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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
Introduction - Materials and methods



Introduction.
Endodontic treatment of teeth with apical periodontitis is directed toward eradication of the intracanal microorganisms. Hence, the efficacy of various combat regimes is often assessed by sampling the root canal for the presence of persisting microbes. Traditionally, identification of microorganisms in the samples has been carried out through various cultivation procedures. However, the accuracy of this methodology has been questioned and the risks of obtaining false positive and false negative recordings have been pointed out (Bender et al . 1964, Engström 1964, Möller 1966, Mikkelsen & Theilade 1969, Morse 1970, Zielke et al . 1976, Safavi et al . 1985, Reit & Dahlén 1988, Molander et al . 1990, Reit et al . 1999). Recently, there has been a focus on the influence of antibacterial dressings on the results of cultivation (Reit et al . 1999). For example, the chemical effects of a substance may cause a temporary loss of the multiplying capacity of surviving microorganisms, resulting in false negative observations. In addition, medicament remnants may enter a sample and inhibit microbial growth in the laboratory and result in a low diagnostic sensitivity. In order to increase the sensitivity of intracanal sampling, other methods of microbial detection and identification need to be explored.
In root canal microbiology alternative diagnostic methods have received limited attention. Nevertheless, when exploring various methods to identify periodontopathic bacteria Ashimoto et al . (1996) found polymerase chain reaction (PCR) to have a higher diagnostic accuracy than culture procedures. PCR has been described to amplify genomic sequences more than 10 million times (Mullis et al . 1986, Saiki et al . 1988) and to have a potential detection level of 10 bacterial cells (Zambon & Haraszthy 1995). Since the method is not dependent on bacterial growth, it may be suitable for analysis of the post-treatment intracanal microbiota.
Amongst bacteria resisting endodontic treatment procedures the frequency and role of enterococci have recently regained considerable attention (Gomes et al . 1996, Sirén et al . 1997, Molander et al . 1998, Sundqvist et al . 1998). PCR has been used extensively for speciation of enterococci, identification of virulence genes and for detecting the drug resistance of enterococci (Dutka- Malen et al . 1995, Tyrell et al . 1997, Shepard & Gilmore 1997, Hirakata et al . 1997, Monstein et al . 1998), but studies focusing on enterococci detection seem to be lacking. Therefore, the aim of the present study was to explore the potential use of PCR in diagnostic root canal microbiology by developing a protocol for the detection of E. faecalis and E. faecium.


Materials and methods.


Bacterial strains.
Type strains of E. faecalis (ATCC 19433, CCUG 19916) and E. faecium (ATCC19434, CCUG 542) were available from the Göteborg University Culture Collection (CCUG). In addition, four isolates of E. faecalis (OMGS 349/98, OMGS 350/98, OMGS 367/98, OMGS 1/97) recovered from infected root canals were also included (Dahlén et al . 2000). OMGS (Oral Microbiology, Göteborg, Sweden) strains are own isolates, if not CCUG, ATCC or NCTC is indicated. Prior to use these strains were transferred by means of sampling solution (VMGA I, Dahlén et al . 1993) from the lyophilized stage onto blood agar plates for incubation overnight in 37 C and air. DNA was prepared both directly from ‘fresh’ cultures and from strains kept frozen.


