In Vitro Antimicrobial Activity and Aggregation Abilities of Probiotic Lactobacillus casei and Lactobacillus salivarius Against Oral Pathogens

ABSTRACT@#The antagonistic effect of probiotics against oral pathogens merits exploration because these bacteria are beneficial to the host’s health. The antimicrobial activity of two probiotic strains, Lactobacillus casei and Lactobacillus salivarius, as well as L. casei and L. salivarius combination (1:1), was investigated against Streptococcus mutans, Streptococcus sobrinus, Candida albicans, Candida glabrata and Candida tropicalis using agar-well diffusion, auto-aggregation and coaggregation assays. L. salivarius cell-free supernatant (CFS) alone exhibited greater inhibitory effect against Streptococci spp. compared to L. casei CFS alone and the combination. However, no inhibition was observed for Candida spp. L. salivarius alone exhibited significantly stronger auto-aggregation than L. casei alone (p ≤ 0.05) and L. casei and L. salivarius combination. L. salivarius exhibited strong coaggregation ability with Candida spp., followed by Streptococci spp. while L. casei exhibited coaggregation only with Streptococci spp. However, L. casei and L. salivarius combination did not display any coaggregation with all strains. L. salivarius alone exhibited a stronger antagonistic effect on the tested organisms than L. casei alone or in combination. Based on the results, both probiotic strains showed good antimicrobial activities against oral pathogens and should be further studied for their human health benefits.

D-Tryptophan governs biofilm formation rates and bacterial interaction in P. mendocina and S. aureus

Biofilm genesis by Pseudomonas and Staphylococcus sp is associated with biofouling in natural settings. D-Tryptophan (DTrp) inhibits bacterial biofilms and have been proposed for biofouling control applications. In this study, D-Trp significantlyinhibited Pseudomonas mendocina and Staphylococcus aureus cell attachment (biofilm formation) rates on polystyrene96-well microtiter plates in comparison with L-Tryptophan (L-Trp) and mixtures of D-/L-Tryptophan (D-/L-Trp). Theinhibitory effect was greater on P. mendocina, where the rate of cell adherence was declined to 8.7 9 105 cells/h from8.0 9 106 cells/h (control) in P. mendocina. In S. aureus it was declined to 4.2 9 107 cells/h from 9.2 9 107 cells/h(control) at 1 mM concentration. It hindered the intracellular communication and adherence in both the strains, as confirmed by SEM and real time PCR analysis. Addition of D-Trp to preformed biofilms also caused partial disassembly. Intraand interbacterial aggregation were decreased subsequently upon treatment with D-Trp. It repressed the genes involved incell–cell communication, which could be responsible for the diminished biofilm formation of the selected strains. HenceD-Tryptophan has proved to be an effective strategy to control biofilm and may support in the development of surfacecoating technologies.

A preliminary study on the effect of histatin 5 inhibiting Porphyromonas gingivalis and Fusobacterium nucleatum co-aggregation / 中华口腔医学杂志

Objective@#To detect the inhibitory ability of histatin 5 on the auto-aggregation of Porphyromonas gingivalis (Pg), and the co-aggregation of Pg with Fusobacterium nucleatum (Fn); and to provide a theoretical basis for the role of oral innate immunity played in the inhibition of chronic periodontitis.@*Methods@#Saliva and supragingival, subgingival plaque samples were collected from 49 chronic periodontitis patients in School of Stomatology, China Medical University and 27 periodontal healthy individuals. Enzyme linked immunosorbent assay was used to assess the amount of histatin 5 in saliva, absolute quantitative real-time PCR (qPCR) was applied to detect the DNA copies of Fn, Pg and total bacteria in supragingival and subgingival plaque samples. The effects of histatin 5 on auto- and co-aggregation were assessed by bacterial adhesion test and scanning electron microscopy. Hemagglutinin gene, arginine-gingipains gene in Pg and FomA gene in Fn were tested by relative qPCR. Independent samples t-test was used to calculate the significance between the experimental group and the control group. P-value<0.05 was considered statistically significant.@*Results@#For chronic periodontitis patients, there was an inverse correlation between the concentration of histatin 5 and Fn and Pg in supragingival plaque samples (r=-0.379, r=-0.624). Similarly, an inverse correlation was also observed between the concentration of histatin 5 and subgingival Fn and Pg, respectively (r=-0.404, r=-0.314). As for periodontally healthy individuals, there was an inverse correlation between the concentration of histatin 5 and supragingival and subgingival Pg (r=-0.572, r=-0.533). Bacterial adhesion test and scanning electron microscopy certified that 25 mg/L histatin 5 inhibited the auto-aggregation of Pg-Pg and the co-aggregation of Pg-Fn. Results of qPCR showed that 25 mg/L histatin 5 up-regulated hemagglutinin gene by (14.52±3.25) fold and down-regulated FomA gene to (0.22±0.10) fold.@*Conclusions@#Histatin 5 could inhibit the auto-aggregation of Pg-Pg and the co-aggregation of Pg-Fn by regulating hemagglutinin gene and FomA gene expression.

