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Bifidobacterium breve is among the first microorganisms colonizing the intestinal tract in humans and is a predominant species in the gut microbiota of newborns and children. This bacterium is widely used in the probiotic industry due to its capacity to improve host health. The search for new targets with probiotic properties is an increasing trend with the help of next-generation sequencing as they facilitate the characterization of the bacterial features. B. breve 1101A was isolated from the faeces of healthy children in Brazil and therefore could play a protective role in the gut. To investigate the beneficial properties of this strain, the present study performed a comprehensive characterization of the genetic features involved in the bacterium resistance and adaptation to gastrointestinal conditions, production of nutrients, and immunomodulatory compounds. Furthermore, this study carried out the prediction of genomic elements (plasmids, prophages, CRISPR-Cas systems, insertion sequences, genomic islands, antibiotic resistance genes) to evaluate the safety of B. breve 1101A. A comparative genomics approach using 45 B. breve complete genomes based on pangenome and phylogenomic analysis was also performed to identify specific genes in B. breve 1101A. The prediction of genetic elements, possibly safety-related, did not detect plasmids, but only one incomplete prophage, two non-functional CRISPR systems, and seven genomic islands. Additionally, three antibiotic resistance genes were identified: ileS (resistance to mupirocin), rpoB, and erm(X). In the comparative genomic analysis, the pangenome was revealed to be open, and B. breve 1101A presented 63 unique genes associated with several processes, such as transmembrane transport, membrane components, DNA processes, and carbohydrate metabolism. In conclusion, B. breve 1101A is potentially safe and well-adapted for intestinal disorder therapeutics, although the role of its unique genetic repertoire needs further investigation.

Staphylococcus aureus is the main etiological agent of mastitis in small ruminants worldwide. This disease has a difficult cure and possible relapse, leading to significant economic losses in production, milk quality and livestock. This study performed comparative genomic analyses between 73 S. aureus genomes from different hosts (human, bovine, pig and others). This work isolated and sequenced 12 of these genomes from ovine. This study contributes to the knowledge of genomic specialization and the role of specific genes in establishing infection in ovine mastitis-associated S. aureus. The genomes of S. aureus isolated from sheep maintained a higher representation when grouped with clonal complexes 130 and 133. The genomes showed high genetic similarity, the species pan-genome consisting of 4200 genes (central = 2008, accessory = 1559 and unique = 634). Among these, 277 unique genes were related to the genomes isolated from sheep, with 39.6 % as hypothetical proteins, 6.4 % as phages, 6.4 % as toxins, 2.9 % as transporters, and 44.7 % as related to other proteins. Furthermore, at the pathogen level, they showed 80 genes associated with virulence factors and 19 with antibiotic resistance shared in almost all isolates. Although S. aureus isolated from ovine showed susceptibility to antimicrobials in vitro, ten genes were predicted to be associated with antibiotic inactivation and efflux pump, suggesting resistance to gentamicin and penicillin. This work may contribute to identifying genes acquired by horizontal transfer and their role in host adaptation, virulence, bacterial resistance, and characterization of strains affecting ovine.

Dietzia strains are widely distributed in the environment, presenting an opportunistic role, and some species have undetermined taxonomic characteristics. Here, we propose the existence of errors in the classification of species in this genus using comparative genomics. We performed ANI, dDDH, pangenome and genomic plasticity analyses better to elucidate the phylogenomic relationships between Dietzia strains. For this, we used 55 genomes of Dietzia downloaded from public databases that were combined with a newly sequenced. Sequence analysis of a phylogenetic tree based on genome similarity comparisons and dDDH, ANI analyses supported grouping different Dietzia species into four distinct groups. The pangenome analysis corroborated the classification of these groups, supporting the idea that some species of Dietzia could be reassigned in a possible classification into three distinct species, each containing less variability than that found within the global pangenome of all strains. Additionally, analysis of genomic plasticity based on groups containing Dietzia strains found differences in the presence and absence of symbiotic Islands and pathogenic islands related to their isolation site. We propose that the comparison of pangenome subsets together with phylogenomic approaches can be used as an alternative for the classification and differentiation of new species of the genus Dietzia.

