinity of genes of interest. The second idea was to remedy the inefficiency of the previous approach by constructing a pipeline that consists of inverse RNA folding considering RNA secondary structure, followed by a BLAST search that is sequence-based and highly efficient. This approach, which relies on inverse RNA folding in general and our own in-house fragment-based inverse RNA folding program called RNAfbinv in particular, shows capability to find attractive candidates that are missed by Infernal and other standard methods being used for riboswitch detection. We demonstrate attractive candidates found by both the moving-window approach and the inverse RNA folding approach performed together with BLAST. We conclude that structure-based methods like the two strategies outlined above hold considerable promise in detecting riboswitches and other conserved RNAs of functional importance in a variety of organisms.

Keywords: riboswitches, RNA folding prediction, RNA structure, structure-based methods

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Authors: Isabel N. Sierra-Garcia, Maria J. Ferreira, Sandro Figuereido, Newton Gomes, Helena Silva, Angela Cunha

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Plant-associated microbial communities are considered crucial in the adaptation of halophytes to coastal environments. The plant microbiota can be horizontally acquired from the environment or vertically transmitted from generation to generation via seeds. Recruiting of the microbial communities by the plant is affected by geographical location, soil source, host genotype, and cultivation practice. There is limited knowledge reported on the microbial communities in halophytes the influence of biotic and abiotic factors. In this work, the microbiota associated with the halophyte Salicornia ramosissima was investigated to determine whether the structure of bacterial communities is influenced by host genotype or soil source. For this purpose, two contrasting sites where S. ramosissima is established in the estuarine system of the Ria de Aveiro were investigated. One site corresponds to a natural salt marsh where S. ramosissima plants are present (wild plants), and the other site is a former salt pan that nowadays are subjected to intensive crop production of S. ramosissima (crop plants). Bacterial communities from the rhizosphere, seeds and root endosphere of S. ramossisima from both sites were investigated by sequencing bacterial 16S rRNA gene using the Illumina MiSeq platform. The analysis of the sequences showed that the three plant-associated compartments, rhizosphere, root endosphere, and seed endosphere, harbor distinct microbiomes. However, bacterial richness and diversity were higher in seeds of wild plants, followed by rhizosphere in both sites, while seeds in the crop site had the lowest diversity. Beta diversity measures indicated that bacterial communities in root endosphere and seeds were more similar in both wild and crop plants in contrast to rhizospheres that differed by local, indicating that the recruitment of the similar bacterial communities by the plant genotype is active in regard to the site. Moreover, bacterial communities from the root endosphere and rhizosphere were phylogenetically more similar in both sites, but the phylogenetic composition of seeds in wild and crop sites was distinct. These results indicate that cultivation practices affect the seed microbiome. However, minimal vertical transmission of bacteria from seeds to adult plants is expected. Seeds from the crop site showed higher abundances of Kushneria and Zunongwangia genera. Bacterial members of the classes Alphaprotebacteria and Bacteroidia were the most ubiquitous across sites and compartments and might encompass members of the core microbiome. These findings indicate that bacterial communities associated with S. ramosissima are more influenced by host genotype rather than local abiotic factors or cultivation practices. This study provides a better understanding of the composition of the plant microbiota in S. ramosissima , which is essential to predict the interactions between plant and associated microbial communities and their effects on plant health. This knowledge is useful to the manipulations of these microbial communities to enhance the health and productivity of this commercially important plant.

Keywords: halophytes, plant microbiome, Salicornia ramosissima, agriculture

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Authors: Zhihao Peng, Qiong Zhang, Xiyao Li, Yongzhen Peng

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Nowadays, conventional nitrogen removal process (nitrification and denitrification) was adopted in most wastewater treatment plants, but many problems have occurred, such as: high aeration energy consumption, extra carbon sources dosage and high sludge treatment costs. The emergence of anammox has bring about the great revolution to the nitrogen removal technology, and only the ammonia and nitrite were required to remove nitrogen autotrophically, no demand for aeration and sludge treatment. However, there existed many challenges in anammox applications: difficulty of biomass retention, insufficiency of nitrite substrate, damage from complex organic etc. Much effort was put into the research in overcoming the above challenges, and the payment was rewarded. It was also imperative to establish an innovative process that can settle the above problems synchronously, after all any obstacle above mentioned can cause the collapse of anammox system. Therefore, in this study, a two-stage process was established that the sequencing batch reactor (SBR) and upflow anaerobic sludge blanket (UASB) were used in the pre-stage and post-stage, respectively. The domestic wastewater entered into the SBR first and went through anaerobic/aerobic/anoxic (An/O/A) mode, and the draining at the aerobic end of SBR was mixed with domestic wastewater, the mixture then entering to the UASB. In the long term, organic and nitrogen removal performance was evaluated. All along the operation, most COD was removed in pre-stage (COD removal efficiency > 64.1%), including some macromolecular organic matter, like: tryptophan, tyrosinase and fulvic acid, which could weaken the damage of organic matter to anammox. And the An/O/A operating mode of SBR was beneficial to the achievement and maintenance of partial nitrification (PN). Hence, sufficient and steady nitrite supply was another favorable condition to anammox enhancement. Besides, the flexible mixing ratio helped to gain a substrate ratio appropriate to anammox (1.32-1.46), which further enhance the anammox. Further, the UASB was used and gas recirculation strategy was adopted in the post-stage, aiming to achieve granulation by the selection pressure. As expected, the granules formed rapidly during 38 days, which increased from 153.3 to 354.3 μm. Based on bioactivity and gene measurement, the anammox metabolism and abundance level rose evidently, by 2.35 mgN/gVss·h and 5.3 x109. The anammox bacteria mainly distributed in the large granules (>1000 μm), while the biomass in the flocs (<200 μm) and microgranules (200-500 μm) barely displayed anammox bioactivity. Enhanced anammox promoted the advanced autotrophic nitrogen removal, which increased from 71.9% to 93.4%, even when the temperature was only 12.9 ℃. Therefore, it was feasible to enhance anammox in the multiple favorable conditions created, and the strategy extended the application of anammox to the full-scale mainstream, enhanced the understanding of anammox in the aspects of culturing conditions.

Keywords: anammox, granules, nitrite accumulation, nitrogen removal efficiency

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Authors: Fawzyah Albaldi

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Urinary tract infections (UTIs) are the most prevalent of infectious diseases and > 80% are caused by uropathogenic E. coli (UPEC). Infection occurs following adhesion to urothelial plaques on bladder epithelial cells, whose major protein constituent are the uroplakins (UPs). Two of the four uroplakins (UPIa and UPIb) are members of the tetraspanin superfamily. The UPEC adhesin FimH is known to interact directly with UPIa. Tetraspanins are a diverse family of transmembrane proteins that generally act as “molecular organizers” by binding different proteins and lipids to form tetraspanin enriched microdomains (TEMs). Previous work by our group has shown that TEMs are involved in the adhesion of many pathogenic bacteria to human cells. Adhesion can be blocked by tetraspanin-derived synthetic peptides, suggesting that tetraspanins may be valuable drug targets. In this study, we investigate the role of tetraspanins in UPEC adherence to bladder epithelial cells. Human bladder cancer cell lines (T24, 5637, RT4), commonly used as in-vitro models to investigate UPEC infection, along with primary human bladder cells, were used in this project. The aim was to establish a model for UPEC adhesion/infection with the objective of evaluating the impact of tetraspanin-derived reagents on this process. Such reagents could reduce the progression of UTI, particularly in patients with indwelling catheters. Tetraspanin expression on the bladder cells was investigated by q-PCR and flow cytometry, with CD9 and CD81 generally highly expressed. Interestingly, despite these cell lines being used by other groups to investigate FimH antagonists, uroplakin proteins (UPIa, UPIb and UPIII) were poorly expressed at the cell surface, although some were present intracellularly. Attempts were made to differentiate the cell lines, to induce cell surface expression of these UPs, but these were largely unsuccessful. Pre-treatment of bladder epithelial cells with anti-CD9 monoclonal antibody significantly decreased UPEC infection, whilst anti-CD81 had no effects. A short (15aa) synthetic peptide corresponding to the large extracellular region (EC2) of CD9 also significantly reduced UPEC adherence. Furthermore, we demonstrated specific binding of that fluorescently tagged peptide to the cells. CD9 is known to associate with a number of heparan sulphate proteoglycans (HSPGs) that have also been implicated in bacterial adhesion. Here, we demonstrated that unfractionated heparin (UFH)and heparin analogs significantly inhibited UPEC adhesion to RT4 cells, as did pre-treatment of the cells with heparinases. Pre-treatment with chondroitin sulphate (CS) and chondroitinase also significantly decreased UPEC adherence to RT4 cells. This study may shed light on a common pathogenicity mechanism involving the organisation of HSPGs by tetraspanins. In summary, although we determined that the bladder cell lines were not suitable to investigate the role of uroplakins in UPEC adhesion, we demonstrated roles for CD9 and cell surface proteoglycans in this interaction. Agents that target these may be useful in treating/preventing UTIs.

