Novel shuttle vector pGMß1 for conjugative chromosomal manipulation of Lactobacillus delbrueckii subsp. bulgaricus.

Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) is widely used as a starter for yogurt and cheese worldwide. Despite the economic importance of this bacterium in the dairy industry, there have been few genetic studies involving knockout or overexpression mutants to identify the functions of L. bulgaricus genes. One of the main reasons for this gap is the low transformation efficiency of available L. bulgaricus chromosome-integrating vectors upon performing conventional electroporation. We previously proposed the conjugal plasmid pAMß1 as an integration vector for L. bulgaricus, as conjugation could avert the need for a restriction modification system; pAMß1 does not replicate and integrate into the chromosome of L. bulgaricus. Here, we describe an effective chromosomal manipulation system involving a novel shuttle vector pGMß1, which could improve the operability of the broad host-range conjugal plasmid pAMß1. We further developed an enhanced filter-mating method for conjugation. To validate this system, the effectiveness of conversion of the lactate dehydrogenase gene D-ldh of L. bulgaricus to the L-ldh form of Streptococcus thermophilus was examined. As pGMß1 and pAMß1 are unable to replicate in L. delbrueckii subsp. delbrueckii, they were chromosomally integrated. However, these plasmids could replicate in L. delbrueckii subsp. indicus and sunkii. This integration system could unearth important gene functions in L. bulgaricus and thus improve its applications in the dairy industry. Moreover, this conjugation system could be used as a stable vector for the transformation of long cluster genes in several species of lactic acid bacteria.

Exopolysaccharides From Streptococcus thermophilus ST538 Modulate the Antiviral Innate Immune Response in Porcine Intestinal Epitheliocytes.

It was reported that exopolysaccharides (EPSs) from lactobacilli are able to differentially modulate mucosal antiviral immunity. Although research has described the ability of EPSs derived from Streptococcus thermophilus to modulate the mucosal immune system, their impact on antiviral immunity was less explored. In this work, we investigated the capacity of the EPS-producing S. thermophilus ST538 to modulate the innate antiviral immune response triggered by the activation of the Toll-like receptor 3 (TLR3) in porcine intestinal epitheliocytes (PIE cells). Moreover, in order to study the immunomodulatory potential of S. thermophilus ST538 EPS, we successfully developed two mutant strains through the knockout of the epsB or epsC genes. High-performance liquid chromatography and scanning electron microscopy studies demonstrated that the wild type (WT) strain produced as high as 595 µg/ml of EPS in the skim milk medium, while none of the mutant strains (S. thermophilus ΔepsB and ΔepsC) were able to produce EPS. Studies in PIE cells demonstrated that the EPS of S. thermophilus ST538 is able to significantly improve the expression of interferon ß (IFN-ß), interleukin 6 (IL-6), and C-X-C motif chemokine 10 (CXCL10) in response to TLR3 stimulation. The role of EPS in the modulation of antiviral immune response in PIE cells was confirmed by comparative studies of cell free culture supernatants and fermented skim milks obtained from S. thermophilus ΔepsB and ΔepsC. These results suggest that S. thermophilus ST538 could be used as an immunobiotic strain for the development of new immunologically functional foods, which might contribute to improve resistance against viral infections.

Lipoteichoic Acid Is Involved in the Ability of the Immunobiotic Strain Lactobacillus plantarum CRL1506 to Modulate the Intestinal Antiviral Innate Immunity Triggered by TLR3 Activation.