Effect of Sub-minimal Inhibitory Concentration of Chlorhexidine on Biofilm Formation and Coaggregation of Early Colonizers, Streptococci and Actinomycetes

Chlorhexidine has long been used in mouth washes for the control of dental caries, gingivitis and dental plaque. Minimal inhibitory concentration (MIC) is the lowest concentration of an antimicrobial substance to inhibit the growth of bacteria. Concentrations lower than the MIC are called sub minimal inhibitory concentrations (sub-MICs). Many studies have reported that sub-MICs of antimicrobial substances can affect the virulence of bacteria. The aim of this study was to investigate the effect of sub-MIC chlorhexidine on biofilm formation and coaggregation of oral early colonizers, such as Streptococcus gordonii, Actinomyces naeslundii and Actinomyces odontolyticus. The biofilm formation of S. gordonii, A. naeslundii and A. odontolyticus was not affected by sub-MIC chlorhexidine. However, the biofilm formation of S. mutans increased after incubation with sub-MIC chlorhexidine. In addition, cell surface hydrophobicity of S. mutans treated with sub-MIC of chlorhexidine, decreased when compared with the group not treated with chlorhexidine. However, significant differences were seen with other bacteria. Coaggregation of A. naeslundii with A. odontolyticus reduced by sub-MIC chlorhexidine, whereas the coaggreagation of A. naeslundii with S. gordonii remained unaffected. These results indicate that sub-MIC chlorhexidine could influence the binding properties, such as biofilm formation, hydrophobicity and coaggregation, in early colonizing streptococci and actinomycetes.

Effect of Sub-Minimal Inhibitory Concentrations of Antibiotics on Biofilm Formation and Coaggregation of Streptococci and Actinomycetes

Minimal inhibitory concentration (MIC) is the lowest antibiotic concentration that inhibits the visible growth of bacteria. Sub-minimal inhibitory concentration (Sub-MIC) is defined as the concentration of an antimicrobial agent that does not have an effect on bacterial growth but can alter bacterial biochemistry, thus reducing bacterial virulence. Many studies have confirmed that sub-MICs of antibiotics can inhibit bacterial virulence factors. However, most studies were focused on Gram-negative bacteria, while few studies on the effect of sub-MICs of antibiotics on Gram-positive bacteria. In this study, we examined the influence of sub-MICs of doxycycline, tetracycline, penicillin and amoxicillin on biofilm formation and coaggregation of Streptococcus gordonii, Streptococcus mutans, Actinomyces naeslundii, and Actinomyces odontolyticus. In this study, incubation with sub-MIC of antibiotics had no effect on the biofilm formation of S. gordonii and A. naeslundii. However, S. mutans showed increased biofilm formation after incubation with sub-MIC amoxicillin and penicillin. Also, the biofilm formation of A. odontolyticus was increased after incubating with sub-MIC penicillin. Coaggregation of A. naeslundii with S. gordonii and A. odontolyticus was diminished by sub-MIC amoxicillin. These observations indicated that sub-MICs of antibiotics could affect variable virulence properties such as biofilm formation and coaggregation in Gram-positive oral bacteria.