Keywords: UTIs, tspan, uroplakins, CD9

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Authors: Asma Naim, Warda Mushtaq

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Staphylococcus species are the most versatile and adaptive organism. They are widespread and naturally found on the skin, mucosa and nose in humans. Among these, Staphylococcus aureus is the most important species. These organisms act as opportunistic pathogens and can infect various organs of the host, causing minor skin infection to severe toxin mediated diseases, and life threatening nosocomial infections. Staphylococcus aureus has acquired resistance against β-lactam antibiotics by the production of β-lactamase, and Methicillin-Resistant Staphylococcus aureus (MRSA) strains have also been reported with increasing frequency. MRSA strains have been associated with nosocomial as well as community acquired infections. Medicinal plants have enormous potential as antimicrobial substances and have been used in traditional medicine. Search for medicinally valuable plants with antimicrobial activity is being emphasized due to increasing antibiotic resistance in bacteria. In the present study, the antibacterial potential of Rosmarinus officinalis (Rosemary) and Murraya koenigii (curry leaves) was evaluated. These are common household herbs used in food as enhancer of flavor and aroma. The crude aqueous infusion, decoction and ethanolic extracts of curry leaves and rosemary and essential oil of rosemary were investigated in the present study for antibacterial activity against multi-drug resistant Staphylococcus strains using well diffusion method. In the present study, 60 Multi-drug resistant clinical isolates of S. aureus (43) and Coagulase Negative Staphylococci (CoNS) (17) were screened against different concentrations of crude extracts of Rosmarinus officinalis and Murraya koenigii. Out of these 60 isolates, 43 were sensitive to the aqueous infusion of rosemary; 23 to aqueous decoction and 58 to ethanolic extract whereas, 24 isolates were sensitive to the essential oil. In the case of the curry leaves, no antibacterial activity was observed in aqueous infusion and decoction while only 14 isolates were sensitive to the ethanolic extract. The aqueous infusion of rosemary (50% concentration) exhibited a zone of inhibition of 21(±5.69) mm. against CoNS and 17(±4.77) mm. against S. aureus, the zone of inhibition of 50% concentration of aqueous decoction of rosemary was also larger against CoNS 17(±5.78) mm. then S. aureus 13(±6.91) mm. and the 50% concentrated ethanolic extract showed almost similar zone of inhibition in S. aureus 22(±3.61) mm. and CoNS 21(±7.64) mm. whereas, the essential oil of rosemary showed greater zone of inhibition against S. aureus i.e., 16(±4.67) mm. while CoNS showed 15(±6.94) mm. These results show that ethanolic extract of rosemary has significant antibacterial activity. Aqueous infusion and decoction of curry leaves revealed no significant antibacterial potential against all Staphylococcal species and ethanolic extract also showed only a weak response. Staphylococcus strains were susceptible to crude extracts and essential oil of rosemary in a dose depend manner, where the aqueous infusion showed highest zone of inhibition and ethanolic extract also demonstrated antistaphylococcal activity. These results demonstrate that rosemary possesses antistaphylococcal activity.

Keywords: antibacterial activity, curry leaves, multidrug resistant, rosemary, S. aureus

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Authors: Reem Alajmi, Hind Al Harbi, Tahany Ayaad, Zainab Al Musawi

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Hard ticks Hyalomma spp. (family: Ixodidae) are obligate ectoparasite in their all life stages on some domestic animals mainly camels and cattle. Ticks may lead to many economic and public health problems because of their blood feeding behavior. Also, they act as vectors for many bacterial, viral and protozoan agents which may cause serious diseases such as tick-born encephalitis, Rocky-mountain spotted fever, Q-fever and Lyme disease which can affect human and/or animals. In the present study, molecular identification of ticks that attack camels in Riyadh region, Saudi Arabia based on the partial sequence of mitochondrial 16s rRNA gene was applied. Also, the present study aims to detect natural infections of collected camel ticks with Rickessia spp. and Borelia spp. using PCR/hybridization of Citrate synthase encoding gene present in bacterial cells. Hard ticks infesting camels were collected from different camels located in a farm in Riyadh region, Saudi Arabia. Results of the present study showed that the collected specimens belong to two species: Hyalomma dromedari represent 99% of the identified specimens and Hyalomma marginatum which account for 1 % of identified ticks. The molecular identification was made through blasting the obtained sequence of this study with sequences already present and identified in GeneBank. All obtained sequences of H. dromedarii specimens showed 97-100% identity with the same gene sequence of the same species (Accession # L34306.1) which was used as a reference. Meanwhile, no intraspecific variations of H. marginatum mesured because only one specimen was collected. Results also had shown that the intraspecific variability between individuals of H. dromedarii obtained in 92 % of samples ranging from 0.2- 6.6%, while the remaining 7 % of the total samples of H. dromedarii showed about 10.3 % individual differences. However, the interspecific variability between H. dromedarii and H. marginatum was approximately 18.3 %. On the other hand, by using the technique of PCR/hybridization, we could detect natural infection of camel ticks with Rickettsia spp. and Borrelia spp. Results revealed the natural presence of both bacteria in collected ticks. Rickettsial spp. infection present in 29% of collected ticks, while 35% of collected specimen were infected with Borrelia spp. The valuable results obtained from the present study are a new record for the molecular identification of camel ticks in Riyadh, Saudi Arabia and their natural infection with both Rickettsia spp. and Borrelia spp. These results may help scientists to provide a good and direct control strategy of ticks in order to protect one of the most important economic animals which are camels. Also results of this project spotlight on the disease that might be transmitted by ticks to put out a direct protective plan to prevent spreading of these dangerous agents. Further molecular studies are needed to confirm the results of the present study by using other mitochondrial and nuclear genes for tick identification.

Keywords: Camel ticks, Rickessia spp. , Borelia spp. , mitochondrial 16s rRNA gene

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Authors: Sonia Kudlacik-Kramarczyk, Anna Drabczyk, Dagmara Malina, Bozena Tyliszczak, Agnieszka Sobczak-Kupiec

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Introduction: In the preparation of polymer materials, compounds of natural origin are currently gaining more and more interest. This is particularly noticeable in the case of synthesis of materials considered for biomedical use. Then, selected material has to meet many requirements. It should be characterized by non-toxicity, biodegradability and biocompatibility. Therefore special attention is directed to substances such as polysaccharides, proteins or substances that are the basic building components of proteins, i.e. amino acids. These compounds may be crosslinked with other reagents that leads to the preparation of polymer matrices. Such amino acids as e.g. cysteine or histidine. On the other hand, previously mentioned requirements may be met by polymers obtained as a result of biosynthesis, e.g. polyhydroxybutyrate. This polymer belongs to the group of aliphatic polyesters that is synthesized by microorganisms (selected strain of bacteria) under specific conditions. It is possible to modify matrices based on given polymer with substances of various origin. Such a modification may result in the change of their properties or/and in providing the material with new features desirable in viewpoint of specific application. Described materials are synthesized using UV radiation. Process of photopolymerization is fast, waste-free and enables to obtain final products with favorable properties. Methodology: Polymer matrices have been prepared by means of photopolymerization. First step involved the preparation of solutions of particular reagents and mixing them in the appropriate ratio. Next, crosslinking agent and photoinitiator have been added to the reaction mixture and the whole was poured into the Petri dish and treated with UV radiation. After the synthesis, polymer samples were dried at room temperature and subjected to the numerous analyses aimed at the determining their physicochemical properties. Firstly, sorption properties of obtained polymer matrices have been determined. Next, mechanical properties have been characterized, i.e. tensile strength. The ability to deformation under applied stress of all prepared polymer matrices has been checked. Such a property is important in viewpoint of the application of analyzed materials e.g. as wound dressings. Wound dressings have to be elastic because depending on the location of the wound and its mobility, such a dressing has to adhere properly to the wound. Furthermore, considering the use of the materials for biomedical purposes it is essential to determine its behavior in environments simulating these ones occurring in human body. Therefore incubation studies using selected liquids have also been conducted. Conclusions: As a result of photopolymerization process, polymer matrices based on natural compounds have been prepared. These exhibited favorable mechanical properties and swelling ability. Moreover, biocompatibility in relation to simulated body fluids has been stated. Therefore it can be concluded that analyzed polymer matrices constitute an interesting materials that may be considered for biomedical use and may be subjected to the further more advanced analyses using specific cell lines.

Keywords: photopolymerization, polymer matrices, simulated body fluids, swelling properties

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Authors: Abheyjit Singh, Kulwant Singh

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There is a gradual depletion of the water table in the earth's crust, and it is required to converse and reduce the scarcity of water. This is only done by rainwater harvesting, recycling of water and by judicially consumption/utilization of water and adopting unique treatment measures. Domestic waste is generated in residential areas, commercial settings, and institutions. Waste, in general, is unwanted, undesirable, and nevertheless an inevitable and inherent product of social, economic, and cultural life. In a cluster, a need-based system is formed where the project is designed for systematic analysis, collection of sewage from the cluster, treating it and then recycling it for multifarious work. The liquid waste may consist of Sanitary sewage/ Domestic waste, Industrial waste, Storm waste, or Mixed Waste. The sewage contains both suspended and dissolved particles, and the total amount of organic material is related to the strength of the sewage. The untreated domestic sanitary sewage has a BOD (Biochemical Oxygen Demand) of 200 mg/l. TSS (Total Suspended Solids) about 240 mg/l. Industrial Waste may have BOD and TSS values much higher than those of sanitary sewage. Another type of impurities of wastewater is plant nutrients, especially when there are compounds of nitrogen N phosphorus P in the sewage; raw sanitary contains approx. 35 mg/l Nitrogen and 10 mg/l of Phosphorus. Finally, the pathogen in the waste is expected to be proportional to the concentration of facial coliform bacteria. The coliform concentration in raw sanitary sewage is roughly 1 billion per liter. The system of sewage disposal technique has been universally applied to all conditions, which are the nature of soil formation, Availability of land, Quantity of Sewage to be disposed of, The degree of treatment and the relative cost of disposal technique. The adopted Thappar Model (India) has the following designed parameters consisting of a Screen Chamber, a Digestion Tank, a Skimming Tank, a Stabilization Tank, an Oxidation Pond and a Water Storage Pond. The screening Chamber is used to remove plastic and other solids, The Digestion Tank is designed as an anaerobic tank having a retention period of 8 hours, The Skimming Tank has an outlet that is kept 1 meter below the surface anaerobic condition at the bottom and also help in organic solid remover, Stabilization Tank is designed as primary settling tank, Oxidation Pond is a facultative pond having a depth of 1.5 meter, Storage Pond is designed as per the requirement. The cost of the Thappar model is Rs. 185 Lakh per 3,000 to 4,000 population, and the Area required is 1.5 Acre. The complete structure will linning as per the requirement. The annual maintenance will be Rs. 5 lakh per year. The project is useful for water conservation, silage water for irrigation, decrease of BOD and there will be no longer damage to community assets and economic loss to the farmer community by inundation. There will be a healthy and clean environment in the community.

