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Antibiotic Resistance Gene Transfer between Streptococcus gordonii and Enterococcus faecalis in Root Canals of Teeth Ex Vivo Christine M. Sedgley, MDSc, MDS, PhD, Esther H. Lee, BS, Matthew J. Martin, BA, and Susan E. Flannagan, MA Abstract Multiple bacterial species coexisting in infected root canals might interact, but evidence for interspecies gene transfer is lacking. This study tested the hypothesis that horizontal exchange of antibiotic resistance can occur between different bacterial species in root canals. Transfer of the conjugative plasmid pAM81 carrying erythromycin resistance between 2 endodontic infection-associated species, Streptococcus gordonii and Enterococcus faecalis, was investigated in an ex vivo tooth model. Equal numbers of each species (one with pAM81 and the other plasmid-free) were combined in prepared root canals of sterilized teeth and incubated at 37°C. At 24 and 72 hours, bidirectional interspecies antibiotic resistance gene transfer was evident in microorganisms recovered from teeth; average transfer frequencies from S. gordonii to E. faecalis were 10 3 transconjugants per donor and from E. faecalis to S. gordonii were 10 6 and 10 7 transconjugants per donor at 24 and 72 hours, respectively. Microbial accumulations were observed on root canal walls with scanning electron microscopy. Horizontal genetic exchange in endodontic infections might facilitate adoption of an optimal genetic profile for survival. (J Endod 2008;34:570 –574)

Key Words Antibiotic resistance, conjugative plasmid, Enterococcus faecalis, ex vivo, filter mating, gene transfer, microbial accumulations, root canal, scanning electron microscopy, Streptococcus gordonii

From the Department of Cariology, Restorative Sciences and Endodontics, The University of Michigan, School of Dentistry, Ann Arbor, Michigan. Address requests for reprints to Dr Christine Sedgley, Department of Cariology, Restorative Sciences and Endodontics, The University of Michigan, School of Dentistry, 1011 N University Ave, Ann Arbor, MI 48109-1078. E-mail address: csedgley@umich.edu. 0099-2399/$0 - see front matter Copyright © 2008 by the American Association of Endodontists. doi:10.1016/j.joen.2008.02.014

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oot canal infections are generally composed of a diverse microflora (1) that can exist as biofilm communities on root canal walls (2, 3). Polymicrobial biofilm communities are well-suited for horizontal gene transfer (4), providing pathogens with the means to adapt rapidly, for example, by the acquisition of genes encoding antibiotic resistance (5). Conjugation is a particularly efficient means of horizontal gene transfer occurring naturally in bacteria, whereby DNA is transferred between cells that are in physical contact, and can involve the crossing of species barriers. Plasmids, some of which are conjugative, are extrachromosomal autonomously replicating elements important for adaptation and survival by the provision of functions not encoded by the chromosome. Conjugative plasmids (“self-transmissible” plasmids) encode the essential functions needed for their own intercellular transmission by conjugation; nonconjugative plasmids do not encode these functions. Plasmids are found in bacteria (5), archaea (6), and yeasts (7) and are of particular clinical importance because they can be involved in the dissemination of antibiotic resistance. Multiple antibiotic resistance in clinical isolates from root canal infections has been reported (8, 9). Persistent root canal infections have been significantly associated with the recovery of Streptococcus spp (10, 11), most commonly S. gordonii (10), and Enterococcus spp (12–15). The latter might include antibiotic resistant isolates carrying up to 4 plasmids per strain, including potential conjugative plasmids (9). It has been speculated that bacterial species in infected root canals could communicate, perhaps resulting in plasmid transfer (16), but evidence for this in root canals is lacking. Specific interactions between different species recovered from root canal infections can vary. For example, endodontic E. faecalis isolates coaggregated with Fusobacterium nucleatum but not with Peptostreptococcus anaerobius, Prevotella oralis, and Streptococcus anginosus (17), a “5-strain” combination associated with apical periodontitis in monkeys (18). Similarly, Porphyromonas gingivalis co-invaded dentinal tubules with S. gordonii but not with Streptococcus mutans (19). However, beyond the concurrent recovery of both Streptococcus and Enterococcus species from infected root canals (10), there are no reports on interactions between these 2 grampositive species in the root canal environment. In this study, the hypothesis was tested that horizontal exchange of antibiotic resistance can occur between different bacterial species in root canals. Bidirectional transfer of an erythromycin resistance determinant on the conjugative plasmid pAM81 between 2 endodontic infection–associated species, S. gordonii and E. faecalis, was investigated using an ex vivo root canal infection model that has been previously described (20).

