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PURPOSE: Clinical and in vitro studies showed selected oral microorganisms to be related to delayed wound healing and ulcerative oral mucositis. However, it is not known whether this effect is due to reduced metabolism and/or the reduced reproductive capacity of epithelial cells. Therefore, we studied the influence of the oral microorganisms Porphyromonas gingivalis, Candida glabrata, and Candida kefyr on cell metabolism and reproductive capacity of oral epithelial cells, aimed to further unravel the pathogenesis of oral mucositis. METHODS: Oral epithelial cells were exposed to different concentrations of P. gingivalis, C. glabrata, and C. kefyr as mono-infections or mixed together. An MTT assay was performed to determine the effect on cell metabolism. A clonogenic assay was used to study the effect on the reproductive capacity of oral epithelial cells. RESULTS: The metabolism of oral epithelial cells was reduced when the microorganisms were present in high concentrations: P. gingivalis at a multiplicity of infection (MOI) of 1000 and the Candida spp. at MOI 100. No statistical difference was observed in the ability of a single epithelial cell to grow into a colony of cells between control and P. gingivalis, C. glabrata, and C. kefyr, independent of the concentrations and combinations used. CONCLUSION: P. gingivalis, C. glabrata, and C. kefyr lowered the metabolic activity of oral epithelial cells in high concentrations, yet they did not influence the reproductive capacity of epithelial cells. Their impact on ulcerative oral mucositis is likely due to an effect on the migration, proliferation, and metabolism of epithelial cells.
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Candida/fisiología , Porphyromonas gingivalis/fisiología , Estomatitis/microbiología , Infecciones por Bacteroidaceae/metabolismo , Infecciones por Bacteroidaceae/microbiología , Infecciones por Bacteroidaceae/patología , Candida glabrata/fisiología , Candidiasis/metabolismo , Candidiasis/microbiología , Candidiasis/patología , Línea Celular , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Células Epiteliales/patología , Humanos , Técnicas In Vitro , Estomatitis/metabolismo , Estomatitis/patologíaRESUMEN
Attachment to human host tissues or abiotic medical devices is a key step in the development of infections by Candida glabrata. The genome of this pathogenic yeast codes for a large number of adhesins, but proteomic work using reference strains has shown incorporation of only few adhesins in the cell wall. By making inventories of the wall proteomes of hyperadhesive clinical isolates and reference strain CBS138 using mass spectrometry, we describe the cell wall proteome of C. glabrata and tested the hypothesis that hyperadhesive isolates display differential incorporation of adhesins. Two clinical strains (PEU382 and PEU427) were selected, which both were hyperadhesive to polystyrene and showed high surface hydrophobicity. Cell wall proteome analysis under biofilm-forming conditions identified a core proteome of about 20 proteins present in all C. glabrata strains. In addition, 12 adhesin-like wall proteins were identified in the hyperadherent strains, including six novel adhesins (Awp8-13) of which only Awp12 was also present in CBS138. We conclude that the hyperadhesive capacity of these two clinical C. glabrata isolates is correlated with increased and differential incorporation of cell wall adhesins. Future studies should elucidate the role of the identified proteins in the establishment of C. glabrata infections.
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Candida glabrata/química , Pared Celular/química , Proteínas Fúngicas/análisis , Proteoma/análisis , Candida glabrata/aislamiento & purificación , Candidiasis/microbiología , Humanos , Espectrometría de Masas , ProteómicaRESUMEN
LFchimera, a construct combining two antimicrobial domains of bovine lactoferrin, lactoferrampin265-284 and lactoferricin17-30, possesses strong bactericidal activity. As yet, no experimental evidence was presented to evaluate the mechanisms of LFchimera against Burkholderia isolates. In this study we analyzed the killing activity of LFchimera on the category B pathogen Burkholderia pseudomallei in comparison to the lesser virulent Burkholderia thailandensis often used as a model for the highly virulent B. pseudomallei. Killing kinetics showed that B. thailandensis E264 was more susceptible for LFchimera than B. pseudomallei 1026b. Interestingly the bactericidal activity of LFchimera appeared highly pH dependent; B. thailandensis killing was completely abolished at and below pH 6.4. FITC-labeled LFchimera caused a rapid accumulation within 15 min in the cytoplasm of both bacterial species. Moreover, freeze-fracture electron microscopy demonstrated extreme effects on the membrane morphology of both bacterial species within 1 h of incubation, accompanied by altered membrane permeability monitored as leakage of nucleotides. These data indicate that the mechanism of action of LFchimera is similar for both species and encompasses disruption of the plasma membrane and subsequently leakage of intracellular nucleotides leading to cell dead.
