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A preliminary study investigating the survival oftetracycline resistant Enterococcus faecalis afterroot canal irrigation with high concentrations oftetracycline G. Rossi-Fedele1 & A. P. Roberts21Endodontic Unit and 2Division of Microbial Diseases, UCL Eastman Dental Institute, London, UK 30 mg mL)1). The roots were sampled by grinding Rossi-Fedele G, Roberts AP. A preliminary study investigat- dentine and canal contents and the debris collected ing the survival of tetracycline resistant Enterococcus faecalis were incubated in broth to assess growth.
after root canal irrigation with high concentrations of tetracy- Results Irrigation with sterile distilled water or 50% cline. International Endodontic Journal.
ethanol did not remove all of the cells present. The Aim To compare the ability of two Enterococcus faecalis tetracycline containing solution was efficient in pre- strains to survive exposure to an irrigation solution venting any growth of sensitive E. faecalis, however the containing a high concentration of tetracycline in the resistant strain was able to survive a 5 min exposure at Methodology The root canals of twelve bovine Conclusions The presence of the Tn916-like conju- incisor root sections were chemo-mechanically pre- gative transposon containing the tetracycline resist- pared using commercially available drills, sodium ance gene tet(M) allowed an E. faecalis strain to survive hypochlorite and ethylenediamine tetra-acetic acid.
irrigation using a solution containing an extremely The root sections were divided into two groups and high concentration of tetracycline in a root canal inoculated with either a tetracycline sensitive or resistant strain of E. faecalis. The strains are isogenic, Keywords: conjugative transposon, root canal irri- however one contains a conjugative transposon related gation, tet(M), tetracycline, tetracycline resistance, to Tn916 which confers resistance to tetracycline, and the other strain is sensitive to the antibiotic. After26 days of incubation the root canals were irrigated Received 27 November 2006; accepted 20 March 2007 using one of three solutions (sterile distilled water, 50% endocarditis, urinary tract infections and endodontic infections (Mejare 1975, Lemoine & Hunter 1987, Enterococcus faecalis is usually found in the human and Boulanger et al. 1991, Jett et al. 1994, Pinheiro et al.
animal gastrointestinal tract, causing no harm to the 2004). E. faecalis can be isolated in relatively low host; however they are responsible for a variety of numbers and at a relatively low frequency in primary human infections including bacteraemia, infective endodontic infections, while it is isolated far morefrequently and in greater numbers in cases of post-treatment endodontic disease (Molander et al. 1988, Correspondence: G. Rossi-Fedele, Endodontic Unit, UCL East- Peciuliene et al. 2000, Hancock et al. 2001). E. faecalis man Dental Institute, 256 Gray’s Inn Road, London WC1X possesses certain characteristics and virulence factors 8LD, UK (Tel.: + 44 (0) 20 7915 1302; fax: + 44 (0) 20 79152371; e-mail: g.rossi-fedele@ucl.ac.uk).
that enable them to survive for long periods of time in Survival of E. faecalis during root canal irrigation Rossi-Fedele & Roberts the root canal. These include the production of proteolytic enzymes, aggregation substances and adhe-sins (Stuart et al. 2006). Additionally E. faecalis has the The two isogenic strains of E. faecalis used throughout ability to survive long periods of starvation (Love 2001, this study were E. faecalis JH2-2 which is tetracycline Figdor et al. 2003), can form biofilms (Distel et al.
sensitive (Jacob & Hobbs 1974) and E. faecalis T1 which 2002) and can invade and live within the dentinal is a JH2-2 derivative containing a Tn916-like con- tubules (Akpata & Blechman 1982). E. faecalis is also jugative transposon conferring tetracycline resistance able to acquire exogenous DNA, which may confer via tet(M) (Rossi-Fedele et al. 2006). The strains were resistance to antimicrobials, such as tetracycline.
grown in Brain Heart Infusion (BHI) broth or on BHI Indeed the recently sequenced genome of E. faecalis agar (Oxoid, Basingstoke, UK). Tetracycline (Sigma, V583 is composed of over 25% DNA which is believed Poole, UK) was used at a concentration of 10 lg mL)1 to either be mobile or from foreign source (Paulsen in all media and at a concentration of 30 mg mL)1 in The tetracycline resistance found in bacteria from Bovine incisors were used throughout this study. The root canals from patients at the Eastman Dental animals were less than 1 year old and slaughtered for Hospital has been recently analysed. A Neisseria sp.
