Epidemiología molecular de aislamientos clínicos de enterococcus faecalis de tres hospitales de tercer nivel de México.

Tesis (Maestría en Ciencias con Especialidad en Microbiología Médica) UANL, 2012.

Eficacia antimicrobiana de microdacyn 60, hipoclorito de sodio al 5.25% y MTAD contra enterococcus faecalis.

Tesis (Maestría en Ciencias Odontológicas con Especialidad en Endodoncia) UANL, 2012.

Efecto antimicrobiano de las nanopartículas de plata contra el microorganismo Enterococcus faecalis

Tesis (Maestría en Odontología Avanzada) UANL, 2014.

Untersuchungen zur Biofilmbildung bei dem klinischen Isolat Enterococcus faecalis 1.10

In der vorliegenden Arbeit wurde die Biofilmbildung bei einem klinischen Isolat von Enterococcus faecalis untersucht. Der Prozess der Biofilmbildung ist in mehrere Abschnitte unterteilt und beinhaltet zu Beginn eine Anhaftung von Zellen an Oberflächen. Dieser adhäsive Schritt wird unter anderem durch Pili vermittelt. Pili bei Grampositiven Mikroorganismen sind kovalent mit der Zellwand verknüpfte Proteinstrukturen, die eine Anheftung an biotische und abiotische Oberflächen sowie den Zell-Zell-Kontakt vermitteln. Bei den Analysen dieser Doktorarbeit lag ein besonderes Interesse bei eben diesen Pili, die für Enterococcus faecalis die Namen Ebp (endocarditis and biofilm associated pili) und Bee (biofilm enhancer in enterococci) tragen. Codiert werden sie durch die entsprechenden ebp-/bee-Loci, deren Aufbau unter den Grampositiven Mikroorganismen hochkonserviert ist. Die Loci bestehen aus Pilusuntereinheiten-codierenden Genen und colokalisierten Pilus-spezifischen Sortase Genen. Während in der Regel drei verschiedene Pilusuntereinheiten vorliegen, kann die Anzahl der Sortasen zwischen einer und zwei variieren. Bei den Experimenten wurde neben einer Komplementationsstudie zu einer Bee-Pilus Defekt-Mutante (1.10.16) das Hauptaugenmerk auf die Analyse des zweiten Pilus (Ebp) gelegt, um die Pilisituation bei Isolat 1.10 im Detail darzustellen Zusätzlich sollten weitere Oberflächenassoziierte Proteinstrukturen bei Isolat 1.10 detektiert werden, die gegebenenfalls an der Biofilmbildung beteiligt sind. Weitere Versuche zur Charakterisierung des Bee-Pilus wurden im Laufe dieser Arbeit durchgeführt, blieben jedoch bisher erfolglos. Die Biofilm-/Pilus-Defekt-Mutante 1.10.16 zeigte aufgrund einer Punktmutation (Pm) in der Pilus-spezifischen Sortase 1 des bee-Locus eine geschwächte Fähigkeit zur Anheftung an abiotische Oberflächen, sowie das Fehlen der Bee2 Untereinheit im Pilus. Nach Komplementation der Mutante (1.10.16K) mit dem Wildtyp-srt1 Gen, wurde die starke Biofilmbildungsfähigkeit zurück erlangt. Die Experimente zeigten, dass der Pilus-Defekt auf die Pm im srt1 Gen zurückzuführen war und der Bee-Pilus in Stamm 1.10.16K wieder korrekt gebildet wurde. Zu sehen war dies in Rasterelektronenmikroskopischen Aufnahmen und ebenfalls im massenspektrometrischen Nachweis aller 3 Pilusuntereinheiten im Bee-Pilus charakteristischen High-Molecular-Weight Komplex (~ 250 kDa). Durch Sequenzierungen konnte gezeigt werden, dass zwei Gene des ebp-Locus (ebpR und ebpC) bei Isolat 1.10 durch die Insertion von IS-Elementen IS1062 und IS6770 inaktiviert wurden. Der proteinbiochemische Nachweis über Pilusspezifische Antikörper gegen die Untereinheiten des Ebp-Pilus verlief negativ. Zusätzlich konnte gezeigt werden, dass die mRNA der beiden inaktivierten Gene nicht gebildet wurde. Dies führte folglich zum vollständigen Verlust des Ebp-Pilus bei Isolat 1.10. Zusammen mit den Ergebnissen der Komplementation konnte somit der große Einfluss mindestens eines intakten Pilus auf die Biofilmbildung gezeigt werden. Sind beide Pili durch Insertionen bzw. Mutationen inaktiviert, kommt es zu einer deutlichen Abnahme der Biofilmbildungsstärke. Dass trotzdem noch ein Biofilm gebildet wurde, zeigt den multifaktoriellen Zusammenhang bzw. Einfluss im Biofilmbildungsprozess. Über das gezielte Markieren von Oberflächenproteinen intakter Zellen mittels der Oberflächenbiotinylierung, konnten in der SDS-PAGE Unterschiede im Bandenmuster im Vergleich zur unbehandelten Probe erkannt werden. Die massenspektrometrische Identifikation dieser Proteine erfolgte bisher nicht, jedoch sind diese vorläufigen Ergebnisse vielversprechender Natur für die Identifikation und Aufklärung der Oberflächenproteinsituation bei Isolat 1.10.

