Estudio in vitro del Efecto Antibacteriano de la Oleorresina de Copaifera reticulata y el Aceite Esencial de Origanum majoricum Frente a Streptococcus mutans y Enterococcus Faecalis Bacterias de Importancia en Patologías Orales / In vitro Study of the Antibacterial Effect of Oleoresin of Copaifera reticulata and the Essential Oil of Origanum majoricum against Streptococcus mutans and Enterococcus faecalis Bacteria of Importance in Oral Pathologies

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.

Incidence of virulence determinants in clinical Enterococcus faecalis and Enterococcus faecium isolates collected in Bulgaria

Abstract Objectives To evaluate the prevalence of some virulence genes among 510 clinical Enterococcus spp. isolates and to assess the association of those genes with the species, infection site, and patient group (inpatients/outpatients). Methods Adhesins genes (aggregation substances agg and asa1 of Enterococcus faecalis and Enterococcus faecium, respectively), enterococcal surface protein (esp), endocarditis-specific antigen A (efaA), collagen-binding proteins (ace/acm)); invasins (hyaluronidase (hyl) and gelatinase (gelE)); cytotoxines (activation of cytolysin (cylA) in E. faecalis); and modulators of the host immunity and inflammation (enhanced expression pheromone (eep) in E. faecalis) were detected by polymerase chain reaction. Results The overall prevalence was: esp – 44.3%, agg/asa1 – 38.4%, ace/acm – 64.3%, efaA – 85.9%, eep – 69.4%, gelE – 64.3%, hyl – 25.1%, and cylA – 47.1%. E. faecalis isolates had significantly higher frequency of adhesin genes (esp and agg/asa1) and gelatinase in comparison to E. faecium. Multiple virulence genes in E. faecalis were significantly more prevalent than in E. faecium isolates. Domination of E. faecium with or without only one gene compared to the isolates of E. faecalis were found. Enterococcus spp. isolates obtained from outpatients compared to inpatients isolates had significantly higher frequency of agg/asa1, eep, gelE and cylA. Some adhesins genes (esp, agg/asa1 and efaA) had higher prevalence among the non-invasive Enterococcus spp. isolates compared to those causing invasive bacteremia, while ace/acm revealed higher dissemination in isolates causing invasive infections compared to non-invasive isolates. Conclusion Most E. faecalis attaches to abiotic surfaces in hospital environment, which correlates with higher prevalence of gene encoding for virulence factors involved in biofilm formation, such as enterococcal surface protein, aggregation substance, and gelatinase. The intestinal tract is an important reservoir for opportunistic enterococcal pathogens and allows them to access infectious sites through different virulence factors, demonstrated in outpatient isolates in this study.

Presence of virulence factors in Enterococcus faecalis and Enterococcus faecium susceptible and resistant to vancomycin

Despite the increasing importance of Enterococcus as opportunistic pathogens, their virulence factors are still poorly understood. This study determines the frequency of virulence factors in clinical and commensal Enterococcus isolates from inpatients in Porto Alegre, Brazil. Fifty Enterococcus isolates were analysed and the presence of the gelE, asa1 and esp genes was determined. Gelatinase activity and biofilm formation were also tested. The clonal relationships among the isolates were evaluated using pulsed-field gel electrophoresis. The asa1, gelE and esp genes were identified in 38%, 60% and 76% of all isolates, respectively. The first two genes were more prevalent in Enterococcus faecalis than in Enterococcus faecium, as was biofilm formation, which was associated with gelE and asa1 genes, but not with the esp gene. The presence of gelE and the activity of gelatinase were not fully concordant. No relationship was observed among any virulence factors and specific subclones of E. faecalis or E. faecium resistant to vancomycin. In conclusion, E. faecalis and E. faecium isolates showed significantly different patterns of virulence determinants. Neither the source of isolation nor the clonal relationship or vancomycin resistance influenced their distribution.

