RESUMEN
Periodontitis is a multi-microbial oral infection with high prevalence among adults. Putative oral pathogens are commonly found in periodontally diseased individuals. However, these organisms can be also detected in the oral cavity of healthy subjects. This leads to the hypothesis, that alterations in the proportion of these organisms relative to the total amount of oral microorganisms, namely their abundance, rather than their simple presence might be important in the transition from health to disease. Therefore, we developed a quantitative molecular method to determine the abundance of various oral microorganisms and the portion of bacterial and archaeal nucleic acid relative to the total nucleic acid extracted from individual samples. We applied quantitative real-time PCRs targeting single-copy genes of periodontal bacteria and 16S-rRNA genes of Bacteria and Archaea. Testing tongue scrapings of 88 matched pairs of periodontally diseased and healthy subjects revealed a significantly higher abundance of P. gingivalis and a higher total bacterial abundance in diseased subjects. In fully adjusted models the risk of being periodontally diseased was significantly higher in subjects with high P. gingivalis and total bacterial abundance. Interestingly, we found that moderate abundances of A. actinomycetemcomitans were associated with reduced risk for periodontal disease compared to subjects with low abundances, whereas for high abundances, this protective effect leveled off. Moderate archaeal abundances were health associated compared to subjects with low abundances. In conclusion, our methodological approach unraveled associations of the oral flora with periodontal disease, which would have gone undetected if only qualitative data had been determined.
Asunto(s)
Archaea/genética , Bacterias/genética , Ácidos Nucleicos/análisis , Periodontitis/microbiología , Adulto , Recuento de Colonia Microbiana , Biología Computacional , Femenino , Alemania , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Oligonucleótidos/genética , ARN Ribosómico 16S/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y Especificidad , Especificidad de la EspecieRESUMEN
The soil bacterium and potential biothreat agent Burkholderia pseudomallei causes the infectious disease melioidosis, which is naturally acquired through environmental contact with the bacterium. Environmental detection of B. pseudomallei represents the basis for the development of a geographical risk map for humans and livestock. The aim of the present study was to develop a highly sensitive, culture-independent, DNA-based method that allows direct quantification of B. pseudomallei from soil. We established a protocol for B. pseudomallei soil DNA isolation, purification, and quantification by quantitative PCR (qPCR) targeting a type three secretion system 1 single-copy gene. This assay was validated using 40 soil samples from Northeast Thailand that underwent parallel bacteriological culture. All 26 samples that were B. pseudomallei positive by direct culture were B. pseudomallei qPCR positive, with a median of 1.84 × 10(4) genome equivalents (range, 3.65 × 10(2) to 7.85 × 10(5)) per gram of soil, assuming complete recovery of DNA. This was 10.6-fold (geometric mean; range, 1.1- to 151.3-fold) higher than the bacterial count defined by direct culture. Moreover, the qPCR detected B. pseudomallei in seven samples (median, 36.9 genome equivalents per g of soil; range, 9.4 to 47.3) which were negative by direct culture. These seven positive results were reproduced using a nested PCR targeting a second, independent B. pseudomallei-specific sequence. Two samples were direct culture and qPCR negative but nested PCR positive. Five samples were negative by both PCR methods and culture. In conclusion, our PCR-based system provides a highly specific and sensitive tool for the quantitative environmental surveillance of B. pseudomallei.
Asunto(s)
Técnicas Bacteriológicas/métodos , Burkholderia pseudomallei/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Microbiología del Suelo , Burkholderia pseudomallei/genética , ADN Bacteriano/genética , Humanos , Proteínas de Transporte de Membrana/genética , Sensibilidad y Especificidad , TailandiaRESUMEN
Environmental surveillance of the Gram-negative soil bacterium Burkholderia pseudomallei, the aetiological agent of melioidosis, is important in order to define human populations and livestock at risk of acquiring the infection. This study aimed to develop a more sensitive method for the detection of B. pseudomallei from soil samples in endemic areas compared with the currently used culture method based on soil dispersion in water. We report the development of a new protocol that involves soil dispersion in a polyethylene glycol (PEG) and sodium deoxycholate (DOC) solution to increase the yield of viable B. pseudomallei from soil samples. Comparative testing of soil samples from Northeast Thailand covering a wide range of B. pseudomallei concentrations demonstrated a significantly higher recovery (P<0.0001) of B. pseudomallei colony-forming units by the new method compared with the conventional method. The data indicate that using the detergents PEG and DOC not only results in a higher recovery of viable B. pseudomallei but also results in a shift in the bacterial species recovered from soil samples. Future studies on the geographical distribution and population structure of B. pseudomallei in soil are likely to benefit from the new protocol described here.