Effect of a calcium hydroxide/chlorhexidine paste as intracanal dressing in human primary teeth with necrotic pulp against Porphyromonas gingivalis and Enterococcus faecalis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of pH, available chlorine content, and antibacterial activity of endodontic irrigants and their combinations against Enterococcus faecalis

Objectives. The objectives of this study were to evaluate pH, available chlorine content, and antibacterial activity of endodontic irrigants and their combinations.Study design. The pH and chlorine content of sodium hypochlorite (NaOCl) were analyzed pure and in combination with 10% citric acid (CA) and apple vinegar (AV). The antibacterial effect of the following solutions was measured by direct contact test against Enterococcus faecalis: 2.5% NaOCl, 2.5% NaOCl + 10% CA (7:3), 2.5% NaOCl + AV (5:5), 10% CA, and AV. Sterile saline was used as control. The colony-forming units were determined by serial decimal dilutions.Results. The combination of 2.5% NaOCl with CA or AV lowered the pH and the chlorine content. NaOCl, alone or in combination was able to eliminate E. faecalis in 30 seconds, and CA, after 10 minutes. AV promoted reduction (32.2%) after 10 minutes.Conclusions. NaOCl with acidic solutions lowered the pH and the chlorine content, but did not alter its antibacterial effect. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011; 112:132-135)

Enterococcus faecalis leakage of root canal sealers: an ex vivo study

The aim of this ex vivo study was to evaluate bacterial penetration after filling root canals using 3 different techniques. Three experimental groups of 25 teeth each, obturated with lateral-warm-vertical condensation of gutta-percha, Microseal technique and EndoREZ (R) system, respectively, were tested in a split chamber model system using Enterococcus faecalis and monitored for 180 days to determine bacterial penetration. A statistical analysis was performed using the Kaplan-Meier method. Median survival time was 25 days for Microseal system, 41 for lateral-warm-vertical condensation and 81 for EndoREZ (R). Significant differences were demonstrated between Microseal and EndoREZ (R) (p<0.001) and between Microseal and lateral-warm-vertical condensation technique (p<0.05). No statistically significant differences were observed between EndoREZ (R) and lateral-warm-vertical condensation. After 180 days of assessment, 20% of the EndoREZ (R) samples resisted bacterial penetration and furthermore, the EndoREZ (R) system has the potential to be a filler system compatible with other currently used systems.

Effectiveness of calcium hydroxide-based intracanal medicaments against Enterococcus faecalis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Antibacterial efficacy of endodontic irrigating solutions and their combinations in root canals contaminated with Enterococcus faecalis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effectiveness of ozonated water on Candida albicans, Enterococcus faecalis, and endotoxins in root canals

The aim of this study was to evaluate the effectiveness of ozonated water in the elimination of Candida albicans, Enterococcus faecalis, and endotoxins from root canals. Twenty-four single-rooted human teeth were inoculated with C. albicans and E. faecalis, and 24 specimens were inoculated with Escherichia coli endotoxin. Ozonated water (experimental group) or physiologic solution (control group) was used as irrigant agent. Antimicrobial effectiveness was evaluated by the reduction of microbial counts. Lipopolissacharide complex presence was assessed by limulus amebocyte lysate test and B-lymphocyte stimulation. Data were analyzed by Wilcoxon and Mann-Whitney tests (5%). Ozonated water significantly reduced the number of C. albicans and E. faecalis at the immediate sampling, but increased values were detected after 7 days. Ozonated water did not neutralize endotoxin. It could be concluded that ozonated water was effective against C. albicans and E. faecalis but showed no residual effect. No activity on endotoxin was observed.

