Use of polymerase chain reaction techniques and sodium dodecyl sulfate-polyacrylamide gel electrophoresis for differentiation of oral Lactobacillus species

BACKGROUND/AIMS: The genus Lactobacillus has been associated with dental caries in humans, although it is seldom speciated due to lack of simple and nonlaborious identification methods. A considerable heterogeneity among Lactobacillus species has been demonstrated. The purpose of this study was to develop simple methods combining restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified 16S rRNA (16S rRNA gene PCR-RFLP) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the identification of 13 reference strains of Lactobacillus. METHODS: The 16S rRNA gene sequences were amplified by PCR using universal primers and digestion of PCR products with the restriction endonucleases, HpaII and HaeIII. The 16S rRNA gene PCR-RFLP is reproducible and has been proved to be useful for differentiating Lactobacillus strains to species level. Seventy-seven Lactobacillus isolates from a Thai population were used to show the applicability of the identification test. RESULTS: PCR-RFLP alone had limitations, because the RFLP patterns of Lactobacillus casei and Lactobacillus rhamnosus and of Lactobacillus acidophilus and Lactobacillus crispatus showed similar patterns; however, these could be differentiated by SDS-PAGE. Of the 77 isolates, 38 were identified as Lactobacillus fermentum, 25 as L. rhamnosus, 5 as Lactobacillus salivarius, 5 as L. casei, 3 as L. acidophilus and 1 as Lactobacillus plantarum. CONCLUSION: 16S rRNA gene PCR-RFLP, using HpaII and HaeIII, together with SDS-PAGE protein profiles could be an alternative method for the identification of oral Lactobacillus strains to species level, and may be applicable for large-scale studies on the association of Lactobacillus to dental caries.     

Antibacterial activity of different generation dentin-bonding systems.

OBJECTIVES: The aim of this in vitro study was to investigate the antibacterial activities of different generation dentin-bonding systems. METHOD AND MATERIALS: The antibacterial activities of dentin-bonding systems Optibond FL Primer, Single Bond, Clearfil SE Bond Primer, and Prompt-L-Pop were evaluated against Streptococcus mutans, Streptococcus salivarius, Lactobacillus casei, and Lactobacillus acidophilus. Cervitec (1% chlorhexidine varnish) was also examined as a positive control material. Disk diffusion tests with filter paper disks (n = 10) containing a drop of each material (20 microL) were used. After incubation at 37 degrees C for 48 hours, the growth inhibition zones were measured in millimeters. RESULTS: Of all the materials tested, Optibond FL Primer, Clearfil SE Bond Primer, and Prompt-LPop showed larger growth inhibition zones than the control material for all bacterial strains. No antibacterial effect was noted for Single Bond. The results indicated that Optibond FL Primer had the strongest effect against the S mutans and L casei, and Clearfil SE Bond Primer produced the largest inhibition zone for S salivarius and L acidophilus among the test materials. CONCLUSION: The antibacterial effects observed for the tested different generation dentin-bonding systems may be related to the acidic nature of the materials.     

In vitro evaluation of antimicrobial property of silver nanoparticles and chlorhexidine against five different oral pathogenic bacteria

Introduction

Numerous antimicrobial agents are used to eliminate oral biofilm. However due to emergence of multi drug resistant microorganisms, the quest to find out biologically safe and naturally available antimicrobial agents continues.

Molecular and biochemical characterizations of human oral lactobacilli as putative probiotic candidates

