Search results for: opportunistic pathogens

Authors: Sabrina Azzi, Michal Iglewski, Véronique Nabelsi

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Diagnosis error problem is frequent and one of the most important safety problems today. One of the main objectives of our work is to propose an ontological representation that takes into account the diagnostic criteria in order to improve the diagnostic. We choose pneumonia disease since it is one of the frequent diseases affected by diagnosis errors and have harmful effects on patients. To achieve our aim, we use a semi-automated method to integrate diverse knowledge sources that include publically available pneumonia disease guidelines from international repositories, biomedical ontologies and electronic health records. We follow the principles of the Open Biomedical Ontologies (OBO) Foundry. The resulting ontology covers symptoms and signs, all the types of pneumonia, antecedents, pathogens, and diagnostic testing. The first evaluation results show that most of the terms are covered by the ontology. This work is still in progress and represents a first and major step toward a development of a diagnosis decision support system for pneumonia.

Keywords: Clinical decision support system, Diagnostic errors, Ontology, Pneumonia

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Authors: Okolie Pius Ifeanyi, Emerenini Emilymary Chima

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Aims: The study investigated the diversity and identities of Lactic Acid Bacteria (LAB) isolated from different fresh fruits using Molecular Nested PCR analysis and the efficacy of cell free supernatants from Lactic Acid Bacteria (LAB) isolated from fresh fruits for in vitro control of some tomato pathogens. Study Design: Nested PCR approach was used in this study employing universal 16S rRNA gene primers in the first round PCR and LAB specific Primers in the second round PCR with the view of generating specific Nested PCR products for the LAB diversity present in the samples. The inhibitory potentials of supernatant obtained from LAB isolates of fruits origin that were molecularly characterized were investigated against some tomato phytopathogens using agar-well method with the view to develop biological agents for some tomato disease causing organisms. Methodology: Gram positive, catalase negative strains of LAB were isolated from fresh fruits on Man Rogosa and Sharpe agar (Lab M) using streaking method. Isolates obtained were molecularly characterized by means of genomic DNA extraction kit (Norgen Biotek, Canada) method. Standard methods were used for Nested Polymerase Chain Reaction (PCR) amplification targeting the 16S rRNA gene using universal 16S rRNA gene and LAB specific primers, agarose gel electrophoresis, purification and sequencing of generated Nested PCR products (Macrogen Inc., USA). The partial sequences obtained were identified by blasting in the non-redundant nucleotide database of National Center for Biotechnology Information (NCBI). The antimicrobial activities of characterized LAB against some tomato phytopathogenic bacteria which include (Xanthomonas campestries, Erwinia caratovora, and Pseudomonas syringae) were obtained by using the agar well diffusion method. Results: The partial sequences obtained were deposited in the database of National Centre for Biotechnology Information (NCBI). Isolates were identified based upon the sequences as Weissella cibaria (4, 18.18%), Weissella confusa (3, 13.64%), Leuconostoc paramensenteroides (1, 4.55%), Lactobacillus plantarum (8, 36.36%), Lactobacillus paraplantarum (1, 4.55%) and Lactobacillus pentosus (1, 4.55%). The cell free supernatants of LAB from fresh fruits origin (Weissella cibaria, Weissella confusa, Leuconostoc paramensenteroides, Lactobacillus plantarum, Lactobacillus paraplantarum and Lactobacillus pentosus) can inhibits these bacteria by creating clear zones of inhibition around the wells containing cell free supernatants of the above mentioned strains of lactic acid bacteria. Conclusion: This study shows that potentially LAB can be quickly characterized by molecular methods to specie level by nested PCR analysis of the bacteria isolate genomic DNA using universal 16S rRNA primers and LAB specific primer. Tomato disease causing organisms can be most likely biologically controlled by using extracts from LAB. This finding will reduce the potential hazard from the use of chemical herbicides on plant.

Keywords: nested pcr, molecular characterization, 16s rRNA gene, lactic acid bacteria

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Authors: Reenu Yadav, Vidhi Guha, Udit N. Soni, Jay Ram Patel

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Chewing gums are mobile novel drug delivery systems, with a potential for administering drugs either for local action or for systemic absorption via the buccal route. An antimicrobial chewing gum delivery system of the methanolic extracts of Beatea monosperma (barks and twigs), Cordia obliqua (leaves and seeds) and Cuminun cyminum (seeds) against periodontal diseases caused by some oral pathogens, was designed and characterized on various parameters.The results of the study support the traditional application of the plants and suggest, plant extracts possess compounds with antimicrobial properties that can be used as potential antimicrobial agents and gums can be a good carrier of herbal extracts. Developed formulation will cure/protect from various periodontal diseases. Further development and evaluations chewing gums including the isolated compounds on the commercial scale and their clinical and toxicological studies are the future challenges.

Keywords: periodontal diseases, herbal chewing gum, herbal extracts, novel drug delivery systems

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Authors: Richard K. Jolley, Jonathan A. Coffman

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Since Enterococci has been implicated in oral disease, we hypothesized the transmission of avian Enterococci to humans via fecal-oral transmission facilitated by adherence to dental plaque. To demonstrate the capability of Enterococci to bind to a dental plaque we filtered avian excreta and incubated the filtrate on a sucrose-induced, Streptococcus mutans biofilm. The biofilm was washed several times with a detergent to remove bacteria binding non-specifically to the biofilm, DNA was isolated from the biofilm, 16S rDNA was amplified, sequenced by Ion Torrent DNA sequencing and analyzed with bioinformatics. Enterococci and other known bacterial pathogens were shown to adhere to the biofilm. Culturing the washed biofilm with Bile Esculin Azide (BEA) agar also confirmed the presence of Enterococci as verified with Sanger sequencing. The results suggest that Enteroccoci in avian excreta has the ability to adhere to human dental plaque and may be a mechanism of entry when humans encounter contaminated aerosols, water or food.

