Search results for: 16S rRNA sequencing

Authors: Noura El-Ahmady El-Naggar

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Among the antitumour drugs, bacterial enzyme L-asparaginase has been employed as the most effective chemotherapeutic agent in pediatric oncotherapy especially for acute lymphoblastic leukemia. Glutaminase free L-asparaginase producing actinomycetes were isolated from soil samples collected from Egypt. Among them, a potential culture, strain NEAE-119, was selected and identified on the basis of morphological, cultural, physiological and biochemical properties, together with 16S rDNA sequence as Streptomyces olivaceus NEAE-119 and sequencing product(1509 bp) was deposited in the GenBank database under accession number KJ200342. The optimization of different process parameters for L-asparaginase production by Streptomyces olivaceus NEAE-119 using Plackett–Burman experimental design and response surface methodology was carried out. Fifteen nutritional variables (temperature, pH, incubation time, inoculum size, inoculum age, agitation speed, dextrose, starch, L-asparagine, KNO3, yeast extract, K2HPO4, MgSO4.7H2O, NaCl and FeSO4. 7H2O) were screened using Plackett–Burman experimental design. The most positive significant independent variables affecting enzyme production (temperature, inoculum age and agitation speed) were further optimized by the central composite face-centered design -response surface methodology. As a result, a medium of the following formula is the optimum for producing an extracellular L-asparaginase in the culture filtrate of Streptomyces olivaceus NEAE-119: Dextrose 3g, starch 20g, L-asparagine 10g, KNO3 1g, K2HPO4 1g, MgSO4.7H2O 0.1g, NaCl 0.1g, pH 7, temperature 37°C, agitation speed 200 rpm/min, inoculum size 4%, v/v, inoculum age 72 h and fermentation period 5 days.

Keywords: Streptomyces olivaceus NEAE-119, glutaminase free L-asparaginase, production, Plackett-Burman design, central composite face-centered design, 16S rRNA, scanning electron microscope

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Authors: D. S. V. Barbieri, R. R. Gomes, G. D. Santos, P. F. Herkert, M. Moreira, E. S. Trindade, V. A. Vicente

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The importance of yeast infections has increased in recent decades. The monitoring of Candida yeasts has been relevant in the study of groups and populations. This research evaluated 31 Candida spp. isolates from oral microbiota of 12 Brazilian schoolchildren coinfected with Streptococcus mutans. The isolates were evaluated for their ability to form biofilm in vitro and molecularly characterized based on the sequencing of intergenic spacer regions ITS1-5,8S-ITS2 and variable domains of the large subunit (D1/D2) regions of the rDNA, as well as ABC system genotyping. The sequencing confirmed 26 lineages of Candida albicans, three Candida tropicalis, one Candida guillhermondii and one Candida glabrata. Genetic variability and differences on in biofilm formation were observed among Candida yeasts lineages. At least one Candida strain from each caries activity child was C.albicans genotype A or Candida non-albicans. C. tropicalis was associated with highest cavities rates. These results indicate that the presence of C. albicans genotype A or multi-colonization by non albicans species seem to be associates to the potentialization of caries risk.

Keywords: biofilm, Candida albicans, oral microbiota, caries

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Authors: Il Ho Park, Joong Seob Lee, Sung Hun Kang, Jae-Min Shin, Il Seok Park, Seok Min Hong, Seok Jin Hong

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Introduction: The human microbiota is the aggregate of microorganisms, and the bacterial microbiome of the human digestive tract contributes to both health and disease. In health, bacteria are key components in the development of mucosal barrier function and in innate and adaptive immune responses, and they also work to suppress the establishment of pathogens. In human upper airway, the sinonasal microbiota might play an important role in chronic rhinosinusitis (CRS). The purpose of this study is to investigate the human upper airway microbiome in CRS patients and to compare the sinonasal microbiome of adults with children. Materials and methods: A total of 19 samples from 19 patients (Group1; 9 CRS in children, aged 5 to 14 years versus Group 2; 10 CRS in adults aged 21 to 59 years) were examined. Swabs were collected from the middle meatus and/or anterior ethmoid region under general anesthesia during endoscopic sinus surgery or tonsillectomy. After DNA extraction from swab samples, we analysed bacterial microbiome consortia using 16s rRNA gene sequencing approach (the Illumina MiSeq platform). Results: In this study, relatively abundance of the six bacterial phyla and tremendous genus and species found in substantial amounts in the individual sinus swab samples, include Corynebacterium, Hemophilus, Moraxella, and Streptococcus species. Anaerobes like Fusobacterium and Bacteroides were abundantly present in the children group, Bacteroides and Propionibacterium were present in adults group. In genus, Haemophilus was the most common CRS microbiome in children and Corynebacterium was the most common CRS microbiome in adults. Conclusions: Our results show the diversity of human upper airway microbiome, and the findings will suggest that CRS is a polymicrobial infection. The Corynebacterium and Hemophilus may live as commensals on mucosal surfaces of sinus in the upper respiratory tract. The further study will be needed for analysis of microbiome-human interactions in upper airway and CRS.

Keywords: microbiome, upper airway, chronic rhinosinusitis, adult and children

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Authors: Nonhlanhla Nkosi, Kulsum Kondiah

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The toxicological manifestation of heavy metals motivates interest towards the development of a reliable, eco-friendly biosorption process. With that being said, the aim of the current study was to characterise the EPS from heavy-metal resistant bacteria isolated from acid mine decant on the West Rand, Gauteng, South Africa. To achieve this, six exopolysaccharide (EPS) producing, metal resistant strains (Pb101, Pb102, Pb103, Pb204, Co101, and Ni101) were identified as Bacillus safensis strain NBRC 100820, Bacillus proteolyticus, Micrococcus luteus, Enterobacter sp. Pb204, Bacillus wiedmannii and Bacillus zhangzhouensis, respectively with 16S rRNA sequencing. Thereafter, EPS was extracted using chemical (formaldehyde/NaOH) and physical (ultrasonification) methods followed by physicochemical characterisation of carbohydrate, DNA, and protein contents using chemical assays and spectroscopy (FTIR- Fourier transformed infrared and 3DEEM- three-dimensional excitation-emission matrix fluorescence spectroscopy). EPS treated with formaldehyde/NaOH showed better recovery of macromolecules than ultrasonification. The results of the present study showed that carbohydrates were more abundant than proteins, with carbohydrate and protein concentrations of 8.00 mg/ml and 0.22 mg/ml using chemical method in contrast to 5.00 mg/ml and 0.77 mg/ml using physical method, respectively. The FTIR spectroscopy results revealed that the extracted EPS contained hydroxyl, amide, acyl, and carboxyl groups that corresponded to the aforementioned chemical analysis results, thus asserting the presence of carbohydrates, DNA, polysaccharides, and proteins in the EPS. These findings suggest that identified functional groups of EPS form surface charges, which serve as the binding sites for suspended particles, thus possibly mediating adsorption of divalent cations and heavy metals. Using the extracted EPS in the development of a cost-effective biosorption solution for industrial wastewater treatment is attainable.