INTRODUCTION: The objective of this study was to characterize the lactobacilli from the human oral cavity as a potential source of probiotic strains. METHODS: Samples were collected from four different locations within the oral cavity: surface of healthy tooth, oral mucous membrane, surface of tooth decay and deep tooth decay. On the basis of morphological and biochemical properties eight categories were formed and 26 isolates were selected for further characterization. The isolates were determined as Lactobacillus sp. using primers specific for 16S rDNA. Sequencing of 16S rDNA genes and repetitive sequence-based polymerase chain reactions were used for determination to species and subspecies levels. RESULTS: Predominant species were Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus salivarius and Lactobacillus paracasei subsp. paracasei, while Lactobacillus acidophilus, Lactobacillus cellobiosus, Lactobacillus delbrueckii subsp. lactis and Lactobacillus gasseri were also present. The isolates Lactobacillus salivarius BGHO1, Lactobacillus fermentum BGHO36 and BGHO64, Lactobacillus gasseri BGHO89 and Lactobacillus delbrueckii subsp. lactis BGHO99 exhibited antagonistic action on the growth of Staphylococcus aureus, Enterococcus faecalis, Micrococcus flavus, Salmonella enteritidis, Streptococcus pneumoniae and Streptococcus mutans, but not on growth of Candida albicans. Moreover, the isolates L. salivarius BGHO1 and L. gasseri BGHO89 were tolerant to low pH and high concentration of bile salts. CONCLUSION: Taken together, these findings imply that L. salivarius BGHO1 and L. gasseri BGHO89 might be subjects for additional investigation as potential probiotic strains.     

Competition between Streptococcus mutans and Lactobacillus casei in mixed continuous culture

In natural oral ecosystems, succession of Streptococcus mutans and Lactobacillus species occurs in bacterial communities which can be associated with the development of carious lesions. The objective of this study was to explore the interaction between 2 strains of S. mutans (SF2452) and Lactobacillus casei (RB1014) when grown together in continuous culture under conditions of varying environmental pH and in the presence of fluoride. When the strains, isolated from approximal carious lesions, were grown initially at a dilution rate of 0.1 h-1 under a glucose limitation at pH 7.0, a stable mixed community developed (Days 5-10) with S. mutans as the dominant population. When the pH control was removed (Day 10), the binary culture lowered the pH to 5.1 causing a small reduction in the numbers of S. mutans and allowing L. casei to predominate. A more dramatic effect was seen when the environmental pH was rapidly lowered to 4.8 by the addition of acid. In this case, the S. mutans population was significantly reduced and the L. casei population rose to the level previously held by S. mutans. Re-adjustment of the pH to 7.0 resulted in a re-establishment of the population ratios prior to the addition of acid. The possibility that S. mutans was capable of aciduric adaptation during a biologically-generated pH reduction was examined by mixing cultures of both organisms after each had been grown to steady state at pH 5.5 in separate chemostats.     

Effect of dentin on the antibacterial activity of dentin bonding agents

Dentin bonding agents with antibacterial effects may inhibit secondary caries formation and pulp inflammation by eliminating residual bacteria in and on dentin. Therefore, the antibacterial effects of Prime & Bond NT (PB), Prime & Bond NT without fluoride (PBNF), Gluma Comfort Bond (GL), ABF, Xeno CF II (XE), 2-hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacrylate (TEG-DMA), and 0.2% chlorhexidine were tested against Streptococcus mutans, S. sobrinus, and Lactobacillus acidophilus using the agar-diffusion method with and without bovine-dentin disks (200 microm and 500 microm thickness) placed between the bacteria and the test substances. Without dentin, ABF Primer showed growth inhibition for all bacterial strains. XE inhibited S. mutans and S. sobrinus, and PB S. sobrinus. ABF Bonding inhibited L. acidophilus. PBNF, HEMA, and TEGDMA did not have any antibacterial effects. Dentin disks of 500 microm thickness reduced the inhibitory effect of chlorhexidine to 23% to 54% compared with direct application. ABF Primer (nonpolymerized) produced inhibition zones against all tester strains regardless of dentin disks interposed or not. XE (against S. mutans and S. sobrinus) and PB (against S. sobrinus) did not produce any inhibition zones on 200 microm thick dentin. After polymerization, the ABF system did not inhibit bacterial growth on 200 microm thick dentin disks. A dentin barrier reduces significantly the antibacterial activity of chlorhexidine and dentin bonding agents.     

The predominant microflora of nursing caries lesions.