Studies have demonstrated that lipoteichoic acid (LTA) is involved in the immunomodulatory properties of some immunobiotic lactobacilli. The aim of this work was to evaluate whether LTA contributes to the capacity of Lactobacillus plantarum CRL1506 in modulating the intestinal innate antiviral immune response. A D-alanyl-lipoteichoic acid biosynthesis protein (dltD) knockout CRL1506 strain (L. plantarumΔdltD) was obtained, and its ability to modulate Toll-like receptor (TLR)-3-mediated immune response was evaluated in vitro in porcine intestinal epithelial (PIE) cells and in vivo in Balb/c mice. Wild-type (WT) CRL1506 (L. plantarum WT) was used as positive control. The challenge of PIE cells with the TLR3 agonist poly(I:C) significantly increased interferon (IFN)-ß, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1 expressions. PIE cells pretreated with L. plantarumΔdltD or L. plantarum WT showed higher levels of IFN-ß while only L. plantarum WT significantly reduced the expression of IL-6 and MCP-1 when compared with poly(I:C)-treated control cells. The oral administration of L. plantarum WT to mice prior the intraperitoneal injection of poly(I:C) significantly increased IFN-ß and IL-10 and reduced intraepithelial lymphocytes (CD3+NK1.1+CD8αα+) and pro-inflammatory mediators (TNF-α, IL-6, and IL-15) in the intestinal mucosa. Similar to the WT strain, L. plantarumΔdltD-treated mice showed enhanced levels of IFN-ß after poly(I:C) challenge. However, treatment of mice with L. plantarumΔdltD was not able to increase IL-10 or reduce CD3+NK1.1+CD8αα+ cells, TNF-α, IL-6, or IL-15 in the intestine. These results indicate that LTA would be a key molecule in the anti-inflammatory effect induced by the CRL1506 strain in the context of TLR3-mediated inflammation.

NADH peroxidase plays a crucial role in consuming H2O2 in Lactobacillus casei IGM394.

The facultative anaerobic bacterium Lactobacillus casei IGM394 is used as a host for drug delivery systems, and it exhibits the same growth rate under aerobic and anaerobic conditions. L. casei strains carry several genes that facilitate oxygen and reactive oxygen species (ROS) tolerance in their genomes, but their complete functions have not been uncovered. To clarify the oxygen and ROS tolerance mechanisms of L. casei IGM394, we constructed 23 deficient mutants targeting genes that confer oxidative stress resistance. Significantly decreased growth and high H2O2 accumulation were observed in the NADH peroxidase gene-mutated strain (Δnpr) compared with the findings in the wild type. The H2O2 degradation capacity of Δnpr revealed that NADH peroxidase is a major H2O2-degrading enzyme in L. casei IGM394. Interestingly, ΔohrR, a mutant deficient in the organic hydroperoxide (OhrA) repressor, exhibited higher H2O2 resistance than the wild-type strain. Increased Npr expression and H2O2 degradation ability were observed in ΔohrR, further supporting the importance of OhrA to ROS tolerance mechanisms. The other mutants did not exhibit altered growth rates, although some mutants had higher growth in the presence of oxygen. From these results, it is presumed that L. casei IGM394 has multiple oxygen tolerance mechanisms and that the loss of a single gene does not alter the growth rate because of the presence of complementary mechanisms. Contrarily, the H2O2 tolerance mechanism is solely dependent on NADH peroxidase in L. casei IGM394.

The membrane phospholipid cardiolipin plays a pivotal role in bile acid adaptation by Lactobacillus gasseri JCM1131T.

Bile acids exhibit strong antimicrobial activity as natural detergents, and are involved in lipid digestion and absorption. We investigated the mechanism of bile acid adaptation in Lactobacillus gasseri JCM1131T. Exposure to sublethal concentrations of cholic acid (CA), a major bile acid in humans, resulted in development of resistance to otherwise-lethal concentrations of CA by this intestinal lactic acid bacterium. As this adaptation was accompanied by decreased cell-membrane damage, we analyzed the membrane lipid composition of L. gasseri. Although there was no difference in the proportions of glycolipids (~70%) and phospholipids (~20%), adaptation resulted in an increased abundance of long-sugar-chain glycolipids and a 100% increase in cardiolipin (CL) content (to ~50% of phospholipids) at the expense of phosphatidylglycerol (PG). In model vesicles, the resistance of PG vesicles to solubilization by CA increased with increasing CL/PG ratio. Deletion of the two putative CL synthase genes, the products of which are responsible for CL synthesis from PG, decreased the CL content of the mutants, but did not affect their ability to adapt to CA. Exposure to CA restored the CL content of the two single-deletion mutants, likely due to the activities of the remaining CL synthase. In contrast, the CL content of the double-deletion mutant was not restored, and the lipid composition was modified such that PG predominated (~45% of total lipids) at the expense of glycolipids. Therefore, CL plays important roles in bile acid resistance and maintenance of the membrane lipid composition in L. gasseri.