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Burkholderia pseudomallei/efectos de los fármacos , Burkholderia/efectos de los fármacos , Lactoferrina/química , Lactoferrina/farmacología , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/farmacología , Burkholderia/metabolismo , Burkholderia/ultraestructura , Burkholderia pseudomallei/metabolismo , Burkholderia pseudomallei/ultraestructura , Bovinos , Membrana Celular/efectos de los fármacos , Técnica de Fractura por Congelación , Hemólisis/efectos de los fármacos , Humanos , Lactoferrina/genética , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/farmacología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Especificidad de la EspecieRESUMEN
The aim of this study was to assess the red autofluorescence from bacterial species related to dental caries and periodontitis in the presence of different nutrients in the growth medium. Bacteria were grown anaerobically on tryptic soy agar (TSA) supplemented with nutrients, including magnesium-porphyrins from spinach and iron-porphyrins from heme. The autofluorescence was then assessed at 405 nm excitation. On the TSA without additives, no autofluorescence was observed from any of the species tested. On the TSA containing sheep blood, red autofluorescence was observed only from Parvimonas micra. When the TSA was supplemented with blood, hemin, and vitamin K, red autofluorescence was observed from Actinomyces naeslundii, Bifidobacterium dentium, and Streptococcus mutans. Finally, on the TSA supplemented with spinach extract, red autofluorescence was observed from Aggregatibacter actinomycetemcomitans, A. naeslundii, Enterococcus faecalis, Fusobacterium nucleatum, Lactobacillus salivarius, S. mutans, and Veillonella parvula. We conclude that the bacteria related to dental caries and periodontal disease exhibit red autofluorescence. The autofluorescence characteristics of the tested strains depended on the nutrients present, such as metalloporphyrins, suggesting that the metabolic products of the oral biofilm could be responsible for red autofluorescence.
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Bacterias Anaerobias/efectos de los fármacos , Bacterias Anaerobias/efectos de la radiación , Bacterioclorofilas/farmacología , Medios de Cultivo/química , Placa Dental/microbiología , Fluorescencia , Hemo/farmacología , Bacterias Anaerobias/metabolismo , Bifidobacterium/fisiología , Biopelículas/efectos de los fármacos , Biopelículas/efectos de la radiación , Caries Dental/diagnóstico , Caries Dental/microbiología , Luz , Periodontitis/microbiología , Streptococcus mutans/fisiologíaRESUMEN
Oral ulcerations often arise as a side effect from chemo- and radiation therapy. In a previous clinical study, Porphyromonas gingivalis was identified as a positive predictor for oral ulcerations after hematopoetic stem cell transplantation, possibly incriminating P. gingivalis in delayed healing of the ulcerations. Therefore, it was tested whether P. gingivalis and its secreted products could inhibit the migration of oral epithelial cells in an in vitro scratch assay. To compare, the oral bacteria Prevotella nigrescens, Prevotella intermedia, Tannerella forsythia, and Streptococcus mitis were included. A standardized scratch was made in a confluent layer of human oral epithelial cells. The epithelial cells were challenged with bacterial cells and with medium containing secretions of these bacteria. Closure of the scratch was measured after 17 h using a phase contrast microscope. P. gingivalis, P. nigrescens, and secretions of P. gingivalis strongly inhibited cell migration. A challenge with 1000 heat-killed bacteria versus 1 epithelial cell resulted in a relative closure of the scratch of 25% for P. gingivalis and 20% for P. nigrescens. Weaker inhibitory effects were found for the other bacteria. The results confirmed our hypothesis that the oral bacteria may be involved in delayed wound healing.
