In Vitro Evaluation of Sodium Hypochlorite, Chlorhexidine, Propolis, and Calcium Hydroxide Effect on Lipoteichoic-Acid-Induced Proinflammatory Cytokines Production.

This study aimed to evaluate the effects of sodium hypochlorite (NaOCl), chlorhexidine (CHX), and the glycolic extract of propolis (GEP) as endodontic irrigants and of calcium hydroxide [Ca(OH)2], CHX, or Ca(OH)2 + CHX as intracanal medications on the capacity of the lipoteichoic acid (LTA) of Enterococcus faecalis in macrophages' proinflammatory cytokines production. Freshly extracted 108 human single-rooted teeth were used in this study. The LTA of E. faecalis was standardized in double-distilled pyrogen-free water (250 µg/mL) and inoculated into the specimens subdivided into nine subgroups (n = 12). Cultures of murine macrophages (RAW 264.7) were treated with 30 µL of each sample collected from root canals and incubated (37 °C, 5% CO2) for 24 h. Lastly, anti-TNF-α, anti-IL-6, anti-IP-10, anti-MIP-1α, anti-G-CSF, and anti-IL-1ß DuoSet kits were used to perform an ELISA assay. Data were analyzed using one-way ANOVA and Tukey test (p ≥ 0.05). It was found that 1% NaOCl was the most effective irrigant in reducing the capacity of LTA in cytokines production, followed by 12% GEP and 2% CHX, respectively. Ca(OH)2 + CHX presented the best results when associated with NaOCl or GEP. Thus, NaOCl or GEP associated with Ca(OH)2 + CHX were effective in reducing the capacity of LTA in different macrophages pro-inflammatory cytokines production.

α-IRAK-4 Suppresses the Activation of RANK/RANKL Pathway on Macrophages Exposed to Endodontic Microorganisms.

Periapical lesions are common pathologies affecting the alveolar bone, often initiated by intraradicular lesions resulting from microbial exposure to dental pulp. These microorganisms trigger inflammatory and immune responses. When endodontic treatment fails to eliminate the infection, periapical lesions persist, leading to bone loss. The RANK/RANKL/OPG pathway plays a crucial role in both the formation and the destruction of the bone. In this study, the objective was to inhibit the RANK/RANKL pathway in vitro within exposed Thp-1 macrophages to endodontic microorganisms, specifically Enterococcus faecalis, which was isolated from root canals of 20 patients with endodontic secondary/persistent infection, symptomatic and asymptomatic, and utilizing an α-IRAK-4 inhibitor, we introduced endodontic microorganisms and/or lipoteichoic acid from Streptococcus spp. to cellular cultures in a culture plate, containing thp-1 cells and/or PBMC from patients with apical periodontitis. Subsequently, we assessed the percentages of RANK+, RANKL+, and OPG+ cells through flow cytometry and measured the levels of several inflammatory cytokines (IL-1ß, TNF-α, IL-6, IL-8, IL-10, and IL-12p70) in the cellular culture supernatant through a CBA kit and performed analysis by flow cytometry. A significant difference was observed in the percentages of RANK+RANKL+, OPG+ RANKL+ cells in thp-1 cells and PBMCs from patients with apical periodontitis. The findings revealed significant differences in the percentages of the evaluated cells, highlighting the novel role of the IRAK-4 inhibitor in addressing this oral pathology, apical periodontitis, where bone destruction is observed.

Identification of Enterococcus spp. by MALDI-TOF mass spectrometry isolated from clinical mastitis and bulk tank milk samples.

BACKGROUND: Throughout a three-year study period, 1,577 bovine clinical mastitis samples and 302 bulk tank samples were analyzed from ten Brazilian dairy herds. Enterococcus spp. was isolated and identified in 93 (5.9%) clinical mastitis samples. In addition, 258 Enterococcus spp. were isolated from the bulk tank samples of the same herds. The identification of Enterococcus spp. isolated from bulk tanks and milk samples of clinical mastitis were accomplished by phenotypic characteristics and confirmed by MALDI-TOF Mass Spectrometry (MS). Fisher test was performed to verify the difference between bulk tanks and mastitis samples. RESULTS: The following species were identified from clinical mastitis: E. saccharolyticus (62.4%), E. faecalis (19.4%), E. faecium (15.1%), E. hirae (1.1%), E. mundtii (1.1%), E. durans (1.1%). Furthermore, from 258 bulk tank milk samples, eight enterococci species were isolated: E. faecalis (67.8%), E. hirae (15.1%), E. faecium (4.6%), E. saccharolyticus (4.6%), E. mundtii (3.1%), E. caseliflavus ( 2.7%), E. durans (1.2%), E. galinarum (0.8%). CONCLUSIONS: The difference in species predominance in bulk tank samples (67.8% of E. faecalis) and clinical mastitis (62.4% of E. saccharolyticus) was unexpected and caught our attention. Although Enterococcus spp. are traditionally classified as an environmental mastitis agent, in the present study, E. saccharolyticus behaved as a contagious agent of mastitis, which consequently changed the control patterns to be implemented.

