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1.
BMC Oral Health ; 22(1): 416, 2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36127648

RESUMO

BACKGROUND: Enterococcus faecalis (E. faecalis) plays an important role in the failure of root canal treatment and refractory periapical periodontitis. As an important virulence factor of E. faecalis, extracellular polysaccharide (EPS) serves as a matrix to wrap bacteria and form biofilms. The homologous rnc gene, encoding Ribonuclease III, has been reported as a regulator of EPS synthesis. In order to develop novel anti-biofilm targets, we investigated the effects of the rnc gene on the biological characteristics of E. faecalis, and compared the biofilm tolerance towards the typical root canal irrigation agents and traditional Chinese medicine fluid Pudilan. METHODS: E. faecalis rnc gene overexpression (rnc+) and low-expression (rnc-) strains were constructed. The growth curves of E. faecalis ATCC29212, rnc+, and rnc- strains were obtained to study the regulatory effect of the rnc gene on E. faecalis. Scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and crystal violet staining assays were performed to evaluate the morphology and composition of E. faecalis biofilms. Furthermore, the wild-type and mutant biofilms were treated with 5% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), and Pudilan. The residual viabilities of E. faecalis biofilms were evaluated using crystal violet staining and colony counting assays. RESULTS: The results demonstrated that the rnc gene could promote bacterial growth and EPS synthesis, causing the EPS-barren biofilm morphology and low EPS/bacteria ratio. Both the rnc+ and rnc- biofilms showed increased susceptibility to the root canal irrigation agents. The 5% NaOCl group showed the highest biofilm removing effect followed by Pudilan and 2% CHX. The colony counting results showed almost complete removal of bacteria in the 5% NaOCl, 2% CHX, and Chinese medicine agents' groups. CONCLUSIONS: This study concluded that the rnc gene could positively regulate bacterial proliferation, EPS synthesis, and biofilm formation in E. faecalis. The rnc mutation caused an increase in the disinfectant sensitivity of biofilm, indicating a potential anti-biofilm target. In addition, Pudilan exhibited an excellent ability to remove E. faecalis biofilm.


Assuntos
Desinfetantes , Enterococcus faecalis , Clorexidina/farmacologia , Desinfecção , Enterococcus faecalis/genética , Violeta Genciana/farmacologia , Humanos , Ribonuclease III/farmacologia , Hipoclorito de Sódio/farmacologia , Fatores de Virulência/farmacologia
2.
Eur J Clin Microbiol Infect Dis ; 41(10): 1245-1261, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36057762

RESUMO

The aim of our study was to characterize the epidemiological situation concerning nosocomial vancomycin-resistant Enterococcus faecalis of VanA-phenotype (VREfs-VanA) in Poland by investigating their clonal relationships and the vanA-associated mobilome. One-hundred twenty-five clinical isolates of VREfs-VanA collected between 2004 and 2016 were studied by phenotypic assays, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), PCR detection of plasmid-specific genes, and Tn1546 structure and localization mapping. Selected isolates were subjected to PFGE-S1, Southern hybridization, genomic sequencing and conjugation experiments. The majority of isolates (97.6%) belonged to clonal complexes CC2 and CC87 of E. faecalis. All isolates were resistant to vancomycin and teicoplanin, and resistance to ciprofloxacin and aminoglycosides (high level) was very prevalent in this group. VanA phenotype was associated with 16 types of Tn1546, carrying insertion sequences IS1216, ISEfa4, IS1251 and IS1542, located on repUS1pVEF1, rep1pIP501, rep2pRE25, rep9pAD1/pTEF2/pCF10 and rep6pS86 replicons. The most common Tn1546 B- and BB-type transposons, harbouring one or two copies of IS1216, were inserted between rep18ap200B and repUS1pVEF1 genes and located on ~ 20 kb and 150-200 kb plasmids. VREfs-VanA in Poland represent a polyclonal group, indicating a number of acquisitions of the vanA determinant. The repUS1pVEF1-vanA plasmids, unique for Poland, were the main factor beyond the acquisition of vancomycin resistance by E. faecalis, circulating in Polish hospitals.


Assuntos
Enterococcus faecium , Infecções por Bactérias Gram-Positivas , Aminoglicosídeos , Proteínas de Bactérias/genética , Carbono-Oxigênio Ligases/genética , Ciprofloxacina , Elementos de DNA Transponíveis , Enterococcus faecalis/genética , Enterococcus faecium/genética , Infecções por Bactérias Gram-Positivas/epidemiologia , Infecções por Bactérias Gram-Positivas/microbiologia , Hospitais , Humanos , Tipagem de Sequências Multilocus , Polônia/epidemiologia , Teicoplanina , Vancomicina
3.
Microb Pathog ; 171: 105745, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36057414

