A spontaneous lactate dehydrogenase deficient mutant of Streptococcus rattus for use as a probiotic in the prevention of dental caries.

AIMS: To study the ability of daily applications of Streptococcus rattus strain JH145 to affect the numbers of an implanted Streptococcus mutans strain in a rat model. METHODS AND RESULTS: A spontaneous L(+)-lactate dehydrogenase (LDH)-deficient mutant of Streptococcus rattus, JH146, was isolated by screening on selective medium and compared with a previously isolated spontaneous LDH deficient strain, JH145. Both strains were shown to have single base pair deletion mutations in the structural gene (ldh) for LDH, and reversion frequencies were approximately the same. Animals treated once daily with >or=10(6) CFU (colony forming units) of JH145 showed a statistically significant decrease in the proportion of implanted S. mutans to total cultivable bacteria in oral swab samples. The rate of decrease in S. mutans levels was dose-dependent. No adverse effects were observed by in-life observation of treated animals, and histopathological, haematological and blood chemistry analyses were unremarkable. CONCLUSIONS: The results presented indicate that daily application of JH145, a naturally occurring LDH-deficient variant of S. rattus, can compete with S. mutans for its habitat on the tooth surface. SIGNIFICANCE AND IMPACT OF THE STUDY: S. rattus JH145 has potential as a probiotic for use in the prevention of dental caries.

Preliminary assessment of safety and effectiveness in humans of ProBiora3, a probiotic mouthwash.

AIMS: To conduct a pilot human clinical trial to assess the safety and to test the ability of a probiotic mouthwash, ProBiora(3), to affect the levels of Streptococcus mutans and certain known periodontal pathogens in the mouth when administered twice daily over a period of 4 weeks. METHODS AND RESULTS: The mouthwash contained three specific strains of naturally occurring oral bacteria and was tested at two dose levels: 10(6) and 10(8) colony forming units each of Strep. oralis strain KJ3sm, Strep. uberis strain KJ2sm, and the spontaneous lactic acid-deficient variant of Strep. rattus, strain JH145. Substantial decreases in the levels of the marker bacteria were observed. No safety issues were noted with the twice daily application of this mouthwash. CONCLUSIONS: Despite the small number of subjects and the use of young, orally healthy adults, along with the inherent variability in the microbiological measurements, the probiotic mouthwash was able to substantially affect the levels of dental pathogens in saliva and periodontal pathogens in subgingival plaque. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this pilot human study suggest that the probiotic mouthwash product may be safe for daily use as an aid in maintaining both dental and periodontal health.

IVIAT: a novel method to identify microbial genes expressed specifically during human infections.

In vivo induced antigen technology (IVIAT) is a novel technology that can quickly and easily identify in vivo induced genes in human infections, without the use of animal models. This technology is expected to facilitate the discovery of new targets for vaccines, antimicrobials and diagnostic strategies in a wide range of microbial pathogens.

Tn917-lac mutagenesis of Streptococcus mutans to identify environmentally regulated genes.

Previously, we demonstrated successful Tn917 mutagenesis of the oral pathogen Streptococcus mutans using pTV1-OK (Km(r), repATs), a temperature conditional replicative delivery vector carrying a lactococcal pWVO1Ts backbone. In this report we describe the construction and utilization of pTV32-OK, a plasmid harboring Tn917-lac (em(r), beta-gal(+)) that was employed to isolate transcriptional fusions of the Escherichia coli lacZ reporter gene with streptococcal promoters in S. mutans strain NG8. Tn917-lac transposition occurred at a frequency of ca. 10(-6) with 20% of the resultant em(r) clones displaying varying levels of lacZ expression. Tn917-lac mutants that expressed beta-galactosidase activity under growth conditions of glucose limitation, acidic pH, 35 mM NaCl, and elevated (42 degrees C) temperature were isolated. Further characterization of one of the mutants with increased beta-gal activity under glucose limitation, strain AS42, revealed maximal activity in batch culture in stationary phase after glucose depletion. The beta-gal activity of AS42 also was found to be repressed 3-fold in medium containing 2% glucose relative to measured activity from cells suspended in the same medium containing no glucose. Further phenotypic analysis revealed that AS42 had a 30% lower growth yield than the parent strain NG8 when grown in pH 5 medium. Sequence analysis of the region harboring the transposon revealed that the lacZ fusion occurred near the 3'-end of a gene encoding a homolog of an ATP binding protein from a family of Gram-positive ABC transporters. These findings demonstrate that Tn917-lac mutagenesis can be used to identify environmentally regulated genes in S. mutans and possibly in other medically relevant streptococcal species.

