Infant temperament characteristics play a critical role in children's developmental pathways and can predict adulthood psychopathology. The diversity and composition of the gut microbiota are associated with human temperament in both adults and young children. However, the relationship between the gut microbiota and temperament in 12-month-old infants is rarely studied; this developmental period is when temperament reaches a relatively stable stage. We used high-throughput sequencing methods to explore whether temperament characteristics were associated with gut microbiota diversity and composition. Infants' fecal samples were collected at 12 months of age for the gut microbiota analysis. Based on the primary caregivers' reports, infants' temperaments were measured using the Infant Behavior Questionnaire-revised (IBQ-R). This study included 51 infants, including 20 boys and 31 girls, with a mean age of 12.25 months. Results showed that soothability was positively correlated with maternal education level (β = 0.29, p = 0.043, adjust p = 0.025) and the abundance of Bifidobacterium genera (β = 0.62, p = 0.004, adjust p = 0.002). Conversely, cuddliness was negatively correlated with the abundance of Hungatella genera. There was no significant difference in temperament based on gender. This study demonstrated that gut microbiota composition was associated with temperament in 12-month-old infants. These results point to the importance of gut microbiota balance. Future studies on the mechanisms behind the gut microbiota affecting temperament are warranted.

(1) Background: The human gut microbiota at early life is shaped by numerous factors, especially factors from mothers, which have huge influence on infants' gut microbiotas. The aim of this study was to investigate the effect of maternal adherence to Chinese traditional postpartum practices of "doing the month" on the development of infant gut microbiota at 6-month postpartum. (2) Methods: A cohort of 62 Chinese women at late pregnancy was recruited from a tertiary general hospital in a central region of China. The participants and their babies were followed up to 6 months postpartum. Finally, 50 mother-infant dyads were enrolled in the study. Women's adherence to the traditional postpartum practices was measured by adherence to doing the month practices (ADP). Infant fecal samples were collected at six months of age and were analyzed using 16S rRNA V3 and V4 gene region sequences. (3) Results: Ruminococcus gnavus was significantly less abundant in infants whose mothers had a better adherence to the traditional postpartum practices of "doing the month." Infants receiving Clostridium-butyricum during the first month after delivery had a significant dominance of Escherichia/Shigella. (4) Conclusions: Adherence to the traditional postpartum practices of "doing the month" can impact an infant's gut microbiota at 6 months of age. Infants receiving probiotics during the first month after delivery had a significant dominance of opportunistic pathogens.

Bifidobacterium longum is one of the most widely distributed and abundant Bifidobacterium in the human intestine, and has been proven to have a variety of physiological functions. In this study, 80 strains of B. longum isolated from human subjects were classified into subspecies by ANI and phylogenetic analyses, and the functional genes were compared. The results showed that there were significant differences in carbohydrate metabolism between the two subspecies, which determined their preference for human milk oligosaccharides or plant-derived carbohydrates. The predicted exopolysaccharide (EPS) gene clusters had large variability within species but without difference at the subspecies level. Four subtype CRISPR-Cas systems presented in B. longum, while the subtypes I-U and II-C only existed in B. longum subsp. longum. The bacteriocin operons in B. longum subsp. infantis were more widely distributed compared with B. longum subsp. longum. In conclusion, this study revealed the similarities and differences between B. longum subsp. infantis and B. longum subsp. longum, which could provide a theoretical basis for further exploring the probiotic characteristics of B. longum.

Gut microbiota composition and functionality are closely linked to host health. In this study, the fecal microbiota and bifidobacterial communities of 111 healthy volunteers from four regions of China of varying age profiles (Child, 1-5 years; Young, 18-50 years; Elder, 60-80 years; Longevity, ≥90 years) were investigated via high-throughput sequencing. Canonical analysis revealed that the gut microbiota, as well as bifidobacteria profiles of the subjects, clustered according to their regions and age. Eight genera were shared among all subjects, however, certain genera distributed differently in subjects grouped by region and age. Faecalibacterium was enriched in samples from Zhongxiang, unclassified Ruminococcaceae and Christensenellaceae were enriched in the Longevity group, and Bifidobacterium was enriched in Child. Within Bifidobacterium, B. longum was the most abundant species in almost all samples except for Child, in which B. pseudocatenulatum was the most abundant. Additionally, the abundances of B. adolescentis and B. dentium were lower in Child. In conclusion, our results suggest that geography and age affect the structure of the gut microbiota, as well as Bifidobacterium composition, and this variation may greatly associate with the metabolic and immune changes that occur during the process of aging.

