Our gut microbiota: a long walk to homeostasis.

The microbiome of the human gastrointestinal tract (GIT) consists of billions of bacteria, fungi and viruses, of which bacteria play the most important role in nutrition, immune development, production of vitamins and maintaining a well-balanced (homeostatic) microbial population. Many papers have been published on the microbiota in the human GIT, but little is known about the first group of bacteria that colonises an infant. The intestinal tract of an unborn is, despite general belief, not sterile, but contains bacteria that have been transferred from the mother. This opens a new research field and may change our understanding about the role bacteria play in early life, the selection of strains with probiotic properties and the treatment of diseases related to bacterial infections. Differences in bacterial populations isolated from meconia may provide answers to the prevention of certain forms of diabetes. More research is now focusing on the effect that a genetically diverse group, versus a much simpler microbial population, may have on the development of a homeostatic gut microbiome. The effect different bacterial species have on the gut-associated lymphoid tissue and cascade of immune responses has been well researched, but we still fail in identifying the ideal group of intestinal bacteria and if we do, it will certainly not be possible to maintain homeostasis with so many challenges the gut faces. Changes in diet, antibiotics, food preservatives and stress are some of the factors we would like to control, but more than often fail to do so. The physiology and genetics of the GIT changes with age and so the microbiome. This review summarises factors involved in the regulation of a gut microbiome.

Safety and persistence of orally administered human Lactobacillus sp. strains in healthy adults.

The aim of the study was to evaluate the safety and persistence of selected Lactobacillus strains in the gastrointestinal tract (GIT) of healthy adult volunteers after oral consumption of high doses of lactobacilli to identify potential candidates for probiotic and biotechnological applications. In the first phase of the study, nine individuals consumed capsules containing Lactobacillus gasseri 177 and E16B7, Lactobacillus acidophilus 821-3, Lactobacillus paracasei 317 and Lactobacillus fermentum 338-1-1 (each daily dose 1×1010 cfu) for 5 consecutive days. Data on gut health, blood parameters, and liver and kidney function were collected. The persistence of Lactobacillus strains was assessed by culturing combined with arbitrarily primed polymerase chain reaction (AP-PCR) and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) on days 0, 5, 8, 10 and 20 from faecal samples. All strains survived gastrointestinal passage and were detected on the 5th day. L. acidophilus 821-3 was detected in four volunteers on the 8th day (4.3 to 7.0 log10 cfu/g) and in two on the 10th day (8.3 and 3.9 log10 cfu/g, respectively). In the second phase of the study, five additional volunteers consumed L. acidophilus 821-3 (daily 1×1010 cfu) for 5 consecutive days. The strain was subsequently detected in faeces of all individuals using real-time PCR on the 10th day (range 4.6-6.7; median 6.0 log10 cell/g) in both phases of the study for at least 5 days after discontinuation of consumption. The administration of high doses of different Lactobacillus strains did not result in any severe adverse effects in GIT and/or abnormal values of blood indices. Thus, the strain L. acidophilus 821-3 is a promising candidate for probiotic and biotechnological applications. Further studies will be performed to confirm the strain persistence and safety in a larger number of individuals.

Characterization of oral lactobacilli as potential probiotics for oral health.