ANTIMICROBIAL ACTIVITY of SODIUM HYPOCHLORITE ASSOCIATED WITH INTRACANAL MEDICATION FOR Candida albicans and Enterococcus faecalis INOCULATED IN ROOT CANALS

Objective: The purpose of this study was to evaluate the action of sodium hypochlorite (NaOCl) associated with an intracanal medication against Candida albicans and Enterococcus faecalis inoculated in root canals. Material and Methods: Thirty-six human single-rooted teeth with single root canals were used. The canals were contaminated with C. albicans and E. faecalis for 21 days and were then instrumented with 1% NaOCl. The roots were divided into 3 groups (n=12) according to the intracanal medication applied: calcium hydroxide paste, 2% chlorhexidine (CHX) gel, and 2% CHX gel associated with calcium hydroxide. The following collections were made from the root canals: a) initial sample (IS): 21 days after contamination (control), b) S1: after instrumentation, c) S2: 14 days after intracanal medication placement; S3: 7 days after intracanal medication removal. The results were analyzed statistically by the Kruskal-Wallis test at 5% significance level. Results and Conclusions: Both 1% NaOCl irrigation and the intracanal medications were effective in eliminating E. faecalis and C. albicans inoculated in root canals.

In vitro antibacterial efficacy of endodontic irrigants against Enterococcus faecalis

Objective. The objective of this study was to compare the in vitro antimicrobial activity of 2% chlorhexidine gel against Enterococcus faecalis with sodium hypochlorite in 2 different concentrations (1.5% and 5.25%).Study design. Eighty human lower premolars with single root canals were prepared, autoclaved, and infected for 7 days with E. faecalis monocultures. The roots were then separated into 5 experimental groups according to the irrigant solution used during the standardized preparation. To assess the antimicrobial action of the irrigant solutions, 3 microbial samples were taken: S1-initial (before the biomechanical preparation), S2-posttreatment (immediately after the biomechanical preparation), and S3-final (7 days after the biomechanical preparation). The microbiological samples were plated to count the colony-forming units (CFU).Results. The 2% chlorhexidine gel and 5.25% sodium hypochlorite significantly reduced the E. faecalis CFU in the posttreatment and final microbiological samples. The 1.5% sodium hypochlorite also reduced the E. faecalis CFU immediately after the root canal instrumentation, but the E. faecalis CFU increased in the final sample showing no statistical difference from the control group.Conclusion. The 2% chlorhexidine gluconate gel and 5.25% sodium hypochlorite were effective in eliminating E. faecalis even 7 days after the instrumentation; moreover, the higher the concentration of sodium hypochlorite the better its antimicrobial action.

Heat-killed Enterococcus faecalis Alters Nitric Oxide and CXCL12 Production but not CXCL8 and CCL3 Production by Cultured Human Dental Pulp Fibroblasts

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

In vitro antimicrobial activity of acroseal, polifl and epiphany against Enterococcus faecalis

Using the agar diffusion method, this study evaluated the in vitro antimicrobial activity of the commercial endodontic sealers Acroseal and Epiphany, a castor-oil based experimental sealer, Polifl, and a primer agent (Epiphany self-etching primer), against Enterococcus faecalis. Zinc oxide and eugenol cement (ZOE) served as control. Five wells per dish were made at equidistant points and immediately flled with the test and control materials. After incubation of the dishes at 37°C for 24 h and 48 h, the diameter of the zones of microbial growth inhibition produced around the wells was measured (in mm) with a millimeter rule. After 48 h, the diameters of the zones of microbial growth inhibition were the same as those observed at 24 h, only the substances continued to diffuse. Epiphany and Polifl did not show antibacterial activity (no formation of zones of microbial growth inhibition). The primer produced the largest zones of inhibition (17.62 mm) followed by Acroseal (7.25 mm) and ZOE (7.12 mm). E. faecalis was resistant to Epiphany and Polifl, while the primer and Acroseal sealer were effective against this microorganism under the tested conditions.