Reducing Apical Bacterial Extrusion: The Impact of Reciproc File Size and Irrigation Technique.

Introduction: This study used different irrigation techniques to compare the levels of apical bacterial extrusion during the preparation of root canals with a reciprocating instrument widely used in endodontics, the Reciproc files 25/0.08 and 40/0.06. Materials and Methods: The irrigation techniques employed were conventional syringe irrigation and passive ultrasonic irrigation (PUI); the latter, with one or two activation cycles. Seventy extracted mandibular human premolars were contaminated with Enterococcus faecalis for 5 days and were distributed into 6 experimental groups (n=10), and the remaining specimens were used as positive and negative control groups (n=5). Group 1: instrumentation performed with Reciproc 25/0.08 and conventional syringe irrigation; Group 2: instrumentation performed with Reciproc 25/0.08 along with PUI for one minute after instrumentation (PUI-1); and Group 3: instrumentation performed with Reciproc 25/0.08 along with PUI for one minute before and after instrumentation (PUI-2). Groups 4, 5 and 6 were instrumented with Reciproc 40/0.06, and irrigation was performed similar to the previous groups, in the aforementioned order. Each root canal was irrigated with saline solution. Extruded debris was collected in microtubes. The contents of the microtubes were homogenized, diluted, and spread on Brain Heart Infusion agar. After 48 hours, the number of colony-forming units was determined for each sample. For statistical analysis, the Kruskal-Wallis test followed by the Dunn's tests were used (α=0.05). Results: The CFU/mL count indicated that the instrumentation with Reciproc 25/0.08 was associated with the highest bacterial extrusion, mainly when PUI was performed (P<0.05). Conclusion: All the instrumentation techniques caused bacterial extrusion through the apical foramen; however, the largest file size of the Reciproc 40/0.06 groups was associated with less apical bacterial extrusion.

Cytocompatibility and Antibiofilm Activity of Calcium Hydroxide Mixed with Cyperus articulatus Essential Oil and Bio-C Temp Bioceramic Intracanal Medicament.

Calcium hydroxide represents the most commonly used intracanal dressing between sessions; however, it may not be effective against all types of microorganisms. Several compounds of plant origin have attracted increasing attention from researchers in recent years. The objective of this study was to evaluate the cytocompatibility and antimicrobial activity of calcium hydroxide associated with the essential oil of Cyperus articulatus and the new bioceramic intracanal medicament Bio-C Temp®. Five experimental groups were designed: group Ca-C. articulatus essential oil; group CHPG-calcium hydroxide associated with propylene glycol; group CHCa-essential oil of C. articulatus associated with calcium hydroxide; and group U-UltraCal® XS; group BCT-Bio-C Temp®. The control group was a culture medium. Cytocompatibility was assessed by the methyltetrazolium (MTT) assay after exposure of the Saos-2 human osteoblast-like cell line to dilutions of commercial products/associations for 24 h and 72 h. The antimicrobial activity against mature Enterococcus faecalis biofilm was evaluated by the crystal violet assay. All commercial products/associations showed a cell viability similar to or even higher than the control group (p > 0.05) for both periods evaluated. C. articulatus essential oil associated or not with calcium hydroxide showed better antibiofilm capacity. C. articulatus associated or not with calcium hydroxide showed superior cytocompatibility and antimicrobial capacity, representing a promissory intracanal medicament.

Enterococcus faecalis: implications for host health.

The microbiota represents a crucial area of research in maintaining human health due to its potential for uncovering novel biomarkers, therapies, and molecular mechanisms relevant to population identification and experimental model characterization. Among these microorganisms, Enterococcus faecalis, a Gram-positive bacterium found in the gastrointestinal tract of humans and animals, holds particular significance. Strains of this bacterial species have sparked considerable debate in the literature due to their dual nature; they can either be utilized as probiotics in the food industry or demonstrate resistance to antibiotics, potentially leading to severe illness, disability, and death. Given the diverse characteristics of Enterococcus faecalis strains, this review aims to provide a comprehensive understanding of their impact on various systems within the host, including the immunological, cardiovascular, metabolic, and nervous systems. Furthermore, we summarize the bacterium-host interaction characteristics and molecular effects to highlight their targets, features, and overall impact on microbial communities and host health.

Diacetylcurcumin: a novel strategy against Enterococcus faecalis biofilm in root canal disinfection.

