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

The Erwinia chrysanthemi phoP-phoQ operon plays an important role in growth at low pH, virulence and bacterial survival in plant tissue

The Erwinia chrysanthemi phoP-phoQ operon plays an important role in growth at low pH, virulence and bacterial survival in plant tissu

The ybiT Gene of Erwinia chrysanthemi Codes for a Putative ABC Transporter and Is Involved in Competitiveness against Endophytic Bacteria during Infection

We investigated the role in bacterial infection of a putative ABC transporter, designated ybiT, of Erwinia chrysanthemi AC4150. The deduced sequence of this gene showed amino acid sequence similarity with other putative ABC transporters of gram-negative bacteria, such as Escherichia coli and Pseudomonas aeruginosa, as well as structural similarity with proteins of Streptomyces spp. involved in resistance to macrolide antibiotics. The gene contiguous to ybiT, designated as pab (putative antibiotic biosynthesis) showed sequence similarity with Pseudomonas and Streptomyces genes involved in the biosynthesis of antibiotics. A ybiT mutant (BT117) was constructed by marker exchange. It retained full virulence in potato tubers and chicory leaves, but it showed reduced ability to compete in planta against the wild-type strain or against selected saprophytic bacteria. These results indicate that the ybiT gene plays a role in the in planta fitness of the bacteria

Strategies to Combat Caries by Maintaining the Integrity of Biofilm and Homeostasis during the Rapid Phase of Supragingival Plaque Formation

Bacteria in the oral cavity, including commensals and opportunistic pathogens, are organized into highly specialized sessile communities, coexisting in homeostasis with the host under healthy conditions. A dysbiotic environment during biofilm evolution, however, allows opportunistic pathogens to become the dominant species at caries-affected sites at the expense of health-associated taxa. Combining tooth brushing with dentifrices or rinses combat the onset of caries by partially removes plaque, but resulting in the biofilm remaining in an immature state with undesirables&rsquo; consequences on homeostasis and oral ecosystem. This leads to the need for therapeutic pathways that focus on preserving balance in the oral microbiota and applying strategies to combat caries by maintaining biofilm integrity and homeostasis during the rapid phase of supragingival plaque formation. Adhesion, nutrition, and communication are fundamental in this phase in which the bacteria that have survived these adverse conditions rebuild and reorganize the biofilm, and are considered targets for designing preventive strategies to guide the biofilm towards a composition compatible with health. The present review summarizes the most important advances and future prospects for therapies based on the maintenance of biofilm integrity and homeostasis as a preventive measure of dysbiosis focused on these three key factors during the rapid phase of plaque formation

Gene expression of Porphyromonas gingivalis ATCC 33277 when growing in an in vitro multispecies biofilm.

Background and objectivePorphyromonas gingivalis, an oral microorganism residing in the subgingival biofilm, may exert diverse pathogenicity depending on the presence of specific virulence factors, but its gene expression has not been completely established. This investigation aims to compare the transcriptomic profile of this pathogen when growing within an in vitro multispecies biofilm or in a planktonic state.Materials and methodsP. gingivalis ATCC 33277 was grown in anaerobiosis within multi-well culture plates at 37°C under two conditions: (a) planktonic samples (no hydroxyapatite discs) or (b) within a multispecies-biofilm containing Streptococcus oralis, Actinomyces naeslundii, Veillonella parvula, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans deposited on hydroxyapatite discs. Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM) combined with Fluorescence In Situ Hybridization (FISH) were used to verify the formation of the biofilm and the presence of P. gingivalis. Total RNA was extracted from both the multispecies biofilm and planktonic samples, then purified and, with the use of a microarray, its differential gene expression was analyzed. A linear model was used for determining the differentially expressed genes using a filtering criterion of two-fold change (up or down) and a significance p-value of ResultsSEM verified the development of the multispecies biofilm and FISH confirmed the incorporation of P. gingivalis. The microarray demonstrated that, when growing within the multispecies biofilm, 19.1% of P. gingivalis genes were significantly and differentially expressed (165 genes were up-regulated and 200 down-regulated), compared with planktonic growth. These genes were mainly involved in functions related to the oxidative stress, cell envelope, transposons and metabolism. The results of the microarray were confirmed by RT-qPCR.ConclusionSignificant transcriptional changes occurred in P. gingivalis when growing in a multispecies biofilm compared to planktonic state

