RESUMO
Among the unfavorable conditions bacteria encounter within the host is restricted access to essential trace metals such as iron. To overcome iron deficiency, bacteria deploy multiple strategies to scavenge iron from host tissues, with abundant examples of iron acquisition systems being implicated in bacterial pathogenesis. Yet the mechanisms utilized by the major nosocomial pathogen Enterococcus faecalis to maintain intracellular iron balance are poorly understood. In this study, we conducted a systematic investigation to identify and characterize the iron acquisition mechanisms of E. faecalis and to determine their contribution to virulence. Bioinformatic analysis and literature surveys revealed that E. faecalis possesses three conserved iron uptake systems. Through transcriptomics, we discovered two novel ABC-type transporters that mediate iron uptake. While inactivation of a single transporter had minimal impact on the ability of E. faecalis to maintain iron homeostasis, inactivation of all five systems (Δ5Fe strain) disrupted intracellular iron homeostasis and considerably impaired cell growth under iron deficiency. Virulence of the Δ5Fe strain was generally impaired in different animal models but showed niche-specific variations in mouse models, leading us to suspect that heme can serve as an iron source to E. faecalis during mammalian infections. Indeed, heme supplementation restored growth of Δ5Fe under iron depletion and virulence in an invertebrate infection model. This study revealed that the collective contribution of five iron transporters promotes E. faecalis virulence and that the ability to acquire and utilize heme as an iron source is critical to the systemic dissemination of E. faecalis.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Bactérias , Transporte Biológico , Enterococcus faecalis , Ferro , Enterococcus faecalis/metabolismo , Enterococcus faecalis/patogenicidade , Virulência , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Ferro/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Bactérias/metabolismo , Heme/metabolismo , Infecções por Bactérias Gram-Positivas/metabolismo , Infecções por Bactérias Gram-Positivas/microbiologia , HumanosRESUMO
Second messenger nucleotides are produced by bacteria in response to environmental stimuli and play a major role in the regulation of processes associated with bacterial fitness, including but not limited to osmoregulation, envelope homeostasis, central metabolism, and biofilm formation. In this study, we uncovered the biological significance of c-di-AMP in the opportunistic pathogen Enterococcus faecalis by isolating and characterizing strains lacking genes responsible for c-di-AMP synthesis (cdaA) and degradation (dhhP and gdpP). Using complementary approaches, we demonstrated that either complete loss of c-di-AMP (ΔcdaA strain) or c-di-AMP accumulation (ΔdhhP, ΔgdpP, and ΔdhhP ΔgdpP strains) drastically impaired general cell fitness and virulence of E. faecalis. In particular, the ΔcdaA strain was highly sensitive to envelope-targeting antibiotics, was unable to multiply and quickly lost viability in human serum or urine ex vivo, and was virtually avirulent in an invertebrate (Galleria mellonella) and in two catheter-associated mouse infection models that recapitulate key aspects of enterococcal infections in humans. In addition to evidence linking these phenotypes to altered activity of metabolite and peptide transporters and inability to maintain osmobalance, we found that the attenuated virulence of the ΔcdaA strain also could be attributed to a defect in Ebp pilus production and activity that severely impaired biofilm formation under both in vitro and in vivo conditions. Collectively, these results demonstrate that c-di-AMP signaling is essential for E. faecalis pathogenesis and a desirable target for drug development.
Assuntos
Fosfatos de Dinucleosídeos/fisiologia , Enterococcus faecalis/patogenicidade , Animais , Biofilmes , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/crescimento & desenvolvimento , Fímbrias Bacterianas/fisiologia , Regulação Bacteriana da Expressão Gênica , Infecções por Bactérias Gram-Positivas/etiologia , Humanos , VirulênciaRESUMO
Infections are the leading cause of failure of osteogenic material implantation. Antibiotic treatment, treatment with bone cement, or collagen sponge placement can result in drug resistance and difficulties in operation. To address this, gellan gum (GG) was selected in this study and prepared as an injectable hydrogel containing chlorhexidine (CHX) and nanohydroxyapatite (nHA) that overcomes these intractable problems. Scanning electron microscopy and micro-computed tomography revealed a three-dimensional polymeric network of the hydrogel. The hydrogel had excellent biocompatibility, as detected by cell counting kit-8 and Live/Dead assay. Bone marrow mesenchymal stem cells could be encapsulated into the network, showing that the structure was suitable for cell growth. Additionally, loading the hydrogel with nHA improved its mechanical, biodegradable, and osteogenic properties. Quantitative alkaline phosphatase and Alizarin Red S staining validated its osteogenic ability. Furthermore, antibacterial activity assessment showed that the hydrogel loaded with 50 µg/mL CHX inhibited Enterococcus faecalis in a concentration-dependent manner. Thus, we report an injectable GG-based hydrogel with superior antibacterial effect against E. faecalis and osteogenesis, which holds promise for treating infectious bone defects caused by refractory periradicular periodontitis.
