Liposomes of probiotic's lyophilized cell free supernatant; a potential cosmeceutical product.

The potential uses of lyophilized cell free supernatant (CFS) of human oral derived Lactobacillus paracasei SD1 and Lactobacillus rhamnosus SD11 as cosmeceutical ingredients were investigated in the present study. Lyophilized CFS of both strains showed the antioxidant activity in concentration dependent manner. They also exhibited antimicrobial activity against P. acne, S. aureus and S. epidermidis. In combination, these two strains produced synergistic responses, not only on antioxidant activity but also on antimicrobial activity. A liposomal delivery system was employed to mask the unpleasant colour and odour of CFS. The optimal liposome formulation was characterized by a particle size of 344 nm, PDI of 0.19, zeta value of -48.05 mV and %EE of 69.45. The cytotoxicity results showed that the lyophilized CFS, which was toxic, became non-toxic after encapsulating into liposomes. Altogether, current findings demonstrate the worthiness of development of liposomes of probiotic's lyophilized CFS for cosmeceutical applications.

Antioxidant activity of various oral Lactobacillus strains.

AIMS: This study aims to evaluate the antioxidant activity of oral Lactobacillus strains in vitro. METHODS AND RESULTS: A total of 201 Lactobacillus strains were investigated for antioxidant activities: free radical scavenging (DPPH), inhibition of linoleic acid peroxidation (TAALA ), superoxide dismutase (SOD) and glutathione (GSH). Resistance to reactive oxygen species was also assessed. Results showed that there was a wide range of individual antioxidant properties of DPPH, TAALA , SOD and GSH between/within the species of Lactobacillus strains. Generally, all tested Lactobacillus, except L. oris and L. gasseri, showed relatively high antioxidative parameters indicating that antioxidative properties are strain specific. Lactobacillus fermentum, L. paracasei and L. rhamnosus strains with high DPPH and TAALA activities (>60%) could survive under oxidative stress (incubated in 1·0 mmol l-1 hydrogen peroxide for 8 h or 1·0 mmol l-1  hydroxyl radicals for 1 h) better than L. salivarius, L. oris and L. gasseri strains. It was also observed that there were significant differences in all antioxidative parameters tested, except DPPH, between the strains from caries-free subjects and those from subjects with caries. CONCLUSIONS: The different antioxidants were found among Lactobacillus strains. The strains with high antioxidants could tolerate well under the oxidative stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to investigate the antioxidant properties of human oral Lactobacillus strains. Significant results found that certain Lactobacillus strains exhibited relatively high antioxidative parameters. Further studies are needed to examine whether there would be any antioxidant effect on oral health.

Effect of fermented milk containing Lactobacillus rhamnosus SD11 on oral microbiota of healthy volunteers: A randomized clinical trial.

The aims of this study were to evaluate whether short-term consumption of fermented milk containing Lactobacillus rhamnosus SD11 affected levels of oral microbiota in vivo and whether L. rhamnosus SD11 could colonize in the human mouth. We also monitored for potential side effects of the probiotic. The applicability of using L. rhamnosus SD11 compared with Lactobacillus bulgaricus as a starter culture for fermented milk was evaluated. After informed consent, 43 healthy young adults were recruited and randomly assigned to either the probiotic or control group and received fermented milk containing L. rhamnosus SD11 or L. bulgaricus, respectively, once daily for 4 wk. The numbers of mutans streptococci, lactobacilli, and total bacteria in saliva were counted at baseline and then after 4 and 8 wk. An oral examination was performed at baseline and after 8 wk. The persistence of L. rhamnosus SD11 was investigated by DNA fingerprinting using arbitrary primer-PCR. Results demonstrated that statistically significant reductions in mutans streptococci and total bacteria were observed in the probiotic group compared with the control group, and the number of lactobacilli was significantly increased in both groups after receiving fermented milks. Lactobacillus rhamnosus SD11 could be detected (in >80% of subjects) up to 4 wk following cessation of dosing among subjects in the probiotic group. No side effects were reported. Thus, L. rhamnosus SD11 could be used as a starter culture for fermented milk. Daily consumption of L. rhamnosus SD11-containing fermented milk for 4 wk may have beneficial effects on oral health by reducing salivary levels of mutans streptococci. The probiotic was apparently able to colonize the oral cavity for a longer time than previously reported. However, the potential benefits of probiotic L. rhamnosus SD11 on oral health require further evaluation with a larger group of volunteers in a longer-term study.

