1 Division of Microbial Diseases, Eastman Dental Institute for Oral Health Care Sciences, University College London, 256 Gray's Inn Road, London, WC1X 8LD, UK; 2 Department of Health, Wellington House, 133135 Waterloo Road, London, SE1 8UG, UK
Received 18 January 2005; returned 12 May 2005; revised 3 June 2005; accepted 23 June 2005
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Abstract |
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Methods: Plaque and saliva samples were taken from 26 children. Tetracycline-resistant bacteria were isolated and identified. The types of resistance genes and their genetic locations were also determined.
Results: Fifteen out of 18 children harboured tetracycline-resistant (defined as having a MIC 8 mg/L) oral bacteria at all three time points. The median percentage of tetracycline-resistant bacteria at 0, 6 and 12 months was 1.37, 1.37 and 0.85%, respectively; these were not significantly different. The MIC50 of the group was 64 mg/L at all three time points compared with the MIC90, which was 64 mg/L at 0 months, and 128 mg/L at 6 and 12 months. The most prevalent resistant species were streptococci (68%), which were isolated at all three time points in 13 children. The most prevalent gene encoding tetracycline resistance was tet(M) and this was found in different species at all three time points. For the first time, tet(32) was found in Streptococcus parasanguinis and Eubacterium saburreum. PCR and Southern-blot analysis (on isolates from three of the children) showed that the tet(M) gene was located on a Tn916-like element and could be detected at all three time points, in four different genera, Streptococcus, Granulicatella, Veillonella and Neisseria.
Conclusions: The results of this study show that tetracycline-resistant bacteria and tet(M) are maintained within the indigenous oral microbiota of children, even though they are unlikely to have been directly exposed to tetracycline.
Keywords: Tn916 , conjugative transposons , tet(M) , tet(32)
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Introduction |
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Packer et al.2 found that amoxicillin-resistant organisms in an individual fluctuated widely over a 6 month period, suggesting that these organisms are frequently present in low numbers in the dental plaque of individuals who have not recently received antibiotics. In a longitudinal study, Nyfors et al.3 demonstrated ß-lactamase production by oral anaerobic Gram-negative rods isolated from the saliva of 44 Caucasian infants at the ages of 2, 6 and 12 months. ß-Lactamase-positive Gram-negative anaerobic species were found in 11, 55 and 89% of each age group, respectively. The presence of ß-lactamase-producing species was significantly associated with the exposure of the infants to antibiotics (the authors did not specify which antibiotics) through antimicrobial treatments given to the infants and/or their mothers.
Tetracycline resistance genes have been shown to be present over a period of time on farmland and in slurry waste lagoons.4,5 Tetracycline resistance determinants are often found on mobile genetic elements, in particular conjugative transposons. Tn916, which contains tet(M), was the first conjugative transposon to be identified; it is highly promiscuous and has been found in >50 different species and in 24 genera of bacteria.6 These figures may now be far higher. Conjugative transposons can transfer from the chromosome of a donor cell to the chromosome of a recipient cell, enabling the element to spread antibiotic resistance genes and persist within a population of bacteria (e.g. the oral cavity).
The aim of this study was to determine if tetracycline-resistant bacteria and the genes encoding resistance are maintained within the oral cavity of children not receiving antibiotic therapy.
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Materials and methods |
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A total of 26 children aged 46 years attending a multi-ethnic school in North London, UK were sampled at time points of 0, 6 and 12 months. The children were given a dental examination and their dental health recorded. The parents were asked to give consent for their child to be included in the study and ethical approval was provided by the Research and Ethics Committee of Barnet Health Authority, London, England. The parents were given a questionnaire to determine their own and their child's antibiotic use and any major illnesses suffered by either child or parents. Children were excluded from the study if they had taken antibiotics 3 months prior to the sample date and if the children had any major illnesses. Those children whose parents had taken antibiotics 3 months prior to sampling were not excluded.