Assuntos
Hidrogéis/química , Osteogênese/efeitos dos fármacos , Polissacarídeos Bacterianos/química , Engenharia Tecidual , Fosfatase Alcalina/química , Animais , Interface Osso-Implante/crescimento & desenvolvimento , Interface Osso-Implante/microbiologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Clorexidina/química , Clorexidina/farmacologia , Modelos Animais de Doenças , Durapatita/química , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/patogenicidade , Humanos , Hidrogéis/síntese química , Hidrogéis/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Polissacarídeos Bacterianos/síntese química , Polissacarídeos Bacterianos/farmacologia , Ratos , Alicerces Teciduais/química , Microtomografia por Raio-XRESUMO
Biogenesis of iron-sulfur (FeS) clusters in an essential process in living organisms due to the critical role of FeS cluster proteins in myriad cell functions. During biogenesis of FeS clusters, multi-protein complexes are used to drive the mobilization and protection of reactive sulfur and iron intermediates, regulate assembly of various FeS clusters on an ATPase-dependent, multi-protein scaffold, and target nascent clusters to their downstream protein targets. The evolutionarily ancient sulfur formation (Suf) pathway for FeS cluster assembly is found in bacteria and archaea. In Escherichia coli, the Suf pathway functions as an emergency pathway under conditions of iron limitation or oxidative stress. In other pathogenic bacteria, such as Mycobacterium tuberculosis and Enterococcus faecalis, the Suf pathway is the sole source for FeS clusters and therefore is a potential target for the development of novel antibacterial compounds. Here we summarize the considerable progress that has been made in characterizing the first step of mobilization and protection of reactive sulfur carried out by the SufS-SufE or SufS-SufU complex, FeS cluster assembly on SufBC2D scaffold complexes, and the downstream trafficking of nascent FeS clusters to A-type carrier (ATC) proteins. Cell Biology of Metals III edited by Roland Lill and Mick Petris.
Assuntos
Evolução Molecular , Proteínas Ferro-Enxofre/genética , Ferro/metabolismo , Enxofre/metabolismo , Adenosina Trifosfatases/genética , Enterococcus faecalis/genética , Enterococcus faecalis/patogenicidade , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas Ferro-Enxofre/metabolismo , Liases/genética , Redes e Vias Metabólicas/genética , Família Multigênica/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidadeRESUMO
Enterococcus faecalis infective endocarditis (EFIE) is a severe disease of increasing incidence. The objective was to analyze whether the outcome of patients with native valve EFIE (NVEFIE) treated with a short course of ampicillin plus ceftriaxone (4wAC) was similar to patients treated according to international guidelines (6wAC). Between January 2008 and June 2018, 1,978 consecutive patients with definite native valve IE were prospectively included in a national registry. Outcomes of patients with NVEFIE treated with 4wAC were compared to those of patients who received 6wAC. Three hundred and twenty-two patients (16.3%) had NVEFIE. One hundred and eighty-three (56.8%) received AC. Thirty-nine patients (21.3%) were treated with 4wAC for four weeks and 70 patients (38.3%) with 6wAC. There were no differences in age or comorbidity. Patients treated 6wAC presented a longer duration of symptoms before diagnosis (21 days, IQR 7-60 days vs. 7 days, IQR 1-22 days; p = 0.002). Six patients presented perivalvular abscess and all of these received 6wAC. Surgery was performed on 14 patients (35.9%) 4wAC and 34 patients (48.6%) 6wAC (p = 0.201). In-hospital mortality, one-year mortality and relapses among 4wAC and 6wAC patients were 10.3% vs. 11.4% (p = 0.851); 17.9% vs. 21.4% (p = 0.682) and 5.1% vs. 4.3% (p = 0.833), respectively. In conclusion, a four-week course of AC may be considered as an alternative regimen in NVEFIE, notably in patients with shorter duration of symptoms and those without perivalvular abscess. These results support the performance of a randomized clinical trial to evaluate the efficacy of this short regimen.
Assuntos
Ampicilina/uso terapêutico , Ceftriaxona/uso terapêutico , Endocardite Bacteriana/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/uso terapêutico , Estudos de Coortes , Quimioterapia Combinada , Endocardite/tratamento farmacológico , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/patogenicidade , Feminino , Gentamicinas/uso terapêutico , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Humanos , Masculino , Estudos Prospectivos , Fatores de TempoRESUMO
The bacterium-bacterium interaction between pathogenic and probiotic Enterococcus as well as the bacterium-host interaction between Enterococcus and intestinal epithelium has drawn increasing attentions, but the influence of those interactions on host pregnancy remains largely unexplored. In the present study, we evaluated the effects of probiotic E. faecalis Symbioflor 1 or/and pathogenic E. faecalis OG1RF on the miscarriage of pregnant mice. Using in vitro assays of competition and exclusion and displacement, antagonistic property of E. faecalis Symbioflor 1 against E. faecalis OG1RF was observed, and the former inhibited the translocation of the later in vivo. The rate of miscarriage induced by E. faecalis OG1RF challenge was significantly reduced by 28% with E. faecalis Symbioflor 1 intervention; and the tissue integrity of ileum, colon, uterus, and placenta and placental blood cell density in pregnant mice were drastically improved by such probiotic intervention. Compared with the controls, probiotic intervention significantly upregulated the level of IL-10 and TGF-ß, downregulated levels of IFN-γ, and increased progesterone level that reversed the trend of being Th1 predominance state reported for adverse pregnancy outcome at early pregnancy stage. In conclusion, E. faecalis Symbioflor 1 decreased the translocation of E. faecalis OG1RF, prevented pathogen-induced tissue damage, and changed Th1-Th2 homeostasis toward Th2 predominance during early pregnancy resulting in decreased miscarriage. KEY POINTS: â¢The mechanism of how probiotic E. faecalis Symbioflor 1 improves pregnancy of mice ⢠Influence of interactions of pathogenic and probiotic Enterococcus on host pregnancy ⢠E. faecalis Symbioflor 1 change Th1-Th2 homeostasis toward Th2 predominance.