Keywords: Enterococci, avian excreta, dental plaque, NGS

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Authors: Okafor Joan, Nwodo Uchechukwu

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Klebsiella pneumoniae (K. pneumoniae) is a significant pathogen responsible for opportunistic and nosocomial infection. One of the most significant antibiotic resistance mechanisms in K. pneumoniae isolates is efflux pumps. Our current study identified efflux genes (AcrAB, OqxAB, MacAB, and TolC) and regulatory genes (RamR and RarA) in multidrug-resistant (MDR) K. pneumoniae isolated from hospital effluents and investigated their relationship with antibiotic resistance. The sum of 145 K. pneumoniae isolates was established by PCR and screened for antibiotic susceptibility. PCR detected efflux pump genes, and their link with antibiotic resistance was statistically examined. However, 120 (83%) of the confirmed isolated were multidrug-resistant, with the largest percentage of resistance to ampicillin (88.3%) and the weakest rate of resistance to imipenem (5.5%). Resistance to the other antibiotics ranged from 11% to 76.6%. Molecular distribution tests show that AcrA, AcrB, MacA, oqxB oqxA, TolC, MacB were detected in 96.7%, 85%, 76.7%, 70.8%, 55.8%, 39.1%, and 29.1% respectively. However, 14.3% of the isolates harboured all seven genes screened. Efflux pump system AcrAB (83.2%) was the most commonly detected in K. pneumonia isolated across all the antibiotics class-tested. In addition, the frequencies of RamR and RarA were 46.2% and 31.4%, respectively. AcrAB and OqxAB efflux pump genes were significantly associated with fluoroquinolone, beta-lactam, carbapenem, and tetracycline resistance (p<0.05). The high rate of efflux genes in this study demonstrated that this resistance mechanism is the dominant way in K. pneumoniae isolates. Appropriate treatment must be used to reduce and tackle the burden of resistant Klebsiella pneumonia in hospital wastewater effluents.

Keywords: Klebsiella pneumonia, efflux pumps, regulatory genes, multidrug-resistant, hospital, PCR

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Authors: Laura Cochrane

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The United States Centers for Disease Control and Prevention has established three categories of biological agents based on their ease of spread and the severity of the disease they cause. Category A biological agents are the highest priority because of their high degree of morbidity and mortality, ease of dissemination, the potential to cause social disruption and panic, special requirements for public health preparedness, and past use as a biological weapon. Despite the threat of Category A biological agents, opportunities for medical intervention exist. This work summarizes public information, consolidated and reviewed across the situational usefulness and disease awareness to offer discussion to three specific Category A agents: anthrax (Bacillus anthracis), botulism (Clostridium botulinum toxin), and smallpox (variola major), and provides an overview on the management of medical countermeasures available to treat these three (3) different types of pathogens. The medical countermeasures are discussed in the setting of pre-exposure prophylaxis, post-exposure prophylaxis, and therapeutic treatments to provide a framework for requirements in public health preparedness.

Keywords: anthrax, botulism, smallpox, medical countermeasures

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Authors: Hattie Hope Makumbe, Bhekisisa Dlamini, Frederick Tabit

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Listeria monocytogenes is one of the most important foodborne pathogens associated with ready-to-eat (RTE) food. This study investigated the incidence of Listeria monocytogenes in 80 RTE food sold in the formal (dairy and processed meat) and informal markets (vegetable salads, beef stew, and rice) of Johannesburg, South Africa. High Enterobacteriaceae, S. aureus, and E. coli counts were obtained, which ranged from 1.9-7.5 log CFU/g. Listeria monocytogenes microbial counts in the food samples ranged from 3.5-6.0 log colony forming unit per gram except in cooked rice. The Listeria monocytogenes isolates were identified using biochemical tests and confirmed with the Biolog identification system and PCR analyses. The percentage incidence for Listeria monocytogenes in ready to eat food was 12.5%. When Minimum Inhibitory Concentrations were under consideration, all disinfectants were effective against Listeria monocytogenes strains. For antimicrobial work, rates of resistance amongst the antibiotics ranged from 17-100%. Therefore, more effective preventive control strategies for Listeria monocytogenes are needed to reduce the prevalence of the pathogen in RTE food that is sold in Johannesburg.

Keywords: Listeria monocytogenes, Listeria species, ready to eat food, sanitiser efficacy

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Authors: Gauravi Agarkar, Mahendra Rai

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Endophytic fungi from medicinal plants are important source of numerous pharmacologically important compounds. In the present investigation, the endophytic fungi were isolated from three medicinal plants; Andrographis paniculata, Rauwolfia serpentina and Tridax procumbens. Endophytic Colletotrichum sp. were identified on the basis of cultural and morphological characteristics as well as internal transcribed spacer (ITS) sequence analysis. Antibacterial and antifungal activity of the ethyl acetate and methanol extract of endophytic Colletotrichum sp. was evaluated against seven different human pathogenic bacteria and six Candida sp. The extracts were effective and showed significant activity against all the test pathogens. In case of yeast Candida, the combined effect of extracts and standard antibiotic was enhanced greatly showing synergistic activity. Further, the extracts were assayed for Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal/Fungicidal Concentration (MBC/MFC) where, MIC values were in the range of 100-250 μg/ml. These results suggest that the endophytic Colletotrichum sp. isolated from the medicinal plants are capable of producing promising antimicrobial metabolites.

Keywords: antimicrobial, colletotrichum, endophytic fungi, medicinal plants

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Authors: Sang-Myung Jung, Gwang Heum Yoon, Hoo Chul Lee, Hwa Sung Shin

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Ceramide, a component of stratum corneum at epidermis, helps to construct a rigid and dense skin barrier to prevent pathogens that cause atopic dermatitis. However, ceramide was too hydrophobic to be directly absorbed into stratum corneum and has risks of side effects by excessive treatment. To overcome the obstacles, ceramide was embedded into PLGA nanoparticles coated with chitosan. PLGA and chitosan have been known as biocompatible materials. PLGA was squeezed when faced with water and pumped ceramide out of PLGA nanoparticle. In addition, the chitosan coating layer helped initial adherence of nanoparticles to skin and regulate ceramide release until removed. This coating was degraded at weakly acid state like skin surface, finally ceramide release could be controlled. Finally, the nanoparticle was demonstrated to be non-cytotoxic and regenerate stratum corneum of atopic dermatitis model. Overall the nanoparticle is suggested as a novel and effective nanodrug to apply atopic dermatitis.

Keywords: nanoparticle, controlled release, atopic dermatitis, chitosan coating, ceramide

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Authors: Susmita Sharma, Pankaj Pathak

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Biosolids are by-products of municipal and industrial wastewater treatment process. The generation of the biosolids is increasing at an alarming rate due to the implementation of strict environmental legislation to improve the quality of discharges from wastewater treatment plant. As such, proper management and safe disposal of sewage sludge have become a worldwide topic of research. Biosolids, rich in organic matter and essential micro and macronutrients; can be used as a soil conditioner, to cut fertilizer costs and create favorable conditions for vegetation. However, it also contains pathogens and heavy metals which are undesirable as they are harmful to both humans and the environment. Therefore, for safe utilization of biosolids for land application purposes, categorization of the contaminant and pathogen is mandatory. In this context, biosolids collected from a wastewater treatment plant in Maharashtra are utilized to determine its physical, chemical and microbiological attributes. This study would ascertain, if the use of these materials from the specific site, are suitable for agriculture. Further, efforts have also been made to present the internationally acceptable legal standards and guidelines for biosolids management or application.

