Rubén León

Antimicrobial therapy in periodontitis: the use of systemic antimicrobials against the subgingival biofilm

OBJECTIVES The aim was to answer three relevant questions: can systemic antimicrobials be efficacious if the biofilm is not disrupted? Can the type of debridement of the subgingival biofilm impact upon the clinical outcomes of the adjunctive antimicrobial therapy? Is the efficacy of the adjunctive systemic antimicrobial therapy dependent on the quality of the debridement of the subgingival biofilm and the sequence debridement-antibiotic usage? MATERIAL AND METHODS Relevant papers were searched, critically analysed and their data were extracted. RESULTS For the first question, studies assessing susceptibility of bacteria in biofilms, and clinical studies evaluating systemic antimicrobials as monotherapy, were reviewed. For the second question, clinical studies comparing systemic antimicrobials as adjuncts to non-surgical debridement or to periodontal surgery and clinical trials using systemic antibiotics with periodontal surgery were evaluated. For the third question, a previous systematic review was updated. CONCLUSION If systemic antimicrobials are indicated in periodontal therapy, they should be adjunctive to mechanical debridement. There is not enough evidence to support their use with periodontal surgery. Indirect evidence suggests that antibiotic intake should start on the day of debridement completion, debridement should be completed within a short time (preferably <1 week) and with an adequate quality, to optimize the results.

Method to quantify live and dead cells in multi-species oral biofilm by real-time PCR with propidium monoazide.

Real-time PCR (qPCR) is a widely used technique in analysing environmental and clinical microbiological samples. However, its main limitation was its inability to discriminate between live and dead cells.Recently, propidium monoazide (PMA) together with qPCR has been used to overcome this problem, with good results for different bacterial species in different types of samples.Our objective was to implement this technique for analysing mortality in multi-species oral biofilms formed in vitro with five oral bacteria: Streptococcus oralis, Streptococcus gordonii, Veillonella parvula, Fusobacterium nucleatum and Prevotella intermedia. We also tested its effectiveness on biofilms treated with an antiseptic solution containing 0.07% w/w cetylpyridinium chloride (CPC).Standardisation of the qPCR-PMA method was performed on pure, heat-killed planktonic cultures of each species, detecting mortality higher than 4 log in S. oralis, S. gordonii and F. nucleatum and higher than 2 for V. parvula and P. intermedia. We obtained similar results for all species when using CPC.When we analysed biofilms with qPCR-PMA, we found that the mortality in the non-CPC treated multi-species biofilms was lower than 1 log for all species. After treatment with CPC, the viability reduction was higher than 4 log in S. oralis and S. gordonii, higher than 3 log in F. nucleatum and P. intermedia and approximately 2 in V. parvula.In short, we standardised the conditions for using qPCR-PMA in 5 oral bacterial species and proved its usefulness for quantification of live and dead cells in multi-species oral biofilms formed in vitro, after use of an antiseptic.

Genetics and electrophoretic karyotyping of wild-type and astaxanthin mutant strains of Phaffia rhodozyma

In this work we establish the chromosomal composition of a wild-type, one astaxanthin and two β-carotene overproducer strains of the red yeast Phaffia rhodozyma. The method used has been pulsed field gel electrophoresis, which has determined 9 DNA chromosomal bands in the yeast genome. The two largest bands are triplets and two other bands, VI and VIII, seem to be doublets. The size of the chromosomal bands varies between 0.35 and 2.5 Mb, suggesting a genome size of 25 Mb. The technique used, complemented with hybridization assays using specific DNA probes, provides direct information about the genomic organization of P. rhodozyma. We have also cloned and located in chromosomal bands different DNA sequences that code for the translation elongation factor 1 alpha (ef-1α), a 7.6 kb BamHI fragment of repetitive DNA (possibly rDNA) and a randomly chosen fragment (named locus R2). Additionally, we have detected a chromosomal length polymorphism between wild-type strains and mutant strains affecting carotenogenesis obtained in our laboratory.

Development of the sexual reproductive cycle of Xanthophyllomyces dendrorhous

Conditions inducing the development of holobasidia with terminal basidiospores in wild-type and astaxanthin mutant strains of Xanthophyllomyces dendrorhous were reexamined. Important factors for the development of holobasidia were the incubation temperature and the medium composition. A temperature of 9 degrees C was demonstrated to enhance holobasidia formation. Minimal growth medium with glucose as sole carbon source at concentrations between 80 and 120 mM, and ammonium nitrate with concentrations of 28 mM gave optimal results. A period of 20 or more days was needed for the formation of holobasidia with basidiospores. Additionally, mutant strains of X. dendrorhous were observed to have different abilities to produce holobasidia and strains obtained after protoplast fusion, which have been called fusant in this study, to have an increased capacity to form holobasidia.