Keywords: biosorbent, exopolysaccharides, heavy metals, wastewater treatment

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Authors: Lohendy Munoz-Vargas, Stephen O. Opiyo, Rose Digianantonio, Michele L. Williams, Asela Wijeratne, Gregory Habing

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Non-typhoidal Salmonella enterica is a zoonotic pathogen with critical importance in animal and public health. The persistence of Salmonella on farms affects animal productivity and health, and represents a risk for food safety. The intestinal microbiota plays a fundamental role in the colonization and invasion of this ubiquitous microorganism. To overcome the colonization resistance imparted by the gut microbiome, Salmonella uses invasion strategies and the host inflammatory response to survive, proliferate, and establish infections with diverse clinical manifestations. Cattle serve as reservoirs of Salmonella, and periparturient cows have high prevalence of Salmonella shedding; however, to author`s best knowledge, little is known about the association between the gut microbiome and the onset of Salmonella shedding during the periparturient period. Thus, the objective of this study was to assess the association between changes in bacterial communities and the onset of Salmonella shedding in cattle approaching parturition. In a prospective cohort study, fecal samples from 98 dairy cows originating from four different farms were collected at four time points relative to calving (-3 wks, -1 wk, +1 wk, +3 wks). All 392 samples were cultured for Salmonella. Sequencing of the V4 region of the 16S rRNA gene using the Illumina platform was completed to evaluate the fecal microbiome in a selected sample subset. Analyses of microbial composition, diversity, and structure were performed according to time points, farm, and Salmonella onset status. Individual cow fecal microbiomes, predominated by Bacteroidetes, Firmicutes, Spirochaetes, and Proteobacteria phyla, significantly changed before and after parturition. Microbial communities from different farms were distinguishable based on multivariate analysis. Although there were significant differences in some bacterial taxa between Salmonella positive and negative samples, our results did not identify differences in the fecal microbial diversity or structure for cows with and without the onset of Salmonella shedding. These data suggest that determinants other than the significant changes in the fecal microbiome influence the periparturient onset of Salmonella shedding in dairy cattle.

Keywords: dairy cattle, microbiome, periparturient, Salmonella

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Authors: Aishik Deb, Rituparna Sinha

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We have developed an algorithm to detect the abnormal segment/"structural variation in the genome across a number of samples. We have worked on simulated as well as real data from the BAM Files and have designed a segmentation algorithm where abnormal segments are detected. This algorithm aims to improve the accuracy and performance of the existing CNV-CH algorithm. The next-generation sequencing (NGS) approach is very fast and can generate large sequences in a reasonable time. So the huge volume of sequence information gives rise to the need for Big Data and parallel approaches of segmentation. Therefore, we have designed a map-reduce approach for the existing CNV-CH algorithm where a large amount of sequence data can be segmented and structural variations in the human genome can be detected. We have compared the efficiency of the traditional and map-reduce algorithms with respect to precision, sensitivity, and F-Score. The advantages of using our algorithm are that it is fast and has better accuracy. This algorithm can be applied to detect structural variations within a genome, which in turn can be used to detect various genetic disorders such as cancer, etc. The defects may be caused by new mutations or changes to the DNA and generally result in abnormally high or low base coverage and quantification values.

Keywords: cancer detection, convex hull segmentation, map reduce, next generation sequencing

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Authors: Vanessa De Carvalho, Chad MacPherson, Julien Tremblay, Julie Champagne, Stephanie-Anne Girard

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Background: The interactions between bacteria and fungi in the human vaginal microbiome are fundamental to the concept of health and disease. The means by which the microbiota and mycobiota interact is still poorly understood and further studies are necessary to properly characterize this complex ecosystem. The aim of this study was to select a DNA extraction method capable of recovering high qualities of fungal and bacterial DNA from a single vaginal swab. Methods: 11 female volunteers ( ≥ 20 to < 55 years old) self-collected vaginal swabs in triplicates. Three commercial extraction kits: Masterpure Yeast Purification kit (Epicenter), PureLink™ Microbiome DNA Purification kit (Invitrogen), and Quick-DNA™ Fecal/Soil Microbe Miniprep kit (Zymo) were evaluated on the ability to recover fungal and bacterial DNA simultaneously. The extraction kits were compared on the basis of recovery, yield, purity, and the community richness of bacterial (16S rRNA - V3-V4 region) and fungal (ITS1) microbiota composition by Illumina MiSeq amplicon sequencing. Results: Recovery of bacterial DNA was achieved with all three kits while fungal DNA was only consistently recovered with Masterpure Yeast Purification kit (yield and purity). Overall, all kits displayed similar microbiota profiles for the top 20 OTUs; however, Quick-DNA™ Fecal/Soil Microbe Miniprep kit (Zymo) showed more species richness than the other two kits. Conclusion: In the present study, Masterpure Yeast purification kit proved to be a good candidate for purification of high quality fungal and bacterial DNA simultaneously. These findings have potential benefits that could be applied in future vaginal microbiome research. Whilst the use of a single extraction method would lessen the burden of multiple swab sampling, decrease laboratory workload and off-set costs associated with multiple DNA extractions, thoughtful consideration must be taken when selecting an extraction kit depending on the desired downstream application.

Keywords: bacterial vaginosis, DNA extraction, microbiota, mycobiota, vagina, vulvovaginal candidiasis, women’s health

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Authors: Zeina Bourhane, Anders Lanzen, Christine Cagnon, Olfa Ben Said, Cristiana Cravo-Laureau, Robert Duran

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The anthropogenic pressure in coastal areas increases dramatically with the exploitation of environmental resources. Biomonitoring coastal areas are crucial to determine the impact of pollutants on bacterial communities in soils and sediments since they provide important ecosystem services. However, relevant biomonitoring tools allowing fast determination of the ecological status are yet to be defined. Microbial ecology approaches provide useful information for developing such microbial monitoring tools reporting on the effect of environmental stressors. Chemical and microbial molecular approaches were combined in order to determine microbial bioindicators for assessing the ecological status of soil and river ecosystems around the Ichkeul Lake (Tunisia), an area highly impacted by human activities. Samples were collected along soil/river/lake continuums in three stations around the Ichkeul Lake influenced by different human activities at two seasons (summer and winter). Contaminant pressure indexes (PI), including PAHs (Polycyclic aromatic hydrocarbons), alkanes, and OCPs (Organochlorine pesticides) contents, showed significant differences in the contamination level between the stations with seasonal variation. Bacterial communities were characterized by 16S ribosomal RNAs (rRNA) gene metabarcoding. Although microgAMBI indexes, determined from the sequencing data, were in accordance with contaminant contents, they were not sufficient to fully explain the PI. Therefore, further microbial indicators are still to be defined. The comparison of bacterial communities revealed the specific microbial assemblage for soil, river, and lake sediments, which were significantly correlated with contaminant contents and PI. Such observation offers the possibility to define a relevant set of bioindicators for reporting the effects of human activities on the microbial community structure. Such bioindicators might constitute useful monitoring tools for the management of microbial communities in coastal areas.