RESUMO

This study aimed to investigate the presence of eight virulence genes (ace, asa1, esp, efaA, gelE, cylA, agg, fsr) in Enterococcus from a variety of animals and to explore the drug resistance and pathogenicity. This could provide a theoretical basis for clinical treatment of Enterococcus infections. Anal swabs from pigs, chickens, cattle, and dogs in farms and pet hospitals were collected for Enterococcus isolation and identification. Eight virulence genes were detected (PCR method), and drug resistance was assessed (drug-sensitive paper method). The strains containing different virulence genes were then divided into EV1, EV2, and EV3 groups. The LD50 and pathogenicity was examined by intra-peritoneal injection to infect mice. Differences were found in the detection rates of virulence genes in Enterococcus from the different animals. The highest overall detection rate was for the esp gene (78.0%), and the lowest for the cylA gene (15.5%). Eight genes were detected most frequently in Enterococcus from dogs and least frequently from cattle. Among the Enterococcus strains from four variety of animals, drug resistance was highest against sulfamethoxazole (100%), cefotaxime (>97%), and cefotaxitin (>93%). Drug resistance was lowest against vancomycin (0%), levofloxacin (<12%) and ciprofloxacin (<13%). The LD50 for each of the three groups was EV1LD50=8.71×109CFU, EV2LD50=2.34×1010CFU,and EV3LD50=9.33×1010CFU. The Enterococcus12LD50 dose group caused significant clinical symptoms in mice, with pathological effects on the heart, liver, lungs, and kidneys, and particularly on the urinary system. The abundance of Enterococcus virulence genes, drug resistance, and pathogenicity vary among different animal origins, and the pathology caused by Enterococcus requires effective treatment protocols based on species and regional characteristics.


Assuntos
Enterococcus faecium , Infecções por Bactérias Gram-Positivas , Animais , Animais Domésticos , Antibacterianos/farmacologia , Bovinos , Cefotaxima/farmacologia , Galinhas , Ciprofloxacina/farmacologia , Cães , Resistência a Medicamentos , Farmacorresistência Bacteriana/genética , Enterococcus , Enterococcus faecalis , Infecções por Bactérias Gram-Positivas/veterinária , Levofloxacino/farmacologia , Camundongos , Testes de Sensibilidade Microbiana , Sulfametoxazol/farmacologia , Suínos , Vancomicina/farmacologia , Virulência/genética , Fatores de Virulência/genética
4.
BMC Oral Health ; 22(1): 407, 2022 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-36117178

RESUMO

BACKGROUND: There is an increasing demand to find a potent antibacterial agent against endodontic pathogens with the least toxic effect. The study aimed to evaluate the antibacterial activity of Lactobacilli probiotics on Enterococcus faecalis (E. faecalis) in comparison to calcium hydroxide paste. METHODS: The study involved two stages; (stage one): determination of the antibacterial properties of three strains of Lactobacilli probiotics supernatants (PS); Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, and a cocktail mix of the three supernatants against E. faecalis using agar well diffusion method in both solution and gel phase. The formed zones of inhibition (ZOI) were measured in millimeters (mm) and compared to each other. PS solution and gel of the largest ZOI were further compared with calcium hydroxide paste (35% Ultra Cal XS Ca(OH)2) This was followed by (stage two): assessment of the minimum inhibitory concentration (MIC) of the PS that showed the largest ZOI against E. faecalis by agar well diffusion assay for both PS solution and gel. RESULTS: All supernatants showed growth inhibition against E. faecalis, and the cocktail mix showed the largest ZOI. However, no significant difference was found between the supernatants in both the solution and gel phases (p > 0.05). Ca(OH)2 showed a significantly lower effect than both the cocktail mix solution and gel (p < 0.05). The MIC of the cocktail mix solution and gel against E. faecalis was 50 mg/ml. (p > 0.05). CONCLUSION: PS has an antibacterial effect on E. faecalis and was more effective than Ca(OH)2. Lactobacilli probiotics could be a promising antibacterial agent used as an irrigant or an intracanal medication.


Assuntos
Enterococcus faecalis , Probióticos , Ágar/farmacologia , Antibacterianos/farmacologia , Hidróxido de Cálcio/farmacologia , Humanos , Lactobacillus , Probióticos/farmacologia
5.
J Dent ; 125: 104278, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36058346

RESUMO

OBJECTIVES: This study aimed to evaluate the antibacterial ability and cytocompatibility of a new irrigant solution for endodontic treatment composed of 10% citric acid (CA) and 1% chlorhexidine (CHX). METHODS: Thirty-five extracted single-canal human teeth were selected and de-crowned. Canal systems (n = 7/group) were infected with Enterococcus faecalis for 4 weeks and subject to irrigation with 1% CHX; 10% CA; irrigating solution 10% CA associated with 1% CHX (CACHX); 2.5% NaOCl or sterile water (control). Microbiological samples were collected immediately and 18 h after irrigation (enriched samples). The canals were filled with culture medium post irrigation to verify the bacterial presence/absence qualitatively and quantitatively through colony counting (log10 CFU/mL). A multiparametric assay was performed after exposure of human periodontal ligament fibroblasts (HPdLF) to the test solutions. The Kruskal-Wallis test with Dunn´s post-test and Fisher's exact test were employed at the 95% confidence level to compare differences among groups. RESULTS: All tested solutions were cytocompatible with human periodontal ligament fibroblasts. No difference was observed on antibacterial activity between 1% CHX, 10% CA, CACHX and 2.5% NaOCl (p > 0.05). Eighteen hours after irrigation, CACHX samples were the only that did not present E. faecalis in the root canal system. CONCLUSIONS: The demonstrated good in vitro biocompatibility and elimination of E. faecalis suggest a potential use of 10% CA associated with 1% CHX as a solution for microbiological control during endodontic treatment. CLINICAL RELEVANCE: Irrigants play an essential role during endodontic therapy. This irrigating solution, based on the association of 10% citric acid with 1% chlorhexidine, seems viable for clinical procedures.