Genetic determination of ploidy level in Xanthophyllomyces dendrorhous

Xanthophyllomyces dendrorhous (formely Phaffia rhodozyma) is a basidiomycetous yeast-like fungus that produces carotenoids useful for the food industry. Recently, its sexual cycle was reported but little is known about its genetic constitution. To inquire into the ploidy state of X. dendrorhous, biased mutant spectrum, genetic complementation and mitotic recombination analysis were used. A wild-type strain was subjected to N-methyl-N′-nitro-N-nitrosoguanidine mutagenic treatment. Auxotrophic and carotene mutants were forced to revert to the wild-type phenotype. Pigment producing and prototroph revertants behaved as diploid except for adenine less mutants. These results are in agreement with the limited spectrum of auxotrophs obtained in this strain for the ADE1 locus. To analyze the genetic characteristic of the adenine genetic marker of X. dendrorhous, protoplast fusion experiments with several adenine less mutants were performed. The experiments presented in this work suggest that the ATCC 2430 (UDC 67-385) strain of X. dendrorhous is diploid and a heterozygous constitution is proposed for the ADE1 locus.

Complementation analysis with new genetic markers in Phaffia rhodozyma

Isolation and characterization of auxotrophic mutants from wild-type and astaxanthin mutant strains of Phaffia rhodozyma is described. Differences in survival were observed when u.v. irradiation of P. rhodozyma wild-type and astaxanthin mutant strains were incubated in the dark or exposed to photoreactivating light. Ultra-violet mutagenesis was not effective to produce auxotrophic mutants in this yeast. Auxotrophic mutants were obtained with high efficiency through a nystatin enrichment procedure after a N-methyl-N′-nitro-N-nitrosoguanidine (NTG) mutagenic treatment with a 0.12% survivor level. Stringent mutagenetic conditions were needed to obtain P. rhodozyma auxotrophs. The most frequent mutants were ade- and met- in a rather narrow auxotroph spectrum. These results may be associated with a possible diploid condition of this yeast. The high number of adenine auxotrophs obtained in relation to other auxotrophic mutants suggests the possibility of some degree of heterozygosity in the wild-type strain UCD 67-385.

Genetic transformation of astaxanthin mutants of Phaffia rhodozyma

Stable red astaxanthin-producing transformants were obtained after genetic transformation of two Phaffia rhodozyma mutants. A yellow mutant, accumulating β-carotene, and an albino mutant, accumulating phytoene, from P. rhodozyma were transformed using a genomic library of wild-type strain UCD 67-385 in the pBluescript vector. Hybridization assays, using the pBluescript DNA as a radioactive probe, indicate integration of vector sequences into the genome of the transformants. Transformants DNA was digested with restriction endonucleases, ligated with T4 DNA ligase and then used to transform E. coli. Ampicillin resistant plasmids, containing 0.1, 0.2, and 2.5 kb DNA inserts of P. rhodozyma, were rescued from the yeast red transformants. The molecular analysis indicate that transformation has occurred by an integration event of donor DNA into the genome of the host strains.

Validation of ATP bioluminescence as a tool to assess antimicrobial effects of mouthrinses in an in vitro subgingival-biofilm model

Objectives. The aim of this investigation was to evaluate whether the adenosine triphosphate (ATP) bioluminescence method is an appropriate tool to assess the efficacy of antiseptic mouthrinses in terms of quantitative reductions of total viable microbial counts in mixed biofilm populations in vitro. Study Design. Three mouthrinses, containing respectively, chlorhexidine and cetylpyridinium chloride (CHX/CPC), essential oils (EO) and amine fluoride/stannous fluoride (AFSF), as well as Phosphate Buffered Saline (PBS) used as control, were tested in an in vitro static biofilm model by ATP bioluminescence and compared to culture method. Biofilms were grown on saliva-coated hydroxyapatite disks for 72 hours and then exposed for 1 minute to the mouthrinse or control by immersion. The antibacterial effect of the rinses was tested by analysis of variance. The reliability of the ATP bioluminescence method was assessed by calculating the Pearson correlation coefficients when compared to the viable cell counts obtained by culture. Results. Using ATP bioluminescence, the antimicrobial activity of the tested mouthrinses was demonstrated when compared to the PBS control. The ATP bioluminescence values were significantly correlated (0.769, p<0.001) to the viable cell counts. CHX/CPC and AFSF showed similar antimicrobial activity, although AFSF had a less homogeneous effect, being both more effective than the EO rinse. Conclusion. ATP bioluminescence viability testing may be considered a useful tool to assess the in vitro efficacy of antibacterial compounds. In the proposed model, CHX/CPC and AFSF containing mouthrinses demonstrated superior antimicrobial activity, as compared to EO rinses, in a multispecies biofilm model. Key words:Biofilm, ATP bioluminescence,mouthrinse, essential oils, chlorhexidine, amine fluoride/stannous fluoride.