Keywords: collection, treatment, utilization, economic

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Authors: Pakize Ozlem Kurt Polat

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GMO (genetically modified organism) is the result of a laboratory process where genes from the DNA of one species are extracted and artificially forced into the genes of an unrelated plant or animal. This technology includes; nucleic acid hybridization, recombinant DNA, RNA, PCR, cell culture and gene cloning techniques. The studies are divided into three groups of properties transferred to the transgenic plant. Up to 59% herbicide resistance characteristic of the transfer, 28% resistance to insects and the virus seems to be related to quality characteristics of 13%. Transgenic crops are not included in the commercial production of each product; mostly commercial plant is soybean, maize, canola, and cotton. Day by day increasing GMO interest can be listed as follows; Use in the health area (Organ transplantation, gene therapy, vaccines and drug), Use in the industrial area (vitamins, monoclonal antibodies, vaccines, anti-cancer compounds, anti -oxidants, plastics, fibers, polyethers, human blood proteins, and are used to produce carotenoids, emulsifiers, sweeteners, enzymes , food preservatives structure is used as a flavor enhancer or color changer),Use in agriculture (Herbicide resistance, Resistance to insects, Viruses, bacteria, fungi resistance to disease, Extend shelf life, Improving quality, Drought , salinity, resistance to extreme conditions such as frost, Improve the nutritional value and quality), we explain all this methods step by step in this research. GMO has advantages and disadvantages, which we explain all of them clearly in full text, because of this topic, worldwide researchers have divided into two. Some researchers thought that the GMO has lots of disadvantages and not to be in use, some of the researchers has opposite thought. If we look the countries law about GMO, we should know Biosafety law for each country and union. For this Biosecurity reasons, the problems caused by the transgenic plants, including Turkey, to minimize 130 countries on 24 May 2000, ‘the United Nations Biosafety Protocol’ signed nudes. This protocol has been prepared in addition to Cartagena Biosafety Protocol entered into force on September 11, 2003. This protocol GMOs in general use by addressing the risks to human health, biodiversity and sustainable transboundary movement of all GMOs that may affect the prevention, transit covers were dealt and used. Under this protocol we have to know the, ‘US Regulations GMO’, ‘European Union Regulations GMO’, ‘Turkey Regulations GMO’. These three different protocols have different applications and rules. World population increasing day by day and agricultural fields getting smaller for this reason feeding human and animal we should improve agricultural product yield and quality. Scientists trying to solve this problem and one solution way is molecular biotechnology which is including the methods of GMO too. Before decide to support or against the GMO, should know the GMO protocols and it effects.

Keywords: biotechnology, GMO (genetically modified organism), molecular marker

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Authors: O. Sauperl, L. Fras Zemljic

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The textile industry is gaining increasing importance in the field of medical materials. Therefore, presented research is focused on textile materials for external (out-of-body) use. Such materials could be various hygienic textile products (diapers, tampons, sanitary napkins, incontinence products, etc.), protective textiles and various hospital linens (surgical covers, masks, gowns, cloths, bed linens, etc.) wound pillows, bandages, orthopedic socks, etc. Function of tampons and sanitary napkins is not only to provide protection during the menstrual cycle, but their function can be also to take care of physiological or pathological vaginal discharge. In general, women's intimate areas are against infection protected by a low pH value of the vaginal flora. High pH inhibits the development of harmful microorganisms, as it is difficult to be reproduced in an acidic environment. The normal vaginal flora in healthy women is highly colonized by lactobacilli. The lactic acid produced by these organisms maintains the constant acidity of the vagina. If the balance of natural protection breaks, infections can occur. In the market, there exist probiotic tampons as a medical product supplying the vagina with beneficial probiotic lactobacilli. But, many users have concerns about the use of tampons due to the possible dry-out of the vagina as well as the possible toxic shock syndrome, which is the reason that they use mainly sanitary napkins during the menstrual cycle. Functionalization of sanitary napkins with probiotics is, therefore, interesting in regard to maintain a healthy vaginal flora and to offer to users added value of the sanitary napkins in the sense of health- and environmentally-friendly products. For this reason, the presented research is oriented in functionalization of the sanitary napkins with the probiotic paste in order to activate the lactic acid bacteria presented in the core of the functionalized sanitary napkin at the time of the contact with the menstrual fluid. In this way, lactobacilli could penetrate into vagina and by maintaining healthy vaginal flora to reduce the risk of vaginal disorders. In regard to the targeted research problem, the influence of probiotic paste applied onto cotton hygienic napkins on selected properties was studied. The aim of the research was to determine whether the sanitary napkins with the applied probiotic paste may assure suitable vaginal pH to maintain a healthy vaginal flora during the use of this product. Together with this, sorption properties of probiotic functionalized sanitary napkins were evaluated and compared to the untreated one. The research itself was carried out on the basis of tracking and controlling the input parameters, currently defined by Slovenian producer (Tosama d.o.o.) as the most important. Successful functionalization of sanitary pads with the probiotic paste was confirmed by ATR-FTIR spectroscopy. Results of the methods used within the presented research show that the absorption of the pads treated with probiotic paste deteriorates compared to non-treated ones. The coating shows a 6-month stability. Functionalization of sanitary pads with probiotic paste is believed to have a commercial potential for lowering the probability of infection during the menstrual cycle.

Keywords: functionalization, probiotic paste, sanitary pads, textile materials

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Authors: L. P. Gomes, G. F. Araújo, Y. M. L. Cordeiro, C. T. Andrade, E. M. Del Aguila, V. M. F. Paschoalin

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The application of nanoscale materials and nanostructures is an emerging area, these since materials may provide solutions to technological and environmental challenges in order to preserve the environment and natural resources. To reach this goal, the increasing demand must be accompanied by 'green' synthesis methods. Chitosan is a natural, nontoxic, biopolymer derived by the deacetylation of chitin and has great potential for a wide range of applications in the biological and biomedical areas, due to its biodegradability, biocompatibility, non-toxicity and versatile chemical and physical properties. Chitosan also presents high antimicrobial activities against a wide variety of pathogenic and spoilage microorganisms. Ultrasonication is a common tool for the preparation and processing of polymer nanoparticles. It is particularly effective in breaking up aggregates and in reducing the size and polydispersity of nanoparticles. High-intensity ultrasonication has the potential to modify chitosan molecular weight and, thus, alter or improve chitosan functional properties. The aim of this study was to evaluate the influence of sonication intensity and time on the changes of commercial chitosan characteristics, such as molecular weight and its potential antibacterial activity against Gram-negative bacteria. The nanoparticles (NPs) were produced from two commercial chitosans, of medium molecular weight (CS-MMW) and low molecular weight (CS-LMW) from Sigma-Aldrich®. These samples (2%) were solubilized in 100 mM sodium acetate pH 4.0, placed on ice and irradiated with an ultrasound SONIC ultrasonic probe (model 750 W), equipped with a 1/2" microtip during 30 min at 4°C. It was used on constant duty cycle and 40% amplitude with 1/1s intervals. The ultrasonic degradation of CS-MMW and CS-LMW were followed up by means of ζ-potential (Brookhaven Instruments, model 90Plus) and dynamic light scattering (DLS) measurements. After sonication, the concentrated samples were diluted 100 times and placed in fluorescence quartz cuvettes (Hellma 111-QS, 10 mm light path). The distributions of the colloidal particles were calculated from the DLS and ζ-potential are measurements taken for the CS-MMW and CS-LMW solutions before and after (CS-MMW30 and CS-LMW30) sonication for 30 min. Regarding the results for the chitosan sample, the major bands can be distinguished centered at Radius hydrodynamic (Rh), showed different distributions for CS-MMW (Rh=690.0 nm, ζ=26.52±2.4), CS-LMW (Rh=607.4 and 2805.4 nm, ζ=24.51±1.29), CS-MMW30 (Rh=201.5 and 1064.1 nm, ζ=24.78±2.4) and CS-LMW30 (Rh=492.5, ζ=26.12±0.85). The minimal inhibitory concentration (MIC) was determined using different chitosan samples concentrations. MIC values were determined against to E. coli (106 cells) harvested from an LB medium (Luria-Bertani BD™) after 18h growth at 37 ºC. Subsequently, the cell suspension was serially diluted in saline solution (0.8% NaCl) and plated on solid LB at 37°C for 18 h. Colony-forming units were counted. The samples showed different MICs against E. coli for CS-LMW (1.5mg), CS-MMW30 (1.5 mg/mL) and CS-LMW30 (1.0 mg/mL). The results demonstrate that the production of nanoparticles by modification of their molecular weight by ultrasonication is simple to be performed and dispense acid solvent addition. Molecular weight modifications are enough to provoke changes in the antimicrobial potential of the nanoparticles produced in this way.

Keywords: antimicrobial agent, chitosan, green production, nanoparticles

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Authors: Karen L. Rincon-Granados, América R. Vázquez-Olmos, Adriana-Patricia Rodríguez-Hernández, Gina Prado-Prone, Margarita Rivera, Roberto Y. Sato-Berrú

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In this work, a hybrid film based on poly-3-hydroxybutyrate (P3HB) and nickel ferrite (NiFe₂O₄) nanoparticles (NPs) was obtained by a simple and reproducible methodology in order to study its antibacterial and cytotoxic properties. The motivation for this research is the current antimicrobial resistance (RAM). This is a threat to human health and development worldwide. RAM is caused by the emergence of bacterial strains resistant to traditional antibiotics that were used as treatment. Due to this, the need to investigate new alternatives for preventing and treating bacterial infections emerges. In this sense, metal oxide NPs have aroused great interest due to their unique physicochemical properties. However, their use is limited by the nanostructured nature, commonly obtained by chemical and physical synthesis methods, as powders or colloidal dispersions. Therefore, the incorporation of nanostructured materials in polymer matrices to obtain hybrid materials that allow disinfecting and preventing the spread of bacteria on various surfaces. Accordingly, this work presents the synthesis and study of the antibacterial properties of the P3HB@NiFe₂O₄ hybrid film as a potential material to inhibit bacterial growth. The NiFe₂O₄ NPs were previously synthesized by a mechanochemical method. The P3HB and P3HB@NiFe₂O₄ films were obtained by the solvent casting method. The films were characterized by X-ray diffraction (XRD), Raman scattering, and scanning electron microscopy (SEM). The XRD pattern showed that the NiFe₂O₄ NPs were incorporated into the P3HB polymer matrix and retained their nanometric sizes. By energy dispersive X-ray spectroscopy (EDS), it was observed that the NPs are homogeneously distributed in the film. The bactericidal effect of the films obtained was evaluated in vitro using the broth surface method against two opportunistic and nosocomial pathogens, Staphylococcus aureus and Pseudomonas aeruginosa. The bacterial growth results showed that the P3HB@NiFe₂O₄ hybrid film was inhibited by 97% and 96% for S. aureus and P. aeruginosa, respectively. Surprisingly, the P3HB film inhibited both bacterial strains by around 90%. The cytotoxicity of the NiFe₂O₄ NPs, P3HB@NiFe₂O₄ hybrid film, and the P3HB film was evaluated using human skin cells, keratinocytes, and fibroblasts, finding that the NPs are biocompatible. The P3HB film and hybrids are cytotoxic, which demonstrated that although P3HB is known and reported as a biocompatible polymer, under our work conditions, P3HB was cytotoxic. Its bactericidal effect could be related to this activity. Its films are bactericidal and cytotoxic to keratinocytes and fibroblasts, the first barrier of human skin. Despite this, the hybrid film of P3HB@NiFe₂O₄ presents synergy with the bactericidal effect between P3HB and NPs, increasing bacterial inhibition. In addition, NPs decrease the cytotoxicity of P3HB to keratinocytes. The methodology used in this work was successful in producing hybrid films with antibacterial activity. However, future challenges are generated to find relationships between NPs and P3HB that allow taking advantage of their bactericidal properties and do not compromise biocompatibility.

