POLYMERASE CHAIN REACTION (PCR) TECHNIQUES FOR THEIDENTIFICATION OF UNCULTIVABLE ORAL MICROORGANISMSHimratul-Aznita WH. Polymerase chain reaction (PCR)techniques for the identification of uncultivable oralmicroorganisms. Annal Dent Univ Malaya 2001; 8:43-49.

ABSTRACT

Until today there are still a high percentage of oralmicroorganisms have not been identified due to inabilityto isolate using the cultural method. However,identification of uncultivable microorganisms associatedwith disease will permits clinicians for a more accuratediagnosis, treatment and preventive measures.Unculturable microorganisms are also involved indisease and may account for treatment failure since theirsusceptibility to antimicrobial agents would be unknown.Thus, the opportunity for a rational approach to thetreatment of disease relies on the state of knowledgeconcerning its aetiology and pathogenesis. Recentlydeveloped molecular methods have made it possible tocharacterise mixed microflora in their entirety, includingthe substantial numbers of unculturable bacteria. Thedevelopment of rapid molecular methods like PCRprovides a reliable identification of unculturablemicroorganisms. This paper will review the currentliterature regarding the PCR techniques used to identifyuncultivable oral microflora.

Key words: Identification, detection, PCR, uncultivableoral microorganisms.

INTRODUCTION

Specific anatomical structures in the oral cavity providebacteria with diverse habitats characterized by differentpH, redox potential and nutrient availability.Additionally, mastication, salivary defence mechanismsand diet exert different influences on bacteria at sitesand as a consequence, the ecosystems support the growthof dissimilar bacterial communities (1). For manydecades, it has been an important part of research toenumerate and identify the oral microflora that has abroad range of genera. It is estimated that 50% of theoral flora is unculturable and the proportion ofunculturable organisms at other body sites remainsunknown (2). The complexicity of bacterial communityand the fastidious nature of some of its populationmembers have hampered the analysis of normal humanflora and of polymicrobial infections in human.Independently of different study designs and detectionmethods, including bacterial culture methods,immunoassays and nucleic acid based techniques usedifferent principles for bacterial identification and theresults obtained by using different methods may vary.

Revievv Article

Himratul-Aznita W.H.

LecturerDepartment of Oral Biology,Faculty of Dentistry,University of Malaya,50603 Kuala Lumpur,Malaysia.

Culture method is frequently used as a referencemethod, which involves the combination of culturedependent and differentiation of isolates. Generally,samples are cultivated on a non-selective agar orenriched media, whereas selective culture employsantimicrobial agents to suppress non-targetmicroorganisms and thereby improve the detection limit.Growth colonies were differentiated and identified afterpassing through a series of physiological and biochemicaltests. Culture methods yield no information onuncultivable forms such as the bacteria and spirochetes(3) and require experienced personnel, relatively time-consuming, expensive and also rely on the presence ofviable microorganisms and are likely to show inherenttechnical limitations (4). Cultural methods have multiplesources of method error relating to the adequacy of thedispersal method, the number of dilutions involved, theplating method, the adequacy of the media, the gasatmosphere used for incubation, the length of incubation,and the reliability of the tests (3). Yet, because ofhistorical precedent, it is generally accepted that thecultural procedure is a reliable reference for determiningthe presence or absence of a cultivable species, howeverthis acceptance has changed with the development ofhighly specific immunological reagents and DNAprobes (3).

In contrast, immunodiagnostic methods do notdiscriminate between viable and non-viable bacteria asin culture method. Most immunodiagnostic methodsprovide a quantitative or semi-quantitative estimate ofthe target microorganism (5). Immunodiagnostic tests aretargeted to specific bacterial species. The immunoassaysuse antibodies to detect antigens on target bacteria.These methods are rapid, relatively inexpensive, andthey can detect non-viable species. Disadvantages ofthese tests include possible cross reactivity betweensimilar species and inability to determine antibioticsensitivity or resistance. In addition, this methodgenerally show poorer detection limits than nucleic acidprobe or PCR assays.