Materials and Methods Microorganisms Bacterial strains used were S. gordonii Challis-Sm (21) (resistant to streptomycin [Sm]), E. faecalis JH2-2 (22) (resistant to rifampin [Rif] and fusidic acid [Fus]), E. faecalis JH2-2/pAM81 (23, 24) (resistant to Rif, Fus, and erythromycin [Em]), and S. gordonii Challis-Sm/pAM81 (resistant to Sm and Em). Conjugative plasmid pAM81 is approximately 27 kb in size and encodes Em resistance (24). S. gordonii Challis-Sm/ pAM81 was created for this study by conjugation between E. faecalis JH2-2/pAM81 and S. gordonii Challis-Sm with overnight filter mating methods as previously described (25).

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Basic Research—Biology Unless otherwise stated, growth conditions were at 37°C in reduced oxygen conditions (candle-jar). Strains were grown in 5 mL of Todd Hewitt Broth (THB; Difco; Becton Dickinson and Company, Sparks, MD) with appropriate antibiotics (Fus 25 ␮g/mL, Rif 25 ␮g/mL for JH2-2; Fus 25 ␮g/mL, Rif 25 ␮g/mL, Em 10 ␮g/mL for JH2-2/ pAM81; Sm 1000 ␮g/mL for Challis-Sm; Sm 1000 ␮g/mL, Em 10 ␮g/mL for Challis-Sm/pAM81). At the mid to late-log stage of growth (optical density at 650 nm ! 0.8), cultures were pelleted by centrifugation at 1300g for 10 minutes, washed twice with sterile phosphate-buffered saline (PBS) (pH 7.4) to remove antibiotics, and resuspended in THB. After direct microscopic count with a Petroff-Hausser counting chamber in which each single cell or short chain of cells was designated as a colony-forming unit (CFU), suspensions were adjusted in THB to a final concentration of 2  107 CFU per 15 ␮L. In addition, by plating 10-fold serial dilutions in triplicate on THB agar with appropriate antibiotics, the concentrations were verified by viable counts.

Teeth Permanent cuspid, single-rooted, extracted, intact human teeth with straight roots, no restorations, and no caries were used (n  85). Tooth length was 22 mm and adjusted if necessary by incisal reduction. Root canal access cavity preparation, working length determination (1 mm short of the apical foramen), biomechanical instrumentation (to apical size 45), and smear layer removal were performed as described elsewhere (20). Root surfaces were coated with nail varnish. Teeth were submerged in water and autoclaved at 125°C for 23 minutes and then aseptically stored in a saturated humid atmosphere at 25°C until use. All later procedures were performed under strict aseptic conditions. Immediately before inoculation with bacteria, root canals were dried with sterile paper points. Viability of Single Species in Teeth Thirty-nine teeth were used in experiments to evaluate viable counts and survival of each of the 4 strains in root canals. Groups of 9 teeth were inoculated with 15 ␮L of bacterial suspension using sterile disposable pipet tips that fit loosely to the working length in root canals. The remaining teeth served as negative controls and were inoculated with 15 ␮L THB. Teeth were transferred to sterile vials and incubated in reduced oxygen conditions in a saturated humid atmosphere at 37°C for up to 72 hours. Triplicate sets of teeth were aseptically crushed, and tooth fragments were suspended in 2 mL PBS and vortexed vigorously to displace bacterial cells into suspension as previously described (26). Viable counts (Fig. 1) enumerated by plating in triplicate onto THB agar containing appropriate antibiotics established that there would be sufficient viability to allow evaluation of antibiotic resistance transfer between species over the duration of the subsequent antibiotic resistance transfer experiments. Transfer of Antibiotic Resistance in Root Canals and on Filters Experiments were conducted with a 1:1 donor strain to recipient strain ratio. In experimental teeth, transfer of pAM81 between S. gordonii and E. faecalis was evaluated in triplicate sets of teeth; equal volumes (7.5 ␮L, corresponding to 1  107 CFU) of a donor and a recipient strain were introduced into canals such that S. gordonii Challis-Sm/pAM81 (donor) was combined with E. faecalis JH2-2 (recipient), and E. faecalis JH2-2/pAM81 (donor) was combined with S. gordonii Challis-Sm (recipient). Triplicate sets of control teeth were inoculated with 15 ␮L of a single bacterial suspension or with 15 ␮L THB. Teeth were transferred to individual sterile vials for incubation and processed as described above at 24 and 72 hours to allow recovery of microorganisms. Filter mating experiments to evaluate plasmid transfer on solid surfaces in vitro were conducted in parallel with tooth experJOE — Volume 34, Number 5, May 2008