Relación entre enfermedad periodontal, infección bacteriana ascendente y patología placentaria con parto prematuro

Objetivo: Determinar la relacion entre enfermedad periodontal, infeccion bacteriana ascendente y patologia placentaria, con parto prematuro. Pacientes y Metodos: Participaron embarazadas entre 24 y 34 semanas de gestacion, con trabajo de parto prematuro sin causa clinica evidente y membranas intactas o con el diagnostico de rotura prematura de membranas (RPM), sin trabajo de parto y sin corioamnionitis clinica. Todas las embarazadas tuvieron estudio periodontal clinico y evaluacion microbiologica de la placa subgingival, del liquido amniotico (LA) y cervicovaginal. R ecibieron corticoesteroides, antibioticos, tocolisis (casos con membranas intactas) y manejo expectante hasta las 35 semanas (casos con RPM) . Las placentas se enviaron a estudio y se diagnostico corioamnionitis, funisitis y vellositis. Se definio invasion microbiana de la cavidad amniotica ( IMCA) el cultivo positivo del liquido amniotico. Infeccion cervicovaginal (ICV) se diagnostico con vaginosis bacteriana (VB) o cultivo positivo para bacteria patogena u oportunista en cervix o vagina, con incremento significativo de los leucocitos polimorfonucleares. Se considero como infeccion bacteriana ascendente (IBA) la presencia de IMCA por bacterias ascendentes y/o ICV. Resultados: Se incluyeron 59 pacientes, 42 con membranas intactas y 17 con RPM . La frecuencia de la enfermedad periodontal fue 93.2%. La IMCA fue 27.1% Se aislaron bacterias patogenas periodontales del LA en el 18.6% y de la placa subgingival en el 71.2% de los casos. La IBA fue 83.1%. La asociacion IBA con enfermedad periodontal fue 72.9%. El parto prematuro ( 82.1% p=0.03 y con la presencia conjunta de infeccion bacteriana ascendente y enfermedad periodontal 74.4% p=0.03. Los casos con nacimiento prematuro y enfermedad periodontal generalizada tuvieron significativa mayor frecuencia de corioamnionitis y funisitis histologica 69.6% p=0.04. Conclusiones : L a enfermedad periodontal generalizada y la presencia conjunta de infeccion bacteriana ascendente y enfermedad periodontal se asocian con parto prematuro . En estos casos, son frecuentes l os marcadores histologicos placentarios de infeccion ascendente .

Variability of the fimA gene in Porphyromonas gingivalis isolated from periodontitis and non-periodontitis patients

Objective: The goal of this study was to determine the genetic variability of the fimA gene in Porphyromonas gingivalis isolates from Spanish patients. Study Design: Pooled subgingival samples were taken, processed and cultured in non-selective blood agar medium. Pure cultures of one to six isolates per patient were obtained and PCR and PCR-RFLP were used for fimbrillin gene (fimA) type determination of the extracted genomic (DNA). Results: Two hundred and twenty four Porphyromonas gingivalis isolates from 65 patients were analyzed consisting of 15 non-periodontitis patients (66 isolates) and 50 with periodontitis (158 isolates). Genotype II was the most prevalent (50.9%), while the other types of fimbriae did not exceed fifteen percent of prevalence. Isolates with types II and IV of fimbriae were significantly more prevalent in periodontitis patients than isolates with genotype I. Co-infection was observed in 17.65% of the patients analyzed. Conclusion: The results suggest that in this population Porphyromonas gingivalis with type II of fimbriae are significantly more predominant in periodontitis patients than genotype I. Key words:Fimbriae, genotype, porphyromonas gingivalis, periodontitis.