Genomic Fingerprinting Research Articles

Draft genome sequence of first Vibrio diabolicus in Mexico strain InDRE-D1-M1, an emergent threat.

The complete genome of the marine environmental bacterium Vibrio diabolicus isolated from raw shrimp in the city of Guadalajara in the state of Jalisco in Mexico is reported here. Vibrio spp. it was isolated and identified using standard microbiological and molecular techniques. Whole genome sequencing was performed using the Miseq system (Illumina, USA). The Multi Locus Sequence Typing profile of the isolated Vibrio bacteria coincided only with 4 specific loci (atpA, gyrB, pyrH and recA) and with a total coverage of the species belonging to Vibrio spp. Analysis of the complete genome of the Vibrio isolate and other closely related species, using the genomic fingerprints of the Virtual Analysis Method for PHylogenomic fingerprint estimation (VAMPHyRe) software, revealed the clustering of this species among the clade Vibrio diabolicus. The antibiogram revealed that this strain of Vibrio diabolicus is resistant to ampicillin, which is consistent with the bioinformatic finding of the β-lactamase enzyme that hydrolyzes carbenicillin class A. This study demonstrated that the environmental marine bacterium Vibrio diabolicus contains carrier genes associated with pathogenicity and ecological function, which could represent a threat to public health.

Comparative proteomics and secretomics revealed virulence, and coresistance-related factors in non O1/O139 Vibrio cholerae recovered from 16 species of consumable aquatic animals

Vibrio cholerae can cause pandemic cholera in humans. The bacterium resides in aquatic environments worldwide. Identification of risk factors of V. cholerae in aquatic products is imperative for assuming food safety. In this study, we determined virulence-associated genes, cross-resistance between antibiotics and heavy metals, and genome fingerprinting profiles of non O1/O139 V. cholerae isolates (n = 20) recovered from 16 species of consumable aquatic animals. Secretomes and proteomes of V. cholerae with distinct genotypes and phenotypes were obtained by using two-dimensional gel electrophoresis (2D-GE) and/or liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques. Comparative secretomic analysis revealed 4 common and 45 differential extracellular proteins among 20 V. cholerae strains, including 13 virulence- and 8 resistance-associated proteins. A total of 21,972 intracellular proteins were identified, and comparative proteomic analysis revealed 215 common and 913 differential intracellular proteins, including 22 virulence- and 8 resistance-associated proteins. Additionally, different secretomes and proteomes were observed between V. cholerae isolates of fish and shellfish origins. A number of novel proteins with unknown function and strain-specific proteins were also discovered in the V. cholerae isolates. SignificanceV. cholerae can cause pandemic cholera in humans. The bacterium is distributed in aquatic environments worldwide. Identification of risk factors of V. cholerae in aquatic products is imperative for assuming food safety. Non-O1/O139 V. cholerae has been reported to cause sporadic cholera-like diarrhea and bacteremia diseases, which indicates virulence factors rather than the major cholera toxin (CT) exist. This study for the first time investigated proteomes and secretomes of non-O1/O139 V. cholerae originating from aquatic animals. This resulted in the identification of a number of virulence and coresistance-related factors, as well as novel proteins and strain-specific proteins in V. cholerae isolates recovered from 16 species of consumable aquatic animals. These results fill gaps for better understanding of pathogenesis and resistance of V. cholerae, and also support the increasing need for novel diagnosis and vaccine targets against the leading waterborne pathogen worldwide.