44 Annals of Dentistry, University of Malaya, Vol. 8 2001

MOLECULAR TECHNIQUES FOR IDENTIFYINGMICROBIAL DIVERSITY

Molecular biology approaches used for the identificationand differentiation of bacterial species can becategorised into main groups such as (i) hybridizationof nucleic acid (DNA/RNA) with DNA probes andgenomic fingerprinting, (ii) sequencing of ribonucleicacid (55, 165, 235). (iii) PCR with species-specificprimers and PCR fingerprint techniques which can detectDNA polymorph isms in the identification of bacterialspecies. The application of modern molecular biologyenables the identification of microorganisms without theneed of bacterial culturing is leading to the identificationof an even more diverse oral microflora, in particularthe unculturable species (6). These molecular techniquesbased on non-amplification nucleic acid probeprocedures are more reliable primary referencestandards than the cultural method. Importantly, itprovides rapid, accurate and inexpensive microbialidentification of pathogens and will probably playincreasing importance in future diagnostics. Applicationof the DNA probe technique in clinical dentistry isuseful for identification of the presence of bacterial DNAfragments within any type of sample based on cross-reactivity patterns with known bacterial types. Also, itallows the examination of a large number of samples thatare difficult to explore with conventional culturaltechniques. Nucleic acid probe assays rely on the uniquedouble stranded structure of the DNA molecule to detectthe target microorganisms. Each species contains uniquesegments of DNA thus, allowing the possibility ofconstructing nucleic acid probes that will hybridize tothese sequences. The detection of target microorganismsis, therefore, based on the binding of the nucleic acidprobes to the complementary strands of nucleic acid inthese unique regions. This technique requires preparingpieces of single-stranded DNA labelled with an enzymeor radioisotope that can locate and bind. to theircomplementary nucleic acid sequences with low cross-reactivity to non-target microorganisms. Probes can beof different types; (i) whole genomic, (ii) cloned or (iii)oligonucleotide probes and may target for whole genomicDNA or individual genes. Whole genomic probes areconstructed from the entire genome of the targetmicroorganism and are more likely to cross react withnon-target organisms due to the presence of homologoussequences between different bacterial species. This typeof probe has several advantages over other nucleic acidprobes. It is the simplest to construct and the leastexpensive to produce. Cloned probes are isolatedsequences of DNA that do not show cross-reactivity andare produced in quantity by cloning in a plasmid vectorwithin a temporary host microorganism such asEscherichia coli. Oligonucleotide probes are usually 10-50 bp whereas the other probe types are often severalkilo base pairs in length. The sequences ofoligonucleotide probes are obtained from unique hypervariable regions of ribosomal RNA of the targetmicroorganism. Oligonucleotide probes for oral speciesare relatively insensitive, detecting only 106 cells,

compared to cloned and whole genomic probes, whichcan routinely identify 103 target cells (4). This probebased on species-specific sequences may display limitedor no cross-reactivity with non-target organisms.Oligonucleotide probes complementary to variableregions of the 165 rRNA genes have been used for thedetection of various bacterial species (5). Thus, DNAprobe technology provides both a sensitive and specificassay and alleviates the concern for transport offastidious microorganisms. The procedure includes (i)disruption of bacterial cells with denaturation of DNA,(ii) immobilization of DNA onto a nitrocellulose filter,(iii) blocking unbound nitrocellulose with non-specificDNA, (iv) hybridization of the filter with labelled probe,(v) washing and detection of bound probe (4).Socransky et. at. (5) developed a checkerboard DNA-DNA hybridization assay for the detection of oralbacteria. In this method, the sample DNA is releasedand immobilized on a nitrocellulose membrane. Themembrane-bound DNA is then allowed to hybridize witheither digoxigenin-Iabelled whole genomic DNA or 165rRNA-based oligonucleotide probes. The checkerboardassay is able to simultaneously screen for the presenceof up to 43 different bacterial species in 43 specimensusing a single nitrocellulose membrane. Thecheckerboard method is particularly suitable for large-scale microbiological studies of the sub gingival microbiota. This method overcomes many of the limitationsof culture and is more rapid, cost-effective thanantibody-based techniques and able to identifyuncultivable or difficult to grow species. However thedisadvantages of this method include the fact thatdetection is limited to species for which probes areavailable, the need for precise quality control and thepossibility of cross reactions (7).

