Bifidobacterium longum subsp. infantis as widespread bacteriocin gene clusters carrier stands out among the Bifidobacterium.

Bifidobacterium is the dominant genus, particularly in the intestinal tract niche of healthy breast-fed infants, and many of these strains have been proven to elicit positive effects on infant development. In addition to its effective antimicrobial activity against detrimental microorganisms, it helps to improve the intestinal microbiota balance. The isolation and identification of bacteriocins from Bifidobacterium have been limited since the mid-1980s, leading to an underestimation of its ability for bacteriocin production. Here, we employed a silicon-based search strategy to mine 354 putative bacteriocin gene clusters (BGCs), most of which have never been reported, from the genomes of 759 Bifidobacterium strains distributed across 9 species. Consistent with previous reports, most Bifidobacterium strains did not carry or carry only a single BGC; however, Bifidobacterium longum subsp. infantis, in contrast to other Bifidobacterium species, carried numerous BGCs, including lanthipeptides, lasso peptides, thiopeptides, and class IId bacteriocins. The antimicrobial activity of the crude bacteriocins and transcription analysis confirmed its potential for bacteriocin biosynthesis. Additionally, we investigated the association of bacteriocins with the phylogenetic positions of their homologs from other genera and niches. In conclusion, this study re-examines a few Bifidobacterium species traditionally regarded as a poor source of bacteriocins. These bacteriocin genes impart a competitive advantage to Bifidobacterium in colonizing the infant intestinal tract. IMPORTANCE Development of the human gut microbiota commences from birth, with bifidobacteria being among the first colonizers of the newborn intestinal tract and dominating it for a considerable period. To date, the genetic basis for the successful adaptation of bifidobacteria to this particular niche remains unclear since studies have mainly focused on glycoside hydrolase and adhesion-related genes. Bacteriocins are competitive factors that help producers maintain colonization advantages without destroying the niche balance; however, they have rarely been reported in Bifidobacterium. The advancement in sequencing methods and bacteriocin databases enables the use of a silicon-based search strategy for the comprehensive and rapid re-evaluation of the bacteriocin distribution of Bifidobacterium. Our study revealed that B. infantis carries abundant bacteriocin biosynthetic gene clusters for the first time, presenting new evidence regarding the competitive interactions of Bifidobacterium in the infant intestinal tract.

Population genomics of Lacticaseibacillus paracasei: pan-genome, integrated prophage, antibiotic resistance, and carbohydrate utilization.

Lacticaseibacillus paracasei has beneficial effects on human health and holds promising potential as a probiotic for use in the development of functional foods, especially dairy products. This species can adapt to a variety of ecological niches and presents fundamental carbohydrate metabolism and tolerance to environmental stresses. However, the population structure, ecology, and antibiotic resistance of Lc. paracasei in diverse ecological niches are poorly understood. Reclassification of Lc. paracasei as a separate species of Lacticaseibacillus has stimulated renewed interest in its research, and a deeper interpretation of it will be important for screening strains beneficial to human health. Here, we collected 121 self-isolated and 268 publicly available Lc. paracasei genomes discussed how genomic approaches have advanced our understanding of its taxonomy, ecology, evolution, diversity, integrated prophage-related element distribution, antibiotic resistance, and carbohydrate utilization. Moreover, for the Lc. paracasei strains isolated in this study, we assessed the inducibility of integrated prophages in their genomes and determined the phenotypes that presented tolerance to multiple antibiotics to provide evidence for safety evaluations of Lc. paracasei during the fermentation processes.

Antibiotic Susceptibility Testing and Establishment of Tentative Species-Specific Microbiological Cut-off Values for Bifidobacteria Isolated from Chinese Population.

