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.

Systematic review of reverse vaccinology and immunoinformatics data for non-viral sexually transmitted infections.

Sexually Transmitted Infections (STIs) are a public health burden rising in developed and developing nations. The World Health Organization estimates nearly 374 million new cases of curable STIs yearly. Global efforts to control their spread have been insufficient in fulfilling their objective. As there is no vaccine for many of these infections, these efforts are focused on education and condom distribution. The development of vaccines for STIs is vital for successfully halting their spread. The field of immunoinformatics is a powerful new tool for vaccine development, allowing for the identification of vaccine candidates within a bacterium's genome and allowing for the design of new genome-based vaccine peptides. The goal of this review was to evaluate the usage of immunoinformatics in research focused on non-viral STIs, identifying fields where research efforts are concentrated. Here we describe gaps in applying these techniques, as in the case of Treponema pallidum and Trichomonas vaginalis.

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.

Unveiling the microbiome during post-partum uterine infection: a deep shotgun sequencing approach to characterize the dairy cow uterine microbiome.

BACKGROUND: The goal of this study was to assess the microbial ecology and diversity present in the uterus of post-partum dairy cows with and without metritis from 24 commercial California dairy farms using shotgun metagenomics. A set subset of 95 intrauterine swab samples, taken from a larger selection of 307 individual cow samples previously collected, were examined for α and ß diversity and differential abundance associated with metritis. Cows within 21 days post-partum were categorized into one of three clinical groups during sample collection: control (CT, n = 32), defined as cows with either no vaginal discharge or a clear, non-purulent mucus vaginal discharge; metritis (MET, n = 33), defined as a cow with watery, red or brown colored, and fetid vaginal discharge; and purulent discharge cows (PUS, n = 31), defined as a non-fetid purulent or mucopurulent vaginal discharge. RESULTS: All three clinical groups (CT, MET, and PUS) were highly diverse, with the top 12 most abundant genera accounting for 10.3%, 8.8%, and 10.1% of mean relative abundance, respectively. The α diversity indices revealed a lower diversity from samples collected from MET and PUS when compared to CT cows. PERMANOVA statistical testing revealed a significant difference (P adjusted < 0.01) in the diversity of genera between CT and MET samples (R2 = 0.112, P = 0.003) and a non-significant difference between MET and PUS samples (R2 = 0.036, P = 0.046). ANCOM-BC analysis revealed that from the top 12 most abundant genera, seven genera were increased in the natural log fold change (LFC) of abundance in MET when compared to CT samples: Bacteroides, Clostridium, Fusobacterium, Phocaeicola, Porphyromonas, Prevotella, and Streptococcus. Two genera, Dietzia and Microbacterium, were decreased in natural LFC of abundance when comparing MET (regardless of treatment) and CT, while no changes in natural LFC of abundance were observed for Escherichia, Histophilus, and Trueperella. CONCLUSIONS: The results presented here, are the current deepest shotgun metagenomic analyses conducted on the bovine uterine microbiome to date (mean of 256,425 genus-level reads per sample). Our findings support that uterine samples from cows without metritis (CT) had increased α-diversity but decreased ß-diversity when compared to metritis or PUS cows, characteristic of dysbiosis. In summary, our findings highlight that MET cows have an increased abundance of Bacteroides, Porphyromonas, and Fusobacterium when compared to CT and PUS, and support the need for further studies to better understand their potential causal role in metritis pathogenesis.

Probiogenomics of Leuconostoc Mesenteroides Strains F-21 and F-22 Isolated from Human Breast Milk Reveal Beneficial Properties.

Bacteria of the Leuconostoc genus are Gram-positive bacteria that are commonly found in raw milk and persist in fermented dairy products and plant food. Studies have already explored the probiotic potential of L. mesenteroides, but not from a probiogenomic perspective, which aims to explore the molecular features responsible for their phenotypes. In the present work, probiogenomic approaches were applied in strains F-21 and F-22 of L. mesenteroides isolated from human milk to assess their biosafety at the molecular level and to correlate molecular features with their potential probiotic characteristics. The complete genome of strain F-22 is 1.99 Mb and presents one plasmid, while the draft genome of strain F-21 is 1.89 Mb and presents four plasmids. A high percentage of average nucleotide identity among other genomes of L. mesenteroides (≥ 96%) corroborated the previous taxonomic classification of these isolates. Genomic regions that influence the probiotic properties were identified and annotated. Both strains exhibited wide genome plasticity, cell adhesion ability, proteolytic activity, proinflammatory and immunomodulation capacity through interaction with TLR-NF-κB and TLR-MAPK pathway components, and no antimicrobial resistance, denoting their potential to be candidate probiotics. Further, the strains showed bacteriocin production potential and the presence of acid, thermal, osmotic, and bile salt resistance genes, indicating their ability to survive under gastrointestinal stress. Taken together, our results suggest that L. mesenteroides F-21 and F-22 are promising candidates for probiotics in the food and pharmaceutical industries.