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Células Epiteliales/citología , Células Epiteliales/microbiología , Mucosa Bucal/microbiología , Cicatrización de Heridas , Línea Celular , Movimiento Celular , Células Cultivadas , Medios de Cultivo Condicionados/química , Humanos , Enfermedades Periodontales/microbiología , Porphyromonas gingivalis/metabolismo , Porphyromonas gingivalis/patogenicidad , Prevotella intermedia/metabolismo , Prevotella intermedia/patogenicidad , Prevotella nigrescens/metabolismo , Prevotella nigrescens/patogenicidad , Streptococcus mitis/metabolismo , Streptococcus mitis/patogenicidadRESUMEN
The water systems inside a dental unit are known to be contaminated with a multi-kingdom biofilm encompassing bacteria, fungi, viruses and protozoa. Aerosolization of these micro-organisms can potentially create a health hazard for both dental staff and the patient. Very little is known on the efficacy of dental unit disinfection products against amoeba. In this study we have examined the effect of four different treatment regimens, with the hydrogen peroxide (H2O2) containing product Oxygenal, on an in-vitro multi-kingdom dental unit water system (DUWS) biofilm. The treatment efficacy was assessed in time using heterotrophic plate counts, the bacterial 16S rDNA, fungal 18S rDNA gene load and the number of genomic units for Legionella spp. the amoeba Vermamoeba vermiformis. The results indicated that a daily treatment of the DUWS with a low dose H2O2 (0.02% for 5 h), combined with a weekly shock dose (0.25% H2O2, 30 min) is necessary to reduce the heterotrophic plate count of a severely contaminated DUWS (>106 CFU.mL-1) to below 100 CFU.mL-1. A daily treatment with a low dose hydrogen peroxide alone, is sufficient for the statistically significant reduction of the total amount of bacterial 16S rDNA gene, Legionella spp. and Vermamoeba vermiformis load (p < 0.005). Also shown is that even though hydrogen peroxide does not kill the trophozoite nor the cysts of V. vermiformis, it does however result in the detachment of the trophozoite form of this amoeba from the DUWS biofilm and hereby ultimately removing the amoeba from the system.
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Porphyromonas gingivalis is associated with the development of periodontitis. Here we describe the development of a highly specific protease-based diagnostic method for the detection of P. gingivalis in gingival crevicular fluid. Screening of a proteolytic peptide substrate library, including fluorogenic dipeptides that contain d-amino acids, led to the discovery of five P. gingivalis-specific substrates. Due to the presence of lysine and arginine residues in these substrates, it was hypothesized that the cleavage was mediated by the gingipains, a group of P. gingivalis-specific proteases. This hypothesis was confirmed by the observation that P. gingivalis gingipain knockout strains demonstrated clearly impaired substrate cleavage efficacy. Further, proteolytic activity on the substrates was increased by the addition of the gingipain stimulators dithiothreitol and l-cysteine and decreased by the inhibitors leupeptin and N-ethylmaleimide. Screening of saliva and gingival crevicular fluid of periodontitis patients and healthy controls showed the potential of the substrates to diagnose the presence of P. gingivalis proteases. By using paper points, a sensitivity of approximately 10(5) CFU/ml was achieved. P. gingivalis-reactive substrates fully composed of l-amino acids and Bz-l-Arg-NHPhNO(2) showed a relatively low specificity (44 to 85%). However, the five P. gingivalis-specific substrates that each contained a single d-amino acid showed high specificity (96 to 100%). This observation underlines the importance of the presence of d-amino acids in substrates used for the detection of bacterial proteases. We envisage that these substrates may improve the specificity of the current enzyme-based diagnosis of periodontitis associated with P. gingivalis.