The number of draft genomes deposited in Genbank from the National Center for Biotechnology Information (NCBI) is higher than the complete ones. Draft genomes are assemblies that contain fragments of misassembled regions (gaps). Such draft genomes present a hindrance to the complete understanding of the biology and evolution of the organism since they lack genomic information. To overcome this problem, strategies to improve the assembly process are developed continuously. Also, the greatest challenge to the assembly progress is the presence of repetitive DNA regions. This article highlights the use of optical mapping, to detect and correct assembly errors in Corynebacterium pseudotuberculosis. We also demonstrate that choosing a reference genome should be done with caution to avoid assembly errors and loss of genetic information.

The Bifidobacterium longum 5

Corynebacterium pseudotuberculosis is a Gram-positive bacterium that causes caseous lymphadenitis, a disease that predominantly affects sheep, goat, cattle, buffalo, and horses, but has also been recognized in other animals. This bacterium generates a severe economic impact on countries producing meat. Gene expression studies using RNA-Seq are one of the most commonly used techniques to perform transcriptional experiments. Computational analysis of such data through reverse-engineering algorithms leads to a better understanding of the genome-wide complexity of gene interactomes, enabling the identification of genes having the most significant functions inferred by the activated stress response pathways. In this study, we identified the influential or causal genes from four RNA-Seq datasets from different stress conditions (high iron, low iron, acid, osmosis, and PH) in C. pseudotuberculosis, using a consensus-based network inference algorithm called miRsigand next identified the causal genes in the network using the miRinfluence tool, which is based on the influence diffusion model. We found that over 50% of the genes identified as influential had some essential cellular functions in the genomes. In the strains analyzed, most of the causal genes had crucial roles or participated in processes associated with the response to extracellular stresses, pathogenicity, membrane components, and essential genes. This research brings new insight into the understanding of virulence and infection by C. pseudotuberculosis.

The genus Corynebacterium includes species of great importance in medical, veterinary and biotechnological fields. The genus-specific families (PLfams) from PATRIC have been used to observe conserved proteins associated to all species. Our results showed a large number of conserved proteins that are associated with the cellular division process. Was not observe in our results other proteins like FtsA and ZapA that interact with FtsZ. Our findings point that SepF overlaps the function of this proteins explored by molecular docking, protein-protein interaction and sequence analysis. Transcriptomic analysis showed that these two (Sepf and FtsZ) proteins can be expressed in different conditions together. The work presents novelties on molecules participating in the cell division event, from the interaction of FtsZ and SepF, as new therapeutic targets.

Transcriptome studies on Corynebacterium pseudotuberculosis have recently contributed to the understanding about this microorganism's survival mechanisms in various hostile conditions. The gene expression profile of the C. pseudotuberculosis strain 1002 (Ovis biovar), has revealed genes that are possible candidates responsible for its maintenance in adverse environments, such as those found in the host. In another strain of this bacterium, 258 (Equi biovar), a high temperature condition was simulated, in order to verify which genes are responsible for promoting the persistence of the bacterium in these conditions, since it tolerates temperatures higher than 40 °C, despite being a mesophilic bacterium. It was possible to generate a list of genes using RNAseq technology that possibly contribute to the survival of the bacteria in this hostile environment. A total of 562 genes were considered as differentially expressed, then, after the fold-change cutoff, 113 were considered induced and 114 repressed, resulting in a total of 227 genes. Therefore, hypothetical proteins presented a fold change above 6, and genes characteristically in control for this type of stress, such as hspR, grpE, and dnaK, presented a fold change above 3. The clpB gene, a chaperone, drew attention due to presenting a fold change above 3 and located in a pathogenicity island. These genes may contribute towards efficient solutions to the effects caused by ulcerative lymphangitis in equines, thus attenuating the damage it causes to agribusiness.