Keywords: bacterial communities, biomonitoring, contamination, human impacts, microbial bioindicators

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Authors: Mohammadreza Hajialigol, Nargues Falahi Charkhabi, Fatemeh Shahryari, Saadat Sarikhani

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BACKGROUND AND OBJECTIVES Iran is the third producer of Persian walnut worldwide. However, its walnut trees have been under threat from decline during last decade. Walnut canker caused by B. nigrifluens and B. rubrifaciens was recorded in multiple regions of Iran. Furthermore, Brenneria rosae subsp. rosae and Gibbsiella quercinecans were recently recognized as responsible for walnut decline in northwestern Iran. This study aimed to identify the causal agent of walnut decline in Kermanshah and Isfahan. MATERIAL AND METHODS Symptomatic samples were collected from affected walnut trees of Kermanshah and Isfahan provinces. The pathogenicity of strains was proved on immature walnut fruits cv. ‘Hartley’ and young green twigs of two-year-old walnut seedling cv. ‘Chandler’. Pathogenic strains were subjected to conventional phenotypic tests. 16S rRNA, gyrB, and infB genes were partially amplified and sequenced. RESULTS Irregular longitudinal cankers and dark lesions were observed in the outer and inner bark, respectively. Twenty-four strains were isolated on EMB-agar media. Fourteen strains were able to cause necrosis and a dark-colored region in the mesocarp and on young green twigs around the inoculation site 14 and 30 days post-inoculation, respectively. Strains were able to hydrolyze Tween 20, Tween 80, gelatin and esculin, however, did not produce indole or urease. Pairwise comparison, the 16S rRNA gene nucleotide sequences of strain I2 were 100% identical with those of Rahnella victoriana FRB 225T. Moreover, a phylogenetic tree reconstructed based on the concatenated sequences of two housekeeping gene fragments, gyrB (601 bp) and infB (615 bp), revealed that the strains I2, I5, and KE6 were clustered with R. victoriana FRB 225T. CONCLUSION To the best of our knowledge, this is the first report of R. victoriana in association with walnut decline. This result is necessary to find resistant genotypes.

Keywords: emerging pathogens, Iran, juglans regia, MLSA

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Authors: Noura El-Ahmady El-Naggar

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L-asparaginase is an important enzyme as therapeutic agents used in combination therapy with other drugs in the treatment of acute lymphoblastic leukemia in children. L-asparaginase producing actinomycete strain, NEAE-K, was isolated from soil sample and identified on the basis of morphological, cultural, physiological and biochemical properties, together with 16S rDNA sequence as new subsp. Streptomyces rochei subsp. chromatogenes NEAE-K and sequencing product (1532 bp) was deposited in the GenBank database under accession number KJ200343. The study was conducted to screen parameters affecting the production of L-asparaginase by Streptomyces rochei subsp. chromatogenes NEAE-K on solid state fermentation using Plackett–Burman experimental design. Sixteen different independent variables including incubation time, moisture content, inoculum size, temperature, pH, soybean meal+ wheat bran, dextrose, fructose, L-asparagine, yeast extract, KNO3, K2HPO4, MgSO4.7H2O, NaCl, FeSO4. 7H2O, CaCl2, and three dummy variables were screened in Plackett–Burman experimental design of 20 trials. The most significant independent variables affecting enzyme production (dextrose, L-asparagine and K2HPO4) were further optimized by the central composite design. As a result, a medium of the following formula is the optimum for producing an extracellular L-asparaginase by Streptomyces rochei subsp. chromatogenes NEAE-K from solid state fermentation: g/L (soybean meal+ wheat bran 15, dextrose 3, fructose 4, L-asparagine 8, yeast extract 2, KNO3 1, K2HPO4 2, MgSO4.7H2O 0.5, NaCl 0.1, FeSO4. 7H2O 0.02, CaCl2 0.01), incubation time 7 days, moisture content 50%, inoculum size 3 mL, temperature 30°C, pH 8.5.

Keywords: streptomyces rochei subsp. chromatogenes neae-k, 16s rrna, identification, solid state fermentation, l-asparaginase production, plackett-burman design, central composite design

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Authors: A. Delimitsou, C. Gouedard, E. Konstanta, A. Koletis, S. Patera, E. Manou, K. Spaho, S. Murray

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Background: There remains a lack of International Standard Control Reference materials for Next Generation Sequencing-based approaches or device calibration. We have designed and validated dsDNA biomimetic reference materials for targeted such approaches incorporating proprietary motifs (patent pending) for device/test calibration. They enable internal single-sample calibration, alleviating sample comparisons to pooled historical population-based data assembly or statistical modelling approaches. We have validated such an approach for BRCA Copy Number Variation analytics using iQRS™-CNVSUITE versus Mixed Ligation-dependent Probe Amplification. Methods: Standard BRCA Copy Number Variation analysis was compared between mixed ligation-dependent probe amplification and next generation sequencing using a cohort of 198 breast/ovarian cancer patients. Next generation sequencing based copy number variation analysis of samples spiked with iQRS™ dsDNA biomimetics were analysed using proprietary CNVSUITE software. Mixed ligation-dependent probe amplification analyses were performed on an ABI-3130 Sequencer and analysed with Coffalyser software. Results: Concordance of BRCA – copy number variation events for mixed ligation-dependent probe amplification and CNVSUITE indicated an overall sensitivity of 99.88% and specificity of 100% for iQRS™-CNVSUITE. The negative predictive value of iQRS-CNVSUITE™ for BRCA was 100%, allowing for accurate exclusion of any event. The positive predictive value was 99.88%, with no discrepancy between mixed ligation-dependent probe amplification and iQRS™-CNVSUITE. For device calibration purposes, precision was 100%, spiking of patient DNA demonstrated linearity to 1% (±2.5%) and range from 100 copies. Traditional training was supplemented by predefining the calibrator to sample cut-off (lock-down) for amplicon gain or loss based upon a relative ratio threshold, following training of iQRS™-CNVSUITE using spiked iQRS™ calibrator and control mocks. BRCA copy number variation analysis using iQRS™-CNVSUITE™ was successfully validated and ISO15189 accredited and now enters CE-IVD performance evaluation. Conclusions: The inclusion of a reference control competitor (iQRS™ dsDNA mimetic) to next generation sequencing-based sequencing offers a more robust sample-independent approach for the assessment of copy number variation events compared to mixed ligation-dependent probe amplification. The approach simplifies data analyses, improves independent sample data analyses, and allows for direct comparison to an internal reference control for sample-specific quantification. Our iQRS™ biomimetic reference materials allow for single sample copy number variation analytics and further decentralisation of diagnostics to single patient sample assessment.

Keywords: validation, diagnostics, oncology, copy number variation, reference material, calibration

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Authors: Niamh Higgins, Dawn Howard

