Endodontic Topics 2004, 9, 514 All rights reserved

Copyright r Blackwell Munksgaard

ENDODONTIC TOPICS 2004
1601-1538

Signicance of bacterial identication by molecular biology methods

DAVID A. SPRATT
Rapid advances in molecular biology over the last 20 years have provided a bewildering array of techniques aimed at helping us to tease apart all aspects of biology. The discipline of microbiology has gained greatly from these advances especially with respect to detection and identication of micro-organisms. Indeed these molecular biology techniques have changed the way we classy all life on Earth. An important part of endodontic microbiology is detection and identication of the micro-organisms associated with initiation and progression of this polymicrobial infection. A range of appropriate molecular techniques are reviewed in the present article and include aspects of comparative 16S rRNA gene sequencing, polymerase chain reaction detection, strategies for identication of unculturable bacteria, and whole community analysis. Some of these techniques are widely used in endodontic microbiology while others are used by only a few workers. The advantages and disadvantages of all the techniques are discussed and put into perspective. All available surfaces in the oral cavity are colonized by different and diverse microbial biolms. Structures present in the mouth but not exposed to the microora are usually sterile e.g. the endodontium the pulp and root canal system within teeth. Endodontic infections are therefore dened as infections of the pulp and periapical tissues. A bacterial cause for these diseases was suggested by Miller (1) at the end of the 19th century when he demonstrated cocci, rods, and spirochaetes in necrotic pulps. However, because of other stronger arguments namely the hollow tube theory, (2) a bacterial cause for these pulpal and periapical diseases has only been attributed since the mid 1960s when pioneering work by Kakehashi et al. (3) demonstrated the importance of bacteria as prerequisites to pulpal inammation and subsequent necrosis. Bacteria and there products gain access to the pulp chamber in the majority of cases as a consequence of caries. Because of signicant demineralization of the enamel, cementum or dentine the pulp can be directly exposed to insult by the biolm associated with the lesion. Additionally, the pulp can be exposed by a number of other mechanisms e.g. trauma, exposed dentinal tubules, congenital conditions, enamel lamellae and possibly anachoresis (47). Before colonization bacterial products e.g. metabolic end products, lipopolysaccharide etc can elicit an inammatory response from the pulp (8). Accurate identication of micro-organisms involved in a disease process is frequently essential not only for effective antimicrobial therapy but also for understanding the disease initiation and progression.

Traditional microbiological identication

Given the microbial nature of the disease, traditional or culture-based microbiology studies were carried out by a number of oral microbiology groups throughout the world. Over the next two decades or so it was shown that the bacteria species associated with these lesions were surprisingly limited (9) given the number of taxa potentially able to colonize and the large number of taxa associated with periodontal lesions (see below). This reduced diversity implies special selective pressures operating within the root canal system. While the culture-based techniques have reported 412 taxa per root canal (10, 11) when the range of taxa isolated from root canal infections as a group as is taken into account 2030 genera are commonly isolated Of these

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the most commonly occurring species are Fusobacterium nucleatum, Streptococcus species, Porphyromonas species, Prevotella intermedia, Peptostreptococcus species, Actimomyces species and Eubacterium species (The genus Eubacterium is very broad and at present undergoing signicant taxonomic revision). The isolation and identication of these taxa lead to large numbers of studies aimed at dening which taxa were responsible for the disease, what mechanisms they used and indeed, associating particular taxa to different aspects of root canal infections e.g. pain, lesion size, etc. From early microscopy studies it was shown that 50% of the oral microbiota was unculturable (12). Therefore, it was very possible that unculturable taxa were present in root canal infections and were potentially playing a role in the disease initiation or progression or both. These unculturable taxa fall into two broad catagories. The rst are taxa that need nutrients or other essential components that conventional sampling techniques, transport conditions or laboratory media do not provide. This could be sensitivity to oxygen (i.e. very strict anaerobes) or the absolute requirement for products provided by other taxa within the root canal (9). These taxa are therefore broadly unknown apart from microscopy studies although; unless distinct morphology is apparent there is no way of knowing what proportion of the taxa are represented in the culture dependant proportion of the sample. The second category contains those taxa that are known, and very often common, but for some reason cannot be cultured, i.e. they are in a dormant state and non-culturable (13). The term viable but not culturable (VBNC) was coined to describe this state. It is thought that cells will go into this state as a protection strategy in response to adverse environmental conditions. It is very possible that adverse conditions exist within root canals especially nutrient deprivation and this may be another explanation for the limited taxa isolated for individual root canal infections. While microbiologists may have suspected that a number of taxa were present and unculturable (for whatever reason) there was very little that could be done other than using complex media to mimic the conditions present at the site of isolation or indeed use co-culture strategies. At the end of the day they had to be able to culture the taxa before they could identify or indeed characterize them.

