Endodontic Topics 2003, 6, 7895 Printed in Denmark.

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Copyright r Blackwell Munksgaard

ENDODONTIC TOPICS 2003

Periapical Actinomycosis and infection with Propionibacterium Propionicum

F. SIQUEIRA JR JOSE

Introduction
Apical periodontitis is an inammatory disease of microbial etiology (1, 2). It is formed in response to intra-radicular infection and comprises an effective barrier against spreading of the infection to the alveolar bone and to other body sites. In most situations, apical periodontitis lesions are free of microorganisms. However, in specic circumstances, the inamed periapical tissues can be invaded by microorganisms, resulting in extra-radicular infection. The most common form of extra-radicular infection is the acute periapical abscess, characterized by purulent inammation in the periapical tissues in response to the egress of virulent bacteria from the root canal (3). There is, however, another form of extra-radicular infection which, unlike the acute abscess, is usually characterized by the absence of overt symptoms. This condition consists of establishment of microorganisms in the periapical tissues, either by their adherence to the apical root surface in the form of biolm-like structures (4) or within the body of the inammatory lesion, usually as cohesive colonies (5). Those extra-radicular microorganisms have been regarded as one of the etiologies of persistence of apical periodontitis in spite of endodontic treatment (46). It can be assumed that the extra-radicular infection may be dependent on, or independent of the root canal infection (Fig. 1). For example, the acute periapical abscess the most common form of extra-radicular infection is clearly dependent on the intra-radicular infection; once the intra-radicular infection is properly treated and drainage of pus is achieved, the extra-

radicular infection subsides. Thus, extra-radicular infections are commonly supported by the intraradicular infection and, except for abscesses, extraradicular infection is a rather rare occurrence (3). Recent studies using culture (79) or molecular methods (10, 11) for microbial identication have reported the extra-radicular occurrence of a complex microbiota associated with post-treatment apical periodontitis, which did not respond favorably to the root canal therapy. Anaerobic bacteria have been reported to be the dominant microorganisms in several of those lesions (711). Although those studies did not evaluate the bacteriological conditions of the apical part of the root canal, it is entirely possible that such extraradicular infections were in fact supported by intracanal microorganisms. This may be another example of extra-radicular infection that is dependent on the presence of an intra-radicular infection. In fact, the aforementioned ndings are very intriguing, as most of the detected species are usually oral opportunistic pathogens that are unlikely to survive in a hostile environment such as the inamed periapical tissues (3). The following questions arise: Were bacteria actually present within the periapical tissues? If so, were they truly established in the inamed periapical tissues or was their presence only transient before elimination by host defenses? Can a mixed infection composed of several species become established in the periapical lesions in a relatively high percentage of cases? If most of the apical periodontitis lesions involve extra-radicular infection, can the nonsurgical (orthograde) endodontic therapy result in a high healing rate? Can we non-surgically treat an

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conceivably independent of the intra-radicular infection. So far, evidence suggests that the main bacterial species implicated in exclusively extra-radicular infections are the members of the genus Actinomyces and the species Propionibacterium propionicum (formerly designated Arachnia propionica), in a pathologic entity named periapical actinomycosis (1215). Given the widely recognized role of these bacteria in causation of post-treatment disease, this review will focus on their involvement with different types of endodontic infections, with special emphasis placed on their association with periapical actinomycosis. The mechanisms of pathogenicity of Actinomyces species and P. propionicum that can play a role in the etiology of periapical actinomycosis, as well as the therapeutic measures to manage this disease, will also be discussed.

Fig. 1. Extra-radicular infections can be dependent on (A) or independent of (B) the intra-radicular infection. In the former, eradication of the intra-radicular infection usually results in healing of the periapical lesion. In the latter, periapical inammation can be sustained, even after thorough elimination of the intra-raticular infection.

extra-radicular infection? Further research is still required before these questions can be answered with a degree of condence. In most teeth associated with apical periodontitis, infection is restricted to the root canal. Most of the microbial species that infect the root canal are opportunistic pathogens (3) that do not have the ability to survive host defense mechanisms in the periapical tissues. Rare exceptions are those microbial species or even strains within a species that possess strategies to survive and thus to infect vital tissues. Such microorganisms must be able to invade tissues, scavenge nutrients, and evade the host defense mechanisms (3). If all these requirements are materialized, an extra-radicular infection may develop. A few oral microorganisms have the ability to overcome host defense mechanisms, thrive in the inamed periapical tissues and, as a consequence, induce an extra-radicular infection. Several species of putative oral pathogens have been detected in posttreatment apical periodontitis lesions (7, 10, 11). Some of them possess an apparatus of virulence that theoretically can allow them to invade and to survive in a hostile environment, such as the periapical lesion (3). However, their involvement in an extra-radicular infectious process independent of the intra-radicular infection is not certain. There are a few conditions in which the extraradicular infection may actually occur, persist even after successful eradication of the intra-radicular infection, and hence be the exclusive etiology of post-treatment disease. In this case, the extra-radicular infection is

Actinomycosis
The term actinomycosis was introduced by Israel, in 1878 (16), in his accurate description of a cervicofacial and thoracic case of the disease. Additional clinical descriptions followed along with the isolation of Actinomyces israelii by Bujwid in 1889 (17). This species was then well described by Wolff and Israel, in 1891 (18). The causative agents of this slowly progressive infection are Gram-positive bacteria of the genera Actinomyces and Propionibacterium, which are normal inhabitants of the oral cavity, colon and vagina (1921). A. israelii is by far the species most commonly involved in causation of actinomycosis, but less common causes of the disease include Actinomyces naeslundii genospecies 1 and 2, Actinomyces odontolyticus, Actinomyces meyeri, Actinomyces gerencseriae, and P. propionicum (19, 20). Actinomycosis is a chronic, granulomatous infectious disease characterized by suppuration, abscess formation and draining sinus tracts, which erupt to the skin or mucosal surfaces and drain pus containing sulfur granules (small colonies of bacteria) (19, 21). The clinical forms of actinomycosis that account for most of these infections in humans are as follows, in decreasing order of prevalence: (1) cervicofacial, (2) abdominal, (3) thoracic, and (4) cerebral forms (19, 21, 22). The cervicofacial form is the most common form of the disease. It is characterized by a slowly evolving induration in the mandibularpreauricular region,