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Técnicas Bacteriológicas/métodos , Péptido Hidrolasas/análisis , Periodontitis/diagnóstico , Periodontitis/microbiología , Porphyromonas gingivalis/enzimología , Porphyromonas gingivalis/aislamiento & purificación , Líquido del Surco Gingival/microbiología , Humanos , Porphyromonas gingivalis/metabolismo , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Oral mucositis is a serious and debilitating side effect of conditioning regimens for hematopoietic stem cell transplant (HSCT). Through HSCT, the homeostasis in the oral cavity is disrupted. The contribution of the oral microflora to mucositis remains to be clarified. The aim of our study was to investigate the relationship between yeasts, bacteria associated with periodontitis, and oral ulcerations in HSCT recipients. METHODS: This prospective observational study included 49 adult HSCT recipients. Twice weekly, oral ulcerations were scored, and oral rinsing samples were obtained. Samples were evaluated for the total bacterial load; the Gram-negative bacteria: Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Parvimonas micra, Fusobacterium nucleatum, Tannerella forsythia, and Treponema denticola; and the yeasts: Candida albicans, Candida glabrata, Candida kefyr, Candida krusei, Candida parapsilosis, and Candida tropicalis using real-time polymerase chain reaction with specific primers and probes. Explanatory variables for oral ulcerations were calculated using the multilevel generalized estimated equations (GEE) technique. RESULTS: None of the samples was positive for A. actinomycetemcomitans, while F. nucleatum was found most often (66 % of samples). C. albicans was the most isolated yeast (88 % of samples), whereas C. parapsilosis was found in only 8 % of the samples. Multivariate GEE analyses identified P. gingivalis, P. micra, T. denticola, F. nucleatum, C. glabrata, and C. kefyr as significant explanatory variables of oral ulcerations. CONCLUSIONS: Our data indicate that P. gingivalis in particular, but also P. micra, T. denticola, F. nucleatum, C. glabrata, and C. kefyr may play a role in ulcerative oral mucositis in patients undergoing HSCT.
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Candida/aislamiento & purificación , Bacterias Gramnegativas/aislamiento & purificación , Trasplante de Células Madre Hematopoyéticas , Boca/microbiología , Úlceras Bucales , Estomatitis , Acondicionamiento Pretrasplante/efectos adversos , Adulto , Carga Bacteriana , Femenino , Neoplasias Hematológicas/terapia , Humanos , Masculino , Persona de Mediana Edad , Úlceras Bucales/etiología , Úlceras Bucales/microbiología , Estudios Prospectivos , Reacción en Cadena en Tiempo Real de la Polimerasa , Estomatitis/etiología , Estomatitis/microbiologíaRESUMEN
Incomplete disinfection of the root canal system is a major cause of post-treatment disease. This study aimed to investigate the disinfecting property of organic acid salts and sodium chloride (NaCl), in a double-hurdle strategy, on Enterococcus faecalis biofilms. First of all, the high-throughput resazurin metabolism assay (RMA) was used to test a range of organic acid salts. Then, to gain more insight into the efficacy of sorbate salt solutions, 48-h E. faecalis biofilms were evaluated in colony-forming unit (CFU) assays. Chlorhexidine (CHX) and calcium hydroxide [Ca(OH)(2) ] were tested in parallel as controls. Sorbate salt produced the largest and most significant reduction of fluorescence intensity in the RMA assay. Neither NaCl nor potassium sorbate (KS) alone induced a clinically relevant reduction of CFU counts after 1 h. Surprisingly, the combination of the two in a single solution had a synergistic effect on the inactivation of E. faecalis. Potassium sorbate amplified the efficacy of NaCl. Of the salts tested, NaCl with KS eradicated E. faecalis biofilms within 1 h. This study showed that the double-hurdle strategy indeed leads to synergistic efficacy and is a possible next step in the complete disinfection of endodontic infections.
