Institute of Dental Research, Westmead Centre For Oral Health, Westmead Hospital, PO Box 533, Wentworthville, NSW 2145, Australia1
Author for correspondence: Mangala A. Nadkarni. Tel: +61 2 9485 7826. Fax: +61 2 9485 7599. e-mail: mnadkarni{at}dental.wsahs.nsw.gov.au
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ABSTRACT |
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Keywords: real-time PCR (TaqMan), detection of bacteria, universal probe, rDNA copy number, carious dentine
Abbreviations: ANGIS, Australian National Genomic Information Service; 6-FAM, 6-carboxyfluorescein; RTF, reduced transport fluid; TAMRA, 6-carboxy-tetramethylrhodamine; CT, threshold cycle; Tm, melting temperature of DNA; td, bacterial doubling time
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INTRODUCTION |
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Fluorescence-based methods can also be used to enumerate bacteria. In the food and biotechnology industries, for instance, the automated counting of pure cultures by flow cytometry is well established (Veal et al., 2000 ). However, most bacteria are optically too similar to resolve from each other or from debris using flow cytometry, without artificially modifying the target bacteria using fluorescent labelling techniques such as fluorescent antibodies or fluorescent dyes (Veal et al., 2000
; Attfield et al., 1999
). Differences in bacterial cell size, coaggregation of bacteria and the presence of different contaminating matrices (e.g. mud, food, dental plaque, dentine) can also make meaningful counting difficult, if not problematic, by interference with direct, or fluorescence, microscopy (Veal et al., 2000
).
Rapid enumeration of bacteria can also be achieved by using a variety of molecular approaches (Ward et al., 1990 ; Amann et al., 1995
; Wintzingerode et al., 1997
; Hugenholtz et al., 1998
). Primers with broad interspecies specificity have been designed to amplify 16S rDNA by PCR and have been used to determine bacterial numbers in complex communities (Wilson et al., 1990
; Relman et al., 1992
; Greisen et al., 1994
; Marchesi et al., 1998
; Klausegger et al., 1999
; Suzuki et al., 2000
). A majority of these studies, however, report the use of more than a single set of primers to detect the bacteria of interest. Other techniques, such as competitive PCR (Blok et al., 1997
; Rupf et al., 1999
), are labour-intensive and require the analysis of results from multiple reactions for each test sample. In contrast, real-time PCR, such as the TaqMan system developed by Applied Biosystems, relies on the release and detection of a fluorescent signal following the cleavage of a fluorescent labelled probe by the 5'-exonuclease activity of Taq polymerase. In the intact state, the fluorescent signal on the probe, such as 6-carboxyfluorescein (6-FAM), is quenched by the close proximity on the probe of a second dye, 6-carboxy-tetramethylrhodamine (TAMRA). The release of the fluorescent dye during each round of amplification allows for the rapid detection and quantification of DNA without the need for post-PCR processing, such as gel electrophoresis and radioactive hybridization (Heid et al., 1996
). In addition, the in-built 96-well format greatly increases the number of samples that can be simultaneously analysed.
In theory, conserved regions of 16S rDNA should provide the means for detecting and enumerating complex bacterial populations by real-time PCR, provided a universal probe can be constructed. However, the final determination of bacterial load by real-time PCR in a multi-species population will be influenced by the variation in the number of rRNA operons in a given species (Farelly et al., 1995 ). Bi-directional replication can further increase the numbers of a given rRNA operon, depending on the number of replication forks and the location of the rRNA operon relative to the origin of replication. The number of replication forks is directly related to the generation time, td, which in turn depends on the metabolic status of the bacteria at the time of sampling (Neidhardt et al., 1990
; Klappenbach et al., 2000
). Not knowing the exact number of copies of 16S rRNA operons in any given species at the time of sampling represents the main limitation to the absolute determination of bacterial numbers by real-time PCR based on 16S rDNA. However, in a variety of complex environmental, industrial and health-related situations in which multi-species populations are sampled along with impurities, or where the bacteria are internalized within a matrix, other methodologies are likely to be far less sensitive or precise.
In this paper, we report the design of a universal probe and primers set which specifically detects 16S rDNA of the Domain Bacteria and which is fully compatible with the TaqMan real-time PCR system. We have further characterized the use of this universal probe and primers set in enumerating bacteria with differing td and rDNA copy and applied this information to the determination of the anaerobic bacterial load in clinical samples derived from carious dentine where colony counting has, historically, been the preferred option.