Figure 1. Bacterial counts of pAM81-containing and plasmid-free strains at initiation and after 24- or 72-hour incubation in root canals. Means " standard errors from 3 experiments.

iments using methods previously described (25). After incubation either in teeth or on filters, donors, recipients, and transconjugant counts were determined in aliquots (100 ␮L) of undiluted and 10-fold serial dilutions of recovered cell suspensions plated in triplicate onto selective media; selective plates for transconjugants prevented growth of both donor and recipient. The presence of pAM81 in transconjugants was confirmed by purification of the plasmid with ethidium bromide–CsCl gradient methods as previously described (27). The frequency of transfer of the plasmid from the donor strain to the recipient strain was calculated as transconjugants per donor. One tooth in each of the 4 experimental groups was evaluated for the presence of microbial accumulations on root canal walls using scanning electron microscopy (SEM). Teeth were split longitudinally, and each half was immersed in 2.5% glutaraldehyde in phosphate buffer, pH 7.4, at 4°C. Fixed samples were rinsed in buffer and dehydrated in ascending strengths of ethanol to 100% followed by 3 changes of hexamethyldisilazane (28), dried, mounted on SEM stubs, sputtercoated with gold, and viewed in an scanning electron microscope (AMRAY 1910FE, Bedford, MA) with an accelerating voltage of 6 kV.

Results Bidirectional transfer of an erythromycin resistance determinant on the conjugative plasmid pAM81 occurred between S. gordonii and E. faecalis. Average frequencies for transfer of erythromycin resistance are shown in Table 1; transfer from S. gordonii to E. faecalis averaged 10 3 transconjugants per donor at both 24 and 72 hours, and transfer from E. faecalis to S. gordonii averaged 10 6 and 10 7 transconjugants per donor at 24 and 72 hours, respectively. Control teeth inoculated with 15 ␮L of a single bacterial suspension or with 15 ␮L THB yielded no transconjugants and no recovered microorganisms, respectively.

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Basic Research—Biology TABLE 1. Transfer of Erythromycin Resistance Between S. gordonii Challis-Sm and E. faecalis JH2-2 in Teeth or on Filters at 24 and 72 Hours Mating type

Donor

Recipient

Transconjugant selection*

24-Hour root canal 24-Hour root canal 24-Hour filter 24-Hour filter 72-Hour root canal 72-Hour root canal 72-Hour filter 72-Hour filter

JH2-2/pAM81 Challis-Sm/pAM81 JH2-2/pAM81 Challis-Sm/pAM81 JH2-2/pAM81 Challis-Sm/pAM81 JH2-2/pAM81 Challis-Sm/pAM81

Challis-Sm JH2-2 Challis-Sm JH2-2 Challis-Sm JH2-2 Challis-Sm JH2-2

Sm, Em Rif, Fus, Em Sm, Em Rif, Fus, Em Sm, Em Rif, Fus, Em Sm, Em Rif, Fus, Em

Em-resistance frequency† 3.8  10 1.1  10 3.8  10 6.9  10 4.0  10 1.1  10 3.6  10 3.2  10

6 3 6 4 7 3 7 3

Representative transconjugant‡ Ch24RC J24RC Ch24F J24F Ch72RC J72RC Ch72F J72F

*Selection for transconjugants involved plating on Todd Hewitt Broth agar plus antibiotics for the plasmid (Em) and recipient marker(s): Fus, Rif, Sm. †Transconjugants per donor; means of triplicates. ‡See Fig. 2 for plasmid DNA purified from representative transconjugants.

