Evaluation of oral microbiota in undernourished and eutrophic children using checkerboard DNA-DNA hybridization.

The aim of this study was to evaluate the relationship among nutritional status, gingival health and the composition of oral microbiota in children of a public school from a very poor area of San Miguel de Tucuman. Forty-five children ranging in age from 6 to 14 years old, 13 males and 32 females were studied. Twenty of these children were undernourished (Lejarraga-Morasso Table) and twenty-five were eutrophic. A clinical study that included DMF and dmf indexes, Löe Silness Plaque Index and bleeding on probing was performed. For microbiological study, saliva samples without stimulation were taken; aliquots of them were immediately placed in TAE buffer pH 7.6, adding NaOH (N and keeping at -70 °C until processed by checkerboard DNA-DNA hybridization method to check the presence of 40 oral microorganism species. Positive bleeding on probing was present in more than 80% of children, without significant differences between eutrophic and undernourished groups. Same result were obtain for the other clinical indexes (p > 0.05, Two Way ANOVA). Significant differences were found for some oral microorganism species, with a higher percentage of undernourished children harboring them. That was the case of S. gordonii (p < 0.05), Capnocitophaga gingivalis and C. ochraceae (p < 0.01 and p < 0.10, respectively), F. nucleatum ss nucleatum (p < 0.05), P. nigrescens (p < 0.10), Campylobacter gracilis (p < 0,05), and T. denticola (p < 0.10, multiple logistic regression). Significant differences were also found between children groups for E. saborreum (p < 0.001), P. acnes (p < 0.10), G. morbillorum (p < 0.05) and L. buccalis (p < 0.10). Gingivitis and bleeding on probing would not be related to nutritional status in the groups of children studied. There were significant differences for the presence of some of the main periodontal pathogen species between eutrophic and undernourished children. It would be important to study the meaning of significant differences found for the other microorganisms more deeply.

[Recovery of Veillonella from saliva]. / Recuperación de veillonellas a partir de saliva.

Veillonella spp. are anaerobic gram-negative cocci associated to oral health. Different types of cultures have been reported for the isolation of these microorganisms. Veillonella spp. colonies produce a red fluorescence, which is made visible through ultraviolet light and disappears in contact with oxygen. This feature would be very useful for rapid presumptive identification. The aims of this study were: 1. to compare the Rogosa selective medium for Veillonella with the cultures recommended by different authors in order to determine best saliva recovery, since this sample is generally used to determine the presence and predominance of this bacteria; 2. to detect red fluorescence production on these different culture media as a rapid method for identification. Selective medium for Veillonella, Schaedler agar for anaerobic bacteria with vitamin K, thioglycollate agar, brain heart infusion agar, Brucella agar, trypticase soy agar, and Columbia agar, all of them with and without the addition of vancomycin, and laked blood were used for this study. The tested sample was a saliva pool. Both, Veillonella colonies, and the total number of microorganisms were counted, and expressed as CFU/ml of saliva. The greatest Veillonella recovery in saliva was obtained with the selective medium for Veillonella with vancomycin and laked blood. The production of fluorescence was only observed in this medium.

Influence of temperature on flavour compound production from citrate by Lactobacillus rhamnosus ATCC 7469.

The citrate utilization by Lactobacillus rhamnosus ATCC 7469 was found to be temperature-dependent. The maximum citrate utilization and incorporation of [1,5-14C]citrate rate were observed at 37 degreesC. At this temperature, maximum citrate lyase activity and specific diacetyl and acetoin production (Y(DA%)) were observed. The high levels of alpha-acetolactate synthase and low levels of diacetyl reductase, acetoin reductase and L-lactate dehydrogenase found at 37 degreesC led to an accumulation of diacetyl and acetoin. Optimum lactic acid production was observed at 45 degreesC, according to the high lactate dehydrogenase activity. The NADH oxidase activity increased with increasing culture temperature from 22 degreesC to 37 degreesC. Thus there are greater quantities of pyruvate available for the production of alpha-acetolactate, diacetyl and aceotin, and less diacetyl and acetoin are reduced.

[Effect of glucose and lactose on the utilization of citrate by Lactobacillus casei subsp. rhamnosus ATCC 7469]. / Efecto de glucosa y lactosa sobre la utilización de citrato por Lactobacillus casei subsp. rhamnosus ATCC 7469.

The utilization of citrate by Lactobacillus casei subsp. rhamnosus ATCC 7469 in a complex medium containing glucose, lactose or citrate was investigated, as an approach to the question of the transport of this acid and the possible relationship with the production of flavour compounds (diacetyl and acetoin). This lactobacillus uses citrate as an energy source in the absence of carbohydrates. External pH and growth increases when citrate is added to complex medium. The presence of citrate does not affect glucose uptake. L. casei ATCC 7469 possibly uses a transport system for citrate utilization, and citrate uptake seems to be under glucose or lactose control. Lactose only inhibits the entrance of citrate at high concentration while the utilization of this acid was negatively regulated by low glucose concentration.

