![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, October 2001, p. 2943-2946, Vol. 45, No. 10
Department of Microbiology, Eastman Dental
Institute for Oral Health Care Sciences,1
and Eastman Dental Hospital,2 University
College London, London WC1X 8LD, United Kingdom
Received 22 January 2001/Returned for modification 28 March
2001/Accepted 16 July 2001
Microcosm dental plaques were grown from an inoculum of human
saliva in a constant-depth film fermentor. The inoculum
contained four tetracycline-resistant streptococcal species, each of
which contained a Tn916-like element. This element was
shown to transfer to other streptococci both in filter-mating
experiments and within the biofilms in the fermentor.
Tetracyclines are used for
the treatment of periodontal disease (15). However,
resistance to the drugs limits their usefulness. The most widespread
resistance gene is tet(M), usually found on conjugative
transposons (cTn) of the Tn916/Tn1545 family
(2). Transfer of Tn916-mediated tetracycline
resistance (Tetr) between oral streptococci during
filter-mating experiments has been observed (6, 7).
Furthermore, the cTn Tn5397 can transfer from a nonoral
organism (Bacillus subtilis) to oral streptococci in a
microcosm dental plaque (13). Tn916-like
elements are present within the oral microflora (1, 3, 6,
8), and in this paper we show that they transfer
Tetr not only in filter-mating experiments but also in a
model oral biofilm.
Bacterial strains are listed in Table 1
and were grown at 37°C anaerobically (80% N2, 10%
H2, and 10% CO2) except Neisseria subflava and Acinetobacter lwoffii, which were grown
aerobically. All strains were grown on brain heart infusion agar
(Oxoid, Basingstoke, United Kingdom) supplemented with 5% defibrinated
horse blood (E and O Laboratories, Bonnybridge, United Kingdom) and
appropriate antibiotics (Sigma-Aldrich, Poole, United Kingdom) at
concentrations of 10 µg ml
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.10.2943-2946.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Transfer of Tn916-Like Elements in
Microcosm Dental Plaques
ABSTRACT
Top
Abstract
Text
References
TEXT
Top
Abstract
Text
References
1 (tetracycline) and 25 µg ml1 (rifampin). Tetr organisms from
human saliva were isolated by spreading four aliquots of pooled saliva
onto selective media and incubating the plates aerobically or
anaerobically for 48 h.
TABLE 1.
Bacterial strains used throughout this study
Filter-mating experiments were carried out as described previously
(17). Transconjugants were selected on media containing tetracycline and rifampin. Multispecies microcosms were produced in a
constant-depth film fermentor (CDFF), with nutrients supplied from
artificial saliva, as described previously (12, 13, 20). Saliva samples from 10 healthy individuals were pooled, aliquoted, and
frozen at 
70°C. The CDFF was inoculated as described previously (12). At 216 h postinoculation the CDFF was pulsed
with tetracycline in artificial saliva at concentrations of 2 µg
ml1 for 1 h and 12 µg ml1 for 2 h
followed by 2 µg ml1 for 1 h. These concentrations
mimic the levels found in gingival crevicular fluid after an oral dose
of tetracycline (5). Selection of Tetr
organisms was carried out as previously described (13).
Growth of the biofilm consortia was carried out essentially as
described above; the inoculum, consisting of 11 species of bacteria
(Table 1) which had been grown overnight in 5 ml of brain heart
infusion broth, was added to 445 ml of artificial saliva, which was
pumped into the CDFF. The fermentor in this experiment was pulsed with tetracycline at 150 h.
Streptococci were identified using API 32 Strep kits (Biomerieux, Basingstoke, United Kingdom) and partial sequencing of the 16S rRNA gene (9) and analyzed using the Ribosomal Database Project II (10). Due to the high degree of homology of the 16S rRNA gene sequences, further differentiation of the streptococcal species was achieved by carbohydrate fermentation, enzyme substrate utilization tests (18), and comparison of 16S rRNA-23S rRNA intergenic nucleotide sequences (19).
PCRs and sequencing reactions were performed as described previously
(17). The positions and sequences of the primers
(Sigma-Genosys Ltd., Pampisford, United Kingdom) used are shown in Fig.
1a and Table 2.