Aim: We evaluated Diacetylcurcumin (DAC), a derivative of curcumin, for its antibacterial and antibiofilm properties against Enterococcus faecalis. Methods: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration were determined, along with antibiofilm potential and toxicity in Galleria mellonella. Additionally, in silico computational analysis was performed to understand its mechanisms of action. Results & conclusion: DAC demonstrated significant antibacterial effects, with MIC and MBC values of 15.6 and 31.25 µg/ml, respectively, and reduced biofilm formation. A synergistic effect, reducing biofilm by 77%, was observed when combined with calcium hydroxide. G. mellonella toxicity tests confirmed DAC's safety at tested concentrations, suggesting its potential for use in root canal disinfection products.

Diacetylcurcumin (DAC) comes from turmeric, a natural spice often used in food. DAC may have the ability to fight germs, including the bacteria Enterococcus faecalis. We tested DAC's ability to kill E. faecalis and stopping the formation of films of the bacteria. We found that a small amount of DAC did kill E. faecalis. When used with calcium hydroxide, DAC works even better to reduce the formation of bacterial films by 77%. DAC is safe to be used on teeth, so may be a useful ingredient for preserving mouth health.

Comparación In vitro de la eficacia antibacteriana de dos medicaciones intraconducto hidróxido de calcio y quitosano / In Vitro comparison of the antibacterial efficacy of two intracanal medications calcium hydroxide and chitosan

Resumen Objetivo: Determinar cuál de estas medicaciones intraconductos (hidróxido de calcio, quitosano e hidróxido de calcio+quitosano), muestra más efecto antibacteriano en la sensibilidad de la bacteria Enterococcus faecalis. Metodología: Se realizó un estudio in vitro por la técnica de susceptibilidad antimicrobiana aplicando el método macrodilución con el objeto de establecer si una concentración de los compuestos tres grupos: Grupo 1: hidróxido de calcio. Grupo 2: quitosano. Grupo 3: hidróxido de calcio+quitosano posee actividad antibacteriana ante la bacteria Enterococcus faecalis y para apoyar esta prueba se realizó la técnica antibiograma por medición de halos de inhibición con el objetivo de determinar la sensibilidad de Enterococcus faecalis a las medicaciones intraconducto hidróxido de calcio y quitosano. Resultados: Existen diferencias estadísticamente significativas en las medicaciones intraconducto de hidróxido de calcio, quitosano e hidróxido de calcio+quitosano frente a la bacteria E. faecalis, mostrando mejor resultado cuando se utiliza el quitosano solo, en la unidad formadora de colonias y en la técnica de antibiograma por halos de inhibición mostro mejores resultados. Limitaciones: No se presentó alguna limitación durante el presente estudio. Valor: No se ha realizado un estudio similar en la Escuela Militar de Graduados de Sanidad. Conclusiones: El quitosano es efectivo contra Enterococcus faecalis, puede reducir significativamente la cantidad de bacterias en una dilución baja de 100 mg/ml, por lo tanto podemos concluir que el quitosano es un candidato prometedor como agente antibacteriano biocompatible alternativo y puede utilizarse como medicación intraconducto a futuro en tratamientos de conductos.

Abstract Objective: Determine which of these intracanal medications (calcium hydroxide, chitosan, and calcium hydroxide+chitosan), shows more antibacterial effect on the sensitivity of Enterococcus faecalis bacterium. Methodology: An in vitro study was carried out using the antimicrobial susceptibility technique applying the microdilution method in order to establish whether a concentration of the compounds was in three groups: Cluster 1: calcium hydroxide. Cluster 2: Chitosan. Cluster 3: calcium hydroxide + chitosan has antibacterial activity against the Enterococcus faecalis bacterium and to support this test, the antibiogram technique was performed by measuring inhibition halos to determine the sensitivity of E. faecalis to intracanal medications calcium hydroxide and chitosan. Results: There are statistically significant differences in the intracanal medications of calcium hydroxide, chitosan, and calcium hydroxide+chitosan against the bacterium Enterococcus faecalis, showing better results when chitosan is used alone, in the colony-forming unit and in the antibiogram technique for inhibition halos showed better results. Limitations: Due to the shortage of Ketamine in hospital units, it is difficult to replicate the study in larger samples. Value: A similar study has not been carried out at the Escuela Militar de Graduados de Sanidad. Conclusions: Chitosan is effective against E. faecalis, it can significantly reduce the number of bacteria at a low dilution of 100 mg/ml, therefore we can conclude that chitosan is a promising candidate for an alternative biocompatible antibacterial agent and can be used as a intracanal medication in root canal treatments.

The effect of root canal preparation tapers on planktonic bacteria and biofilm reduction in the apical third: A correlative microtomography and microbiological laboratory study.

