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.     

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.     

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.     

Lactobacillus plantarum for oral peptide delivery

AIMS: To evaluate strains of lactobacilli for their ability to persist and secrete heterologous protein in the oral cavity. METHODS AND RESULTS: Four different strains of common oral lactobacilli, Lactobacillus brevis, Lactobacillus johnsonii, Lactobacillus murinus and Lactobacillus plantarum, were transformed with the plasmid pKTH2121, which contains a secretion cassette for beta-lactamase. Lactobacilli isolated from the mouth of host mice were also transformed with pKTH2121 for later feeding. Lactococcus lactis, transformed with pKTH2121, was also fed to mice as a negative control. All transformed isolates were fed to C57Black mice in varying schedules. The number of transformed bacteria persisting in the mouth was reported as a percentage of total oral bacteria recovered by swabbing. CONCLUSIONS: The transformed L. lactis, L. brevis, L. johnsonii, L. murinus, and the endogenous murine lactobacillus strain failed to persist in the mouth. Transformed L. plantarum, however, persisted in the mouth and comprised up to 25% of the total lactobacilli at 18 h and 10% at 24 h after feeding. L. plantarum recovered after feeding retained its ability to secrete beta-lactamase into culture medium efficiently. Beta-lactamase activity could be detected in oral secretions at 8 h after feedings. After repeated feedings, however, the L. plantarum containing pKTH2121 gradually lost its ability to persist after feedings. This experiment demonstrates that L. plantarum can transiently colonize the oral mucosa in large numbers, while continuously secreting foreign proteins, raising the possibility of using lactobacilli as a vector for delivery of oral mucosal peptides.     

Biofilm formation by antagonistic strains of oral Lactobacillus

Highantagonistic strains of lactobacillus isolated from the oral cavity of healthy people, genetically identified as L. fermentum 39, L. rhamnosus 50, L. rhamnosus 24, showed high degree of autoaggregation, surface hydrophobicity, coaggregation, adhesion, and the ability to form biofilms. These strains can be used to create new probiotic drugs for the prevention and correction the dysbiosis of the oral cavity.     

婴儿口腔早期定植菌群的一年动态观察

目的　观察婴儿口腔早期定植细菌的动态变化。方法　收集12例健康新生儿出生后第1天和1、3、6、9、 12个月的口腔样本,选择适当稀释度接种于BA、MSA、BHI培养基,经需氧、微需氧及厌氧条件培养后行细菌形态学及生化鉴定。结果　婴儿口腔中检出率最高的细菌为S.salivarius,其次为S.mitis;乳牙萌出后S.sanguis,S.gordo- nii和S.mutans有一定的检出;Veillonella spp.在出生后1月的新生儿口腔即有一定的检出,A.odontolyticus在3个月时开始有8.3%的检出;L.acidophilus在婴儿口腔一直维持在较低的检出水平;少数有部分乳牙萌出的婴儿口腔中检出L.buccalis与Capnocytophaga spp.的存在。结论　S.salivarius与S.mitis是新生儿及婴儿口腔的优势细菌,Veil- lonella是最早、最常检出的厌氧菌,A.odontolyticus是最早检出的放线菌,随着婴儿月龄的增加,其口腔中检出细菌的种类和数量均明显增加。     

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.     

高龋及无龋者变形链球菌临床分离株致龋性研究Ⅰ对唾液包被羟磷灰石的粘附实验

目的：探讨来自不同龋敏感者的变形链球菌（血清型ｃ）临床分离株的粘附能力。方法：采用液体闪烁计测法测量＾３Ｈ－ＴＤＲ标记的变形链球菌临床分离株对唾液包被羟磷灰石（ＳＨＡ）的粘附。结果：同一个体所带不同基因型菌株对ＳＨＡ粘附量差异较大;高龋组个体定植的粘附能力强的菌株所占比较显著高于无龋组。结论：高龋组变形链球菌（血清型ｃ）临床菌株的高致龋力与其携带有粘附能力强的菌株密切相关。     