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The Irish agri-food sector is of major importance to Ireland’s manufacturing sector and to the Irish economy through employment and the exporting of animal products worldwide. Infectious diseases and parasites have an impact on farm animal health causing profitability and productivity to be affected. For the sustainability of Irish dairy farming, there must be the highest standard of animal health. There can be a lack of information in accounting for > 1% of complete microbial diversity in an environment. There is the tendency of culture-based methods of microbial identification to overestimate the prevalence of species which grow easily on an agar surface. There is a need for new technologies to address these issues to assist with animal health. Metagenomic approaches provide information on both the whole genome and transcriptome present through DNA sequencing of total DNA from environmental samples producing high determination of functional and taxonomic information. Nanopore Next Generation Technologies have the ability to be powerful sequencing technologies. They provide high throughput, low material requirements and produce ultra-long reads, simplifying the experimental process. The aim of this study is to use a metagenomics approach to analyze dairy cattle faecal samples using the Oxford Nanopore MinION Next Generation Sequencer and to establish an in-house pipeline for metagenomic characterization of complex samples. Faecal samples will be obtained from Irish dairy farms, DNA extracted and the MinION will be used for sequencing, followed by bioinformatics analysis. Of particular interest, will be the parasite Buxtonella sulcata, which there has been little research on and which there is no research on its presence on Irish dairy farms. Preliminary results have shown the ability of the MinION to produce hundreds of reads in a relatively short time frame of eight hours. The faecal samples were obtained from 90 dairy cows on a Galway farm. The results from Oxford Nanopore ‘What’s in my pot’ (WIMP) using the Epi2me workflow, show that from a total of 926 classified reads, 87% were from the Kingdom Bacteria, 10% were from the Kingdom Eukaryota, 3% were from the Kingdom Archaea and < 1% were from the Kingdom Viruses. The most prevalent bacteria were those from the Genus Acholeplasma (71 reads), Bacteroides (35 reads), Clostridium (33 reads), Acinetobacter (20 reads). The most prevalent species present were those from the Genus Acholeplasma and included Acholeplasma laidlawii (39 reads) and Acholeplasma brassicae (26 reads). The preliminary results show the ability of the MinION for the identification of microorganisms to species level coming from a complex sample. With ongoing optimization of the pipe-line, the number of classified reads are likely to increase. Metagenomics has the potential in animal health for diagnostics of microorganisms present on farms. This would support wprevention rather than a cure approach as is outlined in the DAFMs National Farmed Animal Health Strategy 2017-2022.

Keywords: animal health, buxtonella sulcata, infectious disease, irish dairy cattle, metagenomics, minION, next generation sequencing

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Authors: Maryam Vaezjalali, Koroush Rahimian, Maryam Asli, Tahmineh Kandelouei, Foad Davoodbeglou, Amir H. Kashi

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Objectives: The present study was conducted to assess the HBV molecular profiles including genotypes, subgenotypes, subtypes & mutations in hepatitis B genes. Materials/Patients and Methods: This study was conducted on 229 intravenous drug users who referred to three Drop- in-Centers and a hospital in Tehran. HBV DNA was extracted from HBsAg positive serum samples and amplified by Nested PCR. HBV genotype, subgenotypes, subtype and genes mutation were determined by direct sequencing. Phylogenetic tree was constructed using neighbor- joining (NJ) method. Statistical analyses were carried out by SPSS 20. Results: HBV DNA was found in 3 HBsAg positive cases. Phylogenetic tree of derived HBV DNAs showed the existence of genotype D (subgenotype D1, subtype ayw2). Also immune escape mutations were determined in S gene. Conclusion: There were a few variations and genotypes and subtypes among infected intravenous drug users. This study showed the predominance of genotype D among intravenous drug users. Our study concurs with other reports from Iran, that all showing currently only genotype D is the only detectable genotype in Iran.

Keywords: drug users, genotype, HBV, phylogenetic tree

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Authors: Shilpi, Indu Gaur, Neha Bhadauria, Susmita Goswami, Prabir K. Paul

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Cultivation and consumption of organically grown fruits and vegetables have increased by several folds. However, the presence of Human Enteric Pathogens on the surface of organically grown vegetables causing Gastro-intestinal diseases, are most likely due to contaminated water and fecal matter of farm animals. Human Enteric Pathogens are adapted to colonize the human gut, and also colonize plant surface. Microbes on plant surface communicate with each other to establish quorum sensing. The cross talk study is important because the enteric pathogens on phylloplane have been reported to mask the beneficial resident bacteria of plant. In the present study, HEPs and bacterial colonizers were identified using 16s rRNA sequencing. Microbial colonization patterns after interaction between Human Enteric Pathogens and natural bacterial residents on tomato phylloplane was studied. Tomato plants raised under aseptic conditions were inoculated with a mixture of Serratia fonticola and Klebsiella pneumoniae. The molecules involved in cross-talk between Human Enteric Pathogens and regular bacterial colonizers were isolated and identified using molecular techniques and HPLC. The colonization pattern was studied by leaf imprint method after 48 hours of incubation. The associated protein-protein interaction in the host cytoplasm was studied by use of crosslinkers. From treated leaves the crosstalk molecules and interaction proteins were separated on 1D SDS-PAGE and analyzed by MALDI-TOF-TOF analysis. The study is critical in understanding the molecular aspects of HEP’s adaption to phylloplane. The study revealed human enteric pathogens aggressively interact among themselves and resident bacteria. HEPs induced establishment of a signaling cascade through protein-protein interaction in the host cytoplasm. The study revealed that the adaptation of Human Enteric Pathogens on phylloplane of Solanum lycopersicum involves the establishment of complex molecular interaction between the microbe and the host including microbe-microbe interaction leading to an establishment of quorum sensing. The outcome will help in minimizing the HEP load on fresh farm produce, thereby curtailing incidences of food-borne diseases.

Keywords: crosslinkers, human enteric pathogens (HEPs), phylloplane, quorum sensing

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Authors: Tanushree Jaitly, Shailendra Gupta, Leila Taher, Gerold Schuler, Julio Vera

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Genomics-based personalized medicine is a promising approach to fight aggressive tumors based on patient's specific tumor mutation and expression profiles. A remarkable case is, dendritic cell-based immunotherapy, in which tumor epitopes targeting patient's specific mutations are used to design a vaccine that helps in stimulating cytotoxic T cell mediated anticancer immunity. Here we present a computational pipeline for epitope-based personalized cancer vaccines using patient-specific haplotype and cancer mutation profiles. In the workflow proposed, we analyze Whole Exome Sequencing and RNA Sequencing patient data to detect patient-specific mutations and their expression level. Epitopes including the tumor mutations are computationally predicted using patient's haplotype and filtered based on their expression level, binding affinity, and immunogenicity. We calculate binding energy for each filtered major histocompatibility complex (MHC)-peptide complex using docking studies, and use this feature to select good epitope candidates further.

Keywords: cancer immunotherapy, epitope prediction, NGS data, personalized medicine

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Authors: Sheng-Yu Lee

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Aim: Opioid use disorder is often characterized by repetitive drug-seeking and drug-taking behaviors with severe public health consequences. Animal model showed that opioid-induced perturbations in the gut microbiota causally relate to neuroinflammation, deficits in reward responding, and opioid tolerance, possibly due to changes in gut microbiota. Therefore, we propose that the dysbiosis of gut microbiota can be associated with pathogenesis of opioid dependence. In this current study, we explored the differences in gut microbiota between patients and normal controls and in patients before and after initiation of methadone treatment program for 12 weeks. Methods: Patients with opioid use disorder between 20 and 65 years were recruited from the methadone maintenance outpatient clinic in 2 medical centers in the Southern Taiwan. Healthy controls without any family history of major psychiatric disorders (schizophrenia, bipolar disorder and major depressive disorder) were recruited from the community. After initial screening, 15 patients with opioid use disorder joined the study for initial evaluation (Week 0), 12 of them completed the 12-week follow-up while receiving methadone treatment and ceased heroin use (Week 12). Fecal samples were collected from the patients at baseline and the end of 12th week. A one-time fecal sample was collected from the healthy controls. The microbiota of fecal samples were investigated using 16S rRNA V3V4 amplicon sequencing, followed by bioinformatics and statistical analyses. Results: We found no significant differences in species diversity in opioid dependent patients between Week 0 and Week 12, nor compared between patients at both points and controls. For beta diversity, using principal component analysis, we found no significant differences between patients at Week 0 and Week 12, however, both patient groups showed significant differences compared to control (P=0.011). Furthermore, the linear discriminant analysis effect size (LEfSe) analysis was used to identify differentially enriched bacteria between opioid use patients and healthy controls. Compared to controls, the relative abundance of Lactobacillaceae Lactobacillus (L. Lactobacillus), Megasphaera Megasphaerahexanoica (M. Megasphaerahexanoica) and Caecibacter Caecibactermassiliensis (C Caecibactermassiliensis) were increased in patients at Week 0, while Coriobacteriales Atopobiaceae (C. Atopobiaceae), Acidaminococcus Acidaminococcusintestini (A. Acidaminococcusintestini) and Tractidigestivibacter Tractidigestivibacterscatoligenes (T. Tractidigestivibacterscatoligenes) were increased in patients at Week 12. Conclusion: In conclusion, we suggest that the gut microbiome community maybe linked to opioid use disorder, such differences may not be altered even after 12-week of cessation of opioid use.