Lactobacillus salivarius has drawn attention because of its promising probiotic functions. Tolerance to the gastrointestinal tract condition is crucial for orally administrated probiotics to exert their functions. However, previous studies of L. salivarius have only focused on the bile salt resistance of particular strains, without uncovering the common molecular mechanisms of this species. Therefore, in this study, we expanded our research to 90 L. salivarius strains to explore their common functional genes for bile salt resistance. First, the survival rates of the 90 L. salivarius strains in 0.3% bile salt solutions were determined. Comparative genomics analysis was then performed to screen for the potential functional genes related to bile salt tolerance. Next, real-time polymerase chain reaction and gene knockout experiments were conducted to further verify the tolerance-related functional genes. The results indicated that the strain-dependent bile salt tolerance of L. salivarius was mainly associated with four peptidoglycan synthesis-related genes, seven phosphotransferase system-related genes, and one chaperone-encoding gene involved in the stress response. Among them, the GATase1-encoding gene showed the most significant association with bile salt tolerance. In addition, four genes related to DNA damage repair and substance transport were redundant in the strains with high bile salt tolerance. Besides, cluster analysis showed that bile salt hydrolases did not contribute to the bile salt tolerance of L. salivarius. In this study, we determined the global regulatory genes, including LSL_1568, LSL_1716 and LSL_1709, for bile salt tolerance in L. salivarius and provided a potential method for the rapid screening of bile salt-tolerant L. salivarius strains, based on PCR amplification of functional genes.

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.

(1) Background: Constipation is a common condition that affects the health and the quality of life of patients. Recent studies have suggested that the gut microbiome is associated with constipation, but these studies were mainly focused on a single research cohort. Thus, we aimed to construct a classification model based on fecal bacterial and identify the potential gut microbes' biomarkers. (2) Methods: We collected 3056 fecal amplicon sequence data from five research cohorts. The data were subjected to a series of analyses, including alpha- and beta-diversity analyses, phylogenetic profiling analyses, and systematic machine learning to obtain a comprehensive understanding of the association between constipation and the gut microbiome. (3) Results: The alpha diversity of the bacterial community composition was higher in patients with constipation. Beta diversity analysis evidenced significant partitions between the two groups on the base of gut microbiota composition. Further, machine learning based on feature selection was performed to evaluate the utility of the gut microbiome as the potential biomarker for constipation. The Gradient Boosted Regression Trees after chi2 feature selection was the best model, exhibiting a validation performance of 70.7%. (4) Conclusions: We constructed an accurate constipation discriminant model and identified 15 key genera, including Serratia, Dorea, and Aeromonas, as possible biomarkers for constipation.

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.

The infant gut microbiota plays a critical role in early life growth and derives mainly from maternal gut and breast milk. This study aimed to analyze the differences in the gut microbiota, namely Bifidobacterium and Lactobacillus communities at species level among breast milk as well as maternal and infant feces at different time points after delivery. Fifty-one mother-infant pairs from Indonesia were recruited, and the breast milk and maternal and infant feces were collected and analyzed by high throughput sequencing (16S rRNA, Bifidobacterium groEL and Lactobacillus groEL genes). PCoA results showed bacterial composition was different among breast milk and maternal and infant feces within the first two years. The abundance of Bifidobacterium and Bacteroides were significantly higher in infant feces compared to their maternal feces from birth to two years of age, and maternal breast milk within six months after birth (p < 0.05), whereas the abundance of Blautia, Prevotella, and Faecalibacterium was higher in maternal feces compared to that in breast milk within six months and infant feces within one year after birth, respectively (p < 0.05). The relative abundances of Bacteroides and Lactobacillus was higher and lower in infant feces compared to that in maternal feces only between one and two years of age, respectively (p < 0.05). For Bifidobacterium community at species level, B. adolescentis, B. ruminantium, B. longum subsp. infantis, B. bifidum, and B. pseudolongum were identified in all samples. However, the profile of Bifidobacterium was different between maternal and infant feces at different ages. The relative abundances of B. adolescentis and B. ruminantium were higher in maternal feces compared to those in infant feces from birth to one year of age (p < 0.05), while the relative abundances of B. longum subsp. infantis and B. bifidum were higher in infant feces compared to those in maternal feces beyond three months, and the relative abundance of B. pseudolongum was only higher in infant feces between three and six months (p < 0.05). For Lactobacillus community, L. paragasseri showed higher relative abundance in infant feces when the infant was younger than one year of age (p < 0.05). This study showed bacterial composition at the genus level and Bifidobacterium and Lactobacillus communities at the species level were stage specific in maternal breast milk as well as and maternal and infant feces.

