RESUMEN
Clostridioides difficile infection (CDI) is the leading cause of nosocomial diarrhea and pseudomembranous colitis in the USA. In addition to these symptoms, patients with CDI can develop severe inflammation and tissue damage, resulting in life-threatening toxic megacolon. CDI is mediated by two large homologous protein toxins, TcdA and TcdB, that bind and hijack receptors to enter host cells where they use glucosyltransferase (GT) enzymes to inactivate Rho family GTPases. GT-dependent intoxication elicits cytopathic changes, cytokine production, and apoptosis. At higher concentrations TcdB induces GT-independent necrosis in cells and tissue by stimulating production of reactive oxygen species via recruitment of the NADPH oxidase complex. Although GT-independent necrosis has been observed in vitro, the relevance of this mechanism during CDI has remained an outstanding question in the field. In this study we generated novel C. difficile toxin mutants in the hypervirulent BI/NAP1/PCR-ribotype 027 R20291 strain to test the hypothesis that GT-independent epithelial damage occurs during CDI. Using the mouse model of CDI, we observed that epithelial damage occurs through a GT-independent process that does not involve immune cell influx. The GT-activity of either toxin was sufficient to cause severe edema and inflammation, yet GT activity of both toxins was necessary to produce severe watery diarrhea. These results demonstrate that both TcdA and TcdB contribute to disease pathogenesis when present. Further, while inactivating GT activity of C. difficile toxins may suppress diarrhea and deleterious GT-dependent immune responses, the potential of severe GT-independent epithelial damage merits consideration when developing toxin-based therapeutics against CDI.
Asunto(s)
Toxinas Bacterianas , Clostridioides difficile , Infecciones por Clostridium , Animales , Anticuerpos Antibacterianos , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Infecciones por Clostridium/patología , Diarrea , Enterotoxinas/metabolismo , Enterotoxinas/toxicidad , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Humanos , Inflamación , Ratones , NecrosisRESUMEN
Enzymatic inactivation of Rho-family GTPases by the glucosyltransferase domain of Clostridioides difficile Toxin B (TcdB) gives rise to various pathogenic effects in cells that are classically thought to be responsible for the disease symptoms associated with C. difficile infection (CDI). Recent in vitro studies have shown that TcdB can, under certain circumstances, induce cellular toxicities that are independent of glucosyltransferase (GT) activity, calling into question the precise role of GT activity. Here, to establish the importance of GT activity in CDI disease pathogenesis, we generated the first described mutant strain of C. difficile producing glucosyltransferase-defective (GT-defective) toxin. Using allelic exchange (AE) technology, we first deleted tcdA in C. difficile 630Δerm and subsequently introduced a deactivating D270N substitution in the GT domain of TcdB. To examine the role of GT activity in vivo, we tested each strain in two different animal models of CDI pathogenesis. In the non-lethal murine model of infection, the GT-defective mutant induced minimal pathology in host tissues as compared to the profound caecal inflammation seen in the wild-type and 630ΔermΔtcdA (ΔtcdA) strains. In the more sensitive hamster model of CDI, whereas hamsters in the wild-type or ΔtcdA groups succumbed to fulminant infection within 4 days, all hamsters infected with the GT-defective mutant survived the 10-day infection period without primary symptoms of CDI or evidence of caecal inflammation. These data demonstrate that GT activity is indispensable for disease pathogenesis and reaffirm its central role in disease and its importance as a therapeutic target for small-molecule inhibition.