HIV感染者口腔念珠菌负荷及生物型研究

目的调查人类免疫缺陷病毒（human immunodeficiency virus，HIV）感染者口腔中念珠菌负荷状况、生物分型及与口腔念珠菌病临床表现的关系。方法采取漱口法对64例HIV感染者和42名健康对照者进行口腔念珠菌的定量分离培养，并综合利用革兰染色、厚壁孢子生成实验、CHROMagar显色培养和API 20C AUX酵母菌鉴定系统对分离株进行生物型鉴定。结果64例HIV感染者中，52例中可分离出念珠菌74株，阳性分离率为81．3％，而42名健康对照者口腔念珠菌阳性分离率仅为16．7％（P〈0．001）。通过对74株念珠菌的生物型进行鉴定，发现有39株白色念珠菌，15株热带念珠菌及其他6个生物型20株。健康对照组中，分离出5株白色念珠菌和其他裂2株。结论HIV感染者口腔念珠菌感染率明显增加，其口腔念珠菌的检出率和负荷量亦明显增加，白色念珠菌和热带念珠菌为其主要分离菌；与健康对照组相比，HIV感染者的口腔念珠菌分离株生物类型旱现多样化。     

Detection of oral streptococci with collagen‐binding properties in saliva specimens from mothers and their children

International Journal of Paediatric Dentistry 2010; 20: 254–260 Background. Approximately 10–20% of Streptococcus mutans strains have been reported to possess collagen‐binding properties, whereas other species in the oral cavity with those properties remain to be elucidated. Aim. To identify strains with collagen‐binding properties and analyse their characteristics in comparison with S. mutans. Design. A total of 110 expectorated saliva specimens were collected from 55 pairs of mothers and their children. Bacterial strains with collagen‐binding properties were isolated and the species specified. In addition, strains with collagen‐binding properties isolated from mother–child pairs were analysed using molecular biological approaches. Results. The detection frequency of strains with collagen‐binding properties was shown to be 40.9%, among which S. salivarius was the most frequently detected, followed by S. mutans. The collagen‐binding activity of the S. mutans group was the highest, followed by S. salivarius. In addition, S. mutans and S. salivarius strains from 3 and 1 mother–child pairs, respectively, were shown to be the same clones. Conclusions. Our results indicate that S. mutans and S. salivarius are major species with collagen‐binding properties in the oral cavity, and that strains with such properties may be related to mother–child transmission.     

Cariogenicity of the probiotic bacterium Lactobacillus salivarius in rats

Probiotic bacteria such as lactobacilli and bifidobacteria are considered to be non-pathogenic and non-toxigenic on the basis of long years of safe usage. However, some species of lactobacilli are thought to be associated with the development of dental caries. The purpose of the present study was to examine the cariogenicity of the probiotic bacterium Lactobacillus salivarius in rats. Rats were divided into six groups, and infected with L. salivarius LS1952R and/or Streptococcus mutans MT8148R. L. salivarius LS1952R became established in the oral cavity of rats and induced significant level of dental caries even when infected for only 5 days from 18 to 22 days of age. In addition, the caries scores of rats superinfected with both Streptococcus mutans MT8148R and L. salivarius LS1952R from 18 days of age were significantly higher than those infected with either L. salivarius LS1952R or S. mutans MT8148R alone. Since strain LS1952R can adhere to saliva-coated hydroxyapatites, it is concluded that L. salivarius strain LS1952R possesses an inherent cariogenic activity following adherence to the tooth surface.     

The prevalence and antibiotic sensitivity of oral Viridans streptococci in healthy children and children with disabilities in Kuwait

The purpose of this study was to compare the antibiotic susceptibility profiles of Viridans streptococci (VS) isolated from the oral cavity of healthy children and children with disabilities in Kuwait. Plaque samples were collected from the tooth and tongue surfaces of 102 healthy children and 102 children who were intellectually disabled and institutionalized. The resistance to seven antibiotics (amoxicillin, cephalothin, clindamycin, erythromycin, penicillin G, tetracycline, and vancomycin) was tested.
A total of 330 (44.5%) VS were isolated from the children who were healthy and 411 (55.5%) from children with disabilities. The most common isolates were S. salivarias (27.3%) in healthy children; S. sanguis (22.6%) was predominant among children who were disabled. S. mutans was found in 12.1% of the healthy children and in 16.5% of the children who were disabled. The combined percentage of resistant strains (healthy and disabled) was found to be highest with amoxicillin (43%) and lowest with vancomycin (12%). S. sanguis, S. mitis and S. oralis were more resistant in healthy children (45%, 56%, and 55% respectively) than in children with disabilities (40%, 47% and 47% respectively). S. mutans was the least resistant species to all antibiotics in both groups of children. About 56% of ali streptococci isolated from both groups were resistant to at least one of the antibiotics tested.
The data showed that there was a diference in the level of resistance of oral VS isolated from healthy children and children with disabilities to some antibiotics commonly used in dentistry.     