Keywords: opioid use disorder, gut microbiota, methadone treatment, follow up study

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Authors: Miaomiao Zhang, Sabrina Beckmann, Haluk Ertan, Rocky Chau, Mike Manefield

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Phenazines are a large class of nitrogen-containing aromatic heterocyclic compounds, which are almost exclusively produced by bacteria from diverse genera including Pseudomonas and Streptomyces. Phenazine-1-carboxylic acid (PCA) as one of 'core' phenazines are converted from chorismic acid before modified to other phenazine derivatives in different cells. Phenazines have attracted enormous interests because of their multiple roles on biocontrol, bacterial interaction, biofilm formation and fitness of their producers. However, in spite of ecological importance, degradation as a part of phenazines’ fate only have extremely limited attention now. Here, to isolate PCA-degrading bacteria, 200 mg L-1 PCA was supplied as sole carbon, nitrogen and energy source in minimal mineral medium. Quantitative PCR and Reverse-transcript PCR were employed to study abundance and activity of functional gene MFORT 16269 in PCA degradation, respectively. Intermediates and products of PCA degradation were identified with LC-MS/MS. After enrichment and isolation, a PCA-degrading strain was selected from soil and was designated as Rhodanobacter sp. PCA2 based on full 16S rRNA sequencing. As determined by HPLC, strain PCA2 consumed 200 mg L-1 (836 µM) PCA at a rate of 17.4 µM h-1, accompanying with significant cells yield from 1.92 × 105 to 3.11 × 106 cells per mL. Strain PCA2 was capable of degrading other phenazines as well, including phenazine (4.27 µM h-1), pyocyanin (2.72 µM h-1), neutral red (1.30 µM h-1) and 1-hydroxyphenazine (0.55 µM h-1). Moreover, during the incubation, transcript copies of MFORT 16269 gene increased significantly from 2.13 × 106 to 8.82 × 107 copies mL-1, which was 2.77 times faster than that of the corresponding gene copy number (2.20 × 106 to 3.32 × 107 copies mL-1), indicating that MFORT 16269 gene was activated and played roles on PCA degradation. As analyzed by LC-MS/MS, decarboxylation from the ring structure was determined as the first step of PCA degradation, followed by cleavage of nitrogen-containing ring by dioxygenase which catalyzed phenazine to nitrosobenzene. Subsequently, phenylhydroxylamine was detected after incubation for two days and was then transferred to aniline and catechol. Additionally, genomic and proteomic analyses were also carried out for strain PCA2. Overall, the findings presented here showed that a newly isolated strain Rhodanobacter sp. PCA2 was capable of degrading phenazines through decarboxylation and cleavage of nitrogen-containing ring, during which MFORT 16269 gene was activated and played important roles.

Keywords: decarboxylation, MFORT16269 gene, phenazine-1-carboxylic acid degradation, Rhodanobacter sp. PCA2

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Authors: Dalia. G. Aseel, E. E. Hafez, S. M. Hammad

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Viral RNAs of both potato leaf roll virus (PLRV) and potato virus Y (PVY) were extracted from infected potato leaves collected from different Egyptian regions. Differential Display Polymerase Chain Reaction (DD-PCR) using (Endogluconase, β-1,3-glucanases, Chitinase, Peroxidase and Polyphenol oxidase) primers (forward strand) for was performed. The obtained data revealed different banding patterns depending on the viral type and the region of infection. Regarding PLRV, a 58 up regulated and 19 down regulated genes were detected, while, 31 up regulated and 14 down regulated genes were observed in case of PVY. Based on the nucleotide sequencing, variable phylogenetic relationships were reported for the three sequenced genes coding for: Induced stolen tip protein, Disease resistance RPP-like protein and non-specific lipid-transfer protein. In a complementary approach, using the quantitative Real-time PCR, the expressions of PRs genes understudy were estimated in the infected leaves by PLRV and PVY of three potato cultivars (Spunta, Diamont and Cara). The infection with both viruses inhibited the expressions of the five PRs genes. On the contrary, infected leaves by PLRV or PVY elevated the expression of some defense genes. This interaction also may be enhanced and/or inhibited the expression of some genes responsible for the plant defense mechanisms.

Keywords: PLRV, PVY, PR genes, DD-PCR, qRT-PCR, sequencing

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Authors: Young Ah Kim, Hyunsoo Kim, Eun-Jeong Yoon, Young Hee Seo, Kyungwon Lee

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Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli from food animals are considered as a reservoir for transmission of ESBL genes to human. The aim of this study is to assess the prevalence and molecular epidemiology of ESBL-producing E. coli colonization in pigs, farm workers, and farm environments to elucidate the transmission of multidrug-resistant clones from animal to human. Nineteen pig farms were enrolled across the country in Korea from August to December 2017. ESBL-producing E. coli isolates were detected in 190 pigs, 38 farm workers, and 112 sites of farm environments using ChromID ESBL (bioMerieux, Marcy l'Etoile, France), directly (stool or perirectal swab) or after enrichment (sewage). Antimicrobial susceptibility tests were done with disk diffusion methods and blaTEM, blaSHV, and blaCTX-M were detected with PCR and sequencing. The genomes of the four CTX-M-55-producing E. coli isolates from various sources in one farm were entirely sequenced to assess the relatedness of the strains. Whole genome sequencing (WGS) was performed with PacBio RS II system (Pacific Biosciences, Menlo Park, CA, USA). ESBL genotypes were 85 CTX-M-1 group (one CTX-M-3, 23 CTX-M-15, one CTX-M-28, 59 CTX-M-55, one CTX-M-69) and 60 CTX-M-9 group (41 CTX-M-14, one CTX-M-17, one CTX-M-27, 13 CTX-M-65, 4 CTX-M-102) in total 145 isolates. The rectal colonization rates were 53.2% (101/190) in pigs and 39.5% (15/38) in farm workers. In WGS, sequence types (STs) were determined as ST69 (E. coli PJFH115 isolate from a human carrier), ST457 (two E. coli isolates PJFE101 recovered from a fence and PJFA1104 from a pig) and ST5899 (E. coli PJFA173 isolate from the other pig). The four plasmids encoding CTX-M-55 (88,456 to 149, 674 base pair), whether it belonged to IncFIB or IncFIC-IncFIB type, shared IncF backbone furnishing the conjugal elements, suggesting of genes originated from same ancestor. In conclusion, the prevalence of ESBL-producing E. coli in swine farms was surprisingly high, and many of them shared common ESBL genotypes of clinical isolates such as CTX-M-14, 15, and 55 in Korea. It could spread by horizontal transfer between isolates from different reservoirs (human-animal-environment).