An assessment of adhesion, aggregation and surface charges of Lactobacillus strains derived from the human oral cavity.

UNLABELLED: There is limited information concerning the adhesion and aggregation of human oral lactobacilli. In this study, the adhesion of 10 Lactobacillus species was investigated using H357 oral keratinocyte cells as an in vitro model for oral mucosa. Coaggregation with the representative oral pathogen, Streptococcus mutans ATCC 25175, and the physicochemical cell properties was also evaluated. The results demonstrated significant variations in adhesion (42-96%) and aggregation (autoaggregation, 14-95%; coaggregation, 19-65%). All strains showed a high affinity for chloroform, and most strains had a moderate-to-high hydrophobicity. All strains, except Lactobacillus casei and Lactobacillus gasseri, showed a moderate affinity for ethyl acetate. There was a strong association of autoaggregation with coaggregation (rs = 0·883, P < 0·001). The highest mean for autoaggregation (74%) and coaggregation (47%) belonged to the Lact. gasseri strains. Correlations between the adhesion and surface characteristics and aggregation were observed among the Lactobacillus fermentum and Lactobacillus paracasei strains; however, there was a variation in the strains properties within and between species. This study indicated that the Lact. gasseri, Lact. fermentum, and Lact. paracasei strains might be potential probiotics for the human oral cavity given their desirable properties. It should also be emphasized that a selective process for probiotic strains is required. SIGNIFICANCE AND IMPACT OF THE STUDY: Adhesion to host tissues and bacterial aggregation (auto- and coaggregation) are the highly important criteria for selecting strains with probiotic potential. These abilities are commonly involved with surface-charged characteristics. This is the first study to investigate the oral Lactobacillus species using an oral keratinocyte cell line. Significant results were found for the correlations between the adhesion and surface charge characteristics and for aggregation among certain strains of Lactobacillus gasseri, Lactobacillus fermentum and Lactobacillus paracasei. This observation could be useful when collecting background information for the selection of probiotic strains for use in oral health.

Site-specific dental plaque pH in 13-year-old Thai schoolchildren.

OBJECTIVE: The aim of this paper was to study pH conditions between dental sites, taking account the presence of caries, calculus, and microbial composition and alkali production. MATERIALS AND METHODS: One hundred 13-year-old Thai schoolchildren were recorded for caries experience (DMFT, DT), calculus, plaque, and gingivitis. Ex vivo urease activity was measured on 11, 26, 31, and 46 (distal aspect) with the rapid urease test and pH at baseline and after rinse with 0.25 % urea solution on mesial site in vivo. Interproximal plaque from contralateral teeth was microbiological analysed with the checkerboard technique. RESULTS: Thirty-four children were caries free. Plaque and calculus were abundant; all children showed a high resting plaque pH and the mandibular incisor showed significantly (p < 0.01) higher pH at baseline, max pH and AOC7.0 after urea challenge, ex vivo urease activity and calculus but lower caries experience than other teeth. A significant inverse correlation (p < 0.02) was found between caries frequency and ex vivo urease activity for tooth 11. Anaerobes predominated over streptococci, but no significant differences between dental sites were found. CONCLUSIONS: The study group had a high baseline plaque pH, in vivo and ex vivo urease activity, and calculus but low caries experience, which was best reflected in the lower incisor region. CLINICAL RELEVANCE: Urease activity and pH on site level may be important determinants for individuals at caries risk.

Purification and characterization of bacteriocin produced by oral Lactobacillus paracasei SD1.