The predominant microflora recovered from infected dentine of 52 carious teeth from 14 children with nursing caries was determined using both selective and non-selective media for the isolation of specific genera and acidified media (pH 5.2) to isolate the predominant aciduric microorganisms, and compared with the microflora of sound enamel surfaces in caries-free children. Streptococcus mutans formed a significantly greater proportion of the lesion flora while Streptococcus oralis, Streptococcus sanguis and Streptococcus gordonii formed a significantly greater proportion of the plaque flora from sound tooth surfaces. The proportions of Actinomyces naeslundii and Actinomyces odontolyticus were significantly greater in the plaque samples than in the lesion samples. Actinomyces israelii formed 18.2% of the flora from the lesions, but was not isolated from the plaque samples. The proportions of Candida albicans, Lactobacillus spp. and Veillonella spp. were also significantly greater in the carious dentine than in the plaque samples. The most frequently isolated lactobacilli were Lactobacillus casei, Lactobacillus fermentum and Lactobacillus rhamnosus. The predominant aciduric flora was S. oralis, S. mutans and A. israelii and these taxa were also isolated from a similar proportion of the lesions at pH 7.0. Strains of S. mutans, L. casei, L. fermentum and L. rhamnosus isolated from individual carious teeth were genotyped using PCR-based methods. Each species was genotypically heterogeneous and different genotypes were recovered from different carious teeth in the same child. These data indicate that the microflora of lesions in the same child is microbiologically diverse and support a non-specific aetiology for nursing caries in which the physiological characteristics of the infecting flora, not its composition, is the major determinant underlying the disease process.     

In vitro antibacterial effect of chlorhexidine added to glass-ionomer cements

Abstract— The aims of the present study were to investigate the antibacterial properties of glass-ionomer cements containing different concentrations of chlorhexidine and the possible release of chlorhexidine from the compound. Chlorhexidine gluconate or diacetate was mixed with glass-ionomer cements and tested in vitro against strains of Lactobacillus casei and mutans streptococci. A spectrophotometric test was used to monitor the release of chlorhexidine from the mixture. In a broth culture test, the higher the concentrations of chlorhexidine in the mixture, the longer the time of antibacterial effect on Streptococcus mutans and the greater the tendency of the material to deteriorate. Agar diffusion tests revealed bacterial inhibition in a dose-response manner. The tested bacterial strains were similarly inhibited and the antibacterial properties decreased with time. A minor portion of added chlorhexidine was released from the cement. The deterioration of the cements indicates that the material could be useful as a varnish-like chlorhexidine carrier.     

In vitro antibacterial effect of chlorhexidine added to glass-ionomer cements.

The aims of the present study were to investigate the antibacterial properties of glass-ionomer cements containing different concentrations of chlorhexidine and the possible release of chlorhexidine from the compound. Chlorhexidine gluconate or diacetate was mixed with glass-ionomer cements and tested in vitro against strains of Lactobacillus casei and mutans streptococci. A spectrophotometric test was used to monitor the release of chlorhexidine from the mixture. In a broth culture test, the higher the concentrations of chlorhexidine in the mixture, the longer the time of antibacterial effect on Streptococcus mutans and the greater the tendency of the material to deteriorate. Agar diffusion tests revealed bacterial inhibition in a dose-response manner. The tested bacterial strains were similarly inhibited and the antibacterial properties decreased with time. A minor portion of added chlorhexidine was released from the cement. The deterioration of the cements indicates that the material could be useful as a varnish-like chlorhexidine carrier.     

Antibodies in normal and diseased pulps reactive with microorganisms isolated from deep caries.

Immunoglobulin molecules in the supernatant fluids (SF) from pulpal explant cultures have been observed to react with microorganisms implicated in infections of root canals. In this study, the reactivity of immunoglobulin molecules in the SF from normal and irreversible pulpitis pulps to six strains of predominant microorganisms isolated from the immediate layer of carious lesions above the pulps used for explant cultures was investigated using an enzyme-linked immunosorbent assay. Two ATCC strains of Eubacterium were also included in this assay. Specific antibodies to Lactobacillus casei subsp. casei, Lactobacillus casei subsp. rhamnosus, Lactobacillus acidophilus (I), (II), Streptococcus mutans, Bacteroides intermedius, Eubacterium brachy, and Eubacterium alactolyticum in the SF from the normal and irreversible pulpitis tissues were observed with a large variation of antibody levels in both groups. Immunodiffusion assays of the SF revealed that IgG was the major class of immunoglobulin in the normal as well as the irreversible groups. The presence of natural antibodies in the normal pulps suggested a possible protective role of antibodies during the invasive process of caries.     