Coexistence of Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola in the Red Bacterial Complex in Chronic Periodontitis Subjects / Coexistencia de Porphyromonas gingivalis, Tannerella forsythia y Treponema denticola en el Complejo Rojo Bacteriano en Sujetos con Periodontitis Crónica

Previous reports showed that periodontitis is associated with different microorganisms rather than individual periodontopathogens in the dental biofilm. The purpose of the current study was to evaluate the coexistence and relationship among Porphyromonas gingivalis, Tanerella forsythia, and Treponema denticola in the red complex, noting its association with the severity of periodontitis. In this cross sectional study, 96 subjects, aged 33 to 82 years (with 18 residual teeth) with chronic periodontitis who attended the dental clinics of the Universidad de Antioquia in Medellín, Colombia were invited to participate. The presence or absence of bleeding on probing and plaque were registered. Probing depth and clinical attachment level were measured at all approximal, buccal and lingual surfaces. Microbial sampling on periodontitis patients was performed on pockets >5 mm. The presence of P. gingivalis, T. forsythia, and T. denticola was detected by PCR using primers designed to target the respective 16S rRNA gene sequences. The coexistence of the three periodontopathogens was the most frequent (25 subjects). A statistically significant association between the three bacteria was observed (P. gingivalis and T. forsythia, P<0.0001; P. gingivalis and T. denticola, P=0.001; T. forsythia and T. denticola, P<0.0001). Similarly, the logistic regression analysis showed a significant association among periodontopathogens. The most relevant was observed between P. gingivalis and T. forsythia (OR=6.1). In conclusion, the present study found a significant association in the coexistence of P. gingivalis, T. forsythia and T. denticola, and they related strongly to clinical parameters of inflammation and periodontal destruction.

Reportes previos mostraron que la periodontitis se asocia con diferentes microorganismos en lugar de periodontopatógenos particulares en la biopelícula dental. El objetivo del presente estudio fue evaluar la coexistencia y relación entre Porphyromonas gingivalis, Tanerella forsythia y Treponema denticola en el complejo rojo, señalando su vinculación con la severidad de la periodontitis. En este estudio transversal, 96 sujetos de 33 a 82 años (con 18 dientes residuales) con periodontitis crónica que asistieron a las clínicas dentales de la Universidad de Antioquia en Medellín, Colombia fueron invitados a participar. Se registraron la presencia o ausencia de sangrado al sondaje y placa. La profundidad de sondaje y nivel de inserción clínica se midieron en todas las superficies proximales, bucal y lingual. El muestreo microbiano en pacientes con periodontitis se realizó en los bolsillos mayores a 5 mm. La presencia de P. gingivalis, T. forsythia, y T. denticola se detectó por PCR usando las bolsas periodontales diseñadas para dirigirse a las respectivas secuencias de genes 16S RNAr. La coexistencia de los tres periodontopatógenos fue la más frecuente (25 sujetos). Se observó una asociación estadísticamente significativa entre las tres bacterias (P. gingivalis y T. forsythia, P<0,0001; P. gingivalis y T. denticola, P=0,001; T. forsythia y T. denticola, P<0,0001). Del mismo modo, el análisis de regresión logística mostró una asociación significativa entre periodontopatógenos; la más relevantes se observó entre P. gingivalis y T. forsythia (OR=6,1). El presente estudio encontró una asociación significativa en la coexistencia de P. gingivalis, T. forsythia y T. denticola, y estuvieron fuertemente relacionadas a los parámetros clínicos de la inflamación y destrucción periodontal.

Adhesion, Autoaggregation and Hydrophobicity of Six Lactobacillus Strains.