Detección de genes de virulencia en cepas de Enterococcus faecalis susceptibles y resistentes a aminoglucósidos / Detection of virulence genes in aminoglycoside susceptible and resistant Enterococcus faecalis

Background: Enterococcus spp. is an important cause of nosocomial infections A number of virulence factors that may enhance its ability to colonize have been described. Enterococcus is capable of acquiring resistance genes, including high-level resistance (HLR) to aminoglycoside antibiotics. Aim: to investigate the prevalence of genes encoding virulence factors in aminoglycosides susceptible and resistant E. faecalis. Materials and Methods: A total of 80 E. faecalis isolates from clinical (n: 52) and poultry samples (n: 28) were included in this study. Bacterial identification was performed by biochemical tests and phenotypificationwas done using the Phene-PlateTM system. Susceptibility to different antimicrobial agents was determined by the agar dilution method. Virulence genes aceI, agg, gelE and efaA were detected by multiplex PCR. Results: All isolates were susceptible to vancomycin and ampicillin. HLR to gentamicin (13.5%) and streptomycin (9.6%) was detected only in clinical isolates. The phenotyping revealed a great diversity of PhP-types, but only one clone with 7 strains of similar characteristics was found. The efaA gen was detected in 100% of the isolates. aceI gene was present in 94.2% and 75%, agg gene in 73.1%, and 67.9%, and gelE gene in 57.5% and 28.6% of the clinical and chicken isolates, respectively. Only 6 strains with HLR to aminoglycosides, belonging to the same phenotype, had the aceI, agg, gelE and efaA genes. Conclusions: E. faecalis with virulence genes and HLR to aminoglycosides were isolated from clinical and chicken samples in Antofagasta. More studies will be necessary to establish an association.

Antecedentes: Enterococcus spp. es una causa importante de infecciones nosocomiales, tanto en Chile como internacional. Se han descrito una serie de factores de virulencia en este microorganismo, que pueden, por ejemplo, aumentar su habilidad para colonizar. Enterococcus tiene capacidad de adquirir genes de resistencia, entre ellos la resistencia de alto nivel (RAN) a los antimicrobianos aminoglucósidos. Objetivo: Investigar la prevalencia de genes de virulencia en cepas de E. faecalis susceptibles y resistentes a aminoglucósidos. Material y Métodos: Un total de 80 cepas de E. faecalis aisladas de muestras clínicas (n: 52) y pollos (n: 28) se incluyeron en este estudio. La identificación se hizo por pruebas bioquímicas y se tipificaron por el sistema Phene-PlateMR. La susceptibilidad a diferentes antimicrobianos fue realizada por test de dilución en agar. Los genes de virulencia aceI, agg, gelE y efaA fueron investigados por RPC múltiple. Resultados: Todas las cepas de E. faecalis fueron susceptibles a vancomicina y ampicilina. Un 13,5% de las cepas clínicas presentaron resistencia de alto nivel a gentamicina y 9,6% a estreptomicina. La tipificación reveló una gran diversidad de fenotipos, pero se encontró un clon con 7 cepas de características similares. El gen efaA estaba presente en 100% de las cepas, gen aceI en 94,2 y 75%, gen agg 73,1 y 67,9% y gen gelE 57,5 y 28,6% de las cepas clínicas y de pollos, respectivamente. Seis cepas con resistencia de alto nivel a aminoglucósidos, que pertenecían a un mismo fenotipo exhibieron los genes efaA, aceI, agg y gelE juntos. Conclusiones: Cepas de E. faecalis que albergan genes de virulencia y con resistencia de alto nivel a aminoglucósidos fueron aisladas de muestras clínicas y de pollos en Antofagasta. Se requieren mayores estudios para establecer una asociación entre estos factores.

