The Chromosomal Gene Manipulation Method for Lactobacillus delbrueckii subsp. bulgaricus Using a Conjugative Shuttle Vector pGMß1.

Lactobacillus bulgaricus is an industrial strain that has been used in the dairy products since ancient times. Because of the difficulty of chromosomal gene manipulation, there have been few reports of gene deletion, insertion, or replacement. We have developed a system that enables chromosomal gene manipulation of L. bulgaricus using a conjugal transfer vector and easily vector construction in E. coli. As an example, we have deleted a regulatory gene for the extracellular polysaccharide synthesis of L. bulgaricus to elucidate the function of the gene in question. Methods for constructing vectors for chromosomal integration, conjugation experiment, and obtaining deletion strains by double recombination were presented in detail. This conjugative shuttle vector, pGMß1, has been deposited at Addgene ( https://www.addgene.org )and can be used by anyone for academic purposes.

Comparative physiological and transcriptomic analysis of sono-biochemical control over post-acidification of Lactobacillus delbrueckii subsp. bulgaricus.

Thermosonication (UT) prestress treatments combining with varied fermentation patterns has been revealed as an effective method to regulate post-acidification as exerted by Lactobacillus delbrueckii subsp. bulgaricus (L. delbrueckii), but sono-biochemical controlling mechanisms remain elusive. This study employed physiological and transcriptomic analysis to explore the response mechanism of L. delbrueckii to UT-induced microstress (600 W, 33 kHz, 10 min). UT stress-induced inhibition of acidification of L. delbrueckii during (post)-fermentation was first confirmed, relying on the UT process parameters such as stress exposure duration and UT power. The significantly enhanced membrane permeability in cells treated by 600 W for 10 min than the microbes stressed by 420 W for 20 min suggested the higher dependence of UT-derived stresses on the treatment durations, relative to the ultrasonic powers. In addition, ultrasonication treatment-induced changes in cell membrane integrity enhanced and/or disrupted permeability of L. delbrueckii, resulting in an imbalance in intracellular conditions associated with corresponding alterations in metabolic behaviors and fermentation efficiencies. UT-prestressed inoculum exhibited a 21.46% decrease in the membrane potential during the lag phase compared to untreated samples, with an intracellular pH of 5.68 ± 0.12, attributed to the lower activities of H+-ATPase and lactate dehydrogenase due to UT stress pretreatments. Comparative transcriptomic analysis revealed that UT prestress influenced the genes related to glycolysis, pyruvate metabolism, fatty acid synthesis, and ABC transport. The genes encoding 3-oxoacyl-[acyl-carrier-protein] reductases I, II, and III, CoA carboxylase, lactate dehydrogenase, pyruvate oxidase, glucose-6-phosphate isomerase, and glycerol-3-phosphate dehydrogenase were downregulated, thus identifying the relevance of the UT microstresses-downregulated absorption and utilization of carbohydrates with the attenuated fatty acid production and energy metabolisms. These findings could contribute to provide a better understanding of the inactivated effects on the post-acidification of L. delbrueckii by ultrasonic pretreatments, thus providing theoretical basis for the targeted optimization of acidification inhibition efficiencies for yogurt products during chilled preservation processes.

Multidrug resistance profile in Lactobacillus delbrueckii: a food industry species with probiotic properties.

Lactobacillus delbrueckii, a widely used lactic acid bacterium in the food industry, has been studied for its probiotic properties and reservoir of antibiotic-resistant genes, raising safety concerns for probiotic formulations and fermented products. This review consolidates findings from 60 articles published between 2012 and 2023, focusing on the global antibiotic resistance profile and associated genetic factors in L. delbrueckii strains. Resistance to aminoglycosides, particularly streptomycin, kanamycin, and gentamicin, as well as resistance to glycopeptides (vancomycin), fluoroquinolones (ciprofloxacin), and tetracyclines was predominant. Notably, although resistance genes have been identified, they have not been linked to mobile genetic elements, reducing the risk of dissemination. However, a significant limitation is the insufficient exploration of responsible genes or mobile elements in 80% of studies, hindering safety assessments. Additionally, most articles originated from Asian and Middle Eastern countries, with strains often isolated from fermented dairy foods. Therefore, these findings underscore the necessity for comprehensive analyses of new strains of L. delbrueckii for potential industrial and biotherapeutic applications and in combating the rise of antibiotic-resistant pathogens.

Functional genomic analysis of the isolated potential probiotic Lactobacillus delbrueckii subsp. indicus TY-11 and its comparison with other Lactobacillus delbrueckii strains.