Assuntos
Aborto Espontâneo/microbiologia , Aborto Espontâneo/prevenção & controle , Antibiose , Enterococcus faecalis/fisiologia , Probióticos/administração & dosagem , Células Th1/imunologia , Células Th2/imunologia , Animais , Translocação Bacteriana , Citocinas/imunologia , Enterococcus faecalis/patogenicidade , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Gravidez , Progesterona/sangue , Organismos Livres de Patógenos EspecíficosRESUMO
BACKGROUND: Recent metagenomic analyses have revealed dysbiosis of the gut microbiota of ulcerative colitis (UC) patients. However, the impacts of this dysbiosis are not fully understood, particularly at the strain level. RESULTS: We perform whole-genome shotgun sequencing of fecal DNA extracts from 13 healthy donors and 16 UC and 8 Crohn's disease (CD) patients. The microbiota of UC and CD patients is taxonomically and functionally divergent from that of healthy donors, with E. faecium being the most differentially abundant species between the two microbial communities. Transplantation of feces from UC or CD patients into Il10-/- mice promotes pathological inflammation and cytokine expression in the mouse colon, although distinct cytokine expression profiles are observed between UC and CD. Unlike isolates derived from healthy donors, E. faecium isolates from the feces of UC patients, along with E. faecium strain ATCC 19434, promotes colitis and colonic cytokine expression. Inflammatory E. faecium strains, including ATCC 19434 and a UC-derived strain, cluster separately from commercially available probiotic strains based on whole-genome shotgun sequencing analysis. The presence of E. faecium in fecal samples is associated with large disease extent and the need for multiple medications in UC patients. CONCLUSIONS: E. faecium strains derived from UC patients display an inflammatory genotype that causes colitis.
Assuntos
Colite Ulcerativa/microbiologia , Enterococcus faecalis/patogenicidade , Microbioma Gastrointestinal , Metagenoma , Animais , Estudos de Casos e Controles , Colite/etiologia , Colite/patologia , Colite Ulcerativa/tratamento farmacológico , Colo/patologia , Doença de Crohn/microbiologia , Modelos Animais de Doenças , Quimioterapia Combinada , Enterococcus faecalis/genética , Transplante de Microbiota Fecal , Fezes/microbiologia , Feminino , Humanos , Interleucina-10/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Orthopaedic implant-associated infections are a devastating complication of orthopaedic surgery with a significant impact on patients and healthcare systems. The aims of this work were to describe the patterns of antimicrobial resistance, pathogenicity and virulence of clinical bacterial isolates from orthopaedic implant-associated infections and to further isolate and characterise bacteriophages that are efficient in controlling these bacteria. Staphylococcus aureus, Enterococcus faecalis and Escherichia coli isolated from orthopaedic infections showed multiresistance patterns to the most frequently used antibiotics in clinical settings. The presence of mobile genetic elements (mecA, Tn916/Tn1545 and intl1) and virulence determinants (icaB, cna, hlb, cylLs, cylM, agg, gelE, fsr and fimA) highlighted the pathogenicity of these isolates. Moreover, the isolates belonged to clonal complexes associated with the acquisition of pathogenicity islands and antimicrobial resistance genes by recombination and horizontal gene transfer. Bacteriophages vB_SauM_LM12, vB_EfaS_LM99 and vB_EcoM_JB75 were characterised and their ability to infect clinical isolates of S. aureus, E. faecalis and E. coli, respectively, was assessed. Morphological and genomic analyses revealed that vB_EfaS_LM99 and vB_EcoM_JB75 belong to the Siphoviridae and Myoviridae families, respectively, and no genes associated with lysogeny were found. The bacteriophages showed low latent periods, high burst sizes, broad host ranges and tolerance to several environmental conditions. Moreover, they showed high efficiency and specificity to infect and reduce clinical bacteria, including methicillin-resistant S. aureus and vancomycin-resistant enterococci. Therefore, the results obtained suggest that the bacteriophages used in this work are a promising approach to control these pathogens involved in orthopaedic implant-associated infections.