Keywords: Escherichia coli, extended-spectrum β-lactamase, prevalence, whole genome sequencing

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Authors: Gianmaria Califano, Júlio Augusto Lucena Maciel, Olfa Zarrouk, Miguel Damasio, Jose Silvestre, Ana Margarida Fortes

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Global warming, with rapid and sudden changes in meteorological conditions, is one of the major constraints to ensuring agricultural and crop resilience in the Mediterranean regions. Several strategies are being adopted to reduce the pressure of drought stress on grapevines at regional and local scales: improvements in the irrigation systems, adoption of interline cover crops, and adaptation of pruning techniques. However, still, more can be achieved if also microbial compartments associated with plants are considered in crop management. It is known that the microbial community change according to several factors such as latitude, plant variety, age, rootstock, soil composition and agricultural management system. Considering the increasing pressure of the biotic and abiotic stresses, it is of utmost necessity to also evaluate the effects of drought on the microbiome associated with the grapevine, which is a commercially important crop worldwide. In this study, we characterize the diversity and the structure of the microbial community under three long-term irrigation levels (100% ETc, 50% ETc and rain-fed) in a drought-tolerant grapevine cultivar present worldwide, Syrah. To avoid the limitations of culture-dependent methods, amplicon sequencing with target primers for bacteria and fungi was applied to the same soil samples. The use of the DNeasy PowerSoil (Qiagen) extraction kit required further optimization with the use of lytic enzymes and heating steps to improve DNA yield and quality systematically across biological treatments. Target regions (16S rRNA and ITS genes) of our samples are being sequenced with Illumina technology. With bioinformatic pipelines, it will be possible to obtain a characterization of the bacterial and fungal diversity, structure and composition. Further, the microbial communities will be assessed for their functional activity, which remains an important metric considering the strong inter-kingdom interactions existing between plants and their associated microbiome. The results of this study will lay the basis for biotechnological applications: in combination with the establishment of a bacterial library, it will be possible to explore the possibility of testing synthetic microbial communities to support plant resistance to water scarcity.

Keywords: microbiome, metabarcoding, soil, vinegrape, syrah, global warming, crop sustainability

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Authors: Barsha Saha, Shouvik Chakravarty, Sukanta Ray, Kshaunish Das, Nidhan K. Biswas, Srikanta Goswami

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Pancreatic Ductal Adenocarcinoma is a well-recognised cause of cancer death with a five-year survival rate of about 9%, and its incidence in India has been found to be increased manifold in recent years. Due to delayed detection, this highly metastatic disease has a poor prognosis. Several molecular alterations happen during the progression of the disease from pre-cancerous conditions, and many such alterations could be investigated for their biomarker potential. MicroRNAs have been shown to be prognostic for PDAC patients in a variety of studies. We hereby used NGS technologies to evaluate the role of small RNA changes during pancreatic cancer development from chronic pancreatitis. Plasma samples were collected from pancreatic cancer patients (n=16), chronic pancreatitis patients (n=8), and also from normal individuals (n=16). Pancreatic tumour tissue (n=5) and adjacent normal tissue samples (n=5) were also collected. Sequencing of small RNAs was carried out after small RNAs were isolated from plasma samples and tissue samples. We find that certain microRNAs are highly deregulated in pancreatic cancer patients in comparison to normal samples. A combinatorial analysis of plasma and tissue microRNAs and subsequent exploration of their targets and altered molecular pathways could not only identify potential biomarkers for disease diagnosis but also help to understand the underlying mechanism.

Keywords: small RNA sequencing, pancreatic cancer, biomarkers, tissue sample

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Authors: Yantao Li, Jun Zheng

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Food poison caused by consumption of contaminated food, especially seafood, is one of most serious public health threats worldwide. Vibrio parahaemolyticus is emerging bacterial pathogen and the leading cause of human gastroenteritis associated with food poison, especially in the southern coastal region of China. To successfully cause disease in host, bacterial pathogens need to overcome the host-derived stresses encountered during infection. One of the toxic chemical species elaborated by the host is nitric oxide (NO). NO is generated by acidified nitrite in the stomach and by enzymes of the inducible NO synthase (iNOS) in the host cell, and is toxic to bacteria. Bacterial pathogens have evolved some mechanisms to battle with this toxic stress. Such mechanisms include genes to sense NO produced from immune system and activate others to detoxify NO toxicity, and genes to repair the damage caused by toxic reactive nitrogen species (RNS) generated during NO toxic stress. However, little is known about the NO resistance in V. parahaemolyticus. In this study, a transposon coupled with next generation sequencing (Tn-seq) technology will be utilized to identify genes for NO resistance in V. parahaemolyticus. Our strategy will include construction the saturating transposon insertion library, transposon library challenging with NO, next generation sequencing (NGS), bioinformatics analysis and verification of the identified genes in vitro and in vivo.

Keywords: vibrio parahaemolyticus, nitric oxide, tn-seq, virulence

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Authors: Francesco Pantano, Marta Fogolari, Michele Iuliani, Sonia Simonetti, Silvia Cavaliere, Marco Russano, Fabrizio Citarella, Bruno Vincenzi, Silvia Angeletti, Giuseppe Tonini

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Background: Although immune checkpoint inhibitors (ICIs) have changed the treatment paradigm of non–small cell lung cancer (NSCLC), these drugs fail to elicit durable responses in the majority of NSCLC patients. The gut microbiota, able to regulate immune responsiveness, is emerging as a promising, modifiable target to improve ICIs response rates. Since the oral microbiome has been demonstrated to be the primary source of bacterial microbiota in the lungs, we investigated its composition as a potential predictive biomarker to identify and select patients who could benefit from immunotherapy. Methods: Thirty-five patients with stage IV squamous and non-squamous cell NSCLC eligible for an anti-PD-1/PD-L1 as monotherapy were enrolled. Saliva samples were collected from patients prior to the start of treatment, bacterial DNA was extracted using the QIAamp® DNA Microbiome Kit (QIAGEN) and the 16S rRNA gene was sequenced on a MiSeq sequencing instrument (Illumina). Results: NSCLC patients were dichotomized as “Responders” (partial or complete response) and “Non-Responders” (progressive disease), after 12 weeks of treatment, based on RECIST criteria. A prevalence of the phylum Candidatus Saccharibacteria was found in the 10 responders compared to non-responders (abundance 5% vs 1% respectively; p-value = 1.46 x 10-7; False Discovery Rate (FDR) = 1.02 x 10-6). Moreover, a higher prevalence of Saccharibacteria Genera Incertae Sedis genus (belonging to the Candidatus Saccharibacteria phylum) was observed in "responders" (p-value = 6.01 x 10-7 and FDR = 2.46 x 10-5). Finally, the patients who benefit from immunotherapy showed a significant abundance of TM7 Phylum Sp Oral Clone FR058 strain, member of Saccharibacteria Genera Incertae Sedis genus (p-value = 6.13 x 10-7 and FDR=7.66 x 10-5). Conclusions: These preliminary results showed a significant association between oral microbiota and ICIs response in NSCLC patients. In particular, the higher prevalence of Candidatus Saccharibacteria phylum and TM7 Phylum Sp Oral Clone FR058 strain in responders suggests their potential immunomodulatory role. The study is still ongoing and updated data will be presented at the congress.