Keywords: biosolids, sewage, heavy metal, sustainable agriculture

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Authors: T. Büyüksırıt, H. Kuleaşan

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Natural antimicrobials are used to preserve foods that can be found in plants, animals, and microorganisms. Antimicrobial substances are natural or artificial agents that produced by microorganisms or obtained semi/total chemical synthesis are used at low concentrations to inhibit the growth of other microorganisms. Food borne pathogens and spoilage microorganisms are inactivated by the use of antagonistic microorganisms and their metabolites. Yeasts can produce toxic proteins or glycoproteins (toxins) that cause inhibition of sensitive bacteria and yeast species. Antimicrobial substance producing phenotypes belonging different yeast genus were isolated from different sources. Toxins secreted by many yeast strains inhibiting the growth of other yeast strains. These strains show antimicrobial activity, inhibiting the growth of mold and bacteria. The effect of antimicrobial agents produced by yeasts can be extremely fast, and therefore may be used in various treatment procedures. Rapid inhibition of microorganisms is possibly caused by microbial cell membrane lipopolysaccharide binding and in activation (neutralization) effect. Antimicrobial agents inhibit the target cells via different mechanisms of action.

Keywords: antimicrobial agents, yeast, toxic protein, glycoprotein

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Authors: A. K. Tursunova, O. V. Chebonenko, A. Zh. Amirkulova, A. O. Abaildayev, O. A. Sapko, Y. M. Dyo, A. Sh. Utarbaeva

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Fusarium solani and its variants cause root and stem rot of plants. Dry rot is the most common disease of potato tubers during storage. The causative agents of fusariosis in contact with plants behave as antagonists, growth stimulants or parasites. The diversity of host-parasite relationships is explained by the parasite’s ability to produce a wide spectrum of biologically active compounds including toxins, enzymes, oligosaccharides, antibiotic substances, enniatins and gibberellins. Many of these metabolites contribute to the creation of compatible relations; others behave as elicitors, inducing various protective responses in plants. An important part of the strategy for developing plant resistance against pathogens is the activation of protein synthesis to produce protective ‘pathogenesis-related’ proteins. The family of PR-proteins known to confer the most protective response is chitinases (EC 3.2.1.14, Cht) and β-1,3-glucanases (EC 3.2.1.39, Glu). PR-proteins also include a large multigene family of peroxidases (EC 1.11.1.7, Pod), and increased activity of Pod and expression of the Pod genes leads to the development of resistance to a broad class of pathogens. Despite intensive research on the role of PR-proteins, the question of their participation in the mechanisms of formation of the F.solani–S.tuberosum pathosуstem is not sufficiently studied. Our aim was to investigate the effect of different classes of F. solani metabolites on the activity of chitinase, β-1,3-glucanases and peroxidases in tubers of Solanum tuberosum. Metabolite culture filtrate (CF) and cytoplasmic components were fractionated by extraction of the mycelium with organic solvents, salting out techniques, dialysis, column chromatography and ultrafiltration. Protein, lipid, carbohydrate and polyphenolic fractions of fungal metabolites were derived. Using enzymatic hydrolysis we obtained oligo glycans from fungal cell walls with different molecular weights. The activity of the metabolites was tested using potato tuber discs (d = 16mm, h = 5mm). The activity of PR-proteins of tubers was analyzed in a time course of 2–24 hours. The involvement of the analysed metabolites in the modulation of both early non-specific and late related to pathogenesis reactions was demonstrated. The most effective inducer was isolated from the CF (fraction of total phenolic compounds including naphtazarins). Induction of PR-activity by this fraction was: chitinase - 340-360%, glucanase - 435-450%, soluble forms of peroxidase - 400-560%, related forms of peroxidase - 215-237%. High-inducing activity was observed by the chloroform and acetonitrile extracts of the mycelium (induction of chitinase and glucanase activity was 176-240%, of soluble and bound forms of peroxidase - 190-400%). The fraction of oligo glycans mycelium cell walls of 1.2 kDa induced chitinase and β-1,3-glucanase to 239-320%; soluble forms and related peroxidase to 198-426%. Oligo glycans cell walls of 5-10 kDa had a weak suppressor effect - chitinase (21-25%) and glucanase (25-28%) activity; had no effect on soluble forms of peroxidase, but induced to 250-270% activity related forms. The CF polysaccharides of 8.5 kDa and 3.1 kDa inhibited synchronously the glucanase and chitinase specific response in step (after 24 hours at 42-50%) and the step response induced nonspecific peroxidase activity: soluble forms 4.8 -5.2 times, associated forms 1.4-1.6 times.

Keywords: fusarium solani, PR-proteins, peroxidase, solanum tuberosum

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Authors: Lachin Mikjtarnejad, Mohsen Farzaneh

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Various microbes, such as fungi and bacteria species, are naturally found in the fruit microbiota, and some of them act as a pathogen and result in fruit rot. Among non-pathogenic microbes, yeasts (single-celled microorganisms belonging to the fungi kingdom) can colonize fruit tissues and interact with them without causing any damage to them. Although yeasts are part of the plant microbiota, there is little information about their interactions with plants in comparison with bacteria and filamentous fungi. According to several existing studies, some yeasts can colonize different plant species and have the biological control ability to suppress some of the plant pathogens. It means those specific yeast-colonized plants are more resistant to some plant pathogens. The major objective of the present investigation is to isolate yeast strains from apple fruit and screen their ability to control Penicillium expansum, the causal agent of blue mold of fruits. In the present study, psychrotrophic and epiphytic yeasts were isolated from apple fruits that were stored at low temperatures (0–1°C). Totally, 42 yeast isolates were obtained and identified by molecular analysis based on genomic sequences of the D1/D2 and ITS1/ITS4 regions of their rDNA. All isolated yeasts were primarily screened by' in vitro dual culture assay against P. expansum by measuring the fungus' relative growth inhibition after 10 days of incubation. The results showed that the mycelial growth of P. expansum was reduced between 41–53% when challenged by promising yeast strains. The isolates with the strongest antagonistic activity belonged to Metschnikowia pulcherrima A13, Rhodotorula mucilaginosa A41, Leucosporidium Scottii A26, Aureobasidium pullulans A19, Pichia guilliermondii A32, Cryptococcus flavescents A25, and Pichia kluyveri A40. The results of seven superior isolates to inhibit blue mold decay on fruit showed that isolates A. pullulans A19, L. scottii A26, and Pi. guilliermondii A32 could significantly reduce the fruit rot and decay with 26 mm, 22 mm and 20 mm zone diameter, respectively, compared to the control sample with 43 mm. Our results show Pi. guilliermondii strain A13 was the most effective yeast isolates in inhibiting P. expansum on apple fruits. In addition, various biological control mechanisms of promising biological isolates against blue mold have been evaluated to date, including competition for nutrients and space, production of volatile metabolites, reduction of spore germination, production of siderophores and production of extracellular lytic enzymes such as chitinase and β-1,3-glucanase. However, the competition for nutrients and the ability to inhibit P. expansum spore growth have been introduced as the prevailing mechanisms among them. Accordingly, in our study, isolates A13, A41, A40, A25, A32, A19 and A26 inhibited the germination of P. expansum, whereas isolates A13 and A19 were the strongest inhibitors of P. expansum mycelia growth, causing 89.13% and 81.75 % reduction in the mycelial surface, respectively. All the promising isolates produced chitinase and β-1,3-glucanase after 3, 5 and 7 days of cultivation. Finally, based on our findings, we are proposing that, Pi. guilliermondiias as an effective biocontrol agent and alternative to chemical fungicides to control the blue mold of apple fruit.

