Transmission of Periodontal Bacteria and Models of Infection

J Clin Periodontol 2005; 32 (Suppl. 6): 16–27 Copyright r Blackwell Munksgaard 2005

A. J. Van Winkelhoff and K. Boutaga Transmission of periodontal Academic Centre for Dentistry Amsterdam, Department of Oral Microbiology, bacteria and models of infection Amsterdam, The Netherlands

Van Winkelhoff AJ, Boutaga K. Transmission of periodontal bacteria and models of infection. J Clin Periodontol 2005; 32 (Suppl. 6): 16–27. r Blackwell Munksgaard, 2005.

Abstract Background: Bacteria play an essential role in the aetiology of periodontitis. Most bacterial species isolated from subgingival plaque are indigenous to the oral cavity. Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis are detected infrequently in periodontal health, which makes these species prime candidates to study person-to-person transmission. The aim of the present study was to review the literature on transmission of these periodontal bacterial species. Method: We review the literature on bacterial typing techniques and summarize the information on clonal distribution of A. actinomycetemcomitans and P. gingivalis in family units based on different typing techniques in order to establish the likelihood for person-to-person transmission of these periodontal pathogens. Results: Vertical transmission of A. actinomycetemcomitans is estimated to be between 30% and 60%, whereas vertical transmission of P. gingivalis has rarely been observed. Horizontal transmission between spouses ranges between 14% and 60% for A. actinomycetemcomitans and between 30% and 75% for P. gingivalis. There is some evidence to show that cohabitation with a periodontitis patient influences the periodontal status of the spouse; however, substantially more information is needed to prove this hypothesis. Conclusions: Transmission of putative periodontal pathogens between family members has been shown. The clinical consequences of these events have been poorly documented. Based on the current knowledge, screening for and prevention of transmission of specific virulent clones of A. actinomycetemcomitans may be feasible and effective in preventing some forms of periodontal disease. P. gingivalis is usually recovered from diseased adult subjects, and transmission of this pathogens seems largely restricted to adult individuals. Horizontal transmission of P. gingivalis may Key words: bacteria; transmission; therefore be controlled by periodontal treatment involving elimination or significant periodontitis; infection; bacterial typing suppression of the pathogen in diseased individuals and by a high standard of oral hygiene. Accepted for publication 1 April 2005

Epidemiology is the study of the factors lococcus aureus can cause hard- and cause opportunistic infections. When the that affect the distribution of diseases in soft-tissue infections when penetrating composition of the normal microﬂora is populations, including infectious dis- the natural barriers of the skin or the disturbed, after or during antimicrobial eases that are spread by pathogenic mucosa. Oral streptococci may cause therapy for instance, resistant microor- organisms. transient bacteraemia following dental ganisms can emerge and cause infection Microorganisms that are indigenous extraction or tonsillectomy and may (Helovuo & Paunio 1989, Helovuo et al. to the host and are referred to as com- cause subacute bacterial endocarditis in 1993). Infections caused by commensal mensal, or resident organisms, can cause susceptible individuals. These microor- microorganisms are called endogenous infections. They can initiate disease ganisms can survive in the new environ- infections for which transmission of when they disseminate from their nor- ment if they are able to attach to the microorganisms from host to host is not mal ecological niche to sterile body sites surface of the host and are able to resist needed. In the oral cavity an increase as, for instance, is the case in urinary the local host immune responses. Often, of the bacterial load and a concomitant tract infection caused by Escherichia a change of the innate immunity is shift in the microbial composition of coli from the colon. Indigenous Staphy- necessary for commensal pathogens to the dental plaque can cause gingivitis. 16 Periodontal bacteria 17