Molecular identication methods for culture dependant techniques

With the advent of molecular biology microbiologists had another avenue to pursue with respect to understanding the microbiology of root canal infections. Shortly after Kary Mullis described a polymerase chain reaction (PCR) technique, for which he received the Nobel Prize in 1993 (14), the ood gates opened with respect to what was possible in the world of microbial detection and identication. The application of PCR and sequencing (and associated database construction and searching software) revolutionized the detection and identication of bacteria. PCR is a technique, which uses a DNA polymerase enzyme to make a huge number of copies of virtually any given piece of DNA or gene. It facilitates a short stretch of DNA (usually fewer than 3000 bp) to be amplied by about a million-fold. In practical terms it amplies enough specic copies to be able to carry out any number of other molecular biology applications e.g. size determination (in bases) and its nucleotide sequence. The particular stretch of DNA to be amplied, called the target sequence, is identied by a specic pair of DNA primers, oligonucleotides usually about 20 nucleotides in length which designate the outer limits of the amplication product. Given that there are about 500 bacterial taxa present in the oral cavity (15) the range and complexity of the techniques utilized to identify this very diverse microbiota is bewildering. Molecular biology techniques have lead to new approaches for bacterial identication. The use of nucleotide sequence data from 16S ribosomal RNA genes (amonge others), now makes it possible not only to identify but to infer phylogeny for all organisms on Earth (16). Phylogeny is dened as the evolutionary relationships within and between taxonomic levels, particularly the patterns of lines of descent, in a sense a family tree spanning 3.5 billion years. Therefore within reason a single methodology can be used to identify any bacterial isolate from any environment. The 16S (small subunit) rRNA gene was selected as a candidate molecule for a number of reasons: (i) it is present in all organisms and performs the same function, (ii) its sequence is sufciently conserved and contains regions of conserved, variable and hypervariable sequence, (iii) it is of sufcient size (ca. 1500 bases) to be relatively easily sequenced but large enough to contain sufcient information for

Bacterial identication by molecular biology methods

identication and phylogenetic analysis. While this technology took a few years to become popular a drawback was how to analyze the sequence information you had from your isolates. Until sufcient data had been deposited in the National Centre for Biotechnology Information (NCBI) Genbank database or specialized ribosomal databases very few comparative identications could be made. This has, however, now been rectied and the Ribosomal Database Project has over 124 000 aligned bacterial small subunit rRNA sequences deposited (as of February 2005). By visiting the RPP website unknown 16S rRNA sequence can be easily uploaded and compared with all the sequences on the database to nd its nearest neighbours, i.e. in most cases an identication to the species level can be ascribed (Fig. 1). This technology has meant that hundreds of isolates can be identied quickly and easily. Indeed in a recent study of 261 isolates from ve infected root isolates 20 taxa were identied by comparative 16S rRNA sequence analysis at an average for 12.6 per sample (17). Using this type of technology (a single methodology) these isolates could have been identied with a couple of weeks on a part time basis. This is compared with probably some months work by an experienced microbiologist using a large number of diverse and complex biochemical techniques. There are a number of advantages and indeed disadvantages associated with the comparative sequencing technique. On the plus side the single protocol is easily learned has relatively high through put and a good level of identication can be expected. On the down side is, however, that microbiologists and molecular biologists with very little experience can perform these techniques. The potential upshot of this is the mis-identication of isolates. While the technique is very straightforward the interpretation of the data
PCR Amplify 16S rRNA gene
30 cycles of: Denaturing Annealing Extension