Keywords: poly-3-hydroxybutyrate, nanoparticles, hybrid film, antibacterial

Procedia PDF Downloads 82

Authors: Celine Shi Ying Lee, Jong Jian Lee

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Background: Infectious keratitis is one of the leading causes of blindness worldwide. Prompt treatment with effective medication will control the infection early, preventing corneal scarring and visual loss. fluoroquinolones ophthalmic medication is used because of its broad-spectrum properties, potency, good intraocular penetration, and low toxicity. The study aims to evaluate the treatment outcome of corneal ulcers using Levofloxacin 1.5% ophthalmic solution (LVFX) with adjuvant oral ciprofloxacin when indicated and apply this treatment strategy in primary health care as first-line treatment. Methods: Patients with infective corneal ulcer treated in an eye center were recruited. Inclusion criteria includes Corneal infection consistent with bacterial keratitis, single or multiple small corneal ulcers. Treatment regime: LVFX hourly for the first 2 days, 2 hourly from the 3rd day, and 3 hourly on the 5th day of review. Adjuvant oral ciprofloxacin 500mg BD was administered for 5 days if there were multiple corneal ulcers or when the location of the cornea ulcer was central or paracentral. Results: 47 subjects were recruited. There were 16 (34%) males and 31 (66%) females. 40 subjects (85%) were contact lens (CL) related to corneal ulcer, and 7 subjects (15%) were non-contact lens related. 42 subjects (89%) presented with one ulcer, of which 20 of them (48%) needed adjuvant therapy. 5 subjects presented with 2 or 3 ulcers, of which 3 needed adjuvant therapy. A total of 23 subjects (49%) was given adjuvant therapy (oral ciprofloxacin 500mg BD for 5 days).21 of them (91%) were CL related. All subjects recovered fully, and the average duration of treatment was 3.7 days, with 49% of the subjects resolved on the 3rd day, 38% on the 5thday of and 13% on the 7thday. All subjects showed symptoms of relief of pain, light-sensitivity, and redness on the 3rd day with full visual recovery post-treatment. No adverse drug reactions were recorded. Conclusion: Our treatment regime demonstrated good clinical outcome as first-line treatment for corneal ulcers. A corneal ulcer is a common eye condition in Singapore, mainly due to CL wear. Pseudomonas aeruginosa is the most frequent and potentially sight-threatening pathogen involved in CL related corneal ulcer. Coagulase-negative Staphylococci, Staphylococcus aureus, and Streptococcus Pneumoniae were seen in non-CL users. All these bacteria exhibit good sensitivity rates to ciprofloxacin and levofloxacin. It is therefore logical in our study to use LVFX Eyedrops and adjuvant ciprofloxacin oral antibiotics when indicated as first line treatment for most corneal ulcers. Our study of patients, both CL related and non-CL related, have shown good clinical response and full recovery using the above treatment strategy. There was also a full restoration of visual acuity in all the patients. Eye-trained primary Healthcare practitioners can consider adopting this treatment strategy as first line treatment in patients with corneal ulcers. This is relevant during the COVID pandemic, where hospitals are overwhelmed with patients and in regions with limited access to specialist eye care. This strategy would enable early treatment with better clinical outcome.

Keywords: corneal ulcer, levofloxacin hydrate, treatment strategy, ciprofloxacin

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Authors: Haftay Abraha Tadesse

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Background: Staphylococcus is a genus of worldwide distributed bacteria correlated to several infectious of different sites in humans and animals. They are among the most important causes of infection that are associated with the consumption of contaminated food. Objective: The objective of this study was to determine the isolates, antimicrobial susceptibility patterns and Public Health Significance of Staphylococcus aureus in raw meat from butchery and abattoir houses of Mekelle, Northern Ethiopia. Methodology: A cross-sectional study was conducted from April to October 2019. Socio-demographic data and Public Health Significance were collected using a predesigned questionnaire. The raw meat samples were collected aseptically in the butchery and abattoir houses and transported using an ice box to Mekelle University, College of Veterinary Sciences, for isolating and identification of Staphylococcus aureus. Antimicrobial susceptibility tests were determined by the disc diffusion method. Data obtained were cleaned and entered into STATA 22.0 and a logistic regression model with odds ratio was calculated to assess the association of risk factors with bacterial contamination. A P-value < 0.05 was considered statistically significant. Results: In the present study, 88 out of 250 (35.2%) were found to be contaminated with Staphylococcus aureus. Among the raw meat specimens, the positivity rate of Staphylococcus aureus was 37.6% (n=47) and (32.8% (n=41), butchery and abattoir houses, respectively. Among the associated risks, factories not using gloves reduces risk was found to (AOR=0.222; 95% CI: 0.104-0.473), Strict Separation b/n clean & dirty (AOR= 1.37; 95% CI: 0.66-2.86) and poor habit of hand washing (AOR=1.08; 95%CI: 0.35 3.35) was found to be statistically significant and have associated with Staphylococcus aureus contamination. All isolates of thirty-seven of Staphylococcus aureus were checked and displayed (100%) sensitive to doxycycline, trimethoprim, gentamicin, sulphamethoxazole, amikacin, CN, Co trimoxazole and nitrofurantoi. Whereas the showed resistance to cefotaxime (100%), ampicillin (87.5%), Penicillin (75%), B (75%), and nalidixic acid (50%) from butchery houses. On the other hand, all isolates of Staphylococcus aureus isolate 100% (n= 10) showed sensitive chloramphenicol, gentamicin and nitrofurantoin, whereas they showed 100% resistance of Penicillin, B, AMX, ceftriaxone, ampicillin and cefotaxime from abattoirs houses. The overall multi-drug resistance pattern for Staphylococcus aureus was 90% and 100% of butchery and abattoir houses, respectively. Conclusion: 35.3% Staphylococcus aureus isolated were recovered from the raw meat samples collected from the butchery and abattoirs houses. More has to be done in the development of hand washing behavior and availability of safe water in the butchery houses to reduce the burden of bacterial contamination. The results of the present finding highlight the need to implement protective measures against the levels of food contamination and alternative drug options. The development of antimicrobial resistance is nearly always a result of repeated therapeutic and/or indiscriminate use of them. Regular antimicrobial sensitivity testing helps to select effective antibiotics and to reduce the problems of drug resistance development towards commonly used antibiotics.

Keywords: abattoir house, AMR, butchery house, S. aureus

Procedia PDF Downloads 98

Authors: V. Schmidt, L. Janovák, N. Wiegand, B. Patczai, K. Turzó

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Large bone defects are not able to heal spontaneously. Bioactive glasses seem to be appropriate (bio)materials for bone reconstruction. Bioactive glasses are osteoconductive and osteoinductive, therefore, play a useful role in bony regeneration and repair. Because of their not optimal mechanical properties (e.g., brittleness, low bending strength, and fracture toughness), their applications are limited. Bioactive glass can be used as a coating material applied on metal surfaces. In this way -when using them as implants- the excellent mechanical properties of metals and the biocompatibility and bioactivity of glasses will be utilized. Furthermore, ion release effects of bioactive glasses regarding osteogenic and angiogenic responses have been shown. Silicate bioactive glasses (45S5 Bioglass) induce the release and exchange of soluble Si, Ca, P, and Na ions on the material surface. This will lead to special cellular responses inducing bone formation, which is favorable in the biointegration of the orthopedic prosthesis. The incorporation of other additional elements in the silicate network such as fluorine, magnesium, iron, silver, potassium, or zinc has been shown, as the local delivery of these ions is able to enhance specific cell functions. Although hip and knee prostheses present a high success rate, bacterial infections -mainly implant associated- are serious and frequent complications. Infection can also develop after implantation of hip prostheses, the elimination of which means more surgeries for the patient and additional costs for the clinic. Prosthesis-related infection is a severe complication of orthopedic surgery, which often causes prolonged illness, pain, and functional loss. While international efforts are made to reduce the risk of these infections, orthopedic surgical infections (SSIs) continue to occur in high numbers. It is currently estimated that up to 2.5% of primary hip and knee surgeries and up to 20% of revision arthroplasties are complicated by periprosthetic joint infection (PJIs). According to some authors, these numbers are underestimated, and they are also increasing. Staphylococcus aureus is the leading cause of both SSIs and PJIs, and the prevalence of methicillin-resistant S. aureus (MRSA) is on the rise, particularly in the United States. These deep infections lead to implant removal and consequently increase morbidity and mortality. The study targets this clinical problem using our experience so far with the Ag-doped polymer coatings on Titanium implants. Non-modified or modified (e.g., doped with antibacterial agents, like Ag) bioactive glasses could play a role in the prevention of infections or the therapy of infected tissues. Bioactive glasses have excellent biocompatibility, proved by in vitro cell culture studies of human osteoblast-like MG-63 cells. Ag-doped bioactive glass-scaffold has a good antibacterial ability against Escherichia coli and other bacteria. It may be concluded that these scaffolds have great potential in the prevention and therapy of implant-associated bone infection.