The present study aimed to purify and characterize the antimicrobial protein from Lactobacillus paracasei SD1, which is a strain from the human oral cavity. Antimicrobial activity was obtained from purifying the culture supernatant of L. paracasei SD1. Purification of the active compound was achieved with ammonium sulfate precipitation followed by chloroform and gel filtration chromatography. As revealed by SDS-PAGE, the active fraction was homogeneous, showing a protein with an approximate molecular weight of 25,000 Da. It was confirmed as having a molecular mass of 24,028.2 Da by mass spectrometry. The antimicrobial compound, named "paracasin SD1", exhibited a broad spectrum against oral pathogens. Paracasin SD1 was stable in a pH range between 3.0 and 8.0 at 100 °C for 5 min, and showed resistance to α-amylase, catalase, lysozyme and whole saliva. However, its activity was lost after proteinase K and trypsin treatment. The results obtained suggest the possibility of using paracasin SD1 for application in prevention/treatment of oral diseases.

Metal-ion susceptibility of oral bacterial species.

AIMS: This study aimed to evaluate the effect of lead (Pb) on growth of bacterial species related to dental diseases in vitro. METHODS AND RESULTS: The effects of lead acetate on representative species of the oral flora were examined at 0.1-10 mmol l(-1) and compared with the effect of silver nitrate and ferrous sulfate. The minimal inhibitory concentration of lead acetate was between 0.15 and 5 mmol l(-1) for the bacterial strains tested. The minimal bactericidal concentration of lead acetate for most oral species was detected in the range of 5-10 mmol l(-1). Silver nitrate at a concentration of 1.25 mmol l(-1) was sufficient to exhibit antibacterial activity against almost all bacteria tested. Ferrous sulfate had the lowest effect. CONCLUSIONS: The study indicated a general antimicrobial effect of lead on oral bacterial species in the range of 0.15-10 mmol l(-1). The toxicity of silver nitrate was the highest, whereas that of ferrous sulfate was the lowest. Gram-positive species had a tendency to be less susceptible for metals than Gram-negatives. SIGNIFICANCE AND IMPACT OF THE STUDY: The study shows that it is possible that microbiological changes may occur in the dental plaque in children because of toxic exposure of environmental lead.

Inhibitory effect of oral Lactobacillus against oral pathogens.

AIMS: To determine the inhibitory effect of oral Lactobacillus against putative oral pathogens. METHODS AND RESULTS: Total 357 strains comprising 10 species of oral Lactobacillus, Lactobacillus fermentum (195), Lactobacillus salivarius (53), Lactobacillus casei (20), Lactobacillus gasseri (18), Lactobacillus rhamnosus (14), Lactobacillus paracasei (12), Lactobacillus mucosae (12), Lactobacillus oris (12), Lactobacillus plantarum (11) and Lactobacillus vaginalis (10) were used as producer strains. Inhibitory effect against a panel of indicators, periodontitis- and caries-related pathogens, was assessed. Most oral Lactobacillus was able to inhibit the growth of both periodontitis- and caries-related pathogens. The strongest inhibitory activity was associated with Lact. paracasei, Lact. plantarum, Lact. rhamnosus, Lact. casei and Lact. salivarius. Lactobacillus SD1-SD6, representing the six species with the strong inhibitory effect, inhibited growth of Streptococcus mutans ATCC 25175 in the biofilm model. Also, it was demonstrated that growth of Strep. mutans was inhibited in a mixture with Lact. paracasei SD1. The inhibition was enhanced in acidic condition and 5% glucose. CONCLUSIONS: The results have shown that oral Lactobacillus SD1-SD6 showed a strong inhibitory effect against Strep. mutans and Streptococcus sobrinus, as well as, Gram-negative periodontal pathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicated that Lactobacillus may be of benefit as probiotics for the prevention of oral diseases.

The gene coding for nigrescin produced by Prevotella nigrescens ATCC 25261.