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often accompanied by sinus tracts to the skin that discharge typical sulfur granules (21). Sometimes it may take the form of acute abscesses (21). The majority of cases have been observed in patients with poor oral hygiene and/or a history of invasive dental procedures or trauma. Pulverer et al. (23) evaluated microbiological and selected clinical data derived from 1997 culture-positive cases of human cervicofacial actinomycoses examined during 19721999; they reported that causative actinomycetes belonged to at least nine different species, among which A. israelii and A. gerencseriae predominated. The highest incidence was found in female patients aged 1140 years and in male patients aged 2150 years. Periapical actinomycosis is a form of cervicofacial actinomycosis, but signs and symptoms are usually different (see discussion below). The abdominal and the thoracic forms are presumably due to aspiration and swallowing of bacteria from the oral cavity (24). The basic microscopic picture in actinomycosis is suppurative, but it can vary from an acute abscess to a chronic lesion in which proliferating connective tissue is commonly seen (19). In tissues, Actinomyces species grow in microscopic or macroscopic clusters, which may reach diameters of up to 34 mm (19). Clusters sometimes exude from soft tissues through sinus tracts, and because of their yellowish appearance, they are commonly referred to as sulfur granules, even though there is no clear evidence that they contain sulfur at all (25). In fact, such clusters or granules consist of a central mass of intertwined branching bacterial laments, held together by an extra-cellular matrix, with the peripheral radiating clubs. Microscopically, the granules give the appearance of rays projecting out from a central mass of laments, which gave origin to the name Actinomyces or ray fungus (19). Granules are very likely to be formed in response to host defenses and can provide the bacteria with protection against phagocytosis or other immunological mechanisms (19, 20). It should be pointed out that not all cases in which granules are observed in the purulent exudate should be indiscriminately diagnosed as actinomycosis. This is because other bacteria also can form aggregates with similar appearance (19). Apparently, this observation is also true for periapical actinomycosis. Sunde et al. (25) reported the occurrence of sulfur granules in nine refractory periapical lesions and found bacteria in seven. Actinomyces species occurred in ve granules and a wide spectrum of other bacterial species was detected in addition to Actinomyces. Many of the sulfur granules were calcied and the source for mineralization may have been the inammatory exudate and/or the activity of extra-radicular bacteria (25). Although sulfur granules have long been considered as suggestive of actinomycosis, that study (25) conrmed that other species can form aggregates that are similar to those formed by Actinomyces species and P. propionicum. Therefore, the mere observation of the presence of sulfur granules does not represent sufcient information for the diagnosis of periapical actinomycosis. It has been suggested that the demonstration of typical ray fungus patterns, or actinomycotic rosettes in tissue sections is sufcient to establish a diagnosis of actinomycosis (26). Although this is really widely accepted, an irrefutable diagnosis is only achieved after the bacterial species involved are properly identied by culture-dependent or -independent approaches.

Actinomyces species
The genus Actinomyces encompasses a heterogeneous group of non-acid fast, non-motile, non-spore forming, obligately anaerobic and facultatively anaerobic, Gram-positive rods. Early classication of Actinomyces was complicated by their histological resemblance to fungi (Actinomyces 5 ray fungus), which occurred due to the radial appearance of laments in the granules found in actinomycotic lesions. Actinomyces cells are 0.41 mm wide, short (1.55 mm long) or longer (5 50 mm long). They can be straight, curved, branched or pleomorphic, and they can occur singly, in pairs, clusters or short chains. Most of the species are facultative anaerobes, while some are obligate anaerobes. Actinomyces species are fermentative, generally utilizing carbohydrates to produce formic, acetic, lactic and succinic acids (20, 27). Recent taxonomic changes have taken place in the genus Actinomyces and new species have been proposed. Strains originally classied as A. israelii serotype II have been designated as a separate species, A. gerencseriae, based on comparisons of 16S rRNA sequences (28). A. gerencseriae is a common but minor component of the microbiota of the healthy gingival crevice. Johnson et al. (28) proposed subdivision of A. naeslundii into three new genospecies: (1) genospecies 1 included A. naeslundii serotype I; (2) genospecies 2

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included A. naeslundii serotypes II and III, and human strains of Actinomyces viscosus; and (3) genospecies 3 comprised Actinomyces WVA 963. A previously unknown bacterium isolated from infected root canals was classied as A. radicidentis based on both phylogenetic and phenotypic evidence (29). Cells of this new species are coccoid, Gram-positive, facultatively anaerobic, non-motile, and catalase positive. Although Actinomyces species have been found to be etiologic agents in infections in diverse body sites, including some forms of actinomycosis, eye infections, abscesses in different sites as well as respiratory, genital and urinary tract infections (30), most species are normal inhabitants of the oral cavity (Table 1). A. odontolyticus and A. naeslundii genospecies 1 and 2 are the primary Actinomyces species in infants mouths as well as in early dental plaque (31, 32). Actinomyces georgiae, A. gerencseriae, A. israelii, A. naeslundii and A. meyeri have been found in gingival crevices of
Table 1. Species of the genus Actinomyces found in humans
Oral A. georgiae A. gerencseriae A. graevenitzii A. israelii A. meyeri A. naeslundii genospecies 1 A. naeslundii genospecies 2 (A. viscosus) A. odontolyticus A. radicidentis Non-oral A. europaeus A. funkei A. houstonensis A. neuii A. radingae A. turicensis A. urogenitalis

periodontally healthy and diseased individuals (33, 34). Actinomyces graevenitzii has been found in infants saliva (31). It has been demonstrated that oral Actinomyces species colonize hard tissues (supra- and sub-gingival plaque) at far higher proportions than soft tissues (33). In general, Actinomyces species are usually isolated from supra-gingival and sub-gingival plaque, tonsils, dentinal and root surface caries, periodontal pockets and infected root canals (33, 3537).