AIM: To evaluate the influence of different preparation tapers on the reduction in planktonic bacteria and biofilms of Enterococcus faecalis and Candida albicans in the apical third (4 mm) of the mesial roots of mandibular molars, correlating decontamination with canal shape. METHODOLOGY: After microtomography analysis for morphological standardization of the canals, 48 mandibular molar roots, each containing two canals (96 canals), were contaminated with E. faecalis and C. albicans and divided into four groups (n = 11) for canal instrumentation using ProDesign Logic 2 files with different tapers G (.03): # 25.03; G (.04): # 25.04; G (.05): # 25.05; and G (.06): # 25.06 and irrigation with 2.5% sodium hypochlorite. Four roots were examined under a scanning electron microscope (SEM) to qualitatively assess biofilm formation. Eight roots were used as the negative control group (samples were not contaminated). Bacteriological samples were taken exclusively from the apical third of the roots before and after chemical-mechanical preparation and bacterial counts were determined (CFU/mL). The final micro-CT scan was used to quantify the volume variation and unprepared canal area in the apical third. Statistical analysis was performed using the Kruskal-Wallis, Student-Newman-Keuls and Wilcoxon tests for analysis of microbiological data. anova and the Tukey or Games-Howell test were used for analysis of micro-CT data and Spearman's test for correlations (α = 5%). RESULTS: All groups showed a significant reduction in bacteria (p < .05), with no statistically significant difference between groups. There was no significant difference in per cent volume increase between groups. The unprepared area (Δ%) was affected by the file used (p = .026) and was significantly lower for G (.06) compared to G (.03). There was no statistically significant correlation among bacterial reduction, volume and unprepared area (p > .05). CONCLUSION: The different preparation tapers influenced root canal shaping in the apical third but did not improve decontamination in this region.

Comparación de la actividad antimicrobiana de cementos selladores en endodoncia / Comparison of the antimicrobial activity of sealing cements in endodontics

Introducción: la persistencia de microorganismos en los conductos radiculares es uno de los principales factores del fracaso endodóncico. Por ello la importancia de conocer las propiedades antimicrobianas de los distintos tipos de selladores. Objetivo: realizar una comparación con base en la evidencia disponible sobre la actividad antimicrobiana de los diferentes cementos selladores en endodoncia. Material y métodos: la información fue recopilada de las bases de datos PubMed y Google Académico en el idioma inglés y español, publicados en el periodo 2014-2023. Resultados: un gran número de microorganismos se encuentran presentes en los diferentes tipos de infecciones de origen endodóncico, se han identificado más de 500 especies microbianas, entre ellas bacterias, hongos, arqueas y virus. Los cementos selladores se pueden clasificar según su composición química, en cementos a base de óxido de zinc-eugenol, hidróxido de calcio, a base de ionómero de vidrio, silicona, resina y biocerámicos. Conclusión: los cementos selladores que mostraron mayor actividad antimicrobiana contra los microorganismos persistentes fueron los cementos a base de óxido de zinc-eugenol, resina y biocerámicos. Sin embargo, se identificó que cada autor utilizó diferentes métodos y tiempos, por lo tanto, no es posible lograr definir con exactitud qué cemento sellador posee la mejor capacidad antimicrobiana (AU)

Introduction: the persistence of microorganisms in root canals is one of the main factors of endodontic failure. Therefore, the importance of knowing the antimicrobial properties of the different types of sealants. Objective: to make a comparison based on the available evidence on the antimicrobial activity of the different endodontics sealers. Material and methods: the information was collected from PubMed and Google Academic databases in English and Spanish, published in the period 2014-2023. Results: a large number of microorganisms are present in the different types of infections of endodontic origin, more than 500 microbiological species have been identified, including bacteria, fungi, archaea and viruses. Sealer cements can be classified according to their chemical composition, into cements based on zinc oxide-eugenol, calcium hydroxide, based on glass ionomer, silicone, resin and bioceramics. Conclusion: sealer cements that showed the highest antimicrobial activity against persistent microorganisms were zinc oxide-eugenol, resin, and bioceramic-based cements. However, it was identified that each author used different methods and times, therefore, it is not possible to accurately define which sealer cement has the best antimicrobial capacity (AU)

Terapia fotodinámica, una nueva tendencia en endodoncia para la eliminación del Enterococcus faecalis / Photodynamic therapy, a new trend in endodontics for the removal of Enterococcus faecalis / Terapia fotodinâmica, uma nova tendência na endodontia para a remoção de Enterococcus faecalis

RESUMEN La desinfección se consigue mediante procedimientos como la instrumentación, la irrigación y la medicación intraconducto; sin embargo, estos no son suficientes, ya que diversos estudios han reportado al E. faecalis como uno de los microorganismos persistentes con mayor prevalencia en tratamientos de conductos que no han conseguido la curación de los tejidos periapicales. Se ha tratado de mejorar los protocolos de desinfección incluyendo diferentes herramientas tecnológicas como en el caso de la terapia fotodinámica, la cual utiliza una fuente de luz y sustancias fotosensibilizantes que favorecen el proceso de eliminación de las bacterias remanentes dentro del sistema de conductos radiculares. La presente revisión de literatura científica profundiza en la importancia clínica de la terapia fotodinámica y su efecto en la desinfección e inhibición del E. faecalis dentro del sistema de conductos, lo cual se ha vuelto una pieza clave para el éxito del tratamiento endodóntico.

