Welcome to Scribd, the world's digital library. Read, publish, and share books and documents. See more
Download
Standard view
Full view
of .
Look up keyword
Like this
1Activity
0 of .
Results for:
No results containing your search query
P. 1
PCR-Based Identification of Bacteria Associated With

PCR-Based Identification of Bacteria Associated With

Ratings: (0)|Views: 43|Likes:
Published by Dr.O.R.GANESAMURTHI

More info:

Published by: Dr.O.R.GANESAMURTHI on Mar 15, 2011
Copyright:Attribution Non-commercial

Availability:

Read on Scribd mobile: iPhone, iPad and Android.
download as PDF, TXT or read online from Scribd
See more
See less

03/15/2011

pdf

text

original

 
J
OURNAL OF
C
LINICAL
M
ICROBIOLOGY
, Sept. 2002, p. 32233231 Vol. 40, No. 90095-1137/02/$04.00
0 DOI: 10.1128/JCM.40.9.3223–3231.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.
PCR-Based Identification of Bacteria Associated withEndodontic Infections
Ashraf F. Fouad,
1
* Jody Barry,
1
Melissa Caimano,
2
Michael Clawson,
3
Qiang Zhu,
1
Rachaele Carver,
1
Karsten Hazlett,
4
and Justin D. Radolf 
4
Department of Endodontology, School of Dental Medicine
1
, Department of Pathology,
2
and Center for Microbial Pathogenesis,
4
University of Connecticut Health Center, Farmington,Connecticut, and U.S. Meat Animal Research Center, Agricultural ResearchService, U.S. Department of Agriculture, Clay Center, Nebraska
3
Received 26 November 2001/Returned for modification 15 February 2002/Accepted 10 June 2002
PCR primers that target the bacterial 16S rRNA genes (or the
tuf 
gene for the genus
Enterococcus
) were usedto identify 10 putative bacterial pathogens in root canals with necrotic pulp. In addition, the associations of these microorganisms with symptoms and a history of diabetes mellitus were investigated. Microbial samplesfrom the root canals of 24 teeth with necrotic pulp were included in the study. PCR with universal bacterialprimers identified bacterial DNA in 22 specimens; the remaining 2 specimens were from intact teeth that hadbeen traumatized 6 months prior to treatment. PCR with specific primers showed that preoperative symptomswere significantly associated with the presence of 
Streptococcus
spp. (
P
< 0.001 by chi-square analysis). Therewas also a nonsignificant trend for symptoms to be associated with
Fusobacterium nucleatum
and
Porphyromo-nas gingivalis
(odds ratio, >2) and for diabetes mellitus to be associated with
P. gingivalis
and
Porphyromonasendodontalis
(odds ratio, >2). Cloning and sequencing of the universal PCR product in one specimen revealedthe presence of an organism related to the genus
Olsenella
, which has not previously been described inendodontic infections.
The presence of bacteria in the root canal leads to thedevelopment of periapical periodontitis (32). Several studieshave shown an association between painful exacerbation of periapical lesions and the presence in the root canal of specificbacteria. Black-pigmented bacteria belonging to the genera
Porphyromonas
,
Prevotella
, and
Bacteroides
have been culturedfrom root canals in a significant proportion of cases in thosestudies (25, 53, 57) and are frequently present in the samecanals as members of the genera
Peptostreptococcus
and