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Antiinfecciosos Locales/farmacología , Enterococcus faecalis/efectos de los fármacos , Cloruro de Sodio/farmacología , Ácido Sórbico/farmacología , Biopelículas/efectos de los fármacos , Hidróxido de Calcio/farmacología , Clorhexidina/farmacología , Recuento de Colonia Microbiana , Combinación de Medicamentos , Irrigantes del Conducto Radicular/farmacologíaRESUMEN
Candida glabrata is a prevalent fungal pathogen in humans, which is able to adhere to host cells and abiotic surfaces. Nicotinic acid (NA) limitation has been shown to promote the adherence of C. glabrata to human epithelial cells. Clinically, the elderly and hospitalized patients who are prone to C. glabrata-related denture stomatitis often suffer from vitamin deficiency. This study aimed to investigate C. glabrata adhesion to abiotic surfaces, including acrylic resin (a denture material) surfaces, cell surface hydrophobicity and adhesion gene expression. C. glabrata CBS138 was grown in media containing decreasing NA concentrations (40, 0.4, 0.04 and 0.004 µM). Adherence of C. glabrata to glass coverslips and acrylic resin was analyzed. C. glabrata adhesion to both surfaces generally increased with decreasing NA concentrations. The highest adhesion was found for the cells grown with 0.004 µM NA. The cell surface hydrophobicity test indicated that NA limitation enhanced hydrophobicity of C. glabrata cells. Quantitative PCR showed that of all adhesion genes tested, EPA1, EPA3 and EPA7 were significantly up-regulated in both 0.004 µM NA and 0.04 µM NA groups compared to those in the 40 µM NA group. No significant up- or down-regulation under NA limitation was observed for the other tested adhesion genes, namely AWP3, AWP4, AWP6 and EPA6. NA limitation resulted in increased expression of some adhesion genes, higher surface hydrophobicity of C. glabrata and enhanced adhesion to abiotic surfaces. NA deficiency is likely a risk factor for C. glabrata-related denture stomatitis in the elderly.
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AIMS: Motor Neuron Disease (MND) is a progressive neurodegenerative neuromuscular disease, which can progressively impair arm-hand function. Needs and barriers of MND patients and their caregivers in performing oral hygiene were studied. METHODS: An online survey was sent to 706 MND patients. The questions of the survey included self-reliance, self-reported oral health, and oral hygiene. The oral health-related quality of life (GOHAI-NL) and the subjective well-being (ALSAQ-5) were also measured. RESULTS: A total of 259 patients responded (36.7%), of which 71.9% stated not to be informed about the importance of maintaining good oral health by their MND treatment team. Moreover, 40.4% would like to receive help concerning oral hygiene from a dental professional. 19.8% were not satisfied about oral care as conducted by themselves or their caregivers. Patients who do not ask for support with their daily oral care had a significantly worse oral health-related quality of life compared to patients who do ask for support. CONCLUSIONS: The support for daily oral hygiene of MND patients and their barriers to requesting support needs more attention from both MND-treatment teams and general dental professionals.
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Esclerosis Amiotrófica Lateral , Enfermedad de la Neurona Motora , Estudios Transversales , Humanos , Higiene Bucal , Calidad de VidaRESUMEN
Aggregatibacter actinomycetemcomitans is strongly associated with severe periodontitis, possibly due to its production of a potent leukotoxin. A genetic variant, the JP2 genotype, was found to produce more leukotoxin than the wild type because of a mutation in the leukotoxin gene, and this genotype is frequently found in African populations. The aim of this study was to investigate whether this JP2 genotype can be found in a randomly selected group of inhabitants of Sal, Cape Verde. Twenty-nine adults between 20 and 59 years of age (58.6% female) participated, and information on their oral health and living standards was collected. An oral examination was performed for each participant, including DMF-T and CPI scores. Plaque and saliva samples were collected and transported to Europe, where DNA was isolated, and the concentration of A. actinomycetemcomitans and its JP2 genotype was determined using dedicated PCR analyses. All 29 plaque and 31% of the saliva samples harboured A. actinomycetemcomitans, and two participants were positive for the JP2 genotype. The presence of this JP2 genotype was not associated with either CPI or DMF-T. This pilot study is the first to describe the presence of the A. actinomycetemcomitans JP2 genotype in a Cape Verdean population living in the Cape Verde Islands, and the findings warrant further research.