Bifidobacteria are commonly used as probiotics in the food industry. The resistance of Bifidobacterium species to antibiotics is closely linked to food safety. However, we still lack a system for the safety evaluation of antibiotic resistance in bifidobacteria, and genus-level microbiological cut-off values remain in use for the determination of phenotypic resistance of Bifidobacterium strains to a given antibiotic. Here, we collected a total of 422 gut-derived bifidobacterial strains isolated from Chinese population and identified their phenotypic resistance profiles against ampicillin, amoxicillin, ciprofloxacin, chloramphenicol, clindamycin, erythromycin, rifampicin, tetracycline, trimethoprim, and vancomycin. Different Bifidobacterium species were found to have varying tolerances to the same antibiotic; therefore, we further established species-specific cut-off values for bifidobacterial species to ten antibiotics. Species-specific rather than genus-specific cut-off values for species belonging to the same taxon were considered more suitable to determine the phenotypic resistance of a Bifidobacterium strain. Moreover, a comprehensive scanning of antibiotic resistance genes in all Bifidobacterium strains tested revealed that the existence of the tetracycline resistance gene tet(W) and the erythromycin/clindamycin resistance gene ErmX is closely related to host phenotypes. Our findings provide guidance and reference values at both phenotype and genotype levels for the safe application of bifidobacteria in the food industry and the development of probiotic resistance evaluation standards.

Clostridium butyricum Reduces Obesity in a Butyrate-Independent Way.

Accumulating evidence from recent studies links the gut microbiota to obesity, and microbiome therapy has been examined as a treatment. Clostridium butyricum (C. butyricum), an intestinal symbiont, protects the host from a range of diseases. Studies have shown a negative correlation between the relative abundance of C. butyricum and a predisposition for obesity. However, the physiological function and material basis of C. butyricum for obesity are unclear. Here, five C. butyricum isolates were administered to mice on a high-fat diet (HFD) to determine their anti-obesity effects. All isolates suppressed the formation and inflammation of subcutaneous fat, and the two effective strains considerably reduced weight gain and ameliorated dyslipidemia, hepatic steatosis, and inflammation. These positive effects were not achieved by increasing the concentration of intestinal butyrate, and the effective strains could not be replaced by sodium butyrate (NaB). We also discovered that oral supplementation with the two most effective strains changed the metabolism of tryptophan and purine and altered the composition of the gut microbiota. In summary, C. butyricum improved the metabolic phenotypes under the HFD by controlling the composition of the gut microbiota and modulating intestinal metabolites, thereby demonstrating its ability to fight obesity and providing a theoretical foundation for microbial preparations production.

Diversity within the species Clostridium butyricum: pan-genome, phylogeny, prophage, carbohydrate utilization, and antibiotic resistance.

AIMS: Clostridium butyricum has been recognized as a strong candidate for the "next generation of probiotics" due to its beneficial roles on humans. Owing to our current understanding of this species is limited, it is imperative to unveil the genetic variety and biological properties of C. butyricum on sufficient strains. METHODS AND RESULTS: We isolated 53 C. butyricum strains and collected 25 publicly available genomes to comprehensively assess the genomic and phenotypic diversity of this species. Average nucleotide identity and phylogeny suggested that multiple C. butyricum strains might share the same niche. Clostridium butyricum genomes were replete with prophage elements, but the CRISPR-positive strain efficiently inhibited prophage integration. Clostridium butyricum utilizes cellulose, alginate, and soluble starch universally, and shows general resistance to aminoglycoside antibiotics. CONCLUSIONS: Clostridium butyricum exhibited a broad genetic diversity from the extraordinarily open pan-genome, extremely convergent core genome, and ubiquitous prophages. In carbohydrate utilization and antibiotic resistance, partial genotypes have a certain guiding significance for phenotypes.

Indole metabolites and colorectal cancer: Gut microbial tryptophan metabolism, host gut microbiome biomarkers, and potential intervention mechanisms.

Tryptophan (Trp) functions in host-disease interactions. Its metabolism is a multi-pathway process. Indole and its derivatives are Trp metabolites unique to the human gut microbiota. Changes in Trp metabolism have also been detected in colorectal cancer (CRC). Here, combined with the existing CRC biomarkers, we ascribed it to the altered bacteria having the indole-producing ability by making a genomic prediction. We also reviewed the anti-inflammatory and possible anti-cancer mechanisms of indoles, including their effects on tumor cells, the ability to repair the gut barrier, regulation of the host immune system, and provide resistance against oxidative stress. Indole and its derivatives, along with related bacteria, could be targeted as auxiliary strategies to restrain cancer development in the future.