ABSTRACT Disinfection is achieved through procedures such as instrumentation, irrigation, and intra-canal medication; however, these are not enough since several studies have reported E. faecalis as one of the most prevalent persistent microorganisms in root canal treatments that have not achieved healing of the periapical tissues. Efforts have been made to improve disinfection protocols by including different technological tools, as in the case of photodynamic therapy, which uses a light source and photosensitizing substances that favor the process of elimination of the remaining bacteria within the root canal system. The present review of scientific literature delves into the clinical importance of photodynamic therapy and its effect on the disinfection and inhibition of E. faecalis within the root canal system, which has become a key element for the success of endodontic treatment.

RESUMO A desinfeção é conseguida através de procedimentos como a instrumentação, a irrigação e a medicação intracanal; no entanto, isto não é suficiente, uma vez que vários estudos relataram a E. faecalis como um dos microrganismos persistentes mais prevalentes nos canais radiculares que não conseguiram cicatrizar os tecidos periapicais. Têm sido feitos esforços para melhorar os protocolos de desinfeção através da inclusão de diferentes ferramentas tecnológicas, como a terapia fotodinâmica, que utiliza uma fonte de luz e substâncias fotossensibilizadoras que favorecem o processo de eliminação das bactérias remanescentes no sistema de canais radiculares. Esta revisão da literatura científica analisa a importância clínica da terapia fotodinâmica e o seu efeito na desinfeção e inibição da E. faecalis no interior do sistema de canais radiculares, o que se tornou um fator chave para o sucesso do tratamento endodôntico.

Antimicrobial activity of Er, Cr: YSGG and Ultrasonic on E. faecalisbiofilm in the mesial root canal systems of lower molars / Atividade antimicrobiana de Er, Cr: YSGG e Ultrassônico no biofilme de E. faecalis nos sistemas de canais radiculares mesiais de molares inferiores

The internal topography of the root canal is complex, especially for the permanent molar's mesial root. In response to such issues, improved irrigation techniques have been created, which use laser pulses to agitate fluids and improve microbial deposit removal. Objective: To assess the effectiveness of the Er,Cr:YSGG laser with a wavelength of 2,780 nm via photon-induced photoacoustic streaming (PIPS) protocol which agitated of 2% chlorohexidine (CHX) in removing mature Enterococcus faecalis (E. faecalis) biofilm in root canal systems of lower molars. Material and Methods: The mesial roots of lower first and second molars were separated and inoculated with E. faecalis bacterial suspension for 30 days. The roots were irrigated with CHX, some of them were agitated with a passive ultrasonic device (PUI), while the other roots were agitated by an Er,Cr:YSGG laser in PIPS at 60 µs/pulse, 5 Hz, (0.25, 0.5, 0.75, and 1) W. An atomic force microscope (AFM) was used as a new method to get the results in the isthmus area; the obtained results from each group were compared with each other. Results: Based on the AFM and SEM analyses, laser and ultrasonic activation groups showed higher antimicrobial efficacy than the conventional syringe irrigation group (P<0.05). Conclusion: Based on the investigation's findings, the activation of 2% CHX solution by Er,Cr:YSGG laser in PIPS and PUI offers better mature bacterial biofilm removal in the mesial root of lower human molars than the same irrigant with the SI technique (AU)

A topografia interna do canal radicular é complexa, especialmente para a raiz mesial do molar permanente. Em resposta a esses problemas, foram criadas técnicas aprimoradas de irrigação, que utilizam pulsos de laser para agitar fluidos e melhorar a remoção de depósitos microbianos. Objetivo: Avaliar a eficácia do laser Er,Cr:YSGG com comprimento de onda de 2.780 nm via protocolo de streaming fotoacústico induzido por fótons (PIPS) que agitou clorohexidina a 2% (CHX) na remoção de Enterococcus faecalis maduro (E. faecalis) biofilme em sistemas de canais radiculares de molares inferiores. Material e Métodos: As raízes mesiais de 28 primeiros e segundos molares inferiores foram separadas e inoculadas com suspensão bacteriana de E. faecalis por 30 dias. As raízes foram irrigadas com CHX, sendo algumas delas agitadas com aparelho ultrassônico passivo (PUI), enquanto as demais raízes foram agitadas com laser Er,Cr:YSGG em PIPS a 60 µs/pulso, 5 Hz (0,25, 0,5, 0,75 e 1) W. Um microscópio de força atômica (AFM) foi utilizado como um novo método para obter os resultados na área do istmo; os resultados obtidos de cada grupo foram comparados entre si. Resultados: Com base nas análises de AFM e SEM, os grupos de ativação por laser e ultrassom apresentaram maior eficácia antimicrobiana do que o grupo de irrigação com seringa convencional (P<0.05). Conclusão: Com base nos achados da investigação, a ativação da solução de CHX a 2% pelo laser Er,Cr:YSGG em PIPS a (60 µs/pulso, 5 Hz, 0,75 W) oferece melhor remoção de biofilme (AU)