Keywords: antibacterial agents, bioactive glass, hip and knee prosthesis, medical implants

Procedia PDF Downloads 193

Authors: Oleg Reva, Ilya Korotetskiy, Marina Lankina, Murat Kulmanov, Aleksandr Ilin

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Molecular docking approaches are widely used for design of new antibiotics and modeling of antibacterial activities of numerous ligands which bind specifically to active centers of indispensable enzymes and/or key signaling proteins of pathogens. Widespread drug resistance among pathogenic microorganisms calls for development of new antibiotics specifically targeting important metabolic and information pathways. A generally recognized problem is that almost all molecular targets have been identified already and it is getting more and more difficult to design innovative antibacterial compounds to combat the drug resistance. A promising way to overcome the drug resistance problem is an induction of reversion of drug resistance by supplementary medicines to improve the efficacy of the conventional antibiotics. In contrast to well established computer-based drug design, modeling of drug resistance reversion still is in its infancy. In this work, we proposed an approach to identification of compensatory genetic variants reducing the fitness cost associated with the acquisition of drug resistance by pathogenic bacteria. The approach was based on an analysis of the population genetic of Mycobacterium tuberculosis and on results of experimental modeling of the drug resistance reversion induced by a new anti-tuberculosis drug FS-1. The latter drug is an iodine-containing nanomolecular complex that passed clinical trials and was admitted as a new medicine against MDR-TB in Kazakhstan. Isolates of M. tuberculosis obtained on different stages of the clinical trials and also from laboratory animals infected with MDR-TB strain were characterized by antibiotic resistance, and their genomes were sequenced by the paired-end Illumina HiSeq 2000 technology. A steady increase in sensitivity to conventional anti-tuberculosis antibiotics in series of isolated treated with FS-1 was registered despite the fact that the canonical drug resistance mutations identified in the genomes of these isolates remained intact. It was hypothesized that the drug resistance phenotype in M. tuberculosis requires an adjustment of activities of many genes to compensate the fitness cost of the drug resistance mutations. FS-1 cased an aggravation of the fitness cost and removal of the drug-resistant variants of M. tuberculosis from the population. This process caused a significant increase in genetic heterogeneity of the Mtb population that was not observed in the positive and negative controls (infected laboratory animals left untreated and treated solely with the antibiotics). A large-scale search for linkage disequilibrium associations between the drug resistance mutations and genetic variants in other genomic loci allowed identification of target proteins, which could be influenced by supplementary drugs to increase the fitness cost of the drug resistance and deprive the drug-resistant bacterial variants of their competitiveness in the population. The approach will be used to improve the efficacy of FS-1 and also for computer-based design of new drugs to combat drug-resistant infections.

Keywords: complete genome sequencing, computational modeling, drug resistance reversion, Mycobacterium tuberculosis

Procedia PDF Downloads 263

Authors: Anil Saini, Jatinder Kumar Ratan

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Now a day, the key issue for the scientific community is to devise the innovative technologies for sustainable control of urban pollution. In urban cities, a large surface area of the masonry structures, buildings, and pavements is exposed to the open environment, which may be utilized for the control of air pollution, if it is built from the photocatalytically active cement-based constructional materials such as concrete, mortars, paints, and blocks, etc. The photocatalytically active cement is formulated by incorporating a photocatalyst in the cement matrix, and such cement is generally known as self-cleaning cement In the literature, self-cleaning cement has been synthesized by incorporating nanosized-TiO₂ (n-TiO₂) as a photocatalyst in the formulation of the cement. However, the utilization of n-TiO₂ for the formulation of self-cleaning cement has the drawbacks of nano-toxicity, higher cost, and agglomeration as far as the commercial production and applications are concerned. The use of microsized-TiO₂ (m-TiO₂) in place of n-TiO₂ for the commercial manufacture of self-cleaning cement could avoid the above-mentioned problems. However, m-TiO₂ is less photocatalytically active as compared to n- TiO₂ due to smaller surface area, higher band gap, and increased recombination rate. As such, the use of m-TiO₂ in the formulation of self-cleaning cement may lead to a reduction in photocatalytic activity, thus, reducing the self-cleaning, depolluting, and antimicrobial abilities of the resultant cement material. So improvement in the photoactivity of m-TiO₂ based self-cleaning cement is the key issue for its practical applications in the present scenario. The current work proposes the use of surface-fluorinated m-TiO₂ for the formulation of self-cleaning cement to enhance its photocatalytic activity. The calcined dolomite, a constructional material, has also been utilized as co-adsorbent along with the surface-fluorinated m-TiO₂ in the formulation of self-cleaning cement to enhance the photocatalytic performance. The surface-fluorinated m-TiO₂, calcined dolomite, and the formulated self-cleaning cement were characterized using diffuse reflectance spectroscopy (DRS), X-ray diffraction analysis (XRD), field emission-scanning electron microscopy (FE-SEM), energy dispersive x-ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), BET (Brunauer–Emmett–Teller) surface area, and energy dispersive X-ray fluorescence spectrometry (EDXRF). The self-cleaning property of the as-prepared self-cleaning cement was evaluated using the methylene blue (MB) test. The depolluting ability of the formulated self-cleaning cement was assessed through a continuous NOX removal test. The antimicrobial activity of the self-cleaning cement was appraised using the method of the zone of inhibition. The as-prepared self-cleaning cement obtained by uniform mixing of 87% clinker, 10% calcined dolomite, and 3% surface-fluorinated m-TiO₂ showed a remarkable self-cleaning property by providing 53.9% degradation of the coated MB dye. The self-cleaning cement also depicted a noteworthy depolluting ability by removing 5.5% of NOx from the air. The inactivation of B. subtiltis bacteria in the presence of light confirmed the significant antimicrobial property of the formulated self-cleaning cement. The self-cleaning, depolluting, and antimicrobial results are attributed to the synergetic effect of surface-fluorinated m-TiO₂ and calcined dolomite in the cement matrix. The present study opens an idea and route for further research for acile and economical formulation of self-cleaning cement.

Keywords: microsized-titanium dioxide (m-TiO₂), self-cleaning cement, photocatalysis, surface-fluorination

Procedia PDF Downloads 170

Authors: Narges Bahmanyar, Masoud Ghorbani

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Rabies is caused by several species of the genus Lyssavirus in the Rhabdoviridae family. The disease is deadly encephalitis transmitted from warm-blooded animals to humans, and domestic and wild carnivores play the most crucial role in its transmission. The prevalence of rabies in poor areas of developing salinities is constantly posed as a global threat to public health. According to the World Health Organization, approximately 60,000 people die yearly from rabies. Of these, 60% of deaths are related to the Middle East. Although rabies encephalitis is incurable to date, awareness of the disease and the use of vaccines is the best way to combat the disease. Although effective vaccines are available, there is a high cost involved in vaccine production and management to combat rabies. Increasing the prevalence and discovery of new strains of rabies virus requires the need for safe, effective, and as inexpensive vaccines as possible. One of the approaches considered to achieve the quality and quantity expressed through the manufacture of recombinant types of rabies vaccine. Currently, livestock rabies vaccines are used only in inactivated or live attenuated vaccines, the process of inactivation of which pays attention to considerations. The rabies virus contains a negatively polarized single-stranded RNA genome that encodes the five major structural genes (N, P, M, G, L) from '3 to '5 . Rabies virus glycoprotein G, the major antigen, can produce the virus-neutralizing antibody. N-antigen is another candidate for developing recombinant vaccines. However, because it is within the RNP complex of the virus, the possibility of genetic diversity based on different geographical locations is very high. Glycoprotein G is structurally and antigenically more protected than other genes. Protection at the level of its nucleotide sequence is about 90% and at the amino acid level is 96%. Recombinant vaccines, consisting of a pathogenic subunit, contain fragments of the protein or polysaccharide of the pathogen that have been carefully studied to determine which of these molecules elicits a stronger and more effective immune response. These vaccines minimize the risk of side effects by limiting the immune system's access to the pathogen. Such vaccines are relatively inexpensive, easy to produce, and more stable than vaccines containing viruses or whole bacteria. The problem with these vaccines is that the pathogenic subunits may elicit a weak immune response in the body or may be destroyed before they reach the immune cells, which requires nanoparticles to overcome. Suitable for use as an adjuvant. Among these, biodegradable nanoparticles with functional levels are good candidates as adjuvants for the vaccine. In this study, we intend to use beta-glucan nanoparticles as adjuvants. The surface glycoprotein of the rabies virus (G) is responsible for identifying and binding the virus to the target cell. This glycoprotein is the major protein in the structure of the virus and induces an antibody response in the host. In this study, we intend to use rabies virus surface glycoprotein conjugated with beta-glucan nanoparticles to produce vaccines.

Keywords: rabies, vaccines, beta glucan, nanoprticles, adjuvant, recombinant protein

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Authors: Robin Anderson, David Nisbet

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Nitrate, 3-nitro-1-propionic acid (NPA) and 3-nitro-1-propanol (NPOH) are biologically active chemicals that can accumulate naturally in rangeland grasses forages consumed by grazing cattle, sheep and goats. While toxic to livestock if accumulations and amounts consumed are high enough, particularly in animals having no recent exposure to the forages, these chemicals are known to be potent inhibitors of methane-producing bacteria inhabiting the rumen. Consequently, there is interest in examining their potential use as anti-methanogenic compounds to decrease methane emissions by grazing ruminants. Presently, rumen microbes, collected freshly from a cannulated Holstein cow maintained on 50:50 corn based concentrate:alfalfa diet were mixed (10 mL fluid) in 18 x 150 mm crimp top tubes with 0.5 of high nitrate-containing barley (Hordeum vulgare; containing 272 µmol nitrate per g forage dry matter), and NPA- or NPOH- containing milkvetch forages (Astragalus canadensis and Astragalus miser containing 80 and 174 soluble µmol NPA or NPOH/g forage dry matter respectively). Incubations containing 0.5 g alfalfa (Medicago sativa) were used as controls. Tubes (3 per each respective forage) were capped and incubated anaerobically (using oxygen free carbon dioxide) for 24 h at 39oC after which time amounts of total gas produced were measured via volume displacement and headspace samples were analyzed by gas chromatography to determine concentrations of hydrogen and methane. Fluid samples were analyzed by gas chromatography to measure accumulations of fermentation acids. A completely randomized analysis of variance revealed that the nitrate-containing barley and both the NPA- and the NPOH-containing milkvetches significantly decreased methane production, by > 50%, when compared to methane produced by populations incubated similarly with alfalfa (70.4 ± 3.6 µmol/ml incubation fluid). Accumulations of hydrogen, which are typically increased when methane production is inhibited, by incubations with the nitrate-containing barley and the NPA- and NPOH-containing milkvetches did not differ from accumulations observed in the alfalfa controls (0.09 ± 0.04 µmol/mL incubation fluid). Accumulations of fermentation acids produced in the incubations containing the high-nitrate barley and the NPA- and NPOH-containing milkvetches likewise did not differ from accumulations observed in incubations containing alfalfa (123.5 ± 10.8, 36.0 ± 3.0, 17.1 ± 1.5, 3.5 ± 0.3, 2.3 ± 0.2, 2.2 ± 0.2 µmol/mL incubation fluid for acetate, propionate, butyrate, valerate, isobutyrate, and isovalerate, respectively). This finding indicates the microbial populations did not compensate for the decreased methane production via compensatory changes in production of fermentative acids. Stoichiometric estimation of fermentation balance revealed that > 77% of reducing equivalents generated during fermentation of the forages were recovered in fermentation products and the recoveries did not differ between the alfalfa incubations and those with the high-nitrate barley or the NPA- or NPOH-containing milkvetches. Stoichiometric estimates of amounts of hexose fermented similarly did not differ between the nitrate-, NPA and NPOH-containing incubations and those with the alfalfa, averaging 99.6 ± 37.2 µmol hexose consumed/mL of incubation fluid. These results suggest that forages containing nitrate, NPA or NPOH may be useful to reduce methane emissions of grazing ruminants provided risks of toxicity can be effectively managed.