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METHODS |
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Source of carious dentine.
Twenty carious teeth were obtained with informed consent from randomly selected patients who presented with pain and requested extraction to relieve their symptoms. Patients were excluded from the study if they reported a history of significant medical disease or antimicrobial therapy within the previous four months. Unrestored teeth with coronal enamel and dentine caries were selected for inclusion in the study on the basis of clinical diagnostic tests which indicated that they were vital, with clinical symptoms of reversible pulpitis (pain and heightened sensitivity to hot and cold stimuli). The study was approved by the Central Sydney Area Health Service Ethics Review Committee, Sydney, Australia (ref. no. 6/96).
Immediately after extraction, each tooth was placed in a container of reduced transport fluid (RTF; Syed & Loesche, 1972 ) and transferred to an anaerobic chamber at 37 °C containing 85% N2, 5% CO2 and 10% H2 (by vol.). Superficial plaque and debris overlying the carious lesion were removed and the surface rinsed several times with RTF. Using sterile sharp excavators, all the softened and carious dentine was collected as small fragments from each tooth. Sampling was completed within 20 min of tooth extraction.
Determination of c.f.u. in carious dentine.
The carious dentine extracted from each tooth was individually weighed and a standard suspension of 10 mg wet wt dentine (ml RTF)-1 was prepared at 37 °C in an anaerobic chamber (see above). The dentine fragments were homogeneously dispersed in RTF by first vortexing for 20 s and then homogenizing by hand in a 2 ml glass homogenizer for 30 s. Samples (100 µl) of 10-310-6 serial dilutions of these suspensions were prepared in RTF and plated in duplicate onto Trypticase Soy agar (Oxoid) containing 2 µg menadione ml-1, 5 µg haemin ml-1, 400 µg L-cysteine ml-1 (Sigma) and 5% (v/v) horse blood (Amyl Media) (US Department of Health and Human Services Centres for Disease Control, 1982 ). The plates were incubated at 37 °C in an anaerobic chamber containing 85% N2, 5% CO2 and 10% H2 (by vol.) for 14 d and the number of c.f.u. counted to determine the total microbial load (mg wet wt dentine)-1. The unused dispersed carious dentine samples were frozen at -80 °C.
Determination of viable bacteria from in vitro cultures.
Viable cell counts of cultures of Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus were determined by plating 100 µl of a 10-6 dilution of the appropriate culture grown in LB broth on LB agar plates and counting the colonies after aerobic incubation at 37 °C for 24 h.
Extraction of DNA from bacterial cultures.
DNA was isolated from individual bacterial species either by using the QIAamp DNA Mini Kit (Qiagen), according to the manufacturers instructions, or by using a freezeboil method. In the latter instance, bacterial cells from a 250 µl aliquot of culture were obtained by centrifugation (14000 g, 2 min, 1820 °C) and resuspended in 45 µl 10 mM phosphate buffer, pH 6·7, prior to freezing at -20 °C. The frozen cells were then heated in a boiling water bath for 10 min.
Extraction of anaerobic bacterial DNA from carious dentine.
Frozen suspensions of homogenized carious dentine were thawed on ice and 80 µl samples removed and combined with 100 µl ATL buffer (Qiagen) and 400 µg proteinase K (Qiagen). The samples were vortexed for 10 s and then incubated at 56 °C for 40 min with periodic vortexing for 10 s every 10 min to allow complete lysis of the cells. Following the addition of 200 µg RNase (Sigma), the samples were incubated for a further 10 min at 37 °C. DNA free of contaminating RNA was then purified using the QIAamp DNA Mini Kit, according to the manufacturers instructions.
Sources of other bacterial DNA.
DNA from Legionella pneumophila serogroup 4 ATCC 33156, serogroup 5 ATCC 33216, serogroup 6 ATCC 33215, serogroup 1 Knoxville-1 ATCC 33153 and Philadelphia-1, as well as Legionella anisa, Legionella bozemanii (now Fluoribacter bozemanae) serogroup 2, Legionella londiniensis, Legionella (now Tatlockia) maceachernii and Legionella waltersii was kindly provided by Mr Rodney Ratcliff, Infectious Diseases Laboratories, Institute of Medical and Veterinary Science, SA, Australia. DNA from Mycobacterium tuberculosis H37RV was kindly provided by Mr Greg James, Microbiology Laboratory, Westmead Hospital, NSW, Australia.