Identification of Putative Gene-Target Modulators of Radiosensitivity in Bladder Cancer Cell Lines (BlaCCL)

We aimed to computationally derive potential modulators of radiosensitivity in an expanded panel of BlaCCL based upon whole-transcriptomic RNA-seq analysis integrated with functional enrichment and interaction analysis.Twenty established BlaCCL, verified by genomic fingerprinting, were profiled with RNA-seq with raw counts normalized via log (TPM+1). We performed colony forming assays on all cell lines in triplicate with radiosensitivity measured via area-under-the-cell-survival curve (AUC) at varying doses of radiation (0-8 Gy). Gene signatures of radiosensitivity and resistance were generated via unsupervised matrix decomposition of the RNA-seq dataset using statistical software run at k = 13 for 10,000 iterations correlated with AUC via Spearman's rho. Putative modulators of radiosensitivity were identified as hub proteins in protein-protein interaction networks (via String-DB) of the native gene signatures and of derived sets of upstream kinases with hypergeometric P ≤ 0.05.RNA-seq analysis identified 18,634 genes for the panel of BlaCCL, of which updated satisfactory colony forming results were obtained for 18 cell lines with AUC (range = 3.40-5.94). Generated signatures of radiosensitivity and radioresistance based on baseline gene expression consisted of 2,192 and 282 unique genes respectively. Our radiosensitivity signature contains 1,543 unique genes compared to previously described signatures of response in patients undergoing definitive chemoradiation, i.e., signatures of T-cell inflamed tumors and interferon-gamma response. Network analyses of the gene signatures reveal hub proteins that connect networks of proteins related to inflammatory response, oxidative stress, DNA-damage repair, cell cycle and others. These include TNF, VNN1, ESR1, H2AC14, RXRG, FPR2, ADCY1, and S1PR4. Kinase enrichment analysis revealed hub proteins previously described in the radiation response including ATM, ATR, CHEK2, AKT1, SRC, JAK2, and ERK-signaling among others.Biologically plausible modulators of radiosensitivity and resistance can be derived from transcriptomic data paired with phenotypic data and functional enrichment analysis. Further mechanistic studies are necessary to confirm potential modulators of the radiation response which may eventually lead to novel radiosensitizing drugs to improve response to chemoradiation in bladder cancer patients.

First Experimental Evidence for the Presence of Potentially Virulent Klebsiella oxytoca in 14 Species of Commonly Consumed Aquatic Animals, and Phenotyping and Genotyping of K. oxytoca Isolates.

Klebsiella oxytoca is a recently emerging pathogen that can cause necrotizing enterocolitis, hemorrhagic colitis, sepsis-associated purpura fulminans, and infective endocarditis in humans. The bacterium is ubiquitous in water and soil environments. Nevertheless, current literature on K. oxytoca in aquatic products is rare. In this study, we surveyed K. oxytoca contamination in 41 species of consumable aquatic animals sold in July, August, and September of 2018 and 2019 in Shanghai, China, 40 of which had no history of carrying this bacterium. K. oxytoca was for the first time isolated from 14 species with high abundance in benthic animals. None of the K. oxytoca isolates (n = 125) harbored toxin genes mviM, tisB, and yqgB. However, a high occurrence of virulence-associated genes was observed, including brkB (73.6%), cdcB (66.4%), pduV (64.8%), and virk (63.2%). Resistance to sulphamethoxazole-trimethoprim (56.0%) was the most predominant among the isolates, followed by chloramphenicol (6.4%), tetracycline (5.6%), and kanamycin (3.2%). Approximately 8.0% of the isolates displayed multidrug resistant phenotypes. Meanwhile, high percentages of the isolates tolerated the heavy metals Cu2+ (84.8%), Pb2+ (80.8%), Cr3+ (66.4%), Zn2+ (66.4%), and Hg2+ (49.6%). Different virulence and resistance profiles were observed among K. oxytoca isolates in 3 types and 14 species of aquatic animals. The ERIC-PCR-based genome fingerprinting of the 125 K. oxytoca isolates revealed 108 ERIC genotypes with 79 singletons, which demonstrated the genetic diversity of the isolates. The results of this study fill gaps for policy and research in the risk assessment of K. oxytoca in consumable aquatic animals.

Viruses in colorectal cancer.