Keywords: yeast, yeast enzymes, biocontrol, post harvest diseases

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Authors: Miroslava Cuperlovic-Culf, Lipu Wang, Ketty Boyle, Nadine Makley, Ian Burton, Anissa Belkaid, Mohamed Touaibia, Marc E. Surrette

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Metabolic changes are one of the major factors in the development of a variety of diseases in various species. Metabolism of agricultural plants is altered the following infection with pathogens sometimes contributing to resistance. At the same time, pathogens use metabolites for infection and progression. In humans, metabolism is a hallmark of cancer development for example. Quantified metabolomics data combined with other omics or clinical data and analyzed using various unsupervised and supervised methods can lead to better diagnosis and prognosis. It can also provide information about resistance as well as contribute knowledge of compounds significant for disease progression or prevention. In this work, different methods for metabolomics quantification and analysis from Nuclear Magnetic Resonance (NMR) measurements that are used for investigation of disease development in wheat and human cells will be presented. One-dimensional 1H NMR spectra are used extensively for metabolic profiling due to their high reliability, wide range of applicability, speed, trivial sample preparation and low cost. This presentation will describe a new method for metabolite quantification from NMR data that combines alignment of spectra of standards to sample spectra followed by multivariate linear regression optimization of spectra of assigned metabolites to samples’ spectra. Several different alignment methods were tested and multivariate linear regression result has been compared with other quantification methods. Quantified metabolomics data can be analyzed in the variety of ways and we will present different clustering methods used for phenotype determination, network analysis providing knowledge about the relationships between metabolites through metabolic network as well as biomarker selection providing novel markers. These analysis methods have been utilized for the investigation of fusarium head blight resistance in wheat cultivars as well as analysis of the effect of estrogen receptor and carbonic anhydrase activation and inhibition on breast cancer cell metabolism. Metabolic changes in spikelet’s of wheat cultivars FL62R1, Stettler, MuchMore and Sumai3 following fusarium graminearum infection were explored. Extensive 1D 1H and 2D NMR measurements provided information for detailed metabolite assignment and quantification leading to possible metabolic markers discriminating resistance level in wheat subtypes. Quantification data is compared to results obtained using other published methods. Fusarium infection induced metabolic changes in different wheat varieties are discussed in the context of metabolic network and resistance. Quantitative metabolomics has been used for the investigation of the effect of targeted enzyme inhibition in cancer. In this work, the effect of 17 β -estradiol and ferulic acid on metabolism of ER+ breast cancer cells has been compared to their effect on ER- control cells. The effect of the inhibitors of carbonic anhydrase on the observed metabolic changes resulting from ER activation has also been determined. Metabolic profiles were studied using 1D and 2D metabolomic NMR experiments, combined with the identification and quantification of metabolites, and the annotation of the results is provided in the context of biochemical pathways.

Keywords: metabolic biomarkers, metabolic network, metabolomics, multivariate linear regression, NMR quantification, quantified metabolomics, spectral alignment

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Authors: Deanna Ruano-Meas, Laura Shenkman

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Variances are seen in the way healthcare personnel (HCP) don and doff PPE risking contamination to self and others. By standardizing practice, variances in technique decrease, and so does the risk of contamination. To implement this change, the Model for Improvement will be used. A system change will be developed that will outline the role of the organizational leader’s support of HCP in the proper donning and doffing of PPE. Interventions will include environmental surveys to assess the safety and work situation ensuring a permissible environment, plan audits to confirm consistency, and the assessment of PPE wear for standardization. The change will also include an educational plan that will involve instruction of the current guidelines recommended by the Centers for Disease Control and Prevention (CDC) to all pertinent HCP, and the incorporation of PPE education in yearly educational training. The goal is a standardized practice and a reduced risk of contamination through education and organizational support. Personal protective equipment has had recent attention with the coming of the SARS-CoV-2. The realization that proper technique is important to decreasing contamination of pathogens has led to the revising of current processes.

Keywords: donning and doffing, HAI, infection control, PPE

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Authors: Aleena Rena Onozuka, Patompong Satapornpong

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Introduction: In a Human Leukocyte Antigen (HLA)’s immune response, HLA alleles (HLA class I and class II) play a crucial role in fighting against pathogens. HLA-B*15:01 allele had a significant association with asymptomatic COVID-19 infection (p-value = 5.67 x 10-5 ; OR = 2.40 and 95% CI = 1.54 - 3.64). There was also a notable linkage between HLA-B*51:01 allele and critically ill patients with COVID-19 (p-value = 0.007 and OR = 3.38). This study has described the distribution of HLA marker alleles in Thais and sub-groups. Objective: We want to investigate the prevalence of HLA-B*15:01 and HLA-B*51:01 alleles in the Thai population. Materials and Methods: 200 healthy Thai population were included in this study from the College of Pharmacy, Rangsit University. HLA-B alleles were genotyped using the sequence-specific oligonucleotides process (PCR-SSOs). Results: We found out that HLA-B*46:01 (12.00%), HLA-B*15:02 (9.25%), HLA-B*40:01 (6.50%), HLA-B*13:01 (6.25%), and HLA-B* 38:02 (5.50%) alleles were more common than other alleles in Thai population. HLA-B*46:01 and HLA-B*15:02 were the most common allele found across four regions. Moreover, the frequency of HLA-B*15:01 and HLA-B*51:01 alleles were similarly distributed in Thai population (0.50, 5.25 %) and (p-value > 0.05), respectively. The frequencies of HLA-B*15:01 and HLA-B*51:01 alleles were not significantly different from other populations compared to the Thai population. Conclusions: We can screen for HLA-B*15:01 and HLA-B*51:01 alleles to determine the symptoms of COVID-19 since they are universal HLA-B markers. Importantly, the database of HLA markers indicates the association between HLA frequency and populations. However, we need further research on larger numbers of COVID-19 patients and in different populations.