Colonization of the human oral cavity by a strain of Streptococcus mutans.

Streptococcus mutans strain JH1001 produces a bacteriocin that can kill virtually all other strains of this micro-organism. The ability of JH1001 to colonize the human oral cavity was tested in a study involving five subjects and three different infection regimens, all of which involved multiple exposures to large numbers of organisms. Two and one-half years after infection, JH1001 was found to have persistently colonized three of the subjects. The indigenous S. mutans in one subject were reduced below the level of detection. Levels of (total) S. mutans and S. sanguis were not affected in persistently colonized subjects. Mutants of indigenous S. mutans resistant to the bacteriocin were not observed. The results indicate the importance of host variability and infection regimen for superinfection by this strain of S. mutans. The efficient replacement of indigenous S. mutans by JH1001 in one subject lends support to the eventual application of replacement therapy to the prevention of dental caries.

Colonization of the human oral cavity by a Streptococcus mutans mutant producing increased bacteriocin.

Streptococcus mutans strain JH1005 is a mutant that produces levels of bacteriocin activity three-fold-elevated than those produced by its parent, JH1001. A single infection regimen with JH1005 was found to result in persistent colonization of the teeth of all three adult subjects tested. This is a significant improvement over JH1001, which required multiple exposures in order to colonize the teeth of humans reliably. The levels of total cultivable bacteria and indigenous S. sanguis were not affected by JH1005 colonization. In two of the three subjects, total (indigenous plus JH1005) S. mutans levels were significantly decreased. The results provide additional support for the role of bacteriocin production as an ecological determinant in colonization by S. mutans. They also indicate that a practical regimen for infection by an effector strain might be achieved for use in the replacement therapy of dental caries.

Intravenous conscious sedation in the periodontal patient.

Intravenous diazepam was evaluated as a preoperative medication prior to periodontal surgery. Ten patients were subjected to two separately scheduled surgical episodes. During one procedure, a placebo was administered via an intravenous drip and during the other procedure, diazepam was titrated until Verrill's sign was reached. Vital signs, skin temperature, galvanic skin response, and serum cortisol were monitored and analyzed during both procedures. Vital functions were more stable in diazepam-sedated patients than in the placebo group. This was manifested by less increase in systolic blood pressure, respiration and heart rate. The galvanic skin resistance was increased and serum cortisol values decreased throughout the sedation phase, suggesting a reduced sympathetic arousal. This study indicates that intravenous diazepam sedation enhances patient comfort and improves physiologic safety during periodontal surgery.

Interaction between wild-type, mutant and revertant forms of the bacterium Streptococcus sanguis and the bacterium Actinobacillus actinomycetemcomitans in vitro and in the gnotobiotic rat.

In vitro, Streptococcus sanguis inhibits the growth of Actinobacillus actinomycetemcomitans, a presumed aetiological agent of localized juvenile periodontitis. When provided with glucose and good aeration, a growing culture of Strep. sanguis was found to produce hydrogen peroxide at concentrations in excess of the maximum LD50 reported for strains of A. actinomycetemcomitans. This concentration of hydrogen peroxide also inhibits the growth of the producer organism. A mutant of Strep. sanguis was isolated that lacked the ability to produce alpha-haemolysis on blood agar. This mutant had less than 3 per cent of its parent's level of pyruvate-oxidase activity, and made no detectable hydrogen peroxide. In vitro, the mutant had also lost the ability to inhibit the growth of A. actinomycetemcomitans. A spontaneous revertant, isolated by its ability to produce alpha-haemolysis, was found to have regained parental levels of pyruvate-oxidase activity and hydrogen-peroxide production and could inhibit the growth of A. actinomycetemcomitans in vitro. A gnotobiotic rat model was used to demonstrate that Strep. sanguis and A. actinomycetemcomitans interact in vivo and that this interaction depends on hydrogen-peroxide production by Strep. sanguis.

Competitive properties of lactate dehydrogenase mutants of the oral bacterium Streptococcus mutans in the rat.

Prior establishment of a lactate dehydrogenase (LDH)-deficient mutant of Streptococcus mutans in the mouths of rats caused a 10-10,000-fold increase in the minimal infectious dose for persistent colonization by wild-type strains of this organism. The finding that LDH-deficient mutants can render their host relatively resistant to infection by decay-causing strains of Strep. mutans while being much less pathogenic themselves, lends support to their prospective usefulness as effector strains in the replacement therapy of dental caries.