DNA sequence analysis and design of the universal primers and probe.
The designed probe and primers set were based on regions of identity within 16S rDNA following the alignment of sequences from most of the groups of bacteria outlined in Bergeys Manual of Determinative Bacteriology (Holt et al., 1994 ). The 16S rDNA sequences (GenBank accession no. in parentheses) from Bacteroides forsythus (AB035460), Porphyromonas gingivalis (POYRR16SC), Prevotella melaninogenica (PVORR16SF), Cytophaga baltica (CBA5972), Campylobacter jejuni (CAJRRDAD), Helicobacter pylori (HPU00679), Treponema denticola (AF139203), Treponema pallidum (TRPRG16S), Leptothrix mobilis (LM16SRR), Thiomicrospira denitrificans (TDE243144), Neisseria meningitidis (AF059671), Actinobacillus (now Haemophilus) actinomycetemcomitans (ACNRRNAJ), Haemophilus influenzae (HIDNA5483), Escherichia coli (ECAT1177T), Salmonella typhi (STRNA16), Vibrio cholerae (VC16SRRNA), Coxiella burnetii (D89791), Legionella pneumophila (LP16SRNA), Pseudomonas aeruginosa (PARN16S), Caulobacter vibrioides (CVI009957), Rhodospirillum rubrum (RR16S107R), Nitrobacter winogradskyi (NIT16SRA), Wolbachia sp. (WSP010275), Myxococcus xanthus (MXA233930), Corynebacterium diphtheriae (CD16SRDNA), Mycobacterium tuberculosis (MTRRNOP), Streptomyces coelicolor (SC16SRNA), Actinomyces odontolyticus (AO16SRD), Bacillus subtilis (AB016721), Staphylococcus aureus (SA16SRRN), Listeria monocytogenes (S55472), Enterococcus faecalis (AB012212), Lactobacillus acidophilus (LBARR16SAZ), Streptococcus mutans (SM16SRNA), Clostridium botulinum (CBA16S), Peptostreptococcus (now Micromonas) micros (PEP16SRR8), Veillonella dispar (VDRRNA16S), Fusobacterium nucleatum (X55401), Chlamydia trachomatis (D89067) and Mycoplasma pneumoniae (AF132741) were aligned using the GCG program PILEUP (Wisconsin Package Version 8, 1994) accessed through the Australian National Genomic Information Service (ANGIS, http://www.angis.org.au).
The Primer Express Software provided by Applied Biosystems was of limited value in determining a universal probe and primers set as the primary selection criterion of the software is the length of the amplicon (50150 bp). The use of this software resulted in a series of best fit suggestions for the universal probe and primers set, leading to unsatisfactory sequence homology for many of the bacterial genera. As a result, the regions of identity within the 16S rDNA had to be assessed manually, with the Primer Express Software being limited to checking for primerdimer or internal hairpin configurations, melting temperature (Tm) and percentage G+C values within possible primer/probe sets. The final chosen set, including the forward primer, 5'-TCCTACGGGAGGCAGCAGT-3' (Tm, 59·4 °C), the reverse primer, 5'-GGACTACCAGGGTATCTAATCCTGTT-3' (Tm, 58·1 °C) and the probe, (6-FAM)-5'-CGTATTACCGCGGCTGCTGGCAC-3'-(TAMRA) (Tm, 69·9 °C), complied with six of the eight guidelines set by Applied Biosystems for the design of primers and probes. These included Tm of the DNA being between 58 and 60 °C for the primers and 68 and 70 °C for the probe; the G+C content being between 30 and 80 mol%; no runs of more than three consecutive Gs in either the primers or the probe; no G on the 5' end of the probe; and the probe selected from the strand with more Cs than Gs. The primers and probe set only deviated from the ideal in that the last 5 nt of the 3' end of the forward primer contained more than two GCs and that the amplicon of 466 bp (based on that generated between residues 331 and 797 on the Escherichia coli 16S rRNA gene) exceeded the 50150 bp that was recommended.