Keywords: HLA-B*15:01, HLA-B*51:01, COVID-19, HLA-B alleles

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Authors: M. Mehani, N. Salhi, T. Valeria, S. Ladjel

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Red River Gum (Eucalyptus camaldulensis) is a tree of the genus Eucalyptus widely distributed in Algeria and in the world. The value of its aromatic secondary metabolites offers new perspectives in the pharmaceutical industry. This strategy can contribute to the sustainable development of our country. Preliminary tests performed on the essential oil of Eucalyptus camendulensis showed that this oil has antibacterial activity vis-à-vis the bacterial strains (Enterococcus feacalis, Enterobacter cloaceai, Proteus microsilis, Escherichia coli, Klebsiella pneumonia, and Pseudomonas aeruginosa) and antifungic (Fusarium sporotrichioide and Fusarium graminearum). The culture medium used was nutrient broth Muller Hinton. The interaction between the bacteria and the essential oil is expressed by a zone of inhibition with diameters of MIC indirectly expression of. And we used the PDA medium to determine the fungal activity. The extraction of the aromatic fraction (essentially oil- hydrolat) of the fresh aerian part of the Eucalyptus camendulensis was performed by hydrodistillation. The average essential oil yield is 0.99%. The antimicrobial and fungal study of the essential oil and hydrosol showed a high inhibitory effect on the growth of pathogens.

Keywords: essential oil, Eucalyptus camendulensis, bacteria and fungi, red river gum

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Authors: Saroj Panthi, Sher Singh Thagunna

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Reliable and sufficient information regarding status, distribution and habitat preference of red panda (Ailurus fulgens fulgens) is lacking in Nepal. The research activities on red panda in the mid-western Nepal are very limited, so the status of red panda in the region is quite unknown. The study conducted during May, 2013 in three Village Development Committees (VDCs) namely Godhemahadev, Malikathata and Tamti of Jumla district was an important step for providing vital information including distribution and habitat preference of this species. The study included the reconnaissance, key informants survey, interviews, and consultation for the most potential area identification, opportunistic survey comprising the direct observation and indirect sign count method for the presence and distribution, habitat assessment consisting vegetation sampling and ocular estimation. The study revealed the presence of red panda in three forests namely Bahirepatan, Imilchadamar and Tyakot of Godhemahadev, Tamti and Malikathata VDCs respectively. The species was found distributed between 2880 and 3244 m with an average dropping encounter rate of 1.04 per hour of searching effort and 12 pellets per dropping. Red panda mostly preferred the habitat in the elevation range of 2900 - 3000 m with southwest facing steep slopes (36˚ - 45˚), associated with water sources at the distance of ≤100 m. Trees such as Acer spp., Betula utilis and Quercus semecarpifolia, shrub species of Elaeagnus parvifolia, Drepanostachyum spp. and Jasminum humile, and the herbs like Polygonatum cirrhifolium, Fragaria nubicola and Galium asperifolium were found to be the most preferred species by red panda. The red panda preferred the habitat with dense crown coverage ( >20% - 100%) and 31% - 50% ground cover. Fallen logs (39%) were the most preferred substrate used for defecation.

Keywords: distribution, habitat preference, jumla, red panda

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Authors: Mahla Azimi

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Introduction: Post-bladder catheter infection is a common and significant healthcare-associated infection that affects individuals with indwelling urinary catheters. These infections can lead to various complications, including urinary tract infections (UTIs), bacteremia, sepsis, and increased morbidity and mortality rates. This article aims to provide a comprehensive review of post-bladder catheter infections, including their causes, risk factors, clinical presentation, diagnosis, treatment options, and preventive measures. Causes and Risk Factors: Post-bladder catheter infections primarily occur due to the colonization of microorganisms on the surface of the urinary catheter. The most common pathogens involved are Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Enterococcus species. Several risk factors contribute to the development of these infections, such as prolonged catheterization duration, improper insertion technique, poor hygiene practices during catheter care, compromised immune system function in patients with underlying conditions or immunosuppressive therapy. Clinical Presentation: Patients with post-bladder catheter infections may present with symptoms such as fever, chills, malaise, suprapubic pain or tenderness, and cloudy or foul-smelling urine. In severe cases or when left untreated for an extended period of time, patients may develop more severe symptoms like hematuria or signs of systemic infection. Diagnosis: The diagnosis of post-bladder catheter infection involves a combination of clinical evaluation and laboratory investigations. Urinalysis is crucial in identifying pyuria (presence of white blood cells) and bacteriuria (presence of bacteria). A urine culture is performed to identify the causative organism(s) and determine its antibiotic susceptibility profile. Treatment Options: Prompt initiation of appropriate antibiotic therapy is essential in managing post-bladder catheter infections. Empirical treatment should cover common pathogens until culture results are available. The choice of antibiotics should be guided by local antibiogram data to ensure optimal therapy. In some cases, catheter removal may be necessary, especially if the infection is recurrent or associated with severe complications. Preventive Measures: Prevention plays a vital role in reducing the incidence of post-bladder catheter infections. Strategies include proper hand hygiene, aseptic technique during catheter insertion and care, regular catheter maintenance, and timely removal of unnecessary catheters. Healthcare professionals should also promote patient education regarding self-care practices and signs of infection. Conclusion: Post-bladder catheter infections are a significant healthcare concern that can lead to severe complications and increased healthcare costs. Early recognition, appropriate diagnosis, and prompt treatment are crucial in managing these infections effectively. Implementing preventive measures can significantly reduce the incidence of post-bladder catheter infections and improve patient outcomes. Further research is needed to explore novel strategies for prevention and management in this field.

Keywords: post-bladder catheter infection, urinary tract infection, bacteriuria, indwelling urinary catheters, prevention

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Authors: Priyanka Sharma, Ranjita Das, Mohan C. Kalita, Debajit Thakur