Bacterial interference in the oral ecology of Actinobacillus actinomycetemcomitans and its relationship to human periodontosis.

The plaque of 7 clinical health subjects was analysed for organisms inhibitory to the growth of A. actinomycetemcomitans strain Y4 on chocolate agar medium. Ten of the 11 sites harboured such organisms which constituted a median of 5.8 per cent of the total cultivable flora. Four to 30 per cent of the isolates in samples from eight sites in periodontitis were also inhibitory to strain Y4. By contrast, 15 of the 16 plaque samples from disease sites of 6 periodontosis (juvenile periodontitis) subjects showed no inhibitors. Four of 5 healthy sites sampled from 3 of the periodontosis subjects showed inhibitors in proportions similar to plaque from healthy subjects. The mother and 3 siblings of a subject with periodontosis lacked inhibitors in one or more plaque samples taken from their 1st molars. This work partially explains the basis for the localized pattern of destruction and the familial tendency of periodontosis.

The relationships between streptococcal species and periodontopathic bacteria in human dental plaque.

The existence of antagonistic and commensal relationships between microorganisms was investigated. The predominant cultivable flora in 172 plaque samples from active and non-active sites in 32 human subjects with destructive periodontitis was determined. The presence of putative periodontopathic organisms (Bacteroides gingivalis, Bacteroides intermedius, Bacteroides forsythus, Wolinella recta, Actinobacillus actinomycetemcomitans, and Eikenella corrodens) in a site was correlated with the absence of certain viridans streptococci (Streptococcus sanguis, Streptococcus uberis and Streptococcus intermedius), and vice versa. A strong commensal relationship was found between B. gingivalis and Strep. intermedius. The second study involved 3 subjects with intractable periodontitis whose plaque harboured large numbers of one or more of these periodontopathic organisms. This plaque contained fewer organisms capable of inhibiting the growth of the periodontopathic strains in vitro when compared with a clinically-healthy control subject. Intermediate levels of inhibitors were found in plaque taken from non-active lesions. The majority of inhibitors in plaque from the healthy control were viridans streptococci. Hydrogen-peroxide production by these organisms appears to be the principal mechanism of growth inhibition for periodontopathic organisms. Bacterial interactions may thus be causally related to both periodontal health and disease.

Elucidation of the antimicrobial mechanism of mutacin 1140.

Mutacin 1140 and nisin A are peptide antibiotics that belong to the lantibiotic family. N-Terminal rings A and B of nisin A and mutacin 1140 (lipid II-binding domain) share many structural and sequence similarities. Nisin A binds lipid II and thus disrupts cell wall synthesis and also forms transmembrane pores. Very little is known about mutacin 1140 in this regard. We performed fluorescence-based studies using a bacteria-mimetic membrane system. The results indicated that lipid II monomers are arranged differently in the mutacin 1140 complex than in the nisin A complex. These differences in complex formation may be attributed to the fact that nisin A uses lipid II to form a distinct pore complex, while mutacin 1140 does not form pores in this membrane system. Further experiments demonstrated that the mutacin 1140-lipid II and nisin A-lipid II complexes are very stable and capable of withstanding competition from each other. Transmembrane electrical potential experiments using a Streptococcus rattus strain, which is sensitive to mutacin 1140, demonstrated that mutacin 1140 does not form pores in this strain even at a concentration 8 times higher than the minimum inhibitory concentration (MIC). Circular complexes of mutacin 1140 and nisin A were observed by electron microscopy, providing direct evidence for a lateral assembly mechanism for these antibiotics. Mutacin 1140 did exhibit a membrane disruptive function in another commonly used artificial bacterial membrane system, and its disruptive activity was enhanced by increasing amounts of anionic phospholipids.

Modification of an effector strain for replacement therapy of dental caries to enable clinical safety trials.