P. propionicum
P. propionicum was formerly assigned to the genus Actinomyces, then transferred to the genus Arachnia and from there to Propionibacterium on the basis of sequence homology of ribosomal RNA (38, 39). Further analysis of its fatty acid pattern supported transfer of this species to the genus Propionibacterium (40). Cells are non-motile and may appear as irregular rods, 0.20.3 mm in diameter and 35 mm in length, which may or may not be branched, often with swollen or clubbed ends. They can also occur as branching laments, 520 mm in length. Occasionally large round cells may be observed (5 mm in diameter). P. propionicum is facultatively anaerobic, but best growth is attained under anaerobic conditions. Propionic and acetic acids are the major end products of the anaerobic fermentation of glucose. CO2 and lesser amounts of lactic and succinic acids are also produced (38). P. propionicum is a normal inhabitant of the human oral cavity, and can be involved in several oral diseases. In addition, this species has been reported to occur in cases of tympanomastoiditis (41), vertebral osteomyelitis (42), epidural abscess (43), lacrimal canaliculitis (44, 45), brain abscess (46), pulmonary infection in patients with hairy cell leukemia (47), and actinomycosis (38, 48). P. propionicum may produce disease clinically indistinguishable from that caused by Actinomyces species (43, 48). Like Actinomyces species, P. propionicum is known to be able to ourish in host tissues for long periods of time without causing symptoms.

Association with primary intraradicular infections

Actinomyces species are normal inhabitants of the oral cavity and their occurrence in endodontic infections is

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thereby not surprising. In fact, they are arguably one of the rst colonizers of the exposed pulp, given their high prevalence in carious dentin (49). In one study (49) Actinomyces species along with species from the genera Eubacterium and Propionibacterium were found to have invaded the pulps of six out of nine teeth with deep dentinal lesions even before pulpal exposure occurred. Primary intra-radicular infections are caused by microorganisms that initially colonize the necrotic pulp tissue (3). Several studies, using different approaches for microbial identication, have reported that Actinomyces species can take part in the microbiota associated with primary intra-radicular infections, irrespective of whether symptoms are present or not (1, 5071) (Table 2). Reported prevalence gures for Actinomyces species can reach up to half of the examined cases (71). Commonly detected species have been A. israelii, A. naeslundii genospecies 1 and 2, A. odontolyticus, A. meyeri, and A. gerencseriae (36, 5071). Siqueira and c Ro as (52) reported a very low prevalence of the recently described A. radicidentis in primary endodontic infections. This species was detected in one tooth associated with chronic apical periodontitis and in another tooth associated with acute apical periodontitis. In general, A. radicidentis was found in 4% of the samples taken from primary endodontic infections. Actinomyces species can be found in the apical segment in about 30% of infected root canals (53). They have also been reported to participate in the microbiota associated with acute periapical abscesses. Sundqvist et al. (54) investigated the microbiota of 72 root canals, 17 of which were associated with periapical abscesses and purulent drainage through the canal. Actinomyces species were found in six of the abscessed teeth, usually in combination with other bacteria, except for one tooth in which the root canal contained only A. israelii and A. naeslundii. Siqueira et al. (51) investigated the prevalence of Actinomyces species in abscessed cases using the checkerboard DNADNA hybridization method and found A. gerencseriae in 15% of the cases, A. israelii in 7% and A. odontolyticus in 4%. In general, the presence of Actinomyces genus was positively associated with abscesses, but no such correlation could be established for any particular species. Khemaleelakul et al. (70) isolated A. naeslundii in 18% and A. odontolyticus in 12% of the cases of abscess/cellulitis of endodontic origin. By using a PCR assay, Xia and Baumgartner (71) found Actinomyces species in 46% of abscesses and 30% of cellulitis cases. Most studies using culture identication procedures have revealed the presence of P. propionicum in primary endodontic infections. The prevalence of such occurrence has been reported to range from 3% to 31% of teeth with apical periodontitis (1, 54, 56, 58, 60, 61, c 65, 66, 69). Recently, Siqueira and Ro as (52) devised a nested PCR assay to detect P. propionicum in endodontic infections associated with different forms of apical periodontitis, and found this species in 36% of the cases (29% of the teeth with chronic periapical lesions, in 50% of the teeth with acute apical periodontitis, and in 37% of the teeth with acute periapical abscesses). Cumulatively, all these ndings suggest that Actinomyces species and P. propionicum have the ability to colonize the necrotic pulp and participate in a mixed microbial consortium that can be involved in causation of different forms of periapical diseases.

Association with post-treatment disease

In addition to being frequently found in teeth with primary endodontic infections, Actinomyces species and P. propionicum have also been found in association with post-treatment disease. They have been reported to occur either in persistent/secondary intra-radicular infection, or as the exclusive etiology of extra-radicular infection, diagnosed as periapical actinomycosis.