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Background: Actinomycetes are the richest source of novel bioactive secondary metabolites such as antibiotics, enzymes and other therapeutically useful metabolites with diverse biological activities. The present study aims at the antimicrobial potential and genetic diversity of culturable Actinomycetes isolated from protected forest ecosystems of Assam which includes Kaziranga National Park (26°30˝-26°45˝N and 93°08˝-93°36˝E), Pobitora Wildlife Sanctuary (26º12˝-26º16˝N and 91º58˝-92º05˝E) and Gibbon Wildlife Sanctuary (26˚40˝-26˚45˝N and 94˚20˝-94˚25˝E) which are located in the North-eastern part of India. Northeast India is a part of the Indo-Burma mega biodiversity hotspot and most of the protected forests of this region are still unexplored for the isolation of effective antibiotic-producing Actinomycetes. Thus, there is tremendous possibility that these virgin forests could be a potential storehouse of novel microorganisms, particularly Actinomycetes, exhibiting diverse biological properties. Methodology: Soil samples were collected from different ecological niches of the protected forest ecosystems of Assam and Actinomycetes were isolated by serial dilution spread plate technique using five selective isolation media. Preliminary screening of Actinomycetes for an antimicrobial activity was done by spot inoculation method and the secondary screening by disc diffusion method against several test pathogens, including multidrug resistant Staphylococcus aureus (MRSA). The strains were further screened for the presence of antibiotic synthetic genes such as type I polyketide synthases (PKS-I), type II polyketide synthases (PKS-II) and non-ribosomal peptide synthetases (NRPS) genes. Genetic diversity of the Actinomycetes producing antimicrobial metabolites was analyzed through 16S rDNA-RFLP using Hinf1 restriction endonuclease. Results: Based on the phenotypic characterization, a total of 172 morphologically distinct Actinomycetes were isolated and screened for antimicrobial activity by spot inoculation method on agar medium. Among the strains tested, 102 (59.3%) strains showed activity against Gram-positive bacteria, 98 (56.97%) against Gram-negative bacteria, 92 (53.48%) against Candida albicans MTCC 227 and 130 (75.58%) strains showed activity against at least one of the test pathogens. Twelve Actinomycetes exhibited broad spectrum antimicrobial activity in the secondary screening. The taxonomic identification of these twelve strains by 16S rDNA sequencing revealed that Streptomyces was found to be the predominant genus. The PKS-I, PKS-II and NRPS genes detection indicated diverse bioactive products of these twelve Actinomycetes. Genetic diversity by 16S rDNA-RFLP indicated that Streptomyces was the dominant genus amongst the antimicrobial metabolite producing Actinomycetes. Conclusion: These findings imply that Actinomycetes from the protected forest ecosystems of Assam, India, are a potential source of bioactive secondary metabolites. These areas are as yet poorly studied and represent diverse and largely unscreened ecosystem for the isolation of potent Actinomycetes producing antimicrobial secondary metabolites. Detailed characterization of the bioactive Actinomycetes as well as purification and structure elucidation of the bioactive compounds from the potent Actinomycetes is the subject of ongoing investigation. Thus, to exploit Actinomycetes from such unexplored forest ecosystems is a way to develop bioactive products.

Keywords: Actinomycetes, antimicrobial activity, forest ecosystems, RFLP

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Authors: M. Peña Aguilar Juan, E. Nava Galván Claudia, Pastrana Palma Alberto

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In this work was developed and implemented a thermal fogging disinfection system to counteract pathogens from poultry feces in agribusiness farms, to reduce mortality rates and increase biosafety in them. The control system consists of two phases for the conditioning of the farm during the sanitary break. In the first phase, viral and bacterial inactivation was performed by treating the stool dry cleaning, along with the development of a specialized product that foster the generation of temperatures above 55 °C in less than 24 hr, for virus inactivation. In the second phase, a process for disinfection by fogging was implemented, along with the development of a specialized disinfectant that guarantee no risk for the operators’ health or birds. As a result of this process, it was possible to minimize the level of mortality of chickens on farms from 12% to 5.49%, representing a reduction of 6.51% in the death rate, through the formula applied to the treatment of poultry litter based on oxidising agents used as antiseptics, hydrogen peroxide solutions, glacial acetic acid and EDTA in order to act on bacteria, viruses, micro bacteria and spores.

Keywords: innovation, triple helix, poultry farms, biosecurity

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Authors: Priscila Ribeiro dos Santos, Luiz Antonio Daniel

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Inadequate access to clean water and sanitation has become one of the most widespread problems affecting people throughout the developing world, leading to an unceasing need for low-cost and sustainable wastewater treatment systems. The UASB technology has been widely employed as a suitable and economical option for the treatment of sewage in developing countries, which involves low initial investment, low energy requirements, low operation and maintenance costs, high loading capacity, short hydraulic retention times, long solids retention times and low sludge production. Whereas dissolved air flotation process is a good option for the post-treatment of anaerobic effluents, being capable of producing high quality effluents in terms of total suspended solids, chemical oxygen demand, phosphorus, and even pathogens. This work presents an evaluation and monitoring, over a period of 6 months, of one compact full-scale system with this configuration, UASB reactors followed by dissolved air flotation units (DAF), operating in Brazil. It was verified as a successful treatment system, and an issue of relevance since dissolved air flotation process treating UASB reactor effluents is not widely encompassed in the literature. The study covered the removal and behavior of several variables, such as turbidity, total suspend solids (TSS), chemical oxygen demand (COD), Escherichia coli, total coliforms and Clostridium perfringens. The physicochemical variables were analyzed according to the protocols established by the Standard Methods for Examination of Water and Wastewater. For microbiological variables, such as Escherichia coli and total coliforms, it was used the “pour plate” technique with Chromocult Coliform Agar (Merk Cat. No.1.10426) serving as the culture medium, while the microorganism Clostridium perfringens was analyzed through the filtering membrane technique, with the Ágar m-CP (Oxoid Ltda, England) serving as the culture medium. Approximately 74% of total COD was removed in the UASB reactor, and the complementary removal done during the flotation process resulted in 88% of COD removal from the raw sewage, thus the initial concentration of COD of 729 mg.L-1 decreased to 87 mg.L-1. Whereas, in terms of particulate COD, the overall removal efficiency for the whole system was about 94%, decreasing from 375 mg.L-1 in raw sewage to 29 mg.L-1 in final effluent. The UASB reactor removed on average 77% of the TSS from raw sewage. While the dissolved air flotation process did not work as expected, removing only 30% of TSS from the anaerobic effluent. The final effluent presented an average concentration of 38 mg.L-1 of TSS. The turbidity was significantly reduced, leading to an overall efficiency removal of 80% and a final turbidity of 28 NTU.The treated effluent still presented a high concentration of fecal pollution indicators (E. coli, total coliforms, and Clostridium perfringens), showing that the system did not present a good performance in removing pathogens. Clostridium perfringens was the organism which suffered the higher removal by the treatment system. The results can be considered satisfactory for the physicochemical variables, taking into account the simplicity of the system, besides that, it is necessary a post-treatment to improve the microbiological quality of the final effluent.

Keywords: dissolved air flotation, municipal sewage, UASB reactor, treatment

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Authors: Muhammad Isah Legbo

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The phytochemical screening and antimicrobial activity of Nauclea lotifolia fruit, leaf and stem-bark extracts at various concentration of (20.0,10.0, 5.0, and 2.5 mg/ml) were evaluated against some pathogenic microorganisms such as Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, Staphylococcus aureus, Aspergillus niger and Candida albicans. The antimicrobial activity was assayed using agar well diffusion method. The result obtained show appreciable inhibitory effort of acetone, aqueous and methanolic extracts of Nauclea lotifolia. However, result obtained was less active compared to that of the control antibiotic (Ciprofloxacillin). The minimum inhibitory concentration (MIC) was determined using serial doubling dilution method and ranged from 5.0-10.0mg/ml, while the minimum bactericidal concentration (MBC) was determined by plating various dilution of extracts without turbidity and the result ranged from 5.0-7.5mg/ml. The phytochemical screening revealed the presence of alkaloid, anthraquinones, flavonoids, resin, steroid and saponin. The activities of the plant extract therefore justify their utilization in the treatment of various ailments associated with the test organism.