Southern blots were carried out using ECL kits (Amersham, Little
Chalfont, United Kingdom). Enzymes were obtained from Promega (Southampton, United Kingdom).
|
|
Before we inoculated the CDFF with the pooled human saliva, it
contained the following Tetr (MIC, 
32 µg
ml1) organisms: Streptococcus oralis,
Streptococcus mitis, Streptococcus gordonii, and
Streptococcus salivarius. At 72 h the Tetr
organisms isolated were Streptococcus parasanguinis and
Streptococcus salivarius. Streptococcus sangiunis
was isolated at 218, 221, and 240 h, and S. salivarius
and S. parasanguinis could no longer be isolated. At 240, 336, and 408 h Tetr S. gordonii could also
be isolated. S. sanguinis was isolated at 408 h.
To determine if a Tn916-like element was present a Southern blot was performed on HincII-digested genomic DNA (Fig. 1b). This blot was probed with a labeled PCR product from the int and xis regions of Tn916 (Fig. 1a and Table 2). As we were looking for functional Tn916 elements, the integration region was the best probe to use, as nonfunctional fragments of Tn916-like elements are found in many different bacteria (14, 16). Digestion with HincII will release a fragment containing the int and xis genes and the transposon-genome junction (Fig. 1). With Tn916 the number of hybridizing bands reflects the number of copies of the transposon. The hybridization showed that all of the streptococci possessed at least one copy of a Tn916-like element. We also demonstrated by PCR (primers RT1 and RT2) that all the Tetr streptococci contained the region from orf13 to tet(M) (results not shown). PCRs to detect the circular form of Tn916 (primers REO and LEO) were positive, indicating that the Tn916-like elements were being excised (results not shown).
Filter-mating experiments were carried out between each original
streptococcal isolate and E. faecalis JH2-2. Transconjugants arose at 6 × 107 per recipient from
S. salivarius SSa10 and S. gordonii
SG0. No spontaneously resistant colonies were isolated. PCR,
using the primers in Fig. 1, and Southern blot hybridization against
the int-xis fragment of Tn916 produced
identically sized products and positive hybridization, respectively, in
the donor and the transconjugants; no PCR products or hybridization to
the recipient DNA was observed (results not shown), indicating that a
Tn916-like element was responsible for the transfer.
We set up a known biofilm consortium containing 10 tetracycline-sensitive (Tets) organisms and one Tetr organism, S. salivarius SSa10 (Table 1). After 150 h a Tetr S. parasanguinis organism could be isolated at every sampling occasion. A Tn916 element indistinguishable (determined by PCR, sequencing, and Southern blotting) from that in the donor was found in S. parasanguinis. The putative transconjugant could not be distinguished from the S. parasanguinis organism present in the initial inoculum by PCR amplification and partial sequence analysis of the 16S rRNA gene. This experiment was repeated, and only the Tetr donor was isolated. The fact that no transfer was detected in the second experiment may be due to differences within the biofilm. The multispecies environment of the biofilm is more variable than the two-species environment used in filter matings.
This is the first demonstration of transfer of a native cTn within a model oral biofilm, an important finding because the oral cavity is one of the most colonized environments within humans and oral bacteria have the opportunity to come into contact with bacteria that pass through the oral cavity; also, oral bacteria can be readily transferred from one human to another. This work demonstrates that oral streptococci are responsible for harboring and disseminating these promiscuous mobile elements to other oral microflora.
| |
ACKNOWLEDGMENTS |
|---|
Thanks go to David Spratt for helpful discussions on the differentiation of streptococcal species.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Microbiology, Eastman Dental Institute for Oral Health Care Sciences, University College London, 256 Gray's Inn Rd., London WC1X 8LD, United Kingdom. Phone: 44 (0) 20 7915 1050. Fax: 44 (0) 20 7915 1127. E-mail: aroberts{at}eastman.ucl.ac.uk.
| |
REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. |
Bentocha, F.,
D. Clermont,
G. de Cespedes, and T. Horaud.
1992.
Natural occurrence of structures in oral streptococci and enterococci with DNA homology to Tn916.
Antimicrob. Agents Chemother.
36:59-63 |
| 2. | Clewell, D. B., S. E. Flannagan, and D. D. Jaworski. 1995. Unconstrained bacterial promiscuity: the Tn916-Tn1545 family of conjugative transposons. Trends Microbiol. 3:229-236[CrossRef][Medline]. |
| 3. |
Fitzgerald, G. F., and D. B. Clewell.
1985.
A conjugative transposon (Tn919) in Streptococcus sanguis.
Infect. Immun.