Actividad antibacteriana del Schinus molle l cultivado en Italia / Antibacterial activity of Schinus molle I grown in Italy

El objetivo de esta investigación fue determinar la actividad antibacteriana del extracto etanolico de las hojas y el extracto hexanoico obtenido de los frutos del Schinus molle L. (Anacardiaceae), cultivado en Italia, con un rendimiento de 32,42 % y 5,63 %, respectivamente. Independientemente del tipo de extracto y de la parte de la planta utilizada, el Schinus molle L., mostró una buena actividad antibacteriana contra bacterias Gram positivas (S. aureus ATCC 29213 y E. faecalis ATCC 29212) con una CMI 16 µg/mL. El rango de CBM de estas bacterias estuvo entre 32 y 64 mg/mL, respectivamente.

The aim of this research was to determine antibacterial activity of etanolic extract of the leaves and hexanoic extract obtained from fruits of Schinus molle L. (Anacardiaceae), grown in Italia. with a yield of 32,42 % and 5,63 %, respectively. Whatever type of extract and the plant part used, Schinus molle L., showed good antibacterial activity against Gram positive bacteria (S. aureus ATCC 29213 and E. faecalis ATCC 29212) with MIC 16 mg/mL. Range of MBC of these bacteria were between 32 and 64 mg/mL respectively.

Genes de virulencia y bacteriocinas en cepas de Enterococcus faecalis aisladas desde diferentes muestras clínicas en la Región del Maule, Chile / Virulence genes and bacteriocins in Enterococcus faecalis strains isolated from different clinical samples in Maule Region, Chile

The presence of virulence genes (VG) and bacteriocins from different clinical samples was studied in Enterococcus faecalis isolated from urinary tract infections (UTI), bacteremia and endodontitis and was correlated with haemolysin and gelatinase activity. We evaluated the presence of VG by PCR in 150 strains of E. faecalis including cylA, aggA, efaA, eep, gelE, esp, as-48, bac31, entL50A/B, entA, entP, entB, enlA andentl071. Haemolysin and gelatinase activity was studied. gelE and cylA genes expressed hemolysin and gelatinase, respectively. This activity was observed in some strains of bacteremia, UTI and endodontitis. The highest number of VG was detected in bacteremic strains, being aggA and entA genes the most frequent. efaA, esp, entA, entL50A/B were associated with their clinical origin (p < 0.05). The most common genetic profile was aggA-eep-enlA-entL50A/B. E. faecalis from UTI, bacteremia and endodontitis presented different gene combinations. Some of the genes studied were related to their clinical origin. The results obtained in this study are similar to those reported in other countries.

Desde diferentes muestras clínicas se determinó la presencia de genes codificantes de factores de virulencia (FV) y bacteriocinas en Enterococcus faecalis aislados desde infecciones del tracto urinario (ITU), bacteriemias y endodontitis, correlacionándose con la actividad hemolisina y gelatinasa. En 150 cepas de E. faecalis fue evaluada mediante RPC la presencia de cylA, aggA, efaA, eep, gelE, esp, as-48, bac31, entL50A/B, entA, entP, entB, enlA, y ent1071 determinándose actividad hemolisina y gelatinasa. Los genes cylA y gelE expresaron hemolisina y gelatinasa, respectivamente. Esta actividad fue observada en algunas de las cepas causantes de bacteriemia, ITU y endodontitis. El mayor número de genes estudiados se detectó en cepas bacteriémicas. Los genes aggA y entA, fueron los más frecuentes. Los genes efaA, esp, entL50/AB y entA se asociaron a su origen clínico (p < 0,05). El perfil genético más recurrente fue aggA-eep-enlA-entL50A/B. Enterococcusfaecalis de ITU, bacteriemias y endodontitis presentaron distintas combinaciones génicas. AAlgunos de los genes estudiados se relacionaron con su origen clínico. Los resultados obtenidos son similares a los reportados en otros países.