produced is not as easy as it rst looks. For example new un-named isolates can be forced into species pigeon holes or indeed isolates from very closely related groups can be misidentied, i.e. mitis group streptococci. Additionally in point (iii) above mention is made of the size of the gene being sufcient to contain enough information for identication and phylogenetic analysis. While this is true (in most cases) it is common practice not to sequence all 1500 bases, since this would take a number of sequencing runs. Commonly a single sequence is used ranging form 300700 bases and identity is conferred on this basis. In most cases this may be acceptable (depending on what conclusions are drawn or claims made) but care must be used in interpreting the data without information on the complimentary sequence (the other DNA strand) or more complete sequence. While the 16S rRNA revolution has undoubtedly been a quantum leap for microbiology it has not been as good as originally hoped. Some bacterial groups are very closely related and the sequence information within the gene is not sufcient to resolve these taxa with any certainty. Difcult groups to resolve which are relevant in endodonotic infections include: mitis group streptococci (S. mitis, S. oralis, S. sangiunis, and S. gordonii), Actinomyces spp, (A. naeslundii, A. israelii, A. meyeri, A. odontolyticus, A. viscosus, A. gerencseriae and A. radicidentis), coagulase-negative staphylococci (S. epidermidis, S. warneri, S. lentus etc) and Veillionella spp (V. parvula, V. atypica and V. dispar). In light of this other candidate genes have been proposed and used for comparative sequence analysis studies. Not all of these follow the rules above but are often useful once 16S rRNA or indeed biochemical characterization has identied the isolate as a difcult group. Manganese dependant superoxide dismutase

Prepare DNA from a pure culture

Agar plate

Check for good PCR product

Gel electrophoresis

Clean Product

Identification
Closest match on database in seconds

Analyse base sequence online

BLAST or RDP

Analyse DNA sequence

Capillary or gel electrophoresis

Sequence PCR product

Fig. 1. Flow diagram showing the steps involved in bacterial identication using a 16S rRNA sequencing approach.

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(sodA) is one such gene that has been successfully used to identify the oral streptococci, including the mitis group (18) and the coagulase-negative staphylococci (19). A number of other genes have been used to identify coagulase-negative staphylococci but not commonly within oral microbiology these are the hsp60 and rpoB. target taxon rather than a similar one or indeed something completely different (but happens to share sequence homology)? The specicity testing only takes into account the strains used in the test and this is usually no more than 1030. Given the potential of any of up to 500 taxa being present, 50% of which are unculturable it is not hard to see where false positives can arise. As long as this is appreciated by the researches than there are some remedies especially if 16S rRNA genes are the target. The simplest of which is to randomly select a proportion of the amplication products (1020%) and subject them to comparative sequence analysis, i.e. identify them. A further consideration is the detection limit of the particular PCR technique (this may vary with user, reagents and equipment). It must always be borne in mind that to fail to amplify a product does not mean the target template was not present in the sample it means that it was not there in sufcient quantity to be amplied. As a rule of thumb the lower detection limit of PCR (single round) is about 1000 cells of target (20). Further modications of PCR can take the detection limit down to about 10 cells this is termed nested PCR. Essentially, following the rst PCR reaction another one is performed with a different set of primers using the product from the rst PCR as a template. This technique, while being a very sensitive, is prone to contamination and false positive reactions are very common. Simply increasing the detection limit does not really answer any further questions like for example, which taxa are there in high proportions and which are there as very small proportions? Information on proportions may provide clues as to which taxa are important in disease initiation and progression bearing in mind that