Intracellular pH Determination for the Study of Acid Tolerance of Lactic Acid Bacteria.

It is important to assess acid tolerance in lactic acid bacteria, particularly for probiotics, although it involves multiple mechanisms. Measuring the difference between intracellular and extracellular pH (ΔpH) using the fluorescent probe CFDA-SE is particularly effective for such assessments because it gives direct information on the level of tolerance in the extracellular acidic pH range from 7 to 2.5. It also enables acid adaptation to be induced and observed by slowly introducing HCl into the medium and decreasing the extracellular pH. The difference of acid tolerance between anaerobic and aerobic conditions in lactic acid bacteria can also be evaluated by measuring ΔpH.

NADH Oxidase of Streptococcus thermophilus 1131 is Required for the Effective Yogurt Fermentation with Lactobacillus delbrueckii subsp. bulgaricus 2038.

We previously reported that dissolved oxygen (DO) suppresses yogurt fermentation with an industrial starter culture composed of Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) 2038 and Streptococcus thermophilus 1131, and also found that reducing the DO in the medium prior to fermentation (deoxygenated fermentation) shortens the fermentation time. In this study, we found that deoxygenated fermentation primarily increased the cell number of S. thermophilus 1131 rather than that of L. bulgaricus 2038, resulting in earlier l-lactate and formate accumulation. Measurement of the DO concentration and hydrogen peroxide generation in the milk medium suggested that DO is mainly removed by S. thermophilus 1131. The results using an H2O-forming NADH oxidase (Nox)-defective mutant of S. thermophilus 1131 revealed that Nox is the major oxygen-consuming enzyme of the bacterium. Yogurt fermentation with the S. thermophilus Δnox mutant and L. bulgaricus 2038 was significantly slower than with S. thermophilus 1131 and L. bulgaricus 2038, and the DO concentrations of the mixed culture did not decrease to less than 2 mg/kg within 3 hr. These observations suggest that Nox of S. thermophilus 1131 contributes greatly to yogurt fermentation, presumably by removing the DO in milk.

Application of the Revised Infant Temperament Questionnaire for evaluating temperament in the Japanese infant: creation of an abridged Japanese version.

The Japanese version of the Revised Infant Temperament Questionnaire (RITQ) was implemented to re-evaluate factor structure of the temperament scale with the objective of simplification. The questionnaire was conducted on 1099 mothers of Japanese infants visiting health centers for their children's health examinations at 6 or 7 months. As a result, a 57-item abridged version was constructed consisting of the seven subscales: 'fear of strangers and strange situations', 'insensitivity to food', 'regularity of rhythm', 'manageability', 'activity level', 'persistence of attention', and 'insensitivity to touch'. Although the items did not break down into the traditional nine temperament dimensions extracted by Carey et al., a certain commonality was seen in factor structure compared with the results of factor analysis using the RITQ in studies in Japan and abroad.

Transient eating disorder in early childhood--a case report.

Although children in infancy present eating problems of multifarious nature, the diagnostic classification of infant eating disorders remains markedly deficient. The authors present a case exhibiting transient eating disorder in early childhood, alongside discussion of some considerations relevant to this age group. The subject was a boy aged 5 years 6 months at first presentation. Starting with an inability to swallow "sushi" at dinner, he was brought to the clinic for inability to ingest food or liquids. Treatment was planned as play therapy for the patient, and psychological interviews for the mother. The child's symptoms were improved in five sessions, but interviews of the mother revealed eating disorders such as bulimia and vomiting, and sexual problems in the marital relationship, necessitating long-term therapy.