Increasing evidence suggests that microorganisms might represent at least highly interesting cofactors in colorectal cancer (CRC) oncogenesis and progression. Still, associated mechanisms, specifically in colonocytes and their microenvironmental interactions, are still poorly understood. Although, currently, at least seven viruses are being recognized as human carcinogens, only three of these – Epstein–Barr virus (EBV), human papillomavirus (HPV) and John Cunningham virus (JCV) – have been described, with varying levels of evidence, in CRC. In addition, cytomegalovirus (CMV) has been associated with CRC in some publications, albeit not being a fully acknowledged oncovirus. Moreover, recent microbiome studies set increasing grounds for new hypotheses on bacteriophages as interesting additional modulators in CRC carcinogenesis and progression. The present Review summarizes how particular groups of viruses, including bacteriophages, affect cells and the cellular and microbial microenvironment, thereby putatively contributing to foster CRC. This could be achieved, for example, by promoting several processes – such as DNA damage, chromosomal instability, or molecular aspects of cell proliferation, CRC progression and metastasis – not necessarily by direct infection of epithelial cells only, but also by interaction with the microenvironment of infected cells. In this context, there are striking common features of EBV, CMV, HPV and JCV that are able to promote oncogenesis, in terms of establishing latent infections and affecting p53‐/pRb‐driven, epithelial–mesenchymal transition (EMT)‐/EGFR‐associated and especially Wnt/β‐catenin‐driven pathways. We speculate that, at least in part, such viral impacts on particular pathways might be reflected in lasting (e.g. mutational or further genomic) fingerprints of viruses in cells. Also, the complex interplay between several species within the intestinal microbiome, involving a direct or indirect impact on colorectal and microenvironmental cells but also between, for example, phages and bacterial and viral pathogens, and further novel species certainly might, in part, explain ongoing difficulties to establish unequivocal monocausal links between specific viral infections and CRC.

Genetic variations analysis for complex brain disease diagnosis using machine learning techniques: opportunities and hurdles.

Background and ObjectivesThis paper presents an in-depth review of the state-of-the-art genetic variations analysis to discover complex genes associated with the brain’s genetic disorders. We first introduce the genetic analysis of complex brain diseases, genetic variation, and DNA microarrays. Then, the review focuses on available machine learning methods used for complex brain disease classification. Therein, we discuss the various datasets, preprocessing, feature selection and extraction, and classification strategies. In particular, we concentrate on studying single nucleotide polymorphisms (SNP) that support the highest resolution for genomic fingerprinting for tracking disease genes. Subsequently, the study provides an overview of the applications for some specific diseases, including autism spectrum disorder, brain cancer, and Alzheimer’s disease (AD). The study argues that despite the significant recent developments in the analysis and treatment of genetic disorders, there are considerable challenges to elucidate causative mutations, especially from the viewpoint of implementing genetic analysis in clinical practice. The review finally provides a critical discussion on the applicability of genetic variations analysis for complex brain disease identification highlighting the future challenges.MethodsWe used a methodology for literature surveys to obtain data from academic databases. Criteria were defined for inclusion and exclusion. The selection of articles was followed by three stages. In addition, the principal methods for machine learning to classify the disease were presented in each stage in more detail.ResultsIt was revealed that machine learning based on SNP was widely utilized to solve problems of genetic variation for complex diseases related to genes.ConclusionsDespite significant developments in genetic diseases in the past two decades of the diagnosis and treatment, there is still a large percentage in which the causative mutation cannot be determined, and a final genetic diagnosis remains elusive. So, we need to detect the variations of the genes related to brain disorders in the early disease stages.