47:415-420 |
| 4. | Flannagan, S. E., L. A. Zitzow, Y. A. Su, and D. B. Clewell. 1994. Nucleotide sequence of the 18 kb conjugative transposon Tn916 from Enterococcus faecalis. Plasmid 32:350-354[CrossRef][Medline]. |
| 5. | Gordon, J. M., C. B. Walker, J. C. Murphy, J. M. Goodson, and S. S. Socransky. 1981. Concentration of tetracycline in human gingival fluid after single doses. J. Clin. Periodontol. 8:117-121[CrossRef][Medline]. |
| 6. |
Hartley, D. L.,
K. R. Jones,
J. A. Tobian,
D. J. LeBlanc, and F. L. Macrina.
1984.
Disseminated tetracycline resistance in oral streptococci: implication of a conjugative transposon.
Infect. Immun.
45:13-17 |
| 7. | Kuramitsu, H. K., and V. Trapa. 1984. Genetic exchange between oral streptococci during mixed growth. J. Gen. Microbiol. 130:2497-2500[Medline]. |
| 8. | Lacroix, J. M., and C. B. Walker. 1995. Detection and incidence of the tetracycline resistance determinant tet(M) in the microflora associated with adult periodontitis. J. Periodontol. 66:102-108[Medline]. |
| 9. | Lane, D. J. 1996. 16S/23S rRNA sequencing, p. 115-175. In E. Stackebrandt, and M. Goodfellow (ed.), Nucleic acid techniques in bacterial systematics. Wiley and Sons Ltd., Chichester, United Kingdom. |
| 10. |
Maidak, B. L.,
J. R. Cole,
T. G. Lilburn,
C. T. Parker, Jr.,
P. R. Saxman,
J. M. Stredwick,
G. M. Garrity,
B. Li,
G. J. Olsen,
S. Pramanik,
T. M. Schmidt, and J. M. Tiedje.
2000.
The RDP (Ribosomal Database Project) continues.
Nucleic Acids Res.
28:173-174 |
| 11. | Marra, D., and J. R. Scott. 1999. Regulation of excision of the conjugative transposon Tn916. Mol. Microbiol. 31:609-621[CrossRef][Medline]. |
| 12. |
Pratten, J.,
A. W. Smith, and M. Wilson.
1998.
Response of single species biofilms and microcosm dental plaques to pulsing with chlorhexidine.
J. Antimicrob. Chemother.
42:453-459 |
| 13. | Roberts, A. P., J. Pratten, M. Wilson, and P. Mullany. 1999. Transfer of a conjugative transposon, Tn5397 in a model oral biofilm. FEMS Microbiol. Lett. 177:63-66[CrossRef][Medline]. |
| 14. |
Roberts, A. P.,
P. A. Johanesen,
D. Lyras,
P. Mullany, and J. I. Rood.
2001.
Comparison of Tn5397 from Clostridium difficile, Tn916 from Enterococcus faecalis and the CW459tet(M) element from Clostridium perfringens shows that they have similar conjugation regions but different insertion and excision modules.
Microbiology
147:1243-1251 |
| 15. | Seymour, R. A., and P. A. Heasman. 1995. Tetracyclines in the management of periodontal diseases. A review. J. Clin. Periodontol. 22:22-35[Medline]. |
| 16. | Swartley, J. S., C. F. McAllister, R. A. Hajjeh, D. W. Heinrich, and D. S. Stephens. 1993. Deletions of Tn916-like transposons are implicated in tetM-mediated resistance in pathogenic Neisseria. Mol. Microbiol. 10:299-310[CrossRef][Medline]. |
| 17. |
Wang, H.,
A. P. Roberts,
D. Lyras,
J. I. Rood,
M. Wilks, and P. Mullany.
2000.
Characterization of the ends and target sites of the novel conjugative transposon Tn5397 from Clostridium difficile: excision and circularization is mediated by the large resolvase, TndX.
J. Bacteriol.
182:3775-3783 |
| 18. | Whiley, R. A., and D. Beighton. 1998. Current classification of the oral streptococci. Oral Microbiol. Immunol. 13:195-216[Medline]. |
| 19. | Whiley, R. A., B. Duke, J. M. Hardie, and L. M. Hall. 1995. Heterogeneity among 16S-23S rRNA intergenic spacers of species within the `Streptococcus milleri group'. Microbiology 141:1461-1467[Abstract]. |
| 20. | Wilson, M. 1999. Use of constant depth film fermentor in studies of biofilms of oral bacteria. Methods Enzymol. 310:264-279[Medline]. |
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Clin. Vaccine Immunol. | Clin. Microbiol. Rev. |
|---|---|
| J. Clin. Microbiol. | ALL ASM JOURNALS |