Probiotics refer to living microorganisms that exert a variety of beneficial effects on human health. On the contrary, they also can cause infection, produce toxins within the body, and transfer antibiotic-resistant genes to the other microorganisms in the digestive tract necessitating a comprehensive safety assessment. This study aimed to conduct functional genomic analysis and some relevant biochemical tests to uncover the probiotic potentials of Lactobacillus delbrueckii subsp. indicus TY-11 isolated from native yogurt in Bangladesh. We also performed transmission electron microscopic (TEM) analysis, comparative genomic study as well as phylogenetic tree construction with 332 core genes from 262 genomes. The strain TY-11 was identified as Lactobacillus delbrueckii subsp. indicus, whose genome (1,916,674 bp) contained 1911 CDS, and no gene was identified for either antibiotic resistance or toxic metabolites. It carried genes for the degradation of toxic metabolites, treatment of lactose intolerance, toll-like receptor 2-dependent innate immune response, heat and cold shock, bile salts tolerance, and acidic pH tolerance. Genes were annotated for inhibiting pathogenic bacteria by inhibitory substances [bacteriocin: Helveticin-J (331 bp) and Enterolysin-A (275 bp), hydrogen peroxide, and acid]; blockage of adhesion sites; and competition for nutrients. The genes involved in its metabolic pathway were detected as suitable for digesting indigestible nutrients in the human gut. The TY-11 genome possessed an additional 37 core genes of subspecies indicus which were deficient in the core genome of the most popular subsp. bulgaricus. During the phenotypic testing, the isolate TY-11 demonstrated high antagonistic activity (inhibition zone of 21.33 ± 1.53 mm) against Escherichia coli ATCC 8739 and was not sensitive to any of the 10 tested antibiotics. This study was the first study to explore the molecular insights into probiotic roles, including antimicrobial activities and antibiotic sensitivity, of a representative strain (TY-11) of Lactobacillus delbrueckii subsp. indicus. IMPORTANCE: This study aimed to conduct functional genomic analysis to uncover the probiotic potential of Lactobacillus delbrueckii subsp. indicus TY-11 isolated from native yogurt in Bangladesh. We also performed transmission electron microscopic (TEM) analysis, comparative genomic study as well as phylogenetic tree construction with 332 core genes from 262 genomes. In our current investigation, we revealed a number of common and unique excellences of the probiotic Lactobacillus delbrueckii subsp. indicus TY-11 that are likely to be important to illustrate its intestinal residence and probiotic roles. This is the first study to explore the molecular insights into intestinal residence and probiotic roles, including antimicrobial activities and antibiotic sensitivity, of a representative strain (TY-11) of Lactobacillus delbrueckii subsp. indicus.

Redefining the bacteriophage mv4 site-specific recombination system and the sequence specificity of its attB and core-attP sites.

Through their involvement in the integration and excision of a large number of mobile genetic elements, such as phages and integrative and conjugative elements (ICEs), site-specific recombination systems based on heterobivalent tyrosine recombinases play a major role in genome dynamics and evolution. However, despite hundreds of these systems having been identified in genome databases, very few have been described in detail, with none from phages that infect Bacillota (formerly Firmicutes). In this study, we reanalyzed the recombination module of Lactobacillus delbrueckii subsp. bulgaricus phage mv4, previously considered atypical compared with classical systems. Our results reveal that mv4 integrase is a 369 aa protein with all the structural hallmarks of recombinases from the Tn916 family and that it cooperatively interacts with its recombination sites. Using randomized DNA libraries, NGS sequencing, and other molecular approaches, we show that the 21-bp core-attP and attB sites have structural similarities to classical systems only if considering the nucleotide degeneracy, with two 7-bp inverted regions corresponding to mv4Int core-binding sites surrounding a 7-bp strand-exchange region. We also examined the different compositional constraints in the core-binding regions, which define the sequence space of permissible recombination sites.

Effect of probiotic administration during pregnancy on the functional diversity of the gut microbiota in healthy pregnant women.