牙龈卟啉单胞菌在龈下菌斑中的分布

研究牙龈卟啉单胞菌在牙周炎患者病变部位和健康部位龈下菌班中的分布情况,方法：选择６４例成年牙周炎患者,取龈下菌斑,经厌氧培养,挑战产黑色素菌落,经多聚酶链反应鉴定牙龈卟啉单胞菌。结果：产黑色素Ｇ＾－厌氧杆菌和牙龈卟啉单胞菌的患者检出率分别是６７．２％和６０．９％。牙龈卟啉单胞菌在病变部位和健康部位的检出率分别是３５．９％和２８．１％,二者差异无统计学意义;牙龈卟啉单胞菌在病变部位和健康部位的检出株     

Sucrase and maltase activities in supragingival dental plaque in humans of streptococcal, actinomyces and lactobacilli species

Abstract – 20 reference strains and 72 isolated strains from dental plaque of streptococci-, actinomyces, and lactobacilli species were examined for sucrase and maltase activities. The type of sucrase in the different strains was determined by use of the α-glucosidase inhibitor, acarbose. The enzyme activities were determined as formation of monosaccharide, and quantitated spectrophotometrically. Although variations occurred in enzyme activities between reference and isolated strains, the same general pattern was noticed. Strains of Streptococcus mutans and S. salivarius showed regularly the highest sucrase activities, followed by strains of Actinomyces viscosus and A. naeslundii. Most lactobacilli belonged to the bacteria with low sucrase activity like S. sanguis and S. mitior. In some lactobacilli strains, however, a high sucrase activity was observed. The level of sucrase activity in S. mutans strains was dependent on biotype/serotype, as strains of biotype V/serotype e showed high activities, biotypes I and IV corresponding to serotypes c, f, and d showed intermediate activity, and biotype III/serotype a always showed low activity. In most of the strains the sucrases were composed of enzymes with specificity against both α-glucosidic linkage and β-fructosidic linkage of the sucrose molecule, but in varying ratios. In all species, except S. sanguis and S. mitior, lower maltase than sucrase activity was observed, but even in the two species mentioned the maltase activities were relatively low. On the basis of observations of selected reference strains in every species examined both sucrases and maltases are to some extent inducible.     

Isolation of Candida species from the oral cavity and fingertips of complete denture wearers.

STATEMENT OF PROBLEM: Candida species usually colonize in the oral cavity of denture wearers and may also colonize on their fingers because of frequent manual manipulation of the dentures. PURPOSE: This study investigated the association between oral and fingertip candidal isolation in a group of denture wearers. MATERIAL AND METHODS: Oral rinse and fingerprints obtained from 25 healthy male complete denture wearers were microbiologically investigated for candidal growth, and isolated Candida species were identified with a germ tube test and a commercially available yeast identification system. Denture cleanliness, hand washing, and denture wearing and handling habits were recorded for each subject. RESULTS: Candida species were isolated from the oral cavity of 15 (60%) and fingertips of 11 (44%) subjects. Ten (66.7%) subjects had concomitant oral and fingertip candidal isolation, whereas 5 (33.3%) subjects had only oral Candida (P=.005). CONCLUSION: The hands of denture wearers who had oral Candida were significantly more colonized with Candida species than oral Candida-free subjects. Further studies identifying Candida species to the strain level and the significance of hand Candida as a source for mouth reinfection are needed.     