Comparison of the antimicrobial activity of six irrigants on primary endodontic pathogens

The purpose of this study was to compare the antimicrobial activities of 6% and 3% sodium hypochlorite (NaOCl), 2% and 0.12% chlorhexidine gluconate (CHX), and 0.01% and 0.005% doxycycline (Doxy) on four microorganisms associated with primary endodontic infections. The agar diffusion test was used to measure antimicrobial activities of these agents against Peptostreptococcus micros, Prevotella intermedia, Streptococcus sanguis, and Lactobacillus acidophilus. Minimum inhibitory concentration analysis was performed using the macrodilution method. For three of the four microorganisms, the general order of antimicrobial effectiveness was 0.01% Doxy >0.005% Doxy >6% NaOCl >3% NaOCl >2% CHX > 0.12% CHX. For L. acidophilus, the order of effectiveness was 6% NaOCl >3% NaOCl >2% CHX > 0.01% Doxy >0.005% Doxy >0.12% CHX. The 6% NaOCl showed significantly greater zones of inhibition than 3% NaOCl for all endopathogens tested.     

Antimicrobial properties of self-etching primer-bonding systems

Self-etching primers are now considered the new generation of dentin bonding systems that modify and incorporate the bacteria-containing smear layer into their bonding mechanism. The antibacterial effects of the self-etching primers Clearfil SE Bond, Mac Bond, Imperva FL Bond, One-Up BondF and Prompt L-Pop were evaluated using the bacteria Streptococcus mutans ATCC25175, Peptostreptococcus anaerobius, Peptostreptococcus prevotii, Peptostreptococcus asaccharolyticus, Lactobacillus acidophilus, Lactobacillus catenaforme, Lactobacillus jensenii, Actinomyces odontolyticus, Porphyromonas endodontalis, Clostridium ramosum, Prevotella oris, Prevotella denticola and Fusobacterium nucleatum, with a disk diffusion method. A single-bottle total-etch dentin adhesive (Excite) was used for comparisons and chlorhexidine (0.2%) was used as the positive control. After incubation, zones of inhibited bacterial growth were observed. One-Up BondF, Prompt L-Pop and Excite showed growth inhibition for all bacterial strains. The bonding agents of Clearfil SE Bond, Mac Bond and Imperva FL Bond were unable to inhibit the growth of Lactobacillus jensenii and Actinomyces odontolyticus, while the primers of these systems produced inhibition halos to all tested microorganisms greater than that of chlorhexidine.     

Growth and acid tolerance of human dental plaque bacteria

Pure cultures of representative strains of cariogenic and non-cariogenic plaque bacteria were assessed for their ability to initiate and maintain growth in broths, adjusted to initial pH levels of 7.0, 5.5 or 5.0, and to produce lactic acid from sucrose or glucose in resting-cell suspensions at pH 6.5, 5.0, 4.5 and 4.0. Streptococcus mutans, Lactobacillus casei and Streptococcus faecalis showed greater acid tolerance than strains of Streptococcus sanguis, Streptococcus salivarius, Streptococcus mitis and Actinomyces viscosus. For all species, growth initiation in broth was more acid sensitive than lactic-acid production in resting-cell suspensions. These data confirm and extend previous observations that the species of plaque bacteria most closely associated with the initiation or progression of dental caries are more aciduric than non-cariogenic species.     

Antibacterial activity of fifth-generation dentin bonding systems.