commercial purpose in a Spanish slaughterhouse. The which was able to transfer tetracycline resistance to a study exerted no influence on the animal’s fate at any strain of E. faecalis has been isolated. This was the first stage. The teeth were extracted and stored in 4% formal time such an intergeneric transfer has been reported saline until the study was performed. The apical 5 mm from a Neisseria sp. donor, furthermore it has been and the crown were dissected and the remaining root shown that the tetracycline resistance was mediated by was cut into 1 cm slices with a diamond disc (Abrasive the tet(M) gene and that this gene was present on a Technology Inc, Westerville, OH, USA). Subsequently, conjugative transposon related to Tn916 (Rossi-Fedele the canal lumen was widened to a minimal diameter of et al. 2006). The protein, Tet(M) confers tetracycline 1.14 mm using the ParaPostÒ XPTM Endodontic post resistance by binding to the 30S subunit of the system drills (Coltene/Whaledent, Konstanz, Germany).
ribosome, removing and preventing the drug from Finally the smear layer was removed by copious binding (Connell et al. 2003). In addition to tetracyc- irrigation in an ethylenediamine tetra-acetic acid line, Tet(M) also confers resistance to minocycline and (EDTA) solution (Smear Clear; SybronEndo, Scafati, doxycycline (Chopra & Roberts 2001).
Italy) (4 min) and sodium hypochlorite (Teepol Bleach; Tetracyclines are used in endodontics in a number of Teepol, Orpington, UK) (4 min) using a 27 gauge treatment regimes. Biopure Mixture of Tetracycline, Monoject syringe (Kendall; Tyco, Mansfield, MA, USA) Acid and Detergent (MTAD) (Dentsply Tulsa Dental, Tulsa, OK, USA) for example is a mixture of a Three different irrigation solutions were used to tetracycline isomer (doxycycline hyclate) in a final irrigate the root canals: sterile distilled water, 50% concentration of 3% (according to manufacturers ethanol (JM Loveridge, Southampton, UK) (diluted with instructions; http://www.tulsadental.com), citric acid sterile distilled water) and tetracycline at a concentra- and a detergent (Tween 80) and is used as a final rinse tion of 30 mg mL)1 dissolved in 50% ethanol accord- for disinfection of the root canal system (Shabahang & ing to manufacturers instructions. This concentration Torabinejad 2003). MTAD has been shown to be of tetracycline was used as it is the same as the final effective against E. faecalis during in vitro experiments concentration of doxycycline used in MTAD.
(Shabahang & Torabinejad 2003, Portenier et al.
Twelve roots were placed individually in 10 mL of 2006) however, the concentration of the drug present BHI broth and autoclaved. These were left to cool to in this medicament does not appear to have been tested room temperature and then incubated overnight at on a defined resistant strain of E. faecalis. The genera- 37 °C to verify the sterility of the samples. The tion of E. faecalis T1 (Rossi-Fedele et al. 2006) has given overnight incubation of the sectioned bovine root us the opportunity to determine, using isogenic strains canals resulted in no growth in any of the samples of E. faecalis, if the presence of an orally derived Tn916- indicating that all the root sections were sterile at the like conjugative transposon could allow the E. faecalis start of the experiment. Six of the broths containing the to survive the effects of high level tetracycline irrigation roots were inoculated with 100 lL of an overnight similar to levels that may be encountered during root culture of E. faecalis JH2-2 (tetracycline sensitive) and six with 100 lL of an overnight culture of T1 Rossi-Fedele & Roberts Survival of E. faecalis during root canal irrigation (tetracycline resistant) and left for 26 days at 37 °C to Flowgen, Nottingham, UK). PCRs were performed using allow for bacterial growth, infiltration of the dentine Taq DNA polymerase in the buffer supplied by the tubules and E. faecalis biofilm formation.
manufacturer (Promega, Southampton, UK). The PCR Next, each group of six was divided into three programme was as follows: 94 °C for 4 min followed by subgroups and roots were irrigated by placement of 30 cycles of 94 °C for 30 s, 50 °C for 90 s and 72 °C 1 mL of one of the three irrigation solutions in the for 60 s, followed by a final incubation at 72 °C for canal, using a 27 gauge Monoject syringe 2 mm short 5 min and a final thermal ramp to 4 °C where the of the root end, having previously sealed the apical samples were held until analysis. PCR products were aspect with autoclaved physiowax (RALamb Ltd, East- cleaned for sequencing using a PCR purification kit bourne, UK); the solution was left in-situ for 5 min.