Emergence of optrA-mediated linezolid resistance in clinical isolates of Enterococcus faecalis from Argentina.

OBJECTIVES: The aim of this study was to characterize the first 14 optrA-carrying linezolid resistant E. faecalis clinical isolates recovered in seven Argentinian hospitals between 2016 and 2021. The epidemiology of optrA-carrying isolates and the optrA genetic context were determined. METHODS: The isolates were phenotypically and genotypically characterized. Susceptibility to 13 antimicrobial agents was performed; clonal relationship was assessed by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Data provided by the whole-genome sequencing were used for identification of sequence types, antimicrobial resistance genes, optrA variants, phylogenetic tree, and mobile genetic elements responsible to the dissemination of these strains. RESULTS: All the optrA-carrying E. faecalis isolates were multidrug-resistant and harboured several antimicrobial resistance genes. They carried three optrA variants and belonged to different lineages; however, three of them belonged to the hyperepidemic CC16. Mobile genetic elements were detected in all the isolates. The analysis of the optrA flanking region suggests the plasmidic localization in most of the isolates. CONCLUSIONS: To the best of our knowledge, this is the first report of optrA-mediated linezolid resistance in Argentina. The emergence and dissemination of the optrA genes in clinical E. faecalis isolates are of concern and highlights the importance of initiating the antimicrobial surveillance of Enterococcus spp. under a One Health strategy.

Propylene Glycol Potentiates the Inhibitory Action of CTZ Paste on Antibiotic-Resistant Enterococcus faecalis Isolated from the Root Canal: An In Vitro Study.

This study aimed to evaluate if the change of vehicle for CTZ (Chloramphenicol, Tetracycline, zinc oxide, and Eugenol) paste improves the inhibition of Enterococcus faecalis in vitro. The vehicles evaluated alone and mixed with CTZ were Eugenol, propylene glycol (PG), super-oxidized solution (SOS), grapefruit-seed extract (GSE), and 0.9% saline solution as a negative control. A clinical isolate of E. faecalis was morphologically and biochemically characterized, and its antimicrobial susceptibility was tested using 20 antimicrobial agents. Once characterized, the clinical isolate was cultivated to perform the Kirby-Bauer disc diffusion method with paper discs embedded with the different vehicles mixed or used alone, and incubated at 37 °C for 24 h. Data were analyzed using one-way ANOVA, and the means were compared using Tukey test with a significance level of p < 0.05. For vehicles used alone, GSE presented the greatest inhibition showing a statistically significant difference with the rest of the vehicles. When vehicles were mixed with the CTZ paste, PG showed a greater inhibition with a statistically significant difference from the rest of the vehicles. In conclusion, the vehicle used to mix the CTZ paste plays an important role in the inhibition of E. faecalis in vitro; therefore, we consider that this can be an important factor to achieve success in the use of this technique.

Alpha-Naphthoflavone as a Novel Scaffold for the Design of Potential Inhibitors of the APH(3')-IIIa Nucleotide-Binding Site of Enterococcus faecalis.

The spread of nosocomial infections caused by antibiotic-resistant Enterococcus faecalis is one of the major threats to global health at present. While aminoglycosides are often used to combat these infections, their effectiveness is reduced by various resistance mechanisms, including aminoglycoside modifying enzymes, and there are currently no drugs to inhibit these enzymes. To address this issue, this study was conducted to identify potential aminoglycoside adjuvants from a database of 462 flavones. The affinity of these molecules with the nucleotide-binding site (NBS) of aminoglycoside phosphotransferase type IIIa of E. faecalis (EfAPH(3')-IIIa) was evaluated, and the five molecules with the highest binding energies were identified. Of these, four were naphthoflavones, suggesting that their backbone could be useful in designing potential inhibitors. The highest-ranked naphthoflavone, 2-phenyl-4H-benzo[h]chromen-4-one, was modified to generate two new derivatives (ANF2OHC and ANF2OHCC) to interact with the NBS similarly to adenine in ATP. These derivatives showed higher binding free energies, better stability in molecular dynamics analysis and superior pharmacokinetic and toxicological profiles compared to the parent molecule. These findings suggest that these alpha-naphthoflavone derivatives are potential inhibitors of EfAPH(3')-IIIa and that this core may be a promising scaffold for developing adjuvants that restore the sensitivity of aminoglycosides.