Assuntos
Bacteriólise , Bacteriófagos/isolamento & purificação , Infecções por Escherichia coli/terapia , Infecções por Bactérias Gram-Positivas/terapia , Terapia por Fagos/métodos , Infecções Relacionadas à Prótese/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Bacteriófagos/crescimento & desenvolvimento , Farmacorresistência Bacteriana Múltipla , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/isolamento & purificação , Enterococcus faecalis/patogenicidade , Enterococcus faecalis/virologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Escherichia coli/patogenicidade , Escherichia coli/virologia , Feminino , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Masculino , Pessoa de Meia-Idade , Procedimentos Ortopédicos/efeitos adversos , Infecções Relacionadas à Prótese/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/patogenicidade , Staphylococcus aureus/virologiaRESUMO
AIM: To determine if bacteria associated with persistent apical periodontitis induce species-specific pro-inflammatory cytokine responses in macrophages, and the effects of this species-specific microenvironment on osteogenic differentiation. METHODOLOGY: Macrophages were exposed to Enterococcus faecalis, Streptococcus oralis, Streptococcus mitis, Fusobacterium nucleatum, Treponema denticola or Tannerella forsythia, and levels of TNF-α and IL-1ß elicited were determined by immunoassay. Following treatment of MG-63 pre-osteoblasts with conditioned media from bacteria-exposed macrophages, osteogenic differentiation and viability of osteoblasts were analyzed by Alizarin Red Staining and MTS assay, respectively. Statistical analysis was carried out by one-way anova with the Tukey post-hoc test. Differences were considered to be significant if P < 0.05. RESULTS: Macrophages exposed to Gram-positive bacteria did not produce significant amounts of cytokines. F. nucleatum-challenged macrophages produced up to four-fold more TNF-α and IL-1ß compared to T. denticola or T. forsythia. Only conditioned media from macrophages treated with Gram-negative bacteria decreased mineralization and viability of osteoblasts. CONCLUSIONS: Gram-positive bacteria did not impact osteogenic differentiation and appeared innocuous. Gram-negative bacteria, in particular F. nucleatum elicited an enhanced pro-inflammatory response in macrophages, inhibited osteogenic differentiation and reduced cell viability. The findings suggest that the presence of this organism could potentially increase the severity of persistent apical periodontitis.
Assuntos
Bactérias/classificação , Diferenciação Celular , Citocinas/metabolismo , Osteogênese , Periodontite Periapical/imunologia , Periodontite Periapical/microbiologia , Calcificação Fisiológica , Sobrevivência Celular , Enterococcus faecalis/patogenicidade , Fusobacterium nucleatum/patogenicidade , Expressão Gênica , Humanos , Inflamação/microbiologia , Interleucina-1beta/metabolismo , Macrófagos/imunologia , Macrófagos/microbiologia , Osteoblastos , Periodontite Periapical/patologia , Especificidade da Espécie , Streptococcus mitis/patogenicidade , Streptococcus oralis/patogenicidade , Tannerella forsythia/patogenicidade , Treponema denticola/patogenicidade , Fator de Necrose Tumoral alfa/metabolismoRESUMO
RESUMEN: El objetivo del estudio fue determinar el efecto antibacteriano in vitro de la oleorresina de Copaifera reticulata (C. reticulata) "copaiba" y del aceite esencial de Oreganum majoricum (O. majoricum) "orégano" frente a Streptococcus mutans (S. mutans) y Enterococcus faecalis (E. faecalis). Se desarrollaron pruebas de sensibilidad activando primero las cepas bacterias a enfrentar. La oleorresina de copaiba fue diluida con dimetilsulfósido (DMSO), obteniéndose al final concentraciones a probar de 100 %, 50 %, 25 %, y 12,5 %. En relación al aceite esencial de orégano este se probó solamente al 100 %. Para la prueba de difusión en agar con discos, se tomaron inóculos 100 µL de cada cepa bacteriana a una turbidez de 0,5 de Mc Farlam, para ser sembrados por diseminación en placas de tripticasa soya agar, para luego colocar los discos de forma equidistante cargados con las diferentes concentraciones de los productos naturales, se utilizaron como control positivo a la clorhexidina al 0,12 % y al DMSO como control negativo. Se incubaron las placas por el método de la vela en extinción a 37 °C, por un periodo de 24 horas, pasado el tiempo se realizó la lectura de los halos de inhibición. Los resultados obtenidos por la copaiba, determinaron un efecto antibacteriano en sus cuatro concentraciones, siendo los mayores halos de inhibición a la concentración del 100 %, copaiba genero mayores halos promedios para S, mutans de 30,00 ± 0,00 mm y para E. faecalis de 8,3 ± 0,50 mm. Para el caso del orégano se producen halos a la concentración del 100 % con un promedio de 25,3 ± 0,96 mm para S. mutans y para E. faecalis de 9,5 ± 1,29 mm. Se concluye del estudio que tanto copaiba como el orégano presentan un efecto antibacteriano para ambas bacterias, siendo su mayor efecto antibacteriano para ambos productos naturales sobre S. mutans.
ABSTRACT: The objective of the study was to determine the in vitro antibacterial effect of the oleoresin of Copaifera reticulata (C. reticulata) "copaiba" and of the essential oil of Oreganum majoricum (O. majoricum) "oregano" against Streptococcus mutans (S. mutans) and Enterococcus faecalis (E. faecalis). Sensitivity tests were developed by first activating the bacteria strains to be confronted. The oleoresin of copaiba was diluted with dimethylsulphoside (DMSO), obtaining final concentrations to be tested of 100 %, 50 %, 25 %, and 12.5 %. In relation to the essential oil of oregano, it was only 100 % tested. For the disk agar diffusion test, 100 mL of each bacterial strain was taken at a turbidity of 0.5 of Mc Farlam, to be planted by dissecting trypticase soy agar plates, and then placing the disks equidistantly loaded with the different concentrations of natural products; 0.12 % chlorhexidine was used as a positive control and DMSO as negative control. The plates were incubated by the candle method in extinction at 37 °C, for a period of 24 hours, after which time the inhibition halos were read. The results obtained by the copaiba, determined an antibacterial effect in its four concentrations, being the biggest halos of inhibition at the concentration of 100 %, copaiba genus higher average halos for S. mutans of 30.00 ± 0.00 mm and for E. faecalis of 8.3 ± 0.50 mm. In the case of oregano, haloes are produced at a concentration of 100 % with an average of 25.3 ± 0.96 mm for S. mutans and for E. faecalis 9.5 ± 1.29 mm. It is concluded from the study that both copaiba and oregano present an antibacterial effect for both bacteria, being its greater antibacterial effect for both natural products on S. mutans.