Molecular identication methods for culture independant techniques

The advent of PCR not only led to gene sequencing and identication of culturable taxa. It also provided a technique to circumvent the whole cultivation aspect of species detection and identication. In its simplest form this developed on the premise that given that bacteria can be identied by differences in certain DNA sequences (16S rRNA gene). It should then be possible, using specic PCR primers, to identify a particular species from any given sample whether that be from a root canal, a periodontal pocket, carious plaque or indeed saliva (Fig. 2). This has led to a large body of literature pertaining to the prevalence of specic taxa or groups in root canal infections (and is not reviewed here). This technique is relatively straightforward once the PCR parameters and the specicities have been ascertained and high throughput approaches can deal with hundreds of samples per day. Indeed, multiplex approaches allow more than one target to be detected in each PCR reaction (Fig. 3). Multiple primer sets can be used for at least three separate taxa (20). As you might expect there are some problems with this approach. The main problem is specicity for example, if a positive result occurs for a sample how do you know that it is actually an amplied product for the

Patient samples C 1 2 3 4 5 6 7 8 9 10 C

Fig. 2. Diagrammatic representation of the visualisation of polymerase chain reaction products from 10 subjects where taxon A was targeted with a specic primer set. It shows a band present in subject samples 1, 2, 4, 5, and 10. This indicates that taxon A was detected in these ve subjects and not in subjects 3, 6, 7, 8, or 9. C denotes a control sample of target taxon alone.

Bacterial identication by molecular biology methods

Patient samples C 1 2 3 4 5 6 7 8 9 10 C

D E F

Fig. 3. Diagrammatic representation of the visualisation of multiplex polymerase chain reaction (PCR). Amplication products from 10 subjects where three specic taxa D, E, and F were targeted in one PCR reaction. All three taxa are only detected in subject 2. None of the target taxa were detected in subjects 6, 7, and 9.
A DNA from 5 root canal samples 1 2 3 4 5 a b DNA from 5 taxa c d e Wash and detect B 1 2 3 4 5 a b c d e

Fig. 4. Diagram of Checkerboard DNDDNA hybridization showing; A vertical lanes containing sample DNA and horizontal lanes with DNA from ve known taxa. B following hybridization, washing and detection the presence or absence of the various taxa in the patient samples can be ascertained. Taxon b is present in all samples, taxon c is not detected in any sample, taxon a is detection in samples 2 and 5, etc.

in a polymicrobial infection the key taxa may be different for each stage. A further development of PCR has meant that not only could specic taxa be detected but they could be quantied as well. This technique, real-time PCR or quantitative PCR, uses uorescence to detect PCR products as they accumulate. Theoretically, there is a quantitative relationship between the amount of starting material and the PCR product at any cycle (21). Therefore using PCR primers from above (or modications of these) specic bacterial taxa can be detected and quantied. Indeed if a global 16S rRNA gene PCR primer (theoretically amplies all 16S rRNA genes from all bacterial taxa) is used as well (in a separate reaction) the total number of bacteria present in the sample can be ascertained. Therefore an estimation of the proportion (as a function of the whole microbiota) of a target taxon can be made.

The technique obviously suffers from similar drawbacks mentioned above but as long as these are carefully controlled for and considered, quantitative PCR will provide crucial information pertaining to the progression and nature of root canal infections. A quantitative PCR approach has been used to study the microbiology of carious dentine (22) and showed a greater bacterial load by quantitative PCR than culturing methods and quantied a number of important taxa (Micromonas micros, Porphyromonas endodontalis and P. gingivalis). Molecular techniques for bacterial detection and identication are not restricted to PCR alone and a notable alternative technique is checkerboard DNA DNA hybridization. This technique involves deposition of bacterial DNA from clinical samples (root canal, plaque etc) in parallel (vertical) lines on a nylon membrane. Digoxigenin-labelled whole genomic DNA probes are run at right angles to the samples (horizontal). Following washing the bound probe is detected and quantied (Fig. 4). This method was pioneered and extensively used by Sigmund Socransky in Boston, MA, USA (2326). The technique utilizes whole genomic DNA for 40 bacterial taxa and 28 patient samples per membrane this makes it a very high throughput technique and thousands of samples can be analyzed very quickly generating huge amounts of data regarding the detection rates of the forty taxa in each sample. The technique is semi-quantitative and standards containing known numbers of cells are used (105 and 106). A potential drawback is however the unknown cross reactivity with unknown taxa present in the sample. Additionally the technique can only provide information on known culturable taxa and while very valuable does not address the unculturable proportion of any sample. The technique has been used to a limited extent in endodontic microbiology studies (for details see (2730)).