The universal probe and primers were checked for possible cross-hybridization with bacterial genes other than 16S rDNA as well as genes from Eucarya and Archaea using the database similarity search program BLAST (Altschul et al., 1990 ) accessed through ANGIS. The BLAST search results showed only one significant hit that of a specific breast cancer cell line (BT029) which was detected only by the reverse primer. However, the universal primers did not amplify the human DNA sample supplied by Applied Biosystems in their Beta-actin Detection Kit probe set, thus confirming the specificity of the probe and primers set for the 16S rDNA of the Domain Bacteria.
PCR conditions.
Amplification and detection of DNA by real-time PCR were performed with the ABI-PRISM 7700 Sequence Detection System (Applied Biosystems) using optical grade 96-well plates. Duplicate samples were routinely used for the determination of DNA by real-time PCR, except in the case of carious dentine where the DNA was amplified in triplicate and mean values calculated. The PCR reaction was performed in a total volume of 25 µl using the TaqMan Universal PCR Master Mix (Applied Biosystems), containing 100 nM of each of the universal forward and reverse primers and the fluorogenic probe, except for the determination of the predominantly anaerobic bacterial load in carious dentine where 300 nM of the forward and reverse primers and 175 nM of the fluorogenic probe were used with the TaqMan PCR Core Reagents Kit. The reaction conditions for amplification of DNA were 50 °C for 2 min, 95 °C for 10 min and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Data analysis made use of Sequence Detection Software version 1.6.3 supplied by Applied Biosystems.
DNA standards used for determining bacterial number by real-time PCR.
Escherichia coli DNA was generally used as the standard for determining bacterial number by real-time PCR. However, to determine the effect of variations in rDNA copy number as well as the multiplying effect of the td on the calculation of bacterial number, DNA standards were also prepared from two rapidly growing aerobic bacteria, Staphylococcus aureus and Pseudomonas aeruginosa, with td in vitro in the order of 2050 min and two slow-growing obligate oral anaerobes, Prevotella melaninogenica and Porphyromonas endodontalis, with td in vitro in the order of 515 h. Standard graphs were always prepared from data accumulated at the same time as the test samples to act as internal controls.
Relative estimation of bacteria in an artificial in vitro mixture and in carious dentine.
To determine the validity of using the universal probe and primers set to estimate the total number of bacteria in a mixed culture, three bacteria, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, were grown separately in vitro to late-exponentialearly-stationary phase and equal volumes of the three cultures (2 ml) mixed together. The number of Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus c.f.u. at stationary phase was determined by serial dilution on agar plates and compared with the relative bacterial load determined by real-time PCR using the universal probe and primers set and Escherichia coli DNA as the standard.
The total number of c.f.u. obtained from carious dentine samples were determined by serial dilution on agar plates in an anaerobic chamber as described above and compared with the relative bacterial load determined by real-time PCR using the universal probe and primers set and Prevotella melaninogenica ATCC 25845 DNA as the standard.
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RESULTS AND DISCUSSION |
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Broad-range detection of bacterial species by the universal probe and primers set
To determine the ability of the universal probe and primers set to detect a broad range of bacteria, samples of DNA extracted from 49 different strains representing 34 different species from the major groups of bacteria listed in Bergeys Manual of Determinative Bacteriology (Holt et al. 1994 ), were subjected to real-time PCR using the probe and primers set. All of the selected species were detected within a CT range of 17·0534·00 (Table 1
). For each species there was little variance in the value of 2·00x102 (range 1·98x1022·06x102) Escherichia coli-equivalent bacteria (pg DNA)-1 when Escherichia coli DNA was used as a standard, indicating that the source of DNA was not influencing the level of detection and that the probe and primers set was equally efficient in detecting the DNA irrespective of the species from which it was extracted. Only in the case Micromonas (formerly Peptostreptococcus) micros was there a mismatch in identity between the probe and primers set and the 16S rDNA. This constituted a single nucleotide deletion in the 16S rDNA compared with the 5' end of the forward primer. This sequence discrepancy was clearly tolerated during real-time PCR detection of Micromonas (formerly Peptostreptococcus) micros DNA (Table 1
).