INTRODUCTION: Intestinal lactobacilli have been successfully used as probiotics to treat gastrointestinal disorders, but only limited data are available for the probiotic properties of oral lactobacilli to combat oral diseases. We aimed to characterize oral lactobacilli for their potential probiotic properties according to the international guidelines for the evaluation of probiotics, and to select potential probiotic strains for oral health. METHODS: The study included 67 salivary and subgingival lactobacilli of 10 species, isolated from healthy humans. All strains were identified using amplified ribosomal DNA restriction analysis, tested for antimicrobial activity against oral pathogens, tolerance of low pH and bile content. Thereafter, the lysozyme tolerance and antibiotic susceptibility of 22 potential probiotic strains were assessed. RESULTS: The majority of strains suppressed the growth of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, and Streptococcus mutans, but none inhibited Candida albicans. The lowest pH tolerated by lactobacilli following 4 h of incubation was pH 2.5, but none of the strains grew at this pH. All strains tolerated a high concentration of lysozyme (10 mg/ml) and half of the strains tolerated a high concentration of human bile [5% volume/volume (V/V)]. Four Lactobacillus plantarum and two Lactobacillus oris strains expressed resistance to tetracycline and/or doxycycline. CONCLUSIONS: Strains of L. plantarum, Lactobacillus paracasei, Lactobacillus salivarius, and Lactobacillus rhamnosus expressed both high antimicrobial activity and high tolerance of environmental stress. The absence of transferable antibiotic-resistance genes in L. plantarum strains remains to be confirmed. These results suggest a potential for oral lactobacilli to be used as probiotics for oral health.

Expression of single-chain antibody against RgpA protease of Porphyromonas gingivalis in Lactobacillus.

AIMS: The monoclonal antibody 61BG1.3, recognizing the RgpA protease, has been reported to confer protection against recolonization by the periodontal pathogen Porphyromonas gingivalis in humans. The aim of this study was to express a functional scFv derived from the monoclonal antibody 61BG1.3 on the surface of Lactobacillus paracasei for potential use in the prevention or treatment of periodontal diseases. METHODS AND RESULTS: The scFv was fused to an E-tag and cloned in the Escherischia coli/Lactobacillus shuttle vector pLP501, which mediates surface expression of the scFv. FACS analysis using an anti-E-tag antibody revealed that the scFv was expressed on the surface of the transformed lactobacilli and binding of the scFv to RgpA was shown by ELISA. Lact. paracasei expressing the scFv against RgpA was able to agglutinate P. gingivalis whereas the Lact. paracasei expressing an irrelevant scFv fragment did not. Scanning electron microscopy demonstrated efficient binding of the lactobacilli expressing the scFv anti-RgpA to P. gingivalis. CONCLUSIONS: We have expressed a functional scFv antibody directed against the RgpA protease of P. gingivalis in Lactobacillus. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest a potential of Lactobacillus expressing scFvs against P. gingivalis to be used to combat periodontal disease.

Oral lactobacilli in chronic periodontitis and periodontal health: species composition and antimicrobial activity.

BACKGROUND/AIMS: Lactobacilli are known to play an important role in the maintenance of health by stimulating natural immunity and contributing to the balance of microflora. However, their role in chronic periodontitis is unclear. We aimed to identify oral lactobacilli in chronic periodontitis and periodontally healthy subjects, and to determine their antimicrobial activity against putative oral pathogens. METHODS: A total of 238 Lactobacillus isolates from the saliva and subgingival sites of 20 chronic periodontitis and 15 healthy subjects were collected. In all, 115 strains were identified using rapid amplified ribosomal DNA restriction analysis. Antimicrobial activity against Streptococcus mutans, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia was assessed. RESULTS: Lactobacilli belonging to 10 species were identified. The most prevalent strains in healthy persons were Lactobacillus gasseri and Lactobacillus fermentum and in chronic periodontitis patients, Lactobacillus plantarum. Obligately homofermentatives, particularly L. gasseri, were less prevalent in chronic periodontitis patients compared with healthy subjects (8% vs. 64% for L. gasseri, P < 0.01). Sixty-nine percent of tested lactobacilli inhibited S. mutans, 88% A. actinomycetemcomitans, 82% P. gingivalis and 65% P. intermedia. The strongest antimicrobial activity was associated with Lactobacillus paracasei, L. plantarum, Lactobacillus rhamnosus, and Lactobacillus salivarius. The strains from periodontally healthy patients showed a lower antimicrobial activity against S. mutans than the strains from chronic periodontitis patients. CONCLUSION: The composition of oral lactoflora in chronic periodontitis and healthy subjects differs, with a higher prevalence of homofermentative lactobacilli, particularly L. gasseri, in the latter group. Both homo- and heterofermentative oral lactobacilli suppress the growth of periodontal pathogens, but the antimicrobial properties are strain, species and origin specific.