Persistent and secondary intra-radicular infection

Secondary intra-radicular infections are caused by microorganisms that were not present in the primary intra-radicular infection, but gained entry into the root canal after some treatment intervention and succeeded in colonizing this environment (3). Breach of asepsis during treatment is one of the major causes of secondary infections. Furthermore, secondary infections can occur after root canals had been lled, and thereby can become a cause of post-treatment disease (72). Persistent intra-radicular infections are caused by microorganisms that have survived the intra-canal antimicrobial procedures associated with root canal therapy. The microorganisms involved in persistent infections can either be members of the primary infection or of a secondary infection (3). Persistent

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Table 2. Data from studies that reported the occurrence of Actinomyces species and Propionibacterium propionicum in primary intra-radicular infections
Study Kantz & Henry (55) Identication method Culture Species A. israelii Actinomyces sp.w Wittgow & Sabiston (56) Culture P. propionicum Actinomyces sp. Sundqvist (1) Culture Actinomyces sp. A. naeslundii P. propionicum Zavistovski et al. (57) Sundqvist et al. (54) Culture Culture A. israelii A. meyeri A. israelii A. odontolyticus A. naeslundii genospecies 2 P. propionicum Baumgartner & Falkler (53) Culture Actinomyces sp. A. naeslundii A. israelii A. naeslundii genospecies 2 Sundqvist (58) Culture A. israelii P. propionicum A. naeslundii A. odontolyticus A. meyeri A. naeslundii genospecies 2 Actinomyces sp. Wasfy et al. (59) Culture A. naeslundii genospecies 2 A. odontolyticus A. israelii A. meyeri Sato et al. (60) Culture P. propionicum A. odontolyticus Debelian et al. (61) Culture P. propionicum Prevalencen 4/16 (25%) 1/16 (6%) 1/32 (3%) 1/32 (3%) 3/18 (17%) 2/18 (11%) 2/18 (11%) 1/10 (10%) 2/22 (9%) 1/22 (5%) 1/22 (5%) 1/22 (5%) 1/22 (5%) 3/10 (30%) 2/10 (20%) 1/10 (10%) 1/10 (10%) 7/65 (11%) 5/65 (8%) 3/65 (5%) 1/65 (2%) 1/65 (2%) 1/65 (2%) 1/65 (2%) 9/78 (12%) 8/78 (10%) 4/78 (5%) 1/78 (1%) 1/6 (17%) 1/6 (17%) 8/26 (31%)

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Table 2. Continued
Study Identication method Species A. israelii A. naeslundii A. meyeri A. odontolyticus Brauner & Conrads (62) Culture Actinomyces sp. A. israelii Weiger et al. (63) Culture A. odontolyticus A. meyeri Actinomyces sp. Gomes et al. (64) Culture A. israelii A. meyeri A. odontolyticus A. naeslundii genospecies 2 Le Goff et al. (65) Culture A. odontolyticus P. propionicum Conrads et al. (50) PCR Actinomyces spp. A. israelii A. naeslundii genospecies 2 Lana et al. (66) Culture A. naeslundii A. meyeri P. propionicum Rolph et al. (67) Culture A. naeslundii A. naeslundii genospecies 2 Siqueira et al. (68) Siqueira et al. (51) PCR A. israelii Prevalencen 5/26 (19%) 2/26 (8%) 1/26 (4%) 1/26 (4%) 5/19 (26%) 1/19 (5%) 1/12 (8%) 1/12 (8%) 1/12 (8%) 4/40 (10%) 4/40 (10%) 4/40 (10%) 1/40 (3%) 3/18 (17%) 1/18 (6%) 5/15 (33%) 3/15 (20%) 1/15 (7%) 3/27 (11%) 2/27 (7%) 1/27 (4%) 2/9 (22%) 1/9 (11%) 2/40 (5%) 4/27 (15%) 2/27 (7%) 1/53 (2%) 11/58 (19%) 6/58 (10%) 3/58 (5%) 2/58 (3%) 3/17 (18%)

Checkerboard DNADNA hybridization A. gerencseriae A. israelii A. odontolyticus

Peters et al. (69)

Culture

A. odontolyticus A. meyeri Actinomyces sp. P. propionicum

Khemaleelakul et al. (70)

Culture

A. naeslundii

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Table 2. Continued
Study Identication method Species A. odontolyticus Actinomyces spp. A. naeslundii genospecies 2 A. israelii A. naeslundii c Siqueira & Ro as (52) Nested PCR P. propionicum A. radicidentis Prevalencen 2/17 (12%) 72/129 (56%) 42/131 (32%) 31/131 (24%) 11/131 (9%) 18/50 (36%) 2/50 (4%)

Xia & Baumgartner (71)

PCR

Number of cases positive for Actinomyces species or P. propionicum/number of cases positive for bacteria. wNot identied to species level.
n

Table 3. Data from studies reporting the occurrence of Actinomyces species and Propionibacterium propionicum in persistent intra-radicular infections
Study Molander et al. (73) Sundqvist et al. (74) Identication method Culture Culture Species Actinomyces sp.w A. israelii P. propionicum Cheung & Ho (78) Hancock et al. (75) Rolph et al. (67) Pinheiro et al. (76) Culture Culture Culture Culture P. propionicum Actinomyces sp. A. israelii A. naeslundii A. odontolyticus A. naeslundii genospecies 2 P. propionicum c Siqueira & Ro as (52) Nested PCR P. propionicum A. radicidentis
n Number of cases positive for Actinomyces species or P. propionicum/number of cases positive for bacteria. wNot identied to species level.