Unlike exogenous bacterial disease, with a complex microflora such as infec- 20,000 contaminated droplets. Talking endogenous infections (1) have no defin- tious bowel diseases (Crohn and ulcera- is also a source of airborne particles, able incubation period, (2) are not com- tive colitis), vaginitis and periodontal although the number of organisms that municable in the usual sense, (3) do not disease, the matter of transmission is is shed is significantly less. Whether result in clinically recognizable immu- more complex. periodontal bacteria can be transmitted nity, and (4) involve bacterial agents First, some general principles of bac- via aerosols is currently not known. found in the normal microflora. These terial transmission are discussed. Then, Staphylococci are often transferred by agents are often characterized by a low the rationale for studying the transmis- dust from contaminated clothing, beds intrinsic pathogenicity and cause disease sion of two periodontal pathogens, Acti- and contaminated objects. when they appear either in unusual body nobacillus actinomycetemcomitans and Some systemic infections are caused sites or in greatly increased concentra- Porphyromonas gingivalis, is provided. by transmission of pathogens from tion in or near their usual habitats. Subsequently, studies on the distribution animals (zoonoses), which serve as a Microorganisms that do not normally of these periodontal species in popula- reservoir. Humans can be infected via occur in a healthy host and can cause tions and in families are summarized. milk (brucellosis, tuberculosis), car- disease upon infection in a significant Different typing methods are briefly casses (anthrax, tularaemia), contami- proportion of the recipients are called described, and data on distribution of nated swimming water (leptospirosis) exogenous pathogens (Salyers & Whitt genotypes among families are pre- or wounds. 1994). Most human pathogens not only sented. Finally, routes of transmission Biting/sticking arthropods can serve occur in diseased subjects, but also in of both pathogens and the possible clin- as vectors and may transfer bacteria, healthy carriers, who provide a reservoir ical consequences of transmission are rickettsiae and viruses from an animal and serve as a focus. It is suspected that discussed. reservoir by a cycle of infection (in lice, epidemics can be initiated by recombi- fleas or ticks). Often, these latter infec- nation between strains from different tions are not readily transmitted from carriers. Exogenous pathogens need to Transmission person to person. overcome the conditions of the environment such as ultraviolet light, drying The behaviour of a pathogenic micro- and temperature. To survive these con- organism depends upon the interaction Vertical and horizontal transmission ditions, they have developed different between the host, the pathogen and the When transmission is directly from par- strategies such as spore formation (Clos- environment. Changes in any of these ents to offspring (via ovum, placenta, tridium, Bacillus), encapsulation (Myco- factors will affect the likelihood of milk, blood, saliva) it is referred to as bacterium) and survival in intermediate transmission. Host factors affect the vertical. Transmission is called horizon- hosts such as the rabies virus in animals chance of exposure to a microorganism, tal when an individual infects unrelated or Legionella pneumophila in amoebae. the possibility for colonization and the individuals by contact, respiratory or Organisms with moderate survival response to colonization, i.e. develop- faecal–oral spread. Examples of diseases potential can be transmitted by spread- ment of infection. Host factors include, that may spread by vertical transmission ing in air on droplet nuclei (viruses). among others, sexual behaviour, hygiene, are rubella, cytomegalovirus, syphilis, Some bacterial pathogens are extremely occupation, nutrition, immunity and gen- toxoplasma (pre-natal), gonococcal/chla- delicate and cannot survive outside the eral health. Microbial factors include mydial conjunctivitis (perinatal) and host, e.g. Neisseria gonorrhoea. Trans- infectiousness, pathogenicity, virulence hepatitis B (post-natal). Polio, influenza, mission of this type of pathogen is and survival in human and animal hosts, tuberculosis and typhoid are examples of achieved by direct mucosal contact including resistance towards immune diseases that are horizontally transmitted. between individuals. responses and drugs. Communicable diseases are infections Environmental factors such as tem- that are capable of spreading from person perature, humidity and oxygen tension Essential factors for transmission are determinants of the survival of to person. This is not the case with all The source infections. A subject with infective endo- microbes outside the host. carditis caused by Streptococcus sanguis When the source of the infection is may suffer from an infection that is not restricted to actively diseased indivi- Mechanisms of transmission able to spread from a patient to another duals, isolation measures are effective person. Communicable microorganisms Many bacteria are transmitted from per- in eradication of the infectious agents. are not always pathogenic and not all son to person primarily by direct con- This is not possible when pathogens pathogens are communicable. Prevention tact, via the hands, mouth, or sexual occur in healthy carriers, who represent of transmission of an infectious agent is a organs. Some bacterial (e.g. syphilis) a reservoir. Eradication is also not fea- major measure to prevent disease in a and viral diseases (e.g. rubella) can sible when pathogens are soilborne or population. Therefore, the study of trans- be transmitted transplacentally from animalborne. mission of infectious agents is a relevant mother to child. Indirect transmission public health issue. In diseases that are occurs via intermediate agents. Enteric Number of microorganisms shed caused by one microbial species (whoop- infections are mostly waterborne or ing cough, rabies) or a very limited foodborne and are because of faecal The more viruses, bacteria or protozoa number of related species (Plasmodium discharges. Respiratory infections are are shed, the greater the chance they species all causing malaria), the rationale transmitted by contact and by droplets have of reaching a new host. The num- to study transmission is clear. In multi- (aerosols) emitted during coughing and ber of organisms required to infect a factorial diseases that are associated sneezing, which can produce as many as host varies greatly between microorgan- 18 Van Winkelhoff & Boutaga isms. It takes approximately 10 Shigella shown that a significant number of spe- scopy. With this technique, however, dysenteriae bacteria (from a human cies that may be demonstrated have still they were not able to demonstrate trans- host) to infect a new host, but 106 not been cultured (Paster et al. 2001). mission of any morphotypes between Salmonella sp. (from animals) to cause The habitat of many of these species is spouses. More recent studies have food poisoning. Also, the route of infec- restricted to the oral cavity. The major- focused on transmission of single, culti- tion is important for the infection dose. ity of subgingival bacteria in perio- vable species. Most studies on bacterial As few as 10 gonocci bacteria are able dontitis can also be isolated from the transmission in periodontitis have to cause infection of the urethra, whereas oral cavity in periodontal health and are focused on A. actinomycetemcomitans thousands are needed to infect the muco- therefore considered indigenous to the and P. gingivalis. These species have sa of the oropharynx or rectum. Cough- oral cavity. These indigenous, commen- been selected on the basis of their strong ing, sneezing and spitting are activities sal bacteria are acquired from other association with different forms of that benefit the host by clearing contami- individuals, such as family members. destructive periodontal disease (Slots nated (foreign) material from the upper Ko¨no¨nen et al. (1994) showed that & Ting 1999) and their documented respiratory tract and the mouth. Most of Prevotella melaninogenica can be trans- pathogenic potentials (Haffajee & the shed microorganisms probably die in mitted from the mother to the child, but Socransky 1994, Fives-Taylor et al. the environment; few are able to colonize sources other than the mother for this 1999, Holt et al. 1999). However, the a new host. Transmission also depends species were also noted. It has been possibility that other, and possibly even on genetic factors of the microorganisms, speculated that maternal saliva may act stronger, bacterial markers for disease i.e. some strains of a given species are as a source of oral anaerobes that are exist in the non-cultivable part of the more readily transmitted than others. transmitted to young children (Ko¨no¨nen subgingival microflora cannot be ruled This property may vary independent of et al. 1992). The natural acquisition of out. the pathogenicity and virulence of a the oral microflora is not the aim of this species. Open and closed lesions can paper, and this review will focus on occur in various cases and stages of the studies that have been published on Detection of A. actinomycetemcomitans and P. gingivalis same disease and may vary greatly in the vertical and horizontal transmission of shedding of organisms. The bubonic selected periodontal pathogens. The natural habitat of A. actinomyce- form of plague with closed infections of temcomitans and P. gingivalis is the oral the lymph nodes and the blood stream is cavity. To date, there is no evidence that Is periodontitis a transmittable disease? transmitted from rat to humans (by fleas) both species occur outside the human but not from person to person. However, It is well established that severe perio- host. Animal isolates are phenotypically open pulmonary lesions of the disease dontitis clusters in families (van der and genetically distinct from human are highly contagious. The same is true Velden et al. 1993, 1996). This suggests isolates (Taichman et al. 1987, Beem for pulmonary (open) tuberculosis. that susceptibility factors for the onset et al. 1991, Karjalainen et al. 1993, and progression of the disease may be Meńard & Mouton 1995). Frequency of effective contacts transferred from person to person in The results of epidemiological studies families. This can involve transfer of depend on the sensitivity and specificity Contacts between infected and suscep- genetic susceptibility factors, common of the techniques used to detect a patho- tible individuals are an important factor behavioural factors and exposure to gen. In periodontal research, micro- in determining how fast an infection environmental factors including trans- scopy, culture methods, immunoassays spreads. Crowding and limited airflow mission of putative pathogenic bacteria. and nucleic acid-based methods have are critical terms in cross-infections, It is commonly assumed that bacteria been applied to detect and quantify especially for respiratory diseases. One play an essential role in the pathogenesis microbial pathogens. Detection limits single contact can transmit gonocci and of destructive periodontal disease. are different for the various methods. cause disease. Therefore, the question of whether bac- Also, selection of study populations terial transmission plays a role in perio- affects the conclusions of epidemiologi- Survival in the environment dontitis is relevant. However, the cal trails to a great extent. Tables 1 and 2 classical meaning of transmission of a summarize the prevalence of A. actino- Drying is a major environmental factor pathogen cannot be applied in perio- mycetemcomitans and P. gingivalis in that kills shed microorganisms. Staphy- dontitis given the complexity of the relation to age and periodontal status lococci, tubercle bacilli and clostridia bacterial component in this disease. based on the review by Slots & Ting and anthrax spores are resistant to dry- This involves a different approach (1999). A. actinomycetemcomitans is ing. Species such as N. meningitidis, when the question of transmission of infrequently detected in periodontal gonococci and most viruses are sensitive pathogenic agents in periodontitis is health in children o19 years but was to drying and will soon die in the posed: Which bacteria represent rele- found in up to 78% in 14 Vietnamese environment. vant risk factors upon transmission, subjects. In periodontally healthy sub- and how should these species, out of jects 419 years, it was frequently not the several hundreds of subgingival spe- detected but could be found in up to 33% Microbes Associated with cies identified, be selected? In an early of the subjects. P. gingivalis is usually Periodontitis study, Offenbacher et al. (1985) have not found in edentulous subjects and in The oral cavity harbours several hun- approached this issue by studying not a periodontal health in persons under the dreds of bacterial species, most of which single species, but by analysing the age of 19 years, and it is infrequently belong to the indigenous oral microflora similarity of the subgingival microflora isolated in periodontal health in persons (Moore & Moore 1994). It has been between spouses using dark-field micro- above the age of 19 years. Periodontal bacteria 19