Assuntos
Clorexidina , Irrigantes do Canal Radicular , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Clorexidina/farmacologia , Ácido Cítrico/farmacologia , Cavidade Pulpar/microbiologia , Enterococcus faecalis , Humanos , Irrigantes do Canal Radicular/farmacologia , Irrigantes do Canal Radicular/uso terapêutico , Hipoclorito de Sódio/farmacologia , Hipoclorito de Sódio/uso terapêutico , Água
6.
Int J Mol Sci ; 23(17)2022 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-36077503

RESUMO

The success of root canal therapy depends mainly on the complete elimination of the root canal bacterial biofilm. The validity and biocompatibility of root canal disinfectant materials are imperative for the success of root canal treatment. However, the insufficiency of the currently available root canal disinfectant materials highlights that more advanced materials are still needed. In this study, a nanozyme-loaded hydrogel (Fe3O4-CaO2-Hydrogel) was modified and analyzed as a root canal disinfectant material. Fe3O4-CaO2-Hydrogel was fabricated and examined for its release profile, biocompatibility, and antibacterial activity against E. faecalis and S. sanguis biofilms in vitro. Furthermore, its efficiency in eliminating the root canal bacterial biofilm removal in SD rat teeth was also evaluated. The results in vitro showed that Fe3O4-CaO2-Hydrogel could release reactive oxygen species (ROS). Moreover, it showed good biocompatibility, disrupting bacterial cell membranes, and inhibiting exopolysaccharide production (p < 0.0001). In addition, in vivo results showed that Fe3O4-CaO2-Hydrogel strongly scavenged on root canal biofilm infection and prevented further inflammation expansion (p < 0.05). Altogether, suggesting that Fe3O4-CaO2-Hydrogel can be used as a new effective biocompatible root canal disinfectant material. Our research provides a broad prospect for clinical root canal disinfection, even extended to other refractory infections in deep sites.


Assuntos
Desinfetantes , Enterococcus faecalis , Animais , Antibacterianos/farmacologia , Biofilmes , Cavidade Pulpar , Desinfetantes/farmacologia , Hidrogéis/farmacologia , Peróxido de Hidrogênio/farmacologia , Ratos , Ratos Sprague-Dawley , Tratamento do Canal Radicular/métodos
7.
FEMS Microbiol Lett ; 369(1)2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36044998

RESUMO

Enterococcus faecalis is able to adapt to alkaline conditions and is commonly recovered from teeth in which endodontic treatment has failed. The role that E. faecalis membrane proteins play in survival strategies to extreme alkaline conditions is unclear. We grew E. faecalis V583 in a chemostat at pH 8 and 11 at one-tenth the organism's relative maximum growth rate. Following membrane shaving, isotope-coding protein labels were added at the peptide level to samples and then combined. The relative proportion of membrane proteins were identified using LC-ESI mass spectrometry and MaxQuant analysis. Ratios of membrane proteins were log2 transformed, with proteins deviating by more than 1 SD of the mean considered to be up- or down-regulated. A total of six proteins were up-regulated in pH 11 including: EF0669 (polysaccharide biosynthesis family); EF1927 (glycerol uptake facilitator), and EF0114 (glycosyl hydrolase). A total of five proteins were down-regulated including: EF0108 (C4-dicarboxylate transporter); EF1838 (PTS system IIC component); EF0456 (PTS system IID component); and EF0022 (PTS mannose-specific IID component). In extreme alkaline conditions, the membrane proteins of E. faecalis seem to be involved in a shift of carbohydrate metabolism from the PTS system to glycerol, which supports the formation of a protective capsule protecting the cell.


Assuntos
Enterococcus faecalis , Proteínas de Membrana , Proteínas de Bactérias/metabolismo , Transportadores de Ácidos Dicarboxílicos/metabolismo , Enterococcus/metabolismo , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Glicerol/metabolismo , Hidrolases/metabolismo , Manose/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Polissacarídeos/metabolismo
8.
Microb Pathog ; 171: 105715, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35973648

RESUMO

In this study, we evaluated the antimicrobial susceptibility, the presence of gene-encoding virulence factors and CRISPR systems, as well as the ability to produce lytic enzymes among clinical E. faecalis and E. faecium isolates (n = 44). All enterococci isolates showed phenotypes of multidrug resistance. E. faecalis and E. faecium isolates exhibited high-level aminoglycoside resistance phenotype, several of them harboring the aac(6')Ie-aph(2″)Ia and aph(3')-IIIa genes. The gene vanA was the most frequent among vancomycin-resistant E. faecium. High prevalence of the virulence genes esp and efaA were observed; hyl gene was more associated with E. faecium, while ace and efaA genes were more frequently detected in E. faecalis. Caseinase activity was frequently detected among the isolates. Gelatinase and DNAse activities predominated among E. faecalis, while hemolytic capability was frequent among E. faecium isolates. Twenty-nine isolates showed at least one CRISPR system investigated. Several enterococci isolates harbored the aac(6')-Ie-aph(2″)-Ia or aph(3')-IIIa genes and a CRISPR loci. CRISPR loci were positively correlated to efaA and gelE genes, and gelatinase and DNAse activities, while CRISPR loci absence was related to hyl gene presence. These results show that clinical isolates of E. faecalis and E. faecium harboring virulence genes show the concomitant presence of CRISPR loci and antibiotic resistance determinants.