Transconjugants, selected on the basis of acquired erythromycin resistance, contained pAM81 DNA that appeared intact with no apparent rearrangements, deletions, or insertions. Restriction analyses revealed an identical plasmid in all cases tested, and one representative transconjugant from each experiment is shown in Fig. 2. To assure that transconjugants were correctly identified, 6 transconjugants from each experiment, including the representative transconjugants, were verified to lack the donor antibiotic resistance marker(s) and to match the recipient strain in cell morphology and ability/inability to grow on Enterococcosel agar (Becton, Dickinson and Co, Sparks, MD), a medium that allows growth of enterococci but not streptococci. SEM showed accumulation of microorganisms on root canal walls and in dentinal tubules at 24 and 72 hours (Fig. 3).

Discussion Plasmids are involved in the dissemination of antibiotic resistance and also encode a diverse range of products that might potentially

contribute toward “virulence” such as cytotoxins and adhesins. Horizontal transfer of plasmids can profoundly influence genome plasticity and evolution by allowing movement of genetic information both within and between species, and conferring traits facilitating survival under atypical conditions (5). The available data concerning plasmids in clinical isolates recovered from infected root canals are limited to Enterococcus species (9, 29). The E. faecalis MC4 strain used in monkey root canal infection studies (18) harbors a 130 kb conjugative, pheromone (cCF10)responding plasmid, pAMS1, conferring chloramphenicol, streptomycin, and tetracycline resistances (29). In human root canal infections, up to 4 plasmids per strain were found in 25 of 33 endodontic enterococcal isolates, with 16 of the 25 plasmid-positive strains exhibiting phenotypic properties consistent with having conjugative plasmids (9); 5 of these strains (GS6, GS12, GS21, GS23, and GS29) had a greater capacity to form biofilms in vitro than 65 other E. faecalis isolates tested (30). The plasmid pAM81 used in this study is a 27 kb conjugative plasmid originally identified in an E. faecalis hospital isolate (24).

Figure 2. pAM81 plasmid DNA from donor and transconjugant strains, digested with HindIII. Lanes 1 and 14, molecular size marker; lane 2, E. faecalis JH2-2/pAM81 (donor); lane 3, S. gordonii Challis-Sm (plasmid-free recipient); lane 4, S. gordonii Ch24RC; lane 5, S. gordonii Ch24F; lane 6, S. gordonii Ch72RC; lane 7, S. gordonii Ch72F; lane 8, S. gordonii Challis-Sm/pAM81 (donor); lane 9, E. faecalis JH2-2 (plasmid-free recipient); lane 10, E. faecalis J24RC; lane 11, E. faecalis J24F; lane 12, E. faecalis J72RC; lane 13, E. faecalis J72F. Plasmid DNA restriction fragments were separated by electrophoresis on 0.8% agarose gels in TBE buffer (3.5 hours at 50 V), stained with ethidium bromide, and visualized under ultraviolet light. Plasmid analyses for additional transconjugants of each type exhibited the same plasmid pattern as presented here (data not shown). See Table 1 for transfer frequencies.

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Figure 3. Scanning electron micrographs showing accumulations of microorganisms (A) at the opening of dentinal tubules 24 hours after E. faecalis JH22/pAM81 and S. gordonii Challis-Sm were introduced into the root canal and (B) within dentinal tubules (extending up to 20 ␮m) 72 hours after S. gordonii Challis-Sm/pAM81 and E. faecalis JH2-2 were introduced into the canal. The mixed species, both gram-positive cocci in each case, cannot be distinguished in these images.