Fuso-bacterium
(20, 21, 26). However, the findings of different stud-ies based on culturing of canal contents vary significantly. Thismay be due, at least in part, to the reduced reliability andsensitivity of culturing techniques. The persistence or furtherexpansion of a periapical lesion, despite seemingly adequateendodontic treatment and timely restoration of the tooth, isusually attributed to the persistence of pathogenic microorgan-isms in the root canal system. Recent investigations have doc-umented that the presence of cultivable bacteria from canals atthe time of obturation was critical in predicting failure of treatment (46, 52). However, the microorganisms most com-monly associated with failed endodontic cases are differentfrom those cultured from canals with pulp necrosis. Studiesreveal that most of these failed cases have gram-positive strainssuch as enterococci, streptococci, and eubacteria, with occa-sional
Candida
, peptostreptococci, and fusobacteria (36, 38,53). Although enterococci were the most prevalent microor-ganisms in the last three studies, being present in 54, 70, and38% of the cases, respectively, the percentages of differentstrains identified again vary significantly among the studies,and in a considerable number of cases there were no cultivablemicroorganisms. Therefore, sensitive and accurate moleculartechniques are necessary to accurately characterize the rootcanal microbial irritants in order to determine their associationwith clinical symptoms and the prognosis of treatment. Forexample, the introduction of molecular methods into analysesof root canal samples has led to the identification of a numberof fastidious organisms such as
Bacteroides forsythus
and
Trepo-nema denticola
(11, 23, 42, 43), which have not previously beendescribed in endodontic infections.PCR amplification of the bacterial 16S or 23S rRNA gene(rDNA) or other rDNAs is more sensitive and more efficientthan culturing and biochemical identification of endodonticflora. In the root canal microbial environment, PCR was shownto be more accurate than sodium dodecyl sulfate-polyacrylam-ide gel electrophoresis in differentiating and identifying thetwo important endodontic pathogens,
Prevotella intermedia
and
Prevotella nigrescens
, which could not be differentiated by cul-turing (4). Although the use of DNA probes can be moresensitive and more efficient than culturing, it still requires thepresence of 
10
4
bacterial cells to ensure detection (42). ThePCR technique can be sensitive enough to detect a few DNAstrands of the microorganisms present if adequate primers areused and the PCR conditions are sufficiently optimized. Wehave recently shown that, after inoculation of three endodon-topathogenic bacteria in mouse pulp exposures, PCR wasmuch more accurate than culturing in detecting the inoculatedanaerobic bacteria (18). Several uncultivable species have beenidentified from dentoalveolar abscesses by PCR (55).Previous studies have shown that the diabetic host may have
* Corresponding author. Mailing address: Department of Endodon-tology, University of Connecticut Health Center, 263 FarmingtonAve., Farmington, CT 06030-1715. Phone: (860) 679-2726. Fax: (860)679-2208. E-mail: fouad@nso.uchc.edu.3223
 