Sample collection and processing
Plaque samples were obtained from the entire dentition of each subject, using a calcium alginate swab (Technical Service Consultants Ltd, Heywood, UK). The swab was immediately placed into 4 mL of Calgon Ringers solution (Oxoid Ltd, Basingstoke, UK) in a sterile 7 mL container that included five sterile 23 mm diameter glass beads (BDH Chemicals, Poole, UK). The samples were vortexed for 30 s to dissolve the calcium alginate. A 10-fold serial dilution of the sample was then prepared in tryptone soya broth (Oxoid Ltd) and spread onto antibiotic-containing agar and antibiotic-free agar (to determine the total viable count). Iso-Sensitest agar (Oxoid Ltd) supplemented with 5% defibrinated horse blood (E&O Laboratories, Bonneybridge, Scotland, UK) was used, as was tetracycline (Sigma-Aldrich, Poole, UK) at a breakpoint concentration of 8 mg/L, based on the recommendations of the National Committee for Clinical Laboratory Standards.7 Staphylococcus aureus NCTC 6571 was used as the quality control organism. Plates were incubated in an anaerobic chamber at 37°C for 4872 h (Don Whitley Scientific Ltd, Shipley, UK); a duplicate set was incubated in air supplemented with 5% carbon dioxide at 37°C for 48 h. After incubation, colonies with different morphologies were counted separately, subcultured and incubated under both aerobic and anaerobic conditions to confirm whether they were obligate aerobes. All obligate aerobes were then subcultured and stored at 70°C (Microbank, Prolab diagnostics, Neston, UK). Colonies with different morphologies on the anaerobic plates were also counted separately; these were then subcultured and stored at 70°C.8
Testing for antibiotic susceptibility
An agar dilution method was employed to determine the MIC of tetracycline using Iso-Sensitest agar supplemented with 5% defibrinated horse blood and the antibiotic at concentrations within the range 2512 mg/L. The inoculum was standardized using a 0.5 McFarland standard in accordance with NCCLS recommendations, and inoculated onto the agar using a multipoint inoculator (Mast Diagnostics, Bootle, UK). S. aureus NCTC 6571 and Iso-Sensitest broth (Oxoid Ltd) were included on the plates as controls, which were incubated overnight at 37°C.
Identification of tetracycline-resistant bacteria
Identification of the isolates was carried out on the basis of atmospheric growth requirements, Gram stain, catalase reaction, sensitivity to metronidazole and optochin, growth on selective media including Veillonella agar (Difco Laboratories, Detroit, MI) for Veillonella spp. and mitis salivarius agar (Difco) for streptococci and by partial 16S ribosomal RNA (rRNA) gene sequencing.9 The 16S rRNA gene was amplified using universal primers 27F (5' AGAATTTGATCMTGGCTCAG 3') and 1492R (5' TACGGYTACCTTGTTACGACTT 3') (Genosys, Sigma, UK). A subsequent partial DNA sequencing of the 16S rRNA gene was carried out using a single primer 357F (5' CTCCTACGGGAGGCAGCAG 3') and the sequence analysed using the Ribosomal Database Project II and BLAST at the National Centre for Biotechnological Information.10,11 DNA sequencing was carried out using an ABI310 Genetic Analyser (PE Biosystems, Warrington, UK).
Detection of tetracycline resistance determinants
All of the isolates were tested for genes encoding ribosomal protection proteins [tet(M), tet(O), tet(Q), tet(S) tet(W) and tet(32)] and efflux pumps [tet(A), tet(B), tet(C), tet(D), tet(E), tet(K) and tet(L)] by single PCR reactions previously described1214 and with degenerate primers for ribosomal protection type proteins. The positive PCR products were sequenced using the Big Dye Terminator ready reaction mixture (PE Biosystems, Warrington, UK) and an ABI310 Genetic Analyser (PE Biosystems).15
Identification of the location of resistance genes that persist
Isolates containing tet(M) from three children at all three time points were tested for the presence of the int and xis genes of Tn916 using PCR, as described by Roberts et al.16 Chromosomal DNA from the isolates was then digested with HindIII (Promega, Southampton, UK) and a Southern-blot analysis carried out using an ECL direct DNA labelling kit (Amersham Biosciences, Amersham, UK) according to the manufacturer's protocol. The blots were probed with pAM120 (plasmid containing a whole copy of Tn916),17 a PCR product containing the int and xis genes of Tn916, tet(M) and tet(W).
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Results |
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Fifteen out of the 18 children harboured tetracycline-resistant bacteria at all three time points (0, 6 and 12 months). The percentage of tetracycline-resistant oral bacteria isolated from the 18 subjects ranged from 036.8%. The median percentages of tetracycline-resistant isolates at 0, 6 and 12 months were 1.37, 1.37 and 0.85%, respectively; these were not significantly different. The MICs of the resistant isolates were in the range 8512 mg/L. The MIC50 for the group remained constant at 64 mg/L over the 12 months, compared with the MIC90, which was 64 mg/L at 0 months and 128 mg/L at 6 and 12 months.
The most prevalent species of tetracycline-resistant bacteria were the oral streptococci (68%), followed by Veillonella spp. (11%) and Neisseria spp. (10%). The tetracycline-resistant oral streptococci and Veillonella spp. could be isolated at all three time points in 13 children and one child, respectively.
The most prevalent gene encoding tetracycline resistance was tet(M) and this was found to persist within different genera of oral bacteria at all three time points in 14 out of 18 children (Table 1). Other genes tet(B), tet(K), tet(O), tet(Q), tet(S), tet(W), could only be detected at one time point. The tet(32) gene was found within two isolates of Eubacterium saburreum in children #14 and #17, and Streptococcus parasanguinis also in child #14. These two children were found to be from the same school class. tet(S) was found in a mitis-group Streptococcus sp. in child #3; this is the first time that this gene has been isolated from an oral Streptococcus sp.