POLYMERASE CHAIN REACTION (PCR) ANDRELATED TECHNIQUES

PCR amplification of DNA has enabled the amplificationof specific genes and allowed the rapid characterisationof both entire genome as well as specific genes in largenumbers of organisms (2) including oral spirochetes thatcannot be grown in artificial culture (6). Genes encodingribosomal RNA have been found to be particularly usefulfor the construction of phylogenetic trees reflecting theevolution of bacteria. Due to the need to preservefunction, these genes have been conserved throughoutevolution (2). This technique amplify the number ofcopies of a specific region of DNA, in order to produceenough DNA to be adequately tested, which can be usedto identify with a very high-probability, disease-causingviruses and/or bacteria, a deceased person, or a criminalsuspect. The DNA of different microorganisms isdifferent because there will alwaysbe genes whose DNAsequences differ among different organisms. Therefore,by identifying the genes which are different, one canuse this information to identify an organism.PCR is based on three simple steps required for any

DNA synthesis reaction;

Polymerase chain reaction (PCR) techniques for the identification of uncultivable oral microorganisms 45

(i) denaturation of the template into single strands,

(ii) annealing of primers to each original strand for newstrand synthesis, and

(iii) extension of new DNA strands from primers.

The advantages of using PCR over any other techniquesare;

(i) DNA can be amplified rapidly and accurately whichallow the identification of bacterial species or strain(8).

(ii) Do not require viable microorganisms forenumeration of bacteria from specimens.

(iii) Allows off-site collection of specimens, due to non-requirement for live bacteria in the enumeration ofspecific species and need less cumbersome transportmaterials.

(iv) PCR is reproducible and extremely sensitiverequiring only small numbers of organisms foranalysis. This sensitivity has been exploited as thebasis for a number of tests, including the detectionof pathogens (9,10,11).

Ribosomal RNAs (rRNAs) are ancient molecules,functionally constant, universally distributed, and wellconserved across broad phylogenetic distances. Inprocaryotes, there are three different sizes of ribosomalRNA molecules, 5S, 16S and 23S (12). 5S rRNA hasbeen used for phylogenetic measurements, but its smallsize (- 120 nucleotides), limits the informationobtainable from this molecule. Because 16S rRNA ismore experimentally manageable than 23S rRNA, it hasbeen used extensively for bacterial identification and todevelop the phylogeny of procaryotes (12). The 16SrRNA gene has been found to be particularly usefulbecause its size of approximately 1500 bases in lengthis an ideal size for molecular evolution studies. It is bothlong enough to provide useful discriminatory informationand also short enough for easy sequencing (2,13). Thus,16S rRNA gene is the most widely used gene to dateand have been amplified from a variety of sources,including the environment and human infections, andsequenced to reveal the diversity of organisms presentat the site (2).

The conserved regions are extremely useful for thedesign of PCR primers, while the variable regionscontain the information useful for strain discrimination.Woese (14) in his environmental studies used 16S rRNAas a tool for dissecting complex bacterial communitiesand which gave rise to the subspecialty of molecularmicrobial ecology. This molecular approach has beenapplied successfully to analyze environmental samples,such as photosynthetic microbial mats (15), oligotrophicsea water (16), soil (17) and uncultivable infectiousagents associated with disease from human, have alsobeen identified in bacillary angiomatosis (18) andWhipple's disease-associated bacterium Tropherymawhippetii (19). The primary amplification step is crucialsince the specificity of the primers used will determinethe organisms whose 16S rRNA genes are amplified.

Later, Lane (20) described a selection of primersof varying specificities which proved extremely usefuland have been widely adopted. However, DNA samplesextracted from various sources, including deep seasediment and oral bacteria were found to be poortemplates for amplification on the 16S rRNA gene withamplimers such as 27f and either 1492r or 1392r(numbering is based on the Escherichia coli 16S rRNAgene (21).