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Stem cell transplantation (SCT) is associated with oral microbial dysbiosis. However, long-term longitudinal data are lacking. Therefore, this study aimed to longitudinally assess the oral microbiome in SCT patients and to determine if changes are associated with oral mucositis and oral chronic graft-versus-host disease. Fifty allogeneic SCT recipients treated in two Dutch university hospitals were prospectively followed, starting at pre-SCT, weekly during hospitalization, and at 3, 6, 12, and 18 months after SCT. Oral rinsing samples were taken, and oral mucositis (WHO score) and oral chronic graft-versus-host disease (NIH score) were assessed. The oral microbiome diversity (Shannon index) and composition significantly changed after SCT and returned to pre-treatment levels from 3 months after SCT. Oral mucositis was associated with a more pronounced decrease in microbial diversity and with several disease-associated genera, such as Mycobacterium, Staphylococcus, and Enterococcus. On the other hand, microbiome diversity and composition were not associated with oral chronic graft-versus-host disease. To conclude, dysbiosis of the oral microbiome occurred directly after SCT but recovered after 3 months. Diversity and composition were related to oral mucositis but not to oral chronic graft-versus-host disease.
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Dental unit water systems are prone to biofilm formation. During use of the dental unit, clumps of biofilm slough off and can subsequently be aerosolized and inhaled by both patient and staff, potentially causing infections. The aim of this study was to determine the microbial load and microbiome of dental unit water, in the Netherlands, and the factors influencing these parameters. In total, 226 dental units were sampled and heterotrophic plate counts (HPC) were determined on the traditional effluent sample. Of all dental units, 61% exceeded the recommended microbiological guidelines of 100 colony forming units per milliliter. In addition, the microbiome, with additional q-PCR analysis for specific species, was determined on an effluent sample taken immediately after an overnight stagnancy period, in which the biofilm is in its relaxed state. These relaxed biofilm samples showed that each dental unit had a unique microbiome. Legionella spp., amoeba and fungi were found in 71%, 43% and 98% of all units, respectively. The presence of amoeba was positively associated with nine bacterial biomarkers and correlated positively with bacterial and fungal DNA and Legionella spp. concentrations, but not with HPC. Only when adhering to disinfection protocols, statistically significant effects on the microbial load and microbiome were seen. The relaxed biofilm sample, in combination with molecular techniques gives better insight in the presence of opportunistic pathogens when compared to the heterotrophic plate counts. Infection control measures should focus on biofilm analysis and control in order to guarantee patient safety.
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Legionella , Microbiota , Biopelículas , Recuento de Colonia Microbiana , Equipo Dental , Desinfección , Contaminación de Equipos , Humanos , Legionella/genética , Países Bajos , Microbiología del AguaRESUMEN
Dental practices were approached to fill out a questionnaire on the infection control protocols in use to control biofilm growth in the dental unit and to send two types of water sample. Sampling of the dental units had to be performed prior to any infection control measures and on the second day of operation, to avoid residual effects of biofilm disinfection protocols performed in the weekend. Instructions were given on how to sample the units. Only samples, accompanied with a completed questionnaire and returned within two days by regular mail, were analysed. Samples were processed for heterotrophic plate counts, 16S (V4) rDNA microbiome sequencing and q-PCR for the concentration of bacterial 16S rDNA, fungal 18S rDNA, Legionella spp. and the presence of amoeba. The files contain the metadata needed to interpret and analyse the microbiome data. This dataset can be used by other scientists, members of infection control units, (trainee) bioinformaticians and policy makers. This dataset can provide leads to further unexplored parameters which could influence the microbial ecology of the dental unit.