Aims: The aim of this study was to assess probiotic attributes such as adhesion, auto aggregation, hydrophobicity and antibacterial activity of Lactobacillus strains from dairy products. Methodology: In this study, the autoaggregation, coaggregation, hydrophobicity and adhering abilities and antimicrobial activities of six Lactobacillus strains belonging to different species were assessed. Hydrophobicity was determined by bacterial adherence to hydrocarbons, xylene, n-hexadecane and chloroform. Results: The percentage of hydrophobicity of the strains range from 29.5% to 77.4% as measured by the described test. The autoaggregation among Lactobacillus strains range from 15.8% to 63.1%, while coaggregation range from 18.6% to 55.1%. Adhesion of the tested strains to buccal epithelial cells range from 8.0% to 50%. The tested Lactobacillus strains demonstrated variable inhibitory activity against pathogenic bacteria. Conclusion: Our findings indicated that one Lactobacillus strain expressed broad antibacterial activities against a group of bacterial pathogens and along 2 other strains exhibited ability to adhere to epithelial cells as shown by aggregation, coaggregation and hydrophobicity, indicating that such isolates can be good candidates for probiotic use.

Evaluation of adhesive properties of presumptive probiotic Lactobacillus plantarum strains / Avaliação das propriedades de adesão de presumíveis estirpes probióticas de Lactobacillus plantarum

Thirty-two strains of Lactobacillus plantarum UFLA SAU from pork sausages, pre-selected for some features for probiotic application, were utilized in this study to evaluate their adhesive properties and compare the results against the three pathogens also tested. Strains were tested for autoaggregation and coaggregation capacity and Microbial Adhesion To Solvents (MATS) at the time intervals of 0, 1, 2, 3 and 4 h. Our findings revealed that UFLA SAU strains have a high autoaggregative capacity and coaggregative ability with pathogens, especially Listeria monocytogenes. In relation to adhesion to solvents, in general, L. plantarum strains showed hydrophilic cell surface properties and an important electron donor and basic character. Adhesive properties were markedly separated for the strains under study by Principal Component Analysis software. UFLA SAU 132, 226 and 87 were differentiated by autoaggregation ability. UFLA SAU 11 and Listeria monocytogenes were characterized by adhesion to solvents. UFLA SAU 14, 18 and 172 showed high coaggregation with Escherichia coli, Salmonella Typhi and Listeria monocytogenes. In comparison to the pathogens tested, many UFLA SAU strains presented higher adhesive capacity. These tests should be used for screening and identifying potentially adherent microorganisms. Adhesive properties are important features for the choice of probiotic strains and confer various applications, such as in the pharmaceutical (therapeutic or prophylactic) and food (functional foods) industries.

Trinta e duas estirpes de linguiça suína, Lactobacillus plantarum UFLA SAU, pré-selecionadas com algumas características para aplicação probiótica, foram utilizadas neste estudo para avaliar suas propriedades adesivas e comparar os resultados com três patógenos também testados. As estirpes foram testadas para autoagregação, coagregação e capacidade de adesão microbiana aos solventes (MATS) nos tempos de 0, 1, 2, 3 e 4 h. Nossos resultados revelaram que estirpes UFLA SAU apresentam alta capacidade autoagregativa e coagregativa com patógenos, especialmente com Listeria monocytogenes. Em relação à adesão aos solventes, de um modo geral, as estirpes de L. plantarum mostraram propriedades hidrofílicas de superfície celular e um importante caráter básico e elétron doador. Propriedades adesivas foram marcadamente separadas para as estirpes em estudo através da Análise de Componentes Principais. UFLA SAU 132, 226 e 87 foram diferenciadas pela capacidade de autoagregação. UFLA SAU 11 e Listeria monocytogenes foram caracterizadas por adesão aos solventes. UFLA SAU 14, 18 e 172 apresentaram coagregação com Escherichia coli, Salmonella Typhi e Listeria monocytogenes. Em comparação aos patógenos testados, muitas estirpes UFLA SAU apresentaram maior capacidade adesiva. Estes testes podem ser úteis para a triagem e identificação de micro-organismos potencialmente aderentes. Propriedades adesivas são importantes características para a escolha de estirpes probióticas e conferem várias aplicações, tais como nas indústrias: farmacêutica (terapêutico ou profilático) e de alimentos (alimentos funcionais).