Table 1. Occurrence (%) of subgingival Actinobacillus actinomycetemcomitans in periodontal hypothesis has also been proposed health and disease in untreated individuals [number of studies] (Socransky et al. 1999). Edentulous Periodontal health (%) Gingivitis (%) Periodontitis (%) Distribution within families o11 years 0 (2) 0 [3] 14 [1] 20–100 [6] 3–78 [10] It has been shown that two major perio- 12–18 years Unknown 0 [2] 15–60 [2] 35–100 [12] dontal pathogens, A. actinomycetemco- 4–37 [3] mitans and P. gingivalis, cluster in 19–35 years Unknown 0 [3] 0 [1] 0 [1] families (Zambon et al. 1983, Slots & 17–33 [5] 3–14 [3] 19–94 [12] 435 years 0 0 [1] 0 [1] 30–83 [15] Listgarten 1988, Gunsolley et al. 1990, 20 [1] van der Velden et al. 1993, Tuite- McDonnell et al. 1997). These observ- Adapted from Slots & Ting (1999). ations suggest that bacteria may be transmitted between family members and that they share susceptibility factors Table 2. Occurrence (%) of subgingival Porphyromonas gingivalis in periodontal health and for colonization by these bacteria or disease in untreated individuals [number of studies] clonal types of these species. Detection Edentulous (%) Periodontal health (%) Gingivitis (%) Periodontitis (%) of the same bacterial species in family members does, however, not prove o11 years 0 [2] 0 [5] 14 [1] 0 [2] transmission. For further evidence, typ- 4–70 [4] 7 [1] ing of the bacterial isolates is necessary. 12–18 years Unknown 0 [1] 2 [1] 38–63 [4] 7 [1] 19–35 years Unknown 0 [2] 0 [2] 8–100 [9] Methods of Typing Microorganisms 10 [1] 435 years 0 [1] 0 [1] 100 [1] 0 [1] Bacterial strain typing is an integral part 8 [1] 100 [1] 27–100 [13] of epidemiological investigations. Meth- Adapted from Slots & Ting (1999). ods of bacterial typing range from simple biotyping to complex genetic characterization. It is common to use more than Table 3 summarizes recent studies on increases with age (Rodenburg et al. one typing method, e.g. L. pneumophila the prevalence of A. actinomycetemco- 1990, Slots et al. 1990, Savitt & Kent is first serotyped into nine groups and mitans and P. gingivalis in periodontal 1991). then further biotyped for final identifica- health and disease. Boutaga et al. (2003) tion. Molecular typing systems are able used a real-time PCR technique to detect Intra-oral cross-contamination to distinguish among epidemiologically A. actinomycetemcomitans and P. gingi- unrelated isolates because of the genetic valis in 111 periodontal healthy subjects A. actinomycetemcomitans and P. gingi- variation in the chromosomal DNA of a and 259 adult patients with perio- valis are not restricted to the subgingival bacterial species. dontitis. A. actinomycetemcomitans and area in periodontitis patients. It has been P. gingivalis were detected in 18% and shown that both species can also be 9.9%, respectively, in subjects without found in saliva, supragingival plaque Phenotypic typing methods periodontitis. In contrast, the prevalence and on various mucous membranes in These methods are based on phenotypic of A. actinomycetemcomitans in Chinese the oral cavity of patients with perio- characteristics of the bacterial species adults without periodontitis amounted to dontal infections, although proportions and can, for instance, be used to type 78%, exemplifying geographical differ- are usually highest in subgingival plaque successive isolates of the same organism ences in the prevalence of A. actino- (Van Winkelhoff et al. 1988, Socransky from an individual patient. mycetemcomitans. In young Japanese et al. 1999). Supragingival plaque usually children (2–12 years) without perio- contains higher numbers of P. gingivalis dontitis, A. actinomycetemcomitans was in periodontitis patients compared with Biotyping found in 4.8% of the subjects. In Brazil, periodontally healthy subjects (Socrans- Biotyping uses the results of biochem- the prevalence of A. actinomycetemco- ky et al. 1999). Current data indicate that ical tests to assign members of the same mitans and P. gingivalis appeared much both species spread intra-orally through species into different groups. In A. actino- higher in subjects without destructive contaminated saliva and colonize supra- mycetemcomitans, six biotypes have been periodontal disease (70% and 78%, gingival plaque before they colonize the described based on sugar fermentation, respectively) in comparison with the subgingival area (Socransky et al. 1999). but the method has a low discriminatory Dutch population. It cannot be excluded It has been shown that subgingival colo- potential in comparison with other meth- that these different findings may have nization of both pathogens not only ods (van Steenbergen et al. 1994). A been caused by different techniques. occurs at sites with but also at sites useful biotyping method for human A relationship between age and dis- without periodontal attachment loss P. gingivalis strains is not available. tribution of A. actinomycetemcomitans (Van Winkelhoff et al. 1994). It has and P. gingivalis has been observed. been speculated that deep pockets of Serological typing The prevalence of A. actinomycetemco- diseased subjects act as reservoirs for mitans seems to decrease with age, spread of infection to healthy sites (Riv- Serotyping uses a series of antibodies, whereas the prevalence of P. gingivalis iere et al. 1996), although an alternative raised in animals, to detect different 20 Van Winkelhoff & Boutaga