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Discussion |
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Five children (#1, #3, #5, #7 and #10) had an elevated diversity of tet genes, with only children #1 and #3 having parents who had taken antibiotics. Therefore this suggests that there was not an association between the diversity of tet genes and antibiotic usage. However, no firm conclusion can be drawn from this observation, as the number of children is too small to enable statistical analysis.
Helicobacter pylori has also been found to have been transmitted to children from older family members, often as a result of cramped living conditions and bed sharing.22,23 Because young children also have very close contact with classmates and learn unhygienic habits from each other, day-care centres and infant schools are a good environment for the spread of bacterial pathogens such as Streptococcus pneumoniae and Haemophilus influenzae through coughing and sneezing.24,25 Other work supports the theory that tetracycline resistance determinants may be acquired from food products due to the use of tetracycline as growth promoters and therapeutics in animal husbandry, and pesticides in the fruit industry.4,2630All of these factors contribute to the transmission of tetracycline-resistant bacteria.
The most common genera harbouring resistance to tetracycline were the oral streptococci, particularly the mitis-group (including Streptococcus oralis, Streptococcus mitis and Streptococcus infantis). None was identified as S. pneumoniae. These organisms constitute the largest proportion of oral bacteria, especially in children of a young age.31 Many oral streptococci, including groups such as mitis and anginosus, are naturally competent;32,33 this may explain why the oral streptococci were more frequently tetracycline-resistant than any other species. Tetracycline resistance has also been commonly found on conjugative transposons in these species. These elements are highly promiscuous and may account for the widespread nature of tetracycline resistance.16,34,35 Out of the 10 different genera resistant to tetracycline, resistance was found to persist over 12 months only in oral streptococci and Veillonella spp.
The gene most associated with the maintenance of tetracycline resistance was tet(M). This was found within six different genera of the oral microbiota. The Southern-blot analysis showed that tet(M) was present on a Tn916-like element.36 This element was found within streptococci, Veillonella spp., Neisseria spp. (where the element was truncated), and Granulicatella adiacens, therefore indicating that the maintenance of tet(M) is due to its presence within a Tn916-like element. Conjugative transposons are very stable within the host chromosome. They are unlikely to be lost as they do not place as great a burden on the host as a plasmid, because they integrate into the host genome and replicate with their host's genome.6,37 The other genes encoding tetracycline resistance [tet(B), tet(K), tet(O), tet(Q), tet(S)] were detected only on a single sampling occasion. However, two isolates in child #17 contained tet(W), one in Veillonella dispar at 0 months and in an Actinomyces sp. at 6 months. These isolates did not hybridize with either pAM120 or int/xis, indicating that tet(W) in these isolates was not contained within Tn916 and more work is required to determine the nature of the genetic elements (if any) that support these genes. tet(W) is much rarer within these oral bacteria isolated from children compared with that reported by Villedieu et al.14 They found that tet(W) was the second most commonly isolated resistance determinant (21%) in tetracycline-resistant oral bacteria from healthy adults and it was found in a variety of species including Streptococcus spp., Neisseria spp., Gram-positive organisms other than Streptococcus spp. and Gram-negative anaerobes. In our study, tet(W) was only found in four Veillonella spp., one Actinomyces sp., one Rothia mucilaginosus and one streptococcus. This variability in host species and the fact that we did not find tet(W) in any Neisseria sp., may account for the difference. But tet(W) was the second most common tetracycline resistance determinant in this study.
In all of the tetracycline-resistant Neisseria spp. isolated in this study, the tet(M) gene was contained within a Tn916-like structure. However, the int and xis genes had been deleted in all but one of the isolates. A similar situation has been observed in pathogenic Neisseria spp.38 Also, experimental introduction of Tn916 into Neisseria spp. resulted in the deletion of the ends of the element.39 Our current work indicates that this may be a general phenomenon in Neisseria spp., resulting in the stabilization of the tet(M) gene in the genome. The tet(32) gene was found for the first time in the oral microbiota. Previously it was found in a Clostridium-related human colonic anaerobe K10 and in bacteria from the rumen of sheep and pig faecal samples, suggesting that tet(32) is abundant in farm animals.12 In child #16, tet(32) was found in S. parasanguinis at 0 months and in E. saburreum at 6 months. This may imply that tet(32) is present on a mobile element as it has been found within two different genera in the same child. Also, the two children who harboured tet(32) were from the same classroom and this suggests that one child may have transferred the bacteria to the other. Melville et al.12 have shown that tet(32) is on a mobile element and is chromosomally encoded. However, it is not found on either a Tn916-like or TnB1230-like mobile element but has not been characterized in detail.
In conclusion, the results of this study have shown that tetracycline-resistant bacteria and tet(M) are maintained within the indigenous oral microbiota of children, even though they are unlikely to have been directly exposed to tetracycline.
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Acknowledgements |
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