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  Microorganismsasriskindicatorsforperiodontaldisease P  aul   J. E zzo  & C hristopher  W. C utler The composition of bacterial plaque has been studiedfor many decades using various microbiological andmolecular techniques. These efforts have been osten-sibly motivated by the ‘‘dream’’ that, once we identify the causative agent(s) of periodontitis, we will be abledevelop a rapid test (e.g. the rapid Strep test used inphysicians’ offices (75)) to assess microbial risk atchairside. This information, along with that obtainedfrom antibiotic susceptibility testing, could be appliedfor treating periodontitis like other infections in thebody – administer the appropriate antibiotic and thepatientgetsbetter.However,attemptstodeveloprapidchairside microbial-risk tests and integrate them intodental practices have not been successful for many reasons. As the technology of bacterial detection hasevolved, from morphologic detection to cultivation tomolecular probe analysis, the sensitivity of detectionhas improved logarithmically. Now over 400 bacterialspecieshave beenidentified,with surely more tocome(52). While it is in our nature to try to identify thosespecific organisms most likely to cause or be asso-ciated with disease, dental practitioners are faced withan increasingly daunting task – how to make rationaldecisions based on an increasingly complex databaseofmicrobialriskfactors.Theymustdecidehowto(andindeed whether to) integrate this information intotheir practices, so they can provide efficient, state-of-the-art patient care. Moreover, while some dentaloffices do use antibiotic susceptibility testing services,the overall concept of antimicrobial therapy for treat-ment of periodontitis has not caught on. The simplefact that conventional therapy (i.e. scaling and rootplaning) is the standard-of- care and appears to beeffective when used alone is an important factor inthisregard.Moreover,thereareaptconcernsingrainedin the dental profession about the use of antibioticsbeing the driving force for antibiotic resistance.This review will attempt to clarify the role thatmicrobes and their products play in periodontitis andtry to provide a clinical rationale for assessing micro-bial risk. It is hoped that this will facilitate an under-standing of the pathogenesis of periodontal diseaseand perhaps allow for enhanced treatment outcomes. Plaque101anddisease-associatedspecies For a historical prospective to this topic, readers arereferredtoearlierreviewsthathavedescribedthenon-specific and specific plaque hypotheses (88). The pla-que biofilm consists largely of microbes and host pro-teins that adhere to the teeth within minutes of a dental prophylaxis. In gingival health, gram-positiveorganisms like  Actinomyces   and streptococci domi-nate the plaque biofilm. In the later stages of plaquebiofilm formation (i.e. days to weeks of poor oralhygiene) the plaque ‘‘matures’’, resulting in a shifttowards gram-negative anaerobes and motile organ-isms. Some of the most common organisms asso-ciated with periodontal diseases are  Porphyromonas  gingivalis, Prevotella intermedia, Bacteroides forsythus,Campylobacter rectus  , and  Actinobacillus actinomyce-temcomitans  , as well as the treponemes. At the 1996 World Workshop on Clinical Periodontics, a groupreviewing specific periodontal pathogens as causativeagents in periodontal disease limited their findings tothree organisms:  A. actinomycetemcomitans  ,  B. for-sythus  , and  P. gingivalis   (46), presumably becausethey met Socransky’s modifications of Koch’s postu-lates (70). Among other criteria, these modificationsrequire that, to be considered a periodontal pathogen:   the organism must occur at higher numbers indisease-active sites than at disease-inactive sites.   elimination of the organism should arrest diseaseprogression.   the organism should possess virulence factorsrelevant to the disease process.  24 Periodontology 2000, Vol. 32, 2003, 24–35 Copyright   #  Blackwell Munksgaard 2003Printed in Denmark. All rights reserved   PERIODONTOLOGY 2000 ISSN 0906-6713    the organism should elicit a humoral or cellularimmune response.   animal pathogenicity testing should infer diseasepotential.Implicit in the  fi rst two requirements is the need toconduct longitudinal clinical studies, which has beenachievedforthesethreespecies.Thisreview willthere-fore begin by focusing on each of these three organ-isms in turn, with the caveat that they do not exist inisolation  in vivo , but are part of a microbial commu-nity. We will brie fl  y discuss the virulence factors they produce and the longitudinal studies that implicatethem as risk indicators in periodontal disease. As theterm virulence could be construed as a microbial risk factor for causing disease, we will emphasize the com-monalities in virulence or pathogenesis exhibited by these organisms. We will discuss microbial productscommon to many of the so-called  ‘‘ high-risk  ’’  speciesand how they might confer risk to periodontal disease.It is important to mention in this context that, unlikemanyoftheotherriskfactorsdescribedinthisvolume, we chose to refer to periodontal pathogens as risk indicators due to the fact that the odds ratios betweenthe presence of these speci fi c bacteria individually,and periodontitis are not high enough to classify themas risk factors, notwithstanding the essential role forthe plaque bio fi lm in the initiation of periodontitis.  