Asunto(s)
Proteínas Bacterianas , Toxinas Bacterianas , Clostridioides difficile , Enterocolitis Seudomembranosa , Glucosiltransferasas , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Clostridioides difficile/enzimología , Clostridioides difficile/genética , Clostridioides difficile/patogenicidad , Cricetinae , Modelos Animales de Enfermedad , Enterocolitis Seudomembranosa/enzimología , Enterocolitis Seudomembranosa/genética , Enterocolitis Seudomembranosa/patología , Femenino , Eliminación de Gen , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Masculino , RatonesRESUMEN
Mesenchymal stem cells (MSCs) and photobiomodulation (PBM) both offer significant therapeutic potential in regenerative medicine. MSCs have the ability to self-renew and differentiate; giving rise to multiple cellular and tissue lineages that are utilised in repair and regeneration of damaged tissues. PBM utilises light energy delivered at a range of wavelengths to promote wound healing. The positive effects of light on MSC proliferation are well documented; and recently, several studies have determined the outcomes of PBM on mineralised tissue differentiation in MSC populations. As PBM effects are biphasic, it is important to understand the underlying cellular regulatory mechanisms, as well as, provide accurate details of the irradiation conditions, to optimise and standardise outcomes. This review article focuses on the use of red, near-infra-red (R/NIR) and blue wavelengths to promote the mineralisation potential of MSCs; and also reports on the possible molecular mechanisms which underpin transduction of these effects. A variety of potential photon absorbers have been identified which are reported to mediate the signalling mechanisms, including respiratory chain enzymes, flavins, and cryptochromes. Studies report that R/NIR and blue light stimulate MSC differentiation by enhancing respiratory chain activity and increasing reactive oxygen species levels; however, currently, there are considerable variations between irradiation parameters reported. We conclude that due to its non-invasive properties, PBM may, following optimisation, provide an efficient therapeutic approach to clinically support MSC-mediated hard tissue repair. However, to optimise application, further studies are required to identify appropriate light delivery parameters, as well as elucidate the photo-signalling mechanisms involved.
Asunto(s)
Terapia por Luz de Baja Intensidad , Células Madre Mesenquimatosas/metabolismo , Humanos , Rayos Infrarrojos , Células Madre Mesenquimatosas/patologíaRESUMEN
Clostridioides difficile R20291 is the most studied PCR-Ribotype 027 isolate. The two predominant lineages of this hypervirulent strain, however, exhibit substantive phenotypic differences and possess genomes that differ by a small number of nucleotide changes. It is important that the source of R20291 is taken into account in research outcomes.
Asunto(s)
Clostridioides/genética , Infecciones por Clostridium/microbiología , Polimorfismo de Nucleótido Simple , Clostridioides/clasificación , Clostridioides/aislamiento & purificación , Clostridioides difficile/clasificación , Clostridioides difficile/genética , Clostridioides difficile/aislamiento & purificación , Genoma Bacteriano , Humanos , FilogeniaRESUMEN
Hydrogels constructed from naturally derived polymers provide an aqueous environment that encourages cell growth, however, mechanical properties are poor and degradation can be difficult to predict. Whilst, synthetic hydrogels exhibit some improved mechanical properties, these materials lack biochemical cues for cells growing and have limited biodegradation. To produce hydrogels that support 3D cell cultures to form tissue mimics, materials must exhibit appropriate biological and mechanical properties. In this study, novel organic-inorganic hybrid hydrogels based on chitosan and silica were prepared using the sol-gel technique. The chemical, physical and biological properties of the hydrogels were assessed. Statistical analysis was performed using One-Way ANOVAs and independent-sample t-tests. Fourier transform infrared spectroscopy showed characteristic absorption bands including amide II, Si-O and Si-O-Si confirming formation of hybrid networks. Oscillatory rheometry was used to characterise the sol to gel transition and viscoelastic behaviour of hydrogels. Furthermore, in vitro degradation revealed both chitosan and silica were released over 21 days. The hydrogels exhibited high loading efficiency as total protein loading was released in a week. There were significant differences between TC2G and C2G at all-time points (p < 0.05). The viability of osteoblasts seeded on, and encapsulated within, the hydrogels was >70% over 168 h culture and antimicrobial activity was demonstrated against Pseudomonas aeruginosa and Enterococcus faecalis. The hydrogels developed here offer alternatives for biopolymer hydrogels for biomedical use, including for application in drug/cell delivery and for bone tissue engineering.