an increased periapical lesion size (34) or may develop moreserious infections in response to virulent root canal bacteria(18). Patients with a history of diabetes mellitus and periapicallesions may have signi
cantly reduced healing following end-odontic therapy compared with that for the nondiabetic pop-ulation (17a).The purpose of this study was to determine the presence of 10 putative root canal microorganisms in samples from rootcanals with necrotic pulp and apical periodontitis by usinguniversal bacterial as well as species- or genus-speci
c PCRprimers. We also determined the association of these organ-isms with clinical symptoms and with a history of diabetesmellitus.
MATERIALS AND METHODSPatient selection and sample collection.
All patient-related procedures used inthis study conformed to protocols approved by the Institutional Review Board of the University of Connecticut Health Center. The purpose and scope of the studywere explained to patients presenting for endodontic treatment for a tooth withpulp necrosis and apical periodontitis. Patients who consented to participate inthe study and who had not been treated with antibiotics in the preceding 3months were included in the study. Patients who indicated that they had diabetesmellitus received a free HbA1c test to determine the degree of their glycemiccontrol. Patients who had no history of diabetes were offered a free fasting bloodglucose test to verify that they did not have diabetes.The teeth involved had a negative pulp test result, had a periapical radiolu-cency on a preoperative radiograph, and had not had any previous endodonticprocedures. Detailed information regarding signs and symptoms and radio-graphic and clinical data were collected. Symptomatic patients were de
ned aspatients who had a preoperative visual analogue score of 30 or more on a100-point scale, had moderate to severe pain to percussion or palpation of thetooth involved, and/or had swelling.The technique for sample collection was as follows: following isolation of thetooth involved with a rubber dam, the
eld was disinfected with 30% H
2
O
2
andthen 5% tincture of iodine. Caries and/or existing restorations, if present, wereremoved, and then the cavity was wiped with a sterile cotton pellet slightly wetwith 1% buffered NaOCl, with care that it did not seep into the canal. Thehalogen disinfectants were then inactivated with 5% sodium thiosulfate. Thepulp chamber was then accessed with a new sterile bur. If purulence or serous
uid was present in the canal, this was directly sampled with three size
ne paperpoints. Otherwise, sterile saline was deposited in the canal, making sure that itdid not over
ow. A size 15 to 30
le (depending on the canal size) was used tonegotiate the canal to the estimated length. If the canal was very calci
ed, GatesGlidden burs sizes 2 and 3 were used so that the paper point could penetrateto a depth close to the estimated canal length. Three
ne paper points werethen used to obtain the sample. The last paper point was left in the canal for 30 s.In multicanaled teeth, one paper point sample was obtained from each ca-nal unless the canals were very calci
ed, in which case sampling of the canalin the root with the largest periapical lesion and the largest canal was done.The paper points were placed in sterile, DNA- and RNA-free vials containing 1ml of 
lter-sterilized 10 mM Tris-HCl, 1 mM EDTA (pH 8), and 0.5 g of sterileglass beads (diameter, 0.71 to 1.18 mm). The vials were frozen at
70
°
C untilused.
DNA extraction.
The vials with paper point specimens were vortexed for 2 minto disperse microbial cellular material into suspension. The suspension wasremoved from the original vial and transferred to 2-ml sterile vials, which werethen centrifuged at 7,500 rpm (all centrifuge procedures were carried out withEppendorf [Westbury, N.Y.] scienti
c microcentrifuge model 5417C) for 10 min,and the supernatant was again removed. DNAs were extracted from the cellularpellet by one of two methods. The
rst method (specimens 1 to 10) used theChelex extraction and boiling technique (13). Brie
y, this method involved theaddition of 75
l of 15% Chelex 100 resin (Bio-Rad) to the pellet resuspendedin 0.5 ml of Tris-HCl buffer and thorough mixing, followed by incubation at 56
°
Cfor 30 min in a dry heat block. The mixture was boiled in a dry heat block for 10min and then chilled on ice for 5 min. It was then centrifuged at 12,000 rpm for2 to 3 min. The supernatant was carefully removed, with the Chelex beingavoided. The DNA was stored at
20
°
C until it was ready for use in the PCR.For the last 14 specimens, we switched to the enzymatic extraction method,according to the protocol described for the QIAamp DNA mini kit (Qiagen,Valencia, Calif.), because of the manufacturer
s claims of improved purity andyield of the extracted DNA and because it allows the extraction of fungal DNA(8) for use in future research. The pellet was suspended in 180
l of enzymesolution (20 mg of lysozyme per ml, 20 mM Tris HCl [pH 8.0], 2 mM EDTA,1.2% Triton) and incubated for 30 min at 37
°
C. Proteinase K (20
l) and RNaseA (4
l at 100 mg/ml) were added, and the specimen was incubated for 2 min atroom temperature. Buffer AL (200
