Transmission and Persistence of Actinobacillus Actinomycetemcomitans in Twins with Advanced Periodontitis

By Peter A. Russo, DDS; Hessam Nowzari, DDS; and Jorgen Slots, DDS, PhD

The arbitrarily primed polymerase chain reaction technique (AP-PCR) was used to fingerprint Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis isolates in 44-year-old African American male and female twins who had not lived in the same household for 26 years. Both twins exhibited severe loss of periodontal attachment on several maxillary and mandibular teeth. All isolates of A. actinomycetemcomitans yielded the same AP-PCR banding pattern, whereas the P. gingivalis isolates from each twin showed different AP-PCR profiles. The finding of the same amplitype of A. actinomycetemcomitans in both twins suggests a single source of the organism and possibly a persistence of the organism in each twin for at least 26 years.

Article copyright 1998 Journal of the California Dental Association.
Photographs copyright of the authors.

Actinobacillus actinomycetemcomitans is a gram-negative, capnophilic coccobacillus that resides in supra- and subgingival plaque, tongue dorsum, buccal mucosa and saliva.1,2 A. actinomycetemcomitans can be isolated from both periodontally healthy and diseased sites and has been implicated as a major pathogen in prepubertal,3 localized juvenile4,5 and advanced adult periodontitis.6,7

Recent studies show that intrafamilial transmission of A. actinomycetemcomitans occurs with high frequency in families with localized juvenile periodontitis patients.8-10 Preus and colleagues11 demonstrated that parents with A. actinomycetemcomitans-associated adult periodontitis transmitted the bacterium to one of their children 30 percent of the time. Also, all infected children revealed the same genetic type of A. actinomycetemcomitans strain as did one of their parents,9,10 suggesting that the parents were the source of the infection. An intriguing means of preventing A. actinomycetemcomitans-associated periodontitis may derive from intervening in the organism's transmission from person to person.9,12,13

Using genetic fingerprinting, the present study showed that identical isolates of A. actinomycetemcomitans were recovered from a pair of dizygotic twins who had not lived together for 26 years and who both demonstrated advanced periodontitis. The findings suggest that A. actinomycetemcomitans can survive for a considerable time in subgingival plaque.

Patient Descriptions

A pair of 44-year-old African American male and female twins were referred for treatment of periodontitis to the Advanced Periodontics Clinic at the University of Southern California. Neither patient had received treatment for periodontitis. The male patient's medical history was noncontributory. The female had a history of hypertension controlled by medication and diet. Both patients were of low socioeconomic status. They had lived in the same household for the first 18 years of their lives, but had lived separately since 1968 when the male patient was drafted for military service in Vietnam, where he remained until 1972. Upon his return, he resided in various parts of the Southern United States until about 10 years ago when he returned to Los Angeles. The female remained in Southern California and had no personal contact with her brother until his return. Since then, personal visits had taken place on only rare occasions.

Information on the dental history of the family is limited. Both parents were edentulous when they died. They had had their remaining teeth extracted when they were in their 40s, for reasons their dentists called "pyorrhea." No other family members were available for clinical or microbiological examinations.

In the anterior maxillary and mandibular teeth, both patients demonstrated loss of periodontal attachment with particularly severe damage in teeth Nos. 6, 8 and 10. In the female, suppuration was present around teeth Nos. 6 and 8; and class II mobility (Miller Classification) was noted on teeth Nos. 7, 8 and 13. In the male, class I mobility was present on tooth No. 6. In the molar areas, both patients showed several deep periodontal pockets. The female revealed severe periodontitis around teeth Nos. 2, 3, 15, 18 and 30. The male presented with severe periodontitis around teeth Nos. 2, 15, 18 and 31.

Microbiological Procedures

Pockets with the deepest probing depth measurements in each quadrant were selected for microbiologic investigations. The sites tested for the female patient were teeth No. 2 distobuccal (probing depth = 7 mm), No. 15 mesiobuccal (PD = 9 mm), No. 18 mesiobuccal (PD = 9 mm), and No. 30 distobuccal (PD = 9 mm). The sites tested for the male patient were teeth No. 2 distobuccal (PD = 7 mm), No. 15 mesiobuccal (PD = 7 mm), No. 18 mesiobuccal (PD = 7 mm), and No. 31 distobuccal (PD = 8 mm).