Marchesi et. at. (22) designed and evaluated PCRprimers 63f and 1387r for amplification of 16S rRNAgenes from bacteria. They were found to be more usefulfor 16S rRNA gene amplification compared to thecurrently used primers 27f and 1392r. This study hadattempted to optimise the PCR conditions for 27f-1392rby altering the annealing temperature, Mg2+concentration and DNA template concentration. Also byincluding the PCR additives bovine serum albumin,triton-X-100, T4 gene 32 protein, polyethylene glycol8000 and glycerol, finally, they were successful whenthe newly redesign amplimers 63f and 1387r were usedsuccessfully with the difficult DNA samples. The wideadoption of amplimers 27f, 1392r and 1492r isempirically based, and their utility for investigatingmolecular ecology of natural bacterial communities isoften assumed, and has not been systematically tested(22). Although 63f and 1387r showed some theoreticalbias in the experiment done by Marchesi et. at. (22).In practical they were more successful than amplimerpair 27f-1392r and able to amplify 16S rRNA genesfrom a wider range of bacteria than other primers whichare commonly used for bacterial community analysis.So far, other primers have not been tested in thesystematic way described for 63f-1387r by Marchesi et.a!. (22). However, the aim of which is to minimise PCRbias, and underline the point that the theoretical designof PCR amplimers is only the beginning and thatsystematic empirical testing of the amplimers is ofparamount importance.

PERIODONTITIS

Given the enormous complexity of the oral flora, analternative approach would be focus in on particulargroups of organisms suspected to be important in oraldisease. Spirochetes have been long associated withperiodontitis (23) but because the majority of speciescannot be cultured, the important of these organisms indisease in unknown. Choi et. at. (24) successfullyscreened a 16S rRNA gene library generated from aperiodontal pocket with universal PCR primers using aTreponema-specific oligonucleotide probe. Riviera et. at.(25) described that some spirochetes have beenparticularly associated with active disease and thus beennamed the pathogen-related oral spirochetes (PROS),while Wade (13) discovered that 91% of PROS areunculturable. Therefore, there is great hope that theapproach will assist in elucidating the role of treponemesin the pathogenesis of periodontal infections byassociating defined species with healthy or diseasedsites (24).

46 Annals of Dentistry, University of Malaya, Vol. 8 2001

Another important group of organisms in oralinfections are the oral asaccharolytic Eubacterium.species which present in large number in periodontitis(2,26), but are rarely found in gingival health. Howeverthey are difficult organisms to cultivate, being highlysensitive to oxygen and also difficult to identify becauseof their general unreactivity in biochemical tests. Studiesby Spratt et. at. (27) and Harper-Owen et. at. (28)showed that the indifferent growth of culturable membersof the group suggests the presence of a substantialunculturable population. This study was t) design andvalidate specific PCR primers for these phylotypes andto determine their incidences in samples collected fromhealthy and diseased periodontal tissues. The need forextensive validation of novel primers was demonstratedby the considerable cross-reactivity with related speciesthat was seen with some of the primers (28). Twospecific reverse primers were devised for eachphylotype, and these were used in duplex PCRs withuniversal forward and reverse primers. Thus multipleprimers used, were to maintain high specificity of thedetection system for uncultivable microorganism

DENTOALVEOLAR ABSCESS

Dentoalveolar abscesses are acute infections associatedwith the teeth, arising from pulpal infection secondaryto dental caries. Cultural analysis has revealed they areassociated with a restricted group of organisms from thegenera Fusobacterium, Peptostreptococcus, Prevotellaand Streptococcus.

Dymock et. at. (29) successfully screened forunculturable bacteria associated with this disease byPCR technique of 16S rRNA amplification. Samples ofpus were obtained from acute abscesses by a needle andsyringe. DNA was extracted from the whole sample andthe 16S rRNA genes amplified. These were singularisedby cloning, re-amplified and digested with restrictionendonucleases to give a RFLP profile. These profileswere then compared with similar profiles obtained fromisolates cultured from the sample. In this way clonedgenes isolated from organisms not represented in theculturable portion were identified (24). A generaloverview of the method is outlined in Figure 1. Fivepredominant uncultured organisms were identified asPorphyromonas gingiva lis , Prevotella oris,Peptostreptococcus micros-related, Zooglea ramigera-related and the other was distantly related to the genusPrevotella (29).Using the same approach, Wade et. at. (2) have

identified uncultivable novel anaerobic species indentoalveolar abscesses with universal primers 27f and1492r for amplification of 16S rRNA genes.