l) was added, and the specimen was vor-texed and incubated at 56
°
C for 30 min and then for 15 min at 95
°
C. Ethanol (200
l at 96 to 100%) was added, followed by vortexing and brief centrifugation. Themixture was then added to a QIAamp spin column and centrifuged at 8,000 rpmfor 1 min. The column was then placed in a clean 2-ml collection tube, 500
l of buffer AW1 was added, and the mixture was centrifuged at 8,000 rpm for 1 min.The column was again placed in a clean 2-ml collection tube, and 500
l of bufferAW2 was added, followed by centrifugation at 14,000 rpm for 3 min. Then, bufferAE (200
l) was added, followed by centrifugation at 8,000 rpm for 1 min. Theelutions were combined for a total yield of 400
l, which was aliquoted in sterile,DNA- and RNA-free conical tubes and frozen at
20
°
C until use.Prior to performing the second extraction method we conducted a pilot ex-periment to determine if the two different extraction methods affected the yieldof extracted DNA from representative stock strains of two gram-positive bacteriaand two gram-negative bacteria. This experiment was also run with one clinicalsample that was divided into two aliquots, and each aliquot was extracted by oneof the two methods. PCR was later run with primers speci
c for all the bacteriaunder study. These experiments did not reveal any perceptible differences inDNA yields or PCR results between the different extraction methods, and there-fore, the results obtained by both methods are considered together.The yield of extracted DNA was quanti
ed for each of the control stockbacterial strains and clinical specimens by using a Hoefer DyNA200
uorometer(Amersham Pharmacia Biotech, Piscataway, N.J.). The yield ranged from 2 to 33ng/
l for the stock bacterial strains and 1 to 19.5 ng/
l for the clinical specimens.
Microorganism selection.
We chose to evaluate the root canals for the pres-ence of 10 microorganisms that have frequently been isolated from root canalswith necrotic pulp (Table 1). Our selection of the bacteria was based on thefollowing criteria: organisms that are highly prevalent in root canals with necroticpulp (black-pigmented bacteria,
Fusobacterium nucleatum
,
Peptostreptococcusmicros
, and
Streptococcus
spp.) (51), organisms that are frequently found inpatients with symptomatic endodontic infections (
P. intermedia
,
P. nigrescens
,
Porphyromonas gingivalis
, and
Porphyromonas endodontalis
) (5, 20, 25, 28, 47, 57),organisms that have been detected in root canals from patients who have failedendodontic treatment (
Enterococcus
spp.) (36, 52), and organisms that are prev-alent in patients with severe periodontitis (48) and that have recently beenidenti
ed in root canals by PCR (
T. denticola
and
B. forsythus
) (11, 31, 42).
PCR ampli
cation of rDNA.
Previously published primer pairs were selectedfor speci
c PCR ampli
cation of 16S rDNAs (or the
tuf 
gene for the genus
Enterococcus
) of the microorganisms listed in Table 1. Initially, a universaleubacterial primer pair was used to detect DNAs from all bacterial speciespresent in the sample. Subsequently, a PCR mixture with oligonucleotide prim-ers speci
c for rDNAs was used. At least duplicate experiments were run foreach specimen. PCR ampli
cation was performed in a thermal cycler (PE9700 orPE2400; Perkin-Elmer Applied Biosystems, Foster City, Calif.). It was carriedout in a volume of 50
l containing 10
l of extracted sample DNA or 5
l of extracted control stock bacterial DNA (see below), 5
l of 10
PCR buffer, 0.25
l of 5 U of 
Taq
DNA polymerase (Eppendorf, Cologne, Germany) per
l or 0.5
l of HotStar
Taq
(Qiagen), 1.5 mM MgCl
2
, 0.2 mM concentrations of each of the four deoxynucleoside triphosphates (Takara, Otsu, Shiga, Japan), and a 0.5
M concentration (500 ng) of each (sense and antisense) primer; the balanceconsisted of sterile ultrapure water. PCR conditions for each primer combinationwere optimized in pilot experiments. The PCR conditions used were generally asfollows: the initial denaturation was at 94
°
C for 2 min for Eppendorf 
Taq
or 15min for HotStar
Taq
. This was followed by 30 cycles of denaturation at 94
°
C for15 s, annealing at a temperature that depended on the primer (Table 1) for 15 s,and extension at 72
°
C for 45 s. The
nal extension was at 72
°
C for 5 min, andthen the products were cooled to 4
°
C until they were removed.The ampli
cation products were analyzed by 2% agarose gel electrophoresis inTAE buffer (40 mM Tris-acetate, 2 mM EDTA [pH 8.3]). The Power Pac 1000apparatus (Bio-Rad, Hercules, Calif.) was set at 110 mA for 2 h or 95 V for 1 h.The gels were stained with 0.5
g of ethidium bromide per ml for 30 min anddestained with water for 20 min. The PCR products were visualized under UVlight with an Alpha Imager (Alpha Innotech Corp., San Leandro, Calif.).For each primer we ran a number of PCR controls. These included the use of DNA from American Type Culture Collection (ATCC) stock strains of therespective bacterial species (
Enterococcus faecalis
for the
Enterococcus
primers)as positive controls. The
Streptococcus
-speci
c primers were reported as being
3224 FOUAD ET AL. J. C
LIN
. M
ICROBIOL
.
 