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BACKGROUND: The Gram negative anaerobic bacterium Porphyromonas gingivalis has long been recognized as a causative agent of periodontitis. Periodontitis is a chronic infectious disease of the tooth supporting tissues eventually leading to tooth-loss. Capsular polysaccharide (CPS) of P. gingivalis has been shown to be an important virulence determinant. Seven capsular serotypes have been described. Here, we used micro-array based comparative genomic hybridization analysis (CGH) to analyze a representative of each of the capsular serotypes and a non-encapsulated strain against the highly virulent and sequenced W83 strain. We defined absent calls using Arabidopsis thaliana negative control probes, with the aim to distinguish between aberrations due to mutations and gene gain/loss. RESULTS: Our analyses allowed us to call aberrant genes, absent genes and divergent regions in each of the test strains. A conserved core P. gingivalis genome was described, which consists of 80% of the analyzed genes from the sequenced W83 strain. The percentage of aberrant genes between the test strains and control strain W83 was 8.2% to 13.7%. Among the aberrant genes many CPS biosynthesis genes were found. Most other virulence related genes could be found in the conserved core genome. Comparing highly virulent strains with less virulent strains indicates that hmuS, a putative CobN/Mg chelatase involved in heme uptake, may be a more relevant virulence determinant than previously expected. Furthermore, the description of the 39 W83-specific genes could give more insight in why this strain is more virulent than others. CONCLUSION: Analyses of the genetic content of the P. gingivalis capsular serotypes allowed the description of a P. gingivalis core genome. The high resolution data from three types of analysis of triplicate hybridization experiments may explain the higher divergence between P. gingivalis strains than previously recognized.
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Genoma Bacteriano , Porphyromonas gingivalis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Hibridación Genómica Comparativa , ADN Bacteriano/genética , Genes Bacterianos , Anotación de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Porphyromonas gingivalis/inmunología , Porphyromonas gingivalis/patogenicidad , Virulencia/genéticaRESUMEN
Dental unit water systems (DUWS) provide an excellent environment for biofilm formation and can form a potential health risk for patients and staff. To control this biofilm formation, better understanding of the DUWS biofilm ecology is needed. Described is a newly developed in-vitro DUWS model which is easy to build, can be inoculated with different water sources and allows for sampling of both the effluent and biofilm. Unlike most models, a dynamic flow pattern, typical for a dental unit is used to provide water as a nutrient source. Microbial growth and composition were analyzed using heterotrophic plate counts (HPC) and 16S rDNA sequencing. Growth was reproducible in all models, reaching quasi-steady state at day 16 in the effluent (105-106 CFUâmL-1) and day 23 in the biofilm (108 and 107 CFUâcm-2) for non-potable and potable water, respectively. Principal component analysis of the microbial composition showed that biofilms originating from either non-potable or potable water were significantly different after 30 days of growth (n = 8, PERMANOVA, F = 35.6, p < .005). Treatment of the biofilms with 1000 ppm active chlorine showed a biological and statistical significant decrease in viable counts in the effluent phase to below the detection limit of 100 CFUâmL-1. The HPC returned to pre-treatment levels within 14 days. Using this model results in inoculum dependent biofilms with a higher bacterial density compared to previously described models. The relative ease in which samples can be taken allows for the monitoring of antimicrobial disinfection efficacy on the effluent, biofilm and matrix.