Assuntos
Enterococcus faecium , Infecções por Bactérias Gram-Positivas , Aminoglicosídeos , Antibacterianos/farmacologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Desoxirribonucleases/genética , Farmacorresistência Bacteriana/genética , Enterococcus/genética , Enterococcus faecalis , Gelatinases , Infecções por Bactérias Gram-Positivas/epidemiologia , Humanos , Canamicina Quinase/genética , Testes de Sensibilidade Microbiana , Vancomicina , Virulência/genética , Fatores de Virulência/genética
9.
World J Microbiol Biotechnol ; 38(10): 177, 2022 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-35934729

RESUMO

The ability of bacteria to adapt to the external environment is fundamental for their survival. A halotolerant microorganism Enterococcus faecalis able to grow under high salt stress conditions was isolated in the present study. The SDS-PAGE analysis of the secretome showed a protein band with a molecular weight of 28 kDa, gradually increased with an increase in salt concentration, and the highest intensity was observed at 15% salt stress condition. LC-MS/MS analysis of this particular band identified fourteen different proteins, out of which nine proteins were uncharacterized. Further, the function of uncharacterized proteins was predicted based on structure-function relationship using a reverse template search approach deciphering uncharacterized protein into type III polyketide synthases, stress-induced protein-1, Eed-h3k79me3, ba42 protein, 3-methyladenine DNA glycosylase, Atxa protein, membrane-bound respiratory hydrogenase, type-i restriction-modification system methylation subunit and ManxA. STRING network analysis further a showed strong association among the proteins. The processes predicted involvement of these proteins in signal transduction, ions transport, synthesis of the protective layer, cellular homeostasis and regulation of gene expression and different metabolic pathways. Thus, the fourteen proteins identified in the secretome play an essential role in maintaining cellular homeostasis in E. faecalis under high-salinity stress. This may represent a novel and previously unreported strategy by E. faecalis to maintain their normal growth and physiology under high salinity conditions.


Assuntos
Enterococcus faecalis , Tolerância ao Sal , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cromatografia Líquida , Enterococcus faecalis/metabolismo , Tolerância ao Sal/genética , Secretoma , Espectrometria de Massas em Tandem
10.
Ethiop J Health Sci ; 32(4): 799-808, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35950059

RESUMO

Background: Enterococci are naturally found in the gastrointestinal (GI) tract of animals and humans, as well as animal-derived foods and vegetables. We here aimed to determine the prevalence, antibiotic resistance, and virulence determinants of E. faecium and E. faecalis in traditional cheese in the North-west of Iran. Materials and Methods: Fifty specimens of popular traditional cheese from dairy stores of Urmia and Tabriz, Iran, were collected. Identification of the genus and species of enterococci was done using molecular and phenotypic techniques. Results: Forty-eight (96 %) of 50 traditional cheese samples were harboring Enterococcus spp, including Enterococcus faecalis (n= 40; 83.33 %) and E. faecium (n= 8; 16.67 %). The prevalence of enterococci ranged from 1.1×105 to 9.7×104 CFU/g, and 1.1×103 to 9.8×103 CFU/g in Urmia and Tabriz samples, respectively. Rifampicin resistance (n= 38; 79.2 %) was the most common pattern observed in the susceptibility test, which was followed by quinupristin/dalfopristin (n= 33; 68.75 %). Among E. faecalis isolates, cpd (100 %), ace (92.5 %) and gelE (87.5 %), and among E. faecium isolates, gelE (100 %) and asa1 (75 %) were found to have the most common virulence genes. Conclusion: E. faecalis was the predominant species, displaying more virulence determinants. It also had high antibiotic resistance, as compared to E. faecium. The enterococci identified here commonly expressed virulence and antibiotic resistance determinants. So, it is required to improve the maintenance and production quality of traditional cheese to avoid enterococci contamination.


Assuntos
Queijo , Infecções por Bactérias Gram-Positivas , Animais , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Enterococcus/genética , Enterococcus faecalis/genética , Humanos , Testes de Sensibilidade Microbiana , Prevalência , Virulência , Fatores de Virulência/genética
11.
BMC Oral Health ; 22(1): 354, 2022 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-35974361