speci
c for
Streptococcus intermedius
, with possible cross-reactivity with
Strepto-coccus milleri
isolates (11). However, our positive control experiments haveshown that these primers reacted with
S. intermedius
,
Streptococcus constellatus
,
Streptococcus anginosus, Streptococcus mutans
,
Streptococcus sanguis
, and
Strep-tococcus bovis
, all at a single band at 500 bp. Thus, these primers were considered
Streptococcus
genus speci
c. The DNAs of the ATCC stock strains were ex-tracted from spectrophotometrically determined concentrations of 3
10
8
bac-terial cells/ml that were cultured under ideal conditions for the particular species.An additional positive control was the universal primer pair speci
c for bacterial16S rDNA, with which positive results were obtained with DNA from all bacteriabut negative results were obtained with DNA from
Candida albicans
. In addition,each primer set was run with DNA extracted from all other bacterial species usedin the study together with DNA extracted from ATCC stock strains of 
Eubac-terium nodatum
and
Actinomyces israelii
and with water (no DNA) as negativecontrols. Representative PCR products obtained by use of each of the species- orgenus-speci
c primers with patient specimens were directly sequenced (see
Cloning and sequencing of novel 16S rDNA sequences
below) to determinethe published sequence closest to that of the organism ampli
ed.
Cloning and sequencing of novel 16S rDNA sequences.
Two specimens, spec-imens SP05 and SP08, yielded a PCR product with the universal primer pair butno product with the 10 speci
c primers tested. Specimen SP08 yielded too littleproduct for cloning and will not be described further. Ampli
cation productsfrom specimen SP05 were cloned into the vector pCR 2.1-TOPO TA (Invitrogen,Carlsbad, Calif.) according to the instructions of the manufacturer or weresequenced directly. For cloning, the PCR product was transformed into
Esche-richia coli
One-Shot TOP10. Colonies containing the insert were used to inoc-ulate Luria-Bertani agar (Miller; Fisher Scienti
c Co., Pittsburgh, Pa.). PlasmidDNA was puri
ed with the QIAprep Spin Miniprep kit (Qiagen) or the ConcertRapid Plasmid puri
cation kit (Life Technologies, Gibco BRL, Rockville, Md.).The puri
ed plasmid DNA from the cloning procedure was sequenced in theUniversity of Connecticut Health Center Molecular Core Facility by using anABI Prism 3100 genetic analyzer (Perkin-Elmer Applied Biosystems) and re-verse primer M13 or T7 (Invitrogen). The universal PCR products were puri
edwith the Concert Rapid PCR puri
cation system (Life Technologies, GibcoBRL) and directly sequenced by using the universal forward and reverse primers(Table 1). The resulting sequences were used to search databases availablethrough the National Center for Biotechnology Information. PCR productsobtained with speci
c primers from representative patient specimens were par-tially sequenced directly after puri
cation as described above to verify the iden-tity of the product. All species-speci
c primers yielded sequences that matchedpublished sequences for the respective species. The two representative productsfor the genus-speci
c primers yielded sequences that had close homology withthe sequences of 
S. sanguis
, unidenti
ed oral streptococci (GenBank accessionno. AB028364),
Streptococcus cristatus
, and
Streptococcus pneumoniae
for the
Streptococcus
genus-speci
c primers and various
Enterococcus
spp. for the
En-terococcus
genus-speci
c primers.