With the development of next-generation sequencing (NGS) technologies, genome sequencing process has become cheaper and faster, making it possible the use of the technology in daily routine and, as a result, the number of registered genome projects is increasing rapidly. The pan-omics approaches have been applied for comparison of several genomes in a multipronged strategy. The pan-genome is composed of the core genome, shared genome, and singleton subsets, where the core genome is composed of all the commonly shared genes by all strains of the species; shared genome contains genes that are present in two or more, but not all strains from a species; and the singletons are strain-specific genes. The subsets from pan-transcriptomics and pan-proteomics may be classified in the same way. The concept of pan-genomics is so deep that it has been perfectly applied in the studies of several organisms and diseases. It has great potential which may bring a closer understanding and combating with prokaryotic and eukaryotic diseases. In this chapter, we review the concepts of omics, pan-omics, and the areas of applications of these methods.

Pan-genome is an approach that contributes to the research of bacterial pathogenesis. From the sequencing data, one can thoroughly study each region of the genome, contributing with unpublished information. Since 2005, with the era of new sequencers, the speed, ease, and reliability of data has been increasing and with them the number of bacterial genomes deposited in public databases. Pan-genome studies can be applied with different goals, such as taxonomy, reverse vaccinology, gene variation, and pathogenesis, among others. This chapter is focused on the pan-genomics studies carried out on pathogenic bacteria that cause veterinary diseases, including the ones responsible for zoonotic diseases. These studies showed a great diversity at the genome level which may reflect the associations of genetic groups that could be involved with the capacity to infect and adaptations in different host. From the genetic repertoire studies, the key points (genes) supposedly involved in the spread of a disease, bacterial resistance, infection, adhesion, can be detected, leading to effective solutions against the disease being studied.

Bacteria of the genusGlutamicibacterare considered ubiquitous because they can be found in soil, water and air. They have already been isolated from different habitats, including different types of soil, clinical samples, cheese and plants. Glutamicibacter creatinolyticus is a Gram-positive bacterium important to various biotechnological processes, however, as a pathogen it is associated to urinary tract infections and bacteremia. Recently,Glutamicibacter creatinolyticusLGCM 259 was isolated from a mare, which displayed several diffuse subcutaneous nodules with heavy vascularization. In this study, sequencing, genomic analysis ofG. creatinolyticusLGCM 259 and comparative analyseswere performedamong 4representatives of different members of genusfromdifferent habitats, available in the NCBI database. The LGCM 259 strain's genome carries important factors of bacterial virulence that are essential in cell viability, virulence, and pathogenicity. Genomic islands were predicted for 4 members of genusGlutamicibacter,showing ahigh number of GEIs,which may reflect a high interspecific diversity and a possible adaptive mechanism responsible for the survival of each species in its specific niche. Furthermore,G. creatinolyticusLGCM 259 sharessyntenicregions, albeit with a considerable loss of genes, in relation to the other species. In addition,G. creatinolyticusLGCM 259 presentsresistancegenes to 6 differentclasses ofantibiotics and heavy metals, such as: copper, arsenic, chromium and cobalt-zinc-cadmium.Comparative genomicsanalysescouldcontribute to the identification of mobile genetic elements particular to the speciesG. creatinolyticuscompared to other members of genus. The presence of specific regions inG. creatinolyticuscould be indicative of their rolesin host adaptation, virulence, and the characterization ofastrain that affects animals.