Assuntos
Humanos , Streptococcus mutans/fisiologia , Extratos Vegetais/farmacologia , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções por Bactérias Gram-Positivas/patologia , Enterococcus faecalis/patogenicidade , Origanum/química , Peru , Streptococcus mutans/imunologia , Técnicas In Vitro , Óleos Voláteis/análise , AntibacterianosRESUMO
Oral microbiota consists of hundreds of different species of bacteria, fungi, protozoa and archaea, important for oral health. Oral mycoses, mostly affecting mucosae, are mainly caused by the opportunistic pathogen Candida albicans. They become relevant in denture-wearers elderly people, in diabetic patients, and in immunocompromised individuals. Differently, bacteria are responsible for other pathologies, such as dental caries, gingivitis and periodontitis, which affect even immune-competent individuals. An appropriate oral hygiene can avoid (or at least ameliorate) such pathologies: the regular and correct use of toothbrush, toothpaste and mouthwash helps prevent oral infections. Interestingly, little or no information is available on the effects (if any) of mouthwashes on the composition of oral microbiota in healthy individuals. Therefore, by means of in vitro models, we assessed the effects of alcohol-free commercial mouthwashes, with different composition (4 with chlorhexidine digluconate, 1 with fluoride, 1 with essential oils, 1 with cetylpyridinium chloride and 1 with triclosan), on several virulence traits of C. albicans, and a group of viridans streptococci, commonly colonizing the oral cavity. For the study here described, a reference strain of C. albicans and of streptococci isolates from pharyngeal swabs were used. Chlorhexidine digluconate- and cetylpyridinium chloride-containing mouthwashes were the most effective in impairing C. albicans capacity to adhere to both abiotic and biotic surfaces, to elicit proinflammatory cytokine secretion by oral epithelial cells and to escape intracellular killing by phagocytes. In addition, these same mouthwashes were effective in impairing biofilm formation by a group of viridans streptococci that, notoriously, cooperate with the cariogenic S. mutans, facilitating the establishment of biofilm by the latter. Differently, these mouthwashes were ineffective against other viridans streptococci that are natural competitors of S. mutans. Finally, by an in vitro model of mixed biofilm, we showed that mouthwashes-treated S. salivarius overall failed to impair C. albicans capacity to form a biofilm. In conclusion, the results described here suggest that chlorhexidine- and cetylpyridinium-containing mouthwashes may be effective in regulating microbial homeostasis of the oral cavity, by providing a positive balance for oral health. On the other side, chlorhexidine has several side effects that must be considered when prescribing mouthwashes containing this molecule.
Assuntos
Anti-Infecciosos Locais/administração & dosagem , Candida albicans/efeitos dos fármacos , Enterococcus faecalis/efeitos dos fármacos , Boca/efeitos dos fármacos , Antissépticos Bucais/administração & dosagem , Estreptococos Viridans/efeitos dos fármacos , Animais , Biofilmes/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Candida albicans/patogenicidade , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecalis/metabolismo , Enterococcus faecalis/patogenicidade , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Homeostase/efeitos dos fármacos , Humanos , Camundongos , Microglia/efeitos dos fármacos , Microglia/microbiologia , Boca/microbiologia , Fagocitose/efeitos dos fármacos , Estreptococos Viridans/crescimento & desenvolvimento , Estreptococos Viridans/metabolismo , Estreptococos Viridans/patogenicidade , Virulência/efeitos dos fármacosRESUMO
The alarmone (p)ppGpp mediates the stringent response and has a recognized role in bacterial virulence. We previously reported a stringent response-like state in Enterococcus faecalis isolated from a rabbit foreign body abscess model and showed that E. faecalis mutants with varying levels of cellular (p)ppGpp [Δrel, ΔrelQ and the (p)ppGpp0 ΔrelΔrelQ] had differential abilities to persist within abscesses. In this study, we investigated whether (p)ppGpp contributes to the pathogenesis of E. faecalis infective endocarditis (IE), a biofilm infection of the heart valves. While the stringent response was not activated in heart valve-associated E. faecalis, deletion of the gene encoding the bifunctional (p)ppGpp synthetase/hydrolase Rel significantly impaired valve colonization. These results indicate that the presence of (p)ppGpp is dispensable for E. faecalis to cause IE, whereas the ability to regulate (p)ppGpp levels is critical for valve colonization. Next, we characterized how basal (p)ppGpp levels affect processes associated with IE pathogenesis. Despite being defective in binding to BSA-coated polystyrene surfaces, the Δrel strain bound to collagen- and fibronectin-coated surfaces and ex vivo porcine heart valves as well as the parent and ΔrelΔrelQ strains, ruling out the possibility that the impaired IE phenotype was due to an attachment defect. Moreover, differences in cellular (p)ppGpp levels did not affect extracellular gelatinase activity but significantly impaired enterococcal invasion of human coronary artery endothelial cells. Taken together, this study uncovers for the first time the fact that differences in basal (p)ppGpp levels, rather than the stringent response, differentially affect processes that contribute to the pathogenesis of IE.