Correlation between bacterial counts in saliva and subgingival plaque.

The aim of this study was to examine the correlation between the number and type of bacteria from periodontal pockets more than 4 mm deep and saliva in 26 patients. Periodontal pocket samples were taken with paper points and transferred to 0.1 ml of enriched thioglicollate broth. Saliva samples were collected simultaneously in aseptic flasks. Both samples were processed within the first hour. They were inoculated in Schaedler agar plus 5 micrograms/ml vitamin K and 5% blood, TSBV agar and MGB agar to perform colony counts and identification. Spirochete counts per microscopic field were obtained by direct light microscopy of Gram-stained preparations. The results show a fair to good correlation between both samples for anaerobic, pigmented gram-negative rods, anaerobic non-pigmented gram-negative rods, spirochetes, facultative gram-negative rods other than Actinobacillus actinomycetemcomitans, anaerobic, gram-positive cocci and anaerobic gram-positive rods (Spearman correlation coefficient 0.51 to 0.96). The correlation coefficient values for A.a., facultative gram-positive rods, facultative gram-positive cocci and facultative gram-negative cocci were lower than 0.21. There were no significant differences between the counts in both samples for all the bacterial groups (Student's t test, p > 0.1). We may conclude that, under the experimental conditions of the present study, saliva samples and periodontal pocket samples are equally useful to detect subgingival organisms associated with periodontal disease in the oral cavity. Saliva samples were useful to evaluate risk and periodontal therapy in individual patients or groups.

A culture medium for simultaneous counts of Streptococcus mutans and lactobacilli in saliva.

The use of a single culture medium that allows the isolation and counts of both Streptococcus mutans and lactobacilli could be of great value in microbiological diagnosis, control and evaluation of prevention programs that are nowadays employed in Odontology. To date there is no method that allows the simultaneous counts of lactobacilli and S. mutans in oral samples using a single culture medium. A single culture medium would allow for a more exact diagnosis of cariogenic risk and activity and a reduction in costs and processing time. We here in propose the selective-differential LAPTg 7% sucrose medium to differentiate oral streptococci and lactobacilli according to colony morphology and dextran production. The choice of this medium was the result of testing culture media such as MRS Agar, Elliker Agar and modified LAPTg Agar.

Mitis salivarius-bacitracin 10% sacarose agar for oral streptococci and Streptococcus mutans counts.

The MSB Agar (mitis salivarius-bacitracin) 20% sacarose medium is frequently used for the isolation and count of total streptococci and Streptococcus mutans. Although it is considered a selective culture medium for this micro-organism, S. mutans recovery in this medium is much lower than in this Mitis Salivarius Agar (MSA). Because the number of S. mutans in saliva is used for estimating caries risk and activity from a microbiological stand point, the aim of this work was to find a modification of the MSB 20% sacarose medium so that it would offer not only selectivity in the isolation but also maximum recovery. This would detect people at risk more efficiency and would evaluate the preventive odontological treatments more accurately. The results show that: 1) the greatest recovery of total streptococci and S. mutans is obtained in the MSB 10% sacarose medium, 2) S. mutans must be incubated in aerobiosis and the total streptococci in a candle jar (10% CO2). MSB 10% sacarose medium is proposed as a choice medium for the microbiological estimation of cariogenic risk and activity, to detect infection levels and evaluate preventive odontological treatments.

Inducible transport of citrate in Lactobacillus rhamnosus ATCC 7469.

Lactobacillus rhamnosus ATCC 7469 exhibited diauxie when grown in a medium containing both glucose and citrate as energy source. Glucose was used as the primary energy source during the glucose-citrate diauxie. Uptake of citrate was carried out by an inducible citrate transport system. The induction of citrate uptake system was repressed in the presence of glucose. This repression was reversible and mediated by cAMP.