Prevalencen 2/68 (3%) 3/24 (13%) 1/24 (4%) 1/12 (8%) 8/33 (24%) 1/9 (11%) 4/51 (8%) 3/51 (6%) 3/51 (6%) 1/51 (2%) 7/12 (58%) 1/12 (8%)

intra-radicular infection has been deemed to be the most common cause of post-treatment endodontic disease (72). Most of the studies that investigated the microbiota present in the lled root canals of teeth associated with post-treatment apical periodontitis have demonstrated the occurrence of Actinomyces species in 324% of the teeth (67, 7376) (Table 3).

Species reported to be present in these teeth include A. israelii, A. naeslundii genospecies 1 and 2, A. odontolyticus and A. radicidentis (67, 7476). rssen and Sundqvist (36) found Actinomyces Bo species in 10.6% of 235 root canal samples that had positive bacterial cultures. Twenty-ve Actinomyces strains were isolated. Of these, 17 strains were derived

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from the root canals of teeth with necrotic pulps, ve from root-lled teeth, and three from teeth with vital pulps. Twenty-three strains appeared in mixed cultures and could be eliminated by means of conventional endodontic treatment. Two A. israelii strains were in pure cultures from teeth with post-treatment disease. Immunouorescence revealed that the A. israelii strains had become established and had survived in the periapical lesion (13). A. radicidentis was rst isolated in pure culture from two endodontic patients who had shown persistent signs and symptoms after conventional root canal treatment (29, 77). When this species was rst described, it was unknown whether it persisted from a primary endodontic infection despite treatment, or was the cause of secondary infection. Strains of A. radicidentis exhibit relatively high tolerance to saturated calcium hydroxide solution when compared with other bacterial species commonly found in infected root canals (77). Such resistance to calcium hydroxide may explain the persistence of A. radicidentis during endodontic treatment. Indeed, in a recent study (52) using nested PCR, A. radicidentis was detected in one out of 12 (8.3%) root-lled teeth associated with post-treatment disease. P. propionicum has also been isolated from root-lled teeth associated with apical periodontitis, with the reported prevalence ranging from 2% to 8% of the teeth (74, 76, 78) (Table 3). Nonetheless, a recent study (52) using nested PCR identied P. propionicum in over 50% of the root canal samples obtained from teeth associated with post-treatment disease. This was the highest prevalence value reported for this bacterial species in persistent intra-radicular infections and the possible explanation for this nding was the higher sensitivity and accuracy of the method used for identication (52). For a given bacterial species to be established in lled root canals, it has to survive intra-canal antimicrobial procedures or to invade the lled canal after treatment, possibly as a result of coronal leakage (72). Whatever the mechanism, the bacterial species surviving in lled root canals should endure periods of nutrient deprivation (5, 72). How exactly Actinomyces species and P. propionicum survive in root-lled teeth is still unknown, but their isolation from root-lled teeth associated with apical periodontitis suggests that these species can contribute to the etiology of post-treatment disease by participating in a persistent or secondary intra-radicular infection.

Extra-radicular infections periapical actinomycosis

As highlighted above, periapical actinomycosis is a cervical form of human actinomycosis, and comprises an extra-radicular infection that can be independent of the bacteriological status of the root canal of the affected tooth. In periapical actinomycosis, the causative bacteria may invade the periapical tissues and establish an equilibrium with the host without inducing acute inammation with overt symptoms. Such equilibrium can be described as a situation in which neither the bacteria nor the host defense mechanisms win the battle. On the one hand, the bacteria cannot be eliminated by the host defenses as a result of some bacterial strategies to be discussed below. On the other hand, the host succeeds in conning colonies to the inamed periapical tissues, preventing the spread of the infection. However, persistent actinomycotic colonies seem to be sufcient to sustain chronic inammation and the periapical disease process. The majority of periapical actinomycosis cases have been diagnosed based on the presence of sulfur granules and bacterial aggregates containing Grampositive branching rods in histologic sections obtained through apical surgery or tooth extraction. Bacterial aggregates may show central necrosis and club-shaped extension of laments, and are most often surrounded by inammatory cells (Fig. 2). In addition, there are also several studies that have identied Actinomyces species and P. propionicum in teeth associated with post-treatment disease (711, 79, 80) (Table 4). In most of these studies, however, there was no clear diagnosis established of periapical actinomycosis. It has been suggested that periapical actinomycosis may be more prevalent than previously believed (14, 81). Reviewing the literature, Sakellariou (81) found only 45 cases on record, including the one described in that report. Most communications were in the form of case reports (22, 8186). As of 1996, several more cases (8789) have been reported. Weir and Buck (90) reported on a case of periapical actinomycosis and reviewed a series of 20 cases, including their own. In their review, the average age of the patients was 27.5 years, ranging from 10 to 64 years. Fifty eight percent of the patients were male and the teeth most commonly affected were the maxillary incisors. Many of these teeth were undergoing endodontic treatment or had treatment completed.

