Departments of 1Microbiology, 2Oral Medicine and 5Paediatric Dentistry, and 4Biostatistics Unit, Eastman Dental Institute, University College London, 256 Grays Inn Road, London WC1X 8LD; 3Department of Biology (Darwin Building), University College London, Gower Street, London WC1E 6BT, UK
Received 10 July 2001; returned 10 December 2001; revised 17 December 2001; accepted 5 February 2002.
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Abstract |
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Introduction |
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Genes encoding resistance to antimicrobial agents are often linked genetically and can be transferred together,6,7 and there are many reports of antibiotic-resistant bacteria also being resistant to mercury.811 Hence, it is possible that the constant exposure of oral bacteria to the mercury released from amalgam fillings could promote the development and maintenance of mercury- and antibiotic-resistant bacteria in the oral cavity. Only a few studies have looked at the combined prevalence of mercury and antibiotic resistance in oral bacteria and most of these have been carried out with primates. Summers et al.11 have demonstrated a relationship between the presence of amalgam fillings and the occurrence of mercury- and antibiotic-resistant bacteria in monkeys. In their longitudinal study they found that placement of amal-gam fillings resulted in a significant increase in the incidence of Hg-resistant bacteria. The Hg-resistant organisms isolated included oral streptococci, members of the Enterobacteriaceae and enterococci. Most of these Hg-resistant isolates were also resistant to one or more antibiotics. The study also revealed that, in some of the enterobacterial isolates, determinants for mercury resistance and antibiotic resistance were carried on the same plasmid and, more importantly, in the same integron. Only two studies appear to have been pub-lished concerning the prevalence of antibiotic- and mercury-resistant bacteria in human subjects,12,13 and, of these, only one has examined the oral microflora.13 The latter study found no association between the presence of mercury amalgam fillings and the prevalence of Hg-resistant oral bacteria.
The fact that mercury from dental amalgams may be promoting an increase in antibiotic resistance in oral bacteria is obviously of concern to dental practitioners, as antibiotics are used in the treatment of a number of oral infections e.g. periodontitis and abscesses.14 Oral bacteria such as the streptococci are also a major cause of bacterial endocarditis and infections in immunocompromised individuals for which antibiotic therapy is essential.15 Furthermore, the ingress of these bacteria into the gut may result in the spread of antibiotic resistance to the normal gut microflora. The aims of this study were to determine whether the presence of mercury amalgam results in an increase in the prevalence, or oral load, of mercury-resistant oral bacteria and to investigate whether such bacteria also exhibit resistance to antibiotics.
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Materials and methods |
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Healthy children aged between 5 and 16 years attending the Department of Paediatric Dentistry at the Eastman Dental Hospital were recruited. These included one group of children without amalgam restorations and a second group of children who had at least two tooth surfaces restored with amalgam. In each child, the number of tooth surfaces restored with amalgam was counted. Children with chronic medical disorders, known viral carriage or who had been treated with antibiotics during the preceding 3 months were excluded.
Plaque was collected from around the gingival margins and the surfaces of all the teeth using an alginate swab. The swab was then placed into a sterile bijou bottle containing 4 mL of CalgonRingers solution (Oxoid Ltd, Basingstoke, UK) and five glass beads. One millilitre of saliva was collected from each child by gently dribbling into a sterile universal container. The samples were taken to the laboratory for pro-cessing within 30 min of collection.
Sample processing
On arrival in the laboratory, the swab from each patient was vortexed for 30 s and then added to the saliva sample from the same patient and vortexed for a further 30 s. Serial 1 in 10 dilutions (up to 107) of the sample were then prepared in Trypt-one Soy broth (Oxoid). Preliminary screening of the samples for the presence of Hg-resistant microbes was carried out as follows. Duplicate 100 µL aliquots of the undiluted sample and of dilutions (101, 102, 103) of the sample were spread over the surfaces of MuellerHinton (MH) agar (Oxoid) containing 40 µM HgCl2. These plates were prepared on the same day that the specimen was collected and were kept out of direct sunlight before use. Four plates were used for each dilution: two were incubated at 37°C anaerobically for 48 h and two aerobically for 48 h. Duplicate 100 µL aliquots of dilutions (104, 105, 106, 107) of each sample were also inoculated on to Hg-free MH plates and these were also incubated under aerobic and anaerobic conditions to determine, respectively, the total aerobic and anaerobic viable counts. Anaerobic incubation was carried out in an anaerobic chamber (Fred Baker Scientific, Runcorn, UK). Following incubation, colonies growing on the Hg-containing and Hg-free media were enumerated. Representative colonies (at least four from both the aerobically and anaerobically incubated plates) from the mercury-containing plates were subcultured on to Hg-containing MH and Hg-free MH plates to obtain pure cultures. The MIC of mercury for each of the isolates was determined using an agar dilution technique employing MH medium containing a range of HgCl2 concentrations (1.01024 µM). Mercury-resistant strains of Staphylococcus aureus NCTC 50581 and Enterococcus faecium 6641H1 (kindly supplied by Professor Anne Summers, University of Georgia, USA) were used as positive controls. The MICs of a range of antibiotics (penicillin, ampicillin, erythromycin, vancomycin and tetracycline) for each isolate were then determined using an agar dilution technique employing Isosensitest agar containing 5% horse blood. The concentrations of antibiotics used were: pencillin, ampicillin and erythromycin, 0.0088.0 mg/L; vancomycin, 0.062516 mg/L; tetracycline, 0.016128 mg/L. The MIC of HgCl2 for each of the control Hg-resistant strains under the test conditions described above was 32 µM. Oral isolates able to grow on the MH agar containing 16 µM HgCl2 or greater were regarded as being mercury resistant and these were identified to genus level on the basis of Grams stain, morphology, atmospheric requirements and standard biochemical tests. The vast majority of mercury-resistant isolates were found to be streptococci and these were further identified to species level on the basis of their carbohydrate-fermenting profiles.16