RESUMO

BACKGROUND: Enterococcus faecalis and Candida albicans are frequently found in persistent endodontic infection and could remain in dentinal tubules despite intracanal medication with calcium hydroxide (Ca(OH)2), a commonly used medication. Thus, an effective and safe antimicrobial medication against such refractory infection is necessary in endodontic retreatment, so we aimed to test the efficacy of chitosan paste against these microorganisms compared with Ca(OH)2 in root canals of extracted human teeth. METHODS: Thirty-six sterilized human root samples prepared from extracted premolars and upper maxillary incisors were infected with E. faecalis for 14 days, while 32 were infected with C. albicans for 48 h, for mature biofilm formation. The samples were assigned to 6 groups of intracanal medications: Group 1: no medication (negative control); Group 2: 20% Polyethylene glycol (PEG); Group 3: 20% Propylene glycol (PG); Group 4: Ca(OH)2; Group 5: Chitosan + PEG; and Group 6: Chitosan + PG. After 7 days, intracanal surface dentin was harvested using Protaper next, resuspended, serially diluted and spread on Brain-Heart-Infusion agar (for E. faecalis) and Yeast Extract-Peptone-Dextrose agar (for C. albicans) for colony count. Antimicrobial efficacy was determined as percentage of remaining colony forming unit (CFUs) relative to negative control and analyzed using One-way ANOVA and post-hoc Games-Howell test. The significance level was set at 0.05. RESULTS: For E. faecalis, chitosan + PG had significantly higher antibacterial activity than Ca(OH)2 (P = 0.039). Chitosan + PEG and chitosan + PG medication significantly reduced viable bacteria compared with negative control, PEG and PG (P = 0.001, 0.003, 0.024, respectively for chitosan + PEG; P = 0.002, 0.003, 0.014, respectively for chitosan + PG). For C.albicans, chitosan + PEG and chitosan + PG were not significantly different from Ca(OH)2. However, Chitosan + PEG and chitosan + PG, but not Ca(OH)2, showed a significantly lower level of remaining CFUs compared with negative control (P = 0.013 and 0.005, respectively). CONCLUSION: Chitosan paste showed better efficacy in reducing viable E. faecalis biofilm when compared to Ca(OH)2 after 7-day intracanal medication in this in vitro root canal model. It could also significantly reduce viable C. albicans, but was not significantly different from Ca(OH)2.


Assuntos
Anti-Infecciosos , Quitosana , Ágar , Antibacterianos , Biofilmes , Hidróxido de Cálcio/farmacologia , Hidróxido de Cálcio/uso terapêutico , Candida albicans , Quitosana/farmacologia , Quitosana/uso terapêutico , Cavidade Pulpar/microbiologia , Enterococcus faecalis , Humanos , Irrigantes do Canal Radicular/farmacologia , Irrigantes do Canal Radicular/uso terapêutico
12.
J Bacteriol ; 204(9): e0020222, 2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-35920666

RESUMO

Enterococcus faecalis encodes two proteins, AcpA and AcpB, having the characteristics of acyl carrier proteins (ACPs). We report that the acpA gene located in the fatty acid synthesis operon is essential for fatty acid synthesis and the ΔacpA strain requires unsaturated fatty acids for growth. The ΔacpA strain could be complemented by a plasmid carrying a wild-type acpA gene, but not by a plasmid carrying a wild-type acpB gene. Substitution of four AcpA residues for those of AcpB resulted in a protein that modestly complemented the ΔacpA strain and restored fatty acid synthesis, although the acyl chains synthesized were unusually short. IMPORTANCE Enterococcus faecalis, as well as related species, has two genes-acpA and acpB-encoding putative acyl carrier proteins (ACPs). It has been assumed that AcpA is essential for fatty acid synthesis whereas AcpB is involved utilization of environmental fatty acids. We report here the first experimental test of the essentiality of acpA and show that it is indeed an essential gene that cannot be replaced by acpB.


Assuntos
Proteína de Transporte de Acila , Enterococcus faecalis , Proteína de Transporte de Acila/genética , Proteína de Transporte de Acila/metabolismo , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Ácidos Graxos/metabolismo , Ácidos Graxos Insaturados , Óperon
13.
Eur J Pharmacol ; 931: 175161, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-35964657

RESUMO

Chronic wounds are associated with inflammation, infections, and hypoxic environment. Macrophages play a crucial role in wound healing removing bacteria and secreting signal molecules to coordinate tissue repair. Recently, dextran-shelled Oxygen-Loaded NanoDroplets (OLNDs) have been proposed as new tools to counteract hypoxia in chronic wounds. Here we investigated the effects of OLNDs on Enterococcus faecalis (E. faecalis) killing and the secretion of inflammatory and angiogenic factors by murine (BMDM) and human (dTHP-1, differentiated THP-1) macrophages, in normoxia and hypoxia. Both OLNDs and Oxygen-Free NanoDroplets (OFNDs) significantly increased reactive oxygen species production by BMDM in normoxia (4.1 and 4 fold increase by 10% OLNDs and OFNDs, respectively, after 120 min) and hypoxia (3.8 and 4 fold increase by 10% OLNDs and OFNDs respectively) but not by dTHP-1. Moreover, only OLNDs induced nitric oxide secretion by BMDM in normoxia. Consequently, both nanodroplets improved E. faecalis killing by BMDM in normoxia (% of killing OLNDs = 44.2%; p < 0.01; OFNDs = 41.4%; p < 0.05) and hypoxia (% of killing OLNDs = 43.1%; p < 0.01; OFNDs = 37.7%; p < 0.05), while dTHP-1-mediated killing was not affected. The secretion of the inflammatory cytokines (TNFα, IL-6, IL-1ß) induced by E. faecalis infection in dTHP-1 was reduced by both types of nanodroplets, suggesting a novel anti-inflammatory activity of the dextran shell. Instead, the increase of VEGF induced by hypoxia was reduced only by OLNDs. These data provide new knowledge on the effects of OLNDs as innovative adjuvant in chronic wounds healing promoting bacterial killing and reducing inflammation.