ENDODONTIC INFECTIONS

There is now considerable evidence that endodonticdiseases (pulpal and periradicular inflammation) aredirectly associated with bacterial invasion of the dentine,root canal system, and periradicular tissue. The primarytarget of these infections is the connective tissues of thepulp, which is completely enclosed within rigid dentine.

amplify 16s rRNA gene

.... +Digest WIth restnctlOn endonucleases to generate RFLP profile

Compare RFLPs of cloned geneJwith RFLPs from cultured bacteria

+Sequence DNA of non-culturables

Analyse Phrl0geneticallY

'f~ .Identl Yspecies

Figure 1. Flow chart showing the strategy for detection ofuncultivable bacteria in dentoalveolar abscess

(Dymock et al. 1996).

Polymerase chain reaction (PCR) techniques for the identification of uncultivable oral microorganisms 47

Dentine structure, permit selected anaerobic bacteria toinvade the dentine, the root canal system, and sometimesthe periradicular tissue (30). Williams et. at. (30) haveidentified uncultivable microbial communities in dentineand cementum. They adapted the same PCR techniqueas Choi et. at. (24), amplifying the 165 ribosomal RNA.Tooth with carious lesions, carious tooth, carious toothassociated with pain, heavily restored tooth and intacttooth produced amplifiable product from PCR analysis.Homologies of 95 %-99 % with several gram positive taxathat have not been previously described in the oral cavitywere apparent in this study. Furthermore, the homologiesof several sequences with any DNA sequences ofEubacterium were low; suggesting that there are bacteriain the dentine and cementum that have not yet beenclassified and are probably unique to the oral cavitymainly because of the limited nutrients available and therestricting environment of the dentinal tubules (30).

LIMITATIONS

Unfortunately, this straightforward and universalapproach is hampered by a number of problems, whichmay lead to erroneous results. One of the drawbacks ofthe molecular technique for this application is that it maydetect dead bacteria which have been killed by the hostimmune system but whose DNA remains present in thesample or even fairly short fragments of remaining DNA(31).

Another problem arise with this technique is withthe several commonly used amplimers which have beenconstructed theoretically, using incomplete database of165 rRNA sequences from cultured organisms and havenot been tested systematically (22). However, the aimof which is to minimise PCR bias, and underline thepoint that the theoretical design of PCR amplimers isonly the beginning and that systematic empirical testingof the amplimers is of paramount importance. There arealso primers which consistently failed to work withdifficult samples such as PCR primers described by Lane(20), although these primers can work efficiently withother culturable organisms (32,33). This may explain thedetection of normally culturable organisms by themolecular method alone. Despite its limitations,comparative rRNA analysis does represent the currentlybest approach for the identification and phylogeneticclassification of unculturable microorganisms and thedescription of the population structure of complexmicrobial communities.

CLINICAL ORAL MICROBIOLOGY

There is an important behaviouralleducational aspectwhich relates to the need for an objective biological testto diagnose oral diseases. A better understanding of thebacterial species involved in each disease category wouldimprove the clinicians ability to permit more accuratediagnosis and provide rational therapeutic endpoints fortesting treatment modalities, such as the need and type