Statistical analysis
The outcome measure to be analysed statistically was the proportion of Hg-resistant microbes in the oral microflora expressed as a percentage of both the aerobic and anaerobic counts. Both outcomes were severely positively skewed and could not be subjected to parametric statistical tests. Initially, therefore, data were analysed using the non-parametric MannWhitney test using SPSS software17 and checks were therefore made that the overall shape and distribution of data from each group did not significantly differ from each other using the KolmogorovSmirnov test. In order to optimize statistical power (i.e. to increase the likelihood of correctly identifying a significant result), the alternative approach of transforming the data into a near-normal distribution and undertaking multivariate multilevel regression18 was also implemented using MLwiN software.19 Any differences in the proportion of Hg-resistant bacteria between the two groups was assessed using the 2 test. All statistical tests were two-tailed, with the 5% level of statistical significance adopted throughout.
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Results |
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Bacteria able to grow on MH medium containing 16 µM HgCl2 were considered to be mercury resistant, as the MIC of HgCl2 for the two control mercury-resistant bacteria (S. aureus and E. faecium) was 32 µM.
Of those children who had amalgam fillings, 32 (78%) harboured mercury-resistant bacteria. A similar number (30) and proportion (71%) of children without amalgam fillings also harboured bacteria resistant to mercury. There was no significant difference between the two groups in terms of the number of individuals harbouring Hg-resistant bacteria.
Of the Hg-resistant bacteria isolated, 88% of those obtained from the group with amalgam fillings were streptococci (Table 1). Streptococci comprised a similar proportion (92%) of the Hg-resistant bacterial isolates from the group without amalgam fillings. Of those streptococci that could be identified to species level, Streptococcus oralis comprised the greatest proportion of Hg-resistant bacteria in both patient groups. However, Hg-resistant strains of this organism were isolated more frequently from children with amalgam fillings than from those without (P < 0.05). No Hg-resistant obligate anaerobes were isolated and only one Hg-resistant Gram-negative bacterium (a pseudomonad) was found.
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Discussion |
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There are, unfortunately, few studies with which the results of this investigation can be compared directly and, to complicate matters, there are many methodological differences between studies. One of the main problems with regard to the latter is that there is no internationally accepted criterion defining what is meant by mercury resistance and a variety of HgCl2 concentrations have been used in media to differentiate between sensitive and resistant strains. These include 7.4, 8.4, 18.4, 20, 37, 50, 59 and 100 µM.1013,2026 The only study in humans that is comparable to the present one appears to be that of Edlund et al.,13 who also found that there was no significant difference in the proportion of Hg-resistant bacteria in the saliva of individuals with and without amalgam fillings. However, a number of differences in experimental design between the two studies make direct comparison difficult. First, the present study used children rather than adults and four times as many individuals were included in each group; this increases the reliability of the data analysis. Secondly, the present study involved the processing of saliva plus dental plaque rather than just saliva: the former approach provides a more representative sample of the oral microflora. Thirdly, in the study by Edlund et al.13 the medium used for isolation of Hg-resistant bacteria contained blood (unlike the medium used in the present study), which is known to neutralize the antibacterial activity of mercury com-pounds.5,21,27 Finally, unlike the present study, Edlund et al.13 did not determine the MICs of mercury for the putative Hg-resistant organisms detected on the primary plates, nor did they use Hg-resistant organisms as controls to ensure that the oral isolates were truly Hg resistant. Interestingly, the proportions of Hg-resistant bacteria in the saliva samples reported by Edlund et al.13 were extremely high (of the order of c. 50%) regardless of whether or not the individual had amalgam fillings. This is in marked contrast with the proportions of Hg-resistant bacteria found in the present study, which were generally <1% of the total viable count, raising the possibility that many of the organisms classified as Hg resistant in the study of Edlund et al.13 were not truly Hg resistant. In the present study, however, all of the putative Hg-resistant organisms isolated on the primary plates were retested and designated as Hg resistant only if their MIC on retesting in pure culture was identical to, or greater than, that of control, Hg-resistant, strains of S. aureus and E. faecium. While there are no other comparable studies in humans, Summers et al.11 carried out a longitudinal study of Hg-resistant oral bacteria in monkeys before and after placement of mercury amalgam fillings and found that the proportion of Hg-resistant oral streptococci (i.e. strains with an MIC > 50 µM HgCl2) increased following amalgam installation.