Keywords: oral microbiota, immune checkpoint inhibitors, non-small cell lung cancer, predictive biomarker

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Authors: Sijie Chen, Xiaofang Chen, Juan Wang, Yingying Zhang, Yu Hong, Wanyu Zhuang, Xinxin Huang, Ping Ou, Longsheng Huang

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Purpose: Autistic symptom improvement can be observed in children treated with acupuncture, but the mechanism is still being explored. In the present study, we used scalp acupuncture to treat autism rat model, and then their improvement in the abnormal behaviors and specific mechanisms behind were revealed by detecting animal behaviors, analyzing the RNA sequencing of the prefrontal cortex(PFC), and observing the ultrastructure of PFC neurons under the transmission electron microscope. Methods: On gestational day 12.5, Wistar rats were given valproic acid (VPA) by intraperitoneal injection, and their offspring were considered to be reliable rat models of autism. They were randomized to VPA or VPA-acupuncture group (n=8). Offspring of Wistar pregnant rats that were simultaneously injected with saline were randomly selected as the wild-type group (WT). VPA_acupuncture group rats received acupuncture intervention at 23 days of age for 4 weeks, and the other two groups followed without intervention. After the intervention, all experimental rats underwent behavioral tests. Immediately afterward, they were euthanized by cervical dislocation, and their prefrontal cortex was isolated for RNA sequencing and transmission electron microscopy. Results: The main results are as follows: 1. Animal behavioural tests: VPA group rats showed more anxiety-like behaviour and repetitive, stereotyped behaviour than WT group rats. While VPA group rats showed less spatial exploration ability, activity level, social interaction, and social novelty preference than WT group rats. It was gratifying to observe that acupuncture indeed improved these abnormal behaviors of autism rat model. 2. RNA-sequencing: The three groups of rats differed in the expression and enrichment pathways of multiple genes related to synaptic function, neural signal transduction, and circadian rhythm regulation. Our experiments indicated that acupuncture can alleviate the major symptoms of ASD by improving these neurological abnormalities. 3. Under the transmission electron microscopy, several lysosomes and mitochondrial structural abnormalities were observed in the prefrontal neurons of VPA group rats, which were manifested as atrophy of the mitochondrial membran, blurring or disappearance of the mitochondrial cristae, and even vacuolization. Moreover, the number of synapses and synaptic vesicles was relatively small. Conversely, the mitochondrial structure of rats in the WT group and VPA_acupuncture was normal, and the number of synapses and synaptic vesicles was relatively large. Conclusion: Acupuncture effectively improved the abnormal behaviors of autism rat model and the ultrastructure of the PFC neurons, which might worked by improving their abnormal synaptic function, synaptic plasticity and promoting neuronal signal transduction.

Keywords: autism spectrum disorder, acupuncture, animal behavior, RNA sequencing, transmission electron microscope

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Authors: David Pinzauti, Simon De Jaegher, Maria D'Aguano, Manuele Biazzo

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The COVID-19 pandemic has left an indelible mark on global health, leading to a pressing need for understanding the intricate interactions between the virus and the human microbiome. This study focuses on characterizing the nasal microbiota of patients affected by COVID-19, with a specific emphasis on the comparison with unaffected individuals, to shed light on the crucial role of the microbiome in the development of this viral disease. To achieve this objective, Nanopore technology was employed to analyze the bacterial 16s rRNA full-length gene present in nasal swabs collected in Malta between January 2021 and August 2022. A comprehensive dataset consisting of 268 samples (126 SARS-negative samples and 142 SARS-positive samples) was subjected to a comparative analysis using an in-house, custom pipeline. The findings from this study revealed that individuals affected by COVID-19 possess a nasal microbiota that is significantly less diverse, as evidenced by lower α diversity, and is characterized by distinct microbial communities compared to unaffected individuals. The beta diversity analyses were carried out at different taxonomic resolutions. At the phylum level, Bacteroidota was found to be more prevalent in SARS-negative samples, suggesting a potential decrease during the course of viral infection. At the species level, the identification of several specific biomarkers further underscores the critical role of the nasal microbiota in COVID-19 pathogenesis. Notably, species such as Finegoldia magna, Moraxella catarrhalis, and others exhibited relative abundance in SARS-positive samples, potentially serving as significant indicators of the disease. This study presents valuable insights into the relationship between COVID-19 and the nasal microbiota. The identification of distinct microbial communities and potential biomarkers associated with the disease offers promising avenues for further research and therapeutic interventions aimed at enhancing public health outcomes in the context of COVID-19.

Keywords: COVID-19, nasal microbiota, nanopore technology, 16s rRNA gene, biomarkers

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Authors: Didem Güven, Oytun Hanhan, Elif Ceren Aksoy, Emine Ubay Çokgör

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This paper presents a biological treatability study ofpaintshopwastewaterof a bus factory by an anoxic/aerobic sequencing batch reactor.A lab scale 14L SBR system was implementedto investigate carbon and nitrogen removal performance frompaint shop waste streams combined with domestic and process wastewater of a bus production factory in Istanbul (Turkey).The wastewater collected from decanters of the paint boots and pre-treatmentplant was usedforthefeeding of SBR. The reactor was operated with a total hydraulic retention time of 24 hrs, and a total sludge age of 18.7 days. Initially the efficiency and stability of the reactor were studied when fed with main wastewater stream to simulate the current wastewater treatment plant. Removal efficiency of 57% nitrogen and 90% COD were obtained. Once the paint shop wastewater was introduced to mainstream feeding with a ratio of 1:5, nitrification completely, carbon removal were partially inhibited. SBR system was successful to handle even at very high COD concentrations of paint shop wastewater after feeding of 2 months, with an average effluent COD of 100 mg/L. For the determination of kinetic parameters, respirometric analysis was also conducted with/without paint shop wastewater addition. Model simulation indicated lower maximum specific growth and hydrolysis rates when paint shop wastewater was mixed with the mainstream wastewater of the factory.

Keywords: biological treatability, nitrogen removal, paint shop wastewater, sequencing batch reactor

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Authors: Rozhgar A. Khailany, Mehri Igci, Emine Bayraktar, Sakip Erturhan, Metin Karakok, Ahmet Arslan

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Kidney cancer is the most lethal urological cancer accounting for 3% of adult malignancies. VHL, a tumor-suppressor gene, is best known to be associated with renal cell carcinoma (RCC). The VHL functions as negative regulator of hypoxia inducible factors. Recent sequencing efforts have identified several novel frequent mutations of histone modifying and chromatin remodeling genes in ccRCC (clear cell RCC) including PBRM1 and SETD2. The PBRM1 gene encodes the BAF180 protein, which involved in transcriptional activation and repression of selected genes. SETD2 encodes a histone methyltransferase, which may play a role in suppressing tumor development. In this study, RNAs of 30 paired tumor and normal samples that were grouped according to the types of kidney cancer and clinical characteristics of patients, including gender and average age were examined by RT-PCR, SSCP and sequencing techniques. VHL, PBRM1 and SETD2 expressions were relatively down-regulated. However, statistically no significance was found (Wilcoxon signed rank test, p > 0.05). Interestingly, no mutation was observed on the contrary of previous studies. Understanding the molecular mechanisms involved in the pathogenesis of RCC has aided the development of molecular-targeted drugs for kidney cancer. Further analysis is required to identify the responsible genes rather than VHL, PBRM1 and SETD2 in kidney cancer.