Assuntos
Enterococcus faecalis , Oxigênio , Animais , Dextranos , Humanos , Hipóxia , Inflamação , Macrófagos , Camundongos
14.
Exp Parasitol ; 241: 108358, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36030886

RESUMO

Nematode infections affect a significant percentage of the human population worldwide, especially in developing countries. There are a small number of drugs available to treat these infections, with variable outcomes. Therefore, the potential use of probiotics to help control parasitic infections has emerged as a suitable option. The main goal of this work was to assess the antinematodic effect of the probiotic Enterococcus faecalis CECT7121 (EFCECT7121) in vitro and in vivo, using Trichinella spiralis as a nematode model of infection. The in vitro assay showed a reduction in T. spiralis larvae viability of 31.6% when compared with the control group (6.3%) after 48 h incubation with EFCECT7121. Nevertheless, the isolated antimicrobial peptide AP7121 when inoculated at different concentrations did not reveal any larvicidal effect. Different EFCECT7121 treatment schemes in mice were evaluated, and the reduction of the enteral and parenteral burden of T. spiralis was determined. In addition, the protective effect of EFCECT7121 combined with the conventional anthelmintic albendazole (ABZ, 5 mg/kg) was also assessed. The oral administration of EFCECT7121 previous T. spiralis infection produced a reduction in the larvae per gram (LPG) of mice muscle tissue ranging from 32.8 to 47.9% on the 28th day post-infection. ABZ alone and the combination EFCECT7121 + ABZ produced a reduction of the LPG of muscle tissue of 62 and 60.7%, respectively. Results obtained in the current work support the hypothesis that probiotics such as EFCECT7121 have an antinematodic effect, and their combination with conventional anthelmintic drugs may result useful for improving clinical and parasitological outcomes.


Assuntos
Anti-Helmínticos , Infecções por Nematoides , Trichinella spiralis , Triquinelose , Animais , Anti-Helmínticos/farmacologia , Anti-Helmínticos/uso terapêutico , Enterococcus faecalis , Humanos , Larva , Camundongos , Infecções por Nematoides/tratamento farmacológico , Triquinelose/tratamento farmacológico , Triquinelose/parasitologia , Triquinelose/prevenção & controle
15.
J Photochem Photobiol B ; 234: 112547, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36030693

RESUMO

Knowledge of photo-oxidative stress responses in bacteria that survive antimicrobial photodynamic therapy (aPDT) is scarce. Whereas aPDT is attracting growing clinical interest, subsequent stress responses are crucial to evaluate as they may lead to the up-regulation of pathogenic traits. Here, we aimed to assess transcriptional responses to sublethal aPDT-stress and identify potential connections with virulence-related genes. Six Enterococcus faecalis strains were investigated; ATCC 29212, three dental root-canal isolates labelled UmID1, UmID2 and UmID3 and two vancomycin-resistant isolates labelled A1 and A2. TMPyP was employed as a photosensitiser. A viability dose-response curve to increasing concentrations of TMPyP was determined by culture plating. Differential expression of genes involved in oxidative stress responses (dps and hypR), general stress responses (dnaK, sigma-factorV and relA), virulence-related genes (ace, fsrC and gelE) and vancomycin-resistance (vanA) was assessed by reverse-transcription qPCR. TMPyP-mediated aPDT inactivated all strains with comparable efficiencies. TMPyP at 0.015 µM was selected to induce sublethal photo-oxidative stress. Despite heterogeneities in gene expression between strains, transcriptional profiles revealed up-regulations of transcripts dps, hypR as well as dnaK and sigma factorV after exposure to TMPyP alone and to light-irradiated TMPyP. Specifically, the alternative sigma factorV reached up to 39 ± 113-fold (median ± IQR) (p = 0.0369) in strain A2. Up-regulation of the quorum sensing operon, fsr, and its downstream virulence-related gelatinase gelE were also observed in strains ATCC-29212, A1, A2 and UmID3. Finally, photo-oxidative stress induced vanA-type vancomycin-resistance gene in both carrier isolates, reaching up to 3.3 ± 17-fold in strain A2 (p = 0.015). These findings indicate that, while aPDT successfully inactivates vancomycin-resistant and naïve strains of E. faecalis, subpopulations of surviving cells respond by co-ordinately up-regulating a network of genes involved in stress survival and virulence. This includes the induction of vancomycin-resistance genes in carrier isolates. These data may provide the mechanistic basis to circumvent bacterial responses and improve future clinical protocols.