AIM: To identify the gene that encodes nigrescin, a bacteriocin produced by Prevotella nigrescens ATCC 25261. METHODS AND RESULTS: Each open reading frame (ORF) of the nig gene cluster (nigA, nigB, nigC and nigD) was transferred into an expression vector. The recombinant proteins encoded by nigA, nigB, nigC and nigD were purified and assayed for bacteriocin activity against Porphyromonas gingivalis. The ORFs of the nig gene cluster in Pr. nigrescens ATCC 25261 were re-analysed. It revealed that the position of nig ORFs was similar to previously designated locations, except that the start codon of nigC was reassigned. The new nigC gene started at the nucleotide base position 2454 and stopped at position 3608 (the position designated is relative to the first nucleotide base of the nig locus) and putatively encoded a protein with a predicted molecular mass of 41.9 kDa. The N-terminal 6xHistidine-tag recombinant proteins of NigA, NigB, NigC and NigD were overexpressed in Escherichia coli BL21 star (DE3) and were purified using Ni-NTA resins. Only recombinant NigC showed inhibitory activity against P. gingivalis A244 with minimal inhibition concentration (MIC) of 40 microg ml(-1). CONCLUSION: These results indicate that nigC is the gene that encodes nigrescin. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that indicates that the gene nigC codes for nigrescin, a bacteriocin produced by Pr. nigrescens ATCC 25261.

Oral Lactobacillus species in type 2 diabetic patients living in southern Thailand.

Although lactobacilli are part of normal oral, gastrointestinal and genitourinary flora, they are an uncommon cause of infections in human. Lactobacillus-associated infections have generally occurred in patients with serious underlying conditions e.g. diabetes and cancer that might favour certain microorganisms. The aim of this study was to characterize species and genotypes of lactobacilli isolated from diabetic patients and non-diabetic subjects. One hundred and five type 2 diabetic patients and 103 non-diabetic subjects were recruited in this study. A total of 170 isolates of Lactobacillus were identified using 16S rRNA gene PCR-RFLP and genotyping were performed using AP-PCR by ERIC primers. It was found that type 2 diabetic patients had a significantly higher prevalence (p = 0.008) and level of lactobacilli than non-diabetic controls (p = 0.030). The most frequently isolated Lactobacillus spp. were L. casei/paracasei and L. fermentum in both the diabetic and non-diabetic groups. Strains of L. casei/paracasei and L. fermentum from between and within individuals were genotyped, and the genotyping of Lactobacillus strains showed diversity between individuals. One up to three genotypes of these two species could be found in the same subject. Interestingly, fewer genotypes were found in the diabetic patients than in the non-diabetic subjects.

Root surface and coronal caries in adults with type 2 diabetes mellitus.

OBJECTIVES: To determine the effect of type 2 diabetes mellitus (DM) on coronal and root surface caries and to investigate some factors suspected of being related to or interacting with DM, that may be associated with coronal and root surface caries. METHODS: A stratified cross-sectional study was conducted in 105 type 2 diabetic patients and 103 non-diabetic subjects of the same age and gender. Coronal and root surface caries, exposed root surfaces, periodontal status, stimulated salivary functions, oral hygiene status, oral health behaviors, and counts of mutans streptococci and lactobacilli were measured. RESULTS: Type 2 diabetic patients compared with non-diabetic subjects had a higher prevalence of root surface caries (40.0% versus 18.5%; P = 0.001), a higher number of decayed/filled root surfaces (1.2 +/- 0.2 versus 0.5 +/- 0.1; P < 0.01) and a higher percentage of generalized periodontitis (98.1% versus 87.4%; P < 0.01); but the prevalence and decayed/filled surface of coronal caries was not significantly different (83.8% versus 72.8% and 8.0 +/- 9.4 versus 6.3 +/- 7.5 respectively). The factors associated with root surface caries included type 2 DM, a low saliva buffer capacity, more missing teeth, and existing coronal caries; whereas wearing removable dentures, more missing teeth, a high number of lactobacilli, and a low saliva buffer capacity were associated with coronal caries. CONCLUSION: Type 2 DM is a significant risk factor for root surface, but not for coronal caries. Periodontal disease should be treated early in type 2 diabetic subjects to reduce the risk of subsequent root surface caries.

Longitudinal study of the presence of mutans streptococci and lactobacilli in relation to dental caries development in 3-24 month old Thai children.