Conjugative plasmids are transferred between cells that are in physical contact and can encode a broad range of genes and mobilize, in some cases, otherwise nontransferable elements such as nonconjugative coresident plasmids and even chromosomal genes; they are ubiquitous in gram-negative and gram-positive bacteria (5). The conjugative transfer process typically involves a single strand of DNA being transferred from the donor cell to the recipient cell, after which a complementary strand is synthesized in the recipient, and the resulting double-stranded DNA is circularized. Conjugative transfer of plasmids is enhanced in biofilms (4), which are sessile microbial communities composed of cells irreversibly attached to a substratum, an interface, or to each other (31). Throughout the biofilm, environmental conditions vary, resulting in altered phenotypes that might facilitate certain survival and virulence characteristics (32), as well as increased potential for horizontal gene transfer of antibiotic resistance determinants (4). On the basis of reported biofilm formation at 72 hours in experimentally infected root canals (33), the microbial accumulations observed on root canal walls (Fig. 3) might correspond to early stages of biofilm formation. Biofilms have been observed in the undebrided parts of the root canal system of surgically resected root apices of endodontically treated teeth (3). The

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conditions under which biofilms might occur in infected root canals in vivo might represent an important adaptive mechanism by which remaining bacteria survive endodontic treatment protocols (2). The bacterial inoculum size for this study was selected on the basis of real-time polymerase chain reaction enumeration of total counts of E. faecalis in samples collected from infected human root canals (12), and after time course studies established that this provided sufficient viable cells for the duration of the experiment (72 hours) to allow calculation of antibiotic resistance gene transfer rates. Transconjugant frequencies from ex vivo root canal and filter mating experiments were comparable, indicating that the root canal environment supported transfer of an antibiotic resistance marker at rates comparable to those obtained under optimal laboratory conditions. The range of average transfer frequencies in filter mating experiments is comparable to those reported by others who have used similar methods (34 –36). In the present study there was a substantially higher calculated rate of transfer of pAM81 from S. gordonii to E. faecalis (1.1  10 3 transconjugants per donor, a high rate of transfer considering that only 1000 donor cells are needed for one transfer event), compared with transfer in the reverse direction from E. faecalis to S. gordonii (10 7 to 10 6 transconjugants per donor). This finding might relate to an inherently greater ability of E. faecalis to take up pAM81, which was originally recovered from an E. faecalis host (23), or to the ability of E. faecalis to better survive in the root canal environment (20), as confirmed by the higher ultimate bacterial counts of E. faecalis compared with S. gordonii (Fig. 1). Antibiotic resistance transfer between Enterococcus faecium isolates has been observed in the gastrointestinal tracts of mice (34) and humans (35) and from porcine to human E. faecium isolates in gnotobiotic mice (36). Transfer of a mobile Tn916-like transposon element encoding tetracycline resistance from a Neisseria sp endodontic isolate to the laboratory strain E. faecalis JH2-2 was recently shown using filter mating (37). In the present study, confirmation of bidirectional transfer of an erythromycin resistance determinant on the conjugative plasmid pAM81 between S. gordonii and E. faecalis supports the hypothesis that horizontal exchange of antibiotic resistance can occur between different bacterial species in root canals. These findings provide proof of concept in an ex vivo tooth model and pave the way for in vivo investigations with polymicrobial biofilms. It is conceivable that if endodontic strains contain conjugative plasmids or transposons with genes that could enhance virulence during or after endodontic treatment, such properties might be transferable to other species in the root canal system. This presents the possibility that genetic exchange between species in infected root canals might facilitate an ability to adopt an optimal genetic profile for survival.