Keywords: Nauclea, lotifolia, antimicrobial, pathogens, saponin, extract

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Authors: Mohammad Reza Hossein Zadeh

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Plants have different layers of defense responses against biotic and abiotic stresses. One of the well-defined defense mechanism in plants is systemic acquired resistance (SAR) against a broad-range of pathogens. Salicylic acid (SA) plays a crucial role in regulation of the SAR pathway. It has been proved that Chemically SA-like compounds can mimic the SA signaling role. Imidocloprid is an insecticide being used to control whiteflies on crop plants. In order to study the possible role of Imidocloprid as an elicitor of SAR in plants, experiments were conducted in a completely randomized design frame with three treatments and duplicates on the detached leaves and whole Nicotiana tabacum var. Samsun NN. plants inoculated with Tobacco mild green mosaic virus (TMGMV). Compared with the effect of other SAR-inducers such as SA, Imidoclorid conferred a robust SAR induction in the infected plants. The results suggested that Imidocloprid even more powerful than SA can be considered as strong SAR inducer in the infected plants with viruses, which develop the local lesion symptoms.

Keywords: imidocloprid, Nicotiana tabacum var. Samsun NN, SAR, tobacco mild green, mosaic virus

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Authors: Amira Kalifa, Faïek Errouissi

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The seasonal changes and the daily activity of Stomoxys calcitrans (Diptera: Muscidae) were examined, using Vavoua traps, in a dairy cattle farm in Zaouiet Sousse, the Sahel area of Tunisia during May 2014 to October 2014. Over this period, a total of 4366 hematophagous diptera were captured and Stomoxys calcitrans was the most commonly trapped species (96.52%). Analysis of the seasonal activity, showed that S.calcitrans is bivoltine, with two peaks: a significant peak is recorded in May-June, during the dry season, and a second peak at the end of October, which is quite weak. This seasonal pattern would depend on climatic factors, particularly the temperature of the manure and that of the air. The activity pattern of Stomoxys calcitrans was diurnal with seasonal variations. The daily rhythm shows a peak between 11:00 am to 15:00 pm in May and between 11:00 am to 17:00 pm in June. These vector flies are important pests of livestock in Tunisia, where they are known as a mechanical vector of several pathogens and have a considerable economic and health impact on livestock. A better knowledge of their ecology is a prerequisite for more efficient control measures.

Keywords: cattle farm, daily rhythm, Stomoxys calcitrans, seasonal activity

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Authors: Hen Friman

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Various categories of aqueous solutions are discharged within residential, institutional, commercial, and industrial structures. To safeguard public health and preserve the environment, it is imperative to subject wastewater to treatment processes that eliminate pathogens (such as bacteria and viruses), nutrients (such as nitrogen and phosphorus), and other compounds. Failure to address untreated sewage accumulation can result in an array of adverse consequences. Israel exemplifies a special case in wastewater management. Appropriate wastewater treatment significantly benefits sectors such as agriculture, tourism, horticulture, and industry. Nevertheless, untreated sewage in settlements lacking proper sewage collection or transportation networks remains an ongoing and substantial threat. Notably, the process of wastewater treatment entails substantial energy consumption. Consequently, this study explores the integration of solar energy as a renewable power source within the wastewater treatment framework. By incorporating renewable energy sources into the process, costs can be minimized, and decentralized facilities can be established even in areas lacking adequate infrastructure for traditional treatment methods.

Keywords: renewable energy, solar energy, innovative, wastewater treatment

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Authors: Nouf A. Aldarmahi, Nesrin I. Tarbiah, Nuha A. Alkhattabi, Huda F. Alshaibi

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Nigella sativa which is known as dark cumin is a well-known example for a widely applicable herbal medicine. Nigella sativa can be effective in a variety of diseases such as hypertension, diabetes, bronchitis, gastrointestinal upset, and cancer. The anticancer effect of Nigella sativa appeared to be mediated by immune-modulatory effect through stimulating human natural killer (NK) cells. This is a type of lymphocytes which is part of the innate immunity, also known as the first line of defense in the body against pathogens. This study investigated the effect of thymoquinone as a major component of Nigella sativa on the molecular cytotoxic pathway of NK cell and the role of thymoquinone therapeutic effect on NK cells. NK cells were cultured with breast tumor cells in different ways and cultured media was collected and the concentration of perforin, granzyme B and interferon-α were measured by ELISA. The cytotoxic effect of NK cells on breast tumor cells was enhanced in the presence of thymoquinone, with increased activity of perforin in NK cells. This improved anticancer effect of thymoquinone on breast cancer cells.

Keywords: breast cancer, cancer cells, natural killer cells, thymoquinone

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Authors: Ndarusanze Berchmans

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This research work focused on the analysis of the observations of tuberculous meningitis in HIV-positive patients who were treated by the Prince Regent Charles Hospital in Bujumbura. A number of 246 seropositive patients were examined by the laboratory of Prince Regent Charles in the period between 2010 and 2015. We did a retrospective study; we used data from the registers of the laboratories mentioned above; the objective was to approach the epidemiological, biological, clinical, and therapeutic characteristics of tuberculosis meningitis infection: 124 women (50.40% of AIDS patients) and 122 men (49.59% of AIDS patients) were subject to the diagnosis by identification of cerebrospinal fluid (CSF). The average age of the patients was 30 years for this period. The population at risk has an average age of between 34 and 42 years for the years between 2010-2015. From 2010 to 2012, cases of opportunistic diseases (e.g., tuberculous meningitis and Cryptococcus neoformans meningitis), often found in immunocompromised, were observed at a high rate; in this period, there was a disturbance of the rhythm providing antiretroviral drugs to people with AIDS. The rate of the two meningitis (tuberculous meningitis and Cryptococcus neoformans meningitis) remained above 10% to gradually decrease until 2015, with the gradual return of antiretrovirals. This period records an overall average of 25 cases of tuberculous meningitis, or a percentage of 10.16%. For the year 2015, there were 4 cases of tuberculous meningitis out of a total of 35 seropositive examined (11.42%). This year's percentage shows that the number of tuberculous meningitis cases has fallen from the rate in previous years. This is the result of the care given by associations against HIV/AIDS to HIV-positive people. This decrease in cases of tuberculous meningitis is due to the acquisition of antiretrovirals by all HIV-positive people treated by hospitals. For the moment, these hospitals are taking care of many AIDS patients by providing them permanently with antiretrovirals; Besides that, there are many patients who are supported by associations whose activities are directed against HIV/AIDS.