The Emergence of Antimicrobial Resistance and Virulence Characteristics in Enterococcus Species Isolated from Bovine Milk.

Enterococcus spp., including E. faecalis and E. faecium, pose risks to dairy farms as opportunistic pathogens. The study evaluates antimicrobial resistance (AMR) and virulence characteristics of Enterococcus spp. isolated from bovine milk. Bile esculin agar was used to assess 1471 milk samples, followed by colony identification, gram staining, catalase tests, and 45 °C incubation. PCR analysis targeted E. faecalis and E. faecium in characteristic Enterococcus spp. colonies, with MALDI-TOF used for negative samples. Multiple tests, including disk diffusion, chromogenic VRE agar for vancomycin resistance, Vancomycin Etest® for MIC determination, and PCR for virulence factors (cylA, esp, efaA, ace, asa1, gelE, and hyl genes), were performed. Out of 100 identified strains, E. durans (30.66%), E. faecium (26.28%), and E. faecalis (18.25%) were predominant. AMR in Enterococcus spp. varied, with the highest rates against rifampicin (27%), tetracycline (20%), and erythromycin (18%). Linezolid (5%), vancomycin, ciprofloxacin, and teicoplanin (3% each) had lower prevalence. E. faecium and E. faecalis showed high AMR to rifampicin, erythromycin, and tetracycline. Thirty-two strains (18.98%) grew on VRE Chromoselect agar, while 4 (2 E. faecalis and 2 E. faecium) showed vancomycin resistance by MIC values. E. faecalis carried gelE (45.5%) and asa1 (36%), and E. gallinarum had 9.1% with the asa1 gene. Detecting resistant Enterococcus in bovine milk supports control strategies for enterococci on dairy farms, highlighting AMR concerns in the food chain.

Elimination of E. faecalis and C. albicans Biofilm: A comparison between single and multiple file systems in an Ex Vivo study

To evaluate whether the WaveOne Gold and Reciproc single file instrumentation systems, are effective in reducing the microbial load of a mixed biofilm and the cleaning of apical third compared to the Twisted File Adaptive system (multiple- file system). Seventy mesial roots of the first and second molars were included and randomly divided into three experimental groups (n=20, n=10 controls). Biofilms were formed inside canals over 31 days. After instrumentation with the unique file systems, WaveOne Gold and Reciproc and the multiple file system Twisted File Adaptive, using 2.25% sodium hypochlorite as an irrigant in all cases, a count of colony forming units was performed using serial dilutions, cleaning of the apical third was evaluated using scanning electron microscopy. Comparisons amongst groups were made by using parametric and non-parametric statistics, according to a normal or non-normal data distribution, respectively. No significant differences in the reduction of the microbial load after employing a single-file system in comparison to the multiple-file system were found; in addition, the cleaning of the apical third was similar for the three different instrumentation systems. The single-file system is equal in effectiveness compared with the multiple-file system in reducing the microbial load.

Evaluar si los sistemas de instrumentación de lima única, como WaveOne Gold y Reciproc son efectivos para reducir la carga microbiana de un biofilm mixto y la limpieza del tercio apical, comparado con los sistemas de limas múltiples, como Twisted File Adaptive. Setenta raíces mesiales de primeros y segundos molares fueron incluidos y divididos de forma aleatoria en tres grupos experimentales (n=20, n=10 controles). El biofilm fue formado en el interior de los conductos durante 31 días. Después se instrumentó con los sistemas de lima única (WaveOne Gold y Reciproc) y el sistema de limas múltiples Twisted File Adaptive, usando hipoclorito de sodio al 2.5% en todos los casos. El conteo de unidades formadoras de colonias se realizó usando diluciones seriales, la limpieza del tercio apical se evaluó empleando el microscopio electrónico de barrido. La comparación entre grupos se realizó con pruebas paramétricas y no paramétricas, de acuerdo con la distribución normal y no normal de los datos, respectivamente. No hubo una diferencia significativa en la reducción de la carga microbiana después de emplear los sistemas de lima única en comparación a los de limas múltiples, además, la limpieza del tercio apical fue similar en los 3 diferentes sistemas de instrumentación. Los sistemas de lima única son igual de efectivos para reducir la carga microbiana comparados con los sistemas de limas múltiples.

Interspecies interactions in mixed-species biofilms formed by Enterococcus faecalis and gram-negative bacteria isolated from polymicrobial diabetic foot ulcers.