A. actinomycetemcomitans  asarisk indicatorforperiodontitis  A. actinomycetemcomitans   is a bacterial species whose association with localized aggressive period-ontal disease (formerly localized juvenile periodon-titis) has been most clearly demonstrated (69, 87).The subgingival prevalence of   A. actinomycetemco-mitans   varies widely yet it typically increases withdisease (20, 82, 87). For example, 0 – 26% of healthy children were found to exhibit  A. actinomycetemco-mitans   subgingivally (71). On the other hand, 40 – 100% of subgingival sites in patients with aggressivedisease have  A. actinomycetemcomitans   (71). Thesedata suggest that  A. actinomycetemcomitans   is a cau-sative agent in localized aggressive periodontal dis-ease, but this has been dif  fi cult to prove, presumably due to the episodic nature of disease activity. Theabsence of the organism in a diseased state may bedue to the  ‘‘  window of cultivability  ’’  being missed.  A. actinomycetemcomitans   has also been implicatedin some cases of chronic periodontitis (2, 50, 60).The distribution of   A. actinomycetemcomitans  serotypes within disease categories may be morespeci fi c and indicative of   A. actinomycetemcomitans  as a true pathogen or high-risk organism. Forinstance, seroype c strains are more commonly found in extraoral infections and in periodontalhealth (3, 89). Furthermore, many serotype b strainsof   A. actinomycetemcomitans  , like JP-2, produceincreased amounts of leukotoxin, an important viru-lence factor, and are found most often associated with periodontal disease (19).In a more sensitive approach to identifying therelatedness of   A. actinomycetemcomitans   isolates indisease, DiRienzo and McKay (18) used restrictionfragment length polymorphism (RFLP) analysis totype over 800 clinical isolates of   A. actinomycetemco-mitans  . The identi fi cation of particular genetic var-iants of   A. actinomycetemcomitans   causing a particular disease (or not) would suggest a high (orlow) risk organism. 13 RFLP patterns were foundusing a 4.7-kb DNA probe from  A. actinomycetemco-mitans   strain Y4. An oligonucleotide homologous tothe 5 ’  end of the  leukotoxin A  gene speci fi cally iden-ti fi ed RFLP group II isolates that exhibited a 530-bpdeletion in the leukotoxin operon. This suggests lessgenetic variability than the oral commensal  P. gingi-valis   (36), consistent with  A. actinomycetemcomitans  being a higher risk or more pathogenic organism,especially in younger individuals. In a separate study,nine out of 36 localized aggressive periodontal dis-ease-susceptible individuals showed evidence of per-iodontal breakdown and six of these nine subjectsexhibited RFLP group II variants of   A. actinomyce-temcomitans   (19) that were later shown to be highleukotoxin producers (26). These studies serve as anexample of how molecular patterns characterized within a species of bacteria can allow us to improvethe reliability of diagnostic information related to risk assessment, thus sorting the commensals from thetrue pathogens.  A. actinomycetemcomitans   interacts with the hostby the production of several virulence factors. Leu-kotoxin, the most studied virulence factor made by   A. actinomycetemcomitans  , is an RTX (Repeats inToxin) toxin (33) and shares sequence similarity withthe  a -hemolysin from  Escherichia coli  , the cytolysinfrom  Pasteurella haemolytica  and the leukotoxinfrom  Actinobacillus pleuropneumoniae   (16).  A. acti-nomycetemcomitans   leukotoxin has been shown tokill human and non-human primate polymorpho-nuclear leukocytes and peripheral blood monocytes(4, 83), thereby evading the innate immune responseby attacking it directly. In the process, tissue-dama-ging granule contents from polymorphonuclear leu-kocytes and monocytes are released. Conversely,  Microorganisms as risk indicators for periodontal disease   25   leukotoxin-mediated killing is related to the induc-tion of apoptosis in HL-60 cells (31). Apoptosis, orprogrammed cell death, is a process where host cellsundergo nuclear degeneration, triggering their clear-ance by phagocytes. Apoptosis, when properly regu-lated, is a normal part of tissue and immune cellhomeostasis and, indeed, of the immune response.For example, apoptosis is induced by cytotoxic Tcells when clearing infected target cells. Similarly,proteins made by   A. actinomycetemcomitans   (mostnotably the leukotoxin) can induce apoptosis in a variety of host immune cells. The signi fi cance of this fi nding may be that, in contrast to necrosis, lysis