Bifidobacteria are among the first colonizers of the human gastrointestinal tract. Different bacterial species use different mechanisms for utilization of various carbon sources in order to establish themselves in the complex microbial ecosystem of the gut. However, these mechanisms still need to be explored. Here, a large gene-phenotype correlation analysis was carried out to explore the metabolic and genetic diversity of bifidobacterial carbohydrate utilization abilities. In this study, we used 21 different carbohydrates to determine the growth phenotypes, the distribution of glycoside hydrolases (GHs), and gene clusters related to the utilization of multiple carbon sources in six human-residential Bifidobacterium species. Five carbohydrates significantly stimulated growth of almost all strains, while the remaining sugars exhibited species- and strain-specificity. Correspondingly, different Bifidobacterium species also had specific GHs involved in fermentation of plant or host glycans. Moreover, we analyzed several carbohydrate utilization gene clusters, such as 2-fucosyllactose (2'FL), sialic acid (SA), and fructooligosaccharide (FOS). In summary, by using 217 bifidobacterial strains and a wide range of growth substrates, our research revealed inter- and intra-species differences in bifidobacterial in terms of carbohydrate utilization. The findings of this study are useful for the process of developing prebiotics for optimum growth of probiotics, especially Bifidobacterium species.

Polyethylene glycol (PEG) is one of the most commonly used bowel cleansing methods. Although the safety of PEG for bowel cleansing has been proven, its impact on intestinal microbiota has not been clearly explained, especially in terms of the dynamic changes in intestinal microbiota after PEG bowel cleansing, and there are no consistent results. In this study, stool samples were collected from 12 participants at six time points before and after bowel cleansing. We obtained data on the microbiota of these samples using 16S rRNA gene sequencing and analysis. The data revealed that the structure and composition of the microbiota changed greatly approximately 7 d after intestinal cleansing. The analysis of the dynamic changes in the microbiota showed that the change was most significant at day 3, but the internal structure of the microbiota was similar to that before bowel cleansing. A comparison of the most significantly changed microbiota at different time points before and after bowel cleansing revealed four bacteria: Bacteroides, Roseburia, Eubacterium, and Bifidobacterium. We also established a humanized mouse model to simulate human bowel cleansing using PEG. The results showed that the mouse model achieved similar effects to human bowel cleansing, but its recovery speed was one stage earlier than that of humans. These findings suggest that the intestinal microbiota after bowel cleansing initially underwent a short-term change and then actively returned to its initial status. The results on key bacteria and establishment of mouse models can provide a reference for subsequent research on bowel cleansing.

Biofilm formation has evolved as an adaptive strategy for bacteria to cope with harsh environmental conditions. Currently, little is known about the molecular mechanisms of biofilm formation in bifidobacteria. A time series transcriptome sequencing analysis of both biofilm and planktonic cells of Bifidobacterium longum FGSZY16M3 was performed to identify candidate genes involved in biofilm formation. Protein-protein interaction network analysis of 1296 differentially expressed genes during biofilm formation yielded 15 clusters of highly interconnected nodes, indicating that genes related to the SOS response (dnaK, groS, guaB, ruvA, recA, radA, recN, recF, pstA, and sufD) associated with the early stage of biofilm formation. Genes involved in extracellular polymeric substances were upregulated (epsH, epsK, efp, frr, pheT, rfbA, rfbJ, rfbP, rpmF, secY and yidC) in the stage of biofilm maturation. To further investigate the genes related to biofilm formation, weighted gene co-expression network analysis (WGCNA) was performed with 2032 transcript genes, leading to the identification of nine WGCNA modules and 133 genes associated with response to stress, regulation of gene expression, quorum sensing, and two-component system. These results indicate that biofilm formation in B. longum is a multifactorial process, involving stress response, structural development, and regulatory processes.

In recent years, Blautia has attracted attention for its role in ameliorating host diseases. In particular, Blautia producta DSM 2950 has been considered a potential probiotic due to its ability to mitigate inflammation in poly(I:C) induced HT-29 cells. Thus, to promote the development of indigenous intestinal microorganisms with potential probiotic function, we conducted a comprehensive experimental analysis of DSM 2950 to determine its safety. This comprised a study of its potential virulence genes, antibiotic resistance genes, genomic islands, antibiotic resistance, and hemolytic activity and a 14-day test of its acute oral toxicity in mice. The results indicated no toxin-related virulence genes in the DSM 2950 genome. Most of the genomic islands in DSM 2950 were related to metabolism, rather than virulence expression. DSM 2950 was sensitive to most of the tested antibiotics but was tolerant of treatment with kanamycin, neomycin, clindamycin, or ciprofloxacin, probably because it possessed the corresponding antibiotic resistance genes. Oral acute toxicity tests indicated that the consumption of DSM 2950 does not cause toxic side effects in mice. Overall, the safety profile of DSM 2950 confirmed that it could be a candidate probiotic for use in food and pharmaceutical preparations.