Asunto(s)
Encapsulación Celular/métodos , Quitosano/química , Sistemas de Liberación de Medicamentos/métodos , Hidrogeles/química , Dióxido de Silicio/química , Antibacterianos/química , Antibacterianos/farmacología , Técnicas de Cultivo Tridimensional de Células/métodos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Enterococcus faecalis/efectos de los fármacos , Humanos , Hidrogeles/farmacología , Microscopía Electrónica de Rastreo , Transición de Fase , Espectroscopía de Protones por Resonancia Magnética , Pseudomonas aeruginosa/efectos de los fármacos , Espectroscopía Infrarroja por Transformada de Fourier , Ingeniería de Tejidos/métodosRESUMEN
Objectives: To establish the role of specific, non-synonymous SNPs in the RNA polymerase ß subunit (rpoB) gene in reducing the susceptibility of Clostridium difficile to fidaxomicin and to explore the potential in vivo significance of rpoB mutant strains. Methods: Allelic exchange was used to introduce three different SNPs into the rpoB gene of an erythromycin-resistant derivative (CRG20291) of C. difficile R20291. The genome sequences of the created mutants were determined and each mutant analysed with respect to growth and sporulation rates, toxin A/B production and cytotoxicity against Vero cells, and in competition assays. Their comparative virulence and colonization ability was also assessed in a hamster infection model. Results: The MIC of fidaxomicin displayed by three mutants CRG20291-TA, CRG20291-TG and CRG20291-GT was substantially increased (>32, 8 and 2 mg/L, respectively) relative to that of the parent strain (0.25 mg/L). Genome sequencing established that the intended mutagenic substitutions in rpoB were the only changes present. Relative to CRG20291, all mutants had attenuated growth, were outcompeted by the parental strain, had lower sporulation and toxin A/B production capacities, and displayed diminished cytotoxicity. In a hamster model, virulence of all three mutants was significantly reduced compared with the progenitor strain, whereas the degree of caecum colonization was unaltered. Conclusions: Our study demonstrates that particular SNPs in rpoB lead to reduced fidaxomicin susceptibility. These mutations were associated with a fitness cost in vitro and reduced virulence in vivo.
Asunto(s)
Antibacterianos/farmacología , Clostridioides difficile/genética , ARN Polimerasas Dirigidas por ADN/genética , Farmacorresistencia Bacteriana , Fidaxomicina/farmacología , Aptitud Genética , Mutación Missense , Animales , Toxinas Bacterianas/metabolismo , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Clostridioides difficile/efectos de los fármacos , Clostridioides difficile/crecimiento & desarrollo , Infecciones por Clostridium/microbiología , Infecciones por Clostridium/patología , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Femenino , Mesocricetus , Pruebas de Sensibilidad Microbiana , Esporas Bacterianas/crecimiento & desarrollo , Células Vero , Virulencia , Secuenciación Completa del GenomaRESUMEN
Teixobactin, a recently discovered depsipeptide that binds to bacterial lipidâ II and lipidâ III, provides a promising molecular scaffold for the design of new antimicrobials. Herein, we describe the synthesis and antimicrobial evaluation of systematically modified teixobactin analogues. The replacement of the Ile11 residue with aliphatic isosteres, the modification of the guanidino group at residueâ 10 and the introduction of a rigidifying residue, that is, dehydroamino acid, into the macrocyclic ring generated useful structure-activity information. Extensive antimicrobial susceptibility assessment against a panel of clinically relevant Staphylococcus aureus and Propionibacterium acnes strains led to the identification of the new lead compound, [Arg(Me)10 ,Nle11 ]teixobactin, with an excellent bactericidal activity (minimum inhibitory concentration (MIC)=2-4â µg mL-1 ). Significantly, the antimicrobial activity of several of the teixobactin analogues against the pathogenic Gram-negative Pseudomonas aeruginosa was "restored" when combined with the sub-MIC concentration of the outer membrane-disruptive antibiotic colistin. The antimicrobial effectiveness of a [Tfn10 ,Nle11 ]teixobactin (32â µg mL-1 )-colistin (2â µg mL-1 ; 0.5×MIC) combination against P.â aeruginosa PAO1 reveals, for the first time, an alternative therapeutic option in the treatment of Gram-negative infections.
