Bacteria, yeasts, and fungi associated with larval food of Brazilian native stingless bees.

Stingless bees are a diverse group with a relevant role in pollinating native species. Its diet is rich in carbohydrates and proteins, by collecting pollen and nectar supplies the development of its offspring. Fermentation of these products is associated with microorganisms in the colony. However, the composition of microorganisms that comprise this microbiome and its fundamental role in colony development is still unclear. To characterize the colonizing microorganisms of larval food in the brood cells of stingless bees Frieseomelitta varia, Melipona quadrifasciata, Melipona scutellaris, and Tetragonisca angustula, we have utilized molecular and culture-based techniques. Bacteria of the phyla Firmicutes, Proteobacteria, Actinobacteria, and fungi of the phyla Ascomycota, Basidiomycota, Mucoromycota, and Mortierellomycota were found. Diversity analysis showed that F. varia had a greater diversity of bacteria in its microbiota, and T. angustula had a greater diversity of fungi. The isolation technique allowed the identification of 189 bacteria and 75 fungi. In summary, this research showed bacteria and fungi associated with the species F. varia, M. quadrifasciata, M. scutellaris, and T. angustula, which may play an essential role in the survival of these organisms. Besides that, a biobank with bacteria and fungus isolates from LF of Brazilian stingless bees was created, which can be used for different studies and the prospection of biotechnology compounds.

Multiplex PCR assay for correct identification of the fish pathogenic species of Edwardsiella genus reveals the presence of E. anguillarum in South America in strains previously characterized as E. tarda.

AIMS: Develop a species-specific multiplex PCR to correctly identify Edwardsiella species in routine diagnostic for fish bacterial diseases. METHODS AND RESULTS: The genomes of 62 Edwardsiella spp. isolates available from the National Center for Biotechnology Information (NCBI) database were subjected to taxonomic and pan-genomic analyses to identify unique regions that could be exploited by species-specific PCR. The designed primers were tested against isolated Edwardsiella spp. strains, revealing errors in commercial biochemical tests for bacterial classification regarding Edwardsiella species. CONCLUSION: Some of the genomes of Edwardsiella spp. in the NCBI platform were incorrectly classified, which can lead to errors in some research. A functional mPCR was developed to differentiate between phenotypically and genetically ambiguous Edwardsiella, with which, we detected the presence of Edwardsiella anguillarum affecting fish in Brazil. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that the misclassification of Edwardsiella spp in Brazil concealed the presence of E. anguillarum in South America. Also, this review of the taxonomic classification of the Edwardsiella genus is a contribution to the field to help researchers with their sequencing and identification of genomes, showing some misclassifications in online databases that must be corrected, as well as developing an easy assay to characterize Edwardsiella species in an end-point mPCR.

Exoproteome and secretome derived broad spectrum novel drug and vaccine candidates in Vibrio cholerae targeted by Piper betel derived compounds.

Vibrio cholerae is the causal organism of the cholera epidemic, which is mostly prevalent in developing and underdeveloped countries. However, incidences of cholera in developed countries are also alarming. Because of the emergence of new drug-resistant strains, even though several generic drugs and vaccines have been developed over time, Vibrio infections remain a global health problem that appeals for the development of novel drugs and vaccines against the pathogen. Here, applying comparative proteomic and reverse vaccinology approaches to the exoproteome and secretome of the pathogen, we have identified three candidate targets (ompU, uppP and yajC) for most of the pathogenic Vibrio strains. Two targets (uppP and yajC) are novel to Vibrio, and two targets (uppP and ompU) can be used to develop both drugs and vaccines (dual targets) against broad spectrum Vibrio serotypes. Using our novel computational approach, we have identified three peptide vaccine candidates that have high potential to induce both B- and T-cell-mediated immune responses from our identified two dual targets. These two targets were modeled and subjected to virtual screening against natural compounds derived from Piper betel. Seven compounds were identified first time from Piper betel to be highly effective to render the function of these targets to identify them as emerging potential drugs against Vibrio. Our preliminary validation suggests that these identified peptide vaccines and betel compounds are highly effective against Vibrio cholerae. Currently we are exhaustively validating these targets, candidate peptide vaccines, and betel derived lead compounds against a number of Vibrio species.

Conserved host-pathogen PPIs. Globally conserved inter-species bacterial PPIs based conserved host-pathogen interactome derived novel target in C. pseudotuberculosis, C. diphtheriae, M. tuberculosis, C. ulcerans, Y. pestis, and E. coli targeted by Piper betel compounds.