Frequency of virulence-associated genes in Enterococccus faecalis isolated in Kuwait hospitals

The objective of this study was to investigate the carriage of 6 virulence-associated genes in Enterococcus faecalis isolates obtained from patients in 8 hospitals in Kuwait. In total, 466 E. faecalis isolates were obtained from 313 urine samples, 68 wound swabs, 36 blood samples, 25 rectal swabs, 12 high vaginal swabs and 12 miscellaneous sources. Genes for gelatinase[gelE],aggregation substance [aggA], hemolysin activation factor [cylA], enhanced expression of pheromone [eep], enterococcal surface protein [esp], and E. faecalis endocarditis antigen A [efaA] were detected in PCR assays. Of 466 isolates, 423 [90.8%] were positive for 1 and up to 5 genes. However, none of the genes was detected in all of the isolates. The prevalence of the individual genes was eep: 31.9%; esp: 31.5%; gelE: 28.5%; efaA: 27.9%; aggA: 23.4%, and cylA: 18.5%. Of the 423 positive isolates, 148 [34.9%] were positive for 2 genes and 52 [12.3%], 15 [3.5%] and 5 [0.9%] isolates were positive for 3, 4 and 5 virulence genes, respectively. The efaA and esp combination was detected in isolates from all clinical sources. The study showed a high prevalence of virulence genes in E. faecalis isolated in Kuwait hospitals. The absence of a dominant gene in all of the isolates suggests that infections by E. faecalis may require the involvement of multiple virulence factors

Enterococcus faecalis na endodontia: um desafio ao sucesso: [revisão] / Enterococcus faecalis in endodontics: a challenge to success: [review]

Enterococcus faecalis habitam normalmente na cavidade oral humana e são patógenos oportunistas. Estes micro-organismos são frequentemente detectado sem infecções endodônticas persistentes e mais frequentemente, assintomáticas. Este estudo se propõe a revisar a literatura científica a respeito desta bactéria, apontando e discutindo suas características estruturais, fatores de virulência e sua resistência a antimicrobianos, buscando justificativas para sua alta prevalência nos casos de fracasso do tratamento endodôntico e sua ampla utilização em pesquisa na Endodontia. Pode-se constatar que as principais características que explicam a prevalência desta espécie em casos de fracasso endodôntico são a sua capacidade de formar biofilme e sua elevada resistência ao hidróxido de cálcio. Diante de sua importância, E. faecalis tem sido utilizado como padrão em estudos na Endodontia.

Enterococcus faecalis are normally inhabitants of the human oral cavity, and are opportunistic pathogens. Thesemicroorganisms are frequently detected in asymptomatic persistent endodontic infections. This study proposes to review the scientific bibliography about these bacteria, pointing out and discussing their structural characteristcs,virulence factors and resistance to antibiotics, justifying their prevalence in cases of insucessful root canal treatmentand their extensive use in Endodontics research. The principal characteristics of E. faecalis which explain their high prevalence in insuccessful endodontic therapy are the ability to form biofilm and their low susceptibility to calciumhydroxide. Given their importance, E. faecalis have been used as standard in Endodontics studies.

Mecanismos de resistência do enterococcus / Mechanisms of resistance of the enterococcus

Os Enterococos são cocos gram-positivos com baixo potencial de causar doenças. No entanto, tornaram-se temidos nas últimas décadas devido ao desenvolvimento de cepas bastante resistentes aos aminoglicosídeos, à ampicilina, penicilina e vancomicina. Os locais de infecções enterocócicas mais comuns são as vias urinárias e a corrente sangüínea. O controle e a prevenção dessas infecções é difícil devido ao elevado grau de resistência, o que torna importante a restrição da antibioticoterapia e o controle de pacientes infectados para reduzir a disseminação dessas bactérias

Papel dos Enterococcus SP nos processos infecciosos de natureza endodôntica / Role of Enterococcus SP on infections process of Endodontic origin

