Mass Spectrometry Applications to Study Human Microbiome.

Microbiotas are an adaptable component of ecosystems, including human ecology. Microorganisms influence the chemistry of their specialized niche, such as the human gut, as well as the chemistry of distant surroundings, such as other areas of the body. Metabolomics based on mass spectrometry (MS) is one of the primary methods for detecting and identifying small compounds generated by the human microbiota, as well as understanding the functional significance of these microbial metabolites. This book chapter gives basic knowledge on the kinds of untargeted mass spectrometry as well as the data types that may be generated in the context of microbiome study. While data analysis remains a barrier, the emphasis is on data analysis methodologies and integrative analysis, particularly the integration of microbiome sequencing data. Mass spectrometry (MS)-based techniques have resurrected culture methods for studying the human gut microbiota, filling in the gaps left by high-throughput sequencing methods in terms of culturing minor populations.

Applications of Proteomics in Probiotics Having Anticancer and Chemopreventive Properties.

Proteomics has grown in importance in molecular sciences because it gives vital information on protein identification, expression levels, and alteration. Cancer is one of the world's major causes of death and is the major focus of much research. Cancer risk is determined by hereditary variables as well as the body's immunological condition. Probiotics have increasing medical importance due to their therapeutic influence on the human body in the prevention and treatment of numerous chronic illnesses, including cancer, with no adverse effects. Several anticancer, anti-inflammatory, and chemopreventive probiotics are studied using different proteomic approaches like two-dimensional gel electrophoresis, liquid chromatography-mass spectrometry, and matrix-assisted laser desorption/ionization mass spectrometry. To gain relevant information about probiotic characteristics, data from the proteomic analysis are evaluated and processed using bioinformatics pipelines. Proteomic studies showed the significance of different proteomic approaches in characterization, comparing strains, and determination of oxidative stress of different probiotics. Moreover, proteomic approaches identified different proteins that are involved in glucose metabolism and the formation of cell walls or cell membranes, and the differences in the expression of critical enzymes in the HIF-1 signaling pathway, starch, and sucrose metabolism, and other critical metabolic pathways.

Resequencing and characterization of the first Corynebacterium pseudotuberculosis genome isolated from camel.

Background: Corynebacterium pseudotuberculosis is a zoonotic Gram-positive bacterial pathogen known to cause different diseases in many mammals, including lymph node abscesses in camels. Strains from biovars equi and ovis of C. pseudotuberculosis can infect camels. Comparative genomics could help to identify features related to host adaptation, and currently strain Cp162 from biovar equi is the only one from camel with a sequenced genome. Methods: In this work, we compared the quality of three genome assemblies of strain Cp162 that used data from the DNA sequencing platforms SOLiD v3 Plus, IonTorrent PGM, and Illumina HiSeq 2500 with an optical map and investigate the unique features of this strain. For this purpose, we applied comparative genomic analysis on the different Cp162 genome assembly versions and included other 129 genomes from the same species. Results: Since the first version of the genome, there was an increase of 88 Kbp and 121 protein-coding sequences, a decrease of pseudogenes from 139 to 53, and two inversions and one rearrangement corrected. We identified 30 virulence genes, none associated to the camel host, and the genes rpob2 and rbpA predicted to confer resistance to rifampin. In comparison to 129 genomes of the same species, strain Cp162 has four genes exclusively present, two of them code transposases and two truncated proteins, and the three exclusively absent genes lysG, NUDIX domain protein, and Hypothetical protein. All 130 genomes had the rifampin resistance genes rpob2 and rbpA. Our results found no unique gene that could be associated with tropism to camel host, and further studies should include more genomes and genome-wide association studies testing for genes and SNPs.

Molecular characterization and whole-genome sequencing of Corynebacterium diphtheriae causing skin lesion.

We present a case of skin lesion caused by nontoxigenic Corynebacterium diphtheriae. Genomic taxonomy analyses corroborated the preliminary identification provided by mass spectrometry. The strain showed a susceptible phenotype with increased exposure to penicillin, the first drug of choice for the treatment. An empty type 1 class integron carrying only the sul1 gene, which encodes sulfonamide resistance, was found flanked by transposases. Virulence factors involved in adherence and iron uptake, as well as the CRISPR-Cas system, were predicted. MLST analysis revealed the ST-681, previously reported in French Guiana, a European territory.