The aggregation-promoting factor of Lactobacillus crispatus M247 and its genetic locus.

AIMS: Characterization of the aggregation-promoting factor (APF) of the human intestinal isolate Lactobacillus crispatus M247 and its homologous nonaggregating mutant Mu5. METHODS AND RESULTS: Western blot analysis revealed that the supernatant of both M247 and Mu5 contains a 28-kDa protein which cross reacts with the antiserum produced against the APF of Lact. gasseri 4B2. The apf genes of M247 and Mu5 strains were identical and were shown to be 672 nucleotides in length and encoding a protein of 223 amino acids with a predicted molecular weight of 24.0 kDa. CONCLUSION: Our results shows that the lost of aggregation in Mu5 is not related to a defect in secretion of the APF protein or a mutation in the apf gene. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that the mutation in Mu5 may be contained in another molecule involved in aggregation such as a possible receptor for APF.

Mucosal and cellular immune responses elicited by recombinant Lactococcus lactis strains expressing tetanus toxin fragment C.

The mucosal and cellular responses of mice were studied, following mucosal-route administration of recombinant Lactococcus lactis expressing tetanus toxin fragment C (TTFC), which is a known immunogen protective against tetanus. A TTFC-specific T-cell response with a mixed profile of T-helper (Th) subset-associated cytokines was elicited in the intestine, with a Th2 bias characteristic of a mucosal response. These results correlated with the humoral response, where equivalent titers of anti-TTFC immunoglobulin G1 (IgG1) and IgG2a in serum were accompanied by an elevated IgA-specific response at more than one mucosal site. The route of vaccination had an important role in determining the immune response phenotype, as evidenced by the fact that an IgG1-biased subclass profile was obtained when lactococci were administered parenterally. Stimulation of splenic or mesenteric lymph node cells with lactococci resulted in their proliferation and the secretion of gamma interferon via antigen-specific and innate immune mechanisms. The data therefore provide further evidence of the potential of recombinant lactococcal vaccines for inducing systemic and mucosal immune responses.

Immunoglobulin-binding domains of peptostreptococcal protein L enhance vaginal colonization of mice by Streptococcus gordonii.

Protein L, an immunoglobulin-binding protein of some strains of the anaerobic bacterium Peptostreptococcus magnus, has been hypothesized to be a virulence determinant in bacterial vaginosis. In order to investigate the role of protein L in peptostreptococcal virulence, the Ig-binding domains of protein L were expressed at the surface of the human oral commensal Streptococcus gordonii. Recombinant streptococci were used in vaginal colonization experiments, and protein L-expressing S. gordonii demonstrated enhanced ability to colonize the vaginal mucosa. Compared to the control strain, they also persisted for a longer period in the murine vagina.

Immunization with recombinant Streptococcus gordonii expressing tetanus toxin fragment C confers protection from lethal challenge in mice.

Tetanus toxin fragment C (TTFC) was expressed on the surface of the vaccine vector Streptococcus gordonii, a Gram-positive commensal bacterium of the human oral cavity. The immunogenicity of recombinant S. gordonii expressing TTFC was assayed in mice immunized by the parenteral and mucosal routes. High serum TTFC-specific IgG responses were induced in both BALB/c and C57BL/6 mice immunized subcutaneously. A total of 82% of vaccinated BALB/c mice were protected from the lethal challenge with 50 LD(50) of tetanus toxin (TT) and a direct correlation between the serum TTFC-specific IgG concentration and survival time of unprotected animals was observed. Intranasal immunization of BALB/c mice was also effective in inducing TTFC-specific serum IgG and local IgA in lung washes. Furthermore, 38% of animals immunized intranasally were protected from the lethal challenge with 10 LD(50) of TT while all control animals died within 24 h. Analysis of the serum IgG subclasses showed that the IgG1 subclass was predominant after parenteral immunization in BALB/c mice (IgG1/IgG2a ratio congruent with6) while following mucosal immunization a mixed IgG1 and IgG2a pattern (IgG1/IgG2a ratio congruent with1) was observed. These data show that TTFC expressed on the surface of S. gordonii is immunogenic by the subcutaneous and mucosal routes and the immune response induced is capable of conferring protection from the lethal challenge with TT.