Uncovering the specificity and predictability of tryptophan metabolism in lactic acid bacteria with genomics and metabolomics.

Tryptophan is metabolized by microorganisms into various indole derivatives that have been proven to alleviate diseases and promote human health. Lactic acid bacteria (LAB) are a broad microbial concept, some of which have been developed as probiotics. However, the capacity of most LAB to metabolize tryptophan is unknown. In this study, the aim is to reveal the rule of tryptophan metabolism in LAB by multi-omics. The findings showed that LAB were rich in genes for tryptophan catabolism and that multiple genes were shared among LAB species. Although the number of their homologous sequences was different, they could still form the same metabolic enzyme system. The metabolomic analysis revealed that LAB were capable of producing a variety of metabolites. Strains belonging to the same species can produce the same metabolites and have similar yields. A few strains showed strain-specificity in the production of indole-3-lactic acid (ILA), indole-3-acetic acid, and 3-indolealdehyde (IAld). In the genotype-phenotype association analysis, the metabolites of LAB were found to be highly consistent with the outcomes of gene prediction, particularly ILA, indole-3-propionic acid, and indole-3-pyruvic acid. The overall prediction accuracy was more than 87% on average, which indicated the predictability of tryptophan metabolites of LAB. Additionally, genes influenced the concentration of metabolites. The levels of ILA and IAld were significantly correlated with the numbers of aromatic amino acid aminotransferase and amidase, respectively. The unique indolelactate dehydrogenase in Ligilactobacillus salivarius was the primary factor contributing to its large production of ILA. In summary, we demonstrated the gene distribution and production level of tryptophan metabolism in LAB and explored the correlation between genes and phenotypes. The predictability and specificity of the tryptophan metabolites in LAB were proven. These results provide a novel genomic method for the discovery of LAB with tryptophan metabolism potential and offer experimental data for probiotics that produce specific tryptophan metabolites.

Phenotype-genotype analysis of Latilactobacills curvatus from different niches: Carbohydrate metabolism, antibiotic resistance, bacteriocin, phage fragments and linkages with CRISPR-Cas systems.

The potential probiotic function of Latilactobacills curvatus has attracted the attention of researchers. To explore the differences in the genomes of L. curvatus, nine strains were isolated from various sources, including feces and fermented vegetables and compared with 25 strains from the NCBI database. The findings indicated that the average genome size, GC content, and CDS of L. curvatus were 1.94 MB, 41.9%, and 1825, respectively. Its core genome is associated with transcription, translation, carbohydrate transport and metabolism, and defense functions. The pan-genome of L. curvatus was in a closed state. The genetic diversity of L. curatus is mainly manifested in its ability to use carbohydrates, antibiotic resistance, bacteriocin operon, and polymeric regularly interspaced short palindromic repeats (CRISPR)-Cas for bacterial immunity. The CRISPR system of 34 strains of L. curvatus was predominantly found to be of the IIA type with a few IIC and IE types. These findings will contribute to a better understanding of this species.

Characterization of CRISPR-Cas systems in Bifidobacterium breve.

The clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) system is an important adaptive immune system for bacteria to resist foreign DNA infection, which has been widely used in genotyping and gene editing. To provide a theoretical basis for the application of the CRISPR-Cas system in Bifidobacterium breve, the occurrence and diversity of CRISPR-Cas systems were analysed in 150 B. breve strains. Specifically, 47 % (71/150) of B. breve genomes possessed the CRISPR-Cas system, and type I-C CRISPR-Cas system was the most widely distributed among those strains. The spacer sequences present in B. breve can be used as a genotyping marker. Additionally, the phage assembly-related proteins were important targets of the type I-C CRISPR-Cas system in B. breve, and the protospacer adjacent motif sequences were further characterized in B. breve type I-C system as 5'-TTC-3'. All these results might provide a molecular basis for the development of endogenous genome editing tools in B. breve.

A low molecular weight brown algae Laminaria japonica glycan modulation of gut microbiota and body weight in mice.