Comprehensive probiogenomics analysis of the commensal Escherichia coli CEC15 as a potential probiotic strain.

BACKGROUND: Probiotics have gained attention for their potential maintaining gut and immune homeostasis. They have been found to confer protection against pathogen colonization, possess immunomodulatory effects, enhance gut barrier functionality, and mitigate inflammation. However, a thorough understanding of the unique mechanisms of effects triggered by individual strains is necessary to optimize their therapeutic efficacy. Probiogenomics, involving high-throughput techniques, can help identify uncharacterized strains and aid in the rational selection of new probiotics. This study evaluates the potential of the Escherichia coli CEC15 strain as a probiotic through in silico, in vitro, and in vivo analyses, comparing it to the well-known probiotic reference E. coli Nissle 1917. Genomic analysis was conducted to identify traits with potential beneficial activity and to assess the safety of each strain (genomic islands, bacteriocin production, antibiotic resistance, production of proteins involved in host homeostasis, and proteins with adhesive properties). In vitro studies assessed survival in gastrointestinal simulated conditions and adhesion to cultured human intestinal cells. Safety was evaluated in BALB/c mice, monitoring the impact of E. coli consumption on clinical signs, intestinal architecture, intestinal permeability, and fecal microbiota. Additionally, the protective effects of both strains were assessed in a murine model of 5-FU-induced mucositis. RESULTS: CEC15 mitigates inflammation, reinforces intestinal barrier, and modulates intestinal microbiota. In silico analysis revealed fewer pathogenicity-related traits in CEC15, when compared to Nissle 1917, with fewer toxin-associated genes and no gene suggesting the production of colibactin (a genotoxic agent). Most predicted antibiotic-resistance genes were neither associated with actual resistance, nor with transposable elements. The genome of CEC15 strain encodes proteins related to stress tolerance and to adhesion, in line with its better survival during digestion and higher adhesion to intestinal cells, when compared to Nissle 1917. Moreover, CEC15 exhibited beneficial effects on mice and their intestinal microbiota, both in healthy animals and against 5FU-induced intestinal mucositis. CONCLUSIONS: These findings suggest that the CEC15 strain holds promise as a probiotic, as it could modulate the intestinal microbiota, providing immunomodulatory and anti-inflammatory effects, and reinforcing the intestinal barrier. These findings may have implications for the treatment of gastrointestinal disorders, particularly some forms of diarrhea.

Analysis of Corynebacterium silvaticum genomes from Portugal reveals a single cluster and a clade suggested to produce diphtheria toxin.

Background: Corynebacterium silvaticum is a pathogenic, gram-positive bacterial species that causes caseous lymphadenitis in wild boars, domestic pigs and roe deer in Western Europe. It can affect animal production and cause zoonosis. Genome analysis has suggested that one strain from Portugal and one from Austria could probably produce the diphtheria toxin (DT), which inhibits protein synthesis and can cause death. Methods: To further investigate the species genetic diversity and probable production of DT by Portuguese strains, eight isolates from this country were sequenced and compared to 38 public ones. Results: Strains from Portugal are monophyletic, nearly identical, form a unique cluster and have 27 out of 36 known Corynebacterium virulence or niche factors. All of them lack a frameshift in the tox gene and were suggested to produce DT. A phylogenetic analysis shows that the species has diverged into two clades. Clade 1 is composed of strains that were suggested to have the ability to produce DT, represented by the monophyletic strains from Portugal and strain 05-13 from Austria. Clade 2 is composed of strains unable to produce DT due to a frameshifted tox gene. The second clade is represented by strains from Austria, Germany and Switzerland. Ten genome clusters were detected, in which strains from Germany are the most diverse. Strains from Portugal belong to an exclusive cluster. The pangenome has 2,961 proteins and is nearly closed (α = 0.968). Exclusive genes shared by clusters 1 and 2, and Portuguese strains are probably not related to disease manifestation as they share the same host but could play a role in their extra-host environmental adaptation. These results show the potential of the species to cause zoonosis, possibly diphtheria. The identified clusters, exclusively shaded genes, and exclusive STs identified in Portugal could be applied in the identification and epidemiology of the species.

WGS-Based Lineage and Antimicrobial Resistance Pattern of Salmonella Typhimurium Isolated during 2000-2017 in Peru.