Keywords: Cryptococcus neoformans meningitis, tuberculosis meningitis, neurological complications, epidemiology of meningitis

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Authors: Tramuta Clara, Masotti Chiara, Pitti Monica, Adriano Daniela, Battistini Roberta, Serraca Laura, Decastelli Lucia

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Oysters are considered filter organisms, and their raw consumption may increase health risks for consumers: it is often associated with outbreaks of gastroenteritis or enteric illnesses. Most of these foodborne diseases are caused by Vibrio strains, enteric pathogens also involved in the diffusion of genetic determinants of antibiotic resistance and their entrance along the food chain. The European Food Safety Authority (EFSA), during the European Union report on antimicrobial resistance in 2017, focused the attention about the role of food as a possible carrier of antibiotic-resistant bacteria or antibiotic-resistance genes that determine health risks for humans. This study wants to determine antibiotic resistance and antibiotic-resistance genes in Vibrio spp. isolated from Crassostrea gigas oysters collected in the Golfo della Spezia (Liguria, Italy). A total of 47 Vibrio spp. strains were isolated (ISO21872-2:2017) during the summer of 2021 from oysters of Crassostrea gigas. The strains were identified by MALDI-TOF (Bruker, Germany) mass spectrometry and tested for antibiotic susceptibility using a broth microdiluition method (ISO20776-1:2019) using Sensititre EUVSEC plates (Thermo-Fisher Scientific) to obtain the Minimum Inhibitory Concentration (MIC). The strains were tested with PCR-based biomolecular methods, according to previous works, to define the presence of 23 resistance genes of the main classes of antibiotics used in human and veterinary medicine: tet (B), tet (C), tet (D), tet (A), tet (E), tet (G ), tet (K), tet (L), tet (M), tet (O), tet (S) (tetracycline resistance); blaCTX-M, blaTEM, blaOXA, blaSHV (β-lactam resistance); mcr-1 and mcr-2 (colistin resistance); qnrA, qnrB, and qnrS (quinolone resistance); sul1, sul2 and sul3 (sulfonamide resistance). Six different species have been identified: V. alginolyticus 34% (n=16), V. harveyi 28% (n=13), V. fortis 15% (n=7), V. pelagius 8% (n=4), V. parahaemolyticus 11% (n=5) e V. chagasii 4% (n=2). The PCR assays showed the presence of the blaTEM gene on 40% of the strains (n=19). All the other genes were not detected, except for a V. alginolyticus positive for anrS gene. The broth microdiluition method results showed an high level of resistance for ciprofloxacin (62%; n=29), ampicillin (47%; n=22), and colistin (49%; n=23). Furthermore, 32% (n=15) of strains can be considered multiresistant bacteria for the simultaneous presence of resistance for three different antibiotic classes. Susceptibility towards meropenem, azithromycin, gentamicin, ceftazidime, cefotaxime, chloramphenicol, tetracycline and sulphamethoxazole reached 100%. The Vibrio species identified in this study are widespread in marine environments and can cause gastrointerstinal infections after the ingestion of raw fish products and bivalve molluscs. The level of resistance to antibiotics such as ampicillin, ciprofloxacin and colistin can be connected to anthropic factors (industrial, agricultural and domestic wastes) that promote the spread of resistance to these antibiotics. It can be also observed a strong correlation between phenotypic (resistant MIC) and genotypic (positive blaTEM gene) resistance for ampicillin on the same strains, probably due to the transfer of genetic material between bacterial strains. Consumption of raw bivalve molluscs can represent a risk for consumers heath due to the potentially presence of foodborne pathogens, highly resistant to different antibiotics and source of transferable antibiotic-resistant genes.

Keywords: vibrio species, blaTEM genes, antimicrobial resistance, PCR

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Authors: Alex J. Summers, Jasmine P. Devadhasan, Douglas Montgomery, Brittany Fischer, Jian Gu, Frederic Zenhausern

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Immunoassays have been utilized for several applications, including the detection of pathogens. Our laboratory is in the development of a tier 1 biothreat panel utilizing Vertical Flow Assay (VFA) technology for simultaneous detection of pathogens and toxins. One method of manufacturing VFA membranes is with non-contact piezoelectric dispensing, which provides advantages, such as low-volume and rapid dispensing without compromising the structural integrity of antibody or substrate. Challenges of this processinclude premature discontinuation of dispensing and misaligned spotting. Preliminary data revealed the Yp 11C7 mAb (11C7)reagent to exhibit a large angle of failure during printing which may have contributed to variable printing outputs. A Design of Experiment (DOE) was executed using this reagent to investigate the effects of hydrostatic pressure and reagent concentration on microarray printing outputs. A Nano-plotter 2.1 (GeSIM, Germany) was used for printing antibody reagents ontonitrocellulose membrane sheets in a clean room environment. A spotting plan was executed using Spot-Front-End software to dispense volumes of 11C7 reagent (20-50 droplets; 1.5-5 mg/mL) in a 6-test spot array at 50 target membrane locations. Hydrostatic pressure was controlled by raising the Pressure Compensation Vessel (PCV) above or lowering it below our current working level. It was hypothesized that raising or lowering the PCV 6 inches would be sufficient to cause either liquid accumulation at the tip or discontinue droplet formation. After aspirating 11C7 reagent, we tested this hypothesis under stroboscope.75% of the effective raised PCV height and of our hypothesized lowered PCV height were used. Humidity (55%) was maintained using an Airwin BO-CT1 humidifier. The number and quality of membranes was assessed after staining printed membranes with dye. The droplet angle of failure was recorded before and after printing to determine a “stroboscope score” for each run. The DOE set was analyzed using JMP software. Hydrostatic pressure and reagent concentration had a significant effect on the number of membranes output. As hydrostatic pressure was increased by raising the PCV 3.75 inches or decreased by lowering the PCV -4.5 inches, membrane output decreased. However, with the hydrostatic pressure closest to equilibrium, our current working level, membrane output, reached the 50-membrane target. As the reagent concentration increased from 1.5 to 5 mg/mL, the membrane output also increased. Reagent concentration likely effected the number of membrane output due to the associated dispensing volume needed to saturate the membranes. However, only hydrostatic pressure had a significant effect on stroboscope score, which could be due to discontinuation of dispensing, and thus the stroboscope check could not find a droplet to record. Our JMP predictive model had a high degree of agreement with our observed results. The JMP model predicted that dispensing the highest concentration of 11C7 at our current PCV working level would yield the highest number of quality membranes, which correlated with our results. Acknowledgements: This work was supported by the Chemical Biological Technologies Directorate (Contract # HDTRA1-16-C-0026) and the Advanced Technology International (Contract # MCDC-18-04-09-002) from the Department of Defense Chemical and Biological Defense program through the Defense Threat Reduction Agency (DTRA).

Keywords: immunoassay, microarray, design of experiment, piezoelectric dispensing

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