Assuntos
Enterococcus faecalis , Estresse Oxidativo , Fotoquimioterapia , Vancomicina , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Enterococcus faecalis/metabolismo , Enterococcus faecalis/patogenicidade , Testes de Sensibilidade Microbiana , Estresse Oxidativo/fisiologia , Fator sigma/metabolismo , Vancomicina/farmacologia , Virulência/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
16.
mBio ; 13(4): e0111922, 2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-35913163

RESUMO

Enterococcus faecalis is an opportunistic pathogen and a major cause of severe nosocomial infections. Treatment options against enterococcal infections are declining due to the resistance of enterococci to numerous antibiotics. A key risk factor for developing enterococcal infections is treatment with cephalosporin antibiotics, to which enterococci are intrinsically resistant. For susceptible organisms, cephalosporins inhibit bacterial growth by acylating the active site of penicillin-binding proteins (PBPs), key enzymes that catalyze peptidoglycan cross-linking. Two specific PBPs of enterococci, Pbp4(5) and PbpA(2b), exhibit low reactivity toward cephalosporins, allowing these PBPs to cross-link peptidoglycan in the presence of cephalosporins to drive resistance in enterococci, but the mechanisms by which these PBPs are regulated are poorly understood. The CroS/R two-component signal transduction system (TCS) is also required for cephalosporin resistance. Activation of CroS/R by cephalosporins leads to CroR-dependent changes in gene expression. However, the specific genes regulated by CroS/R that are responsible for cephalosporin resistance remain largely unknown. In this study, we characterized CroR-dependent transcriptome remodeling by RNA-seq, identifying pbp4(5) as a CroR regulon member in multiple, diverse lineages of E. faecalis. Through genetic analysis of the pbp4(5) and croR promoters, we uncovered a CroR-dependent regulatory motif. Mutations in this motif to disrupt CroR-dependent upregulation of pbp4(5) in the presence of cell wall stress resulted in a reduction of resistance to cephalosporins in E. faecalis, demonstrating that enhanced production of Pbp4(5) and likely other proteins involved in peptidoglycan biogenesis by the CroS/R system drives enterococcal cephalosporin resistance. IMPORTANCE Investigation into molecular mechanisms used by enterococci to subvert cephalosporin antibiotics is imperative for preventing and treating life-threatening infections. In this study, we used genetic means to investigate the functional output of the CroS/R TCS required for enterococcal resistance to cephalosporins. We found that enhanced production of the penicillin-binding protein Pbp4(5) upon exposure to cell wall stress was mediated by CroS/R and was critical for intrinsic cephalosporin resistance of E. faecalis.


Assuntos
Resistência às Cefalosporinas , Enterococcus faecalis , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Cefalosporinas/farmacologia , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Testes de Sensibilidade Microbiana , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano/metabolismo
17.
J Med Microbiol ; 71(8)2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35972461

RESUMO

Introduction. Vaginal colonization of Group B Streptococcus (GBS) is associated with preterm births and neonatal sepsis. Thus routine screening of GBS in prenatal care is recommended.Hypothesis. Chromogenic media (carrot broth) aids in specific and rapid detection of GBS.Aim. To investigate the efficiency of Strep B Carrot Broth for detection of GBS in high vaginal swabs from pregnant women.Methods. In this study 201 vaginal swab samples were collected from pregnant women. Swabs were inoculated in chromogenic media (Strep B Carrot Broth). The positive and negative cultures were inoculated on blood agar and crome agar plates. The colonies were subjected to 16S rRNA sequencing and gene-specific PCR for confirmation. The Christie Atkins Munch Peterson (CAMP) and bile esculin agar (BEA) tests were used for biochemical confirmation. PCR was performed on genomic DNA isolated from uncultured vaginal swabs.Results. It was found that 20/201 (9.9 %) vaginal swab samples were positive in the Strep B Carrot Broth and 17/20 (85 %) and 19/20 (95 %) of these samples yielded colonies on blood agar and crome agar, respectively. Of the 181 carrot broth-negative samples, 1 (0.5 %) and 38 (20.9 %) yielded colonies on blood agar and crome agar plates, respectively. However, 16 s rRNA sequencing revealed that none of the 20 carrot broth-positive cultures were GBS and had sequence similarities to Enterococcus faecalis. This was also confirmed by using gene-specific PCR and BEA positivity. Furthermore, E. faecalis was detected by PCR in DNA isolated from 57 uncultured vaginal swabs samples, GBS could only be detected by PCR in four samples.Conclusion. Carrot broth-based culture can lead to false-positive detection due to the presence of E. faecalis. Thus GBS-positive results in carrot broth must be confirmed by the other molecular and biochemical tests before making a final diagnosis.


Assuntos
Daucus carota , Complicações Infecciosas na Gravidez , Infecções Estreptocócicas , Ágar , Meios de Cultura/química , Enterococcus faecalis/genética , Feminino , Humanos , Recém-Nascido , Gravidez , Complicações Infecciosas na Gravidez/diagnóstico , RNA Ribossômico 16S/genética , Infecções Estreptocócicas/diagnóstico , Streptococcus agalactiae , Vagina
18.
Sci Rep ; 12(1): 14045, 2022 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-35982076