References 1. Munson MA, Pitt-Ford T, Chong B, Weightman A, Wade WG. Molecular and cultural analysis of the microflora associated with endodontic infections. J Dent Res 2002;81:761– 6. 2. Chavez de Paz LC. Redefining the persistent infection in root canals: possible role of biofilm communities. J Endod 2007;33:652– 62. 3. Nair PN, Henry S, Cano V, Vera J. Microbial status of apical root canal system of human mandibular first molars with primary apical periodontitis after “one-visit” endodontic treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;99:231–52. 4. Sorensen SJ, Bailey M, Hansen LH, Kroer N, Wuertz S. Studying plasmid horizontal transfer in situ: a critical review. Nat Rev Microbiol 2005;3:700 –10. 5. Clewell DB, Francia MV. Conjugation in gram-positive bacteria. In: Funnell BE, Phillips GJ, eds. Plasmid biology. Washington, DC: ASM Press, 2004:227–56. 6. Brugger K, Redder P, She Q, Confalonieri F, Zivanovic Y, Garrett RA. Mobile elements in archaeal genomes. FEMS Microbiol Lett 2002;206:131– 41. 7. Jayaram M, Mehta S, Uzri D, Velmurugan S. Segregation of the yeast plasmid: similarities and contrasts with bacterial plasmid partitioning. Plasmid 2004;51:162–78.

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Basic Research—Biology 8. Pinheiro ET, Gomes BP, Ferraz CC, Teixeira FB, Zaia AA, Souza Filho FJ. Evaluation of root canal microorganisms isolated from teeth with endodontic failure and their antimicrobial susceptibility. Oral Microbiol Immunol 2003;18:100 –3. 9. Sedgley CM, Molander A, Flannagan SE, et al. Virulence, phenotype and genotype characteristics of endodontic Enterococcus spp. Oral Microbiol Immunol 2005;20:10 –9. 10. Chavez de Paz L, Svensater G, Dahlen G, Bergenholtz G. Streptococci from root canals in teeth with apical periodontitis receiving endodontic treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;100:232– 41. 11. Fouad AF, Barry J, Caimano M, et al. PCR-based identification of bacteria associated with endodontic infections. J Clin Microbiol 2002;40:3223–31. 12. Sedgley C, Nagel A, Dahlen G, Reit C, Molander A. Real-time quantitative polymerase chain reaction and culture analyses of Enterococcus faecalis in root canals. J Endod 2006;32:173–7. 13. Reynaud Af Geijersstam AH, Ellington MJ, Warner M, Woodford N, Haapasalo M. Antimicrobial susceptibility and molecular analysis of Enterococcus faecalis originating from endodontic infections in Finland and Lithuania. Oral Microbiol Immunol 2006;21:164 – 8. 14. Williams JM, Trope M, Caplan DJ, Shugars DC. Detection and quantitation of E. faecalis by real-time PCR (qPCR), reverse transcription-PCR (RT-PCR), and cultivation during endodontic treatment. J Endod 2006;32:715–21. 15. Schirrmeister JF, Liebenow AL, Braun G, Wittmer A, Hellwig E, Al-Ahmad A. Detection and eradication of microorganisms in root-filled teeth associated with periradicular lesions: an in vivo study. J Endod 2007;33:536 – 40. 16. Sedgley CM, Clewell DB. Bacterial plasmids in the oral and endodontic microflora. Endod Topics 2004;9:37–51. 17. Johnson EM, Flannagan SE, Sedgley CM. Coaggregation interactions between oral and endodontic Enterococcus faecalis and bacterial species isolated from persistent apical periodontitis. J Endod 2006;32:946 –50. 18. Moller AJ, Fabricius L, Dahlen G, Sundqvist G, Happonen RP. Apical periodontitis development and bacterial response to endodontic treatment: experimental root canal infections in monkeys with selected bacterial strains. Eur J Oral Sci 2004;112:207–15. 19. Love RM, McMillan MD, Park Y, Jenkinson HF. Coinvasion of dentinal tubules by Porphyromonas gingivalis and Streptococcus gordonii depends upon binding specificity of streptococcal antigen I/II adhesin. Infect Immun 2000;68:1359 – 65. 20. Sedgley CM, Lennan SL, Appelbe OK. Survival of Enterococcus faecalis in root canals ex vivo. Int Endod J 2005;38:735– 42. 21. Lunsford RD, London J. Natural genetic transformation in Streptococcus gordonii: comX imparts spontaneous competence on strain wicky. J Bacteriol 1996;178: 5831–5.

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Antibiotic Resistance Gene Transfer  

Multiple bacterial species coexisting in infected root