Diabetic foot ulcers (DFU) are exacerbated by bacterial colonisation. Here, a high prevalence of Enterococcus faecalis was observed in DFU patients from an Argentinean hospital. E. faecalis was frequently co-isolated with Escherichia coli, Morganella morganii, and Pseudomonas aeruginosa. The effect of interspecies interactions on bacterial growth was investigated in mixed-species macrocolony biofilms developed in Lubbock-Glc-agar. Similar cell counts were found for E. faecalis and M. morganii growing in mixed and single-species biofilms. An E. faecalis strain showed 1 Log higher cell counts in mixed biofilms with E. coli. Remarkably, E. faecalis strains showed 2 to 4 Log higher cell counts in mixed biofilms with P. aeruginosa. This effect was not observed in planktonic growth or biofilms developed in tryptic soy agar. The present findings reveal bacterial interactions that benefit E. faecalis in mixed-species biofilms, mainly with P. aeruginosa, in a medium that partially mimics the nutrients found in DFU.

Evaluation of antibacterial and antioxidant activity of purple araçá essential oil (Psidium rufum, Myrtaceae) / Avaliação das atividades antibacteriana e antioxidante do óleo essencial do araçá-roxo (Psidium rufum, Myrtaceae)

The aim of this study was to evaluate the antibacterial and antioxidant activities of essential oil (EO) from fresh leaves of Psidium rufum. The EO was extracted by hydrodistillation and identified by gas chromatography coupled to mass spectrometry. The antibacterial activity was evaluated by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Antioxidant activity was determined by ß-carotene/linoleic acid co-oxidation system, 2,2-diphenyl-1-picrylhydrazyl radical scavenging and iron reduction methods. Hydrocarbon sesquiterpenes were the predominant class, indicating 1,8 cineole, α-longipinene as major. The EO was tested against the bacteria Staphylococcus aureus and Pseudomonas aeruginosa (MIC = 2,500 µg/mL and MBC = 20,000 µg/mL); Enterococcus faecalis (MIC = 2,500µg/mL and MBC > 20,000µg/mL) and Escherichia coli (MIC > 20,000µg/mL and MBC > 20,000µg/mL). The EO showed antioxidant potential due to ß-carotene/linoleic acid co-oxidation system, with 76.63% of oxidation inhibition (1.0mg/mL) and due to the iron reduction power (5,38 µmol Fe 2+ /mg sample). The results are promising in recommending this species for the development of food, cosmetic and pharmaceutical products.

O objetivo deste estudo foi avaliar as atividades antibacteriana e antioxidante do óleo essencial (OE) das folhas frescas de Psidium rufum. O OE foi extraído por hidrodestilação e identificado por cromatografia gasosa acoplada à espectrometria de massas. Foi avaliada a atividade antibacteriana, determinando-se a concentração inibitória mínima (CIM) e a concentração bactericida mínima (CBM). A atividade antioxidante foi determinada pelo sistema de co-oxidação ß-caroteno/ácido linoleico, pelos métodos de sequestro do radical 2,2-difenil-1-picrilhidrazil e de redução do ferro. Sesquiterpenos hidrocarbonetos foram a classe predominante, indicando 1,8 cineol, α-longipineno como majoritário. O OE foi testado contra as bactérias Staphylococcus aureus e Pseudomonas aeruginosa (CIM = 2,500µg/mL e CBM = 20.000µg/mL); Enterococcus faecalis (CIM=2,500µg/mL e CBM > 20.000µg/mL) e Escherichia coli (CIM > 20.000µg/mL e CBM > 20.000µg/mL). O OE apresentou potencial antioxidante pelo sistema co-oxidação ß-caroteno/ácido linoleico com 76,63% de inibição da oxidação (1,0mg/mL) e pelo poder de redução de ferro (5,38 µmol Fe2+/mg amostra). Os resultados são promissores em indicar essa espécie para o desenvolvimento de produtos alimentícios, cosméticos e farmacêuticos.

Optimization of a medium composition for the heterologous production of Alcaligenes faecalis penicillin G acylase in Bacillus megaterium.

Penicillin G acylase (PGA) is a strategic enzyme in the production processes of beta-lactam antibiotics. High demand for ß-lactam semisynthetic antibiotics explain the genetic and biochemical engineering strategies devoted towards novel ways for PGA production and application. This work presents a fermentation process for the heterologous production of PGA from Alcaligenes faecalis in Bacillus megaterium with optimization. The thermal stability from A. faecalis PGA is considerably higher than other described PGA and the recombinant enzyme is secreted to the culture medium by B. megaterium, which facilitates the separation and purification steps. Media optimization using fractional factorial design experiments was used to identify factors related to PGA activity detection in supernatant and cell lysates. The optimized medium resulted in almost 6-fold increased activity in the supernatant samples when compared with the basal medium. Maximum enzyme activity in optimized medium composition achieves values between 135 and 140 IU/ml. The results suggest a promising model for recombinant production of PGA in B. megaterium with possible extracellular expression of the active enzyme.