Evidence of episodic positive selection in Corynebacterium diphtheriae complex of species and its implementations in identification of drug and vaccine targets.

BACKGROUND: Within the pathogenic bacterial species Corynebacterium genus, six species that can produce diphtheria toxin (C. belfantii, C. diphtheriae, C. pseudotuberculosis, C. rouxii, C. silvaticum and C. ulcerans) form a clade referred to as the C. diphtheria complex. These species have been found in humans and other animals, causing diphtheria or other diseases. Here we show the results of a genome scale analysis to identify positive selection in protein-coding genes that may have resulted in the adaptations of these species to their ecological niches and suggest drug and vaccine targets. METHODS: Forty genomes were sampled to represent species, subspecies or biovars of Corynebacterium. Ten phylogenetic groups were tested for positive selection using the PosiGene pipeline, including species and biovars from the C. diphtheria complex. The detected genes were tested for recombination and had their sequences alignments and homology manually examined. The final genes were investigated for their function and a probable role as vaccine or drug targets. RESULTS: Nineteen genes were detected in the species C. diphtheriae (two), C. pseudotuberculosis (10), C. rouxii (one), and C. ulcerans (six). Those were found to be involved in defense, translation, energy production, and transport and in the metabolism of carbohydrates, amino acids, nucleotides, and coenzymes. Fourteen were identified as essential genes, and six as virulence factors. Thirteen from the 19 genes were identified as potential drug targets and four as potential vaccine candidates. These genes could be important in the prevention and treatment of the diseases caused by these bacteria.

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.

Safety Evaluation of Lactobacillus delbrueckii subsp. lactis CIDCA 133: a Health-Promoting Bacteria.

Lactobacillus delbrueckii subsp. lactis CIDCA is a new potential probiotic strain whose molecular basis attributed to the host's benefit has been reported. This study investigated the safety aspects of Lactobacillus delbrueckii subsp. lactis CIDCA 133 based on whole-genome sequence and phenotypic analysis to avoid future questions about the harmful effects of this strain consumption. Genomic analysis showed that L. delbrueckii subsp. lactis CIDCA 133 harbors virulence, harmful metabolites, and antimicrobial resistance-associated genes. However, none of these genetic elements is flanked or located within prophage regions and plasmid sequence. At a phenotypic level, it was observed L. delbrueckii subsp. lactis CIDCA 133 antimicrobial resistance to aminoglycosides streptomycin and gentamicin antibiotics, but no hemolytic and mucin degradation activity was exhibited by strain. Furthermore, no adverse effects were observed regarding mice clinical and histopathological analysis after the strain consumption (5 × 107 CFU/mL). Overall, these findings reveal the safety of Lactobacillus delbrueckii subsp. lactis CIDCA 133 for consumption and future probiotic applications.

The Space-Exposed Kombucha Microbial Community Member Komagataeibacter oboediens Showed Only Minor Changes in Its Genome After Reactivation on Earth.

Komagataeibacter is the dominant taxon and cellulose-producing bacteria in the Kombucha Microbial Community (KMC). This is the first study to isolate the K. oboediens genome from a reactivated space-exposed KMC sample and comprehensively characterize it. The space-exposed genome was compared with the Earth-based reference genome to understand the genome stability of K. oboediens under extraterrestrial conditions during a long time. Our results suggest that the genomes of K. oboediens IMBG180 (ground sample) and K. oboediens IMBG185 (space-exposed) are remarkably similar in topology, genomic islands, transposases, prion-like proteins, and number of plasmids and CRISPR-Cas cassettes. Nonetheless, there was a difference in the length of plasmids and the location of cas genes. A small difference was observed in the number of protein coding genes. Despite these differences, they do not affect any genetic metabolic profile of the cellulose synthesis, nitrogen-fixation, hopanoid lipids biosynthesis, and stress-related pathways. Minor changes are only observed in central carbohydrate and energy metabolism pathways gene numbers or sequence completeness. Altogether, these findings suggest that K. oboediens maintains its genome stability and functionality in KMC exposed to the space environment most probably due to the protective role of the KMC biofilm. Furthermore, due to its unaffected metabolic pathways, this bacterial species may also retain some promising potential for space applications.

Molecular Characterization and Survive Abilities of Salmonella Heidelberg Strains of Poultry Origin in Brazil.