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Fig. 2. Bacterial aggregate in an epithelialized periapical lesion, suggestive of actinomycosis. Inset, higher magnication of the actinomycotic aggregate, which is surrounded by inammatory cells (courtesy of Drs D. Ricucci and E.A. Pascon).

gren et al. (12) reported on a tooth with postSjo treatment disease, where the periapical tissue harbored P. propionicum. This tooth healed completely after apical surgery. Happonen et al. (15) used immunocytochemical methods for the demonstration of Actinomyces species and P. propionicum in seven routine periapical specimens. Co-infection of P. propionicum and Actinomyces species was found in four specimens, while P. propionicum occurred in ve of the seven specimens. In another study, Happonen (14) reported on 16 surgically treated cases that had immunocytochemically veried periapical actinomycosis. A. israelii was detected in 13 biopsy specimens, P. propionicum in 10, and A. naeslundii in six. More than one of these species was present in nine specimens. Nair et al. (88) described three cases of ciliated epithelium-lined periapical cysts and reported the

presence of typical ray-fungus actinomycotic colonies in the lumen of one of the lesions. Because the lesion happened to be a periapical pocket cyst, the authors suggested that Actinomyces cells could have advanced from the infected root canal directly into the lumen of the cyst. Rush et al. (89) reported several cases of actinomycosis, including the periapical form of the disease. The study was based on records of a diagnostic pathology service. Fifty-six percent of the submitting clinicians indicated a clinical impression of non-specic periapical granuloma or cyst. Patient ages ranged from 13 to 86 years, with an average age of 59.8 years. The gender of the patients was almost evenly distributed between males and females. Distribution of cases per race was reported as 73% Caucasian, 7% Hispanic, 3% African American and 17% unstated. Biopsy specimens were more common in the maxilla (53% of the cases).

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Table 4. Data from studies reporting the occurrence of Actinomyces species and Propionibacterium propionicum or evidence of actinomycotic colonies in periapical lesions
Study Tronstad et al. (7) Identication method Culture Species A. israelii A. naeslundii genospecies 2 Iwu et al. (9) Culture A. naeslundii A. naeslundii genospecies 2 Wayman et al. (79) Culture A. odontolyticus A. meyeri Abou-Rass & Bogen (80) Gatti et al. (10) Culture Checkerboard DNA-DNA hybridization Actinomyces sp.w A. naeslundii genospecies 2 Prevalencen 1/8 (13%) 1/8 (13%) 5/16 (31%) 2/16 (13%) 1/24 (4%) 1/24 (4%) 3/13 (23%) 16/20 (80% IS)

A. naeslundii genospecies 2 A. odontolyicus Sunde et al. (8) Culture Actinomyces sp. A. naeslundii A. odontolyticus P. propionicum Sunde et al. (11) Checkerboard DNADNA hybridization A. naeslundii

16/16 (100% SM) 5/16 (31% SM) 2/15 (13% IS) 1/15 (7% IS) 1/15 (7% IS) 1/15 (7% SM) 16/17 (94% IS)

A. naeslundii A. israelii A. israelii A. naeslundii genospecies 2 A. naeslundii genospecies 2 A. odontolyticus A. odontolyticus A. gerencseriae A. gerencseriae Nair & Schroeder (26) Light and transmission electron microscopy Culture Actinomycotic colonies

4/17 (24% SM) 16/17 (94% IS) 14/17 (82% SM) 16/17 (94% IS) 14/17 (82% SM) 12/17 (71% IS) 9/17 (53% SM) 10/17 (59% IS) 7/17 (41% SM) 2/45 (4%)

Sunde et al. (25)

A. naeslundii genospecies 2 A. israelii A. naeslundii A. meyeri P. propionicum

7/36 (19%) 6/36 (17%) 5/36 (14%) 3/36 (8%) 2/36 (6%)

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Table 4. Continued
Study Identication method Species Actinomyces sp. Hirshberg et al. (87)
n

Prevalencen 1/36 (3%) 17/963 (1.8%)

Light microscopy

Actinomycotic colonies

Number of cases positive for Actinomyces species, P. propionicum or actinomycotic colonies/number of cases evaluated. wNot identied to species level. IS, samples taken when an intra-sulcular incision was made; SM, samples taken when a sub-marginal incision was made.

While most cases of periapical actinomycosis have been reported as isolated cases, data regarding the actual frequency of periapical actinomycosis among periapical lesions is still limited to a few studies (6, 26, 87). Nair & Schroeder (26) examined 45 periapical specimens using light and transmission electron microscopy and reported the occurrence of typical actinomycotic colonies in two lesions diagnosed as periapical granuloma. In one specimen the colonies were restricted to the apical root canal, while in the other a typical actinomycotic colony was observed within the body of the lesion. Polymorphonuclear leukocytes were seen surrounding the colonies. m et al. (6) followed the outcome of endoBystro dontic treatment in 79 teeth with apical periodontitis for 25 years post-treatment. Only ve teeth showed little or no improvement after treatment. Of these, two teeth were diagnosed as periapical actinomycosis. Histological examination of periapical specimen obtained from one of the two teeth revealed a periapical cyst with A. israelii and P. propionicum present. The other tooth with periapical actinomycosis was clinically diagnosed as a periapical abscess with A. israelii present. Hirshberg et al. (87) evaluated the incidence and clinical outcome of lesions histologically diagnosed as periapical actinomycosis. The study included 963 periapical biopsy specimens submitted for histologic examination and the diagnosis of periapical actinomycosis was based on the presence of typical branching colonies of lamentous bacteria staining positive for periodic acid Schiff and Gram stain. They reported the occurrence of actinomycotic colonies in 17 (1.8%) of the examined lesions. The maxilla was the most frequently involved site (11 cases, 65%), with equal distribution in the anterior and posterior areas. Males were predominat (11 cases, 65%). Radiographi-