BACTERIAL DIVERSITY WITH PLANT GROWTH-PROMOTING POTENTIAL ISOLATED FROM Agave americana RHIZOSPHERE

ABSTRACT
 Objective: Study the diversity of cultivable rhizospheric bacteria associated to Agave americana, and select native strains with potential as plant growth-promoting bacteria (PGPB).
 Design/methodology/approach: The isolated bacteria were phenotypically characterized. The genetic diversity and identity of the strains were revealed by genomic fingerprints and by sequencing of 16S rRNA gene. Plant growth promoting ability and plant inoculation assays were evaluated to know the potential as PGPB.
 Results: A total of 235 strains were isolated from A. americana rhizosphere and were classified within of 10 different bacterial genera. Rhizobium, Pseudomonas, Acinetobacter had high potential as PGPB.
 Study limitations/implications Cultivable approach was used to study rhizobacteria. A metagenomic study could expand the knowledge about the structure and diversity of bacterial community associated to A. americana.
 Findings/conclusions Rhizosphere bacteria have potential use as biofertilizer for the cultivation and propagation of A. americana and other agave species.

Genetic and physiological diversity of white Spanish broom (Chamaecytisus albus) endophytes.

Chamaecytisus albus (Spanish broom) is a legume shrub that can be found in only one natural locality in Poland. This specimen is critically endangered; therefore, different actions focusing on protection of this plant in the natural habitat are undertaken, and one of them involves studies of the population of Chamaecytisus albus bacterial endophytes, which in the future could be used as bioprotectants and/or biofertilizers. A collection of 94 isolates was obtained from Spanish broom nodules, and the physiological and genetic diversity of these strains was studied. A few potentially beneficial traits were detected, i.e. secretion of cellulases (66 isolates), production of siderophores (60 isolates), phosphate solubilization (25 isolates), and production of IAA (58 isolates), indole (16 isolates), or HCN (3 isolates). Twenty-nine of the 94 tested isolates were able to induce the development of root nodules in plants grown in vitro and can therefore be assumed as Chamaecytisus albus symbionts. Genome fingerprinting by BOX-PCR, as well as gyrB and nodZ gene sequencing revealed a great genetic diversity of specimens in the collection. The symbiotic isolates were classified in different clades, suggesting they could belong to different species, however, most of them revealed sequence similarity to Bradyrhizobium genus.

Whole Genome Sequencing of Pediatric Klebsiella pneumoniae Strains Reveals Important Insights Into Their Virulence-Associated Traits.

Klebsiella pneumoniae is recognized as a common cause of nosocomial infections and outbreaks causing pneumonia, septicemia, and urinary tract infections. This opportunistic bacterium shows an increasing acquisition of antibiotic-resistance genes, which complicates treatment of infections. Hence, fast reliable strain typing methods are paramount for the study of this opportunistic pathogen’s multi-drug resistance genetic profiles. In this study, thirty-eight strains of K. pneumoniae isolated from the blood of pediatric patients were characterized by whole-genome sequencing and genomic clustering methods. Genes encoding β-lactamase were found in all the bacterial isolates, among which the blaSHV variant was the most prevalent (53%). Moreover, genes encoding virulence factors such as fimbriae, capsule, outer membrane proteins, T4SS and siderophores were investigated. Additionally, a multi-locus sequence typing (MLST) analysis revealed 24 distinct sequence types identified within the isolates, among which the most frequently represented were ST76 (16%) and ST70 (11%). Based on LPS structure, serotypes O1 and O3 were the most prevalent, accounting for approximately 63% of all infections. The virulence capsular types K10, K136, and K2 were present in 16, 13, and 8% of the isolates, respectively. Phylogenomic analysis based on virtual genome fingerprints correlated with the MLST data. The phylogenomic reconstruction also denoted association between strains with a higher abundance of virulence genes and virulent serotypes compared to strains that do not possess these traits. This study highlights the value of whole-genomic sequencing in the surveillance of virulence attributes among clinical K. pneumoniae strains.

