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Azerbaycan Saytlari

 »  Home  »  Endodontic Articles 1  »  A protocol for polymerase chain reaction detection of Enterococcus faecalis and Enterococcus faecium from the root canal
A protocol for polymerase chain reaction detection of Enterococcus faecalis and Enterococcus faecium from the root canal
Discussion and References



Discussion.

Polymerase chain reaction amplification of the 16S/23S ribosomal spacer region (ITS-PCR) produced characteristic and identical amplicon profiles for E. faecalis and E. faecium . When the same pair of primers were used on DNA extracted from S. equinus , S. uberis , S. milleri , S. mutans , S. salivarius , S. sanguis , S. anginosus , S. pyogenes and G. morbillorum profiles were produced that were easily distinguished from the enterococci when run in separate lanes to high separation on 2.5% TAE agarose gels. Enterococcus species have, until recently, been classified as streptococci, according to Lancefield as group D. S. uberis and S. equinus are still classified as streptococcal species, belonging to Lancefield group D (Hardie 1986). Thus S. equinus and S. uberis have a close relationship to enterococci and if these two species should be PCR amplified together and subsequently run in the same lane of the gel they could be expected to resemble the two-band pattern of E. faecalis and E. faecium . However, optimal electrophoresis conditions clearly separated these two species from the enterococci. Moreover, S. equinus and S. uberis are not relevant in root canal infections but were included in order to challenge the methodology. It thus seems as if the pair of primers used in this study is suitable for identification of enterococci at the genus level. This is in concordance with the findings of Tyrell et al . (1997). When PCR amplifying highly purified E. faecalis DNA a third amplicon of 600 bp became evident. The sequence of this DNA fragment showed a partial strong homology to a 16S-23S intergenic spacer sequence from the proteobacter X. campestris. This high homology to another 16S- 23S intergenic spacer sequence leads us to conclude that this is probably a third E. faecalis 16S-23S intergenic spacer sequence even though the E. faecalis and X. campestris are only distantly related. To unequivocally assign this DNA sequence as an E. faecalis 16S-23S intergenic spacer, identification of flanking regions and Southern blots on E. faecalis need to be performed. Meanwhile, in the scope of the present study, the 600 bp amplicon poses no problem to identification and detection of E. faecalis by the PCR technique developed. In a root canal sample of the posttreatment microbiota a low number of microorganisms can be expected. Consequently, a very low detection level of the identification methodology is essential. Crucial for DNA-techniques is the extraction of DNA from the cells. Lysis of the cells by boiling, a technique favoured in identification of periodontopathic bacteria (Ashimoto et al. 1996, Papapanou et al. 1997), was not successful in our study. In contrast to samples obtained from the negotiated root canal, samples from gingival pockets contain a large number of microorganisms. Also, in that context the species of interest are anaerobic and mostly Gram negative. Such bacteria are easily disrupted by physical influence and sufficient amounts of DNA are rather easily extracted. In the present study a great number of protocols for extraction of DNA from serial dilutions of cells of E. faecalis were unsuccessfully tested. Traditional techniques such as boiling, enzymatic cell lysis followed by proteinase K digestion and phenol-chizam extraction gave detection levels in the range of hundreds to thousands of bacteria per sample. Finally, using the method described above, a detection level of 10 cells was reached. This level is in concordance with what has been described elsewhere (Zambon & Haraszthy 1995). The practical results correspond to the theoretically calculated potential of the PCR protocols to detect approximately 20 bacterial genomes from 10–13 grams of DNA. Using conventional culturing identification methodology on plaque samples, Loesche et al. (1992) reported a detection level of 2 103 cells. Zambon & Haraszthy (1995) detected 104–105 cells using non-selective media and 103 cells when selective media were used. Contrasting these findings from mixed samples, Möller (1966), using broth, was able to disclose 5 101 cells for several root canal species when cultured as monocultures. Although enterococci easily grow on selective media, PCR might be the slightly superior technology regarding the detection level. However, the advantages of PCR over culturing above all are associated with its low sensitivity to physical and chemical influence. In a clinical situation the use of various medicaments like chloroform, interappointment dressings and irrigants are unlikely to bias the test performance. An apparent limitation of a species specific PCR-based bacterial detection is its inability to detect ‘unexpected’ bacteria. In other words, the technique can only identify selected microorganisms for which specific primers are available. Moreover, it may not be as useful for ‘broadrange’ microbiological analysis of the root canal, although a few different species can be simultaneously detected from samples of small volume by utilizing a multiplex PCR protocol. Such broad range detection is possible using primer pairs targeted to conserved gene sequences. Extensive subcloning and sequencing must then, however, be performed to identify species present in the sample, which, from practical reasons, will limit its use in a clinical situation. In addition, PCR does not discriminate dead from viable cells. The dead microbe will degrade in the canal due to lyzosomal activities but the fate and significance of DNA in a non-vascular confinement is poorly explored.

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