antigenic determinants on the bacterial cell surface. It remains a valuable method for typing isolates of P. gingivalis (Van Winkelhoff et al. 1993, Laine et al. 1996) and A. actinomycetemcomitans (Zambon et al. 1983).

Phage typing Different strains within a species have different sensitivities for the lytic activ- ity of bacteriophages. Phage typing is not available for A. actinomycetemcomitans and P. gingivalis. Prevalence of bacteria (%)

Molecular typing methods Protein typing periodontitis healthy periodontitis healthy A. actinomycetemcomitans P. gingivalis This is a relatively crude method of typing in which the proteins of an organism are simultaneously extracted. The patterns of proteins are demonstrated by polyacrylamide gel electrophoresis (PAGE). The SDS-PAGE typing techni- in healthy subjects and adult patients with periodontitis by culture and PCR que has a low discriminatory potential. Culture 31 12.8 59.5 10.6 Real-time PCRCulture 27 19.7 18 4.5 43 53 9.9 7.2 PCRCulture ND 24 ND 79 24 25 PCRCulture 26.5 22.4 ND 70 18.4 ND CulturePCR 6.3 18.8 0 6.7 84.4 81.3 20 13.3 PCRCulture 1.33 8.6 ND 97.1 16.10 ND PCR 20 4.8 20 4.8 PCR 69 78 ND ND Multilocus enzyme electrophoresis (MLEE) MLEE is based on metabolic enzymes that are highly conserved and unlikely to change quickly in a given species. The

Porphyromonas gingivalis method is highly reproducible with a moderate level of discriminatory power. and It is useful for epidemiological studies that compare isolates from geographically distant areas over time. 25 years old): 94 healthy, 116 25 years old): 111 healthy, 259 Restriction enzyme analysis (REA) 4 4 REA uses endonucleases to cleave DNA at a particular sequence of nucleotides that may be present on different places on the bacterial chromosome. The number in size of the generated restriction periodontitis, the Netherlands periodontitis, the Netherlands periodontitis, USA Japan periodontitis, Spain periodontitis, 35 generalized chronichealthy, periodontitis, Japan 18 periodontitis, Japan moderate periodontitis, 27 severe periodontitis, China fragments reﬂects the frequency and

Actinobacillus actinomycetemcomitans distribution of the restriction sites. Fragments are separated by size using agarose gel electrophoreses. Different strains of the same bacterial species have different REA proﬁles because of variations in their DNA sequences. This technique has been applied to type A. actinomycetemcomitans (Petit et al. 1993a, b) and P. gingivalis (van Steen- bergen et al. 1993). Reference Study group Technique used Restriction fragment length polymorphism (RFLP)