(Qiagen, Crawley, UK). Sequencing was carried out Irrigation was completed by a final flush using a further using Big Dye Ready Reaction Mix V.3.1 and analysed on a 310 genetic analyser (Applied Biosystems, Foster After removal of the apical seal to allow for the City, CA, USA). All sequence data were edited using irrigation solution to drain, the coronal 5 mm portion of the specimen was sampled by grinding dentine and canal mas.html) and DNAMAN 5.2.2 software (Lynnon contents using ParaPostÒ XPTM Endodontic post system Biosoft, Quebec, Canada). The 16S sequence homology drills of diameters 1.25 and 1.40 mm, obtaining a was determined using the ribosomal database project II dentine surface and deep dentine sample for each root.
Debris collected in the flutes of each drill was placed www.ncbi.nlm.nih.gov/BLAST/) tools. The identity of in 10 mL of BHI broth (without antibiotics) and all the bacteria was confirmed, by partial sequencing of incubated overnight at 37 °C to assess growth. A loop full (approximately 10 lL) of each broth (regardless of Cultures of each isolate were made up to McFarland whether or not growth could be observed) was plated standard 1 and a multipoint inoculator used to onto both antibiotic free and tetracycline containing inoculate plates containing various concentrations of (10 lg mL)1) BHI agar plates and incubated overnight tetracycline (8, 16, 32, 64 and 128 lg mL)1). The MIC was taken as the concentration that completely inhib- To control for tetracycline carry-over (with the den- ited bacterial growth in an aerobic environment after tine debris) into the 10 mL BHI broths five additional sterile bovine teeth were irrigated with 30 mg mL)1tetracycline and sampled as described above. The dentine shavings from surface and deep samples for each toothwere transferred to an individual 10 mL BHI broth.
The results of growth of E. faecalis following irrigation These broths were immediately inoculated with 100 lL with various solutions in the bovine root model are aliquots of a dilution of an overnight culture of E. faecalis shown in Table 1. All broths that contained growth JH2-2 containing approximately 100 viable cells (deter- showed heavy growth. All of these cultures were mined by plating an aliquot onto fresh antibiotic free BHI shown to be pure upon subculturing to a fresh plate.
agar plates) and incubated overnight at 37 °C. Two JH2-2 containing cultures grew on the antibiotic free additional broths (containing no debris) were inoculated plates but not on plates containing tetracycline. The T1 with aliquots of E. faecalis JH2-2 and incubated as cultures grew on both antibiotic free and tetracycline containing plates. The identity of the bacteria in all of To compare the susceptibility of planktonically the cultures was confirmed as E. faecalis by partial growing cells to this concentration of tetracycline 10 mL of an overnight culture (containing approxi- The assays designed to detect carry-over of tetracyc- mately 1.410 cells mL)1) of each strain of E. faecalis line with the dentine debris showed no growth in the were spun down and resuspended in 5 mL of tetracyc- 10 test samples (five containing surface debris and five line (30 mg mL)1) dissolved in 50% ethanol. These containing deep debris) and heavy growth in the two cells were incubated at room temperature for 5 min, control samples (containing no debris).
re-pelleted and resuspended in fresh BHI broth.
The test for the susceptibility of planktonically Genomic DNA extraction was performed using the growing cells of each strain of E. faecalis showed no PureGene Gram-Positive and Yeast DNA Isolation Kit growth of either strain after incubation in the tetra- (Gentra Systems, Minneapolis, MN, USA supplied by Survival of E. faecalis during root canal irrigation Rossi-Fedele & Roberts samples (Table 1). Again the growth in the surface 32 lg mL)1, while sensitive strains showed no growth sample and absence in the cognate deep sample can be at any of the tetracycline concentrations tested.
explained by lack of dentinal tubule penetration at thatdepth in those roots. An important consideration is thecarry-over of tetracycline with the debris collected in the drill flute. Tetracycline has been shown to bind very These experiments aimed to assess the survival of two well to dentine (Bjorvatn et al. 1985) and carry-over of isogenic E. faecalis strains that differ only in the the antibiotic with dentine shavings has been demon- presence of a Tn916-like conjugative transposon in strated and alluded to in previous studies (Shabahang & strain T1. The bovine root model used here is a Torabinejad 2003, Portenier et al. 2006). Therefore, a variation of the dentine block model suggested previ- control experiment was undertaken to determine if any ously (Haapasalo & Ørstavik 1987), although in this carried-over tetracycline would be present in sufficient investigation it was decided not to remove the concentrations to inhibit the sensitive strain. All of the cementum to avoid bacterial invasion from the 10 test samples (five surface and five deep) showed external surface through dentinal tubules, and to inhibition of growth when compared with the controls allow the use of the root as an irrigation reservoir, in indicating that the negative results for the growth of order to obtain a dynamic that resembles more closely the sensitive strain JH2-2 after tetracycline irrigation may be explained by either the complete killing during The distilled water irrigation was used to evaluate the irrigation of the tooth, by the inhibition of growth the flushing action during the procedures, while 50% in the BHI broth by the tetracycline carried over with ethanol is used in laboratories to dissolve tetracycline the dentine shavings or by a combination of both of and might have an antimicrobial effect. The results these factors. Therefore, there is a possibility that the show that the irrigation with sterile distilled water did sensitive strain did survive the 5 min irrigation period not remove all of the E. faecalis present. One of the deep within the tooth and was prevented from growing in samples showed no growth (Table 1) and this is most the broth by the carried-over tetracycline. However, likely due to the lack of penetration of the E. faecalis this distinction is not possible using the current model.
