1Department of Microbiology, Eastman Dental Hospital, University College London Hospitals NHS Trust; 2Eastman Dental Institute for Oral Health Care Sciences, University College London, 256 Grays Inn Road, London WC1X 8LD, UK
Received 28 August 2001; returned 5 December 2001; revised 19 December 2001; accepted 20 January 2002.
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Abstract |
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Introduction |
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There is growing concern over the emergence of antibiotic-resistant bacteria. Lacroix & Walker7 showed that from 68 patients with adult periodontitis, the numbers of tetracycline-resistant bacteria from subgingival plaque samples represented c. 12% of the total viable count. In a separate study, the median percentage of the oral microflora resistant to doxycycline, a tetracycline derivative, was shown to be 4.3%, ranging from 0% to 26% in the saliva of 20 human subjects.8 The extensive use of antimicrobials both in the community and hospitals has accelerated the emergence of antibiotic-resistant organisms.911 This has caused increas-ing concern in the medical and scientific community as to the level of antibiotic use in the general population and the degree of inappropriate antibiotic prescription. The biofilm mode of growth can also affect antibiotic sensitivity. Bacteria living in biofilms in general are considered less susceptible to antibiotics than free-living bacteria.12 When Pseudomonas aeruginosa cells that were susceptible to tobramycin were grown as a biofilm, there was a c. 1000-fold reduction in susceptibility to this antibiotic.13 The attachment of Klebsiella pneumoniae to the surface of a glass slide resulted in a 150-fold decrease in susceptibility to hypochlor-ous acid.14 When treating biofilm-associated Porphyromonas gingivalis with metronidazole the MICs were 160 times higher than those obtained for planktonic cells.15 Another study has reported that in order to eliminate bacteria grown in biofilms, MICs greater than 500 times the MIC determined for planktonic culture may be required.16
The purpose of this study was to determine the bacterial composition and the antibiotic susceptibility of microcosm dental plaques. Resistance to penicillin, ampicillin, erythromycin, chloramphenicol, tetracycline and vancomycin was investigated before and after the addition of tetracycline in concentrations that could be found in the oral cavity following administration of a single systemic dose of the antibiotic.
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Materials and methods |
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Human saliva was used as an inoculum to provide a multi-species biofilm consisting of organisms found in the oral cavity. The saliva was collected from 10 healthy individuals who had not taken antibiotics in the previous 3 months, and an equal volume from each of the subjects was pooled. Ali-quots of 1 mL were stored at 80°C for subsequent use.
Production of biofilms
Biofilms were grown in a constant depth film fermenter (CDFF; University of Wales, Cardiff, UK) as described by Wilson.17 The CDFF is a sophisticated means of generating large numbers of identical biofilms and has been used to investigate factors that may influence the growth of bacterial communities in the oral ecosystem.18 Previous studies involv-ing the growth of microcosm plaques in the CDFF from saliva samples have shown good reproducibility between runs.19,20 Briefly, the CDFF is comprised of a stainless steel turntable, which rotates under polytetrafluoroethylene (PTFE) scraper blades. The turntable holds 15 PTFE pans flush around its rim, each having five vertical holes containing 5 mm diameter PTFE plugs. The biofilms were grown on bovine enamel discs, 5 mm in diameter and 1 mm in depth, which sit on the plugs and were recessed 300 µm below the height of the pan. Media and nutrients enter through a stainless steel plate at the top of the fermenter, which also has an air inlet and sample port, whereas the base plate has an effluent outlet. The nutrient source for the experiments was a mucin-containing artificial saliva, the composition of which has been described previously.21
Inoculation of the CDFF
Seven hundred and fifty microlitres of the pooled saliva were added to 500 mL of artificial saliva. This was mixed and pumped into the CDFF for 8 h. After this time, the inoculation vessel was disconnected and the medium reservoir containing sterile artificial saliva was connected to the CDFF. The artificial saliva was delivered via a peristaltic pump (Watson-Marlow, Falmouth, UK) at a rate of 0.5 mL/min, corresponding to the resting salivary flow rate in man.2224
Antibiotic delivery