cally, most cases presented as radiolucent lesions with well-dened borders. Of the cases diagnosed as periapical actinomycosis, four lesions (24%) were true periapical cysts and 11 (65%) were epithelialized granulomas. One case was diagnosed as residual cyst and another as periapical granuloma. The actinomycotic colonies presented as isolated masses of lamentous bacteria with a central area of necrosis and radiating laments. Most colonies were surrounded by an inammatory inltrate composed of polymorphonuclear neutrophils, lymphocytes and plasma cells. On the basis of these few studies on the prevalence of periapical actinomycosis, one can realize that the percentage of apical periodontitis lesions infected by Actinomyces species and/or P. propionicum is low. However, their importance should not be underrated since periapical actinomycosis is usually refractory to conventional endodontic procedures and as such, it can be one of the etiologies of post-treatment disease. The bacterial source for periapical actinomycosis is conceivably the intra-radicular infection. Since Actinomyces species and P. propionicum are more prevalent in intra-radicular than in extra-radicular infections, it can be assumed that only a small percentage of the cases in which these species are in the root canal evolve into an extra-radicular infection. Situations that can permit these bacteria to reach periapical tissues and establish an extra-radicular infection are likely to be the following: (1) apical extrusion of debris during root canal instrumentation; (2) direct advance from the infected root canal into the lumen of the pocket cysts; or (3) previous participation in acute periapical abscesses followed by persistence after the acute response subsides. Moreover, the virulence of the involved strains and the host resistance to the infection appear to be important factors dictating whether an extra-radicular infection will develop or not.

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Mechanisms of pathogenicity
Actinomyces species have a low pathogenicity (potential to produce disease) in their normal habitats. However, when normal mucosal barriers are disrupted by trauma, surgery or preceding infection, these bacteria can establish a chronic, pus-forming infection that can spread unchecked through host tissues. Under specic circumstances, an acute abscess can occur after invasion of the host tissues by these bacteria. Several studies have demonstrated the pathogenic potential of Actinomyces species and P. propionicum in animal models. Brown and von Lichtenberg (91) evaluated the pathogenicity of A. israelii in mice and observed a pattern of infection that was similar to the course of other chronic infections. Initially, there was an acute phase of growth and expansion of the lesions, followed by a static period during which some animals aborted the infection. In most cases, animals then entered a prolonged chronic phase characterized by a balance between aggression and defense, with slow growth of the lesions. Behbehani and Jordan (92) compared the pathogenicity of different species of Actinomyces and of P. propionicum using a mouse model. They reported that intraperitoneal injection of strains of A. israelii, A. naeslundii genospecies 1 and 2, and P. propionicum caused numerous abscesses in the intestine, mesentery, liver, and at the site of injection. A. odontolyticus did not cause any lesions. Differences in pathogenicity among Actinomyces species were much less pronounced during the initial acute stage of the infection. A. naeslundii genospecies 1 and 2 (A. viscosus) produced acute lesions that resolved after a few weeks. Abscesses caused by rough strains of A. israelii and P. propionicum persisted and led to a slowly progressive chronic infection. The other species apparently lacked the virulence to survive the transitional phase of the infection from acute to chronic stage. In an experimental study in mice, Siqueira et al. (93) reported that a strain of A. naeslundii induced abscess formation when inoculated subcutaneously in pure culture or in association with Prevotella intermedia or with Prevotella nigrescens. Buchanan & Pine (94) injected 16 mice intraperitoneally with two strains of P. propionicum and observed that abscesses developed in all animals. Georg & Coleman (95) inoculated mice with two strains of P. propionicum and reported that lesions resembling those produced by A. israelii occurred in all animals. Sumita et al. (96) packed A.

israelii cells in alginate gel particles and injected them intraperitoneally into BALB/c mice. Actinomycotic lesions were induced efciently in nine out of 12 mice after 3 or 9 weeks. Serum IgG levels against A. israelii were signicantly elevated, indicating the activation of the animals humoral immunological response. The researchers suggested that the bacteria might have become resistant to phagocytosis. Co-infection with other bacteria can play a role in Actinomyces pathogenicity, by enhancing their virulence. In a study of experimental mixed infections (97), A. israelii was a component of mixtures of bacteria that produced abscesses but it was not essential for abscess formation. In another study (98), A. meyeri grew better in mixed cultures than alone. In addition, its presence stimulated growth of non-protein-cleaving oral bacteria, most likely due to the ability of A. meyeri to degrade serum proteins and thus to provide peptides for the growth of amino acid-fermenting bacteria that cannot cleave intact proteins by themselves. Their ndings indicated that protein-degrading A. meyeri might play an important role in mixed oral infections, by providing nutrients for growth of other species present in the bacterial consortium. Although the exact mechanism by which Actinomyces species exert their pathogenicity has not been totally claried, there is some evidence that can help explain infections caused by these microorganisms. Most Actinomyces species are of low virulence and their mere invasion into tissues does not usually sufce to establish an infection. However, necrotic pulps do not offer resistance to invasion by microorganisms, except for selective pressures exerted by the environmental conditions, which are arguably adequate for most Actinomyces species. Some Actinomyces species have mbrial structures that may play a role in bacterial coaggregation within the root canal and can be important for bacterial survival in the ecosystem. In addition, mbriae would enable Actinomyces cells to adhere to the root canal wall and to dentinal debris forced out through the apical foramen during treatment, and to cling to other bacteria or host cells as they advance into the periapical tissues (99). Actinomyces species usually have a hydrophobic cell surface character, which facilitates uptake by leukocytes. Figdor and Davies (100) investigated the ultrastructure of A. israelii by electron microscopy and reported that strains can have hair-like mbriae protruding through a thick surface coat. Thin sectioning revealed a Gram-positive cell wall surrounded by a