. Studies on the frequency of detection of With RFLP, chromosomal DNA is digested by frequent cutting enzymes Van Winkelhoff et al. (2002) 210 adult subjects ( techniques Boutaga et al. (2003) 370 adult subjects ( Table 3 Griffen et al. (1998) 311 adult subjects (49.2–51.4 years old): 181 healthy, 130 Riggio et al. (1996)Ashimoto et al. (1996) 43 periodontitis patients, UK 49 subjects (15–75 years old): 49 advanced periodontitis, PCR 15 ND 11 ND Lau et al. (2004) 92 subjects (30–71 years old): 30 healthy, 30 gingivitis, 32 Takeuchi et al. (2003) 103 (412 sites) subjects (15–70 years old): 50 aggressive Avila-Campos & Velasquez-Melendez (2002)Okada et al. (2004) 100 subjects: 50 healthy, 50 periodontitis, Brazil PCR 104 subjects (2–12 years old): 10 healthy, 73 gingivitis, 21 90 70 66 78 Tan et al. (2001)and separated by agarose 92 adult subjects (25–65 years old): 50 healthy, 15 gel electro- Periodontal bacteria 21 phoresis. The DNA fragments are trans- Arbitrarily primed (AP)-PCR of distinct bacterial strains among indi- ferred onto a membrane. The DNA on viduals. Strains within a bacterial spe- the membrane is then hybridized with a This is a kind of PCR technique using a cies may display variable virulence, and specific labelled probe, which binds to short primer that is not targeted to typing of these strains may distinguish a limited number of fragments on the amplify any specific bacterial DNA between pathogenic and less pathogenic membrane that have complementary sequence. The resulting AP-PCR pro- representatives of a species. nucleotide acid sequences. Variations in ducts will represent a variety of differ- Using the AP-PCR method, 95 the number and size of the fragments ent-sized DNA fragments that are A. actinomycetemcomitans strains from detected by hybridization are referred to visualized by agarose gel electrophor- 92 unrelated Finnish subjects yielded 17 as RFLPs. This method has been success- esis. This method has been applied to genotypes, and 29 P. gingivalis isolates A. actinomycetemcomitans P. gingi- fully used to type A. actinomycetemcomi- and from 29 unrelated subjects yielded 27 valis tans (DiRienzo & Slots 1990, DiRienzo (Dogan et al. 1999a). genotypes (Asikainen et al. 1996). van & McKay 1994, DiRienzo et al. 1994). Steenbergen et al. (1993) used the REA Amplified fragment length method to type P. gingivalis and found, Ribotyping polymorphism (AFLP) with one exception, that each individual was colonized with one clonal type of This method uses chromosomal DNA AFLP is a PCR-based fingerprinting P. gingivalis only. The DNA patterns of and a ribosomal RNA probe to provide technology. AFLP involves the restric- all P. gingivalis isolates from unrelated different profiles. Because all bacterial tion of genomic DNA, followed by individuals were found to be distinct. isolates have one or more chromosomal ligation of adaptors complementary to This was also found for A. actinomyce- rRNA operons distributed on the chro- the restriction sites and selective PCR temcomitans using the REA technique mosome, and because these sequences amplification of a subset of the adapted (Petit et al. 1993a, b). are highly conserved, essentially all restriction fragments. Compared with bacterial isolates can be typed using other marker technologies, AFLP pro- probes directed to the DNA sequences vides equal or greatly enhanced perfor- Genetic diversity of A. actinomycetem- that encode the rRNA loci using a single mance in terms of reproducibility and comitans and P. gingivalis within rRNA probe. This method has been resolution. Probably the single greatest individuals successfully used to type A. actinomy- advantage of the AFLP technology is its Asikainen & Chen (1999) have recently cetemcomitans (Alaluusua et al. 1993, sensitivity to polymorphism detection at analysed all studies dealing with the Saarela et al. 1993a). the total genome level. This sensitive number of clonal types of A. actinomy- technique has occasionally been applied cetemcomitans and P. gingivalis within Pulse field gel electrophoresis (PFGE) to type A. actinomycetemcomitans and individuals (Table 4). They found dif- P. gingivalis (Fig. 1; Van Winkelhoff ferences between studies performed in With the PFGE method, the bacterial et al. 2000). Europe and the United States on the one genome is digested with a restriction hand and Asian studies on the other. In enzyme that has relatively few recogni- Genetic diversity of A. actinomycetem- Europe and the USA, the majority of tion sites and thus generates 10–30 patients studied harboured one single restriction fragments and then is sepa- comitans and P. gingivalis between individuals genotype of A. actinomycetemcomitans rated by PFGE to produce a pattern of (75%, range 67–80%), and 80% (range bands. This method has been applied to Unique genetic or phenotypic markers 75–85%) of the tested patients har- A. actinomycetemcomitans (Valcarcel between independent bacterial isolates boured one genotype of P. gingivalis. et al. 1997). are a prerequisite to studying the spread These observations confirmed earlier observations of Loos et al. (1990), Petit et al. (1993b) and van Steenbergen et al. (1993), who found only one clonal type of A. actinomycetemcomitans and P. gingivalis in most study subjects using the REA typing method. Recently, P. gingivalis strains, isolated from 106 Indonesian subjects were typed using the AFLP method. The number of subjects with one single genotype amounted to 65%. In 27% of the subjects two genotypes were detected and 7.5% (eight subjects) harboured three clonal types. The presence of 42 clonal types of P. gingivalis has Fig. 1. Example of amplified fragment length polymorphism (AFLP) typing profiles of not been observed so far in any Western Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis isolates from a 13- year-old Turkish patient with congenital periodontitis and their mother and father. (a) The study population (Table 5, Van Win- A. actinomycetemcomitans isolates from the patient (n 5 2) and the father (n 5 2) were kelhoff et al. unpublished data, Timmer- indistinguishable, suggesting transmission. (b) The P. gingivalis isolates from the patient man et al. 1998). This exemplifies that (n 5 ...), the mother (n 5 1) and the father (n 5 2) show different AFLP profiles (similarity the distribution of clonal types of o70%) (Van Winkelhoff et al. 2000). P. gingivalis in Eastern populations is 22 Van Winkelhoff & Boutaga