Asunto(s)
Burkholderia pseudomallei/aislamiento & purificación , Melioidosis/microbiología , Microbiología del Suelo , Burkholderia pseudomallei/genética , Recuento de Colonia Microbiana , Femenino , Humanos , Masculino , Melioidosis/epidemiología , Tailandia/epidemiologíaRESUMEN
A novel thermophilic, hydrogen-oxidizing bacterium, designated strain CP.B2(T), was isolated from a terrestrial hot spring in Waiotapu, New Zealand. Cells were motile, slightly rod-shaped, non-spore-forming and Gram-negative. Isolate CP.B2(T) was an obligate chemolithotroph, growing by utilizing H(2) as electron donor and O(2) as corresponding electron acceptor. Elemental sulfur (S(0)) or thiosulfate ( ) was essential for growth. Microbial growth occurred under microaerophilic conditions in 1.0-10.0 % (v/v) O(2) [optimum 4-8 % (v/v) O(2)], between 45 and 75 degrees C (optimum 70 degrees C) and at pH values of 4.8-5.8 (optimum pH 5.4). The DNA G+C content was 29.3 mol%. 16S rRNA gene sequence analysis demonstrated that strain CP.B2(T) belonged to the order Aquificales, with a close phylogenetic relationship to Sulfurihydrogenibium azorense (94 % sequence similarity to the type strain). However, genotypic and metabolic characteristics differentiated the novel isolate from previously described genera of the Aquificales. Therefore, CP.B2(T) represents a novel species in a new genus, for which the name Venenivibrio stagnispumantis gen. nov., sp. nov. is proposed. The type strain of Venenivibrio stagnispumantis is CP.B2(T) (=JCM 14244(T) =DSM 18763(T)).
Asunto(s)
Bacterias Gramnegativas Quimiolitotróficas/clasificación , Bacterias Gramnegativas Quimiolitotróficas/aislamiento & purificación , Manantiales de Aguas Termales/microbiología , Calor , Hidrógeno/metabolismo , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/análisis , Genes de ARNr , Bacterias Gramnegativas Quimiolitotróficas/genética , Bacterias Gramnegativas Quimiolitotróficas/crecimiento & desarrollo , Datos de Secuencia Molecular , Nueva Zelanda , Oxidación-Reducción , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
Surveys of Champagne Pool, one of New Zealand's largest terrestrial hot springs and rich in arsenic ions and compounds, have been restricted to geological and geochemical descriptions, and a few microbiological studies applying culture-independent methods. In the current investigation, a combination of culture and culture-independent approaches were chosen to determine microbial density and diversity in Champagne Pool. Recovered total DNA and adenosine 5'-triphosphate (ATP) content of spring water revealed relatively low values compared to other geothermal springs within New Zealand and are in good agreement with low cell numbers of 5.6 +/- 0.5 x 10(6) cells/ml obtained for Champagne Pool water samples by 4',6-diamidino-2-phenylindole (DAPI) staining. Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA (small-subunit ribosomal nucleic acid) gene clone library analyses of environmental DNA indicated the abundance of Sulfurihydrogenibium, Sulfolobus, and Thermofilum-like populations in Champagne Pool. From these results, media were selected to target the enrichment of hydrogen-oxidizing and sulfur-dependent microorganisms. Three isolates were successfully obtained having 16S rRNA gene sequences with similarities of approximately 98% to Thermoanaerobacter tengcongensis, 94% to Sulfurihydrogenibium azorense, and 99% to Thermococcus waiotapuensis, respectively.
Asunto(s)
Archaea/aislamiento & purificación , Bacterias/aislamiento & purificación , Biodiversidad , Sedimentos Geológicos/microbiología , Manantiales de Aguas Termales/microbiología , Microbiología del Agua , Adenosina Trifosfato/análisis , Archaea/clasificación , Archaea/crecimiento & desarrollo , Bacterias/clasificación , Bacterias/crecimiento & desarrollo , Medios de Cultivo , ADN de Archaea/análisis , ADN Bacteriano/análisis , ADN Ribosómico/análisis , Biblioteca de Genes , Sedimentos Geológicos/química , Manantiales de Aguas Termales/química , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Nueva Zelanda , Filogenia , ARN Ribosómico 16S , Ribotipificación , Sulfolobus/aislamiento & purificación , Temperatura , Thermoanaerobacter/aislamiento & purificación , Thermococcus/aislamiento & purificación , Thermofilaceae/aislamiento & purificaciónRESUMEN
This study reports surface complexation models (SCMs) for quantifying metal ion adsorption by thermophilic microorganisms. In initial cadmium ion toxicity tests, members of the genus Geobacillus displayed the highest tolerance to CdCl2 (as high as 400 to 3,200 microM). The thermophilic, gram-positive bacteria Geobacillus stearothermophilus and G. thermocatenulatus were selected for further electrophoretic mobility, potentiometric titration, and Cd2+ adsorption experiments to characterize Cd2+ complexation by functional groups within and on the cell wall. Distinct one-site SCMs described the extent of cadmium ion adsorption by both studied Geobacillus sp. strains over a range of pH values and metal/bacteria concentration ratios. The results indicate that a functional group with a deprotonation constant pK value of approximately 3.8 accounts for 66% and 80% of all titratable sites for G. thermocatenulatus and G. stearothermophilus, respectively, and is dominant in Cd2+ adsorption reactions. The results suggest a different type of functional group may be involved in cadmium biosorption for both thermophilic strains investigated here, compared to previous reports for mesophilic bacteria.