A few studies have also reported on the effect of amalgam fillings on mercury resistance in the intestinal microflora, although the results are conflicting. Osterblad et al.12 found that there was no difference between individuals with and without amalgam fillings with respect to the proportion of Hg-resistant Gram-negative aerobic bacilli, whereas Wire-man et al.10 reported that installation of amalgam fillings in monkeys increased the number of faecal bacteria (including aerobic Gram-negative bacilli) resistant to mercury. Edlund et al.13 found a significant increase in the proportion of Hg-resistant Bacteroides spp. (but not in the proportion of Hg-resistant Escherichia coli or enterococci) in the intestinal microflora of adults with amalgam fillings compared with those without.
In the present study the majority of Hg-resistant bacteria isolated from both subject groups were streptococci. Furthermore, in the case of the amalgam group, most of these were S. oralis. Even in the amalgam-free group, S. oralis comprised a high proportion of the Hg-resistant isolates. S. oralis, together with Streptococcus mitis, are common members of the microflora of several oral habitats including supragingival plaque, saliva and mucosal surfaces.28 Unfortunately, very little is known concerning the susceptibility of oral streptococci (or, indeed, any oral bacteria) to mercury. In a study of the susceptibility of oral streptococci and Actinomyces spp. to mercuric chloride, Lyttle & Bowden5 found that the former were more resistant to mercury than the latter. All 10 of the streptococci tested were able to grow in a medium containing 5 mg/L (18.4 µM) HgCl2 but only eight and five strains were able to grow at 36.8 and 110.4 µM HgCl2, respectively. Surprisingly, in the present study, no Hg-resistant species from other genera encountered frequently in the oral microflora of children were isolated. Although the MH medium used for isolation of Hg-resistant organisms was not supplemented with blood (because of its neutralizing effect on the HgCl2) it is able to support the growth of the predominant organisms present in plaque and saliva, i.e. Streptococcus spp., Actinomyces spp., Haemophilus spp. and Veillonella spp.28,29
Many of the Hg-resistant isolates (41% and 33%, respectively, in the amalgam-containing and amalgam-free groups) were also resistant to one or more antibiotics. While no similar study on human subjects has been carried out, Summers et al.,11 in an analysis of antibiotic resistance in Hg-resistant oral streptococci from monkeys, also found that a high proportion (59%) was resistant to one or more antibiotics; resistance to streptomycin was encountered most frequently. In both groups of children in the present study, resistance to tetracycline and erythromycin was encountered most frequently. Resistance to these two antibiotics appears to be common, and occurs at high frequencies, in oral streptococci. For example, Ioannidou et al.30 reported that 38.5% and 23% of 200 oral viridans group streptococci isolated from the oropharynx of healthy children were resistant to erythromycin and tetracycline, respectively. Resistance to tetracycline and erythromycin was found to be common among viridans group streptococci responsible for a variety of infections in a survey carried out by Teng et al.31 Of the 207 isolates tested, 53% were resistant to tetracycline while 40% were resistant to erythromycin. A number of other studies have re-ported similar high frequencies of resistance to erythromycin (3853.3%),3234 and to tetracycline (1241%);32,35 in viridans group streptococci isolated from patients with serious infections. Of particular interest, and concern, was the finding that most of the Hg- and antibiotic-resistant isolates from the amalgam group were identified as being strains of S. oralis, a bacterium that is very closely related to Streptococcus pneumoniae, with which it shares over 99% sequence homology.36 This bacterium, as well as being associated with bacterial endocarditis, has been shown to be the causative agent of a variety of infections in immunocompromised patients (par-ticularly neutropenic patients and individuals undergoing transplants) and so may be regarded as a significant human pathogen.16,3739 In keeping with the results of this study, other investigations have shown that this bacterium is frequently resistant to tetracycline and erythromycin. Hence, Teng et al.31 found that 60% of 40 isolates were resistant to tetracycline while 55% were resistant to erythromycin. Wisplinghoff et al.40 found that 26% of 19 strains of S. oralis isolated from blood samples from neutropenic patients were resistant to tetracycline.
The results of this study have shown that there was no statistically significant difference in the prevalence, or the proportion, of Hg-resistant bacteria in the oral microflora of children with and without amalgam fillings. Surprisingly, mercury-resistant oral bacteria were isolated frequently from children regardless of their exposure to amalgam fillings and many of these bacteria were found to be resistant to antibiotics, principally tetracycline and erythromycin. We are currently undertaking studies to investigate the nature of the genes encoding Hg resistance and antibiotic resistance in these bacteria and possible linkage between them.
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Acknowledgements |
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Footnotes |
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References |
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