## 3 Results
### Processing and Bacteriological Analysis of Infected Tenth
Because of safety concerns, cytotoxic reactions and the potential side effects of the synthetic irrigants, the use of herbal agents has been increased recently; studies reported that the herbal agents have an excellent biocompatibility, anti-inflammatory, antioxidant properties and increased antimicrobial activity [31; 34; 35; 36; 37]. The most effective method to combat _E. faecalis_ is to use 2% chlorhexidine in combination with sodium hypochlorite as endodontic irrigant, which is most often recommended [8]. Nonetheless, NaOCl is known to be highly toxic to periapical tissues [9]. Severe complications were also described following NaOCl extrusion during root canal treatment [38]. Essential oils are complex, concentrated natural bioactive compounds with documented antibacterial activity, acting with similar effectiveness on antibiotic-susceptible or on multidrug-resistant bacteria, even if organized in biofilms [39]. The EO mechanism of action is complex, depending on the content of bioactive compounds; for example, they may interfere with bacterial enzymes, respiratory pathways, membrane integrity, protein synthesis or transmembrane transportation functions [9]. Also, EO have the ability to achieve high concentrations inside the bacterial biofilm, and thus are able to efficiently eradicate _E. faecalis_, even if aggregated and organized in biofilms. Moreover, the review findings of [PERSON] et al. show that chlorhexidine or NaOCl present a low ability to eliminate _E. faecalis_[40]. In intracanal endodontic biofilms, _E. faecalis_ form colonies on the canal walls and in the root canal dentin tubules, either as surface aggregates (in rich nutrient environment) or adherent cell clumps with many dead cells and islands of active bacteria that degrade the dentin (in nutrient-deprived environment). _E. faecalis_ has the ability to precipitate the apatite from dentin, and develop a \"calcified\" biofilm. In addition, other bacteria and fungi are capable of forming colonies on root canal walls. All these contribute to _E. faecalis_ persistence in root canals [33; 41]. Even if all these are supporting the pathogenicity of _E. faecalis_ in root canal infections, other studies question its relevancy as the predominant pathogen in the etiology of secondary endodontic infections, because enterococci are not common colonizers of oral cavity [42]. Thus, the origin of _E. faecalis_, as responsible for secondary intraradicular infection, is most probably exogenous, rather than the patient's oral microbiome [43]. Our results suggest that both mechanical cleaning, completed by chemical decontamination, are needed in order to properly control the canal microflora and to prevent infection relapse. When comparing the root canal treatment with and without instrumentation, the results show that this procedure significantly contributes to bacterial clearance. Without instrumentation, which is described as part of the root canal treatment protocol, bacteria manage to survive in a more or less latent state in the tubular structure of the dentin, with chance of later reactivation. _Enterococcus_ spp., but also other species such as _Streptococcus mutans_, _Streptococcus sanguis_ or _Fusobacterium nucleatum_ (an important co-aggregator of _E. faecalis_) have the ability to strongly bind underlying tissues and organize in biofilms, which are more resilient to antibacterial agents [33]. Our results are in concordance with other findings [44; 45; 46], that showed the importance of instrumentation in clearing the bacterial biohazard in root canals. Following instrumentation and irrigation, the AqEOs of oregano, thyme and clove showed no bacterial growth after processing the samples harvested with sterile paper points from the root canal. We have to mention here the limitation of the paper-point sampling method, where the accuracy of sampling and the absorbance rate may vary despite the controlled experimental conditions; also, contamination is possible during the handling.