into the dentinal tubules of this tooth. The cognate The fact that there is enough carry-over of the surface sample resulted in growth of E. faecalis follow- antibiotic to inhibit the growth of the sensitive strain ing irrigation with the sterile distilled water demon- does not affect the results of the resistant T1 strain as strating that the irrigation had not cleared all of the these grew well in the BHI broths. Furthermore, viable cells from the area that was drilled. The second tetracycline containing medications are likely to be irrigant (50% ethanol, diluted in sterile distilled water) active after the irrigation stage in clinical situations due was included as a control because the tetracycline to the strong affinity between tetracycline and calcium would be dissolved in this solution. All samples resulted containing enamel and dentine (Bjorvatn et al. 1985).
in growth of E. faecalis following irrigation with 50% Additionally no attempt is made to neutralize the ethanol demonstrating that this solution was unable to antimicrobial effect following the irrigation regime in eradicate viable E. faecalis cells. The third irrigant (tetracycline dissolved in 50% ethanol) was efficient in Another consideration is that while it can be said preventing any growth of the sensitive E. faecalis in that the killing or inhibition of growth was 100% in all both the surface and deep samples; however the the negative samples, theoretically only a single cell resistant strain was able to survive in both surface needs to survive to result in heavy growth in broth samples and showed positive growth in one of the deep after the drill debris have been incubated for 18 h at Rossi-Fedele & Roberts Survival of E. faecalis during root canal irrigation 37 °C. Therefore these results are not quantitative, fundamental difference in the outcome of treatment however a similar situation exists clinically where it might be preferable that all bacteria are cleared,considering that the success in treatment of chronic apical periodontitis depends on the control of themicrobial infection present in the root canal system Under the conditions of this preliminary study, it can be concluded that the presence of a tetracycline resistance The MIC of E. faecalis T1 when grown in plates is encoding conjugative transposon in E. faecalis enabled 32 lg mL)1, while in the root model they survived it to survive root canal irrigation with tetracycline at a 30 mg mL)1, almost 1000 times more than the con-centration present in plates. There are certain factors that may go some way to explain this extraordinarysurvival; interactions between the dentine and the We are particularly grateful to Jose´ Ignacio Zalba Elizari tetracycline may have reduced the overall concentra- for the provision of the bovine teeth.
tion of the drug in contact with the cells. It haspreviously been demonstrated that the presence of dentine caused a delay in E. faecalis killing in vitro whenMTAD and 0.2% chlorhexidine was used (Portenier Akpata ES, Blechman H (1982) Bacterial invasion of pulpal et al. 2006). Additionally, it is likely that the majority dentin wall in vitro. Journal of Dental Research 61, 435–8.
of the cells were present as a biofilm on and within the Bjorvatn K, Skaug N, Selvig KA (1985) Tetracycline-impreg- tooth. Biofilm growing cells are often up to 1000 times nated enamel and dentin: duration of antimicrobial capa- more resistant to antimicrobial agents than plankton- city. Scandinavian Journal of Dental Research 93, 192–7.
Boulanger JM, Ford-Jones EL, Matlow AG (1991) Enterococcal ically growing counterparts (Walker et al. 2004). In an bacteremia in a pediatric institution: a four-year review.
additional experiment during this study it was also Reviews of Infectious Diseases 13, 847–56.
demonstrated that planktonically growing cells of Chopra I, Roberts M (2001) Tetracycline antibiotics: mode of either strain were killed efficiently by incubation in action, applications, molecular biology, and epidemiology of 30 mg mL)1 of tetracycline for 5 min. The metabolic bacterial resistance. Microbiology and Molecular Biology activity of the cells may also have an effect on the susceptibility of the cell to tetracycline as those in a Connell SR, Tracz DM, Nierhaus KH, Taylor DE (2003) metabolically inactive growth phase (as the E. faecalis Ribosomal protection proteins and their mechanism of are likely to be after 26 days incubation) are less tetracycline resistance. Antimicrobial Agents and Chemother- susceptible to antimicrobials (Mah & O’Toole 2001).