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Detection and identication of unculturable taxa using molecular methods

To determine the unculturable microbiota in a sample, culture and PCR approaches are often used in a subtractive technique. A given root canal sample is processed routinely by culture that is to culture on a range of media (selective and non-selective) in both aerobic and anaerobic atmospheres. Isolates are identied by 16S rRNA gene sequence analysis (as above). Additionally an aliquot can be analyzed using a PCRcloning approach. DNA is isolated and puried from this aliquot. Using a similar PCR technique used for bacterial identication (see above), 16S rRNA genes are amplied. This will produce a mixed product i.e. instead of amplifying DNA from a pure culture (one taxon) this amplies DNA from all the taxa present in the sample. Because of this the PCR product cannot be simply sequenced since, sequencing a number of different 16S rRNA genes from different bacteria at the same time will produce nonsense sequence data
Amplicon A C B from taxon B C B A B A B A B C D C C B C C A

which cannot be analyzed. Therefore the different 16S rRNA genes present need to be separated. This is done by cloning the PCR products (see Fig. 5). Once the PCR products are singularized (each one separated into a plasmid and transformed into a host cell) they can be sequenced and identied as for culturable taxa (above). At this point there are two lists of bacteria identied from the sample; a culture dependent list and a culture independent list. Those taxa present on the culture independent list but not on the culture dependent list are therefore counted as unculturable (Fig. 6). The taxa determined as unculturable maybe either new unknown taxa or indeed well known and usually culturable taxa (possibly in a VBNC state). To understand the prevalence of these newly detected taxa in the infection specic PCR primers can be obtained or designed for straightforward PCR detection assays as detailed above. The subtractive PCR cloning approach is very powerful but is very time consuming and expensive to perform on large numbers of samples. It can however provide detailed information on the richness of the microbiota at any given site and provide targets for further studies.
Insert

C C A A B

D Plasmid B

Mixed PCR product

Universal primers used to amplify 16S rRNA genes from a 4-membered community (A-D)

Ligate products into a Plasmid

Plasmid contains insert and gene encoding antibiotic resistance amongst other things

Cells can now be replicated and stored. Insert can be sequenced and identified Transformed colony Each colony consists of cells with one plasmid containing one 16S rRNA gene
Cells can grow only if the plasmid is present since it contains resistance to the antibiotic used

A Plasmid B

E. coli cell

Transform E. coli cells with vector

One plasmid per cell. Culture on antibiotic containing media

Fig. 5. Diagramatic representation of the polymerase chain reaction (PCR) cloning process used to singularise mixed PCR products.

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Bacterial identication by molecular biology methods

Root canal Sample Paper point or pus aspirate

Culture (+/ enrichment) on agar media

Non-selective or selective Aerobic & Anaerobic

Isolate and purify DNA directly from clinical sample

Randomly Select 30 50 isolates

PCR amplify 16S rRNA gene and sequence See Figure 1

Singularise by TA cloning Characterise

Colony morphology Gram morphology Catalase test Oxidase test

Randomly Select 30 50 clones containing 16S rDNA insert

PCR amplify 16S rRNA gene and sequence

See Figure 1

Amplify 16S rRNA gene and sequence

See Figure 1

Compare Identify
Any taxon present as a clone and not as an isolate is considered unculturable

Identify

Fig. 6. Strategy dening the unculturable microbiota in a root canal sample.

The main perceived drawback of this technique is a concern that the universal primers used in the PCR are not as universal as once hoped for example, selective amplication of templates with a low GC content (31, 32). Additionally previous studies have also reported that all of the steps involved in the production of a gene library may have some biases (3336). Therefore, although it is often boasted that this technique negates the biases inherent in culture it is less frequently mentioned that it might have a number of biases itself! A number of studies have been carried out with root canal samples (3739, 17) and pus from alveolar abscesses (40, 41). In most of these studies novel taxa were detected and described. Indeed, in a culture and cloning study similar to that described in Fig. 6 Munson et al. (17) detected 65 taxa from only ve root canal samples, 27 of which were novel.