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fuzzy outer coat. They suggest that both the mbriaelike structures and the matrix of the outer coat surrounding the bacteria can help the cells to aggregate into cohesive colonies of tangled laments. Moreover, strains associated with post-treatment disease were demonstrated to grow as intertwining laments, forming granulae within host tissues (99). It is believed that the ability to form branching, lamentous microcolonies may be critical for the establishment of these bacteria in the tissue. The size of bacterial aggregates is important for phagocytosis to occur. The presence of a hyaloid or hyaline layer in actinomycotic colonies may provide protection against host defenses, and it may also serve to embed the lamentous and branching microorganisms in a cohesive mass (99). Thus, the bacteria appear to be able to evade collectively host defenses by building in host tissues cohesive colonies consisting of large numbers of branching and lamentous bacteria enmeshed in a matrix of protein polysaccharide complex (99). Actinomycotic colonies may live in equilibrium with host tissues without necessarily inducing an acute response, but rather maintaining a chronic periapical inammation. Very high numbers of Actinomyces cells are usually needed to form persistent infections (92). The low pathogenicity of these microorganisms and the consequent minimal host response may be the reasons for the perpetuation of the chronic periapical lesion. Although P. propionicum is also recognized to be pathogenic, its mechanisms of pathogenicity have yet to be claried. Figdor et al. (99) addressed the question as to what allows P. propionicum to cause extraradicular infections by evaluating the ability of this bacterium to induce experimental infections in guineapigs, its surface properties, as well as in vitro phagocytosis and intracellular killing by polymorphonuclear leukocytes. Their results showed that P. propionicum declined in number during the entire period of infection and did not form colonies. P. propionicum cells were hydrophobic, readily phagocytosed and efciently killed by leukocytes. The authors were not able to draw signicant conclusions about the mechanisms of pathogenicity of this bacterial species. swelling, induration of soft tissues, multiple abscesses and draining sinus tracts. If the clinician faces such clinical picture, he/she should suspect actinomycosis and look for the laboratory conrmation of the disease in the pus collected from abscesses (81). However, periapical actinomycosis is rather different as both the clinical and radiographic manifestations are usually indistinguishable from common apical periodontitis. The occurrence of multiple sinus tracts may suggest but is not a prerequisite for diagnosis of periapical actinomycosis, since such has not been associated with many reported cases (12, 23, 87). Some cases can present a painless swelling (85). The mere occurrence of persistent exudation and/or symptomatology, associated or not with persistent sinus tract, is not exclusively indicative of periapical actinomycosis. Such clinical picture can be caused by many etiological factors, of which a persistent intra-radicular infection (not necessarily containing Actinomyces species) is arguably the most common one. Therefore, diagnosis is usually achieved only after surgical removal of the lesion, followed by histopathological and microbiological examination of the specimen (81). Most forms of actinomycosis are usually treated with systemic antibiotic therapy. Studies (20, 101) have demonstrated that Actinomyces species and P. propionicum are commonly susceptible to the most widely used antibiotics. Actinomyces species are usually highly sensitive to the b-lactam antibiotics and have a high-tomoderate sensitivity to tetracyclines, macrolides, lincomycins and vancomycin (20). They are generally resistant to aminoglycosides and metronidazole (20). Holmberg et al. (101) tested the susceptibility to several antibiotics of 46 reference strains and clinical isolates of A. israelli and eight strains of P. propionicum, using the agar dilution method in vitro. All strains were susceptible to benzylpenicillin. Erythromycin, tetracycline, clindamycin and lincomycin possessed in vitro activity at concentrations readily attainable in serum. In vitro resistance to metronidazole was observed. Prolonged systemic antibiotic therapy has been the treatment of choice for all clinical forms of the disease (21), except for periapical actinomycosis. As far as we are aware, the vast majority of the reported cases of periapical actinomycosis have been successfully treated either by apical surgery or by extraction of the affected tooth. In several reported cases, no systemic antibiotic therapy was prescribed and healing was uneventful (6, 12, 14). Happonen (14) reported only one clear case of

Clinical manifestation and treatment

The typical clinical manifestation of the cervicofacial actinomycosis is characterized by the presence of

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persistent disease after apical surgery had been performed on 16 teeth with periapical actinomycosis. It was concluded that a prolonged administration of antibiotics, as generally recommended for actinomycotic infections elsewhere in the body, might be unnecessary for the treatment of periapical actinomycosis (14). In reality, there appears to be no need for prolonged use of systemic antibiotics provided the infected periapical lesion is entirely removed during surgery. The use of systemically administered antibiotics alone to treat periapical actinomycosis does not appear to be an effective alternative to surgical procedures. First, as already mentioned, the accurate diagnosis of periapical actinomycosis is only possible after surgical removal of the lesion. A question therefore arises when should one consider the prescription of antibiotics? Because the incidence of periapical actinomycosis is rather low when compared with other forms of apical periodontitis (where the primary cause of disease is usually intra-radicular infection, not affected by systemic antibiotics), and taking into account that periapical actinomycosis is virtually impossible to diagnose based only on clinical and radiographic ndings, prescription of antibiotics in all suspected cases is not warranted, and would not guarantee healing. In addition, such indiscriminate use would enhance the deleterious effects of the abuse of antibiotics, such as selection of resistant microorganisms. Finally, even if periapical actinomycosis is suspected, there is no evidence as to which antibiotic agent, dosage, and duration of therapy is effective (if ever) in treating this disease. apical surgery, including thorough curettage of the periapical inammatory lesion.

References
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Conclusions
Actinomyces species and P. propionicum can be found in primary, persistent and secondary intra-radicular infections, as constituents of a polymicrobial consortium. With regard to extra-radicular infection, however, Actinomyces species and P. propionicum can be the exclusive pathogens sustaining the post-treatment disease process associated with root-lled teeth. The latter may be characterized as an independent pathologic entity termed periapical actinomycosis. The prevalence of periapical actinomycosis appears to be low; therefore, it is one of several etiological factors of post-treatment disease. Once periapical actinomycosis is established, it can only be successfully treated by

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51.

52.

53.

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55.

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58.

59.

60.

61.

62.

63.

64.

65.

66.

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