Molecular diversity and phylogenetic analysis of Azerbaijan oaks (Quercus spp.) revealed by RAPD markers

Although Azerbaijan is a center of natural distribution and diversity of genus Quercus around the world, less published data can be found about PCR-based genetic diversity in prevalent oak accessions. The present study aims at finding molecular variation among the Quercus species using RAPD markers. In this study, genomic fingerprinting in five Azerbaijan oak species (Quercus spp.) was carried out using 10 RAPD markers. The result showed that a total of 113 DNA fragments were amplified, of those, 91 bands were polymorphic. The highest PIC, EMR and MI values was observed in the primers OPD-05, OPB-01 and OPB-03. The genetic similarity among the genotypes examined ranged from 0.333 to 0.818 with an average of 0.498. Species-specific DNA fragments were found in all species examined. Based on the cluster analysis using UPGMA method and Jaccard’s similarity index, all the examined oak species fell into three main groups and principal coordinate analysis (PCoA) supported the clustering result. High genetic diversity was found in the tested genetic germplasm of oak species. The results of this study would be very useful for oak improvement program.

Culture-Independent and Culture-Dependent Characterization of the Black Soldier Fly Gut Microbiome Reveals a Large Proportion of Culturable Bacteria with Potential for Industrial Applications.

Black soldier fly larvae (BSFL) are fast-growing, resilient insects that can break down a variety of organic substrates and convert them into valuable proteins and lipids for applications in the feed industry. Decomposition is mediated by an abundant and versatile gut microbiome, which has been studied for more than a decade. However, little is known about the phylogeny, properties and functions of bacterial isolates from the BSFL gut. We therefore characterized the BSFL gut microbiome in detail, evaluating bacterial diversity by culture-dependent methods and amplicon sequencing of the 16S rRNA gene. Redundant strains were identified by genomic fingerprinting and 105 non-redundant isolates were then tested for their ability to inhibit pathogens. We cultivated representatives of 26 genera, covering 47% of the families and 33% of the genera detected by amplicon sequencing. Among these isolates, we found several representatives of the most abundant genera: Morganella, Enterococcus, Proteus and Providencia. We also isolated diverse members of the less-abundant phylum Actinobacteria, and a novel genus of the order Clostridiales. We found that 15 of the isolates inhibited at least one of the tested pathogens, suggesting a role in helping to prevent colonization by pathogens in the gut. The resulting culture collection of unique BSFL gut bacteria provides a promising resource for multiple industrial applications.

Combination of high-throughput microfluidics and FACS technologies to leverage the numbers game in natural product discovery.

SummaryHigh‐throughput platforms facilitating screening campaigns of environmental samples are needed to discover new products of natural origin counteracting the spreading of antimicrobial resistances constantly threatening human and agricultural health. We applied a combination of droplet microfluidics and fluorescence‐activated cell sorting (FACS)‐based technologies to access and assess a microbial environmental sample. The cultivation performance of our microfluidics workflow was evaluated in respect to the utilized cultivation media by Illumina amplicon sequencing of a pool of millions of droplets, respectively. This enabled the rational selection of a growth medium supporting the isolation of microbial diversity from soil (five phyla affiliated to 57 genera) including a member of the acidobacterial subgroup 1 (genus Edaphobacter). In a second phase, the entire diversity covered by 1071 cultures was used for an arrayed bioprospecting campaign, resulting in > 6000 extracts tested against human pathogens and agricultural pests. After redundancy curation by using a combinatorial chemical and genomic fingerprinting approach, we assigned the causative agents present in the extracts. Utilizing UHPLC‐QTOF‐MS/MS‐guided fractionation and microplate‐based screening assays in combination with molecular networking the production of bioactive ionophorous macrotetrolides, phospholipids, the cyclic lipopetides massetolides E, F, H and serratamolide A and many derivatives thereof was shown.