Assuntos
Endocardite Bacteriana/microbiologia , Enterococcus faecalis/patogenicidade , Guanosina Pentafosfato/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Células Cultivadas , Modelos Animais de Doenças , Endocardite Bacteriana/metabolismo , Endocardite Bacteriana/patologia , Células Endoteliais/metabolismo , Células Endoteliais/microbiologia , Enterococcus faecalis/genética , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecalis/metabolismo , Gelatinases/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Humanos , Ligases/genética , Ligases/metabolismo , Coelhos , Suínos , Virulência/genéticaRESUMO
BACKGROUND/AIM: Enterococcus faecalis is responsible for most cases of endodontic treatment failure. Despite various conventional disinfection methods, root canals are not completely free of microorganisms. Photodynamic therapy (PDT) is a new antimicrobial strategy that involves the use of a non-toxic photosensitizer (PS) and a light source. The aim of this study was to evaluate the antimicrobial effect of PDT using diode laser and pyoktanin blue (PB) and confirm the nontoxicity of PB as a PS. MATERIALS AND METHODS: Laser irradiation with an output power of 3 W was performed with PB as the PS to a bacterial solution containing E. faecalis. Then, the number of colony-forming units was counted. PB cytotoxicity was also assessed by the MTT assay. RESULTS: E. faecalis counts were reduced after laser irradiation, laser irradiation with PB, or the combination thereof compared to the control, non-irradiation or water. The 50% cytotoxic concentration value for adult human dermal fibroblasts incubated with PB for 1 min was 108 µg/ml. CONCLUSION: Diode laser irradiation in combination with PB as the PS is efficacious for the elimination of E. faecalis without toxic effects to human dermal fibroblasts. This strategy might be useful for root canal irrigants.
Assuntos
Enterococcus faecalis/efeitos da radiação , Lasers Semicondutores/uso terapêutico , Fotoquimioterapia , Dente não Vital/radioterapia , Biofilmes/crescimento & desenvolvimento , Biofilmes/efeitos da radiação , Cavidade Pulpar/microbiologia , Cavidade Pulpar/efeitos da radiação , Desinfecção/métodos , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecalis/patogenicidade , Humanos , Irrigantes do Canal Radicular/uso terapêutico , Dente não Vital/microbiologiaRESUMO
OBJECTIVES: Infections are major causes of morbidity and mortality in the early postoperative period after liver transplant. We observed a high rate of enterococcal infections at our center. Therefore, we added an intraoperative single shot of vancomycin to the standard regimen of meropenem given over 5 days. The aim of this study was to determine the prevalence of both Enterococcus faecium and Enterococcus faecalis infections during the first 28 days after surgery depending on the type of antibiotic prophylaxis and their implications on mortality and morbidity. MATERIALS AND METHODS: Our retrospective cohort analysis included 179 patients: 93 patients received meropenem only and 86 patients were treated with meropenem plus vancomycin. RESULTS: During the first 28 days after transplant, microbiological tests showed that 51 patients (28.5%) were positive for Enterococcus faecium and 25 patients (14.0%) were positive for Enterococcus faecalis. Enterococcus faecium infections appeared significantly more often in patients without vancomycin (P = .013). In the second week after transplant, there was a significant reduction in Enterococcus faecium infections in the meropenem plus vancomycin group (P = .015). Enterococcus faecalis infections occurred more often in the patients receiving meropenem alone, but results were not statistically significant (P = .194). There was a trend toward more frequent renal replacement therapy in the meropenem plus vancomycin group. We found no differences between the groups regarding survival after 1 and 2 years, length of hospital stay, or duration in the intensive care unit. Overall 1-year survival was 78.8% (141/179 patients). CONCLUSIONS: Although postoperative Enterococcus species infections can be reduced after liver transplant by adding vancomycin to the intraoperative antibiotic regimen, it does not improve the long-term outcomes.
Assuntos
Antibacterianos/administração & dosagem , Antibioticoprofilaxia/métodos , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecium/efeitos dos fármacos , Infecções por Bactérias Gram-Positivas/prevenção & controle , Transplante de Fígado/efeitos adversos , Vancomicina/administração & dosagem , Adulto , Antibacterianos/efeitos adversos , Antibioticoprofilaxia/efeitos adversos , Antibioticoprofilaxia/mortalidade , Enterococcus faecalis/patogenicidade , Enterococcus faecium/patogenicidade , Feminino , Alemanha/epidemiologia , Infecções por Bactérias Gram-Positivas/diagnóstico , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções por Bactérias Gram-Positivas/mortalidade , Humanos , Injeções Intravenosas , Cuidados Intraoperatórios , Transplante de Fígado/mortalidade , Masculino , Meropeném/administração & dosagem , Pessoa de Meia-Idade , Prevalência , Estudos Retrospectivos , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Vancomicina/efeitos adversosRESUMO
Enterococcus faecalis (E. faecalis) infection is considered an important etiological factor for the development of persistent apical periodontitis (PAP), but the exact mechanisms of autophagy between E. faecalis and immune cells remain unknown. In this study, we elucidated how E. faecalis lipoteichoic acid (LTA) is associated with macrophages autophagy. We found that E. faecalis LTA apparently activated macrophage autophagy with significant increase of autophagosomes and autophagy relative protein. Meanwhile, we noticed significantly decreasing expression of p-Akt and p-mTOR. However, these effect were absent in macrophages knockdown of Beclin1. In summary, these findings suggested E. faecalis LTA may increased macrophages autophagy via inhibiting PI3K/Akt/mTOR pathway and this process was Beclin1 dependent.