AIMS: To construct a genetically modified strain of Streptococcus mutans for dental caries prevention. The strain has significantly reduced cariogenicity owing to a deletion of the entire open reading frame for lactate dehydrogenase, and has excellent colonization potential through the production of a natural antibiotic called mutacin 1140. For use in human clinical trials, additional mutations were introduced to enable rapid elimination of the strain in case of adverse side effects and to increase genetic stability. METHODS: Deletion mutations were introduced into the dal gene for d-alanine biosynthesis and the comE gene for genetic transformation. The resulting strain, A2JM, was tested for dependence on exogenous d-alanine and its ability to be eradicated from colonized rats. The strain was also tested for its ability to exchange DNA with another strain of S. mutans in in vitro and in vivo models. CONCLUSIONS: A2JM was completely dependent on exogenous d-alanine, but could colonize the oral cavity of rats in low numbers in the absence of dietary d-alanine. Results indicated that A2JM can scavenge d-alanine from other plaque bacteria. Lowering of the total oral bacterial load through daily application of chlorhexidine enabled virtually complete eradication of A2JM. The introduction of the comE gene did not significantly decrease the transformability of A2JM in in vitro or in vivo models. The addition of a deletion in the comE gene does, nonetheless, provide additional safety as it has a very low reversion frequency. SIGNIFICANCE AND IMPACT OF THE STUDY: Based on the safety and efficacy profiles established in vitro and in animal models, A2JM appears suitable for safe use in human clinical trials.

An alternative bactericidal mechanism of action for lantibiotic peptides that target lipid II.

Lantibiotics are polycyclic peptides containing unusual amino acids, which have binding specificity for bacterial cells, targeting the bacterial cell wall component lipid II to form pores and thereby lyse the cells. Yet several members of these lipid II-targeted lantibiotics are too short to be able to span the lipid bilayer and cannot form pores, but somehow they maintain their antibacterial efficacy. We describe an alternative mechanism by which members of the lantibiotic family kill Gram-positive bacteria by removing lipid II from the cell division site (or septum) and thus block cell wall synthesis.

Mutant analysis of glyceraldehyde 3-phosphate dehydrogenase in Escherichia coli.

NAD(+)-specific glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from Escherichia coli was purified to homogeneity by a relatively simple procedure involving affinity chromatography on agarose-hexane-NAD(+) and repeated crystallization. Rabbit antiserum directed against this protein produced one precipitin line in double-diffusion studies against the pure enzyme, and two lines against crude extracts of wild-type E. coli strains. Both precipitin lines represent the interaction of antibody with determinants specific for glyceraldehyde 3-phosphate dehydrogenase. Nine independent mutants of E. coli lacking glyceraldehyde 3-phosphate dehydrogenase activity all possessed some antigenic cross-reacting material to the wild-type enzyme. The mutants could be divided into three groups on the basis of the types and amounts of precipitin lines observed in double-diffusion experiments; one group formed little cross-reacting material. The cross-reacting material in crude cell-free extracts of several of the mutant strains were also tested for alterations in their affinity for NAD(+) and their phosphorylative activity. The cumulative data indicate that the protein in several of the mutant strains is severely altered, and thus that glyceraldehyde 3-phosphate dehydrogenase is unlikely to have an essential, non-catalytic function such as buffering nicotinamide nucleotide or glycolytic-intermediate concentrations. Others of the mutants tested have cross-reacting material which behaved like the wild-type enzyme for the several parameters studied; the proteins from these strains, once purified, might serve as useful analogues of the wild-type enzyme.

Principles of microbial ecology and their application to xerostomia-associated opportunistic infections of the oral cavity.

The human oral flora is normally composed of hundreds of different types of bacteria. This high degree of complexity is largely responsible for the observation that a state of health is the predominant condition of the oral cavity: Myriad bacterial interactions between innocuous and potentially pathogenic species prevent the latter from attaining sufficient numbers to initiate clinically observable diseases. However, protracted or profound perturbations of the oral environment may lead to ecological upsets fundamentally characterized by extreme simplification of the resident flora. Radiation- or autoimmune-induced xerostomia is a striking example of such a perturbation. During the period of floral simplification, as the bacterial interactions diminish, impediments to the outgrowth of pathogens are lost, ultimately leading to the onset of disease. In the absence of methods to reverse the conditions responsible for the ecological upset, as is currently the case in many forms of xerostomia, the possibility of restoring a complex, balanced flora is not imminently practical. However, as our understanding of microbial ecology and pathogenesis develops, application of a replacement therapy approach may become plausible to prevent or retard particular diseases prevalent in xerostomia patients.

Lactate dehydrogenase mutants of Streptococcus mutans: isolation and preliminary characterization.

Mutants of Streptococcus mutans were isolated which lack the enzyme activity L (+)-lactate dehydrogenase. Reversion studies indicate that the genetic defects are in the structural gene for the enzyme. The mutants produce less titratable acid from glucose, adhere better to hydroxyapatite, and accumulate more plaque when grown in the presence of sucrose than does the parent strain. These findings suggest a possible use for the mutants as effector strains in the replacement therapy of dental caries.