The Bifidobacterium longum 51A strain of isolated from feces of a healthy child, has demonstrated probiotic properties by in vivo and in vitro studies, which may be assigned to its production of metabolites such as acetate. Thus, through the study of comparative genomics, the present work sought to identify unique genes that might be related to the production of acetate. To perform the study, the DNA strain was sequenced using Illumina HiSeq technology, followed by assembly and manual curation of coding sequences. Comparative analysis was performed including 19 complete B. longum genomes available in Genbank/NCBI. In the phylogenetic analysis, the CECT 7210 and 157F strains of B. longum subsp. infantis aggregated within the subsp. longum cluster, suggesting that their taxonomic classification should be reviewed. The strain 51A of B. longum has 26 unique genes, six of which are possibly related to carbohydrate metabolism and acetate production. The phosphoketolase pathway from B. longum 51A showed a difference in acetyl-phosphate production. This result seems to corroborate the analysis of their unique genes, whose presence suggests the strain may use different sources of carbohydrates that allow a greater production of acetate and consequently offer benefits to the host health.

Corynebacterium pseudotuberculosis is the etiologic agent of veterinary relevance diseases, such as caseous lymphadenitis, affecting different animal species causing damage to the global agribusiness. So far, there are no completely effective treatment methods to overcome the impacts caused by this pathogen. Several genomes of the species are deposited on public databases, allowing the execution of studies related to the pan-genomic approach. In this study, we used an integrated in silico workflow to prospect novel putative targets using the core genome, a set of shared genes among 65 C. pseudotuberculosis strains. Subsequently, through RNA-Seq data of the same abiotic stresses in two strains, we selected only induced genes to compose the reverse vaccinology workflow based in two different strategies. Our results predicted six probable antigens in both analysis, which indicates that they have a strong potential to be used in further studies as vaccine targets against this bacterium.

Corynebacterium pseudotuberculosis is a pathogenic bacterium which has been rapidly spreading all over the world, causing economic losses in the agricultural sector and sporadically infecting humans. Six C. pseudotuberculosis strains were isolated from goats, sheep, and horses with distinct abscess locations. For the first time, Mexican genomes of this bacterium were sequenced and studied in silico. All strains were sequenced using Ion Personal Genome Machine sequencer, assembled using Newbler and SPAdes software. The automatic genome annotation was done using the software RAST and in-house scripts for transference, followed by manual curation using Artemis software and BLAST against NCBI and UniProt databases. The six genomes are publicly available in NCBI database. The analysis of nucleotide sequence similarity and the generated phylogenetic tree led to the observation that the Mexican strains are more similar between strains from the same host, but the genetic structure is probably more influenced by transportation of animals between farms than host preference. Also, a putative drug target was predicted and in silico analysis of 46 strains showed two gene clusters capable of differentiating the biovars equi and ovis: Restriction Modification system and CRISPR-Cas cluster. Electronic supplementary material The online version of this article (10.1186/s40793-018-0325-z) contains supplementary material, which is available to authorized users.