Asunto(s)
Antibacterianos/farmacología , Depsipéptidos/química , Diseño de Fármacos , Propionibacterium acnes/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/síntesis química , Antibacterianos/química , Depsipéptidos/síntesis química , Depsipéptidos/farmacología , Pruebas de Sensibilidad Microbiana , Propionibacterium acnes/crecimiento & desarrollo , Pseudomonas aeruginosa/crecimiento & desarrollo , Staphylococcus aureus/crecimiento & desarrollo , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Clostridium difficile (Peptoclostridium difficile) is a common health care-associated infection with a disproportionately high incidence in elderly patients. Disease symptoms range from mild diarrhea to life-threatening pseudomembranous colitis. Around 20% of patients may suffer recurrent disease, which often requires rehospitalization of patients. C. difficile was isolated from stool samples from a patient with two recurrent C. difficile infections. PCR ribotyping, whole-genome sequencing, and phenotypic assays were used to characterize these isolates. Genotypic and phenotypic screening of C. difficile isolates revealed multiple PCR ribotypes present and the emergence of rifamycin resistance during the infection cycle. Understanding both the clinical and bacterial factors that contribute to the course of recurrent infection could inform strategies to reduce recurrence. (This study has been registered at ClinicalTrials.gov under registration no. NCT01670149.).
Asunto(s)
Antibacterianos/farmacología , Clostridioides difficile/clasificación , Clostridioides difficile/efectos de los fármacos , Infecciones por Clostridium/microbiología , Coinfección/microbiología , Farmacorresistencia Bacteriana , Rifamicinas/farmacología , Anciano de 80 o más Años , Técnicas de Tipificación Bacteriana , Clostridioides difficile/genética , Clostridioides difficile/aislamiento & purificación , Heces/microbiología , Humanos , Masculino , Recurrencia , Ribotipificación , Análisis de Secuencia de ADNRESUMEN
Clostridium difficile infection is the leading cause of healthcare-associated diarrhoea in Europe and North America. During infection, C. difficile produces two key virulence determinants, toxin A and toxin B. Experiments with purified toxins have indicated that toxin A alone is able to evoke the symptoms of C. difficile infection, but toxin B is unable to do so unless it is mixed with toxin A or there is prior damage to the gut mucosa. However, a recent study indicated that toxin B is essential for C. difficile virulence and that a strain producing toxin A alone was avirulent. This creates a paradox over the individual importance of toxin A and toxin B. Here we show that isogenic mutants of C. difficile producing either toxin A or toxin B alone can cause fulminant disease in the hamster model of infection. By using a gene knockout system to inactivate the toxin genes permanently, we found that C. difficile producing either one or both toxins showed cytotoxic activity in vitro that translated directly into virulence in vivo. Furthermore, by constructing the first ever double-mutant strain of C. difficile, in which both toxin genes were inactivated, we were able to completely attenuate virulence. Our findings re-establish the importance of both toxin A and toxin B and highlight the need to continue to consider both toxins in the development of diagnostic tests and effective countermeasures against C. difficile.
Asunto(s)
Toxinas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Clostridioides difficile/patogenicidad , Infecciones por Clostridium/microbiología , Enterotoxinas/metabolismo , Animales , Anticuerpos Neutralizantes , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/genética , Chlorocebus aethiops , Clostridioides difficile/clasificación , Clostridioides difficile/genética , Cricetinae , Modelos Animales de Enfermedad , Enterotoxinas/antagonistas & inhibidores , Enterotoxinas/genética , Eliminación de Gen , Células HT29 , Humanos , Pruebas de Neutralización , Células Vero , Virulencia/genéticaRESUMEN
CsrA/RsmA homologs are an extensive family of ribonucleic acid (RNA)-binding proteins that function as global post-transcriptional regulators controlling important cellular processes such as secondary metabolism, motility, biofilm formation and the production and secretion of virulence factors in diverse bacterial species. While direct messenger RNA binding by CsrA/RsmA has been studied in detail for some genes, it is anticipated that there are numerous additional, as yet undiscovered, direct targets that mediate its global regulation. To assist in the discovery of these targets, we propose a sequence-based approach to predict genes directly regulated by these regulators. In this work, we develop a computer code (CSRA_TARGET) implementing this approach, which leads to predictions for several novel targets in Escherichia coli and Pseudomonas aeruginosa. The predicted targets in other bacteria, specifically Salmonella enterica serovar Typhimurium, Pectobacterium carotovorum and Legionella pneumophila, also include global regulators that control virulence in these pathogens, unraveling intricate indirect regulatory roles for CsrA/RsmA. We have experimentally validated four predicted RsmA targets in P. aeruginosa. The sequence-based approach developed in this work can thus lead to several testable predictions for direct targets of CsrA homologs, thereby complementing and accelerating efforts to unravel global regulation by this important family of proteins.