Molecular characterization and aggressiveness of the Ralstonia solanacearum species complex from Eucalyptus spp. in Brazil

AbstractBacterial wilt, one of the world's most destructive diseases in many crops, including eucalypt, is caused by four distinct phylogenetic lineages of the Ralstonia solanacearum species complex, recently classified in three distinct species: R. solanacearum (phylotype II), R. pseudosolanacearum (phylotype I and III) and R. syzygii (phylotype IV). In this study, we characterized 93 Ralstonia isolates obtained from eucalypt grown in different Brazilian regions using phylotype and sequevar designations and genomic fingerprinting with BOX‐PCR. In addition, we evaluated the aggressiveness of a select group of isolates in two eucalypt clones differing in resistance. We found that 89 isolates belong to R. solanacearum (phylotype II) and four to R. pseudosolanacearum (phylotype I). Unlike R. pseudosolanacearum, R. solanacearum was phylogenetically diverse and no correlation was found between sequevar and geographic origin. Most isolates grouped with reference isolates of phylotype IIA sequevar 41, whereas a few others clustered in phylotype IIB, mainly sequevar 4NPB, which is an emergent variant described affecting eucalypt for the first time. Isolates of R. solanacearum phylotype IIB were less aggressive to clone CLR371 (susceptible) whereas the R. pseudosolanacearum isolate tested was the only one pathogenic to the CLR172 (resistant) clone. Isolate aggressiveness varied between the eucalypt clones tested. The results of this study reinforce the importance of conducting molecular and aggressiveness characterization of the pathogen population to develop management strategies aimed at the deployment of host resistance in eucalypt breeding programmes.

A pilot study revealing host-associated genetic signatures for source attribution of sporadic Campylobacter jejuni infection in Egypt.

Campylobacter jejuni (C. jejuni), is considered among the most common bacterial causes of human bacterial gastroenteritis worldwide. The epidemiology and the transmission dynamics of campylobacteriosis in Egypt remain poorly defined due to the limited use of high-resolution typing methods. In this pilot study, we evaluated the discriminatory power of multiple typing 'gene-by-gene based' techniques to characterize C. jejuni obtained from different sources and estimate the relative contribution of different potential sources of C. jejuni infection in Egypt. Whole genome sequencing (WGS) was performed on 90 C. jejuni isolates recovered from clinical samples, retail chicken, and dairy products in Egypt from 2017 to 2018. Comparative genomic analysis was performed using conventional seven-locus multilocus sequence typing (MLST), ribosomal MLST (rMLST), core genome MLST (cgMLST), allelic variation in 15 host-segregating (HS) markers, and comparative genomic fingerprinting (CGF40). The probabilistic source attribution was performed via STRUCTURE software using MLST, CGF40, cgMLST and allelic variation in HS markers. Comparison of the discriminatory power of the aforementioned genotyping methods revealed cgMLST to be the most discriminative method, followed by HS markers. The source attribution analysis showed the role of retail chicken as a source of infection among clinical cases in Egypt when HS and cgMLST were used (64.2% and 52.3% of clinical isolates were assigned to this source, respectively). Interestingly, the cattle reservoir was also identified as a contributor to C. jejuni infection in Egypt; 35.8% and 47.7% of clinical isolates were assigned to this source by HS and cgMLST, respectively. Here, we provided evidence of the importance of using WGS typing methods to facilitate source tracking of C. jejuni. Our findings suggest the importance of non-poultry sources, together with the previously reported role of retail chicken in human campylobacteriosis in Egypt that can provide insights to inform national control measures.

First Experimental Evidence for the Presence of Potentially Toxic Vibrio cholerae in Snails, and Virulence, Cross-Resistance and Genetic Diversity of the Bacterium in 36 Species of Aquatic Food Animals.

