Dental caries is the most common bacterial disease of humans and occurs when oral bacteria produce acids, following their fermentation of dietary carbohydrates. This acid can then cause a localised demineralisation of the tooth surface. A group of seven species of bacteria, collectively known as the mutans streptococci, have been predominantly implicated in the onset of dental caries. In particular, Streptococcus mutans and Streptococcus sobrinus have been shown to be the main aetiological agents of this disease in humans. Most attempts to control the microbial component of caries target these bacteria. The past 50 years has provided considerable information about the pathogenesis of dental caries, the likely route and time of transmission of cariogenic bacteria to susceptible hosts and possible ways of either treating or controlling the onset of this disease. In regards to the latter, many techniques (such as the use of tooth brushes, mouth washes, dental floss and tooth paste) for the control of plaque build-up exist and the examples listed are generally part of a daily routine. However, these techniques need to be applied regularly, and as such only highly-motivated individuals generally experience improved oral health. Therefore, the search for more effective less labour-intensive approaches continues. One area of research is into the potential application of small ribosomally-synthesised antimicrobial peptides, known as bacteriocins. Bacteriocins generally inhibit closely-related species that occupy the same ecological niche. Their relatively-specific targeting, plus the fact that many are remarkably heat and chemically-stable molecules, makes them excellent candidates for possible anti-caries applications. Numerous bacteriocins produced by the lactic acid bacteria have now been identified. Most can be broadly categorised into one of four main classes, of which Class I, the lantibiotics and Class II, the small (<10 kDa) non-modified peptides, contain the most examples. Many screens for anti-mutans streptococcal (MS) bacteriocins have been carried out and it appears that the best source of anti-MS bacteriocins are the mutans streptococci themselves. Research in this laboratory has identified examples of anti-mutans streptococcal bacteriocins produced by both mutans streptococci and non-mutans streptococci. The present study investigated the anti-MS inhibitors produced by two streptococcal strains, S. mutans N and Streptococcus sanguis K11. During the course of this study a third strain, S. mutans UA159, was also studied for its bacteriocinogenic properties. Although S. sanguis K11 produces anti-mutans streptococcal inhibitory activity, this appears only effective against Streptococcus rattus. In addition however, the inhibitory activity of this strain is also directed against all tested strains of Streptococcus agalactiae and ca. 50% of Streptococcus pyogenes. In the present study a 5069 Da novel inhibitory agent (sanguicin K11) was characterised and shown responsible for this unusual inhibitory spectrum. Through reverse genetics the sanK11 locus was identified and shown to encode a Class II type bacteriocin, the first shown to be produced by S. sanguis. Following screens of additional S. sanguis, sanK11 was shown to be present only in strains producing the same type of inhibitory pattern (P-type) as strain K11. The cysteine residues at positions 7 and 38 of the sanguicin K11 propeptide were shown to form a disulphide bridge essential for sanguicin K11 inhibitory activity. S. mutans N and eight other S. mutans strains have been found to have what appears to be the same inhibitory spectrum, which includes members of the mutans streptococci and several other oral streptococcal species. One strain (UA140) of the eight has previously been shown to produce the lantibiotic mutacin I and the non-lantibiotic mutacin IV. S. mutans N was known to produce the non-lantibiotic mutacin N. The current study set out to investigate how two strains, apparently producing completely different bacteriocins could have the same inhibitory spectrum. Reverse genetics identified the mutacin N structural gene (mutN) and mutagenesis studies showed that this bacteriocin was responsible only for the inhibitory activity against mutans streptococci. Further sequencing around the mutN locus identified a second bacteriocin-like locus (mutO) adjacent to mutN. mutO was also identified to have anti-mutans streptococcal inhibitory activity and because of