We adopted a bioinformatics-based technique to identify strain-specific markers, which were then used to quantify the abundances of three distinct B. longum sup. longum strains in fecal samples of humans and mice. A pangenome analysis of 205 B. longum sup. longum genomes revealed the accumulation of considerable strain-specific genes within this species; specifically, 28.7% of the total identified genes were strain-specific. We identified 32, 14, and 49 genes specific to B. longum sup. longum RG4-1, B. longum sup. longum M1-20-R01-3, and B. longum sup. longum FGSZY6M4, respectively. After performing an in silico validation of these strain-specific markers using a nucleotide BLAST against both the B. longum sup. longum genome database and an NR/NT database, RG4-1_01874 (1331 bp), M1-20-R01-3_00324 (1745 bp), and FGSZY6M4_01477 (1691 bp) were chosen as target genes for strain-specific quantification. The specificities of the qPCR primers were validated against 47 non-target microorganisms and fecal baseline microbiota to ensure that they produced no PCR amplification products. The performance of the qPCR primer-based analysis was further assessed using fecal samples. After oral administration, the target B. longum strains appeared to efficiently colonize both the human and mouse guts, with average population levels of >108 CFU/g feces. The bioinformatics pipeline proposed here can be applied to the quantification of various bacterial species.

The production of bacteriocin is considered to be a probiotic trait of lactic acid bacteria (LAB). However, not all strains of LAB harbour bacteriocin genes, even within the same species. Moreover, the effects of bacteriocins on the host gut microbiota and on host physiological indicators are rarely studied. This study evaluated the effects of the bacteriocin-producing Lactobacillus acidophilus strain JCM1132 and its non-producing spontaneous mutant, L. acidophilus CCFM720, on the physiological statuses and gut microbiota of healthy mice. Mice that received the bacteriocin-producing strain JCM1132 exhibited reduced water and food intake. Furthermore, the administration of these strains induced significant changes in the compositional abundance of faecal microbiota at the phylum and genus levels, and some of these changes were more pronounced after one week of withdrawal. The effects of CCFM720 treatment on the gut microbiota seemed to favour the prevention of metabolic diseases to some extent. However, individuals that received JCM1132 treatment exhibited weaker inflammatory responses than those that received CCFM720 treatment. Our results indicate that treatment with bacteriocin-producing or non-producing strains can have different effects on the host. Accordingly, this trait should be considered in the applications of LAB.

Lactiplantibacillus plantarum CCFM8724 is a probiotic with the potential to prevent dental caries in vitro and in vivo. To explore the effects of this probiotic at inhibiting Streptococcus mutans-Candida albicans mixed-species biofilm and preventing dental caries, multi-omics, including metabolomics and transcriptomics, was used to investigate the regulation of small-molecule metabolism during biofilm formation and the gene expression in the mixed-species biofilm. Metabolomic analysis revealed that some carbohydrates related to biofilm formation, such as sucrose, was detected at lower levels due to the treatment with the L. plantarum supernatant. Some sugar alcohols, such as xylitol and sorbitol, were detected at higher levels, which may have inhibited the growth of S. mutans. In transcriptomic analysis, the expression of the virulence genes of C. albicans, such as those that code agglutinin-like sequence (Als) proteins, was affected. In addition, metabolomics coupled with a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and RNA-seq revealed that the L. plantarum supernatant had an active role in sugar metabolism during the formation of the S. mutans-C. albicans mixed-species biofilm, and the L. plantarum supernatant was also related to carbohydrate utilization, glucan biosynthesis, and mycelium formation. Hence, L. plantarum CCFM8724 decreased the mixed-species biofilm mass from the perspective of gene expression and metabolic reprogramming. Our results provide a rationale for evaluating L. plantarum CCFM8724 as a potential oral probiotic for inhibiting cariogenic pathogen biofilm formation and improving dental caries.