Asunto(s)
Algoritmos , Pseudomonas aeruginosa/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/metabolismo , Análisis de Secuencia de ARN/métodos , Sitios de Unión , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Pseudomonas aeruginosa/metabolismo , ARN Mensajero/químicaRESUMEN
Clostridium difficile infection is one of the leading causes of healthcare associated diarrhoea in the developed world. Although the contribution of C. difficile toxins to disease pathogenesis is now well understood, many facets of host-pathogen interactions between the human intestinal epithelia and the C. difficile bacterium that may contribute to asymptomatic carriage and/or clinical disease remain less clear. Herein, we tested the hypothesis that C. difficile strains mediate intestinal epithelial cell (IEC) antimicrobial immunity via toxin dependent and independent means and that the 'anaerobic' environment has a significant impact on bacterial-IEC interactions. Crosstalk between three C. difficile PCR ribotypes (RT) [RT027 (strain R20291), RT012 (strain 630) and RT017 (strains M68 and CF5)] and IEC cell-lines were investigated. All RTs showed significant engagement with human Toll-like receptors (TLR)-5, TLR2-CD14 and TLR2/6 as measured by IL-8 release from TLR-transfected HEK cells. Co-culture studies indicated minimal impact of R20291 and 630 TcdA and TcdB on bacterial adherence to Caco-2 cells. An apical anaerobic environment had a major effect on C. difficile-T84 crosstalk as significantly greater cytokine immunity and trans-epithelial electrical resistance (TEER) dysfunction was recorded when co-cultures were performed in an Ussing chamber system compared to standard 5% CO2 conditions. Overall, this study suggests that anaerobic C. difficile engagement with human IECs is a complex interplay that involves bacterial and toxin-mediated cellular events.
Asunto(s)
Clostridioides difficile/fisiología , Mucosa Intestinal/microbiología , Adhesión Bacteriana , Toxinas Bacterianas , Células CACO-2 , Clostridioides difficile/inmunología , Citocinas/biosíntesis , Cámaras de Difusión de Cultivos , Enterotoxinas , Células HEK293 , Humanos , Inmunidad Innata , Mucosa Intestinal/inmunología , Modelos BiológicosRESUMEN
Point mutations conferring resistance to fluoroquinolones were introduced in the gyr genes of the reference strain Clostridium difficile 630. Only mutants with the substitution Thr-82âIle in GyrA, which characterizes the hypervirulent epidemic clone III/027/NAP1, were resistant to all fluoroquinolones tested. The absence of a fitness cost in vitro for the most frequent mutations detected in resistant clinical isolates suggests that resistance will be maintained even in the absence of antibiotic pressure.
Asunto(s)
Clostridioides difficile/efectos de los fármacos , Clostridioides difficile/genética , Farmacorresistencia Bacteriana/genética , Fluoroquinolonas/farmacología , Antibacterianos/farmacología , Girasa de ADN/genética , Mutación Puntual/genéticaRESUMEN
Clostridium difficile infection is the main cause of healthcare-acquired diarrhea in the developed world. In addition to the main virulence factors toxin A and B, epidemic, PCR Ribotype 027 strains, such as R20291, produce a third toxin, CDT. To develop effective medical countermeasures, it is important to understand the importance of each toxin. Accordingly, we created all possible combinations of isogenic toxin mutants of R20291 and assessed their virulence. We demonstrated that either toxin A or toxin B alone can cause fulminant disease in the hamster infection model and present tantalizing data that C. difficile toxin may also contribute to virulence.