Oral microbial ecology and the role of salivary immunoglobulin A.

In the oral cavity, indigenous bacteria are often associated with two major oral diseases, caries and periodontal diseases. These diseases seem to appear following an imbalance in the oral resident microbiota, leading to the emergence of potentially pathogenic bacteria. To define the process involved in caries and periodontal diseases, it is necessary to understand the ecology of the oral cavity and to identify the factors responsible for the transition of the oral microbiota from a commensal to a pathogenic relationship with the host. The regulatory forces influencing the oral ecosystem can be divided into three major categories: host related, microbe related, and external factors. Among host factors, secretory immunoglobulin A (SIgA) constitutes the main specific immune defense mechanism in saliva and may play an important role in the homeostasis of the oral microbiota. Naturally occurring SIgA antibodies that are reactive against a variety of indigenous bacteria are detectable in saliva. These antibodies may control the oral microbiota by reducing the adherence of bacteria to the oral mucosa and teeth. It is thought that protection against bacterial etiologic agents of caries and periodontal diseases could be conferred by the induction of SIgA antibodies via the stimulation of the mucosal immune system. However, elucidation of the role of the SIgA immune system in controlling the oral indigenous microbiota is a prerequisite for the development of effective vaccines against these diseases. The role of SIgA antibodies in the acquisition and the regulation of the indigenous microbiota is still controversial. Our review discusses the importance of SIgA among the multiple factors that control the oral microbiota. It describes the oral ecosystems, the principal factors that may control the oral microbiota, a basic knowledge of the secretory immune system, the biological functions of SIgA, and, finally, experiments related to the role of SIgA in oral microbial ecology.

Comparison of the indigenous oral microbiota and immunoglobulin responses of athymic (nu/nu) and euthymic (nu/+) mice.

The role of the immune system in the homeostasis of indigenous oral bacterial populations is poorly understood. In this study, we compared the evolution of the indigenous oral microbiota of specific pathogen-free athymic nude (nu/nu) BALB/c mice with that of their corresponding phenotypically normal (nu/-) littermates. We also evaluated corresponding salivary and serum antibody activities (IgA and IgG) against the predominant indigenous oral bacteria. The bacterial species recovered from the two mouse strains were Lactobacillus murinus, Enterococcus faecalis, Streptococcus oralis and Staphylococcus epidermidis. From 27 days of age, nu/+ and nu/nu mice had significantly different proportions of oral bacterial populations. When the microbiota stabilized (at 40 days of age), the total cultivable microbiota of nu/+ mice was dominated by L. murinus (65-85%), while that of nu/nu mice was dominated by E. faecalis (40-60%). The precise factors that alter the oral resident microbiota in nu/nu mice are unknown. We found that total salivary IgA levels were significantly lower in nu/nu mice, but no association were observed between the level of salivary IgA antibody against indigenous bacteria and the proportion of these indigenous bacteria in the oral microbiota. The change in the microbiota of nude mice may have been caused by other factors such as defects in other immune functions or cold stress.

No apparent influence of immunoglobulins on indigenous oral and intestinal microbiota of mice.

The role of secretory immunoglobulin A (sIgA) in the control of the indigenous microbiota is not well understood. In this study, we compared the oral and intestinal microbiota of transgenic B-cell-deficient (microMT) mice with their heterozygous (microMT/+) normal littermates. The levels of salivary IgA and serum IgA and IgG were normal in microMT/+ mice, while no immunoglobulins were detected in microMT/microMT mice. The acquisition and proportions of the different species of the oral and intestinal indigenous bacterial populations were not significantly different between the two groups of mice. Our results thus suggest that secretory IgA does not play a major role in the regulation of the indigenous microbiota of mice.