the close proximity of mutO and mutN and given the homology they share with other known two-peptide bacteriocins it seemed probable that mutacins O and N are components of a new member of this special class of bacteriocins (Class IIb, the two peptide bacteriocins) in which the optimal inhibitory activity is dependent on the co-operative activity of the two peptides. Further investigations of strain N examined the expression of mutacins O and N. During a search for a suitable heterologous non-mutacinogenic S. mutans strain to act as an expression host, the genome reference strain, S. mutans UA159 was given consideration. However, contrary to previous reports, this strain was found to exhibit bacteriocin-like inhibitory activity. During a follow-up investigation, strain UA159 was found to inhibit 84 strains representing 11 different species of bacteria, but no inhibition of mutans streptococci was detected. The locus (nlmAB) encoding the two-peptide bacteriocin mutacin IV was identified within the UA159 genome. Using genetic dissection of nlmA and nlmB, the contribution of each peptide was examined and it was found that only the NlmA* propeptide appears to be active, raising doubts as to whether mutacin IV is a bona fide two-peptide bacteriocin. Deletion of the entire nlmAB locus created a mutant strain that exhibited a loss of inhibitory activity against the same 64 strains as was found for the nlmA mutant. A BLASTP search for the consensus leader sequence that precedes the propeptide of Class II bacteriocins, identified ORFs encoding 9 more putative bacteriocin-like peptides. Further genetic dissection identified the SMU.1914c locus as being responsible for the inhibitory activity against a further 15 strains not already sensitive to mutacin IV. SMU.1914c was renamed mutacin V. However, it appears that another as yet unidentified mutacin(s) is also produced by strain UA159 given that three indicator strains still remained sensitive to a double mutant [UA[Delta](1914/NlmAB)] in which both the mutacin IV and putative mutacin V loci were inactivated. Export of Class II bacteriocins has been found to occur by either a SEC-dependent system or via a dedicated peptide ATP Binding Cassette (ABC) transporter. Three potential ABC transporter ORFs were identified in S. mutans UA159. Two (comA and cslA) had the characteristic accessory factor ORF (comB and cslB respectively) located adjacent to the main ABC transporter ORF, while the third ORF763 appeared to lack this. Mutagenesis of each of these five ORFS was carried out and confirmed cslAB to be the ABC transporter involved in the export of the competence stimulating factor, while the function of ORF763 could not be established in this study. Mutagenesis of either comA or comB resulted in a complete cessation of bacteriocin production by the respective mutant strains. Historically, comA and comB is the nomenclature used for loci encoding the exporter of the competence inducing factors in streptococci. In light of this new information, comA and comB were renamed nlmT and nlmE respectively, to account for the newly defined role of this ABC transporter. The present study investigated four bacteriocins two of which (sanguicin K11 and mutacin ON) appear to have some potential for application to anti-caries control, and the others (mutacins IV and V) being shown to be produced by the genome reference strain (UA159). All three mutacins were shown to be exported from their respective producer cells by the NlmTE ABC transporter, while sanguicin K11 is predicted to be exported by a peptide ABC transporter located adjacent to sanK11. Bacteriocins may yet provide a novel alternative for the treatment and control of dental caries. In their favour is that fact that they have relatively narrow defined inhibitory spectra and thus are unlikely to produce widespread changes to plaque ecosystems. Potential uses include as topical agents where bacteriocin preparations could be incorporated into dentrifices such as toothpastes or mouthwashes. Alternatively, streptococci producing anti-mutans streptococcal bacteriocins could be implanted into the oral cavity in strain replacement therapy strategies. There are pros and cons to each technique and the most effective anti-caries control appears more likely to result from "cocktail therapy" where bacteriocins are combined with a number of other anti-mutans streptococcal agents to achieve long-lasting protection against mutans streptococcus proliferation.