Evaluation and comparison of lactobacilli characteristics in the mouths of patients with or without cavities

Lactobacilli were considered the prime cariogenic microorganisms until phylogenetic related bacteria, mutans streptococci, were associated with caries. Today, lactobacilli are still considered a factor in determining the predisposition to develop cavities. As a first step in colonization, microorganisms adhere to oral tissues. Based on this fact, the purpose of our study was to determine if there was a statistical association or difference related to the state of oral health with the surface characteristics of lactobacilli. Patients were classified as caries-free and caries-active. Interviews were performed to establish the nutritional and oral hygiene habits. The previously reported characteristics of isolated lactobacilli's quantification and association to dental tissues were determined. Although the nutritional habits for caries-free and caries-active patients were similar, the patients' oral hygiene and dental care determined differences in risk indices. The number of lactobacilli was statistically lower in saliva of subjects with good oral health. Certain species of lactobacilli could not be associated to specific areas of the mouth, although some species could be localized. Lactobacilli from caries active (CA) subjects showed a greater ability to adhere to hydrophobic substances, had a greater salt agglutination property, and showed lower production of inhibitory substances. Lactobacilli from caries free (CF) subjects were better able to inhibit oral, potentially pathogenic, microorganisms. These studies prove that preliminary differences between oral lactobacilli in CF and CA patients exist. Non-specific and specific adhesion mechanisms in bacteria should be further demonstrated.     

同一口腔中c血清型变形链球菌高、低毒力株的筛选

目的：由同一高龋者口腔中分离出c血清型变形链球菌高致龋毒力的菌株和低致龋毒力的菌株，为变形链球菌的比较基因组研究奠定基础。方法：从4个方面比较24株c血清型变形链球菌临床分离株对唾液包被羟基磷灰石的黏附能力、产酸和耐酸能力以及合成胞外多糖的能力。结果：筛选出位于同一口腔中的高、低毒力株。结论：同一口腔中存在着2株或2株以上致龋能力不同的c血清型变形链球菌。     

Presence of Lactobacillus casei in saliva from children and adults using a new medium

The aim of this study was to devise a medium for cultivation of L. casei and to compare the presence of L. casei in saliva from children and adults. The new medium was produced by omitting the sugars from Rogosa SL agar medium and supplementing this medium with melezitose. Although all tested strains of lactobacilli grew on the new medium, the facultatively homofermentative lactobacilli, L. casei and L. plantarum, could be identified by a typical colonial morphology. L. casei was the most common species of oral lactobacilli in saliva from children and made up a significantly higher proportion of the salivary lactobacillus flora in children than in adults.     

Presence of Lactobacillus casei in saliva from children and adults using a new medium

The aim of this study was to devise a medium for cultivation of L. casei and to compare the presence of L. casei in saliva from children and adults. The new medium was produced by omitting the sugars from Rogosa SL agar medium and supplementing this medium with melezitose. Although all tested strains of lactobacilli grew on the new medium, the facultatively homofermentative lactobacilli, L. casei and L. plantarum, could be identified by a typical colonial morphology. L. casei was the most common species of oral lactobacilli in saliva from children and made up a significantly higher proportion of the salivary lactobacillus flora in children than in adults.     

Identification and characterization of a collagen-binding protein, Cbm, in Streptococcus mutans

Streptococcus mutans, a major pathogen of dental caries, is occasionally isolated from the blood of patients with infective endocarditis. Bacterial attachment of exposed collagen tissue in the impaired endothelium is an important step in the onset of infective endocarditis. In our previous studies, some S. mutans strains were shown to possess collagen-binding activities and most of them had an approximately 120-kDa cell-surface collagen-binding protein called Cnm. However, several strains without Cnm proteins show collagen-binding properties. In the present study, another collagen-binding protein, Cbm, was characterized and its coding gene cbm was sequenced in these strains. The amino acid alignment in the putative collagen-binding domain of Cbm was shown to have approximately 80% identity and 90% similarity to the comparable region of Cnm. Cbm-deficient isogenic mutant strains constructed by insertional inactivation of the cbm gene, lacked collagen-binding properties, which were recovered in the complemented mutant. Analyses of a large number of clinical isolates from Japan, Thailand and Finland revealed that cbm-positive strains were present in all of these countries and that cnm-positive and cbm-positive strains were detected in the oral cavity of approximately 10 and 2% of systemically healthy subjects, respectively. In addition, cnm-positive strains were predominantly identified in the serotype f group, whereas cbm-positive strains were frequently detected in serotype k. These results suggest that Cbm as well as Cnm are major cell surface proteins of S. mutans associated with binding to type I collagen and predominantly identified in serotype k strains.