of the cells does not occur. Although cell lysis spillstissue-degrading enzymes, it also releases antimicro-bial peptides (e.g. defensins) that can kill bacteria,and attract other in fl ammatory cells. Thus  A. actino-mycetemcomitans   might bene fi t at some stage inthe disease process (e.g. when it emerges from aninfected gingival epithelial cell) by obtunding itsleukotoxin production and thereby blunting thein fl ammatory response. Considering that  A. actino-mycetemcomitans   has been shown to penetrate hostcells (40), one might presume that this organismmight bene fi t from eliciting apoptosis.The  A. actinomycetemcomitans   lipopolysaccharideor endotoxin also imparts virulence capabilities tothis organism (30, 63). Like the lipopolysaccharidemade by   E. coli   and  Salmonella typhimurium ,  A. actinomycetemcomitans   endotoxin has the poten-tial to modulate host responses and contribute totissue destruction. Most pertinent may be the ability of the  A. actinomycetemcomitans   lipopolysaccharidetostimulatemacrophagestoreleaseinterleukin(IL)-1,IL-1 b , and tumor necrosis factor (TNF); these cyto-kines, among other activities, are capable of stimu-lating bone resorption (63). Very little is known aboutthe biological activity of   A. actinomycetemcomitans  lipopolysaccharide. We do not understand the typeof immune response it elicits (i.e. Th1/Th2) and how it signals the innate immune cell response (i.e.through CD14, Toll-like receptor [TLR] 4 or TLR 2).In general, the structural features of   A. actinomyce-temcomitans   lipopolysaccharide are typical andinclude a surfaceantigen composed of various sugars,a lipid A region deep within the outer membrane, andaninnercorepolysaccharide(30).Whilethecorepoly-saccharide and lipid A structure remain conservedbetween the different serotypes of   A. actinomycetem-comitans  , there is a distinct difference between thesurface or O-antigen component (22). As an example,theserotypebO-antigenhasbeende fi nedasarepeat-ing unit containing D-fructose, L-rhamnose, andN-acetyl-D-galactosamine (53). The O-antigens of the other serotypes of   A. actinomycetemcomitans   aredistinct from serotype b (22). As the serotype b strainsseem to produce more leukotoxin (19) and produce a different O-antigen, it would seem that diagnosticefforts to identify this high risk organism and immu-notherapeutic efforts to combat its infection  in vivo should focus on serotype b strains.Perhaps most relevant to the ability of   A. actinomy-cetemcomitans   to evade the innate defenses and sur-vive mechanical periodontal therapy is its ability toinvade gingival tissues (8, 64) and, in particular, toinvade epithelial cells (40). It has been speculated thatonce  A. actinomycetemcomitans   gains entry into theconnective tissue, its production of collagenase willallow it to acquire nutrients and possibly disseminate(59). One of the classic  fi ndings in periodontitis, col-lagenbreakdown,couldresult(49).However,thedirectproteolytic activity of   A. actinomycetemcomitans  enzymes are likely to be eclipsed by host metallopro-teinases released upon  A. actinomycetemcomitans   lysisof polymorphonuclear leukocytes and monocytes.Many, but not all, strains of   A. actinomycetemcomi-tans   have been found to invade mammalian cell lines(6, 40, 76). Invasion ef  fi ciency can be affected by themammalian celltype used and by thestrain of   A. acti-nomycetemcomitans   tested (40). In a human epider-moid carcinoma cell line (KB), invasion is initiated by the contact of   A. actinomycetemcomitans   with micro-villi.Onceinthecell,  A. actinomycetemcomitans  SUNY 465 can spread intracellularly by utilizing host cellmicrotubules (41). Recently,  A. actinomycetemcomi-tans  , among the other periodontal pathogens dis-cussed in this review, have been detected in humanbuccal epithelial cells. This group was able to show   A. actinomycetemcomitans  ,  B. forsythus  , and  P. gingi-valis  activelygrowinginthesecells.Arguably,theabil-ity of a microorganism to penetrate host cells is animportant trait common to those organisms mostlikely to be associated with disease. The ability of per-iodontalpathogenstoconcealthemselvesinhostcellsmightenablethemtosurviveconventionalperiodontaltherapy and emerge to cause disease again.  