Estudio de la sensibilidad a los antimicrobianos de patógenos periodontales / Study of antimicrobial susceptibility of periodontal pathogens

Considerando que las diversas fallas en la implementación de la terapia antimicrobiana para el tratamiento de las periodontitis conducen a la aparición de cepas resistentes a los mismos, el objetivo de este trabajo fue realizar un estudio preliminar del patrón de resistencia a los antimicrobianos de uso común en la clínica de cepas de Porphyromonas gingivalis, Prevotella intermedia y Fusobacterium nucleatum. La prueba de sensibilidad a los antimicrobianos se realizó por el método de dilución en agar descrito por la NCCLS (National Committee for Clinical Laboratory Standards). Se probaron antimicrobianos seleccionados entre los más utilizados en la práctica clínica en nuestro medio: amoxicilina, clindamicina, doxiciclina, eritromicina, metronidazol y tetraciclina. Los resultados mostraron que el 100 por ciento de las cepas de P. gingivalisfueron resistentes a metronidazol y sensibles a tetraciclina, y un 33 por ciento sensibles a clindamicina. Las CIMs obtenidas para amoxicilina estuvieron entre 1 y 32 mg/ml, para doxiciclina entre 0,125 y 5 mg/ml y para eritromicina entre 8 y >32 mg/ml. En el caso de P. intermedia, se observó también un 100 por ciento de resistencia a metronidazol, un 67 por ciento de sensibilidad a tetraciclina y un 62,5 por ciento a clindamicina. Las CIMs para amoxicilina estuvieron entre 0,125 y 16 mg/ml, para doxicilina entre 0,125 y 4 mg/ml, y para eritromicina entre 8 y > 32 mg/ml. El 100 por ciento de las cepas de F. nucleatum resultaron sensibles a tetraciclina y resistentes a metronidazol, y el 25 por ciento fueron sensibles a clindamicina. En cuanto a amoxicilina, las CIMs estuvieron entre 0,125 y 16 mg/ml, para doxicilina entre 0,125 y 4 mg/ml, y eritromicina entre 16 y >32 mg/ml. Los patrones de resistencia obtenidos con estas cepas mostraron en general mayores porcentajes de resistencia que lo reportado por otros autores, lo que podría deberse a la falta de políticas de control en el uso de antimicrobianos en nuestro país...

Taking into account that several failures in antibiotic treatments used in periodontitis are driving to the appearance of resistant strains, the aim of this work was to realize a preliminary study of the resistance pattern to commonly used antimicrobials of regional Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum strains. Antibiotic susceptibility test was performed by NCCLS (National Committee for Clinical Laboratory Standards) agar dilution method. Proof antibiotics were selected among the most used in the clinical practice in our region: amoxicillin, clindamycin, doxycycline, erythromycin, metronidazole and tetracycline. The results showed that 100 percent of P.gingivalis strains were resistant to metronidazole and susceptible to tetracycline, and 33 percent susceptible to clindamycin. The MICs obtained for amoxicillin were between 1 and 32 mg/ml, for doxycycline between 0,125 and 5 mg/ml and for erythromycin between 8 and >32 mg/ml. In the case of P. intermedia, were also observed a 100 percent of resistance to metronidazole, 67 percent of susceptibility to tetracycline, and 62,5 percent of susceptibility to clindamycin.. The MICs for amoxicillin were between 0,125 y 16 mg/ml, for doxycycline between 0,125 and 4 mg/ml, and for erythromycin between 8 and >32 mg/ml. 100 percent of F. nucleatum strains were susceptible to tetracycline and resistant to metronidazole, and a 25 percent susceptible to clindamycin. MICs to amoxicillin were between 0,125 y 16 mg/ml, for doxycycline between 0,125 and 4 mg/ml, and to erythromycin between 16 and >32 mg/ml. Resistance patterns obtained with strains of our region showed higher percentages of resistance than those reported by other authors, which might owe to the lack of control politicise for the use of antimicrobials in our country. However, MICs values founded for the 50 and 90 percent of strains are similar to tose reported by NCCLS, except in the case of doxycycline, ...

Recuperación de veillonellas a partir de saliva / [Recovery of Veillonella from saliva]

Veillonella spp. are anaerobic gram-negative cocci associated to oral health. Different types of cultures have been reported for the isolation of these microorganisms. Veillonella spp. colonies produce a red fluorescence, which is made visible through ultraviolet light and disappears in contact with oxygen. This feature would be very useful for rapid presumptive identification. The aims of this study were: 1. to compare the Rogosa selective medium for Veillonella with the cultures recommended by different authors in order to determine best saliva recovery, since this sample is generally used to determine the presence and predominance of this bacteria; 2. to detect red fluorescence production on these different culture media as a rapid method for identification. Selective medium for Veillonella, Schaedler agar for anaerobic bacteria with vitamin K, thioglycollate agar, brain heart infusion agar, Brucella agar, trypticase soy agar, and Columbia agar, all of them with and without the addition of vancomycin, and laked blood were used for this study. The tested sample was a saliva pool. Both, Veillonella colonies, and the total number of microorganisms were counted, and expressed as CFU/ml of saliva. The greatest Veillonella recovery in saliva was obtained with the selective medium for Veillonella with vancomycin and laked blood. The production of fluorescence was only observed in this medium.