Asunto(s)
ADP Ribosa Transferasas/fisiología , Proteínas Bacterianas/fisiología , Clostridioides difficile/patogenicidad , Infecciones por Clostridium/microbiología , Enterotoxinas/fisiología , ADP Ribosa Transferasas/genética , Animales , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Muerte Celular , Clostridioides difficile/genética , Infecciones por Clostridium/patología , Cricetinae , Enterotoxinas/genética , Femenino , Células HT29 , Humanos , Mesocricetus , Virulencia/genética , Virulencia/fisiologíaRESUMEN
Effective isolation of Clostridium difficile from stool samples is important in the research setting, especially where low numbers of spores/vegetative cells may be present within a sample. In this study, three protocols for stool culture were investigated to find a sensitive, cost effective and timely method of C. difficile isolation. For the initial enrichment step, the effectiveness of two different rich media, cycloserine-cefoxitin fructose broth (CCFB) and cycloserine-cefoxitin mannitol broth with taurocholate and lysozyme (CCMB-TAL) were compared. For the comparison of four different, selective solid media; Cycloserine-cefoxitin fructose agar (CCFA), Cycloserine-cefoxitin egg yolk agar (CCEY), ChromID C. difficile and tryptone soy agar (TSA) with 5% sheep's blood with and without preceding broth enrichment were used. As a means to enable differentiation between C. difficile and other fecal flora, the effectiveness of the inclusion of a pH indictor (1% Neutral Red), was also evaluated. The data derived indicated that CCFB is more sensitive than CCMB-TAL, however, the latter had an improved recovery rate. A broth enrichment step had a reduced sensitivity over direct plating. ChromID C. difficile showed the best recovery rate whereas CCEY egg yolk agar was the most sensitive of the four. The addition of 1% Neutral Red did not show sufficient colour change when added to CCEY egg yolk agar to be used as a differential medium. For a low cost, timely and sensitive method of isolating C. difficile from stool samples we recommend direct plating onto CCEY egg yolk agar after heat shock.
Asunto(s)
Técnicas Bacteriológicas/métodos , Clostridioides difficile/aislamiento & purificación , Heces/microbiología , Animales , Técnicas Bacteriológicas/economía , Clostridioides difficile/crecimiento & desarrollo , Análisis Costo-Beneficio , Medios de Cultivo/química , Mesocricetus , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
INTRODUCTION: Biological characterization of root canal sealers is important as it assesses the ability of the root canal sealer to exert antimicrobial properties thus avoiding treatment failures caused by microbial challenge and also assess the cytotoxic effect on the periapical tissues. Assessment of the biological testing of root canal sealers necessitates the sterilisation of the materials prior to evaluation. This study aims to analyse the influence of various sterilisation techniques conducted prior to biological testing on the microstructure and surface properties of endodontic sealers. Assessment of the initial microbial contamination on the material was also undertaken. METHODS: Four commercial sealers were investigated. The sealers were either prepared in a laminar flow cabinet or on a laboratory bench top under ambient conditions. Each group was further divided into 5 groups (n = 3) based on the sterilization technique:1) ethanol-10 mins, 2) ultraviolet-1 h, 3) ethanol-10 mins + ultraviolet-1 h, 4) autoclave, and 5) no sterilisation (control). Microbial levels in the materials were assessed by plate streaking technique. The materials were characterized by scanning electron microscopy and energy dispersive spectroscopy, and Fourier transform infrared spectroscopy, before and after sterilisation, to assess any changes in microstructure and chemical composition. RESULTS: All the materials did not exhibit contamination when prepared in laminar flow chamber in sterile conditions compared with sealers prepared on the bench top. Three of the commercial materials showed changes in microstructure while one (TotalFill) was not affected by the sterilisation. AH Plus and BioRoot RCS exhibited alterations in water and alcohol peaks in FT-IR while the single syringe sealers (TotalFill and BioRoot Flow) showed no changes. CONCLUSIONS: Sterilisation methods cause physical and chemical alterations to sealers. Material preparation should be performed in a laminar flow cabinet and a test for sterility should be performed prior to any biological testing being undertaken. If the materials are not sterile, assessment of the effects of the sterilization methods is recommended.