Response to antiseptic agents of periodontal pathogens in in vitro biofilms on titanium and zirconium surfaces

Depto. de Especialidades Clínicas OdontológicasFac. de OdontologíaTRUEpu

Use of probiotics in preventing and treating excess weight and obesity. A systematic review

Abstract Background The prevalence of excess weight and obesity is increasing in an extremely concerning manner worldwide, with highly diverse therapies for current treatment. This review evaluated the scientific evidence of the past 10 years on the use of probiotics in treating excess weight and obesity in the absence of dieting. Materials A systematic review was conducted by searching for clinical trials on humans published in English in the PubMed, Scopus and Cochrane Central databases, using the combination of keywords “Overweight”, “Probiotics” and “Obesity”, and published between 2012 and 2022. Results Six published studies met the inclusion criteria. The review showed that, although there is a lack of consensus in the literature, the use of probiotics in the absence of dieting produced a significant reduction in body weight and body mass index in 66.6% of the reviewed studies, a significant reduction in waist circumference in 80.0% of the reviewed studies, and an improvement in total body fat mass and waist circumference. Conclusions This review showed evidence of a trend in preventing body weight gain and reducing weight through the use of probiotics in individuals with excess weight or obesity. A combination of various strains of the genera Bifidobacterium and Lactobacillus was the most effective

Comparative gene expression analysis of planktonic Porphyromonas gingivalis ATCC 33277 in the presence of a growing biofilm versus planktonic cells

Abstract Background Porphyromonas gingivalis, a microorganism residing in the oral cavity within complex multispecies biofilms, is one of the keystone pathogens in the onset and progression of periodontitis. In this in vitro study, using DNA microarray, we investigate the differential gene expression of Porphyromonas gingivalis ATCC 33277 when growing in the presence or in absence of its own monospecies biofilm. Results Approximately 1.5% of genes (28 out of 1909 genes, at 1.5 fold change or more, p-value < 0.05) were differentially expressed by P. gingivalis cells when in the presence of a biofilm. These genes were predominantly related to the metabolism of iron, bacterial adhesion, invasion, virulence and quorum-sensing system. The results from microarrays were consistent with those obtained by RT-qPCR. Conclusion This study provides insight on the transcriptional changes of planktonic P. gingivalis cells when growing in the presence of a biofilm. The resulting phenotypes provide information on changes occurring in the gene expression of this pathogen

<i>Aggregatibacter actinomycetemcomitans</i> Growth in Biofilm versus Planktonic State: Differential Expression of Proteins

<i>Aggregatibacter actinomycetemcomitans (Aa)</i> is a pathogenic bacterium residing in the subgingival plaque biofilm strongly associated with the pathogenesis of periodontitis. The aim of this investigation was to study the protein differential expression of <i>Aa</i> when growing on biofilm compared with planktonic state using proteomic analysis by the 2D-DIGE system. Eighty-seven proteins were differentially expressed during biofilm growth (1.5-fold, <i>p</i> < 0.05), with 13 overexpressed and 37 down-expressed. Those repressed were mainly proteins involved in metabolism, biosynthesis, and transport. The overexpressed proteins were outer membrane proteins (OMPs) and highly immunogenic proteins such as YaeT (OMP), FtsZ, OMP39, OMP18/16, the chaperone GroEL, OMPA, adenylate kinase (Adk), and dihydrolipoamide acetyltransferase. The enrichment fractions of the OMPs from biofilm and planktonic states were obtained, and these proteins were analyzed by Western blotting with human serum from a periodontitis patient and one healthy control. These immunogenic proteins overexpressed in the biofilm may represent candidate virulence factors