Experiments were carried out with and without the addition of tetracycline. For the administration of tetracycline, 100 mL of sterile artificial saliva containing 2 mg/L tetracycline (Sigma, Poole, UK) were pumped into the CDFF for 2 h via a second peristaltic pump. After this period, the concentration of tetracycline was increased to 12 mg/L for 1 h, which was subsequently followed by a 2 h addition of 2 mg/L tetracycline. These concentrations of antibiotic were selected as they mimic the peak concentration of tetracycline found in the oral cavity after a single systemic dose.25
Culture methods
Pans were removed from the CDFF before and after the addition of tetracycline, the bovine enamel discs were aseptically removed and placed into 1 mL of phosphate-buffered saline (Sigma), and then vortexed for 1 min to disrupt the biofilm. Three discs from each pan were used for determining the bacterial composition of the biofilms; spread-plating of each biofilm was carried out in duplicate. The total anaerobic count was carried out on Fastidious anaerobic agar (FAA) (LabM, Bury, UK) containing 5% defibrinated horse blood (E and O Laboratories, Bonnybridge, UK). Lactobacilli were isolated from FAA plates on the basis of colonial morphology, Actinomyces spp. were isolated on Cadmium fluoride/acriflavin/tellurite agar plates26 and streptococci were isolated on Mitis Salivarius agar (Difco Laboratories, Detroit, MI, USA). All of the plates were incubated anaerobically for 3 days at 37°C. Antibiotic-resistant organisms were isolated on Isosensitest agar (Oxoid, Basingstoke, UK) containing 5% defibrinated horse blood supplemented with antibiotics (Sigma) at the following concentrations: penicillin 4 mg/L, ampicillin 8 mg/L, erythromycin 1 mg/L, tetracycline 10 mg/L, chloramphenicol 5 mg/L and vancomycin 8 mg/L.27 Plates were incubated either anaerobically or aerobically at 37°C for 3 days. Organisms were identified by Gram-reaction, biochemical analysis and PCR amplification with subsequent partial DNA sequencing of their 16S rRNA gene as described previously28 using universal primers.29 Identifications were obtained by the use of the ribosomal database.30 DNA sequencing was carried out using an ABI310 Genetic Analyser (PE Biosystems, Warrington, UK). Further differentiation of the various streptococcal species was achieved by carbohydrate fermentation and enzyme substrate utilization tests.31
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Results |
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Before the addition of tetracycline at 221 h, the microcosm plaques had a mean total viable anaerobic count of 7 x 107 ± 3.6 x 107 cfu per biofilm (Figure 1). This value changed after the pulse of tetracycline to the fermenter, there was an immediate reduction in the total viable anaerobic count to 1.3 x 107 cfu per biofilm. This was further reduced with a 94% kill to 6 x 106 cfu per biofilm 24 h (240 h) after the addition of tetracycline. The total anaerobic viable count never fully recovered to the pre-tetracycline level (Figure 1). There was no obvious effect on the numbers of lactobacilli immediately after pulsing with tetracycline; however, 24 h after pulsing, the numbers of viable lactobacilli reduced to 1.5 x 106 cfu per biofilm, which was followed by an increase in the viable lactobacilli count up to 8 days post-delivery. The greatest reductions in the viable counts were seen for the streptococci, with a reduction of 98.5%, decreasing from 1.3 x 107 to 1.9 x 105 cfu per biofilm (Figure 1) immediately after tetracycline delivery. The number of Actinomyces spp. also decreased after the addition of tetracycline with a reduction of 96% 24 h post-antibiotic delivery.
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There were 11 vancomycin-resistant isolates, all identified as Lactobacillus fermentum, which are intrinsically resistant to this antibiotic. The 21 tetracycline-resistant isolates were iden-tified as L. fermentum, Streptococcus gordonii, Streptococcus mitis, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus salivarius and Streptococcus sanguinis. Multi-antibiotic-resistant bacteria were readily isolated; of the tetracycline-resistant isolates, 67% were also resistant to erythromycin and 11% were intrinsically vancomycin resistant.
Immediately before and after tetracycline delivery all antibiotic-resistant viable counts were of a similar level. However, the antibiotic resistance profile of the community dramatically changed from 24 h post-tetracycline delivery (240 h). The numbers of viable erythromycin-resistant isolates had decreased by 92.6%, and remained depressed throughout the study. The tetracycline-resistant microflora mirrored the response of the erythromycin-resistant isolates; at 8 days post-tetracycline addition the numbers of viable tetracycline-resistant isolates had reduced by 92%.