Asunto(s)
Materiales de Obturación del Conducto Radicular , Resinas Epoxi/química , Gutapercha/química , Espectroscopía Infrarroja por Transformada de Fourier , Cavidad Pulpar , Compuestos de Calcio/química , Ensayo de Materiales , Etanol , Silicatos/químicaRESUMEN
INTRODUCTION: Peri-implantitis is an inflammatory process around dental implants that is characterised by bone loss that may jeopardize the long-term survival of osseo integrated dental implants. The aim of this study was to create a surface coating on titanium abutments that possesses cellular adhesion and anti-microbial properties as a post-implant placement strategy for patients at risk of peri-implantitis. MATERIALS AND METHODSMETHODS: Titanium alloy Grade V stubs were coated with gold particles and then subjected to ceramic conversion treatment (CCT) at 620 °C for 3, 8 and 80 h. The surface characteristics and chemistry were assessed using scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), and X-ray diffraction (XRD) analysis. The leaching profile was investigated by inductively coupled plasma mass spectroscopy (ICP-MS) for all groups after 7, 14 and 28 days in contact with distilled water. A scratch test was conducted to assess the adhesion of the gold coating to the underlying titanium discs. Two bacterial species (Staphylococcus aureus (SA) & Fusobacterium nucleatum (FN)) were used to assess the antibacterial behaviour of the coated discs using a direct attachment assay test. The potential changes in surface chemistry by the bacterial species were investigated by grazing angle XRD. RESULTS: The gold pre-coated titanium discs exhibited good stability of the coating especially after immersion in distilled water and after bacterial colonisation as evident by XRD analysis. Good surface adhesion of the coating was demonstrated for gold treated discs after scratch test analysis, especially titanium, following a 3-hour (3 H) ceramic conversion treatment. All coated discs exhibited significantly improved antimicrobial properties against both tested bacterial species compared to untreated titanium discs. CONCLUSIONS: Ceramic conversion treated titanium with a pre-deposited gold layer showed improved antimicrobial properties against both SA and FN species than untreated Ti-C discs. Scratch test analysis showed good adherence properties of the coated discs the oxide layer formed is firmly adherent to the underlying titanium substrate, suggesting that this approach may have clinical efficacy for coating implant abutments.
Asunto(s)
Cerámica , Pilares Dentales , Fusobacterium nucleatum , Oro , Microscopía Electrónica de Rastreo , Staphylococcus aureus , Propiedades de Superficie , Titanio , Difracción de Rayos X , Titanio/química , Oro/química , Fusobacterium nucleatum/efectos de los fármacos , Cerámica/química , Staphylococcus aureus/efectos de los fármacos , Ensayo de Materiales , Espectrometría por Rayos X , Periimplantitis , Implantes Dentales/microbiología , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/farmacología , Antiinfecciosos/farmacología , Antiinfecciosos/química , Antibacterianos/farmacología , Antibacterianos/químicaRESUMEN
Bacteria within biofilms are protected from multiple stresses, including immune responses and antimicrobial agents. The biofilm-forming ability of bacterial pathogens has been associated with increased antibiotic resistance and chronic recurrent infections. Although biofilms have been well studied for several gut pathogens, little is known about biofilm formation by anaerobic gut species. The obligate anaerobe Clostridium difficile causes C. difficile infection (CDI), a major health care-associated problem primarily due to the high incidence of recurring infections. C. difficile colonizes the gut when the normal intestinal microflora is disrupted by antimicrobial agents; however, the factors or processes involved in gut colonization during infection remain unclear. We demonstrate that clinical C. difficile strains, i.e., strain 630 and the hypervirulent strain R20291, form structured biofilms in vitro, with R20291 accumulating substantially more biofilm. Microscopic and biochemical analyses show multiple layers of bacteria encased in a biofilm matrix containing proteins, DNA, and polysaccharide. Employing isogenic mutants, we show that virulence-associated proteins, Cwp84, flagella, and a putative quorum-sensing regulator, LuxS, are all required for maximal biofilm formation by C. difficile. Interestingly, a mutant in Spo0A, a transcription factor that controls spore formation, was defective for biofilm formation, indicating a possible link between sporulation and biofilm formation. Furthermore, we demonstrate that bacteria in clostridial biofilms are more resistant to high concentrations of vancomycin, a drug commonly used for treatment of CDI. Our data suggest that biofilm formation by C. difficile is a complex multifactorial process and may be a crucial mechanism for clostridial persistence in the host.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Clostridioides difficile/fisiología , Antibacterianos/farmacología , Adhesión Bacteriana , Biopelículas/efectos de los fármacos , Clostridioides difficile/clasificación , Clostridioides difficile/efectos de los fármacos , Flagelos , Proteínas de la Membrana/fisiología , Pruebas de Sensibilidad Microbiana , Percepción de Quorum , Esporas Bacterianas , Factores de Tiempo , Vancomicina/farmacología , Resistencia a la VancomicinaRESUMEN