Pneumocystis carinii infection in transgenic B cell-deficient mice.

Pneumocystis carinii is an important cause of pneumonia in immunocompromised hosts. Both cellular and humoral immunity seem important in resistance to this pathogen, but the specific role of each component is poorly understood. An outbreak of P. carinii pneumonia in transgenic B cell-deficient mice (muMT) was studied. Over 4 months, >50% of 41 muMT/muMT mice maintained in a sterile environment died of pneumonia. Some mice had concurrent infection with Pasteurella pneumotropica. Homozygous muMT/muMT mice had no detectable serum immunoglobulins, while their heterozygous muMT/+ counterparts had normal levels of IgM, IgG, and IgA and did not develop pneumonia. The infection was controlled by treating the mice with trimethoprim-sulfamethoxazole, and the pathogen was eliminated by cesarean rederivation. These observations suggest an important role for B cells in the host defense against P. carinii.

Colonization of the oral cavity of mice by an unidentified streptococcus.

While studying the oral bacterial biota of mice, we observed an unidentified streptococcus (TG) that eventually became the dominant species of the oral cavities of all other mice in our animal facility. We found that the strain is indigenous to Jackson Laboratory mice but is absent in animals from Charles River Laboratories. TG was also transmitted from artificially contaminated BALB/c mice to the oral cavities of 4 other mouse strains. Streptococcus sp. TG stimulated the secretory and systemic immune systems of artificially contaminated Charles River BALB/c mice but did not provoke clinical symptoms. The increase in antibody level to TG did not prevent its colonization and persistence in these mice. In mice from Jackson Laboratory, the secretory and systemic immune response to TG was significantly lower. In vitro, Streptococcus sp. TG inhibited murine oral lactobacilli and staphylococci, probably due to the production of hydrogen peroxide.

Evaluation of mouse salivary IgA directed against indigenous oral bacteria.

We are developing an ELISA to follow the evolution of specific salivary IgA directed against the indigenous oral bacteria of the BALB/c mouse. To reduce the variability of the IgA levels detected between different mice, we standardized the method used for sampling saliva and the method used for bacterial cell fixation. Incubation of whole bacteria for one hour at 4 degrees C in poly-L-lysine-treated plates followed by glutaraldehyde fixation increased ELISA reactivities by improving cell fixation. Our results also indicate that salivary IgA concentrations in BALB/c mice peak at the age of three months and that biweekly carbachol-stimulated saliva sampling does not significantly affect the amount of salivary IgA detected.

Bacterial colonization of the oral cavity of the BALB/c mouse.

The acquisition of the human oral bacterial flora follows a relatively well known sequence of succession that can be influenced by various host factors. These factors have not been studied in the mouse. In the present work, we followed the bacterial colonization of the oral cavity of mice from birth, and tested our mouse model for its suitability in studying the influence of weaning and puberty on the indigenous oral bacterial flora. We observed that the first colonizers were staphylococci, followed by lactobacilli. The proportions of these two predominant bacteria fluctuated for a period of 30-50 days, but evolved towards the proportions previously observed among the indigenous bacterial species of 6-8 week-old BALB/c male mice (predominantly Lactobacillus murinus and Staphylococcus aureus). The weaning period significantly altered the equilibrium among the oral bacterial flora. This equilibrium was not significantly modified during puberty.

Computer simulation of human mitral valve mechanics and motion.

The human mitral valve is the left atrio-ventricular valve which is composed of several components including leaflets, chordae tendineae, papillary muscles, and the valve annulus. Any or all of these components may fail and contribute to various valvular diseases including mitral regurgitation and mitral valve prolapse. A computer simulation of mitral valve mechanics and motion was written in BASIC for micro-computers. This program allows valvular geometry and biomechanical parameters to be varied and records time varying motion of the valve and all components during systole including a graphic display of the valve leaflets.