Keywords: nitrate, nitropropanol, nitropropionic acid, rumen methane emissions

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Authors: J. Gabrielczyk, J. Kluitmann, T. Dammeyer, H. J. Jördening

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High-level expression of proteins in bacteria often causes production of insoluble protein aggregates, called inclusion bodies (IB). They contain mainly one type of protein and offer an easy and efficient way to get purified protein. On the other hand, proteins in IB are normally devoid of function and therefore need a special treatment to become active. Most refolding techniques aim at diluting the solubilizing chaotropic agents. Unfortunately, optimal refolding conditions have to be found empirically for every protein. For large-scale applications, a simple refolding process with high yields and high final enzyme concentrations is still missing. The constructed plasmid pASK-IBA63b containing the sequence of fructosyltransferase (FTF, EC 2.4.1.162) from Bacillus subtilis NCIMB 11871 was transformed into E. coli BL21 (DE3) Rosetta. The bacterium was cultivated in a fed-batch bioreactor. The produced FTF was obtained mainly as IB. For refolding experiments, five different amounts of IBs were solubilized in urea buffer with protein concentration of 0.2-8.5 g/L. Solubilizates were refolded with batch or continuous dialysis. The refolding yield was determined by measuring the protein concentration of the clear supernatant before and after the dialysis. Particle size was measured by dynamic light scattering. We tested the solubilization properties of fructosyltransferase IBs. The particle size measurements revealed that the solubilization of the aggregates is achieved at urea concentration of 5M or higher and confirmed by absorption spectroscopy. All results confirm previous investigations that refolding yields are dependent upon initial protein concentration. In batch dialysis, the yields dropped from 67% to 12% and 72% to 19% for continuous dialysis, in relation to initial concentrations from 0.2 to 8.5 g/L. Often used additives such as sucrose and glycerol had no effect on refolding yields. Buffer screening indicated a significant increase in activity but also temperature stability of FTF with citrate/phosphate buffer. By adding citrate to the dialysis buffer, we were able to increase the refolding yields to 82-47% in batch and 90-74% in the continuous process. Further experiments showed that in general, higher ionic strength of buffers had major impact on refolding yields; doubling the buffer concentration increased the yields up to threefold. Finally, we achieved corresponding high refolding yields by reducing the chamber volume by 75% and the amount of buffer needed. The refolded enzyme had an optimal activity of 12.5±0.3 x104 units/g. However, detailed experiments with native FTF revealed a reaggregation of the molecules and loss in specific activity depending on the enzyme concentration and particle size. For that reason, we actually focus on developing a process of simultaneous enzyme refolding and immobilization. The results of this study show a new approach in finding optimal refolding conditions for inclusion bodies at high concentrations. Straightforward buffer screening and increase of the ionic strength can optimize the refolding yield of the target protein by 400%. Gentle removal of chaotrope with continuous dialysis increases the yields by an additional 65%, independent of the refolding buffer applied. In general time is the crucial parameter for successful refolding of solubilized proteins.

Keywords: dialysis, inclusion body, refolding, solubilization

Procedia PDF Downloads 294

Authors: Shagufta Jabeen, Faez Jesse Firdaus Abdullah, Zunita Zakaria, Nurulfiza Mat Isa, Yung Chie Tan, Wai Yan Yee, Abdul Rahman Omar

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Pasteurella multocida is associated with acute, as well as, chronic infections in avian and bovine such as pasteurellosis and hemorrhagic septicemia (HS) in cattle and buffaloes. Iron is one of the most important nutrients for pathogenic bacteria including Pasteurella and acts as a cofactor or prosthetic group in several essential enzymes and is needed for amino acid, pyrimidine, and DNA biosynthesis. In our recent study, we showed that 2% of Pasteurella multocida serotype A strain PMTB2.1 encode for iron regulating genes (Accession number CP007205.1). Genome sequencing of other Pasteurella multocida serotypes namely PM70 and HB01 also indicated up to 2.5% of the respective genome encode for iron regulating genes, suggesting that Pasteurella multocida genome comprises of multiple systems for iron uptake. Since P. multocida PMTB2.1 has more than 40 CDs out of 2097 CDs (approximately 2%), encode for iron-regulated. The gene expression profiling of four iron-regulating genes namely fbpb, yfea, fece and fur were characterized under iron-restricted environment. The P. multocida strain PMTB2.1 was grown in broth with and without iron chelating agent and samples were collected at different time points. Relative mRNA expression profile of these genes was determined using Taqman probe based real-time PCR assay. The data analysis, normalization with two house-keeping genes and the quantification of fold changes were carried out using Bio-Rad CFX manager software version 3.1. Results of this study reflect that iron reduced environment has significant effect on expression profile of iron regulating genes (p < 0.05) when compared to control (normal broth) and all evaluated genes act differently with response to iron reduction in media. The highest relative fold change of fece gene was observed at early stage of treatment indicating that PMTB2.1 may utilize its periplasmic protein at early stage to acquire iron. Furthermore, down-regulation expression of fece with the elevated expression of other genes at later time points suggests that PMTB2.1 control their iron requirements in response to iron availability by down-regulating the expression of iron proteins. Moreover, significantly high relative fold change (p ≤ 0.05) of fbpb gene is probably associated with the ability of P. multocida to directly use host iron complex such as hem, hemoglobin. In addition, the significant increase (p ≤ 0.05) in fbpb and yfea expressions also reflects the utilization of multiple iron systems in P. multocida strain PMTB2.1. The findings of this study are very much important as relative scarcity of free iron within hosts creates a major barrier to microbial growth inside host and utilization of outer-membrane proteins system in iron acquisition probably occurred at early stage of infection with P. multocida. In conclusion, the presence and utilization of multiple iron system in P. multocida strain PMTB2.1 revealed the importance of iron in the survival of P. multocida.

Keywords: iron-related genes, real-time PCR, gene expression profiling, fold changes

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Authors: Geremew Geidare Kailo, Igor Gáspár, András Koris, Ivana Pajčin, Flóra Vitális, Vanja Vlajkov

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Three-dimension (3D) printing, a very popular additive manufacturing technology, has recently undergone rapid growth and replaced the use of conventional technology from prototyping to producing end-user parts and products. The 3D Printing technology involves a digital manufacturing machine that produces three-dimensional objects according to designs created by the user via 3D modeling or computer-aided design/manufacturing (CAD/CAM) software. The most popular 3D printing system is Fused Deposition Modeling (FDM) or also called Fused Filament Fabrication (FFF). A 3D-printed object is considered food safe if it can have direct contact with the food without any toxic effects, even after cleaning, storing, and reusing the object. This work analyzes the processing timeline of the filament (material for 3D printing) from unboxing to the extrusion through the nozzle. It is an important task to analyze the growth of bacteria on the 3D printed surface and in gaps between the layers. By default, the 3D-printed object is not food safe after longer usage and direct contact with food (even though they use food-safe filaments), but there are solutions for this problem. The aim of this work was to evaluate the 3D-printed object from different perspectives of food safety. Firstly, testing antimicrobial 3D printing filaments from a food safety aspect since the 3D Printed object in the food industry may have direct contact with the food. Therefore, the main purpose of the work is to reduce the microbial load on the surface of a 3D-printed part. Coating with epoxy resin was investigated, too, to see its effect on mechanical strength, thermal resistance, surface smoothness and food safety (cleanability). Another aim of this study was to test new temperature-resistant filaments and the effect of high temperature on 3D printed materials to see if they can be cleaned with boiling or similar hi-temp treatment. This work proved that all three mentioned methods could improve the food safety of the 3D printed object, but the size of this effect variates. The best result we got was with coating with epoxy resin, and the object was cleanable like any other injection molded plastic object with a smooth surface. Very good results we got by boiling the objects, and it is good to see that nowadays, more and more special filaments have a food-safe certificate and can withstand boiling temperatures too. Using antibacterial filaments reduced bacterial colonies to 1/5, but the biggest advantage of this method is that it doesn’t require any post-processing. The object is ready out of the 3D printer. Acknowledgements: The research was supported by the Hungarian and Serbian bilateral scientific and technological cooperation project funded by the Hungarian National Office for Research, Development and Innovation (NKFI, 2019-2.1.11-TÉT-2020-00249) and the Ministry of Education, Science and Technological Development of the Republic of Serbia. The authors acknowledge the Hungarian University of Agriculture and Life Sciences’s Doctoral School of Food Science for the support in this study

Keywords: food safety, 3D printing, filaments, microbial, temperature

Procedia PDF Downloads 142

Authors: Hady Hamidyan

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As the global population continues to grow, the demand for clean water and effective wastewater treatment becomes increasingly critical. By 2030, global water demand is projected to exceed supply by 40%, driven by population growth, increased water usage, and climate change. Currently, about 4.2 billion people lack access to safely managed sanitation services. The wastewater treatment sector faces numerous challenges, including the need for energy-efficient solutions, cost-effectiveness, ease of use, and low maintenance requirements. This abstract presents a groundbreaking wastewater treatment technology that addresses these challenges by offering an energy-saving approach, wastewater recovery capabilities, and a ready-made, affordable, and user-friendly package with minimal maintenance costs. The unique design of this ready-made package made it possible to eliminate the need for pumps, filters, airlift, and other common equipment. Consequently, it enables sustainable wastewater treatment management with exceptionally low energy and cost requirements, minimizing investment and maintenance expenses. The operation of these packages is based on continuous aeration, which involves injecting oxygen gas or air into the aeration chamber through a tubular diffuser with very small openings. This process supplies the necessary oxygen for aerobic bacteria. The recovered water, which amounts to almost 95% of the input, can be treated to meet specific quality standards, allowing safe reuse for irrigation, industrial processes, or even potable purposes. This not only reduces the strain on freshwater resources but also provides economic benefits by offsetting the costs associated with freshwater acquisition and wastewater discharge. The ready-made, affordable, and user-friendly nature of this technology makes it accessible to a wide range of users, including small communities, industries, and decentralized wastewater treatment systems. The system incorporates user-friendly interfaces, simplified operational procedures, and integrated automation, facilitating easy implementation and operation. Additionally, the use of durable materials, efficient equipment, and advanced monitoring systems significantly reduces maintenance requirements, resulting in low overall life-cycle costs and alleviating the burden on operators and maintenance personnel. In conclusion, the presented wastewater treatment technology offers a comprehensive solution to the challenges faced by the industry. Its energy-saving approach, combined with wastewater recovery capabilities, ensures sustainable resource management and enhances environmental stewardship. This affordable, ready-made, and low-maintenance package promotes broad adoption across various sectors and communities, contributing to a more sustainable future for water and wastewater management.