Salmonella Typhimurium is associated with foodborne diseases worldwide, including in Peru, and its emerging antibiotic resistance (AMR) is now a global public health problem. Therefore, country-specific monitoring of the AMR emergence is vital to control this pathogen, and in these aspects, whole genome sequence (WGS)­based approaches are better than gene-based analyses. Here, we performed the antimicrobial susceptibility test for ten widely used antibiotics and WGS-based various analyses of 90 S. Typhimurium isolates (human, animal, and environment) from 14 cities of Peru isolated from 2000 to 2017 to understand the lineage and antimicrobial resistance pattern of this pathogen in Peru. Our results suggest that the Peruvian isolates are of Typhimurium serovar and predominantly belong to sequence type ST19. Genomic diversity analyses indicate an open pan-genome, and at least ten lineages are circulating in Peru. A total of 48.8% and 31.0% of isolates are phenotypically and genotypically resistant to at least one antibiotic, while 12.0% are multi-drug resistant (MDR). Genotype−phenotype correlations for ten tested drugs show >80% accuracy, and >90% specificity. Sensitivity above 90% was only achieved for ciprofloxacin and ceftazidime. Two lineages exhibit the majority of the MDR isolates. A total of 63 different AMR genes are detected, of which 30 are found in 17 different plasmids. Transmissible plasmids such as lncI-gamma/k, IncI1-I(Alpha), Col(pHAD28), IncFIB, IncHI2, and lncI2 that carry AMR genes associated with third-generation antibiotics are also identified. Finally, three new non-synonymous single nucleotide variations (SNVs) for nalidixic acid and eight new SNVs for nitrofurantoin resistance are predicted using genome-wide association studies, comparative genomics, and functional annotation. Our analysis provides for the first time the WGS-based details of the circulating S. Typhimurium lineages and their antimicrobial resistance pattern in Peru.

Comparative genomics and in silico gene evaluation involved in the probiotic potential of Bifidobacterium longum 51A.

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.

Complete genome sequence of the biocontrol agent Serratia marcescens strain N4-5 uncovers an assembly artefact.

Serratia marcescens are gram-negative bacteria found in several environmental niches, including the plant rhizosphere and patients in hospitals. Here, we present the genome of Serratia marcescens strain N4-5 (=NRRL B-65519), which has a size of 5,074,473 bp (664-fold coverage) and contains 4840 protein coding genes, 21 RNA genes, and an average G + C content of 59.7%. N4-5 harbours a plasmid of 11,089 bp and 43.5% G + C content that encodes six unique CDS repeated 2.5× times totalling 13 CDS. Our genome assembly and manual curation uncovered the insertion of two extra copies of the 5S rRNA gene in the assembled sequence, which was confirmed by PCR and Sanger sequencing to be a misassembly. This artefact was subsequently removed from the final assembly. The occurrence of extra copies of the 5S rRNA gene was also observed in most complete genomes of Serratia spp. deposited in public databases in our comparative analysis. These elements, which also occur naturally, can easily be confused with true genetic variation. Efforts to discover and correct assembly artefacts should be made in order to generate genome sequences that represent the biological truth underlying the studied organism. We present the genome of N4-5 and discuss genes potentially involved in biological control activity against plant pathogens and also the possible mechanisms responsible for the artefact we observed in our initial assembly. This report raises awareness about the extra copies of the 5S rRNA gene in sequenced bacterial genomes as they may represent misassemblies and therefore should be verified experimentally.

Taxonomic classification of strain PO100/5 shows a broader geographic distribution and genetic markers of the recently described Corynebacterium silvaticum.

The bacterial strain PO100/5 was isolated from a skin abscess taken from a pig (Sus scrofa domesticus) in the Alentejo region of southern Portugal. It was identified as Corynebacterium pseudotuberculosis using biochemical tests, multiplex PCR and Pulsed Field Gel Electrophoresis. After genome sequencing and rpoB phylogeny, the strain was classified as C. ulcerans. To better understand the taxonomy of this strain and improve identification methods, we compared strain PO100/5 to other publicly available genomes from C. diphtheriae group. Taxonomic analysis reclassified it and three others strains as the recently described C. silvaticum, which have been isolated from wild boar and roe deer in Germany and Austria. The results showed that PO100/5 is the first sequenced genome of a C. silvaticum strain from livestock and a different geographical region, has the unique sequence type ST709, and could be could produce the diphtheriae toxin, along with strain 05-13. Genomic analysis of PO100/5 showed four prophages, and eight conserved genomic islands in comparison to C. ulcerans. Pangenome analysis of 38 C. silvaticum and 76 C. ulcerans genomes suggested that C. silvaticum is a genetically homogeneous species, with 73.6% of its genes conserved and a pangenome near to be closed (α > 0.952). There are 172 genes that are unique to C. silvaticum in comparison to C. ulcerans. Most of these conserved genes are related to nutrient uptake and metabolism, prophages or immunity against them, and could be genetic markers for species identification. Strains PO100/5 (livestock) and KL0182T (wild boar) were predicted to be potential human pathogens. This information may be useful for identification and surveillance of this pathogen.