OBJECTIVE: To study the acquisition of mutans streptococci (MS) and lactobacilli in relation to dental caries development in 3-24 month old Thai children. METHODS: Salivary samples were collected from 169 children using sterile wooden tongue depressors at the ages of 3, 9, 12, 18 and 24 months. The blades were pressed onto selective media for MS or lactobacilli. After incubation, the colony forming units of MS/lactobacilli were counted. Dental status was recorded from 9 months old using modified WHO criteria. RESULTS: The number of children with caries and number and severity of decayed teeth significantly increased with age. The presence of MS/lactobacilli was detectable at an early age and the cumulative prevalence of MS/lactobacilli increased with age. Children who were colonised early by MS or lactobacilli showed a higher number of decayed teeth than of those who were colonised later. The children with no MS/lactobacilli had significantly fewer decayed teeth and there was a significant correlation between MS/lactobacilli level and tooth decay. CONCLUSIONS: This longitudinal study shows early colonisation of the mouths of Thai children by MS/lactobacilli and where there are persistently high levels of the bacterias increased risk of development of dental caries.

Molecular fingerprinting of Porphyromonas gingivalis by PCR of repetitive extragenic palindromic (REP) sequences and comparison with other fingerprinting methods.

Knowledge of the genetic structure of populations of potentially pathogenic bacteria is important in understanding the epidemiology of diseases. Porphyromonas gingivalis is thought to be an important aetiological agent in periodontal diseases and several methods have been used for typing strains of this species. Here, PCR with primers to repetitive extragenic palindromic sequences (REP-PCR) was compared with three other widely used molecular fingerprinting techniques -- restriction endonuclease analysis (REA), ribotyping and PCR with arbitrary primers (AP-PCR) -- to type P. gingivalis isolates from healthy and diseased periodontal sites. The data obtained with all four methods were in broad agreement and, with one exception, each subject harboured a single unique genotype of P. gingivalis. REP-PCR of P. gingivalis resulted in the production of 5-10 amplicons, which gave unique electrophoretic patterns in each individual (10 REP-PCR types in 10 patients) and similar results were obtained with AP-PCR. Two isolates from one subject appeared identical by REP-PCR and AP-PCR, but could be differentiated by ribotyping, although there was only minor polymorphism. Thus, ribotyping and REA were the most discriminating methods; however, these are time-consuming and expensive relative to the PCR-based techniques. REP-PCR has the advantage that the same pair of primers is used for all species, whereas AP-PCR needs to be optimised by screening a range of primers. These results show that REP-PCR is a useful and rapid technique for typing P. gingivalis.

Production and sensitivity of bacteriocin-like activity among Porphyromonas gingivalis, Prevotella intermedia and Pr. nigrescens strains isolated from periodontal sites.

The production of and sensitivity to bacteriocin-like activity among 44 strains of black-pigmented anaerobes isolated from periodontal sites were evaluated by both an overlay and an agar diffusion method. The species studied were Porphyromonas gingivalis, Prevotella intermedia and the closely related species Pr. nigrescens. Pr. intermedia strains (90%) produced bacteriocin-like activity against Pr. nigrescens and all Pr. nigrescens were active against Pr. intermedia. Both species showed a high degree of activity against P. gingivalis, whereas only one P. gingivalis strain produced bacteriocin-like activity against either of the other two species. Both Pr. nigrescens and Pr. intermedia showed some activity (40% and 20%, respectively) against other strains of the same species. Such bacteriocin production might be expected to influence the distribution of these black-pigmented species in vivo. Of 224 periodontal sites sampled, only 2.6% yielded mixed cultures of black-pigmented species and of these only one strain, a P. gingivalis isolate, produced bacteriocin-like activity against any of the other strains isolated from these sites. These data support the concept that local production of bacteriocin-like activity in vivo may contribute to the selection of the black-pigmented bacterial profile in subgingival sites.

Binding and degradation of lactoferrin by Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens.

The ability of laboratory and clinical strains of Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens to bind and to degrade lactoferrin (Lf) has been assessed. Lf bound readily to whole cells of each species apparently via high-affinity site and one or more low-affinity sites. P. gingivalis showed a lower affinity for Lf than the other two species (P < 0.001). Virtually all strains of P. gingivalis completely degraded Lf under the conditions employed, whereas P. intermedia and P. nigrescens showed only partial degradation. These data suggest that Lf binds to a high-affinity receptor on all these bacteria and, particularly in the case of P. gingivalis, is then degraded by cell-associated proteases. This property may provide protection to the cell against the effects of Lf in periodontal sites and so is a possible virulence factor in disease. There was no association between the ability to degrade Lf and whether the strains had originated from healthy or diseased oral sites.