Vascular reactive oxygen species (ROS) play an essential role in cardiovascular diseases and the antioxidative effects of probiotics have been widely reported. To screen the probiotic strains that may prevent cardiovascular diseases, we tested the antioxidative effects of supernatants of different Bifidobacterium and Lactobacillus strains on A7R5 cells. Preincubation with supernatants of B. longum CCFM752, L. plantarum CCFM1149, or L. plantarum CCFM10 significantly suppressed the angiotensin II-induced increases in ROS levels and increased catalase (CAT) activity in A7R5, whereas CCFM752 inhibited NADPH oxidase activation and CCFM1149 enhanced the intracellular superoxide dismutase (SOD) activity simultaneously. Treatment with CCFM752, CCFM1149, or CCFM10 supernatants had no significant impact on transcriptional levels of Cat, Sod1, Sod2, Nox1, p22phox, or p47phox, but altered the overall transcriptomic profile and the expression of genes relevant to protein biosynthesis, and up-regulated the 60S ribosomal protein L7a (Rpl7a). A positive correlation between Rpl7a expression and intracellular CAT activity implied that Rpl7a may participate in CAT synthesis in A7R5. Supernatant of CCFM752 could also down-regulate the expression of NADPH oxidase activator 1 (Noxa1) and angiotensinogen in A7R5. Collectively, the probiotic strains CCFM752, CCFM1149, and CCFM10 exhibited antioxidative attributes on A7R5 cells and might help to reduce the risk of cardiovascular diseases.

Artificial fishery habitats have been extensively used for fishery resource protection and water habitat restoration, and they could attract a large number of omnivorous fishes to gather together. This study intended to reveal the relationship between bacterial communities in the habitats (water and sediment) and intestines of omnivorous fishes (Oreochromis mossambicus, Toxabramis houdemeri and Hemiculter leucisculus). Therefore, we investigated the bacterial communities of samples collected from intestines, water, and sediments in artificial fishery habitats via 16S rRNA metabarcoding high-throughput sequencing technology. The results showed that there were significant differences in the composition, core indicators, diversity and prediction functions in water, sediments, and intestinal microbial communities of the three omnivorous fish. The microbial diversities were significantly higher in habitats than in intestines. The analysis of similarity (ANOSIM) and nonmetric multidimensional scaling (NMDS) results indicated that the intestine microbial communities (T. houdemeri and H. leucisculus) were more similar to the water microbiota, but the intestine microbial communities (O. mossambicus) were more similar to the sediments. Source tracking analysis also confirmed that the contribution of habitat characteristics to omnivorous fish intestinal microorganisms was different; the sediment had a greater contribution than water to the intestinal microbiota of O. mossambicus, which was consistent with their benthic habit. Moreover, the functional prediction results showed that there were unique core indicators and functions between the bacterial community of habitats and intestines. Altogether, these results can enhance our understanding of the bacterial composition and functions about omnivorous fish intestines and their living with habitats, which have provided new information for the ecological benefits of artificial fishery habitats from the perspective of bacterial ecology and contributed to apply artificial fishery habitats in more rivers.

Eating disorders are directly or indirectly influenced by gut microbiota and innate immunity. Probiotics have been shown to regulate gut microbiota and stimulate immunity in a variety of species. In this study, three kinds of probiotics, namely, Lactobacillus plantarum, Lactobacillus rhamnosus and Clostridium butyricum, were selected for the experiment. The results showed that the addition of three probiotics at a concentration of 108 colony forming unit/mL to the culture water significantly increased the ratio of the pellet feed recipients and survival rate of mandarin fish (Siniperca chuatsi) under pellet-feed feeding. In addition, the three kinds of probiotics reversed the decrease in serum lysozyme and immunoglobulin M content, the decrease in the activity of antioxidant enzymes glutathione and catalase and the decrease in the expression of the appetite-stimulating regulator agouti gene-related protein of mandarin fish caused by pellet-feed feeding. In terms of intestinal health, the three probiotics reduced the abundance of pathogenic bacteria Aeromonas in the gut microbiota and increased the height of intestinal villi and the thickness of foregut basement membrane of mandarin fish under pellet-feed feeding. In general, the addition of the three probiotics can significantly improve eating disorders of mandarin fish caused by pellet feeding.

Lactobacillus acidophilus is a common kind of lactic acid bacteria usually found in the human gastrointestinal tract, oral cavity, vagina, and various fermented foods. At present, many studies have focused on the probiotic function and industrial application of L. acidophilus. Additionally, dozens of L. acidophilus strains have been genome sequenced, but there has been no research to compare them at the genomic level. In this study, 46 strains of L. acidophilus were performed comparative analyses to explore their genetic diversity. The results showed that all the L. acidophilus strains were divided into two clusters based on ANI values, phylogenetic analysis and whole genome comparison, due to the difference of their predicted gene composition of bacteriocin operon, CRISPR-Cas systems and prophages mainly. Additionally, L. acidophilus was a pan-genome open species with a difference in carbohydrates utilization, antibiotic resistance, EPS operon, surface layer protein operon and other functional gene composition. This work provides a better understanding of L. acidophilus from a genetic perspective, and offers a frame for the biotechnological potentiality of this species.