In contrast, the number of vancomycin-resistant isolates remained constant up to 24 h post-antibiotic delivery, being 6.5 x 105 cfu per biofilm. The viable counts for the vancomycin-resistant bacteria then increased over time reaching 2.7 x 106 cfu per biofilm. The microcosm altered in composition from one where a tetracycline- and erythromycin-resistant microflora predominated, to one at 8 days post-puls-ing where the vancomycin-resistant bacteria were the greatest component. There was a slight increase in the number of ampicillin-resistant bacteria immediately after tetracycline delivery. Penicillin-resistant and chloramphenicol-resistant isolates were undetectable.
The percentage of the total microflora that was resistant to the four different antibiotics changed substantially after the addition of tetracycline (Figure 2). Tetracycline-resistant isolates showed an immediate increase after the 5 h tetracycline pulse from 6% to 45% of the total microflora; however, this subsequently reduced until 8 days after the pulse the percentage had reduced to a level just below that obtained before the pulse (4.5%). The percentage of erythromycin-resistant isolates also increased after the tetracycline pulse, increasing from 5% to 28%, which also subsequently reduced over time. Initially, there was a slight increase in the percentage of vancomycin-resistant isolates from 1% to 5% immediately after the addition of tetracycline. This increase continued with the vancomycin-resistant isolates reaching a maximum of 30% of the total viable microflora. Duplicate test runs were carried out, each over a 408 h period. For each run, the addition of tetracycline altered the composition and increased the proportions of antibiotic-resistant bacteria. In the control biofilms grown in the absence of tetracycline, the proportions of antibiotic-resistant bacteria did not vary over 408 h. The mean percentage of the oral microflora resistant to each of the antibiotics in the control biofilms was: ampicillin 0.01% (range 00.02%), erythromycin 0.7% (0.32.0%), tetracycline 2.6% (1.45.8%) and vancomycin 2.3% (1.43.8%).
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Discussion |
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The presence of tetracycline also resulted in a change in the antibiotic resistance profile of the biofilms. The emergence of antibiotic-resistant bacteria has frequently been reported to be a direct result of antibiotic usage9,35 and in the present study the addition of tetracycline resulted in an increase in the proportions of ampicillin-, erythromycin-, tetracycline- and vancomycin-resistant isolates; these antibiotics are members of four different classes of antibiotics. The ampicillin-resistant microflora before the addition of tetracycline comprised <0.5% of the total anaerobic count, consistent with results obtained from other studies which demonstrated that ampicillin-resistant bacteria only constitute a low percentage of the oral microflora.8,36 In the present study, it was not until the addition of tetracycline to the system that the percentage of ampicillin-resistant isolates increased, reaching a maximum of 4% of the total anaerobic microflora.
Our data show that a pulse of tetracycline is selecting for organisms that are resistant to multiple antibiotics. This is not surprising, as multiply resistant bacteria are frequently isolated so that selection for one resistance will increase the overall level of resistance in a population. Furthermore, in additional work we have demonstrated the presence of Tn916-like elements in the tetracycline-resistant streptococci isolated.37 These conjugative transposons have been shown to transfer within the model oral biofilms37 and this may also account for the observed increase in the proportions of antibiotic-resistant bacteria. These elements commonly contain resistances to antibiotics other than tetracycline (especially erythromycin), and tetracycline can stimulate the transfer of these elements. Therefore, pulsing the oral cavity with tetracycline could lead to the spread of these genetic elements through the oral microflora. It has been shown that if people are fed low doses of tetracycline, their faecal enteric microflora can acquire tetracycline resistance, and may also become multi-resistant.38
The antibiotic profile data demonstrate that there was also a dramatic increase in the percentage of vancomycin-resistant bacteria after the addition of tetracycline. This change reflects the increase in proportions of lactobacilli. The vancomycin-resistant isolates in the study were members of the intrinsically resistant Lactobacillus genus, in particular L. fermentum.
Tetracyclines may be administered for the treatment of a range of bacterial infections and for their non-antibacterial properties. This investigation has demonstrated that the addition of tetracycline at clinically obtainable levels to a model oral biofilm not only substantially alters the bacterial composition, but also the antibiotic resistance profile of the microbial community.
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Footnotes |
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References |
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