Vibrio cholerae is the most common waterborne pathogen that can cause pandemic cholera in humans. Continuous monitoring of V. cholerae contamination in aquatic products is crucial for assuring food safety. In this study, we determined the virulence, cross-resistance between antibiotics and heavy metals, and genetic diversity of V. cholerae isolates from 36 species of aquatic food animals, nearly two-thirds of which have not been previously detected. None of the V. cholerae isolates (n = 203) harbored the cholera toxin genes ctxAB (0.0%). However, isolates carrying virulence genes tcpA (0.98%), ace (0.5%), and zot (0.5%) were discovered, which originated from the snail Cipangopaludina chinensis. High occurrences were observed for virulence-associated genes, including hapA (73.4%), rtxCABD (68.0–41.9%), tlh (54.2%), and hlyA (37.9%). Resistance to moxfloxacin (74.9%) was most predominant resistance among the isolates, followed by ampicillin (59.1%) and rifampicin (32.5%). Approximately 58.6% of the isolates displayed multidrug resistant phenotypes. Meanwhile, high percentages of the isolates tolerated the heavy metals Hg2+ (67.0%), Pb2+ (57.6%), and Zn2+ (57.6%). Distinct virulence and cross-resistance profiles were discovered among the V. cholerae isolates in 13 species of aquatic food animals. The ERIC-PCR-based genome fingerprinting of the 203 V. cholerae isolates revealed 170 ERIC-genotypes, which demonstrated considerable genomic variation among the isolates. Overall, the results of this study provide useful data to fill gaps for policy and research related to the risk assessment of V. cholerae contamination in aquatic products.

A Review: Genetics Algorithms in Bioinformatics Tools

Bioinformaticians study biological questions by analyzing molecular data with various programs and tools. Today, bioinformatics is used in large number of fields such as microbial genome applications, biotechnology, waste cleanup, Gene therapy, fingerprint and eye detection. The field of bioinformatics, is one of the most prominent areas that our need is increasing, and the demand for it is increasing day by day. Where dealing with this vital and biological information using advanced computer technologies to generate useful information and new discoveries. For this reason, vital bioinformatics is one of the domains that combines both interested and programming at the same time. It provides you with resources for self-learning, the most important information in the field of vital information, and asked questions of those wishing to learn this field. The term bioinformatics was first used in 1968 by Margret Dayhoff, which is a pioneer in this field, but its definition appeared for the first time in 1978. This science arose and developed in conjunction with the emergence and development of computers. It is also referred to as "computational biology."

Evaluation of the production of antifungal metabolites against Colletotrichum gloeosporioides in Streptomyces 5.1 by random mutagenesis

Streptomyces 5.1 is a bacterium isolated from rice soils in the south of the Tolima department (Colombia). This microorganism is characterized by its antagonistic activity against rubber tree phytopathogens like Colletotrichum gloeosporioides, the causal agent of leaf anthracnose. The antifungal activity of this Streptomyces isolate has been associated with secondary metabolites production. However, the identity of those metabolites is unknown because its purification and identification have not been possible through classic chemical studies. Therefore, aiming to contribute in the study of the secondary metabolites produced by 5.1 from a molecular approach, this research seeks to identify -preliminarily- the genomic fingerprint changes associated with the production of antifungal secondary metabolites produced by Streptomyces 5.1 through the evaluation of a mutant library of 5.1 obtained by random mutagenesis using controlled ultraviolet light exposure. The antifungal activity of obtained mutants was evaluated using Colletotrichum gloeosporioides (C1) fungus as a biosensor, isolated by the Biotechnology Institute of Universidad Nacional de Colombia. In this way, the library of mutants of 5.1, initially formed by 300 isolations, was classified into two phenotypic groups of interest: enhanced mutants (1 isolate) and null mutants (11 isolates) of secondary metabolites. The genomic changes in both groups were analyzed by obtaining the genomic profile of the isolates using Repetitive Extragenic Palindromic (Rep-PCR). The obtained profiles evidenced the presence of one additional band in the enhanced mutant, and the absence of a specific band in the non-producing mutants, both in comparison with the original strain. These bands are proposed for a future sequencing study which will define their role in the production process of metabolites with antifungal activity in Streptomyces 5.1.

Diversity and Virulence of Streptomyces spp. Causing Potato Common Scab in Prince Edward Island, Canada.