of antibiotics prescribed, adjusting maintenanceschedules. Also, the informations obtained will help theclinicians to evaluate current and predict future diseasestatus in order to determine preventive measures and theendpoint of treatment (5). Thus, the microbiologicalspecificity paradigm has already changed the way thatthe research community and some clinician view oraldiseases. The goal of therapy is to target and then reduceand/or the eliminate the specific pathogens. Theopportunity for a rational approach to the treatment ofdisease depends in large measure on the state ofknowledge concerning its aetiology and pathogenesis.Although most of the commonest oral diseases such asdental caries, periodontitis etc are rarely life-threatening,the direct cost of treating these common oral diseasesis enormous and they are the most expensive infectionsthat the majority of individual will have to contend within their lifetime (34). The bacteria associated withdentoalveolar abscesses are principally viridan groupstreptococci, particularly the Streptococcus millerigroup, Fusobacterium sp., Prevotella sp.,Porphyromonas sp. and Peptostreptococcus sp. However,it is estimated that approximately 50% of the oral florais unculturable (29). Today, we are aware thatunculturable microorganisms have been isolated andidentified from common oral diseases such asperiodontitis, dentoalveolar abscesses and endodonticinfections. However, it is not impossible that other oralinfection harbours unculturable microorganisms as well.Currently, there has been increasing reports and concernamong clinicians despite the widespread use ofantibiotics in Ludwig's angina disease, one of the oralinfections that resulted with a very high mortality rateamong patients. Although with the introduction ofantibiotic therapy, mortality rate has decreased to lessthan 10% from 50 % (35,36) scientists and dentalclinicians must have a thorough study/research relatingto microorganisms involved, in order to overcome thisdeadly disease. Ngeow (37) reported that Streptococcusspecies have been isolated from a 3 years old child withLudwig's angina. This indicates that further study isneeded as there are possibilities of other microorganismsinvolved including unculturable microorganisms. It islikely therefore, that at least some members of theunculturable part of the flora are involved in disease andindeed, may account for treatment failure since theirsusceptibility to antimicrobial agents would be unknown.

CONCLUSIONS AND FUTURE DEVELOPMENT

The failure to grow an organism on artificial culturemedia obviously means that the correct environment forgrowth has not been provided. If an organism cannotbe cultured then obviously its biological propertiescannot be determined. Therefore, methods for the studyof unculturable bacteria have long been sought. The keyto the development of techniques for the investigationof unculturable organisms came from the realisation thatthe sequence information contained in biologicalmacromolecules could be deciphered and would include

48 Annals af Dentistry, University af Malaya, Val. 8 2001

regions specific to the whole organism itself. Thus,molecular methods are now available which allow thecharacterisation of the unculturable portion of bacterialcommunities. There are a number of possible reasonsfor this. Artificial culture media may lack the nutrientsrequired by the organism or incubation atmosphere ortemperature may be sub-optimal. Alternatively, mediamay contain substances toxic for growth or other bacteriain the sample may produce inhibitory substances.Currently, the lower limit of sensitivity of DNA probes·which are commercially available is approximately 6000bacteria and may not provide the sensitivity necessaryfor detection of pathogens in those specific sites suchas dentine, cementum or periodontal pocket in whichthe number of pathogens are low or fluctuates (38). Thedevelopment of rapid molecular methods like PCR notonly it is more sensitive for bacterial identification (i.e.100 cells), it also enable the investigation of mixedmicroflora associated with disease in their entiretywithout the inherent biases of culture. The proceduresare obviously applicable to infections at all body sitesand should lead to a more complete understanding ofassociations between bacteria and disease. AlthoughPCR method is relatively time consuming at present, itis still considered as one of the most rapid detectionmethod for unculturable bacteria. With these PCRapproaches, a number of novel sequences, which do notcorrespond to known, culturable organisms, have beenidentified. The ability to identify both the culturab1e .andunculturable microorganisms associated with disease willimprove our knowledge of the aetiology of oral pathogenrelated diseases and allow the discovery of additionalmarker organisms of value in disease diagnosis andtreatment monitoring. These can be used to determinethe prevalence of the organisms in healthy and diseasedtissues and, if specific associations are found, might beuseful markers of disease activity. In conclusion,molecular techniques such as those described in thisreview are a new, culture independent era of medicalmicrobiology and there is great hope that the approachwill assist in elucidating the role of microorganisms inthe pathogenesis of oral infections. Clearly, further workis required to characterize the microbial populationsassociated with oral health and disease. Thus, molecularmethods provide the tools for the detection ofunculturable microorganisms and hopefully will allowa far better understanding of oral diseases.

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