Streptococcus sanguis 12 adhesins mediating attachment to saliva-coated hydroxyapatite beads (S-HA) were isolated and characterized. Cell surface fibrils were released from this organism by a method of freeze-thawing followed by brief homogenization. Fibrils in the homogenate were precipitated by ultracentrifugation or ammonium sulphate precipitation. This precipitate was shown to contain fibrils by electron microscopy. Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis of fibrils showed a single band which stained with Coomassie blue and periodate-Schiff. The molecule had a Mr in excess of 300,000. This protein has been given the name long-fibril protein (LFP). Antibody raised against the LFP reacted with long fibrils of S. sanguis 12. LFP was degraded by subtilisin, pronase, papain, and trypsin, but not by chymotrypsin and muramidases. Fibrils were hydrolyzed by subtilisin into discrete lower Mr protein bands which reacted with both anti-fibril and anti-LFP serum. F(ab')₂ prepared from anti-fibril IgG inhibited adhesion of S. sanguis 12 to pH modified S-HA, indicating that fibrils were acting as an adhesin mediating attachment via the neuraminidase-sensitive receptor on S-HA. Five recombinant clones expressing surface antigens of S. sanguis 12 were isolated by ligating a partial digest of S. sanguis 12 chromosomal DNA with the plasmid vector pUC 18, and transforming into Escherichia coli JM83. Recombinant clones were screened by a colony immunoassay with antisera raised against either S. sanguis 12 whole cells or with anti-fibril serum. Positive clones were then analyzed by SDS-PAGE, Western blotting and restriction endonuclease digestion of recombinant plasmids. One recombinant plasmid, pSA2 expressed two proteins of Mrs of 20,000 and 36,000. The 36,000-Mr protein has been designated as SsaB (S. sanguis adhesin B). Both proteins were purified to homogeneity by gel filtration and ion exchange chromatography. Anti-SsaB serum was used in an immunogold bead labelling experiment to demonstrate that this protein was present on the surfaces of S. sanguis 12 and in the non-saliva-aggregating variant 12na, but not on the non-adhering non-aggregating hydrophilic variant 12L. Western blot analysis with anti-SsaB and anti-20 kd sera showed that both SsaB and the 20 kd proteins were present in cell extracts of S. sanguis 12 and its variants. SsaB inhibited adhesion of S. sanguis 12na to S-HA, indicating that it was the adhesin which mediates the binding to the pH-sensitive receptor. SsaB was found to be present on all S. sanguis strains tested, but not on other oral streptococci. Chemical cross-linking studies of SsaB on S. sanguis 12 cell surface suggested that this protein may be present in a higher Mr complex. This study provides direct evidence that binding of S. sanguis 12 to S-HA involves at least two adhesin-receptor interactions. The adhesin mediating binding to the neuraminidase-sensitive receptor on S-HA involves the long fibrils and the adhesin binding to the acid labile receptor is a 36,000 Mr protein. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Objective: The aim of this study was to evaluate the antibacterial effect of Camellia sinensis (green tea) on Streptococcus mutans (ATCC 25175) and Streptococcus sanguinis (ATCC10556). Materials and Methods: The methanolic extract of green tea (commercial and bulk) was examined on Streptococcus mutans (ATCC 25175) and Streptococcus sanguinis (ATCC10556), using the well assay method. A total of 24 plates for the first extract (Commercial) and the same amount for the extract No. 2 (Bulk) were used and were divided into groups of 12 disks for each bacterium, with a total of 4 disks per group. In addition, each plate contained 3 disks embedded with tea and 1 disc with 0.12% chlorhexidine as a control group. Results: It was found antibacterial effect in both methanolic extracts of green tea. No statistically significant differences when comparing the natural green tea with the other extract, in every single microorganism. A p value of 0.18 and 0.63 respectively were obtained. The methanol extract of the commercial green tea had better antibacterial effect than the second extract with an average of 19.72 mm, for the second extract of green tea was 18.1 mm against Streptococcus mutans, whereas for Streptococcus sanguinis was 17.94mm and 16.46 mm respectively. The minimum inhibitory concentration (MIC) for the commercial and bulk extract was 0.08 