Table 4. Number of genotypes of Actinobacillus actinomycetemcomitans and Porphyromonas phylogenetic trees derived from sequen- gingivalis in subjects with periodontitis cing fragments of four housekeeping Species Total # of Mean number of Number of clones genes. They concluded that P. gingivalis subjects strains typed/subject has a non-clonal population structure n 5 1 n 5 2 n 5 3 n 5 4 characterized by frequent recombination. Their results also suggest that A. actinomycetemcomitans 120 10.3 (range: 1–115) 75% 19% 5% 0.8% particular genotypes with possible P. gingivalis 41 5.6 (range: 1–15) 80% 20% 0 0 increased pathogenic potential may Adapted from Asikainen & Chen (1999). spread in the human population. AP-PCR genotyping of P. gingivalis has also not been able to associate Table 5. Number of genotypes of Porphyromonas gingivalis in an Indonesian study population specific genotypes and disease status (Meńar & Mouton 1995). Serotyping Species Total # of Mean number Number of clones subjects strains typed/subject of P. gingivalis, based on capsular poly- n 5 1 n 5 2 n 5 3 n 5 4 saccharide antigens, has identified at least six different serotypes, of which P. gingivalis 106 3.4 (range: 1–6) 65% 27% 7.5% 0 the uncapsulated variant (K-) appeared less pathogenic in an animal model (Laine & Van Winkelhoff 1998). different in comparison with Western showed a deletion in the promoter Whether the different K serotypes of populations. The phenomenon of poly- region (Bueno et al. 1998), which may P. gingivalis are associated with differ- clonal colonization has also been found be associated with a high leucotoxine ent forms or variation in progression of for A. actinomycetemcomitans in non- production (Brogan et al. 1994). This periodontitis in humans is currently Western populations. Using the serotyp- type of A. actinomycetemcomitans unknown. ing method, it has been found that, in seems to occur specifically in patients contrast to European and USA studies originating from the Northern part of the (Zambon et al. 1983, Ebersole et al. African continent (Haubek et al. 1996). Distribution of A. actinomycetem- 1994, Dogan et al. 1999), subjects Specific AP-PCR A. actinomycetemcomitans and P. gingivalis among family members from Eastern countries may frequently comitans genotypes have been identified harbour more than one serotype of in periodontitis patients in the USA One important study on the distribution A. actinomycetemcomitans (Chung et al. (Asikainen et al. 1995) and in Finnish of A. actinomycetemcomitans and 1989, Ho¨ltta et al. 1994). This discre- localized juvenile periodontitis patients, P. gingivalis among family members is pancy in intra-individual colonization which were rarely found in Finnish adult from Asikainen et al. (1996). They by periodontal pathogens may be caused patients with periodontitis and perio- studied 47 families for the occurrence by socio-economic status, higher fre- dontally healthy subjects (Asikainen of A. actinomycetemcomitans and 31 quency of contacts with infected indivi- et al. 1997). Recently, a study suggested families for P. gingivalis. Probands duals and individual oral hygiene level. increased periodontal destruction among were selected on the basis of the pre- Moroccan early-onset periodontitis sence of periodontal diseases (adult patients who were culture positive for periodontitis) or on the presence of Distribution of clonal types of A. actino- A. actinomycetemcomitans of the highly A. actinomycetemcomitans-positive chil- mycetemcomitans leucotoxic JP2 type compared with EOP dren (n 5 6). They found that spouses Not all representatives of a species may patients without the JP2 clone (Haubek of periodontitis patients with A. actino- display identical pathogenic properties. et al. 2002). mycetemcomitans were seven times as Within a species, special pathogenic MLEE has been applied to study the frequently infected with the organism in clones can be present (Selander et al. population genetic structure of P. gingi- comparison with spouses of bacterium- 1987, Musser 1996). Population studies valis (Loos et al. 1993). A total of 78 negative patients (Table 6). Spouses of on A. actinomycetemcomitans using distinct electrophoretic types (ETs), patients culture positive for P. gingivalis MLEE revealed that the species com- representing multilocus genotypes, were twice as frequently positive com- prises distinct genetic clusters that cor- were identified, and cluster analysis pared to spouses of patients negative relate with serotype distribution, but an identified three major phylogenetic divi- for this species. Thirty-two percent of association between genetic clusters and sions. Division I (71 ETs) included all the children with a proband culture type of disease was not found (Poulsen 88 human isolates examined. The strains positive for A. actinomycetemcomitans et al. 1994, Haubek et al. 1995). In con- in division II and division III, isolated also had detectable levels of this species trast, DiRienzo et al. (1994), using the from different animal species, were (Table 6). Only one of 19 children with RFLP method, have identified a specific strongly differentiated from those in a P. gingivalis-positive proband had A. actinomycetemcomitans genotype division I. They found no evidence of detectable P. gingivalis. that appeared to be associated with association between specific genetic In a large study on the transmission of juvenile periodontitis and was asso- lineages or clusters and the type of P. gingivalis within families, Tuite- ciated with conversion from periodontal disease (periodontitis or root canal McDonnell et al. (1997) used the PCR health to disease. They also identified infections), invasive potential, ser- technique to detect P. gingivalis in 564 two genotypes that were exclusively ogroup or fimbrial RFLP group. Frand- members of 104 multigeneration isolated from periodontally healthy sub- sen et al. (2001) studied the population families recruited from church and com- jects. The disease-associated genotypes structure of P. gingivalis by comparing munity organizations in Columbus, OH, Periodontal bacteria 23

Table 6. Occurrence of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in family members of adult patients with periodontitis Proband Family OR p-value % of %of member positive family positive family members when members when proband is positive proband is negative

Adult patient with A. actinomycetemcomitans Spouse: 7/11 17.5 0.024 64 9 Parent with A. actinomycetemcomitans periodontitis Child: 6/19 8.2 0.14 32 0 Adult patient with P. gingivalis Spouse 9/12 5 0.17 75 37.5

From Asikainen et al. (1996).