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Bacterias/crecimiento & desarrollo , Biopelículas/crecimiento & desarrollo , Equipo Dental/microbiología , Contaminación de Equipos , Recuento de Colonia Microbiana , Desinfección/métodos , Humanos , Hidrodinámica , Investigación Biomédica Traslacional , Microbiología del AguaRESUMEN
OBJECTIVE: Clinical diagnostics often requires the detection of multiple bacterial species in limited clinical samples with a single DNA extraction method. This study aimed to compare the bacterial DNA extraction efficiency of two lysis methods automated with the MagNA-Pure LC instrument. The samples included five oral bacterial species (three Gram-positive and two Gram-negative) with or without human saliva background. METHODS: Genomic DNA (gDNA) was extracted from bacterial cultures by bead-beating lysis (BMP) or chemical lysis (MP), followed by automated purification and measurement by quantitative PCR. RESULTS: For pure bacterial cultures, the MP method yielded higher quantities of extracted DNA and a lower detection limit than the BMP method, except where the samples contained high numbers of Gram-positive bacteria. For bacterial cultures with a saliva background, no difference in gDNA extraction efficacy was observed between the two methods. CONCLUSIONS: The efficiency of a bacterial DNA extraction method is not only affected by the bacterial cell wall structure but also by the sample milieu. The MP method provided superior gDNA extraction efficiency when the samples contained a single bacterial species, whereas either of the BMP and MP methods could be applied with similar efficiencies to samples containing multiple species of bacteria.
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Técnicas de Tipificación Bacteriana/métodos , ADN Bacteriano/aislamiento & purificación , Genoma Bacteriano/genética , Bacterias Gramnegativas/aislamiento & purificación , Bacterias Grampositivas/aislamiento & purificación , Técnicas de Tipificación Bacteriana/instrumentación , ADN Bacteriano/genética , Bacterias Gramnegativas/genética , Bacterias Grampositivas/genética , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Saliva/microbiología , Manejo de Especímenes/métodosRESUMEN
BACKGROUND: Biofilm formation in dental unit waterlines (DUWL) may lead to health risks for dental staff and patients. Therefore, dental unit waterlines need to be disinfected, for instance by using chemical disinfectants. However, the application of chemical disinfectants may lead to the selection of specific microorganisms. Therefore, the aim of our study was to assess the microbial composition of water-derived biofilms, after a continuous exposure to maintenance doses of commercially available chemical disinfectants, in vitro. METHODS: The AAA-model was used to grow water derived biofilms. The biofilms were subjected to the maintenance dose of each disinfectant. To determine the microbial composition, the V4 hypervariable region of the 16S rRNA gene was sequenced. The sequences were clustered in operational taxonomic units (OTUs). RESULTS: The bacterial composition of biofilms in all treatment groups differed significantly (PERMANOVA F = 4.441, p = 0.001). Pairwise comparisons revealed Anoxyl treated biofilms were significantly different from all groups (p = 0.0001). In the Anoxyl-treated biofilms, the relative abundance of Comamonadaceae and Sphingopyxis was high compared to the Dentosept, Green and Clean and Oxygenal groups. CONCLUSION: We concluded that exposure to low doses of the chlorine-based chemical disinfectant Anoxyl led to a substantially different composition of water derived biofilms compared to biofilms exposed to H2O2-based chemical disinfectants.
RESUMEN
The aim of this prospective, two center study was to investigate the dynamics of the microbial changes in relation to the development of ulcerative oral mucositis in autologous SCT (autoSCT) recipients. Fifty-one patients were diagnosed with multiple myeloma and treated with high-dose melphalan followed by autoSCT. They were evaluated before, three times weekly during hospitalization, and three months after autoSCT. At each time point an oral rinse was collected and the presence or absence of ulcerative oral mucositis (UOM) was scored (WHO scale). Oral microbiome was determined by using 16S rRNA amplicon sequencing and fungal load by qPCR. Twenty patients (39%) developed UOM. The oral microbiome changed significantly after autoSCT and returned to pre-autoSCT composition after three months. However, changes in microbial diversity and similarity were more pronounced and rapid in patients who developed UOM compared to patients who did not. Already before autoSCT, different taxa discriminated between the 2 groups, suggesting microbially-driven risk factors. Samples with high fungal load (>0.1%) had a significantly different microbial profile from samples without fungi. In conclusion, autoSCT induced significant and reversible changes in the oral microbiome, while patients who did not develop ulcerative oral mucositis had a more resilient microbial ecosystem.