Chronic wounds are a drain on global health services and remain a major area of unmet clinical need. Chronic wounds are characterised by a stable and stubborn bacterial biofilm which hinders innate immune response and delays or prevents wound healing. Bioactive glass (BG) fibres offer a promising novel treatment for chronic wounds by targeting the wound-associated biofilm. In this study, the antimicrobial properties of silver-doped BG fibres were tested against Pseudomonas aeruginosa biofilms, which are commonly found in chronic wound infections. Results showed that BG fibres doped with silver resulted in a 5log10 reduction in biofilm formation whereas silver-free fibres only reduced formation by log10, therefore silver-doped fibres possess stronger antimicrobial effects. Moreover, there appeared to be a synergistic effect between the fibres and the silver as the application of the silver-doped fibres placed directly in contact with the forming biofilm resulted in a higher reduction in biofilm formation compared to treatments either: using the dissolution ions, using BG powder, or when the fibres were placed in an insert above the biofilm, inhibiting physical contact, instead. This suggests that the physical properties of the fibres, as well as silver, influence biofilm formation. Finally, results demonstrated that silver chloride, which is not antimicrobial, forms and the concentrations of antimicrobial silver species, namely silver ions and nanoparticles, reduce over time when fibres are soaked in cell culture media, which partially explains why the silver-doped dissolution ions contained lower antimicrobial activity compared to the fibres. As silver chloride is more likely to form with increased temperature and time, the antimicrobial activity of silver-containing dissolution ions is highly dependent on the length of ageing and storage conditions. Many studies investigate the antimicrobial and cytotoxic properties of biomaterials through their dissolution products. However, instability of antimicrobial silver species due to silver chloride formation and its effect on antimicrobial properties of silver-based biomaterials has not been reported before and could influence past and future dissolution-based assays as results showed that the antimicrobial activity of silver-based dissolution ions can vary greatly depending on post processing steps and can therefore produce misleading data.
RESUMEN
Quorum sensing molecules (QSMs) in the oral cavity regulate biofilm formation, the acquisition of iron, stress responses, and the expression of virulence factors. However, knowledge of the direct QSM-host interactions in the oral environment is limited, although their understanding could provide greater insight into the cross-kingdom communication occurring during oral disease development. This review aims to explore the literature on oral QSM-host interactions and to highlight areas of advancement in this field. The studies included in this review encompass an array of cell types and oral QSMs, with particular emphasis on immune cells and their relationship to periodontal diseases. It can be inferred from the current literature that QSMs are utilised by host cells to detect bacterial presence and, in the majority of cases, elicit an immune response towards the environmental QSMs. This may provide a base to target QSMs as a novel treatment of oral diseases. However, N-acyl homoserine lactone (AHL) detection methods remain an area for development, through which a greater understanding of the influence of oral QSMs on host cells could be achieved.
RESUMEN
The gut microbiota is a reservoir for antimicrobial resistance genes (ARGs). With current sequencing methods, it is difficult to assign ARGs to their microbial hosts, particularly if these ARGs are located on plasmids. Metagenomic chromosome conformation capture approaches (meta3C and Hi-C) have recently been developed to link bacterial genes to phylogenetic markers, thus potentially allowing the assignment of ARGs to their hosts on a microbiome-wide scale. Here, we generated a meta3C dataset of a human stool sample and used previously published meta3C and Hi-C datasets to investigate bacterial hosts of ARGs in the human gut microbiome. Sequence reads mapping to repetitive elements were found to cause problematic noise in, and may importantly skew interpretation of, meta3C and Hi-C data. We provide a strategy to improve the signal-to-noise ratio by discarding reads that map to insertion sequence elements and to the end of contigs. We also show the importance of using spike-in controls to quantify whether the cross-linking step in meta3C and Hi-C protocols has been successful. After filtering to remove artefactual links, 87 ARGs were assigned to their bacterial hosts across all datasets, including 27 ARGs in the meta3C dataset we generated. We show that commensal gut bacteria are an important reservoir for ARGs, with genes coding for aminoglycoside and tetracycline resistance being widespread in anaerobic commensals of the human gut.