Os Enterococcus Faecalis são os microorganismos anaeróbicos facultativos mais comumente isolados nas infecções refratárias à terapia endodôntica. Incluem-se entre os fatores de virulência produzidos por essa espécie a habilidade em formar biofilmes, a capacidade de invandir túbulos dentinários além da atividade proteolítica e o desenvolvimento de resistência aos agentes antimicrobianos. O controle das infecções endodônticas causadas por E. faecalis requer preparo químico-cirúrgico adequado, com utilização de irrigantes em abundância, medicação intracanal e obturação efetiva com materiais dotados de atividade antimicrobiana. Evidências indicam a necessidade de novos estudos acerca do papel das infecções endodônticas na disseminação sistêmica dos E. Faecalis.

in-vitro evaluation of survival of entrococcus faecalis [E.F.] in infected root canals after root canal obturation with or without using of calcium hydroxide

The main goal of root canal therapy is total elimination of bacteria from root canals. Studies have shown that the cleaning and shaping of root canals can not complexly eliminate these bacteria from the root canals. Obturation may kill the bacteria due to entrapment of them into the canals and prevent their access to nutrition. On the other hand, using of Ca [OH][2] before obturation may help to promise a better reduction of bacteria from root canals. The purpose of this study is an in vitro evaluation of antibacterial effect of obturaiton solely, or with using Ca [OH][2] as an intracanal dressing on E.F. in infected human root canals. For this experimental study, 50 single rooted human teeth without significant curvature were chosen. After cutting the crowns and preparing root canals, the samples were sterilized and then contaminated with Entrococcus faecalis bacteria for 5 days. The samples were divided randomly into 4 groups. The first experimental group which included 20 teeth, were filled by Ca [OH][2] for one week before obturation by lateral condensation technique. The second experimental group [20 samples] were obturated by ZOE sealer and gutta percha with lateral condensation for 2 weeks. Five roots considered as positive control were contaminated with bacteria but were not obturated, and 5 other roots were obturated after sterilization without contamination as negative control group. All samples were incubated in 35°C and 10% moisture for 2 weeks. Then the coronal and apical part of roots were removed and the dentin powder were collected from the middle parts by drilling the canals in sterile condition. Two mg of dentin powder was weighed from each root and cultured in blood agar for 48 hours. The results were recorded and compared with each other by Chi-Square test. All samples were positive in culture of positive control group, and negative in culture of negative control group. First experimental group did not show any positive growth after the incubation period and there was only one positive culture in the second experimental group. The findings of this study illustrated no significant difference between the two experimental groups. In other words, the obturation of canals with gutta percha and sealer merely could kill the E.F. in the infected root canals and application of Ca [OH][2] before obturation was not necessary for disinfection of canals

Estudo comparativo da atividade in vitro da apicilina/sulbactam e outros agentes antimicrobianos frente a bactérias isoladas de diversos materiais clínicos / Comparative study of in vitro activity of ampicillin/sulbactam and other antimicrobics aginst bacteria isolated from several clinical specimens

Um total de 4.813 amostras bacterians, recentemente isoladas de diferentes materiais clínicos, foram testadas frente a 33 diferentes antimicrobianos, pelo método da difusão e disco e analisados de acordo com os padrões estabelecidos pelo National Commitee for Clinical Laboratory Standards (NCCLS). Os resultados mostraram que a Ampicilina/Sulbactam apresentou uma ação extremamente eficaz frente à grande maioria das bactérias estudadas, 92,16 por cento de amostras sensíveis, quando comparado com a Ampicilina (54,63 por cento) e com os demais antimicrobianos testados. A Ampiclilina/Sulbactam mostrou-se também bastante eficiente frente a microorganismos fastidiosos, como o H. influenzae e N. gonorrhoeae, com 100 por cento das amostras testadas sensíveis. Concluímos que a Ampicilina/Sulbactam é um antibiótico com potente atividade "in vitro" frente às bactérias mais comumente isoladas de processos infecciosos.