Corynebacterium pseudotuberculosis has been widely studied in an effort to understand its biological evolution. Transcriptomics has revealed possible candidates for virulence and pathogenicity factors of strain 1002 (biovar Ovis). Because C. pseudotuberculosis is classified into two biovars, Ovis and Equi, it was interesting to assess the transcriptional profile of biovar Equi strain 258, the causative agent of ulcerative lymphangitis. The genome of this strain was re-sequenced; the reassembly was completed using optical mapping technology, and the sequence was subsequently re-annotated. Two growth conditions that occur during the host infection process were simulated for the transcriptome: the osmotic and acid medium. Genes that may be associated with the microorganism's resilience under unfavorable conditions were identified through RNAseq, including genes present in pathogenicity islands. The RT-qPCR was performed to confirm the results in biological triplicate for each condition for some genes. The results extend our knowledge of the factors associated with the spread and persistence of C. pseudotuberculosis during the infection process and suggest possible avenues for studies related to the development of vaccines, diagnosis, and therapies that might help minimize damage to agribusinesses. Fil: Gomide, Anne Cybelle Pinto. Universidade Federal de Minas Gerais; Brasil Fil: Ibraim, Izabela Coimbra. Universidade Federal de Minas Gerais; Brasil Fil: Alves, Jorianne T. C.. Universidade Federal do Pará; Brasil Fil: de Sá, Pablo Gomes. Federal Rural University Of Amazonia; Brasil Fil: de Oliveira Silva, Yuri Rafael. Universidade Federal do Pará; Brasil Fil: Santana, Mariana Passos. Universidade Federal de Minas Gerais; Brasil Fil: Marques Da Silva, Wanderson. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Folador, Edson Luiz. Universidade Federal Da Paraiba; Brasil Fil: Mariano, Diego C. B.. Universidade Federal de Minas Gerais; Brasil Fil: de Paula Castro, Thiago Luiz. Universidade Federal da Bahia; Brasil Fil: Barbosa, Silvanira. Universidade Federal do Pará; Brasil Fil: Dorella, Fernanda Alves. Universidade Federal de Minas Gerais; Brasil Fil: Carvalho, Alex F.. Universidade Federal de Minas Gerais; Brasil Fil: Pereira, Felipe L.. Universidade Federal de Minas Gerais; Brasil Fil: Leal, Carlos A. G.. Universidade Federal de Minas Gerais; Brasil Fil: Figueiredo, Henrique C. P.. Universidade Federal de Minas Gerais; Brasil Fil: Azevedo, Vasco. Universidade Federal de Minas Gerais; Brasil Fil: Silva, Artur. Universidade Federal do Pará; Brasil Fil: Folador, Adriana Ribeiro Carneiro. Universidade Federal do Pará; Brasil