Past concepts that the pulp does not become infected until an actual carious exposure takes place have been challenged. The antibacterial effects of the dentin bonding systems Single Bond, Prime&Bond NT, and Excite were evaluated using the bacteria Streptococcus mutans ATCC 25175, Streptococcus intermedius, Lactobacillus acidophilus, Prevotella oris, Prevotella bivia, Prevotella denticola, Porphyromonas gingivalis, Porphyromonas endodontalis, and Clostridium ramosum with a disk diffusion method. Chlorhexidine (0.2%) was used as a positive control. After incubation zones of inhibited bacterial growth were measured. Prime&Bond NT showed growth inhibition for all bacterial strains. Lactobacillus acidophilus and Streptococcus mutans were remarkably resistant to Single Bond, whereas EX produced no inhibitory effect on Porphyromonas endodontalis, although the adhesive produced the maximum halo inhibition to Streptococcus mutans (15+/-1 mm), showing an antibacterial effect closest to chlorhexidine. The variety of results obtained in this study suggest that antibacterial properties of current dentin adhesives may depend on components that are originally incorporated to promote adhesion.     

对氨基苯甲酸对乳杆菌生长影响的体外实验

目的:探讨对氨基苯甲酸菌株(PABA)对牙本质龋、根面龋致病菌之一的乳杆菌生长的影响。方法:10倍系列稀释法配制浓度为10-3~10-10gPL的PABA液,将其分别加入改良的Carlsson培养基中,再将乳杆菌ATCC4356放入该培养基厌氧培养48 h(80%N2、10%H2、10%CO2),用紫外可见分光光度仪测定细菌浓度OD值(K=540 nm);并在琼脂平板培养观察细菌生长的情况,进行菌落计数。结果:PABA也有促进乳杆菌生长的能力,当PABA的浓度从 10-10~10-4gPL增加时,其促进乳杆菌生长的能力增强(P<0.05),且当浓度为10-5gPL时,促进乳杆菌生长的能力达最强,随着PABA的浓度增加到10-4gPL时,此促进作用开始下降,PABA的浓度再增加至10-3gPL时,对乳杆菌的生长无明显的促进作用。结论:PABA的浓度为10-5gPL时,促进乳杆菌生长的能力达最强,高于或低于此浓度,这种促生长的作用均下降。     

Effect of monoclonal antibodies against lipoteichoic acid from the oral bacterium Streptococcus mutans on its adhesion and plaque-accumulation in vitro

Five monoclonal antibodies directed against Streptococcus mutans strain JBP lipoteichoic acid (LTA) were characterized. They were all similarly reactive with the immunizing LTA-containing extract, with intact Strep. mutans JBP cells and with LTA purified from Lactobacillus casei. Immobilized anti-LTA antibodies removes LTA from LTA-containing extracts. The binding of antibodies to LTA was inhibited by the aqueous extract but not by the organic extract of de-acylated LTA, indicating reactivity with the polyglycerol-phosphate portion of the molecule. Antibodies were reactive with all serotypes of Strep. mutans, as well as with strains of Streptococcus salivarius, Streptococcus sanguis and L. casei, but not with LTA-negative species Streptococcus mitis or Actinomyces viscosus. Anti-LTA antibodies at doses of 0.3 or 3.0 micrograms/ml, had no effect on the adherence of Strep. mutans JBP to experimental salivary pellicles formed on hydroxyapatite, but enhanced adherence 150-300 per cent at 30 micrograms/ml. There was no effect of anti-LTA antibodies in a chemostat model which measured sucrose-dependent plaque accumulation by Strep. mutans. The results argue against a major role for LTA in Strep. mutans adherence or plaque accumulation in vitro.     

Salivary Actinomyces naeslundii genospecies 2 and Lactobacillus casei levels predict pregnancy outcomes

BACKGROUND: Gravida's poor periodontal health is emerging as a modifiable independent risk factor for preterm delivery and low birth weight. METHODS: To test the hypothesis that oral bacteria other than periodontal pathogens are also associated with pregnancy outcomes, specific oral bacterial levels measured during pregnancy were evaluated in relation to gestational age and birth weight while controlling for demographic, medical, and dental variables. The study population consisted of 297 predominantly African- American women who were pregnant for the first time. The salivary bacterial levels evaluated were Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguinus, Lactobacillus acidophilus, Lactobacillus casei, Actinomyces naeslundii genospecies (gsp) 1 and 2, total streptococci, and total cultivable organisms. RESULTS: For 1 unit increase in log(10) A. naeslundii gsp 2 levels, there was a 60 gm decrease in birth weight (beta = -59.7 g; SE = 29.1; P = 0.04), and a 0.17 week decrease in gestational age (beta = -0.17 wk; SE = 0.09; P = 0.05). In contrast, per 1 unit increase in log(10) L. casei levels, there was a 42 gm increase in birth weight (beta = 42.2 g; SE = 19.3; P = 0.03), and a 0.13 week increase in gestational age (beta = 0.13 week; SE = 0.06; P = 0.04). CONCLUSIONS: We conclude that other oral bacterial species can also be related to pregnancy outcomes in addition to previously reported periodontal pathogens. These organism levels may not only predict poor pregnancy outcomes, but also be used as modifiable risk factors in reducing prematurity and low birth weight.     