Although attempts have been made to unveil protein-protein and host-pathogen interactions based on molecular insights of important biological events and pathogenesis in various organisms, these efforts have not yet been reported in Corynebacterium pseudotuberculosis (Cp), the causative agent of Caseous Lymphadenitis (CLA). In this study, we used computational approaches to develop common conserved intra-species protein-protein interaction (PPI) networks first time for four Cp strains (Cp FRC41, Cp 316, Cp 3/99-5, and Cp P54B96) followed by development of a common conserved inter-species bacterial PPI using conserved proteins in multiple pathogens (Y. pestis, M. tuberculosis, C. diphtheriae, C. ulcerans, E. coli, and all four Cp strains) and E. Coli based experimentally validated PPI data. Furthermore, the interacting proteins in the common conserved inter-species bacterial PPI were used to generate a conserved host-pathogen interaction (HP-PPI) network considering human, goat, sheep, bovine, and horse as hosts. The HP-PPI network was validated, and acetate kinase (Ack) was identified as a novel broad spectrum target. Ceftiofur, penicillin, and two natural compounds derived from Piper betel were predicted to inhibit Ack activity. One of these Piper betel compounds found to inhibit E. coli O157:H7 growth similar to penicillin. The target specificity of these betel compounds, their effects on other studied pathogens, and other in silico results are currently being validated and the results are promising.

Tips and tricks for the assembly of a Corynebacterium pseudotuberculosis genome using a semiconductor sequencer.

New sequencing platforms have enabled rapid decoding of complete prokaryotic genomes at relatively low cost. The Ion Torrent platform is an example of these technologies, characterized by lower coverage, generating challenges for the genome assembly. One particular problem is the lack of genomes that enable reference-based assembly, such as the one used in the present study, Corynebacterium pseudotuberculosis biovar equi, which causes high economic losses in the US equine industry. The quality treatment strategy incorporated into the assembly pipeline enabled a 16-fold greater use of the sequencing data obtained compared with traditional quality filter approaches. Data preprocessing prior to the de novo assembly enabled the use of known methodologies in the next-generation sequencing data assembly. Moreover, manual curation was proved to be essential for ensuring a quality assembly, which was validated by comparative genomics with other species of the genus Corynebacterium. The present study presents a modus operandi that enables a greater and better use of data obtained from semiconductor sequencing for obtaining the complete genome from a prokaryotic microorganism, C. pseudotuberculosis, which is not a traditional biological model such as Escherichia coli.

Whole-genome sequence of Corynebacterium pseudotuberculosis strain Cp162, isolated from camel.

Corynebacterium pseudotuberculosis is a pathogen of great veterinary and economic importance, since it affects livestock, mainly sheep and goats, worldwide, together with reports of its presence in camels in several Arabic, Asiatic, and East and West African countries, as well as Australia. In this article, we report the genome sequence of Corynebacterium pseudotuberculosis strain Cp162, collected from the external neck abscess of a camel in the United Kingdom.

Complete genome sequence of Corynebacterium pseudotuberculosis strain Cp267, isolated from a llama.

In this work we report the genome of Corynebacterium pseudotuberculosis strain 267, isolated from a llama. This pathogen is of great veterinary and economic importance, as it is the cause of caseous lymphadenitis in several livestock species around the world and causes significant losses due to the high cost of treatment.

Whole-genome sequence of Corynebacterium pseudotuberculosis PAT10 strain isolated from sheep in Patagonia, Argentina.

In this work, we report the complete genome sequence of a Corynebacterium pseudotuberculosis PAT10 isolate, collected from a lung abscess in an Argentine sheep in Patagonia, whose pathogen also required an investigation of its pathogenesis. Thus, the analysis of the genome sequence offers a means to better understanding of the molecular and genetic basis of virulence of this bacterium.

Complete genome sequence of Corynebacterium pseudotuberculosis strain CIP 52.97, isolated from a horse in Kenya.

In this work, we report the whole-genome sequence of Corynebacterium pseudotuberculosis bv. equi strain CIP 52.97 (Collection Institut Pasteur), isolated in 1952 from a case of ulcerative lymphangitis in a Kenyan horse, which has evidently caused significant losses to agribusiness. Therefore, obtaining this genome will allow the detection of important targets for postgenomic studies, with the aim of minimizing problems caused by this microorganism.

Complete genome sequence of type strain Campylobacter fetus subsp. venerealis NCTC 10354T.

Campylobacter fetus subsp. venerealis is the etiologic agent of bovine genital campylobacteriosis, a sexually transmitted disease of cattle that is of worldwide importance. The complete sequencing and annotation of the genome of the type strain C. fetus subsp. venerealis NCTC 10354(T) are reported.