Keywords: kidney cancer, molecular biomarker, expression analysis, mutation screening

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Authors: M. Suwaibah, S. W. Tan, I. Aiini, K. Yusoff, A. R. Omar

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Respiratory diseases are the most important infectious diseases affecting poultry worldwide. One of the avian respiratory virus of global importance causing significant economic losses is Infectious Bronchitis Virus (IBV). The virus causes a wide spectrum disease known as Infectious Bronchitis (IB), affecting not only the respiratory system but also the kidney and the reproductive system, depending on its strain. IB and Newcastle disease are two of the most prevalent diseases affecting poultry in Malaysia. However, a study on the molecular characterization of Malaysian IBV is lacking. In this study, an IBV strain IBS130 which was isolated in 2015 was fully sequenced using next-gene sequencing approach. Sequence analysis of IBS130 based on the complete genome, polyprotein 1ab and S1 genes were compared with other IBV sequences available in Genbank, National Center for Biotechnology Information (NCBI). IBV strain IBS130 is characterised as QX-like strain based on whole genome and S1 gene sequence analysis. Comparisons of the virus with other IBV strains showed that the nucleotide identity ranged from 67% to 99.2%, depending on the region analysed. The similarity in whole genome nucleotide ranging from 84.9% to 90.7% with the least similar was from Singapore strains (84.9%) and highly similar with China QX-like strains. Meanwhile, the similarity in polyprotein 1ab ranging from 85.3% to 89.9% with the least similar to Singapore strains (85.3%) and highly similar with Mass strains from USA.

Keywords: infectious bronchitis virus, phylogenetic analysis, chicken, Malaysia

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Authors: Adriana Sosa, Susana Rincon, Chérif Ben, Diana Cabañas, Juan E. Ruiz, Alejandro Zepeda

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The complex chemical composition (mixtures of ammonium and recalcitrant compounds) of the effluents from the chemical, pharmaceutical and petrochemical industries represents a challenge in their biological treatment. This treatment involves nitrification process that can suffer an inhibition due to the presence of aromatic compounds giving as a result the decrease of the process efficiency. The inhibitory effects on nitrification in the presence of aromatic compounds have already been studied; however a few studies have considered the presence of phenolic compounds in the form of mixtures, which is the form that they are present in real context. For this reason, we realized a kinetic study on the nitrifying process in the presence of different concentrations of a mixture of phenol, o-cresol, m-cresol and p-cresol (0 - 320 mg C/L) in a sequencing batch reactor (SBR). Firstly, the nitrifying process was evaluated in absence of the phenolic mixture (control 1) in a SBR with 2 L working volume and 176 mg/L of nitrogen of microbial protein. Total oxidation of initial ammonium (efficiency; ENH4+ of 100 %) to nitrate (nitrifying yield; YNO3- of 0.95) were obtained with specific rates of ammonium consumption (qN-NH4+) and nitrate production (qN-NO3-) (of 1.11 ± 0.04 h-1 and 0.67 h-1 ± 0.11 respectively. During the phase of acclimation with 40 mg C/L of the phenolic mixture, an inhibitory effect on the nitrifying process was observed, provoking a decrease in ENH4+ and YNO3- (11 and 54 % respectively) as well as in the specific rates (89 y 46 % respectively), being the ammonia oxidizing bacteria (BAO) the most affected. However, in the next cycles without the phenolic mixture (control 2), the nitrifying consortium was able to recover its nitrifying capacity (ENH4+ = 100% and YNO3-=0.98). Afterwards the SBR was fed with 10 mg C/L of the phenolic mixture, obtaining and ENH4+ of 100%, YNO3- and qN-NH4+ 0.62 ± 0.006 and 0.13 ± 0.004 respectively, while the qN-NO3- was 0.49 ± 0.007. Moreover, with the increase of the phenolic concentrations (10-160 mg C/L) and the number of cycles the nitrifying consortium was able to oxidize the ammonia with ENH4+ of 100 % and YNO3- close to 1. However a decrease in the values of the nitrification specific rates and increase in the oxidation in phenolic compounds (70 to 94%) were observed. Finally, in the presence of 320 mg C/L, the nitrifying consortium was able to simultaneously oxidize the ammonia (ENH4+= 100%) and the phenolic mixture (p-cresol>phenol>m-cresol>o-cresol) being the o-cresol the most recalcitrant compound. In all the experiments the use of a SBR allowed a respiratory adaptation of the consortium to oxidize the phenolic mixture achieving greater adaptation of the nitrite-oxidizing bacteria (NOB) than in the ammonia-oxidizing bacteria (AOB).

Keywords: cresol, inhibition, nitrification, phenol, sequencing batch reactor

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Authors: Sugandha Asthana, Geetika Vajpayee, Pratibha Kumari, Shanthy Sundaram

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An economically important disease of wheat in North Western region of India is Karnal Bunt caused by smut fungus Tilletia indica. This fungal pathogen spreads by air, soil and seed borne sporodia at the time of flowering, which ultimately leads to partial bunting of wheat kernels with fishy odor and taste to wheat flour. It has very serious effects due to quarantine measures which have to be applied for grain exports. Chemical fungicides such as mercurial compounds and Propiconazole applied to the control of Karnal bunt have been only partially successful. Considering the harmful effects of chemical fungicides on man as well as environment, many countries are developing biological control as the superior substitute to chemical control. Repeated use of fungicides can be responsible for the development of resistance in fungal pathogens against certain chemical compounds. The present investigation is based on the isolation and evaluation of antifungal properties of some isolated (from natural manure) and commercial bacterial strains against Tilletia indica. Total 23 bacterial isolates were obtained and antagonistic activity of all isolates and commercial bacterial strains (Bacillus subtilis MTCC8601, Bacillus pumilus MTCC 8743, Pseudomonas aeruginosa) were tested against T. indica by dual culture plate assay (pour plate and streak plate). Test for the production of antifungal volatile organic compounds (VOCs) by antagonistic bacteria was done by sealed plate method. Amongst all s1, s3, s5, and B. subtilis showed more than 80% inhibition. Production of extracellular hydrolytic enzymes such as protease, beta 1, 4 glucanase, HCN and ammonia was studied for confirmation of antifungal activity. s1, s3, s5 and B. subtilis were found to be the best for protease activity and s5 and B. subtilis for beta 1, 4 glucanase activity. Bacillus subtilis was significantly effective for HCN whereas s3, s5 and Bacillus subtilis for ammonia production. Isolates were identified as Pseudomonas aeruginosa (s1) and B. licheniformis (s3, s5) by various biochemical assays and confirmed by16s rRNA sequencing. Use of microorganisms or their secretions as biocontrol agents to avoid plant diseases is ecologically safe and may offer long term of protection to crop. The above study reports the promising effects of these strains in better pathogen free crop production and quality maintenance as well as prevention of the excessive use of synthetic fungicides.

Keywords: antagonistic, antifungal, biocontrol, Karnal bunt

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