gr/ml against Streptococcus mutans and 0.08 gr/ml and 0.25 gr/ml against the strains of Streptococcus sanguinis respectively. Conclusions: Both methanolics extracts, commercial and bulk have anti Streptococcus mutans (ATCC 25175) and Streptococcus sanguinis (ATCC10556) activity in vitro. The activity of commercial tea had better effects than the bulk methanolic extract. / Objetivo: Evaluar in vitro el efecto antibacteriano de la Camellia sinensis (té verde) frente al Streptococcus mutans (ATCC 25175) y al Streptococcus sanguinis (ATCC10556). Materiales y métodos: Se probaron dos extractos de té verde, uno comercial y otro a granel. Se utilizaron 24 discos para el primer extracto (Comercial) y la misma cantidad para el segundo extracto metanólico (Granel), se dividieron en grupos de 12 discos para cada bacteria, con un total de 4 placas petri por cada uno. Además, cada placa contenía 3 discos embebidos de té y 1 disco con Clorhexidina al 0.12% como grupo control. Estas muestras fueron analizadas con el método de difusión en agar con discos y los halos de inhibición se midieron a las 72 horas. Resultados: Se encontró efecto antibacteriano para ambos extractos probados. El promedio del halo de inhibición para el extracto de té verde comercial fue de 19.72 mm y para el extracto de té verde a granel fue de 18.1 mm frente al Streptococcus mutans, mientras que para el Streptococcus sanguinis la media obtenida fue de 17.94 mm y 16.46 mm respectivamente. Con respecto a la Concentración mínima inhibitoria (CMI), para el caso de Streptococcus mutans se determinó una CMI de 0.08 gr/ml para el extracto comercial y al extracto a granel. Mientras que para el caso de Streptococcus sanguinis la CMI fue de 0.08 gr/ml para el extracto comercial y de 0.25 gr/ml para el extracto a granel. Conclusiones: Ambos extractos metanólicos de té verde presentaron efecto antibacteriano contra las cepas del Streptococcus mutans (ATCC 25175) y Streptococcus sanguinis (ATCC10556). El té verde comercial fue el que presentó mayor efecto antibacteriano que el extracto a granel. / Tesis

The target was evaluate the “in vitro” antibacterial effect about five peruvian propolis on strains of Streptococcus mutans (ATCC 25175) and Streptococcus sanguinis (ATCC 10556), Having an experimental study design “in vitro” made in laboratorie of Microbiology of the Peruvian University of Applied Sciences. Materials and Methods: Sample size were about ten holes for each of the five extracts of propolis, either for Streptococcus mutans and Streptococcus sanguinis individualy. The antibacterial effect was developed with the technical “Agar overlay interference test”, which is used 200ml of Agar BHI homogenized with bacteria indispensably (a bottle for bacteria). This agar is distributed in the plates, once solidified the holes are made with 150μL of different kinds of propolis and the control group called Clorhexidine 0.12%. Completed this process is placed in the anaerobiosis chamber on 37º C, for 72 hours. Finally, the measurement of the inhibitory halo is made. Results: The methanolic extract of propolis of Oxapampa-Perú present inhibition halos in larger averaging of 33.15 + 3.26 mm against strains of Streptococcus mutans (ATCC 25175) and Streptococcus sanguinis (ATCC 10556), their average was about 23.23 + 0.82mm. In case of 4 extracts of commercial, just 3 of them (tincture of propolis Farmagel, Madre Nature y Kaita®), had bacterian activity in front of the studies strains. In all cases the antibacterian activity is less than positive control. Conclusions: The methanolic extract of propolis of Oxapampa-Peru, made in the laboratory has better antibacterian activity than commercial extracts against strains of Streptococcus mutans (ATTC 25175) and Streptococcus sanguinis (ATCC 10556). About the four commercial propolis evaluated on this study, propolis tincture Farmagel, Kaita®, Madre Natura and Max propolis tincture, only three of this have antibacterial activity against strains of de Streptococcus mutants (ATCC 25175) and Streptococcus sanguinis (ATCC 10556). / El objetivo fue evaluar in vitro el efecto antibacteriano de cinco propóleos peruanos sobre cepas de Streptococcus mutans (ATCC 25175) y Streptococcus sanguinis (ATCC 10556) teniendo un diseño de estudio experimental in vitro, realizado en el laboratorio de Microbiología de la Universidad Peruana de Ciencias Aplicadas. Materiales y métodos: se comparó el efecto antibacteriano de cuatro