USA. One hundred and one of the Table 7. w2 test results for observed and expected concordances of colonization by Porphyr- families contained two parents, at least omonas gingivalis of pairs within families according to Tuite-McDonnell et al. (1997) one child and at least one grand parent. Pair No. of pairs % concordance Probability The child had lived with the parents at the time of sampling, the parents had observed expectedn lived together for at least 2 years and the parents had lived with the grand parents Parental spouses 102 78 51 o 0.001 at least up to the age of 15 years. 53.4% Grandparental spouses 62 73 51 o 0.001 Oldest, second oldest children 63 71 59 0.05 were females, 98.2% were white sub- o Mother, oldest child 103 72 54 o 0.001 jects and 1.8% were African Americans. Father, oldest child 101 76 54 o 0.01 The mean age of the 564 subjects was Adults, grandmother 98 76 51 o 0.005 37.5 years (SD 24 years, range 0.7–95.4 Adults, grandfathers 69 64 53 o 0.10 years). The overall prevalence of nCalculated based on the prevalence of P. gingivalis in the group being considered. P. gingivalis was 37.1%, 46.1% in the grand parents, 42.3% in the parents and 28.2% in the children (children versus Table 8. Relative risk for colonization with Porphyromonas gingivalis for pairs within families grand parents po0.02). Table 7 sum- according to Tuite-McDonnell et al. (1997) marizes the relative risks for coloniza- Pairs Generation N RR (95% confidence interval) tion of P. gingivalis within pairs of family members. Individuals whose Wife–husband F2–F2 102 4.0 (2.3–7) spouses were colonized were four times Mother–oldest child F2–F3 103 4.7 (3.1–7) more likely to be colonized than those Grandmother–adult F1–F2 98 2.4 (1.5–3.7) married to persons who were not colo- Oldest child–second child F3–F3 63 2.4 (1.1–5.3) nized. No relationship was found Father–oldest child F2–F3 101 3 (2–4.5) Grandfather–adult F1–F2 69 1.8 (1–3.3) between the length of time a couple had been married (mean 14.1 years) RR, relative risk calculated w2 with Bonferroni’s correction for multiple testing. and their concordance of colonization (p 5 0.61 by logistic regression). Chil- dren and mothers and their fathers were these observations, Tuite-McDonnell cal genotypes (36% probability for also significantly more frequently con- et al. concluded that ‘‘contact with an transmission between spouses) and cordant in colonization than would be infected family member substantially 32% of the children harboured A. acti- expected if P. gingivalis were randomly increased the relative risk of coloniza- nomycetemcomitans that was always of distributed in the study population tion for spouses, children and their the same genotype as the parent. Petit (Table 7). A P. gingivalis-colonized parents, adults and their mother, and et al. (1993a, 1993b), using REA typing mother would give a child a risk for siblings’’ (Table 8). and an A. actinomycetemcomitans-posi- colonization that was 4.7 times greater tive periodontitis patient as the proband, than a child with a mother who was not observed that A. actinomycetemcomi- colonized. This risk was 2.98 for colo- Genotyping of A. actinomycetemcomitans tans can be transmitted among family and P. gingivalis within families nized fathers and a child. The risk for members but at low occurrence (15% colonization was highest when a child Asikainen et al. (1996) used AP-PCR to for children, 8% for spouses). Preus had both parents colonized by P. gingi- genotype A. actinomycetemcomitans et al. (1994) found essentially the same valis. The likelihood of whole families isolates from family members. In 11 using AP-PCR genotyping of isolates (grandfather/mother, both parents and a of 12 families, they found identical from family members of which one child, N 5 À 101 with 404 subjects) was A. actinomycetemcomitans genotypes parent suffered from periodontitis. tested. Concordance in colonization sta- among family members, which could They found that in six of seven families, tus was highly significantly more fre- not be explained by chance alone. This the husband and wife did not harbour quent among family members than suggests transmission of this microor- the same A. actinomycetemcomitans would be expected if P. gingivalis ganism among family members. Four of genotype. Furthermore, most often, chil- were randomly distributed in the popu- 11 couples with an A. actinomycetemco- dren carried a genotype identical to one lation studied (p 5 0.0000). Based on mitans-positive proband showed identi- of the parents, indicating vertical trans- 24 Van Winkelhoff & Boutaga mission (Alaluusua et al. 1991). The (1988) found P. gingivalis in six of eight mitans. It is not as clear which microbial phenomenon of vertical transmission of saliva samples from untreated patients and host factors determine the onset of A. actinomycetemcomitans was not with periodontitis with numbers ranging periodontal breakdown upon infection observed in a study with Brazilian from 6 to 20 Â 106/ml. Kissing may with these pathogens. patients with juvenile periodontitis, therefore be one possible way of direct One study has dealt with the issue of who served as probands (Tinoco et al. inoculation of this pathogen into another bacterial transmission between spouses 1998). None of the parent–child pairs person. The survival of periodontal and the clinical status of the spouse (von showed identical A. actinomycetemco- pathogens in saliva outside the oral Troil-Linden et al. 1995b). In this study, mitans genotypes. The presence of mul- cavity is not known. Indirect inoculation two groups of married couples were tiple clones in the study subjects and may occur when a contaminated tooth- involved to assess whether spouses social–economic situation may explain brush is used within several hours after show similarities in their periodontal these discrepant results. use (Mu¨ller et al. 1989, Quirynen et al. status. One group consisted of probands P. gingivalis was isolated from both 2001). The minimal infection dose and with periodontitis and their spouses spouses in 10 couples, two of which the required number of exposures to (n 5 10); a second group consisted of showed identical AP-PCR profiles (20% transfer A. actinomycetemcomitans or periodontally healthy proband and their probability for sharing the same geno- P. gingivalis from one person to another spouses. Clinical, radiographic and type, Asikainen et al. 1996). This was are unknown, but it seems likely that microbiological data were obtained significantly lower in comparison with both parameters will influence the odds from all subjects. It was found that the findings of van Steenbergen et al. for transmission. Periodontal treatment spouses of the diseased probands had a (1993), who found identical REA pro- reduces the salivary load of both species significantly lower percentage of 43 files in six of eight couples positive for significantly (von Troil-Lindeń et al. and 4–5 mm pockets and more sites P. gingivalis (75% probability of shar- 1995a). Periodontally healthy subjects with alveolar bone (o1/3 of root length) ing the same genotype). Saarela et al. may have detectable salivary A. actino- than spouses of the healthy probands. (1993b) found two of four couples shar- mycetemcomitans and may therefore Microbiological analysis showed that in ing the same serotype and ribotype of remain a source of transmission (Asi- the diseased group, four couples (pro- A. actinomycetemcomitans and in two kainen et al. 1991). Salivary A. actino- band and spouse) were infected with of four couples the same ribotype of mycetemcomitans and P. gingivalis have A. actinomycetemcomitans, and six cou- P. gingivalis. These findings support the shown to be of the same clonal type as ples with P. gingivalis, whereas in the phenomenon of transmission of perio- those found in the subgingival plaque in healthy group no A. actinomycetemco- dontal pathogens among family mem- the majority of patients (Petit et al. mitans-positive couple (proband and bers. Both different and identical 1993a, 1993b, van Steenbergen et al. spouse) and one P. gingivalis-positive genotypes of A. actinomycetemcomitans 1993, von Troil-Lindeń et al. 1996, couple was found. These observations and P. gingivalis were found among Asikainen et al. 1997). suggest that cohabitation with a person spouses with a probability of 20–75% with periodontitis may influence the of transmission (identical clones). Chil- microbiological and the periodontal sta- Consequences of transmission dren of a parent with an A. actinomyce- tus of the spouse. One factor that may temcomitans-associated form of The clinical consequence of transmis- explain these observations is the higher periodontitis had an 8–30% probability sion of A. actinomycetemcomitans or occurrence of A. actinomycetemcomi- of acquiring the A. actinomycetemcomi- P. gingivalis from a diseased person to tans and P. gingivalis in subjects in the tans from the proband. Vertical trans- another person is not known. It is evi- diseased group, suggesting person-to- mission of P. gingivalis has rarely been dent that both bacterial species can be person transmission of these pathogens. observed. found in subjects without overt perio- More of these studies, with larger num- dontal breakdown (Petit et al. 1993a, bers of couples and genotyping of the 1993b, van Steenbergen et al. 1993, bacterial isolates, are needed to provide Possible ways of transmitting Griffen et al. 1998, Van Winkelhoff et further evidence for a possible transmis- periodontal bacteria al. 2002). These species can probably sion of destructive periodontal disease Nearly all microorganisms are shed persist in equilibrium with the host and among spouses. from human body surfaces and dissemi- the resident microflora for years. Strik- nated into the outside world. Most of ingly, van Steenbergen et al. (1993) Summary and conclusions these microbes will not survive in the found high percentages of P. gingivalis environment; few can find a next host. not only in the periodontitis probands Periodontitis is a collection of clinically There is no evidence that periodontal (ranging from 5% to 48% of the culti- different diseases associated with a sub- bacteria can be transmitted via aerosols, vable subgingival microflora) but also in gingival microflora that significantly and it seems likely that saliva and direct the spouses (0.2–61%). Van Winkelhoff differs among different patient groups. mucosal contact are the transmission et al. (2002) also found only a marginal The majority of subgingival bacterial routes of periodontal bacteria. Also, difference in the percentage of P. gingi- species are indigenous to the oral cavity, inanimate objects (dental probes, tooth valis between periodontitis patients and and periodontitis associated with indi- brushes) may serve as a vehicle for subjects without periodontitis (28.7% genous bacteria can be looked upon as transmission. A. actinomycetemcomi- versus 17.4% of the cultivable subgin- an opportunistic infection. A. actinomy- tans and P. gingivalis can be cultured gival microflora, p 5 0.052), although cetemcomitans and P. gingivalis are from saliva from periodontitis patients the absolute numbers were significantly infrequently detected in periodontal (Van Winkelhoff et al. 1988, Asikainen higher in patients. The same phenomen- health in Western populations and have et al. 1991). Van Winkelhoff et al. on was found for A. actinomycetemco- characteristics of exogenous pathogens. Periodontal bacteria 25