The aim of the study was to evaluate the genotypic and phenotypic characteristics of 20 strains of S. Heidelberg (SH) isolated from broilers produced in southern Brazil. The similarity and presence of genetic determinants linked to virulence, antimicrobial resistance, biofilm formation, and in silico-predicted metabolic interactions revealed this serovar as a threat to public health. The presence of the ompC, invA, sodC, avrA, lpfA, and agfA genes was detected in 100% of the strains and the luxS gene in 70% of them. None of the strains carries the bla SHV, mcr-1, qnrA, qnrB, and qnrS genes. All strains showed a multidrug-resistant profile to at least three non-ß-lactam drugs, which include colistin, sulfamethoxazole, and tetracycline. Resistance to penicillin, ceftriaxone (90%), meropenem (25%), and cefoxitin (25%) were associated with the presence of bla CTX-M and bla CMY-2 genes. Biofilm formation reached a mature stage at 25 and 37°C, especially with chicken juice (CJ) addition. The sodium hypochlorite 1% was the least efficient in controlling the sessile cells. Genomic analysis of two strains identified more than 100 virulence genes and the presence of resistance to 24 classes of antibiotics correlated to phenotypic tests. Protein-protein interaction (PPI) prediction shows two metabolic pathways correlation with biofilm formation. Virulence, resistance, and biofilm determinants must be constant monitoring in SH, due to the possibility of occurring infections extremely difficult to cure and due risk of the maintenance of the bacterium in production environments.

Probiogenomics of Lactobacillus delbrueckii subsp. lactis CIDCA 133: In Silico, In Vitro, and In Vivo Approaches.

Lactobacillus delbrueckii subsp. lactis CIDCA 133 (CIDCA 133) has been reported as a potential probiotic strain, presenting immunomodulatory properties. This study investigated the possible genes and molecular mechanism involved with a probiotic profile of CIDCA 133 through a genomic approach associated with in vitro and in vivo analysis. Genomic analysis corroborates the species identification carried out by the classical microbiological method. Phenotypic assays demonstrated that the CIDCA 133 strain could survive acidic, osmotic, and thermic stresses. In addition, this strain shows antibacterial activity against Salmonella Typhimurium and presents immunostimulatory properties capable of upregulating anti-inflammatory cytokines Il10 and Tgfb1 gene expression through inhibition of Nfkb1 gene expression. These reported effects can be associated with secreted, membrane/exposed to the surface and cytoplasmic proteins, and bacteriocins-encoding genes predicted in silico. Furthermore, our results showed the genes and the possible mechanisms used by CIDCA 133 to produce their beneficial host effects and highlight its use as a probiotic microorganism.

Characterization of a new multidrug-resistant Brazilian K. pneumoniae isolate and 172 Klebsiella spp. sequenced strains: Genomic island, multilocus sequence typing and capsule locus dataset.

The genus Klebsiella comprises species that cause nosocomial and community-acquired infections. A dataset was created to compile the sequence type (ST) and capsule type (K-locus) information predicted for 172 worldwide isolates of Klebsiella spp. whose complete genomes could be retrieved from the GenBank (NCBI) repository. The dataset also includes information related to one multidrug-resistant strain (B31) isolated from a patient who was admitted to an intensive care unit in the Northeast region of Brazil. This strain was phenotypically characterized and submitted to whole-genome sequencing and comparative genomics analysis as we recently reported [1]. The dataset also compiles information on Pathogenicity Islands (PIs), Resistance Islands (RIs) and Miscellaneous Islands (MIS) present in the genome of strain B31. The information provided here may support outbreak prevention policies and future epidemiological studies involving Klebsiella spp.

Comparative genomics with a multidrug-resistant Klebsiella pneumoniae isolate reveals the panorama of unexplored diversity in Northeast Brazil.

The emergence of community acquired infections increases the public health concern on K. pneumoniae and closely related bacteria among which antimicrobial resistance spreads. We report a multidrug-resistant K. pneumoniae isolate, B31, of a patient infected in the community and admitted to an intensive care unit in Northeast Brazil. Antimicrobial susceptibility and genome information were thoroughly investigated to characterize B31 in front of 172 sequenced strains of different countries. Assigned to the Sequence Type 15, which is globally spread, B31 presented extended spectrum beta-lactamase, tigecycline and ciprofloxacin resistance. Genome sequencing revealed most resistance genes being carried by plasmids with high dissemination potential. The absence of main virulence factors, like yersiniabactin and colibactin, apparently suggests a mild pathogenic strain which, on the contrary, persisted and caused severe infection in a previously healthy patient. The present study contributes to unveil the unclear genomic scenario of virulent and multidrug-resistant K. pneumoniae in Brazil.

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.

Complete genome analysis of Glutamicibacter creatinolyticus from mare abscess and comparative genomics provide insight of diversity and adaptation for Glutamicibacter.

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.

Re-sequencing and optical mapping reveals misassemblies and real inversions on Corynebacterium pseudotuberculosis genomes.

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.