marcas comerciales de propóleo Tintura de propóleo Farmagel, Tintura de propóleo Max, Madre Natura, Kaita® y un extracto metanólico de propóleo de Oxapampa, el cual se elaboró en el laboratorio de Bioquímica de la UPC, como control (+) la clorhexidina al 0.12%. Para este estudio se utilizó 10 pocillos por cada extracto de propóleo, para el Streptococcus mutans y para el Streptococcus sanguinis individualmente. Se desarrolló con la técnica “Agar overlay interference test”, para lo cual se utilizó 200ml de Agar BHI homogenizado con las bacterias de manera independiente (un frasco por bacteria). Se distribuyó este agar en las placas, una vez solidificado se realizaron los pocillos con 150μL de los distintos tipos de propóleo y para el grupo control, se utilizó clorhexidina al 0.12%. Terminado este proceso se colocó en la cámara de anaerobiosis a 37°C, durante 72 horas. Por último, se realizó la medición del halo inhibitorio con una regla Vernier. Resultados: El extracto metanólico de propóleo de Oxapampa presentó halos de inhibición de mayor tamaño con una media de 33.15 + 3.26 mm frente a las cepas de Streptococcus mutans (ATCC 25175), para el Streptococcus sanguinis (ATCC 10556) su media fue de 23.23 + 0.82 mm. En el caso de los 4 extractos de propóleo comerciales, sólo 3 de ellos (Tintura de propóleo Farmagel, Madre Natura y Kaita®), tuvieron actividad antibacteriana frente a las cepas estudiadas, en todos los casos la actividad antibacteriana es menor que el control (+). Conclusiones: El extracto metanólico de propóleo de Oxapampa elaborado en el laboratorio tiene mayor actividad antibacteriana que los extractos comerciales frente a las cepas Streptococcus mutans (ATCC 25175) y Streptococcus sanguinis (ATCC 10556). De los 4 propóleos comerciales evaluados en el estudio, Tintura de propóleo Farmagel, Kaita®, Madre Natura y Tintura de propóleo Max, sólo tres de ellos tiene actividad antibacteriana frente a las cepas de Streptococcus mutans (ATCC 25175) y Streptococcus sanguinis (ATCC 10556). / The target was evaluate the “in vitro” antibacterial effect about five peruvian propolis on strains of Streptococcus mutans (ATCC 25175) and Streptococcus sanguinis (ATCC 10556), Having an experimental study design “in vitro” made in laboratorie of Microbiology of the Peruvian University of Applied Sciences. Materials and Methods: Sample size were about ten holes for each of the five extracts of propolis, either for Streptococcus mutans and Streptococcus sanguinis individualy. The antibacterial effect was developed with the technical “Agar overlay interference test”, which is used 200ml of Agar BHI homogenized with bacteria indispensably (a bottle for bacteria). This agar is distributed in the plates, once solidified the holes are made with 150μL of different kinds of propolis and the control group called Clorhexidine 0.12%. Completed this process is placed in the anaerobiosis chamber on 37º C, for 72 hours. Finally, the measurement of the inhibitory halo is made. Results: The methanolic extract of propolis of Oxapampa-Perú present inhibition halos in larger averaging of 33.15 + 3.26 mm against strains of Streptococcus mutans (ATCC 25175) and Streptococcus sanguinis (ATCC 10556), their average was about 23.23 + 0.82mm. In case of 4 extracts of commercial, just 3 of them (tincture of propolis Farmagel, Madre Nature y Kaita® ), had bacterian activity in front of the studies strains. In all cases the antibacterian activity is less than positive control. Conclusions: The methanolic extract of propolis of Oxapampa-Peru, made in the laboratory has better antibacterian activity than commercial extracts against strains of Streptococcus mutans (ATTC 25175) and Streptococcus sanguinis (ATCC 10556). About the four commercial propolis evaluated on this study, propolis tincture Farmagel, Kaita® , Madre Natura and Max propolis tincture, only three of this have antibacterial activity against strains of de Streptococcus mutants (ATCC 25175) and Streptococcus sanguinis (ATCC 10556). / Tesis