A. actinomycetemcomitans  asa riskindicatorforperiodontitis–longitudinalstudies The presence of   A. actinomycetemcomitans   and theprogression or initiation of localized aggressive per-iodontal disease has been evaluated longitudinally.In the 1996 World Workshop, it was acknowledged  26  Ezzo & Cutler   that the presence of   A. actinomycetemcomitans   in younger patients was a risk indicator for the devel-opment of periodontal disease (46). Since that time,many studies have investigated whether the subgin-gival presence of   A. actinomycetemcomitans   in adultscoincides with periodontal disease. The majority of these studies have been unable to show that thepresence of   A. actinomycetemcomitans   puts adultsat further risk of disease progression. This supportsmost of the work done prior to 1996 that suggeststhat it is the presence of   P. gingivalis   or  B. forsythus  that more frequently correlates with progressivechronic disease. Therefore, one can describe  A. acti-nomycetemcomitans   as being an initiator of aggres-sive disease, but not essential for it to occur. The truemediator of disease is likely to be how the hostresponds  –  other bacterial species may play surro-gate roles in initiating a similar response.Since 1996, one group has investigated longitudin-ally whether the presence of   A. actinomycetemcomi-tans   puts adolescents at higher risk of aggressivedisease (80, 81). This group described three risk mar-kers: age, presence of local factors and subgingival  A. actinomycetemcomitans  . When  A. actinomycetem-comitans   was found subgingivally in Indonesian ado-lescents the odds ratio for progressive disease was4.61. Like other studies analyzing the natural history of disease, these subjects had no access to dental careand therefore the prevalence of disease was high. Inanother study where army recruits were analyzed forthe presence of   A. actinomycetemcomitans   and theprogression of disease, signi fi cant numbers of   A. acti-nomycetemcomitans  -positive samples were found in ‘‘ active ’’  patients after 12 months (44). However, nostatistical difference was found at the patient level,leading these investigators to conclude that  A. actino-mycetemcomitans   wasnotariskfactorinthesepatients.In general, few longitudinal studies describing the risk of subgingival  A. actinomycetemcomitans   tothe progression of aggressive disease have been donesince 1996. Further studies will be needed to provethe cause and effect relationship of   A. actinomyce-temcomitans   and aggressive disease. However, thesestudies should be approached using a systems biol-ogy approach, taking into account the genetics of thehost and of the pathogen. Bacteroides forsythus  asarisk indicatorofperiodontitis Of the three periodontal pathogens discussed in thisreview,  B. forsythus   is the least understood, possibly because it is the most dif  fi cult to cultivate  in vitro . Itsassociation with periodontitis hasgained more atten-tion in recent years.  B. forsythus   possesses severalvirulence traits including the production of a tryp-sin-like protease and lipopolysaccharide (43, 77) butmore recently its ability to penetrate host cells orinduce apoptosis has received attention (1, 62).These recent  fi ndings have given rise to new ideasfor how this organism, as well as the others discussedin this review, cause disease. As we begin to under-stand the mechanisms by which this organism cancause disease, a clear view of the commonalitiesbetween different periodontal pathogens emerges.It is widely accepted that many of the periodontalpathogens may attach and penetrate oral epithelialcells. Since  A. actinomycetemcomitans   and  P. gingi-valis   are perhaps the most studied periodontalpathogens, their ability to invade cells  in vitro  and in vivo  has been well described. Understanding that B. forsythus   is almost always detected where  P. gin- givalis   is present, investigators have hypothesizedthat  B. forsythus   might also penetrate oral epithelialcells. Recently, investigators found  B. forsythus   inbuccal epithelial cells  fi rst using polymerase chainreaction methodology and later using   fl uorescent  insitu  hybridization (FISH) (61, 62). This group hasbeen able to show, using this technology, that  B. for-sythus  , in addition to  P. gingivalis   and  A. actinomy-cetemcomitans  , may be actively growing within thesecells. This would imply that the bacteria maintain anintracellular  ‘‘ reserve ’’  in areas otherwise dif  fi cult tocolonize due to their anaerobic characteristics. Inaddition, it is possible that these infected cells may act to transmit bacteria from site to site and host tohost during cellular turnover, possibly protecting them from the harsh hypotonic conditions of saliva (5). The ability of   B. forsythus   to attach and penetratecells may be related to its surface (S-) layer produc-tion. This protein component of   B. forsythus   showshemagglutination activity and is important inabscess formation in animal models (34). Conversely,other groups have not seen  B. forsythus   intracellu-larly in primary cultures of human gingival epithelialcells (HGEC) (25). The authors went on to mentionthat other invasive strains of   B. forsythus   may existand be more capable of intracellular penetration. Of the pathogens tested in these studies,  Fusobacteriumnucleatum , however, was found to be highly invasivein HGEC and KB cells.Perhaps the most intriguing aspect of   B. forsythus  virulence is its ability to induce apoptosis. When B. forsythus   extract was added to HL-60 and otherhuman leukemic cells, a cytocidal activity was  27   Microorganisms as risk indicators for periodontal disease 
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