Brown algae glycan from Laminaria japonica (LJNP) is a heterogeneous glycan with two apparent molecular weights of 1.1 and 37.3 kDa, and is mainly composed of α ß-glucan and a few fucosyl residues. To explore the regulation of gut microbiota and the host, LJNP and 2'-fucosyllactose (2'FL) were compared to investigate their effect on mice via oral administration. Using metagenomic and metabolomic analyses, we found that 2'FL mainly relied on Adlercreutzia equolifaciens and Akkermansia muciniphila to improve gut amino acid and bile acid metabolism, whereas LJNP mainly drove Bacteroides vulgatus and Bacteroides uniformis to regulate gut amino acid metabolism and glycometabolism. Moreover, LJNP showed a weight loss effect and better protection of the intestinal barrier than 2'FL. We further employed LJNP and 2'FL on a germ-free mice model. Interestingly, the body weight management was not microbiome mediated. This study showed that LJNP can ameliorate the intestinal barrier through modulation of the gut microbiota, maintain the blood glucose level, and regulate body weight and the antioxidant function. Although the benefits of LJNP on host health were partly revealed, mechanisms such as the weight loss effect require further study.

Evaluation of Tetracycline Resistance and Determination of the Tentative Microbiological Cutoff Values in Lactic Acid Bacterial Species.

Lactic acid bacteria (LAB) are widely used as probiotics in the food industry owing to their beneficial effects on human health. However, numerous antibiotic resistance genes have been found in LAB strains, especially tetracycline resistance genes. Notably, the potential transferability of these genes poses safety risks. To comprehensively evaluate tetracycline resistance in LAB, we determined the tetracycline susceptibility patterns of 478 LAB strains belonging to four genera and eight species. By comparing phenotypes with genotypes based on genome-wide annotations, five tetracycline resistance genes, tet(M), tet(W/N/W), tet(L), tet(S), and tet(45), were detected in LAB. Multiple LAB strains without tetracycline resistance genes were found to be resistant to tetracycline at the currently recommended cutoff values. Thus, based on the minimum inhibitory concentrations of tetracycline for these LAB strains, the species-specific microbiological cutoff values for Lactobacillus (para)gasseri, Lactobacillus johnsonii, and Lactobacillus crispatus to tetracycline were first developed using the Turnidge, Kronvall, and eyeball methods. The cutoff values for Lactiplantibacillus plantarum were re-established and could be used to better distinguish susceptible strains from strains with acquired resistance. Finally, we verified that these five genes play a role in tetracycline resistance and found that tet(M) and tet(W/N/W) are the most widely distributed tetracycline resistance genes in LAB.

Comparative Genomic Analysis of Bifidobacterium bifidum Strains Isolated from Different Niches.

The potential probiotic benefits of Bifidobacterium bifidum have received increasing attention recently. We used comparative genomic analysis to explore the differences in the genome and the physiological characteristics of B. bifidum isolated from the fecal samples of Chinese adults and infants. The relationships between genotypes and phenotypes were analyzed to assess the effects of isolation sources on the genetic variation of B. bifidum. The phylogenetic tree results indicated that the phylogeny of B. bifidum may be related to the geographical features of its isolation source. B. bifidum was found to have an open pan-genome and a conserved core genome. The genetic diversity of B. bifidum is mainly reflected in carbohydrate metabolism- and immune/competition-related factors, such as the glycoside hydrolase gene family, bacteriocin operons, antibiotic resistance genes, and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas. Additionally, the type III A CRISPR-Cas system was discovered in B. bifidum for the first time. B. bifidum strains exhibited niche-specific characteristics, and the results of this study provide an improved understanding of the genetics of this species.

Comprehensive Scanning of Prophages in Lactobacillus: Distribution, Diversity, Antibiotic Resistance Genes, and Linkages with CRISPR-Cas Systems.