Additionally, access to the dentinal tubules by the drug Dahle´n G, Haapasalo M (1998) Microbiology of apical periodontits. In: Ørstavik D, Pitt-Ford TR, eds. Essential may not have been possible (Shabahang & Torabinejad Endodontology, 1st edn. Osney Mead, Oxford, UK: Blackwell 2003) therefore some of the bacterial cells may have failed to come into contact with the tetracycline or the Distel JW, Hatton JF, Gillepsie MJ (2002) Biofilm formation in exposure time of these cells may have been reduced.
medicated root canals. Journal of Endodontics 28, 689–93.
However, all of these factors apply to both strains of Figdor D, Davies JK, Sudqvist G (2003) Starvation, survival, E. faecalis tested in these experiments, the only growth and recovery of Enterococcus faecalis in human difference being the presence of an orally derived serum. Oral Microbiology and Immunology 18, 234–9.
Tn916-like conjugative transposon. Therefore, it is Haapasalo M, Ørstavik D (1987) In vitro infection and likely that one or more of these above factors reduced disinfection of dentinal tubules. Journal of Dental Research the level of tetracycline and possibly reduced the exposure time to a level where the T1 strain could Hancock HH , Sigurdsson A, Trope M, Moiseiwitsch J (2001) Bacteria after unsuccessful endodontic treatment in a North survive for 5 min due to the presence of the tet(M) gene American population. Oral Surgery, Oral Medicine, Oral on the conjugative transposon. If this happened in the Pathology, Oral Radiology, and Endodontics 91, 579–86.
clinical setting it is possible that treatment failure could Jacob AE, Hobbs SJ (1974) Conjugal transfer of plasmid-borne follow. It also demonstrates the point that resistance multiple antibiotic resistance in Streptococcus faecalis var.
mechanisms which may appear to confer a relatively zimogenes. Journal of Bacteriology 117, 360–72.
low MIC on a bacterium in the laboratory may make a Survival of E. faecalis during root canal irrigation Rossi-Fedele & Roberts Jett BD, Huycke MM, Gilmore MS (1994) Virulence of Pinheiro ET, Gomes BP, Drucker DB, Zaia AA, Ferraz CC, Enterococci. Clinical Microbiology Reviews 7, 462–78.
Lemoine L, Hunter PR (1987) Enterococcal urinary tract Enterococcus faecalis isolated from canals of root filled teeth infections in a teaching hospital. European Journal of Clinical with periapical lesions. International Endodontic Journal 37, Love RM (2001) Enterococcus faecalis – a mechanism for its Portenier I, Waltimo T, Ørstavik D, Haapasalo M (2006) role in endodontic failure. International Endodontic Journal Killing of Enterococcus faecalis by MTAD and chlorexidine digluconate with or without cetrimide in the presence or Mah TF, O’Toole GA (2001) Mechanisms of biofilm resistance absence of dentine powder or BSA. Journal of Endodontics 32, to antimicrobial agents. Trends in Microbiology 9, 34–9.
Mejare B (1975) Streptococcus faecalis and Streptococcus Rossi-Fedele G, Scott W, Spratt D, Gulabivala K, Roberts AP faecium in infected dental root canals at filling and their (2006) Incidence and behaviour of Tn916-like elements susceptibility to azidocillin and some comparable antibiotics.
within tetracycline-resistant bacteria isolated from root canals. Oral Microbiology and Immunology 21, 218–22.
Molander A, Reit C, Dahle´n G, Kvist T (1988) Microbiological Shabahang S, Torabinejad M (2003) Effect of MTAD on status of root-filled teeth with apical periodontitis. Interna- Enterococcus faecalis-contaminated root canals of extracted tional Endodontic Journal 31, 1–7.
human teeth. Journal of Endodontics 29, 576–9.
Paulsen IT, Banerjei L, Myers GS et al. (2003) Role of mobile Stuart CH, Schwartz SA, Beeson TJ, Watz CB (2006) Entero- DNA in the evolution of vancomycin-resistant Enterococcus coccus faecalis: its role in root canal treatment failure and current concepts in retreatment. Journal of Endodontics 32, Peciuliene V, Balciuniene I, Eriksen HM, Haapasalo M (2000) Isolation of Enterococcus faecalis in previously root-filled Walker CB, Karpinia K, Baehni P (2004) Chemotherapeutics: canals in a Lithuanian population. Journal of Endodontics 26, antibiotics and other antimicrobials. Periodontology 2000

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