Rapidly evolving changes and gene loss associated with host switching in Corynebacterium pseudotuberculosis.

Phylogenomics and genome scale positive selection analyses were performed on 29 Corynebacterium pseudotuberculosis genomes that were isolated from different hosts, including representatives of the Ovis and Equi biovars. A total of 27 genes were identified as undergoing adaptive changes. An analysis of the clades within this species and these biovars, the genes specific to each branch, and the genes responding to selective pressure show clear differences, indicating that adaptation and specialization is occurring in different clades. These changes are often correlated with the isolation host but could indicate responses to some undetermined factor in the respective niches. The fact that some of these more-rapidly evolving genes have homology to known virulence factors, antimicrobial resistance genes and drug targets shows that this type of analysis could be used to identify novel targets, and that these could be used as a way to control this pathogen.

Pan-genomic approach shows insight of genetic divergence and pathogenic-adaptation of Pasteurella multocida.

Pasteurella multocida is a gram-negative, non-motile bacterial pathogen, which is associated with chronic and acute infections as snuffles, pneumonia, atrophic rhinitis, fowl cholera and hemorrhagic septicemia. These diseases affect a wide range of domestic animals, leading to significant morbidity and mortality and causing significant economic losses worldwide. Due to the interest in deciphering the genetic diversity and process adaptive between P. multocida strains, this work aimed was to perform a pan-genome analysis to evidence horizontal gene transfer and positive selection among 23 P. multocida strains isolated from distinct diseases and hosts. The results revealed an open pan-genome containing 3585 genes and an accessory genome presenting 1200 genes. The phylogenomic analysis based on the presence/absence of genes and islands exhibit high levels of plasticity, which reflects a high intraspecific diversity and a possible adaptive mechanism responsible for the specific disease manifestation between the established groups (pneumonia, fowl cholera, hemorrhagic septicemia and snuffles). Additionally, we identified differences in accessory genes among groups, which are involved in sugar metabolism and transport systems, virulence-related genes and a high concentration of hypothetical proteins. However, there was no specific indispensable functional mechanism to decisively correlate the presence of genes and their adaptation to a specific host/disease. Also, positive selection was found only for two genes from sub-group hemorrhagic septicemia, serotype B. This comprehensive comparative genome analysis will provide new insights of horizontal gene transfers that play an essential role in the diversification and adaptation mechanism into P. multocida species to a specific disease.

Comparative genomic analysis between Corynebacterium pseudotuberculosis strains isolated from buffalo.

Corynebacterium pseudotuberculosis is a Gram-positive, pleomorphic, facultative intracellular pathogen that causes Oedematous Skin Disease (OSD) in buffalo. To better understand the pathogenic mechanisms of OSD, we performed a comparative genomic analysis of 11 strains of C. pseudotuberculosis isolated from different buffalo found to be infected in Egypt during an outbreak that occurred in 2008. Sixteen previously described pathogenicity islands (PiCp) were present in all of the new buffalo strains, but one of them, PiCp12, had an insertion that contained both a corynephage and a diphtheria toxin gene, both of which may play a role in the adaptation of C. pseudotuberculosis to this new host. Synteny analysis showed variations in the site of insertion of the corynephage during the same outbreak. A gene functional comparison showed the presence of a nitrate reductase operon that included genes involved in molybdenum cofactor biosynthesis, which is necessary for a positive nitrate reductase phenotype and is a possible adaptation for intracellular survival. Genomes from the buffalo strains also had fusions in minor pilin genes in the spaA and spaD gene cluster (spaCX and spaYEF), which could suggest either an adaptation to this particular host, or mutation events in the immediate ancestor before this particular epidemic. A phylogenomic analysis confirmed a clear separation between the Ovis and Equi biovars, but also showed what appears to be a clustering by host species within the Equi strains.