Prophage integration, release, and dissemination exert various effects on host bacteria. In the genus Lactobacillus, they may cause bacteriophage contamination during fermentation and even regulate bacterial populations in the gut. However, little is known about their distribution, genetic architecture, and relationships with their hosts. Here, we conducted prophage prediction analysis on 1,472 genomes from 16 different Lactobacillus species and found prophage fragments in almost all lactobacilli (99.8%), with 1,459 predicted intact prophages identified in 64.1% of the strains. We present an uneven prophage distribution among Lactobacillus species; multihabitat species retained more prophages in their genomes than restricted-habitat species. Characterization of the genome features, average nucleotide identity, and landscape visualization presented a high genome diversity of Lactobacillus prophages. We detected antibiotic resistance genes in more than 10% of Lactobacillus prophages and validated that the occurrence of resistance genes conferred by prophage integration was possibly associated with phenotypic resistance in Lactobacillus plantarum. Furthermore, our broad and comprehensive examination of the distribution of CRISPR-Cas systems across the genomes predicted type I and type III systems as potential antagonistic elements of Lactobacillus prophage. IMPORTANCE Lactobacilli are inherent microorganisms in the human gut and are widely used in the food processing industries due to their probiotic properties. Prophages were reportedly hidden in numerous Lactobacillus genomes and can potentially contaminate entire batches of fermentation or modulate the intestinal microecology once they are released. Therefore, a comprehensive scanning of prophages in Lactobacillus is essential for the safety evaluation and application development of probiotic candidates. We show that prophages are widely distributed among lactobacilli; however, intact prophages are more common in multihabitat species and display wide variations in genome feature, integration site, and genomic organization. Our data of the prophage-mediated antibiotic resistance genes (ARGs) and the resistance phenotype of lactobacilli provide evidence for deciphering the putative role of prophages as vectors of the ARGs. Furthermore, understanding the association between prophages and CRISPR-Cas systems is crucial to appreciate the coevolution of phages and Lactobacillus.

The Diversity of the CRISPR-Cas System and Prophages Present in the Genome Reveals the Co-evolution of Bifidobacterium pseudocatenulatum and Phages.

Diverse CRISPR-Cas systems constitute an indispensable part of the bacterial adaptive immune system against viral infections. However, to escape from this immune system, bacteriophages have also evolved corresponding anti-defense measures. We investigated the diversity of CRISPR-Cas systems and the presence of prophages in the genomes of 66 Bifidobacterium pseudocatenulatum strains. Our findings revealed a high occurrence of complete CRISPR-Cas systems (62%, 41/66) in the B. pseudocatenulatum genomes. Subtypes I-C, I-U and II-A, were found to be widespread in this species. No significant association was found between the number of bacterial CRISPR spacers and its host's age. This study on prophages within B. pseudocatenulatum genomes revealed that prophage genes related to distinct functional modules became degraded at different levels, indicating that these prophages were not likely to enter lytic cycle spontaneously. Further, the evolutionary analysis of prophages in this study revealed that they might be derived from different phage ancestors. Notably, self-targeting phenomenon within B. pseudocatenulatum and Anti-CRISPR (Acr) coding genes in prophages was observed. Overall, our results indicate that the competition between B. pseudocatenulatum and phages is a major driving factor for the genomic diversity of both partners.

Identification, characterization, and phylogenetic analysis of eight new inducible prophages in Lactobacillus.

Lysogenic bacterial strains abound in the Lactobacillus genus and contain dormant prophages inserted within their genomes. To evaluate the prophage-induction potential of the Lactobacillus strains of six species, 142 randomly selected strains from these species were induced with Mitomycin C. Eight newly-induced phages were identified and found to be diverse in morphology. Among the six species assessed, Lactobacillus plantarum and Lactobacillus rhamnosus strains were generally insensitive to induction. The genomic characterizations of eight phages were performed via whole genome sequencing and protein prediction. Meanwhile, genome comparison of the induced phages and predicted prophages demonstrated that the prediction software PHASTER can accurately locate major prophage regions in Lactobacillus. A phylogenetic tree of the Lactobacillus phage population was constructed to obtain further insights into the clustering of individuals, two major groups were found, one of which consisted mostly of L. plantarum virulent phages, the other was represented by Lactobacillus casei/paracasei temperate phages. Finally, it was confirmed via genomic collinear analysis, which seven of the eight Lactobacillus temperate phages were newly discovered, and two Lactobacillus brevis temperate phages belonged to a novel lineage.