Common scab (CS) is a potato disease that significantly decreases the market value of potato tubers after the development of necrotic lesions on their surface. Streptomyces scabiei is the main causal agent of CS; however, other closely related species, including S. acidiscabies and S. turgidiscabies, have also been shown to cause the disease. In this study, we characterized the genetic and phenotypic diversity of Streptomyces spp. causing CS in Prince Edward Island, the main potato-producing province in Canada. Two hundred and ninety-six pathogenic Streptomyces spp. isolates were retrieved from diseased tubers harvested from six fields located across a longitudinal geographical gradient. Genome fingerprinting analyses using repetitive elements PCR (ERIC- and BOX-PCR) revealed 14 distinct genetic groups. Thirteen groups were taxonomically affiliated with S. scabiei, whereas the fourteenth group was affiliated with S. acidiscabies. Their geographical distribution was characterized and revealed that on average between six and eight different genetic groups were detected per field, with variable abundance. Virulence assays showed strong differences in virulence between the genetic groups, ranging from low to highly virulent. Interestingly, pathogenic Streptomyces spp. populations in each field seem to be dominated by the most virulent genetic groups. The results obtained will contribute to better understanding of the population dynamic of pathogenic Streptomyces spp. causing CS of potato and promoting the development of more efficient detection and intervention tools to manage this important potato disease.

Microbiological and physicochemical characterisation of green table olives of Halkidiki and Conservolea varieties processed by the Spanish method on industrial scale

AbstractThe microbiological and physicochemical changes of industrially fermented Halkidiki and Conservolea green table olives were determined. Samples were analysed to monitor the population of lactic acid bacteria (LAB), yeasts and Enterobacteriaceae, together with changes in pH, acidity, salinity, colour, lactic acid, acetic acid and ethanol. LAB and yeast species diversity was evaluated at the beginning (1 day), middle (75 days) and final (135 days) stages of fermentation by RAPD‐PCR genomic fingerprinting. Results revealed vigorous lactic acid processes as indicated by the dominance of LAB over yeasts. No Enterobacteriaceae could be detected after 30 days. Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) dominated in the beginning of fermentation in both varieties. In the end, Lactiplantibacillus pentosus (formerly Lactobacillus pentosus) and Pediococcus ethanolidurans prevailed in Halkidiki and Conservolea varieties, respectively. As for yeasts, Kluyveromyces lactis/marxianus and Pichia manshurica prevailed at the onset of fermentation in Halkidiki and Conservolea varieties, whereas in the end Pichia membranifaciens dominated in both varieties.

Survival of escherichia coli in Water Microcosm Study and Rethinking its Use as Indicator

We investigated the fate of Escherichia coli in natural waters, addressing survival, viable but non-culturable (VBNC) state, changes in phenotype and genomic diversification under laboratory microcosm environment. Five E. coli isolates (three marine, one clinical and one laboratory strain) were inoculated in microcosms of sterile distilled water, as well as, water collected from river, pond, sea, and estuary. A viable count was measured every week for up to 33 weeks. Strains which undergone VBNC state in microcosms were resuscitated. The revived isolates were tested to determine changes in antibiotic susceptibility, thermotolerance and genetic fingerprinting. We found extended persistence of five E. coli strains in water microcosms at room temperature for durations ranging from four to 33 weeks. Even in distilled water, some isolates persisted up to seven weeks; proving E. coli have excellent tenacity to survive. At VBNC state in microcosms, bacteria were small coccoid in shape and formed biofilms in most cases. Resuscitated laboratory strains showed changes in antibiotic susceptibility and thermotolerance compared to the original counterpart. However, the revived marine isolates showed very little to no difference in those tests. Resuscitation media could not revived the pathogenic isolates. The REP- and BOX-PCR genomic fingerprinting also showed some genetic diversification among resuscitated strains during the extended survival and VBNC state. The overall observation supports for the E. coli to survive in natural waters and to enter into VBNC state. It ultimately proves that their presence in water does not necessarily indicate faecal contamination but still beyond acceptance.