RESUMO

Protective microbes have a major role in shaping host-pathogen interactions, but their relative importance in the structure of the host microbiota remains unclear. Here, we used a network approach to characterize the impact of a novel, experimentally evolved 'protective microbial symbiont' (Enterococcus faecalis) on the structure and predicted function of the natural microbiota of the model organism Caenorhabditis elegans. We used microbial network analysis to identify keystone taxa and describe the hierarchical placement of protective and non-protective symbionts in the microbiota. We found that early colonization with symbionts produce statistically significant changes in the structure of the community. Notably, only the protective E. faecalis became a keystone taxon in the nematode microbiota. Non-protective lineages of the same bacterial species remained comparatively unimportant to the community. Prediction of functional profiles in bacterial communities using PICRUSt2 showed that the presence of highly protective E. faecalis decreased the abundance of ergothioneine (EGT) biosynthesis pathway involved in the synthesis of the antioxidant molecule EGT, a potential public good. These data show that in addition to direct antagonism with virulent pathogens, keystone protective symbionts are linked to modified bacterial community structure and possible reductions in public goods, potentially driving decreased antioxidant defense. We suggest that this response could suppress infection via wholesale microbial community changes to further benefit the host. These findings extend the concept of protective symbionts beyond bodyguards to ecosystem engineers.


Assuntos
Caenorhabditis elegans , Microbiota , Animais , Antioxidantes , Bactérias/genética , Caenorhabditis elegans/microbiologia , Enterococcus faecalis
19.
Indian J Dent Res ; 33(1): 69-74, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35946248

RESUMO

Aims: To determine the cytokine expression by human gingival fibroblasts in response to different calcium hydroxide (Ca(OH)2) dilutions and test the effectiveness of these dilutions in root canal dentin infected with Enterococcus faecalis (E. faecalis). Methods: UltraCal XS Ca(OH)2 dilutions were prepared (60, 10, and 1 mg\mL) and co-cultured with gingival fibroblasts for 24 and 48 hours. Untreated cells were used as controls. Expressions of interleukin (IL-1ß), tumour necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-ß), and IL-10 were analysed with real-time polymerase chain reaction (PCR). Root canals of extracted human teeth were inoculated with E. faecalis. After 21 days, canals were medicated with Ca(OH)2 dilutions for 7 days. Samples were taken to determine bacterial reduction using quantitative PCR. Analysis of variance, Tukey post-test, and Wilcoxon matched pair test were used for statistics. Results: IL-1ß and TNF-α expressions of all Ca(OH)2 dilutions were higher at 24 and 48 hours compared to the control. Similarly, all Ca(OH)2 dilutions induced TGF-ß expression at 24 hours compared to the control and continued to be higher in 60 mg/mL groups at 48 hours. In contrast, IL-10 was constitutively expressed by untreated cells in the control group and was down-regulated significantly by all Ca(OH)2 dilutions at 24 and 48 hours. All dilutions demonstrated a significant E. faecalis reduction (P < 0.001) with no significant difference between dilution groups (P > 0.05). Conclusions: All Ca(OH)2 dilutions had a differential inflammatory effect on fibroblasts and had a down-regulation effect to IL-10. All dilutions tested were effective against E. faecalis, with 60 mg/mL having the highest bacterial reduction.


Assuntos
Hidróxido de Cálcio , Irrigantes do Canal Radicular , Hidróxido de Cálcio/farmacologia , Hidróxido de Cálcio/uso terapêutico , Clorexidina/farmacologia , Citocinas , Cavidade Pulpar/microbiologia , Dentina , Enterococcus faecalis , Humanos , Interleucina-10/farmacologia , Irrigantes do Canal Radicular/farmacologia , Irrigantes do Canal Radicular/uso terapêutico , Fator de Crescimento Transformador beta/farmacologia , Fator de Necrose Tumoral alfa/farmacologia
20.
Photobiomodul Photomed Laser Surg ; 40(8): 559-564, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35917508

RESUMO

Objective: To carry out a histological and morphometric analysis of the antimicrobial effect of Er:YAG laser irradiation combined with photodynamic therapy (PDT) on root canals infected with Enterococcus faecalis. Background: PDT and Er:YAG laser irradiation may be alternatives for effective endodontic disinfection but there are no data on the combination of these therapies. Materials and methods: Forty single-rooted bovine teeth had their roots contaminated with E. faecalis for 72 h. The teeth were randomly divided into four groups (n = 10): group 1, irrigation with 2.5% sodium hypochlorite (NaOCl); group 2, Er:YAG laser (λ2940 nm, 15 Hz, 100 mJ); group 3, PDT with 0.07% methylene blue as photosensitizer and laser irradiation (λ660 nm, power 40 mW, 5 min); and group 4, Er:YAG laser + PDT. After treatment, the teeth were examined by confocal laser scanning microscopy to verify bacterial viability, and morphometric analysis of the images was performed. Results: The PDT and Er:YAG + PDT treatments promoted the greatest reduction in bacteria among the proposed therapies, whereas 2.5% NaOCl was the least effective in bacterial elimination. A statistically significant difference (p < 0.05) was observed among the groups studied, except between the group combining Er:YAG and PDT and the group treated with PDT alone. Conclusions: PDT combined or not with Er:YAG laser was found to be more effective in root canal disinfection when compared with the other groups.


Assuntos
Anti-Infecciosos , Lasers de Estado Sólido , Fotoquimioterapia , Animais , Anti-Infecciosos/farmacologia , Biofilmes , Bovinos , Cavidade Pulpar , Enterococcus faecalis/efeitos da radiação , Lasers de Estado Sólido/uso terapêutico , Fotoquimioterapia/métodos
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