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico O gene ciuA codifica uma proteína de superfície associada a um sistema de captação de ferro dependente de sideróforos, porém seu papel na aquisição de ferro nessa bactéria ainda é pouco conhecido. O ferro é crucial no crescimento e desenvolvimento de bactérias, porém no hospedeiro as frações livres disponíveis de ferro são inferiores às necessárias para o crescimento bacteriano. Em bactérias intracelulares como C. pseudotuberculosis, a homeostase de ferro no interior do hospedeiro é controlada transcricionalmente pela concentração de ferro no meio e envolve a regulação de genes voltados para a subversão dos sistemas de restrição nutricional imposto pelo hospedeiro. Trabalhos anteriores realizados por nosso grupo, levaram a construção de uma linhagem mutante (Cp13) para o gene ciuA, através de mutagênese aleatória utilizando o transposon TnFuZ na linhagem T1 selvagem de Corynebacterium pseudotuberculosis. A interrupção deste gene, contribuiu para a redução da virulência e viabilidade intracelular da linhagem, além de induzir níveis significativos de anticorpos e alta taxa de sobrevivência em camundongos imunizados. Diante do exposto, este trabalho teve como objetivo principal avaliar o crescimento e a resposta transcricional de C. pseudotuberculosis em resposta à restrição de ferro, bem como a caracterização funcional de genes associados a virulência, entre linhagem T1 e mutante Cp13. Assim, a análise de expressão diferencial de ambas as linhagens, foi realizada a partir de culturas incubadas em meio com baixa disponibilidade de ferro (low iron – LI), obtido pelo agente quelante 2,2-bipiridina (BIP), e meio com alta disponibilidade de ferro (high iron – HI). Neste contexto, os dados aqui apresentados demonstraram uma redução significativa na densidade celular e na taxa de crescimento da linhagem mutante em comparação a linhagem parental em culturas LI. No total, 120 e 86 genes diferencialmente expressos (GDEs) foram identificados nas cepas T1 e Cp13 em resposta à restrição de ferro, respectivamente. Dentre esses genes, 36 GDEs foram compartilhados entre as duas linhagens, sendo que 26 apresentaram um perfil de expressão semelhante entre as linhagens. Dois fatores transcricionais ripA e hrrA, regulados por DtxR, foram identificados entre os GDEs compartilhados, demonstrando uma complexa rede regulatória controlada pela disponibilidade de ferro. Sete dos 36 GDEs codificam proteínas de superfície e transportadores de hemina, sendo quatro genes localizados em ilhas genômicas associados a fatores de virulência. Nossos resultados demonstraram que a restrição intracelular da utilização de ferro é fundamental para a resposta de C. pseudotuberculosis à disponibilidade do metal. Alterações significativas também foram observadas em relação aos GDEs identificados exclusivamente entre as linhagens, indicando divergência entre a resposta adaptativa da linhagem mutante em relação a linhagem parental. Em conjunto, os resultados do presente trabalho fornecem uma análise abrangente da rede regulatória e da resposta transcricional da linhagem T1 selvagem e Cp13 mutante de C. pseudotuberculosis, acrescentando informações importantes envolvendo o processo de adaptação desse importante patógeno à restrição de ferro. The ciuA gene codes for a surface protein which is associated to a siderophore-base iron acquisition system; however, its role in the acquisition of iron in C. pseudotuberculosis is still largely unknown. Iron is crucial for the growth and development of many bacterial pathogens; however, within the host, the concentration that is freely accessible is much lower than the concentration required for bacterial growth. In intracellular bacteria, like C. pseudotuberculosis, iron homeostasis is transcriptionally controlled by the availability of iron in the environment, which allows the bacteria to counteract the limitation of the metal imposed by the host. In a previous work conducted by our group, we described the construction of a ciuA gene mutant strain (Cp13), generated by using the in vivo insertional mutagenesis of the reporter transposon-based system TnFuZ in the T1 strain of C. pseudotuberculosis. The disruption of the ciuA gene led to a decrease in virulence and intracellular viability of the mutant strain. In addition, immunization of mice with the Cp13 strain elicited significantly IgG titers and higher survival rate (80%). The ciuA gene codes for a surface protein which is associated to a siderophore-base iron acquisition system; however, its role in the acquisition of iron in C. pseudotuberculosis is still largely unknown. Iron is crucial for the growth and development of many bacterial pathogens; however, within the host, the concentration that is freely accessible is much lower than the concentration required for bacterial growth. In intracellular bacteria, like C. pseudotuberculosis, iron homeostasis is transcriptionally controlled by the availability of iron in the environment, which allows the bacteria to counteract the limitation of the metal imposed by the host. Herein, our primary goal was to comprehensively characterize the transcriptional response of C. pseudotuberculosis under iron restriction. Differential gene expression analyses of the wild-type strain (T1 strain) and the (ciuA) Cp13 mutant strain (Cp13 strain) were conducted in low iron (LI) cultures, supplemented with the iron-chelator 2,2’-dipyridyl-DIP, in relation to high iron (HI) cultures in both strains. In this context, the data presented here shows a significant reduction in cellular density and the growth rate of the Cp13 mutant strain in relation to the parental strain under iron restriction. In total, 120 and 86 differentially expressed genes (DEGs) were identified in the T1 and Cp13 strains in response to iron restriction, respectively. 36 of these genes were identified in both strains and 26 of these genes had a similar expression pattern in both strains. Two transcription factors, known to be regulated by DtxR, were also identified between the overlapped DEGs of both strains, indicating a complex regulatory network controlled by the availability of iron in C. pseudotuberculosis. Out of the 26 genes, 7 DEGs encode high-affinity hemin-binding proteins. Interestingly, 4 hemin-binding genes were identified located within genomic islands harboring known virulence factors, corroborating with the association of iron acquisition and virulence in pathogenic bacteria. We have also observed that the intracellular restriction of iron usage is key to the iron starvation response of C. pseudotuberculosis. Significant differences between mutant and parental strains were also observed in relation to the strain-specific DEGs, indicating divergence in the adaptive responses of the mutant and parental strains in response to iron restriction. Altogether, these findings provide a comprehensive analysis of the regulatory network and transcriptional response of the Cp13 mutant and T1 parental strain of C. pseudotuberculosis, adding relevant insights into the transcriptional adaptation of this important pathogen within an iron-restricted environment.