Efecto de glucosa y lactosa sobre la utilización de citrato por Lactobacillus casei subsp. rhamnosus ATCC 7469 / [Effect of glucose and lactose on the utilization of citrate by Lactobacillus casei subsp. rhamnosus ATCC 7469]

The utilization of citrate by Lactobacillus casei subsp. rhamnosus ATCC 7469 in a complex medium containing glucose, lactose or citrate was investigated, as an approach to the question of the transport of this acid and the possible relationship with the production of flavour compounds (diacetyl and acetoin). This lactobacillus uses citrate as an energy source in the absence of carbohydrates. External pH and growth increases when citrate is added to complex medium. The presence of citrate does not affect glucose uptake. L. casei ATCC 7469 possibly uses a transport system for citrate utilization, and citrate uptake seems to be under glucose or lactose control. Lactose only inhibits the entrance of citrate at high concentration while the utilization of this acid was negatively regulated by low glucose concentration.

Efecto de glucosa y lactosa sobre la utilización de citrato por Lactobacillus casei subsp. rhamnosus ATCC 7469 / [Effect of glucose and lactose on the utilization of citrate by Lactobacillus casei subsp. rhamnosus ATCC 7469]

The utilization of citrate by Lactobacillus casei subsp. rhamnosus ATCC 7469 in a complex medium containing glucose, lactose or citrate was investigated, as an approach to the question of the transport of this acid and the possible relationship with the production of flavour compounds (diacetyl and acetoin). This lactobacillus uses citrate as an energy source in the absence of carbohydrates. External pH and growth increases when citrate is added to complex medium. The presence of citrate does not affect glucose uptake. L. casei ATCC 7469 possibly uses a transport system for citrate utilization, and citrate uptake seems to be under glucose or lactose control. Lactose only inhibits the entrance of citrate at high concentration while the utilization of this acid was negatively regulated by low glucose concentration.

Correlation between bacterial counts in saliva and subgingival plaque

The aim of this study was to examine the correlation between the number and type of bacteria from periodontal pockets more than 4 mm deep and saliva in 26 patients. Periodontal pocket samples were taken with paper points and transferred to 0.1 ml of enriched thioglicollate broth. Saliva samples were collected simultaneously in aseptic flasks. Both samples were processed within the first hour. They were inoculated in Schaedler agar plus 5 micrograms/ml vitamin K and 5

blood, TSBV agar and MGB agar to perform colony counts and identification. Spirochete counts per microscopic field were obtained by direct light microscopy of Gram-stained preparations. The results show a fair to good correlation between both samples for anaerobic, pigmented gram-negative rods, anaerobic non-pigmented gram-negative rods, spirochetes, facultative gram-negative rods other than Actinobacillus actinomycetemcomitans, anaerobic, gram-positive cocci and anaerobic gram-positive rods (Spearman correlation coefficient 0.51 to 0.96). The correlation coefficient values for A.a., facultative gram-positive rods, facultative gram-positive cocci and facultative gram-negative cocci were lower than 0.21. There were no significant differences between the counts in both samples for all the bacterial groups (Student’s t test, p > 0.1). We may conclude that, under the experimental conditions of the present study, saliva samples and periodontal pocket samples are equally useful to detect subgingival organisms associated with periodontal disease in the oral cavity. Saliva samples were useful to evaluate risk and periodontal therapy in individual patients or groups.

Recuento simultáneo de Streptococcus mutans y lactobacilos en placa bacteriana y saliva en medio LAPTg sacarosa al 7 por ciento / Simultaneous count of Streptococcus mutans and lactobacilli in dental plaque and saliva samples in LAPTg sucrose 7 por ciento medium

Una de las finalidades del tratamiento odontológico preventivo es reducir el riesgo biológico de caries, lo que debería traducirse desde el punto de vista microbiológico en una disminución en el número de Streptococcus mutans y lactobacilos en cavidad bucal. El objetivo de este trabajo fue evaluar el efecto del tratamiento preventivo en 33 pacientes con edades comprendidas entre 12 y 27 años, a los que se les tomó muestras de placa dental y saliva al iniciar y al finalizar el mismo. Con estas muestras se realizó el recuento simultáneo de Streptococccus mutans y lactobacilos sembrando en un solo medio de cultivo (LAPTg sacarosa 7 por ciento), teniendo en cuenta las diferencias morfológicas de las colonias. La identificación de especies fue confirmada por medio de pruebas bioquímicas. Se observó que el tratamiento odontológico preventivo disminuye significativamente el número de Streptococcus mutans y lactobacilos presentes en la placa dental, mientras que no existe variación en saliva. Se propone el medio de cultivo LAPTg sacarosa 7 por ciento para el aislamiento y recuento simultáneo de Streptococcus mutans y lactobacilos