Influência da co-agregação entre candida. albicans e lactobacillus acidophilus na capacidade de adesão destes microrganismos às células epiteliais vaginais humanas (CEVH) / Influence of the co-aggregation between candida. albicans e lactobacillus acidophilus on the adhesion capacity these microorganisms in the human ephitelial vaginal cells (HEVC)

Este trabalho teve por objetivo avaliar a influência da co-agregação in vitro entre Candida albicans e Lactobacillus acidophilus na capacidade de adesão destes microrganismos às células epiteliais vaginais humanas (CEVH). Foram utilizados um isolado vaginal de C. albicans e uma cepa ATCC de L. acidophilus. Uma suspensão de cada microrganismo isoladamente e do coagregado foram incubados com as CEVH obtidas de uma doadora saudável. Foram feitos esfregaços por cristal violeta e Papanicolaou, e o número de leveduras, lactobacilos ou coagregados aderidos às células foi contado (em 300 células superficiais-CS e 300 intermediárias- CI). A Microscopia eletrônica de varredura (MEV) foi realizada em todas as situações dos ensaios. Leveduras e lactobacilos aderiram fortemente as CEVH, tanto em CS quanto em CI. A coagregação levou a um aumento na capacidade de adesão das leveduras (p < 0,001) e diminuiu a dos lactobacilos (p < 0,001). A adesão dos microrganismos isolados ou co-agregados não apresentou diferença entre CS e CI (p > 0,05). Havendo correlação com o que acontece in vivo, probióticos à base de L. acidophillus e mesmo uma flora lactobacilar vaginal não surtiriam efeito protetor contra a adesão de C. albicans as CEVH e do possível desenvolvimento de candidíase vulvovaginal.

This work has aimed to evaluate the influence of the L. acidophilus and Candida albicans co-aggregation on the adhesion capacity this microorganisms in the human ephitelial vaginal cells (HEVC). One vaginal isolated of C. albicans and one ATCC strain of L. acidophilus was used. A suspension of the isolated and co-aggregated microorganisms was incubated with HVEC obtained from a healthy donor. After one hour, smears were made with crystal violet and Papanicolaou, and the number of yeasts adhered to HVEC was evaluated (300 superficial-SC and 300 intermediate cells-IC). Scanning electron microscopy (SEM) was made in all situations of the assays. Yeasts and lactobacilli adhered strongly to the HEVC, both SC and IC. The co-aggregation there was an increase in the adhesion capacity of the yeasts (p < 0.001) and a diminished adhesion of the lactobacilli (p < 0.001) in SC and IC. The adhesion of isolated and co-aggregated microorganisms was not significantly different between SC and IC (p > 0.05). Supposing that of these findings correlated with the conditions in vivo, the use of probiotics based on L. acidophilus or its presence in the vaginal microbiota would not protect against the adhesion of C. albicans to the HVEC and possible consequent vulvovaginal candidiasis.

Coaggregation between Porphyromonas gingivalis and Tannerella forsythia / 대한치주과학회지

Dental plaque, a biofilm consisting of more than 500 different bacterial species, is an etiological agent of human periodontal disease. It is therefore important to characterize interactions among periodontopathic microorganisms in order to understand the microbial pathogenesis of periodontal disease. Previous data have suggested a synergistic effect of tow major periodontal pathogens Porphyromonas gingivalis and Tannerella forsythia in the periodontal lesion. In the present study, to better understand interaction between P. gingivalis and T. forsythia, the coaggregation activity between these bacteria was characterized. The coaggregation activity was observed by a direct visual assay by mixing equal amount (1 x 10(9)) of T. forsythia and P. gingivalis cells. It was found that the first aggregates began to appear after 5-10 min, and that the large aggregates completely settled within 1 h. Electron and epifluorescence microscopic studies confirmed cell-cell contact between two bacteria. The heat treatment of P. gingivalis completely blocked the activity, suggesting an involvement of a heat-labile component of P. gingivalis in the interaction. On the other hand, heat treatment of T. forsythia significantly increased the coaggregation activity; the aggregates began to appear immediately. The coaggregation activity was inhibited by addition of protease, however carbohydrates did not inhibit the activity, suggesting that coaggregation is a protein-protein interaction. The results of this study suggest that coaggregation between P. gingivalis and T. forsythia is a result of cell-cell physical contact, and that coaggregation is mediated by a heat-labile component of P. gingivalis and T. forsythia component that can be activated on heat treatment.