ThyA as a selection marker in construction of food-grade host-vector and integration systems for Streptococcus thermophilus.

We constructed food-grade host-vector and integration systems for Streptococcus thermophilus by using a thymidylate synthase gene (thyA) as the selection marker. Two thyA genes, thyA(St) and thyA(Lb), were cloned from S. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, respectively. Thymidine-requiring mutants of S. thermophilus were obtained after successive cultures in the presence of trimethoprim, and one of them, TM1-1, was used as the host. Food-grade vectors were constructed by using either thyA(St) or thyA(Lb) as the selection marker. Transformants of TM1-1 created by using these vectors were selected for thymidine autotrophy as efficiently as for erythromycin resistance. By using the host-vector system developed in this way, a foreign amylase gene (amyA) was expressed in TM1-1 and was also integrated into the chromosome by use of a temperature-sensitive integration vector constructed with thyA(Lb) as the selection marker via a double-crossover event. The results obtained show that thyA is an efficient and safe selection marker for S. thermophilus that is suitable for food applications.

Antenatal depression and maternal-fetal attachment.

BACKGROUND: In recent years, attention has been turned to maternal mental health in relation to the mother-child relationship accompanying a widening in focus, i.e. taking into account not only the puerperium, but also the stage of pregnancy. This applies to studies that have revealed a connection between depression and maternal attachment in the postpartum period and late pregnancy. This study, however, was designed to evaluate the maternal-fetal relationship in the first and second trimesters, being the first one to address this issue in these early stages. SAMPLING AND METHODS: Zung's Self-Rating Depression Scale (ZSDS), the original Antenatal Maternal Attachment Scale (AMAS), and a questionnaire addressing peripheral factors were given to 216 pregnant women (3-6 months of gestation) who visited the Nagoya University Hospital between September 1998 and June 2001. RESULTS: Contrary to reports on the latter stages of pregnancy, no direct association was observed between depression in mothers and maternal-fetal attachment before fetal movement was perceived. CONCLUSION: However, education, form of employment, planning of pregnancy, and premenstrual mood changes were found to be associated with the ZSDS score (mean: 41.9), while form of employment, feelings regarding pregnancy, and sources of support were extracted as factors associated with the AMAS, which are of interest in terms of the subsequent association between depression and maternal-fetal attachment in the peri- and postnatal periods.

Study on feelings of school avoidance, depression, and character tendencies among general junior high and high school students.

School refusal is a phenomenon that first drew attention in Japan around 1960 and it remains one of the major issues in child psychiatry today. Moreover, it is now said that there exists a large group of latent school refusers currently attending school but harboring feelings of school avoidance. To address this issue, a questionnaire survey was conducted on students enrolled in a junior high and high school affiliated with the Nagoya University School of Education. The questionnaire consisted of the Children's Depression Inventory (CDI), a scale for evaluating feelings of school avoidance (School Avoidance Scale), and a scale for assessment of personality characteristics associated with school refusal (School Refusal Personality Scale). The subjects were 425 first-year junior high to second-year high school students. Factor analysis of each scale revealed the CDI to consist of three factors: 'core depression', 'feelings of interpersonal maladaptation', and 'self-revulsion', and the School Avoidance Scale to consist of two factors: 'school dislike', and 'school avoidance'. The School Refusal Personality Scale consisted of three factors: 'obsessive-compulsive', 'passive/unsocial', and 'socially introverted'. Mean CDI score and standard deviation (SD) was 19.44 +/- 7.49, and that for 'feelings of school avoidance' was 20.18 +/- 5.61. The two subordinate factors of the School Avoidance Scale were intimately associated with both 'feelings of interpersonal maladaptation' and 'core depression' of the CDI, and negatively correlated with the 'obsessive-compulsive' factor of the School Refusal Personality Scale.