Coaggregation of oral lactobacilli with streptococci from the oral cavity

The ability of oral lactobacilli to coaggregate with streptococci and actinomycetes was investigated. Of the 7 species of lactobacilli studied, only two were capable of Coaggregation and the Coaggregation was restricted to streptococci. Lactobacillus salivarius strains (2/4) coaggregated with Streptococcus salivarius, Streptococcus gordonii, Streptococcus crista and tufted Streptococcus sanguis II strains. Lactobacillus fermentum (2/3) coaggregated with S. gordonii and S. sanguis. The Coaggregation between L. salivarius and S. salivarius, S. gordonii or tufted S. sanguis II strains was mediated by a protein on the surface of the lactobacilli and was not inhibited by lactose. The Coaggregation between L. fermentum and the streptococci was mediated by protein on the surface of the streptococci and was inhibited by lactose.     

Antibacterial activity of dental gels containing combinations of amine fluoride, stannous fluoride, and chlorhexidine against cariogenic bacteria

The in vitro susceptibility of Streptococcus mutans, S. sobrinus and Lactobacillus casei to dental gels containing various combinations of amine fluoride (AmF 297), stannous fluoride (SnF2), and chlorhexidine (CHX) was studied. The combination of AmF-SnF2 with a total fluoride content of 1.2% was the most effective against mutans streptococci but not against L. casei. At notably lower total fluoride concentration (0.4%), AmF as such or combined with SnF2, was significantly less effective against mutans streptococci than CHX or CHX-AmF-SnF2 combinations. CHX-AmF combination was a slightly more potent inhibitor of streptococcal growth than CHX-NaF. With L. casei the differences between various gels were small but CHX alone seemed to be the most effective. Of the studied agents, CHX seemed to be the most potent individual chemotherapeutic compound whose activity against S. mutans could be enhanced by combining it with AmF. However, clinical experiments are required to test the in vivo antibacterial efficacy of CHX-AmF and AmF-SnF2 combinations which were most effective in our in vitro experiments.     

Antibacterial activities of MDPB and fluoride in dentin bonding agents

The aim of this study was to compare in vitro antibacterial activity of MDPB containing bonding system ABF with activities of three Fluoride containing bonding systems (Fuji Bond LC (FBLC), Prime&Bond NT (PBNT), and FluoroBond (FLB). Two bacterial strains were tested: Streptococcus mutans and Lactobacillus acidophilus. The study was performed on Muller Hinton Agar by Agar Well Technique. The bacterial agar was evenly distributed over the surface of petri dishes. Standard wells were punched into the agar. The test materials were placed in the wells of Muller Hinton agar plates, inoculated with Streptococcus mutans NCTC10449 and Lactobacillus acidophilus. The diameters of inhibition zones produced around the materials were measured after 24 h of incubation at 37 +/- 1 degrees C. For statistical analysis, Duncan's multiple range test was used. The primers of bonding agents were highly effective against to Streptococcus mutans. The MDPB containing primer of ABF Bond produced the greatest inhibition zones against to Lactobacillus acidophilus. It was followed by FLB primer. Lactobacillus acidophilus was resistant to FBLC primer and bonding, ABF, PBNT and FLB bonding agents. The results of this study indicated that, incorporation of MDPB in primer of self-etch system increased its antibacterial activity especially against to Lactobacillus acidophilus. However, all bonding systems except for bonding agent of ABF showed some antibacterial activity against to Streptococcus mutans.