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Effect of the source of standard DNA on the measurement of relative DNA concentration
To confirm that a DNA standard other than that of Escherichia coli should result in a difference in the relative amount of DNA detected due to variations in rDNA copy number and the effect of the td on this number, the relative amounts of DNA from the rapidly growing aerobic bacteria Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa were compared with the slow-growing obligate oral anaerobes Prevotella melaninogenica and Porphyromonas endodontalis. In each instance the relative amount of DNA was estimated by real-time PCR using each of the five DNAs as standards and compared with the amount of DNA determined at A260 (set at 100%). It would be expected that comparison of like DNA by real-time PCR with the known amount of added DNA would be approximately 100%. In two instances this was not the case. For both Pseudomonas aeruginosa and Prevotella melaninogenica approximately twice the amount of DNA was detected. This was due in part to the fact that the relative amounts of DNA were calculated by Sequence Detection System version 1.6.3 software supplied by Applied Biosystems based upon the arbitrary placement of the horizontal threshold line used to determine the CT (cf. Fig. 1a). The horizontal threshold line was therefore adjusted to bring these two values as close to 100% as possible and the relative amount of DNA recalculated (Table 2
).
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The data in Table 2 allowed an estimation of the ratio of the number of copies of the 16S rRNA operons in the different species. A mean ratio of 20:10:9:1:1 (to the nearest integer) for the copy numbers in Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Porphyromonas endodontalis and Prevotella melaninogenica, respectively, fitted the data. This implied that the fast-growing aerobes, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa possessed approximately twice the known chromosomal complement of 16S rRNA operons. The data also predicted that the obligate anaerobes possess only one or two 16S rRNA operons per chromosome. The exact copy numbers are currently unknown.
These results demonstrate that failure to compare DNA from similar groups of bacteria possessing similar growth rates readily leads to an under or over estimation of the amount of DNA by one order of magnitude. Our analyses, however, show that if the ratio of the estimated amount of DNA measured against a rapidly growing bacterium, such as Staphylococcus aureus, to that measured against a slow-growing bacterium, such as Prevotella melaninogenica, is <1·0, then the number of bacteria in the sample should be estimated using the DNA extracted from the fast-growing bacterium. If the ratio is >1·0, the alternative standard DNA from the slow-growing bacterium should be used. In practice, this may simply require reference to a standard curve where the DNA is derived from a bacterium considered to represent the predominant species in the sample. Others, however, have come to different conclusions. For instance, Lyons et al. (2000) found no difference in the number of rDNAs per bacterial cell for Haemophilus (formerly Actinobacillus) actinomycetemcomitans, Porphyromonas gingivalis, Escherichia coli and group G streptococci and therefore assumed that the mean number of 16S rDNA operons in each bacterial cell was similar in all dental plaque samples. Thus, they made no attempt to compensate for differences in 16S rDNA copy number.
Comparison of viable cell numbers and the relative estimation of bacteria in an artificial in vitro mixture using real-time PCR
To determine the validity of using the universal probe and primers set to estimate the total number of bacteria in a mixed culture, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus were grown separately and equal volumes of the three cultures mixed together. The number of bacteria was similar irrespective of whether the estimation was made using real-time PCR or colony counting (Table 3), despite the fact that the number of copies of the 16S rRNA operons in a single chromosome of Escherichia coli is seven while that in Pseudomonas aeruginosa is four (Farelly et al., 1995
) and Staphylococcus aureus is nine (Gurtler & Stanisich, 1996
), and the further expectation that Pseudomonas aeruginosa would be underestimated (as was apparently the case) and Staphylococcus aureus overestimated against the Escherichia coli standard DNA.
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In conclusion, the universal probe and primers set that we have developed for the TaqMan system enables the sensitive detection of numerous bacterial species and strains belonging to the major groups of bacteria defined in Bergeys Manual of Determinative Bacteriology (Holt et al., 1994 ), without cross-detection of DNA from Eucarya or Archaea. Detection was achieved in minimum time and with no additional handling of the PCR product, thereby reducing the chances of contamination. We therefore believe that our designed universal probe and primers set should universally estimate total bacteria by real-time PCR in the shortest possible time. The greatest potential of our probe and primers set lies in its ability to detect bacteria from environmental samples which are difficult to cultivate and that would in all practicality remain undetected or underestimated by viable culture count methods or, alternatively, bacteria that are in an aggregated or coaggregated state or contained within matrix material, such as the carious dentine samples examined in this study, where fluorescent detection and/or microscopic enumeration are also impractical. In addition, the application of this universal probe and primers set could enable rapid differentiation of bacterial from viral infections within the limited time constraints sometimes experienced in life-threatening clinical situations.
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ACKNOWLEDGEMENTS |
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Received 1 June 2001;
revised 19 August 2001;
accepted 4 September 2001.