Background: The antibacterial activity of restorative materials has an important role in the prevention of recurrent caries. Objectives: To compare the antibacterial activity of three glass ionomer cements Ketac Molar®, Fuji II® and Maxxion R® against Streptococcus mutans (ATCC 25175) and Streptococcus sanguinis (ATCC 10556). Materials and methods: The study was experimental in vitro and consisted of the evaluation of the antibacterial activity of three glass ionomer cements: Ketac Molar®, Fuji II LC ® and Maxxion R®. he which were prepared in accordance with the manufacturer's recommendations and was inserted into wells made with sterile disposable tips using the CENTRIX® system in petris plaques with agar BHI. Furthermore, the antibacterial activity was evaluated by using a caliper to measure the diameter of the growth inhibition zones of strains tested; also, tested; Also, the survival and proliferation of bacteria on ionomers was evaluated by the method of the reduction of 3- (4,5- dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT) analysis. As the viability of Streptococcus mutans was 1.221nm for Ketac Molar®, 0.457nm for Maxxion R® and 0.804nm for Fuji II®. Also the viability of Streptococcus sanguinis was Ketac 0.3175nm for Molar®, for 1.195nm and 0.276nm Maxxion R® for Fuji II®. Results: Ketac Molar®, Fuji II® and Maxxion R® showed inhibition halos 16.27mm ± 1.62, 11.95 ± 1.51 mm and 12.38 mm ± 2.55 against strains of Streptococcus mutans respectively with p = 0.0001, which means that there statistically significant difference. Furthermore, inhibition halos Ketac Molar®, Fuji II® and Maxxion R® against Streptococcus sanguinis were 10.11mm ± 1.42, 2.34 ± 13.51mm and 10.83mm ± 1.22 respectively with p =0.0008, demonstrating that there is also significant difference. Conclusion: The three GIC under evaluation showed halos of growth inhibition of cariogenic bacteria assayed. However, Ketac Molar® had greater antibacterial activity against Streptococcus mutans; while the Fuji II® had greater antibacterial activity against Streptococcus sanguinis. / Introducción: la actividad antibacteriana de los materiales de restauración tiene un papel importante en la prevención de caries recidivante. Objetivos: comparar la actividad antibacteriana de tres ionómeros de vidrio Ketac Molar®, Fuji II LC® y Maxxion R® contra cepas Streptococcus mutans (ATCC 25175) y Streptococcus sanguinis Materiales y métodos: el estudio fue experimental in vitro y consistió en la evaluación de la actividad antibacteriana de tres ionómeros de vidrio: Ketac Molar®, Fuji II LC® y Maxxion R®, los cuáles fueron preparados en cumplimiento de las recomendaciones del fabricante y se insertaron en pozos confeccionados con puntas desechables estériles utilizando el sistema Centrix® en placas de petri con agar BHI. Por otro lado, la actividad antibacteriana se evaluó mediante el uso de un vernier para medir el diámetro de los halos de inhibición del crecimiento de las cepas ensayadas; asimismo, se evaluó la supervivencia y proliferación de las bacterias sobre los ionómeros mediante el método de la reducción de 3-(4,5-Dimetiltiazol-2-yl)-2,5-difenil tetrazolio bromuro (MTT).En cuanto al análisis de la viabilidad del Streptococcus mutans fue de 1.221nm para el Ketac Molar®, 0.457nm para el Maxxion R® y 0.804nm para el Fuji II®. Asimismo, la viabilidad del Streptococcus sanguinis fue de 0.3175nm para el Ketac Molar®, 1.195nm para el Maxxion R® y 0.276nm para el Fuji II®. El estudio se realizó por triplicado y el análisis estadístico utilizó la prueba de ANOVA a 3 criterios. Resultados: el Ketac Molar®, Fuji II® y Maxxion R® mostraron halos de inhibición de 16.27mm ± 1.62, 11.95 mm ± 1.51 y 12.38 mm ±2.55 contra cepas de Streptococcus mutans respectivamente con un p= 0.0001, lo que significa que existe diferencia estadísticamente significativa. Asimismo, se encontraron halos de inhibición del Ketac Molar®, Fuji II® y Maxxion R® contra cepas de Streptococcus sanguinis de 10.11mm ±1.42, 13.51mm ±2.34 y 10.83m m ±1.22 respectivamente con un p= 0.0008, lo que demuestra que también existe diferencia significativa. Conclusión: los tres cementos de ionómero de vidrios evaluados, presentaron halos de inhibición del crecimiento de las bacterias cariogénicas ensayadas. Sin embargo, el Ketac Molar® tuvo mayor actividad antibacteriana contra el Streptococcus mutans ; mientras que el Fuji II® tuvo mayor actividad antibacteriana contra el Streptococcus sanguinis. / Tesis