Assuntos
Autofagia/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Ácidos Teicoicos/farmacologia , Animais , Proteína Beclina-1 , Enterococcus faecalis/patogenicidade , Macrófagos/efeitos dos fármacos , Camundongos , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
This study investigated the host response to a polymicrobial pulpal infection consisting of Streptococcus anginosus and Enterococcus faecalis, bacteria commonly implicated in dental abscesses and endodontic failure, using a validated ex vivo rat tooth model. Tooth slices were inoculated with planktonic cultures of S. anginosus or E. faecalis alone or in coculture at S. anginosus/E. faecalis ratios of 50:50 and 90:10. Attachment was semiquantified by measuring the area covered by fluorescently labeled bacteria. Host response was established by viable histological cell counts, and inflammatory response was measured using reverse transcription-quantitative PCR (RT-qPCR) and immunohistochemistry. A significant reduction in cell viability was observed for single and polymicrobial infections, with no significant differences between infection types (â¼2,000 cells/mm2 for infected pulps compared to â¼4,000 cells/mm2 for uninfected pulps). E. faecalis demonstrated significantly higher levels of attachment (6.5%) than S. anginosus alone (2.3%) and mixed-species infections (3.4% for 50:50 and 2.3% for 90:10), with a remarkable affinity for the pulpal vasculature. Infections with E. faecalis demonstrated the greatest increase in tumor necrosis factor alpha (TNF-α) (47.1-fold for E. faecalis, 14.6-fold for S. anginosus, 60.1-fold for 50:50, and 25.0-fold for 90:10) and interleukin 1ß (IL-1ß) expression (54.8-fold for E. faecalis, 8.8-fold for S. anginosus, 54.5-fold for 50:50, and 39.9-fold for 90:10) compared to uninfected samples. Immunohistochemistry confirmed this, with the majority of inflammation localized to the pulpal vasculature and odontoblast regions. Interestingly, E. faecalis supernatant and heat-killed E. faecalis treatments were unable to induce the same inflammatory response, suggesting E. faecalis pathogenicity in pulpitis is linked to its greater ability to attach to the pulpal vasculature.
Assuntos
Coinfecção/patologia , Enterococcus faecalis/patogenicidade , Interações Hospedeiro-Parasita , Pulpite/microbiologia , Pulpite/fisiopatologia , Ratos/microbiologia , Streptococcus anginosus/patogenicidade , Animais , Modelos AnimaisRESUMO
Several ATPases in the ATP-binding cassette F (ABCF) family confer resistance to macrolides, lincosamides and streptogramins (MLS) antibiotics. MLS are structurally distinct classes, but inhibit a common target: the peptidyl transferase (PTC) active site of the ribosome. Antibiotic resistance (ARE) ABCFs have recently been shown to operate through direct ribosomal protection, but the mechanistic details of this resistance mechanism are lacking. Using a reconstituted translational system, we dissect the molecular mechanism of Staphylococcus haemolyticus VgaALC and Enterococcus faecalis LsaA on the ribosome. We demonstrate that VgaALC is an NTPase that operates as a molecular machine strictly requiring NTP hydrolysis (not just NTP binding) for antibiotic protection. Moreover, when bound to the ribosome in the NTP-bound form, hydrolytically inactive EQ2 ABCF ARE mutants inhibit peptidyl transferase activity, suggesting a direct interaction between the ABCF ARE and the PTC. The likely structural candidate responsible for antibiotic displacement by wild type ABCF AREs, and PTC inhibition by the EQ2 mutant, is the extended inter-ABC domain linker region. Deletion of the linker region renders wild type VgaALC inactive in antibiotic protection and the EQ2 mutant inactive in PTC inhibition.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Resistência Microbiana a Medicamentos/genética , Peptidil Transferases/genética , Biossíntese de Proteínas/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Antibacterianos/química , Antibacterianos/farmacologia , Sítios de Ligação , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/genética , Enterococcus faecalis/patogenicidade , Humanos , Lincosamidas/farmacologia , Macrolídeos/química , Macrolídeos/farmacologia , Peptidil Transferases/química , Ligação Proteica , Ribossomos/efeitos dos fármacos , Ribossomos/genética , Staphylococcus haemolyticus/efeitos dos fármacos , Staphylococcus haemolyticus/genética , Staphylococcus haemolyticus/patogenicidade , Estreptograminas/química , Estreptograminas/farmacologiaRESUMO
Recent studies of honeybees and bumblebees have examined combinatory effects of different stressors, as insect pollinators are naturally exposed to multiple stressors. At the same time the potential influences of simultaneously occurring agricultural agents on insect pollinator health remain largely unknown. Due to different farming methods, and the drift of applied agents and manure, pollinators are most probably exposed to insecticides but also bacteria from organic fertilizers at the same time. We orally exposed honeybee workers to sub-lethal doses of the insecticide thiacloprid and two strains of the bacterium Enterococcus faecalis, which can occur in manure from farming animals. Our results show that under laboratory conditions the bees simultaneously exposed to the a bacterium and the pesticide thiacloprid thiacloprid had significant higher survival rates 11 days post exposure than the controls, which surprisingly showed the lowest survival. Bees that were exposed to diet containing thiacloprid showed decreased food intake. General antibacterial activity is increased by the insecticide and the bacteria, resulting in a higher immune response observed in treated individuals compared to control individuals. We thus propose that caloric restriction through behavioural and physiological adaptations may have mediated an improved survival and stress resistance in our tests. However, the decreased food consumption could in long-term also result in possible negative effects at colony level. Our study does not show an additive negative impact of sub-lethal insecticide and bacteria doses, when tested under laboratory conditions. In contrast, we report seemingly beneficial effects of simultaneous exposure of bees to agricultural agents, which might demonstrate a surprising biological capacity for coping with stressors, possibly through hormetic regulation.