Genome Sequences of Two Brucella suis Strains Isolated from the Same Patient, 8 Years Apart.

Brucella suis is a Gram-negative, facultative intracellular pathogen that has pigs as its preferred host, but it can also infect humans. Here, we report the draft genome sequences of two B. suis strains that were isolated from the same patient, 8 years apart.

Genome Sequences of Three Brucella canis Strains Isolated from Humans and a Dog.

Brucella canis is a facultative intracellular pathogen that preferentially infects members of the Canidae family. Here, we report the genome sequencing of two Brucella canis strains isolated from humans and one isolated from a dog host.

Complete genome sequence of Streptococcus agalactiae strain GBS85147 serotype of type Ia isolated from human oropharynx.

Streptococcus agalactiae, also referred to as Group B Streptococcus, is a frequent resident of the rectovaginal tract in humans, and a major cause of neonatal infection. The pathogen can also infect adults with underlying disease, particularly the elderly and immunocompromised ones. In addition, S. agalactiae is a known fish pathogen, which compromises food safety and represents a zoonotic hazard. This study provides valuable structural, functional and evolutionary genomic information of a human S. agalactiae serotype Ia (ST-103) GBS85147 strain isolated from the oropharynx of an adult patient from Rio de Janeiro, thereby representing the first human isolate in Brazil. We used the Ion Torrent PGM platform with the 200 bp fragment library sequencing kit. The sequencing generated 578,082,183 bp, distributed among 2,973,022 reads, resulting in an approximately 246-fold mean coverage depth and was assembled using the Mira Assembler v3.9.18. The S. agalactiae strain GBS85147 comprises of a circular chromosome with a final genome length of 1,996,151 bp containing 1,915 protein-coding genes, 18 rRNA, 63 tRNA, 2 pseudogenes and a G + C content of 35.48 %.

Complete Genome Sequence of Corynebacterium pseudotuberculosis Strain 12C.

We present here the complete genome sequence of Corynebacterium pseudotuberculosis strain 12C, isolated from a sheep abscess in the Brazil. The sequencing was performed with the Ion Torrent Personal Genome Machine (PGM) system, a fragment library, and a coverage of ~48-fold. The genome presented is a circular chromosome with 2,337,451 bp in length, 2,119 coding sequences, 12 rRNAs, 49 tRNAs, and a G+C content of 52.83%.

Genome Sequence of Corynebacterium ulcerans Strain FRC11.

Here, we present the genome sequence of Corynebacterium ulcerans strain FRC11. The genome includes one circular chromosome of 2,442,826 bp (53.35% G+C content), and 2,210 genes were predicted, 2,146 of which are putative protein-coding genes, with 12 rRNAs and 51 tRNAs; 1 pseudogene was also identified.

Inside the Pan-genome - Methods and Software Overview.

The number of genomes that have been deposited in databases has increased exponentially after the advent of Next-Generation Sequencing (NGS), which produces high-throughput sequence data; this circumstance has demanded the development of new bioinformatics software and the creation of new areas, such as comparative genomics. In comparative genomics, the genetic content of an organism is compared against other organisms, which helps in the prediction of gene function and coding region sequences, identification of evolutionary events and determination of phylogenetic relationships. However, expanding comparative genomics to a large number of related bacteria, we can infer their lifestyles, gene repertoires and minimal genome size. In this context, a powerful approach called Pan-genome has been initiated and developed. This approach involves the genomic comparison of different strains of the same species, or even genus. Its main goal is to establish the total number of non-redundant genes that are present in a determined dataset. Pan-genome consists of three parts: core genome; accessory or dispensable genome; and species-specific or strain-specific genes. Furthermore, pan-genome is considered to be "open" as long as new genes are added significantly to the total repertoire for each new additional genome and "closed" when the newly added genomes cannot be inferred to significantly increase the total repertoire of the genes. To perform all of the required calculations, a substantial amount of software has been developed, based on orthologous and paralogous gene identification.

Genome Sequence of Corynebacterium ulcerans Strain 210932.

In this work, we present the complete genome sequence of Corynebacterium ulcerans strain 210932, isolated from a human. The species is an emergent pathogen that infects a variety of wild and domesticated animals and humans. It is associated with a growing number of cases of a diphtheria-like disease around the world.