Objective: The aim of this study was to evaluate the antibacterial and cytotoxic effect of the methanol extract of the seed and pulp of Myrciaria dubia (camu camu) against Streptococcus mutans (ATCC 25175) and Streptococcus sanguinis (ATCC 10556). Materials and Methods: For the present in vitro experimental study two methanolic extracts were prepared from the seed and pulp of Myrciaria dubia (camu camu) to be tested against Streptococcus mutans and Streptococcus sanguinis. Ten independents tests were prepared for each type of extract, using Clorhexidine at 0.12% solution as a positive control. The method used was agar diffusion test preparing wells with the experimental solutions cultivated in anaerobic conditions for 48 hours at 37°C, after this time the growth inhibition halo was analysed in millimeters using a vernier. Meanwhile, the Minimum Inhibitory Concentration (MIC) and the cytotoxic effect (CC50) over Jurkat T cell line was found. Results: The methanolic seed extract had more antibacterial effect over Streptococcus mutans with an inhibitory halo of 21.36mm ± 6.35, while the pulp had 16.20mm ± 2.08. For the Streptococcus sanguinis group the inhibitory halo were 19.21mm ± 5.18 and 19.34mm ± 2.90 for the methanolic seed and pulp extract respectively. The MIC for the pulp extract was 125µg/ml for both strains, whereas for the seed antibacterial activity was observed even at low concentrations. Finally the CC50 for the seed extract was at a higher concentration than 800 µg/ml and 524.37µg/ml for the pulp exactract. Conclusions: The methanolic seed and pulp extract of Myrciaria dubia (camu camu) had antibacterial effect against Streptococcus mutans and Streptococcus sanguinis. These extracts were not cytotoxic. / Objetivo: evaluar in vitro el efecto antibacteriano y citotóxico del extracto metanólico de semilla y pulpa de la Myrciaria dubia (camu camu) sobre cepas de Streptococcus mutans (ATCC 25175) y Streptococcus sanguinis (ATCC 10556). Materiales y métodos: el estudio fue experimental in vitro. Se utilizaron los extractos metanólicos de la semilla y pulpa de Myrciaria dubia (camu camu) sobre las cepas de Streptococcus mutans y Streptococcus sanguinis. Para cada extracto se realizaron 10 pruebas independientes y como control positivo se utilizó la Clorhexidina al 0.12%. Se utilizó el método de difusión en agar con pocillos con las soluciones experimentales en condiciones de anaerobiosis por 48 horas a 37°C, para luego proceder a la lectura de los diámetros del halo de inhibición con un vernier. Asimismo, se halló la concentración mínima inhibitoria (CMI) de los extractos y se determinó el efecto citotóxico (CC50) sobre la línea celular Jurkat. Resultados: el extracto metanólico de la semilla tuvo mayor efecto antibacteriano sobre el Streptococcus mutans con halos de 21.36mm ± 6.35, mientras que la pulpa tuvo 16.20mm ± 2.08. Para el grupo de Streptococcus sanguinis, se observó que los halos de inhibición fueron de 19.21mm ± 5.18 y 19.34mm ± 2.90 para los extractos de semilla y pulpa respectivamente. La CMI del extracto de la pulpa se encontró en la concentración de 125µg/ml para ambas cepas, mientras que para el caso de la semilla se pudo observar actividad antibacteriana aún en bajas concentraciones. Finalmente, la CC50 del extracto de la semilla fue en una concentración mayor a 800 µg/ml y de la pulpa fue 524.37µg/ml. Conclusiones: se determinó que existe efecto antibacteriano del extracto metanólico de la semilla y pulpa de la Myrciaria dubia (camu camu) sobre cepas de Streptococcus mutans y Streptococcus sanguinis. Los extractos metánolicos de este fruto no fueron citotóxicos. / Tesis

Objective: The aim of this study was to evaluate the antibacterial effect of Physalis peruviana on Streptococcus mutans (ATCC 25175) and Streptococcus sanguinis (ATCC 10556). Materials y Methods: The present in vitro experimental study was to determinate the antibacterial effect of the fruit of Physalis Peruviana’s methanolic extract against Streptococcus mutans (ATCC 25175) and Streptococcus sanguinis (ATCC 10556) and Clorhexidine (CHX) as control group, the minimum inhibitory concentration (MIC) and citotoxicity. For the antibacterial assay, 14 wells were prepared from the extract and Clorhexidine 0.12% as a positive control for each bacteria. The method used was agar diffusion test preparing wells with the experimental solutions cultivated in anaerobic conditions for 48 hours at 37ºC, after this time, the growth inhibition was analysed in milimiters. with wells. Results: It was found antibacterial effect of the metanolic extract of the fruit in Streptococcus mutans and Streptococcus sanguinis. In the first group, the inhibicion halo of the extract was 19.15mm ± 1.83 and for Clorhexidine 25.78mm ± 2.27, while for the group of Streptococcus sanguinis, the Physalis Peruviana’s extract had 18.56mm ± 1.59 for