Phylogenetic analyses of the novel 16S rDNA sequence.
Signi
cant databasehits were aligned with our unknown sequence by using ClustalW software inMacVector (Genetics Computer Group, Oxford Molecular Co.). A neighbor-joining phylogenetic tree was constructed from the alignment by using MacVec-tor (Genetics Computer Group, Oxford Molecular Co.). A distance matrix wasconstructed by using a Tamura-Nei model without gamma correction and withgaps distributed proportionately. Neighbor-joining bootstrap values were derivedfrom 1,000 replications and were added to the tree.
Data analysis.
The associations between the positive identi
cation of a bac-terial species or genus and symptoms or a history of diabetes were analyzed byodds ratio (OR) analysis. OR associations of 2 or more were considered positiveassociations (49, 50). These positive associations were further analyzed by achi-square analysis to determine their statistical signi
cance.
Nucleotide sequence accession number.
The sequence that forms a basallineage in the
Olsenella
clade detected in this study has been deposited inGenBank under accession number AF426827.
RESULTS
Of 24 patients participating in the study, 8 were consideredto be symptomatic and 6 had a history of diabetes mellitus (2with type 1 diabetes mellitus and 4 with type 2 diabetes mel-litus) (Table 2). The HbA1c results revealed that three diabeticpatients had moderate glycemic control (7 to 10%) and threehad poor glycemic control (
10%). Of the 18 nondiabeticpatients, 9 agreed to take the fasting blood glucose test, and allhad results below 126 mg/dl, which is generally accepted as thethreshold value for the diagnosis of diabetes mellitus (1).Twenty-two of the 24 specimens tested reacted positivelywith the universal bacterial primer pair. Two specimens, spec-imen SP09 (from a nondiabetic individual) and specimen SP12(from an individual with type 2 diabetes), had no identi
ablePCR amplicons with the universal primers. Retrospective anal-ysis of the clinical conditions of the root canals from which
TABLE 1. Oligonucleotide primers used
Primer pair or organism Sequence (5
to 3
)
a
Size (bp) Annealing temp (
°
C) Reference
Universal 16S rRNA gene-speci
c primer pairAGA GTT TGA TCC TGG CTC AG 1,500 56 56ACG GCT ACC TTG TTA CGA CTT
Prevotella intermedia
CGT GGA CCA AAG ATT CAT CGG TGG A 259 64 7CCG CTT TAC TCC CCA ACA AA
Prevotella nigrescens
ATG AAA CAA AGG TTT TCC GGT AAG 804 60 9CCC ACG TCT CTG TGG GCT GCG A
Porphyromonas endodontalis
GCT GCA GCT CAA CTG TAG TC 672 60 9CCG CTT CAT GTC ACC ATG TC
Porphyromonas gingivalis
AGG CAG CTT GCC ATA CTG CG 404 60 9ACT GTT AGC AAC TAC CGA TGT
Peptostreptococcus micros
AGA GTT TGA TCC TGG CTC AG 207 60 13ATA TCA TGC GAT TCT GTG GTC TC
Streptococcus
spp. AGA GTT TGA TCC TGG CTC AG 500 55 11GTA CCG TCA CAG TAT GAA CTT TCC
Fusobacterium nucleatum
AGA GTT TGA TCC TGG CTC AG 360 60 11GTC ATC GTG CAC ACA GAA TTG CTG
Bacteroides forsythus
TAC AGG GGA ATA AAA TGA GAT ACG 745 59 54ACG TCA TCC CCA CCT TCC TC
Enterococcus
spp. TAC TGA CAA ACC ATT CAT GAT G 112 55 33AAC TTC GTC ACC AAC GCG AAC
Treponema denticola
TAA TAC CGA ATG TGC TCA TTT ACA T 316 60 2TCA AAG AAG CAT TCC CTC TTC TTC TTA
a
The top primer is the sense primer, and the bottom primer is the antisense primer.
V
OL
. 40, 2002 ENDODONTIC BACTERIAL DETECTION BY PCR 3225

You're Reading a Free Preview

Download
scribd
/*********** DO NOT ALTER ANYTHING BELOW THIS LINE ! ************/ var s_code=s.t();if(s_code)document.write(s_code)//-->