The usefulness of the modified extra-oral vacuum aspirator (EOVA) from household vacuum cleaner in reducing bacteria in dental aerosols.

AIMS AND OBJECTIVES: Currently, as a preventive measure against air contamination caused by dental procedures, dust-collecting aspirators such as an extra-oral vacuum aspirator (EOVA) are coming into general use. In this study, we tested the ability of a modified EOVA that uses a household vacuum machine. DESIGN AND SETTING: The study was conducted in two parts. First, the modified EOVA was tested for its ability to eliminate an Escherichia coli aerosol generated in a manikin. Second, the device was tested for its ability to remove the aerosol generated by dental treatment procedures, scaling and drilling a tooth, on human subjects. RESULTS: There was a statistically significant reduction in both the E. coli aerosol and the oral bacterial aerosol during dental treatment procedures. CONCLUSION: The modified EOVA machine is highly effective in preventing air contamination by dental procedures. Its main advantages are that it can easily be modified for use with any dental unit and it is relatively inexpensive.

Oral histoplasmosis associated with candidiasis in HIV-infected patients: a report of two cases.

Oral histoplasmosis, a rare condition, is reported in two HIV-infected patients. The lesions showed different characteristics. One was an ulcerative lesion presenting as the only manifestation of the disease. The other was nodular, with disseminated histoplasmosis. In both cases infection with Candida was also noted within the lesions.

16S rRNA PCR-Denaturing Gradient Gel Electrophoresis of Oral Lactobacillus casei Group and Their Phenotypic Appearances.

This study aimed to develop a 16S rRNA PCR-denaturing gradient gel electrophoresis (DGGE) to identify the species level of Lactobacillus casei group and to investigate their characteristics of acid production and inhibitory effect. PCR-DGGE has been developed based on the 16S rRNA gene, and a set of HDA-1-GC and HDA-2, designed at V2-V3 region, and another set of CARP-1-GC and CARP-2, designed at V1 region, have been used. The bacterial strains included L. casei ATCC 393, L. paracasei CCUG 32212, L. rhamnosus ATCC 7469, L. zeae CCUG 35515, and 46 clinical strains of L. casei/paracasei/rhamnosus. Inhibitory effect against Streptococcus mutans and acid production were examined. Results revealed that each type species strain and identified clinical isolate showed its own unique DGGE pattern using CARP1-GC and CARP2 primers. HDA1-GC and HDA2 primers could distinguish the strains of L. paracasei from L. casei. It was found that inhibitory effect of L. paracasei was stronger than L. casei and L. rhamnosus. The acid production of L. paracasei was lower than L. casei and L. rhamnosus. In conclusion, the technique has been proven to be able to differentiate between closely related species in L. casei group and thus provide reliable information of their phenotypic appearances.

Lactobacillus species and genotypes associated with dental caries in Thai preschool children.

Lactobacilli have been associated with the presence and progression of dental caries. Nevertheless, the relation between certain species or genotypes of Lactobacillus and caries is unclear and there are no data available for the Thai population. This study aimed to examine the distribution of species and genotypes of oral Lactobacillus among children with rather high caries prevalence, and to investigate whether certain species or genotypes were more related to caries activity than others. One hundred and sixty-five children were examined for caries status. Saliva samples were collected and the numbers of lactobacilli were counted. A total of 357 Lactobacillus isolates from 59 children were identified to species level by 16S ribosomal RNA genes polymerase chain reaction (PCR) -restriction fragment length polymorphism and 16S ribosomal RNA gene sequencing. Furthermore, 304 isolates from 56 children were genotyped using arbitrarily primed PCR. Significant correlation was found between levels of lactobacilli and dental caries (P < 0.001). Among the 10 identified species of Lactobacillus, L. salivarius was more prevalent in children with moderate to high caries prevalence compared with children with low caries prevalence, while L. fermentum was the most predominant species in all study groups. Moreover, a genetic heterogeneity of Lactobacillus species was found among the children and those with high caries prevalence tended to be colonized with more than one clonal type. In summary, L. salivarius may be a putative caries pathogen among preschool Thai children.