Keywords: wastewater treatment, energy saving, wastewater recovery, affordable package, low maintenance costs, sustainable resource management, environmental stewardship

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Authors: Megan L. Srinivas

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Individuals with underlying rheumatologic diseases such as dermatomyositis may not adequately respond to tuberculin (PPD) skin tests, creating false negative results. These illnesses are frequently treated with immunosuppressive therapy making proper identification of TB infection imperative. A 59-year-old Filipino man was diagnosed with dermatomyositis on the basis of rash, electromyography, and muscle biopsy. He was initially treated with IVIG infusions and transitioned to oral prednisone and mycophenolate. The patient’s symptoms improved on this regimen. Six months after starting mycophenolate, the patient began having fevers, night sweats, and productive cough without hemoptysis. He moved from the Philippines 5 years prior to dermatomyositis diagnosis, denied sick contacts, and was PPD negative both at immigration and immediately prior to starting mycophenolate treatment. A third PPD was negative following the onset of these new symptoms. He was treated for community-acquired pneumonia, but symptoms worsened over 10 days and he developed watery diarrhea and a growing non-tender, non-mobile mass on the left side of his neck. A chest x-ray demonstrated a cavitary lesion in right upper lobe suspicious for TB that had not been present one month earlier. Chest CT corroborated this finding also exhibiting necrotic hilar and paratracheal lymphadenopathy. Neck CT demonstrated the left-sided mass as cervical chain lymphadenopathy. Expectorated sputum and stool samples contained acid-fast bacilli (AFB), cultures showing TB bacteria. Fine-needle biopsy of the neck mass (scrofula) also exhibited AFB. An MRI brain showed nodular enhancement suspected to be a tuberculoma. Mycophenolate was discontinued and dermatomyositis treatment was switched to oral prednisone with a 3-day course of IVIG. The patient’s infection showed sensitivity to standard RIPE (rifampin, isoniazid, pyrazinamide, and ethambutol) treatment. Within a week of starting RIPE, the patient’s diarrhea subsided, scrofula diminished, and symptoms significantly improved. By the end of treatment week 3, the patient’s sputum no longer contained AFB; he was removed from isolation, and was discharged to continue RIPE at home. He was discharged on oral prednisone, which effectively addressed his dermatomyositis. This case illustrates the unreliability of PPD tests in patients with long-term inflammatory diseases such as dermatomyositis. Other immunosuppressive therapies (adalimumab, etanercept, and infliximab) have been affiliated with conversion of latent TB to disseminated TB. Mycophenolate is another immunosuppressive agent with similar mechanistic properties. Thus, it is imperative that patients with long-term inflammatory diseases and high-risk TB factors initiating immunosuppressive therapy receive a TB blood test (such as a quantiferon gold assay) prior to the initiation of therapy to ensure that latent TB is unmasked before it can evolve into a disseminated form of the disease.

Keywords: dermatomyositis, immunosuppressant medications, mycophenolate, disseminated tuberculosis

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Authors: Haftay Abraha Tadesse

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Background: Staphylococcus is a genus of worldwide distributed bacteria correlated to several infectious of different sites in human and animals. They are among the most important causes of infection that are associated with the consumption of contaminated food. Objective: The objective of this study was to determine the isolates, antimicrobial susceptibility patterns and public health significance for Staphylococcus aureus in raw meat from butchery and abattoir houses of Mekelle, Northern Ethiopia. Methodology: A cross-sectional study was conducted from April to October 2019. Sociodemographic data and public health significance were collected using predesigned questionnaire. The raw meat samples were collected aseptically in the butchery and abattoir houses and transported using ice box to Mekelle University, College of Veterinary Sciences for isolating and identification of Staphylococcus aureus. Antimicrobial susceptibility tests were determined by disc diffusion method. Data obtained were cleaned and entered in to STATA 22.0 and logistic regression model with odds ratio were calculated to assess the association of risk factors with bacterial contamination. P-value < 0.05 was considered as statistically significant. Results: In present study, 88 out of 250 (35.2%) were found to be contamination with Staphylococcus aureus. Among the raw meat specimens to be positivity rate of Staphylococcus aureus were 37.6% (n=47) and (32.8% (n=41), butchery and abattoir houses, respectively. Among the associated risk factories not using gloves reduces risk was found to (AOR=0.222; 95% CI: 0.104-0.473), Strict Separation b/n clean & dirty (AOR= 1.37; 95% CI: 0.66-2.86) and poor habit of hand washing (AOR=1.08; 95%CI: 0.35-3.35) were found to be statistically significant and ha ve associated with Staphylococcus aureus contamination. All isolates thirty sevevn of Staphyloco ccus aureus were checked displayed (100%) sensitive to doxycycline, trimethoprim, gentamicin, sulphamethoxazole, amikacin, CN, Co trimoxazole and nitrofurantoi. whereas the showed resistance of cefotaxime (100%), ampicillin (87.5%), Penicillin (75%), B (75%), and nalidixic acid (50%) from butchery houses. On the other hand, all isolates of Staphylococcus aur eu isolate 100% (n= 10) showed sensitive chloramphenicol, gentamicin and nitrofurantoin whereas the showed 100% resistance of Penicillin, B, AMX, ceftriaxone, ampicillin and cefotaxime from abattoirs houses. The overall multi drug resistance pattern for Staphylococcus aureus were 90% and 100% of butchery and abattoirs houses, respectively. Conclusion: 35.3% Staphylococcus aureus isolated were recovered from the raw meat samples collected from the butchery and abattoirs houses. More has to be done in the developed of hand washing behavior, and availability of safe water in the butchery houses to reduce burden of bacterial contamination. The results of the present finding highlight the need to implement protective measures against the levels of food contamination and alternative drug options. The development of antimicrobial resistance is nearly always as a result of repeated therapeutic and/or indiscriminate use of them. Regular antimicrobial sensitivity testing helps to select effective antibiotics and to reduce the problems of drug resistance development towards commonly used antibiotics. Key words: abattoir houses, antimicrobial resistance, butchery houses, Ethiopia,

Keywords: abattoir houses, antimicrobial resistance, butchery houses, Ethiopia, staphylococcus aureuse, MDR

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Authors: Sumeet Rai, Anuj Tyagi, B. T. Naveen Kumar, Shubhkaramjeet Kaur, Niraj K. Singh

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Since their discovery, indiscriminate use of antibiotics in human, veterinary and aquaculture systems has resulted in global emergence/spread of multidrug-resistant bacterial pathogens. Thus, the need for alternative approaches to control bacterial infections has become utmost important. High selectivity/specificity of bacteriophages (phages) permits the targeting of specific bacteria without affecting the desirable flora. In this study, a lytic phage (Ahp1) specific to Aeromonas hydrophila subsp. hydrophila was isolated from finfish aquaculture pond. The host range of Ahp1 range was tested against 10 isolates of A. hydrophila, 7 isolates of A. veronii, 25 Vibrio cholerae isolates, 4 V. parahaemolyticus isolates and one isolate each of V. harveyi and Salmonella enterica collected previously. Except the host A. hydrophila subsp. hydrophila strain, no lytic activity against any other bacterial was detected. During the adsorption rate and one-step growth curve analysis, 69.7% of phage particles were able to get adsorbed on host cell followed by the release of 93 ± 6 phage progenies per host cell after a latent period of ~30 min. Phage nucleic acid was extracted by column purification methods. After determining the nature of phage nucleic acid as dsDNA, phage genome was subjected to next-generation sequencing by generating paired-end (PE, 2 x 300bp) reads on Illumina MiSeq system. De novo assembly of sequencing reads generated circular phage genome of 42,439 bp with G+C content of 58.95%. During open read frame (ORF) prediction and annotation, 22 ORFs (out of 49 total predicted ORFs) were functionally annotated and rest encoded for hypothetical proteins. Proteins involved in major functions such as phage structure formation and packaging, DNA replication and repair, DNA transcription and host cell lysis were encoded by the phage genome. The complete genome sequence of Ahp1 along with gene annotation was submitted to NCBI GenBank (accession number MF683623). Stability of Ahp1 preparations at storage temperatures of 4 °C, 30 °C, and 40 °C was studied over a period of 9 months. At 40 °C storage, phage counts declined by 4 log units within one month; with a total loss of viability after 2 months. At 30 °C temperature, phage preparation was stable for < 5 months. On the other hand, phage counts decreased by only 2 log units over a period of 9 during storage at 4 °C. As some of the phages have also been reported as glycerol sensitive, the stability of Ahp1 preparations in (0%, 15%, 30% and 45%) glycerol stocks were also studied during storage at -80 °C over a period of 9 months. The phage counts decreased only by 2 log units during storage, and no significant difference in phage counts was observed at different concentrations of glycerol. The Ahp1 phage discovered in our study had a very narrow host range and it may be useful for phage typing applications. Moreover, the endolysin and holin genes in Ahp1 genome could be ideal candidates for recombinant cloning and expression of antimicrobial proteins.