Assuntos
Abelhas/efeitos dos fármacos , Abelhas/microbiologia , Enterococcus faecalis/patogenicidade , Inseticidas/toxicidade , Neonicotinoides/toxicidade , Tiazinas/toxicidade , Animais , Abelhas/fisiologia , Ingestão de Alimentos/efeitos dos fármacos , Hormese , Inseticidas/administração & dosagem , Neonicotinoides/administração & dosagem , Polinização , Estresse Fisiológico , Tiazinas/administração & dosagem , VirulênciaRESUMO
BACKGROUND: The role of enterococci in the context of peritonitis and surgical site infections (SSI) has not yet been definitively clarified but enterococci are being detected more frequently. Numerous resistances reduce the available antibiotic options. OBJECTIVE: This article gives an overview of the pathogenic importance of enterococci and of current recommendations for therapy and prophylaxis. On the basis of our own data we discuss the relevance of enterococci for SSI. MATERIAL AND METHODS: All colorectal resections carried out between January 2008 and September 2016 were retrospectively documented. Revision surgery, SSI and intra-abdominally or subcutaneously detected pathogens were recorded. RESULTS: A total of 2713 interventions were evaluated with 28.3% having primary peritonitis. In 587 patients (21.6%) SSI followed, and pathogen determination was possible in 431 cases (73.4%). Enterococci were frequently found in re-operations (58.4%) and SSI (46.1%), with E. faecalis and E. faecium in approximately equal proportions. If intra-abdominal enterococci were detectable in patients with primary peritonitis, it was more common to develop SSI and enterococci were more frequently detected subcutaneously. Enterococci in SSI were found to be significantly more frequent in left hemicolectomies as well as in pre-existing renal insufficiency. CONCLUSION: It can be inferred that enterococci are not adequately covered by commonly used perioperative antibiotic therapy or preoperative prophylaxis, which increases the risk for SSI by enterococci. This could be favored by selection of these pathogens due to the use of antibiotics without enterococcal efficacy (e. g. cephalosporins). The consideration in the choice of perioperative antibiotic prophylaxis by the additional administration of ampicillin or vancomycin could be advantageous.
Assuntos
Cirurgia Colorretal , Enterococcus faecalis/patogenicidade , Enterococcus faecium/patogenicidade , Infecções por Bactérias Gram-Positivas/diagnóstico , Infecções por Bactérias Gram-Positivas/microbiologia , Peritonite/diagnóstico , Infecção da Ferida Cirúrgica/diagnóstico , Infecção da Ferida Cirúrgica/microbiologia , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana Múltipla , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecium/efeitos dos fármacos , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/prevenção & controle , Humanos , Peritonite/tratamento farmacológico , Peritonite/microbiologia , Peritonite/prevenção & controle , Reoperação , Fatores de Risco , Infecção da Ferida Cirúrgica/tratamento farmacológico , Infecção da Ferida Cirúrgica/prevenção & controle , VirulênciaRESUMO
OBJECTIVE: Photodynamic therapy (PDT) is considered a promising adjunct to the currently available endodontic disinfection techniques leading to more effective reduction of intracanal bacteria. The present ex vivo study aimed to assess the antimicrobial effect of PDT using indocyanine green (ICG) as photosensitizer and a near-infrared (NIR) diode laser in root canals of human teeth infected with Enterococcus faecalis. MATERIALS AND METHODS: Ninety single-rooted teeth after chemomechanical preparation and sterilization were contaminated with an E. faecalis strain. The specimens were divided, randomly, into eight experimental groups: (1) PDT with ICG and laser (0.5 W output power-medium-energy fluence), (2) PDT with ICG and laser (1 W output power-high-energy fluence), (3) only laser emission, (4) only ICG, (5) 2.5% sodium hypochlorite (NaOCl) as irrigant, (6) 2.5% NaOCl and PDT with ICG and laser, (7) no treatment (positive control), and (8) no bacterial biofilm growth (negative control). The root canal contents were sampled by flushing and the collected washings were plated on an appropriate culture medium, which was incubated for 48 h at 35°C ± 2.0. The colony-forming units (CFUs) were determined to assess the bactericidal effect of the tested experimental combinations. RESULTS: The microbiological tests revealed that PDT groups, regardless of the overall power, showed significant lower mean log10 CFU levels, than groups 3 and 4 (p < 0.001) and similar reduction of viable counts with group 5. The combination treatment (group 6) achieved adequate reduction of log10 CFU levels in the viable counts. However, no significant difference (p > 0.05) was observed between groups 1, 2, 5, and 6 and significant difference was noticed between groups 3, 4, and 5 (p < 0.001). CONCLUSIONS: ICG-mediated PDT activated by an NIR diode laser provided increased disinfection of the root canal system, but the overall benefit in total bacterial elimination should be further investigated.