inhibicion halos and 22.92mm ± 1.96 for Clorhexidine. On the other hand, the extract’s MIC was 50mg/ml. Finally, the methanolic extract of Physalis peruviana inhibited 50% of viability cells (CC50) at 241.01ug/ml concentration. Conclusions: The methanolic extract of Physalis Peruviana had antibacterial effect against Streptococcus mutans and Streptococcus sanguinis. Besides, this fruit wasn’t citotoxic for Jurkat’s line cell. / Objetivo: evaluar in vitro el efecto antibacteriano del extracto metanólico de Physalis peruviana sobre cepas de Streptococcus mutans (ATCC 25175) y Streptococcus sanguinis (ATCC 10556). Materiales y Métodos: el presente estudio fue de tipo experimental in vitro. Se evaluó el efecto antibacteriano del extracto metanólico del fruto de Physalis peruviana frente a cepas de Streptococcus mutans (ATCC 25175) y Streptococcus sanguinis (ATCC 10556) y la Clorhexidina (CHX) como grupo control, determinando la concentración mínima inhibitoria (CMI) y citotoxicidad. Para realizar la actividad antibacteriana se trabajó con una muestra de 14 pocillos por grupo, mediante el método de difusión en agar con pocillos. Por otro lado, para la MIC se utilizó el método de dilución en caldo en medio Brain Heart infusión Broth (BHI) y para evaluar la citotoxicidad se empleó la línea celular Jurkat mediante el ensayo de MTT. Resultados: se observaron halos de inhibición de 19.15mm ± 1.83 para el extracto de physalis peruvina y 25.78mm ± 2.27 para la clorhexidina sobre Streptococcus mutans, mientras que para el grupo de Streptococcus sanguinis, se observaron halos de inhibición de 18.56mm ± 1.59 del extracto de la planta y 22.92mm ± 1.96 para la clorhexidina. Por otro lado, la MIC del extracto fue de 50mg/ml. Finalmente, el extracto metanólico de la Physalis peruviana inhibió la viabilidad celular al 50% (CC50) en una concentración de 241.01ug/ml. Conclusiones: el extracto metanólico de Physalis Peruviana tuvo efecto antibacteriano sobre cepas de Streptococcus mutans y Streptococcus sanguinis. Además, no fue citotóxico para la línea celular Jurkat. / Tesis

Indiana University-Purdue University Indianapolis (IUPUI) / Streptococcus mutans is a key contributor to dental caries. Smokers have increased caries, but the association between tobacco, nicotine, caries and S. mutans growth is little investigated. In the first section, seven S. mutans strains were used for screening. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum biofilm inhibitory concentration (MBIC) were 16 mg/ml (0.1 M), 32 mg/ml (0.2 M), and 16 mg/ml (0.1 M), respectively, for most of the S. mutans strains. Growth of planktonic S. mutans cells was significantly repressed by 2.0-8.0 mg/ml nicotine concentrations. Biofilm formation and metabolic activity of S. mutans was increased in a nicotine-dependent manner up to 16.0 mg/ml. Scanning electron microscopy (SEM) revealed higher nicotine-treated S. mutans had thicker biofilm and more spherical bacterial cells than lower concentrations of nicotine. In the second section, confocal laser scanning microscopy (CLSM) results demonstrated that both biofilm bacterial cell numbers and extracellular polysaccharide (EPS) synthesis were increased by nicotine. Glucosyltransferase (Gtf) and glucan binding protein A (GbpA) protein expression of S. mutans planktonic cells were upregulated, while GbpB protein expression of biofilm cells were downregulated by nicotine. The mRNA expression of those genes were mostly consistent with their protein results. Nicotine was not directly involved in S. mutans LDH activity. However, since it increased the total number of bacterial cells in biofilm; total LDH activity of S. mutans biofilm was increased. In the third section, a PCR-based multiple species cell counting (PCR-MSCC) method was designed to investigate the effect of nicotine on S. mutans in a ten mixed species culture. The absolute S. mutans number in mixed biofilm culture was increased but the percentage of S. mutans in the total number of bacterial cells was not changed. In conclusion, nicotine enhanced biofilm formation and biofilm metabolism of S. mutans, through stimulating S. mutans planktonic cell Gtfs and Gbps expression. This leads to more planktonic cells attaching to dental biofilm. Increased S. mutans cell numbers, in biofilms of single species or ten mixed species, resulted in higher overall LDH activity. More lactic acid may be generated and contribute to caries development in smokers.