DNA preparation.
(i) For the serial dilutions of chromosomal DNA from 10 7 cells, DNA was simply extracted by boiling for 5 min.
(ii) To mimic a clinical sample, serial dilutions of E. faecalis cells in TE buffer (10 mmol L 1 Tris-HCl, 1 mmol L 1 EDTA, pH 8.0) were prepared, ranging from 10 7 to 10 per 100 L; samples were processed in triplicates. DNA was extracted from these samples by using the Wizard Genomic DNA Purification System (Promega, Madison, WI, USA), except as noted according to the manufacturer’s instructions, scaled down to a sample size of 100 L. This kit uses a salt-based, selective precipitation step to remove proteins and cell debris. Phenol-chizam extraction was thus not required to obtain pure DNA. Initial cell wall degradation was performed by adding lysozyme, 450 g, achromopeptidase, 150 g, and mutanolysin, 15 g (all from Sigma Chemical Co., St. Louis, MO, USA), to the samples. The samples were incubated at 37 C for 1 h, after which DNA isolation proceeded according to the manufacturer’s instructions. RNAse treatment of lysed cells was postponed, allowing the bacterial RNA to act as a carrier for the precipitation of the chromosomal DNA. In addition, 0.5 g sonicated salmon sperm DNA (Stratagene, La Jolla, CA, USA) was added to each sample to act as carrier when precipitating DNA. DNA from these preparations were resuspended in 20 L of TE buffer overnight at 4 C. The resuspended DNA was treated with RNAse A, 5 g, for 45 min at 37 C. The entire 20 L of purified chromosomal DNA was added to the subsequent PCR reaction.


PCR conditions.
The chromosomal DNA was amplified using the primers CAA GGC ATC CAC CGT and GAA GTC GTA ACA AGG targeted against the 16S/23S rDNA intergenic region (Barry et al . 1991, Jensen et al . 1993). PCR reactions were set up containing 0.1 mol L 1 of each primer, 0.2 mmol L 1 dNTPs, 3 mmol L 1 Mg 2+ and 1.5 units of TaqGold polymerase (Perkin-Elmer, Foster City, CA, USA) in a volume of 50 L and amplified using the following sequence: 95 C for 2 min succeeded by 40 cycles of 95 C 60 s, 55 C 60 s, 72 C 60 s followed by a final elongation step at 72 C for 10 min. As a positive control of the PCR reaction a type strain of E. faecalis (ATCC 19433, CCUG 19916) was used. A negative control devoid of template DNA was included in all experiments. All components used in preparation of DNA was also amplified in the same manner to ascertain that no contamination or cross reactivity had been introduced by the preparation method.


Electrophoresis and imaging.
Polymerase chain reaction products were run on 1% or 2.5% TBE-agarose (Seakem GTG agarose, FMC Bioproducts, Rockland, ME, USA) gel and visualized by ethidium bromide staining under UV light and photographed.

Subcloning and sequencing.
When PCR amplifying highly purified chromosomal DNA from E. faecalis , a previously undetected band of 600 bp became evident. To ascertain the origin of this band it was excised from the gels and the DNA purified using the QIAEX II gel extraction kit (Qiagen, Valecia, CA, USA). Purified PCR product was cloned into the pGEMT vector (Promega, Madison, WI, USA) and transformed into JM109 competent cells (Promega) according to the manufacturer’s instructions. Positive colonies were isolated and plasmids purified with the Wizard Plus Sv Minipreps (Promega) plasmid purification system. Clones were sequenced by cycle sequencing using the Big Dye terminator sequencing kit (ABI Prism, Perkin Elmer, MA, USA) and T7 and Sp6 sequencing primers (Promega). Reactions were then analysed on an ABI 377 automated DNA sequencer (Perkin Elmer). Four individual clones were sequenced. Accuracy of the

PCR system.
Using strains of E. faecalis , the sensitivity of the PCR system was studied by
(i) titrating bacterial suspensions of 10 7 cells mL 1 , estimated by turbidimetry at 605 mm, in 10-fold dilutions series,
and by
(ii) 10-fold dilution series of extracted DNA from 10 7 cells. The original suspension and the dilutions were thoroughly mixed by vortexing. The series were run in triplicates.
The specificity of the method was tested against type strains of Streptococcus equinus (OMGS 2297), Streptococcus uberis (OMGS 2999), Streptococcus milleri (OMGS 1773), Streptococcus anginosus (OMGS 2479, NCTC 10713), Streptococcus pyogenes (OMGS 1775, CCUG 23117), Streptococcus mutans (OMGS 2428, ATCC 25175), Streptococcus salivarius (OMGS 2293), Streptococcus sanguis (OMGS 2478, ATCC 10556), and Gemella morbillorum (OMGS 2415).