Therefore, periodontitis associated with transmission of specific virulent clones Boutaga, K., Van Winkelhoff, A. J., Vanden- these pathogens can be looked upon as may be feasible and effective in pre- broucke-Grauls, C. M. J. E. & Savelkoul, P. exogenous infections. In A. actinomyce- venting some forms of periodontal dis- H. M. (2003) Comparison of real-time PCR temcomitans, but not in P. gingivalis, ease. P. gingivalis is usually recovered and culture for detection of Porphyromonas special clones associated with localized from diseased adult subjects and trans- gingivalis in subgingival plaque samples. Journal of Clinical Microbiology 41, juvenile periodontitis have been identi- mission of this pathogen seems largely 4950–4954. fied. Vertical transmission of A. actino- restricted to adult individuals. Horizon- Brogan, J., Lally, E., Poulsen, K., Kilian, M. & mycetemcomitans but not of P. tal transmission of P. gingivalis may Demuth, D. (1994) Regulation of Actinoba- gingivalis has been established. Most therefore be controlled by periodontal cillus actinomycetemcomitans leukotoxin studies have shown that if children treatment involving elimination or sig- expression: analysis of the promoter regions harbour A. actinomycetemcomitans, nificant suppression of the pathogen in of leukotoxic and minimally leukotoxic usually one or two parents harbour the diseased individuals and by a high stan- strains. Infection and Immunity 62, 501–508. same genotype. From these observa- dard of oral hygiene. Bueno, L., Mayert, M. & DiRienzo, J. (1998) tions, it is assumed that the parent is Relationship between conversion of localized juvenile periodontitis-susceptible children the source of transmission. However, from health to disease and Actinobacillus identical genotypes in family members actinomycetemcomitans leukotoxin promoter are not 100% proof of transmission, as References structure. 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Department of Oral Microbiology S., Abbas, F., Winkel, E., Van Winkelhoff, A. von Troil-Lindeń, B., Torkko, H., Alaluusua, S., Van der Boechorststraat 7 J. & van der Velden, U. (1998) Untreated Jousimies-Somer, H. & Asikainen, S. (1995b) 1081 BT Amsterdam periodontal disease in Indonesian adoles- Periodontal findings in spouses. A clinical, The Netherlands cents. Clinical and microbiological baseline radiographic and microbiological study. E-mail: [email protected]