Our study aims to investigate the impact of probiotic consumption during pregnancy on gut microbiota functional diversity in healthy pregnant women. Thirty-two pregnant women were randomly assigned to two groups. The probiotic group (PG) consisted of pregnant women who consumed triple viable Bifidobacterium longum, Lactobacillus delbrueckii bulgaricus, and Streptococcus thermophilus tablets from the 32nd week of pregnancy until delivery. The functional profiles of the gut microbiota were predicted through high-throughput 16S rRNA sequencing results using PICRUSt software and referencing the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. In the gut microbiota of the PG, the genera Blautia and Ruminococcus, as well as the species Subdoligranulum, showed significantly higher relative abundances compared to the control group (CG) (P < 0.05). At Level 1 of the KEGG signaling pathways, there was a significant reduction in the functional genes of the gut microbiota involved in Organismal Systems in the PG (P < 0.05). In Level 2 of the KEGG signaling pathways, there was a significant reduction in the functional genes of the gut microbiota involved in Infectious Disease in the PG (P < 0.05). In Level 3 of the KEGG signaling pathways, the PG exhibited a significant increase in the functional genes of the gut microbiota involved in ABC transporters, Oxidative phosphorylation, Folate biosynthesis, and Biotin metabolism (P < 0.05). The CG showed a significant increase in the functional genes related to Cysteine and methionine metabolism, Vitamin B6 metabolism, Tuberculosis, and Vibrio cholerae pathogenic cycle (P < 0.05). In conclusion, our findings suggest that probiotic supplementation during pregnancy has a significant impact on functional metabolism in healthy pregnant women. IMPORTANCE: Probiotics are considered beneficial to human health. There is limited understanding of how probiotic consumption during pregnancy affects the functional diversity of the gut microbiota. The aim of our study is to investigate the impact of probiotic consumption during pregnancy on the functional diversity of the gut microbiota. Our findings suggest that probiotic supplementation during pregnancy has a significant impact on functional metabolism. This could potentially open up new avenues for preventing various pregnancy-related complications. This also provides new insights into the effects of probiotic consumption during pregnancy on the gut microbiota and offers a convenient method for exploring the potential mechanisms underlying the impact of probiotics on the gut microbiota of pregnant women.

New Genetic Determinants for qPCR Identification and the Enumeration of Selected Lactic Acid Bacteria in Raw-Milk Cheese.

Lactic acid bacteria (LAB) play an important role in the ripening of cheeses and contribute to the development of the desired profile of aroma and flavor compounds. Therefore, it is very important to monitor the dynamics of bacterial proliferation in order to obtain an accurate and reliable number of their cells at each stage of cheese ripening. This work aimed to identify and conduct a quantitative assessment of the selected species of autochthonous lactic acid bacteria from raw cow's milk cheese by the development of primers and probe pairs based on the uniqueness of the genetic determinants with which the target microorganisms can be identified. For that purpose, we applied real-time quantitative PCR (qPCR) protocols to quantify Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and Lactococcus lactis subsp. cremoris cells in cheese directly after production and over three-month and six-month ripening periods. While L. lactis subsp. cremoris shows good acidification ability and the ability to produce antimicrobial compounds, L. delbrueckii subsp. bulgaricus has good proteolytic ability and produces exo-polysaccharides, and S. thermophilus takes part in the formation of the diacetyl flavor compound by metabolizing citrate to develop aroma, they all play an important role in the cheese ripening. The proposed qPCR protocols are very sensitive and reliable methods for a precise enumeration of L. delbrueckii subsp. bulgaricus, S. thermophilus, and L. lactis subsp. cremoris in cheese samples.

In vitro investigation of the effects of Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 exopolysaccharides on tight junction damage caused by influenza virus infection.

It is a problem that influenza virus infection increases susceptibility to secondary bacterial infection in lungs leading to lethal pneumonia. We previously reported that exopolysaccharides (EPS) derived from Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (OLL1073R-1) could prevent against influenza virus infection followed by secondary bacterial infection in vitro. Therefore, the present study assessed whether EPS derived OLL1073R-1 protects the alveolar epithelial barrier disfunction caused by influenza virus infection. After A549 cells treated with EPS or without EPS were infected influenza virus A/Puerto Rico/8/34 (IFV) for 12 h, the levels of tight junction genes expression and inflammatory genes expression were measured by reverse transcription polymerase chain reaction. As results, EPS treatment could protect against low-titer IFV infection, but not high-titer IFV infection, followed by suppression of the increased expression of inflammatory cytokine gene levels and recovery of the decrease in the expression level of ZO-1 gene that was caused by low-titer IFV infection, leading to an improvement trend in the barrier function. Our findings showed that EPS derived from OLL1073R-1 could inhibit low-titer IFV infection leading to maintenance of the epithelial barrier function through the suppression of inflammatory cytokine genes expression.

Deciphering the metabolism of Lactobacillus delbrueckii subsp. delbrueckii during soy juice fermentation using phenotypic and transcriptional analysis.