Keywords: Aeromonas hydrophila, endolysin, phage, narrow host range

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Authors: Fernanda Bravo Cornejo, Camilo Cerda Sarabia, Belén Díaz Díaz, Diego Santibañez Oyarce, Esteban Gómez Terán, Hugo Osses Prado, Raúl Caulier-Cisterna, Jorge Vergara-Quezada, Ana Moya-Beltrán

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Streptococcus pyogenes is a gram-positive bacteria involved in a wide range of diseases and is a major-human-specific bacterial pathogen. In Chile, this year the 'Ministerio de Salud' declared an alert due to the increase in strains throughout the year. This increase can be attributed to the multitude of factors including antimicrobial resistance (AMR) and Virulence Factors (VF). Understanding these VF and AMR is crucial for developing effective strategies and improving public health responses. Moreover, experimental identification and characterization of these pathogenic mechanisms are labor-intensive and time-consuming. Therefore, new computational methods are required to provide robust techniques for accelerating this identification. Advances in Machine Learning (ML) algorithms represent the opportunity to refine and accelerate the discovery of VF associated with Streptococcus pyogenes. In this work, we evaluate the accuracy of various machine learning models in predicting the virulence factors and antimicrobial resistance of Streptococcus pyogenes, with the objective of providing new methods for identifying the pathogenic mechanisms of this organism.Our comprehensive approach involved the download of 32,798 genbank files of S. pyogenes from NCBI dataset, coupled with the incorporation of data from Virulence Factor Database (VFDB) and Antibiotic Resistance Database (CARD) which contains sequences of AMR gene sequence and resistance profiles. These datasets provided labeled examples of both virulent and non-virulent genes, enabling a robust foundation for feature extraction and model training. We employed preprocessing, characterization and feature extraction techniques on primary nucleotide/amino acid sequences and selected the optimal more for model training. The feature set was constructed using sequence-based descriptors (e.g., k-mers and One-hot encoding), and functional annotations based on database prediction. The ML models compared are logistic regression, decision trees, support vector machines, neural networks among others. The results of this work show some differences in accuracy between the algorithms, these differences allow us to identify different aspects that represent unique opportunities for a more precise and efficient characterization and identification of VF and AMR. This comparative analysis underscores the value of integrating machine learning techniques in predicting S. pyogenes virulence and AMR, offering potential pathways for more effective diagnostic and therapeutic strategies. Future work will focus on incorporating additional omics data, such as transcriptomics, and exploring advanced deep learning models to further enhance predictive capabilities.

Keywords: antibiotic resistance, streptococcus pyogenes, virulence factors., machine learning

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Authors: Dalia Ambrazaitienė, Monika Vilkienė, Danute Karcauskienė, Gintaras Siaudinis

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The soil is a complex ecosystem that is part of our biosphere. The ability of soil to provide ecosystem services is dependent on microbial diversity. T Tillage is one of the major factors that affect soil properties. The no-till systems or shallow ploughless tillage are opposite of traditional deep ploughing, no-tillage systems, for instance, increase soil organic matter by reducing mineralization rates and stimulating litter concentrations of the top soil layer, whereas deep ploughing increases the biological activity of arable soil layer and reduces the incidence of weeds. The role of soil organisms is central to soil processes. Although the number of microbial species in soil is still being debated, the metagenomic approach to estimate microbial diversity predicted about 2000 – 18 000 bacterial genomes in 1 g of soil. Despite the key role of bacteria in soil processes, there is still lack of information about the bacterial diversity of soils as affected by tillage practices. This study focused on metagenomic analysis of bacterial diversity in long-term experimental plots of Dystric Epihypogleyic Albeluvisols in western part of Lithuania. The experiment was set up in 2013 and had a split-plot design where the whole-plot treatments were laid out in a randomized design with three replicates. The whole-plot treatments consisted of two tillage methods - deep ploughing (22-25 cm) (DP), ploughless tillage (7-10 cm) (PT). Three subsamples (0-20 cm) were collected on October 22, 2015 for each of the three replicates. Subsamples from the DP and PT systems were pooled together wise to make two composition samples, one representing deep ploughing (DP) and the other ploughless tillage (PT). Genomic DNA from soil sample was extracted from approximately 200 mg field-moist soil by using the D6005 Fungal/Bacterial Miniprep set (Zymo Research®) following the manufacturer’s instructions. To determine bacterial diversity and community composition, we employed a culture – independent approach of high-throughput pyrosequencing of the 16S rRNA gene. Metagenomic sequencing was made with Illumina MiSeq platform in Base Clear Company. The microbial component of soil plays a crucial role in cycling of nutrients in biosphere. Our study was a preliminary attempt at observing bacterial diversity in soil under two common but contrasting tillage practices. The number of sequenced reads obtained for PT (161 917) was higher than DP (131 194). The 10 most abundant genus in soil sample were the same (Arthrobacter, Candidatus Saccharibacteria, Actinobacteria, Acidobacterium, Mycobacterium, Bacillus, Alphaproteobacteria, Longilinea, Gemmatimonas, Solirubrobacter), just the percent of community part was different. In DP the Arthrobacter and Acidobacterium consist respectively 8.4 % and 2.5%, meanwhile in PT just 5.8% and 2.1% of all community. The Nocardioides and Terrabacter were observed just in PT. This work was supported by the project VP1-3.1-ŠMM-01-V-03-001 NKPDOKT and National Science Program: The effect of long-term, different-intensity management of resources on the soils of different genesis and on other components of the agro-ecosystems [grant number SIT-9/2015] funded by the Research Council of Lithuania.

Keywords: deep ploughing, metagenomics, ploughless tillage, soil community analysis

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Authors: N. Perini, M. C. Thaller, F. Mercuri, S. Orlanducci, A. Rubechini, L. Migliore

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The preservation of cultural heritage is one of the major challenges of today’s society, because of the fundamental right of future generations to inherit it as the continuity with their historical and cultural identity. Parchments, consisting of a semi-solid matrix of collagen produced from animal skin (i.e., sheep or goats), are a significant part of the cultural heritage, being used as writing material for many centuries. Due to their animal origin, parchments easily undergo biodeterioration. The most common biological damage is characterized by isolated or coalescent purple spots that often leads to the detachment of the superficial layer and the loss of the written historical content of the document. Although many parchments with the same biodegradative features were analyzed, no common causative agent has been found so far. Very recently, a study was performed on a purple-damaged parchment roll dated back 1244 A.D, the A.A. Arm. I-XVIII 3328, belonging to the oldest collection of the Vatican Secret Archive (Fondo 'Archivum Arcis'), by comparing uncolored undamaged and purple damaged areas of the same document. As a whole, the study gave interesting results to hypothesize a model of biodeterioration, consisting of a microbial succession acting in two main phases: the first one, common to all the damaged parchments, is characterized by halophilic and halotolerant bacteria fostered by the salty environment within the parchment maybe induced by bringing of the hides; the second one, changing with the individual history of each parchment, determines the identity of its colonizers. The design of this model was pivotal to this study, performed by different labs of the Tor Vergata University (Rome, Italy), in collaboration with the Vatican Secret Archive. Three documents, belonging to a collection of dramatically damaged parchments archived as 'Faldone Patrizi A 19' (dated back XVII century A.D.), were analyzed through a multidisciplinary approach, including three updated technologies: (i) Next Generation Sequencing (NGS, Illumina) to describe the microbial communities colonizing the damaged and undamaged areas, (ii) RAMAN spectroscopy to analyze the purple pigments, (iii) Light Transmitted Analysis (LTA) to evaluate the kind and entity of the damage to native collagen. The metagenomic analysis obtained from NGS revealed DNA sequences belonging to Halobacterium salinarum mainly in the undamaged areas. RAMAN spectroscopy detected pigments within the purple spots, mainly bacteriorhodopsine/rhodopsin-like pigments, a purple transmembrane protein containing retinal and present in Halobacteria. The LTA technique revealed extremely damaged collagen structures in both damaged and undamaged areas of the parchments. In the light of these data, the study represents a first confirmation of the microbial succession model described above. The demonstration of this model is pivotal to start any possible new restoration strategy to bring back historical parchments to their original beauty, but also to open opportunities for intervention on a huge amount of documents.

Keywords: biodeterioration, parchments, purple spots, ecological succession

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Authors: Isaac Martin, Abigail Lark, Sandra Morales, Eric W. Alton, Jane C. Davies

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Objectives: The cystic fibrosis (CF) lung is a unique microbiological niche, wherein harmful bacteria persist for many years despite antibiotic therapy. Pseudomonas aeruginosa (Pa), the major culprit leading to lung decline and increased mortality, thrives in the lungs of patients with CF due to several factors that have been linked with poor antibiotic performance. Our group is investigating alternative therapies including bacteriophage cocktails with which we have previously demonstrated efficacy against planktonic organisms. In this study, we explored the effects of a 4-phage cocktail on Pa grown in two different conditions, intended to mirror the CF lung: a) alongside standard antibiotic treatment in pre-formed biofilms (structures formed by Pa-secreted exopolysaccharides which provide both physical and cell division barriers to antimicrobials and host defenses and b) in an acidic environment postulated to be present in the CF airway due both to the primary defect in bicarbonate secretion and secondary effects of inflammation. Methods: 16 Pa strains from CF patients at the Royal Brompton Hospital were selected based on sensitivity to a) ceftazidime/ tobramycin and b) the phage cocktail in a conventional plaque assay. To assess efficacy of phage in biofilms, 96 well plates with Pa (5x10⁷ CFU/ ml) were incubated in static conditions, allowing adherent bacterial colonies to form for 24 hr. Ceftazidime and tobramycin (both at 2 × MIC) were added, +/- bacteriophage (4x10⁸ PFU/mL) for a further 24 hr. Cell viability and biomass were estimated using fluorescent resazurin and crystal violet assays, respectively. To evaluate the effect of pH, strains were grown planktonically in shaking 96 well plates at pH 6.0, 6.6, 7.0 and 7.5 with tobramycin or phage, at varying concentrations. Cell viability was quantified by fluorescent resazurin assay. Results: For the biofilm assay, treatment groups were compared with untreated controls and expressed as percent reduction in cell viability and biomass. Addition of the 4-phage cocktail resulted in a 1.3-fold reduction in cell viability and 1.7-fold reduction in biomass (p < 0.001) when compared to standard antibiotic treatment alone. Notably, there was a 50 ± 15% reduction in cell viability and 60 ± 12% reduction in biomass (95% CI) for the 4 biofilms demonstrating the most resistance to antibiotic treatment. 83% of strains tested (n=6) showed decreased bacterial killing by tobramycin at acidic pHs (p < 0.01). However, 25% of strains (n=12) showed improved phage killing at acidic pHs (p < 0.05), with none showing the pattern of reduced efficacy at acidic pH demonstrated by tobramycin. Conclusion: The 4-phage anti-Pa cocktail tested against Pa performs well in pre-formed biofilms and in acidic environments; two conditions intended to mimic the CF lung. To our knowledge, these are the first data looking at the effects of subtle pH changes on phage-mediated bacterial killing in the context of Pa infection. These findings contribute to a growing body of evidence supporting the use of nebulised lytic bacteriophage as a treatment in the context of lung infection.

Keywords: biofilm, cystic fibrosis, pH, Pseudomonas aeruginosa, lytic bacteriophage

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