Whole community analysis

Rather than trying to dissect the microbial nature of root canal infections (or indeed any polymicrobial infection) an alternative approach can be taken. This involves dening the community and its characteristics as a whole from a root canal and comparing these characteristics with other root canals. While the above techniques can be considered as community analysis the only one, which really counts with respect to whole is the culture/PCRcloning approach and this is impractical for detailed comparisons between a number of samples. The precise nature of these techniques is still being developed for oral microbiology but a good example is denaturing gradient gel electrophoresis (DGGE). DGGE is a PCR based technique with a difference; rather than using the sequence of bases in the amplied

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Patient samples 1 2 3 4 5 6 7 8 9 10

Fig. 7. Diagrammatic representation of the visualization of a DGGE gel. Directly amplied 16S rRNA genes from root canal samples from 10 subjects showing different banding patterns (ngerprints). Lane 2 and 3 show complex patterns indicating a high species richness while lanes 4 and 6 show a less complex pattern indicating low species richness.

product for identication (i.e. sequencing a 16S rRNA gene) DGGE separates DNA fragments according to their sequence information (42). The basis of this technique is that DNA fragments of the same size but with differing base-pair sequence can be separated (43). This separation by DGGE relies on the electrophoretic mobility of partially denatured DNA molecules in a polyacrylamide gel, which is encumbered in comparison with the completely helical form of the molecule (43). A banding pattern is formed based on the number of taxa present in the sample (Fig. 7). If 16S rRNAgene is used as the target for the PCR then these bands can be cut out from the gel and sequenced to provide an identity for the particular band. Since a PCR amplication step is used then the biases previously mentioned may be operating, however, the rst step towards overcoming these is acknowledging their existence. A further problem is the interpretation of the data at a community ngerprint level. While cutting out bands and sequencing them gives valuable information it is also time consuming and costly. What is needed is a way to compare banding patterns within and between gels (samples) such that a particular pattern or specic bands in a pattern is indicative of certain clinical parameter. A number of techniques have been used but none have been broadly adopted perhaps because the most applicable one has not been developed to date. DGGE has been applied in environmental microbiology (4446) and in the analysis of microbial communities in the human body (4750) Recently DGGE has also been applied to analyse the bacterial diversity of human subgingival plaque (51, 52) as well as laboratory-grown dental plaque microcosms (53). Siqueira et al. (54) have

successfully used this technique for root canal samples and found differences in banding pattern between symptomatic and asymptomatic infections assigning a mean of seven taxa to asymptomatic endodontic infections and 12 taxa to symptomatic infections.

Polyphasic approaches
The molecular biology approaches described above have given endodontic microbiologists a range of powerful tools to understand the complex nature of root canal infections. There is now a simple technique to identify isolates, in most cases, to species level and sometimes beyond. High throughput techniques have been developed to detect specic taxa in large numbers of samples. Even those taxa, which we have previously termed unculturable are now being detected and characterized. Indeed, the concept of community is also being explored. However, given the large number and variety of molecular techniques available for the detection, quantication and identication of micro-organisms from root canal infections one might be forgiven for thinking that traditional culture is redundant and destined for microbiology history textbooks this is however far from the case. As I hope I have demonstrated here there are a large number of molecular biology techniques used to detect and identify bacteria but none of them is without aw. A major draw back with most of these techniques is that, because of there very nature as culture independent techniques, they do not provide access to the whole genome. This has major implications if the molecular detection technique of choice shows a very strong correlation with an as yet

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Bacterial identication by molecular biology methods

unculturable taxon. In the past using culture dependent techniques the isolate in question can be subjected to a battery of biochemical tests to ascertain what virulence factors it has. On the basis of this information further biochemical and molecular biology techniques are used to characterize the nature of these factors with respect to their role in disease initiation and progression. A culture independent technique to proved whole cells or whole genomes is therefore eagerly awaited. These techniques (culture dependant and culture independent) are not exclusive to each other and should be used together by endodontic microbiologists in an informed polyphasic manner to understand the complex nature of root canal infections.
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