In the context of sustainable diet, the development of soy-based yogurt fermented with lactic acid bacteria is an attractive alternative to dairy yogurts. To decipher the metabolism of Lactobacillus delbrueckii subsp. delbrueckii during soy juice (SJ) fermentation, the whole genome of the strain CIRM-BIA865 (Ld865) was sequenced and annotated. Then Ld865 was used to ferment SJ. Samples were analyzed throughout fermentation for their cell number, carbohydrate, organic acid, free amino acid, and volatile compound contents. Despite acidification, the number of Ld865 cells did not rise, and microscopic observations revealed the elongation of cells from 3.6 µm (inoculation) to 36.9 µm (end of fermentation). This elongation was observed in SJ but not in laboratory-rich medium MRS. Using transcriptomic analysis, we showed that the biosynthesis genes of peptidoglycan and membrane lipids were stably expressed, in line with the cell elongation observed, whereas no genes implicated in cell division were upregulated. Among the main sugars available in SJ (sucrose, raffinose, and stachyose), Ld865 only used sucrose. The transcriptomic analysis showed that Ld865 implemented the two transport systems that it contains to import sucrose: a PTS system and an ABC transporter. To fulfill its nitrogen needs, Ld865 probably first consumed the free amino acids of the SJ and then implemented different oligopeptide transporters and proteolytic/peptidase enzymes. In conclusion, this study showed that Ld865 enables fast acidification of SJ, despite the absence of cell division, leads to a product rich in free amino acids, and also leads to the production of aromatic compounds of interest. IMPORTANCE: To reduce the environmental and health concerns related to food, an alternative diet is recommended, containing 50% of plant-based proteins. Soy juice, which is protein rich, is a relevant alternative to animal milk, for the production of yogurt-like products. However, soy "beany" and "green" off-flavors limit the consumption of such products. The lactic acid bacteria (LAB) used for fermentation can help to improve the organoleptic properties of soy products. But metabolic data concerning LAB adapted to soy juice are lacking. The aim of this study was, thus, to decipher the metabolism of Lactobacillus delbrueckii subsp. delbrueckii during fermentation of a soy juice, based on a multidisciplinary approach. This result will contribute to give tracks for a relevant selection of starter. Indeed, the improvement of the organoleptic properties of these types of products could help to promote plant-based proteins in our diet.

Comparative transcriptomic analysis of the flavor production mechanism in yogurt by traditional starter strains.

The synergistic fermentation of milk by Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus is one of the key factors that determines the quality of yogurt. In this study, the mechanism whereby yogurt flavor compounds are produced by a mixture of S. thermophilus SIT-20.S and L. delbrueckii ssp. bulgaricus SIT-17.B were investigated by examining the flavor production, growth, and gene transcription of these strains. The results showed that yogurt produced by a 10:1 mixture of the aforementioned strains had the highest abundance of acetoin, whereas yogurt produced by a 1:1 mixture had the highest abundance of diacetyl and acetaldehyde. In addition, the growth of S. thermophilus SIT-20.S was enhanced in the 10:1 mixture. Transcriptomic analysis revealed differentially expressed genes in the flavor-compound-related pathways of S. thermophilus SIT-20.S and L. delbrueckii ssp. bulgaricus SIT-17.B in yogurts produced by 10:1 and 1:1 mixtures compared with those produced by either strain alone. Mixed fermentations regulated the expression of genes related to glycolysis, resulting in an increase of pyruvate, which is an important precursor for diacetyl and acetoin synthesis. The gene encoding the acetoin reductase (SIT-20S_orf01454) was decreased in S. thermophilus SIT-20.S, which ensured the accumulation of acetoin. In addition, the gene encoding the acetaldehyde dehydrogenase (SIT-20S_orf00949) was upregulated in S. thermophilus SIT-20.S, and the expression of alcohol dehydrogenase (SIT-20S_orf01479; SIT-17B_orf00943) was downregulated in both strains, maintaining the abundance of acetaldehyde. In addition, the gene encoding the NADH oxidase (SIT-17B_orf00860) in L. delbrueckii ssp. bulgaricus SIT-17.B were upregulated, which promoted the accumulation of diacetyl and acetoin. Overall, we characterized the mechanism by which S. thermophilus and L. delbrueckii ssp. bulgaricus synergistically generated yogurt flavor compounds during their production of yogurt and highlighted the importance of appropriate proportions of fermentation starters for improving the flavor of yogurts.