After supragingival plaque was removed and each site thoroughly dried with cotton gauze, three sterile paper points were used to obtain subgingival plaque from each study site. The paper points were inserted to the bottom of the pocket, retained for 10 seconds, and then transferred to a 2 ml vial containing VMGA III anaerobic transport medium (Moller 1966).
The microbiological samples were processed within 60 minutes of collection. Microorganisms were mechanically dispersed with a Vortex mixer at the maximal setting for 45 seconds, and the microbial suspension was tenfold serially diluted in Moller's VMG I anaerobic dispersion solution.14 With a sterile bent glass rod, 0.1 ml aliquots from appropriate dilutions were plated onto nonselective 4.3 percent brucella agar (BBL Microbiology Systems, Cockeysville, Md.) supplemented with 0.3 percent Bactoagar, 5 percent defibrinated sheep blood, 0.2 percent hemolyzed sheep red blood cells, 0.0005 percent hemin, and 0.00005 percent menadione, and onto TSBV (Trypticase soy agar-Serum- Bacitracin-Vancomycin) selective medium for A. actinomycetemcomitans, enteric gram- negative rods, pseudomonads, and yeasts.15 The nonselective blood agar was incubated at 35 degrees Celsius in a Coy anaerobic chamber (Coy Laboratory Products, Ann Arbor, Mich.) containing 85 percent N2, 10 percent H2, and 5 percent CO2 for 10 days. TSBV medium was incubated in 10 percent CO2 in air at 35 degrees Celsius for four days. Presumptive identification was performed according to methods described by Slots16 and by use of commercial micromethod systems.

Microorganisms examined included A. actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, Bacteroides forsythus, Campylobacter rectus, Fusobacterium species, Peptostreptococcus micros, Capnocytophaga species, beta-hemolytic Steptococcus species, Staphylococcus species, Enterobacteriaceae species and Candida species.
Following identification of bacterial species, A. actinomycetemcomitans and P. gingivalis isolates from each patient were analyzed using the arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting technique. PCR is a cyclical, enzymatic reaction in which DNA strands are copied and used as templates for subsequent cycles, resulting in an exponential increases of the desired target(s). The resulting amplicons can be visualized in agarose gel electrophoresis. The AP-PCR study was carried out as described previously.17,18 The primers used for both the A. actinomycetemcomitans and the P. gingivalis isolates were 5'AGTCAGCCAC 3' (primer OPA-3) and 5'AATCGGGCTG 3'(primer OPA-4).

Results

Figures 1 and 2 demonstrate the severity of the radiographic bone loss in the female and male patients, respectively.

Figure 1. A radiographic representation of the bone loss in the mandibular left posterior sextant in the female patient. Severe vertical radiographic bone loss is demonstrated.

Figure 2. A radiographic representation of the bone loss in the maxillary left posterior sextant in the male patient. Moderately severe radiographic bone loss with molar furcation involvement is demonstrated.

Table 1 shows the cultivable subgingival microbiota of the two study patients. Both individuals harbored subgingival A. actinomycetemcomitans, P. gingivalis, B. forsythus, Fusobacterium species, P. micros, and Capnocytophaga species.

Table 1 Subgingival microorganisms in four deep pockets in each of two dizygotic twins with advanced periodontitis
Species	Percent of cultivable microorganisms
	Female patient	Male patient
A. actinomycetemcomitans	18.2	0.004
P. gingivalis	36.4	22.2
B. forsythus	1.7	0.6
Capnocytophaga spp.	1.0	0.6
Fusobacterium spp.	18.2	6.9
P. micros	9.1	4.1
P. intermedia	0.0	11.1

Figure 3. AP-PCR profile of A. actinomycetemcomitans for both patients. Lanes 1 and 5: A. actinomycetemcomitans primers. Lane 2: profile of male isolates using OPA 3 primer. Lane 3: profile of male isolates using OPA 4 primer. Lanes 4 and 8 are molecular size markers. Lane 6: profile of female isolates using OPA 3 primer. Lane 7: profile of female isolates using OPA 4 primer.

Figure 3 demonstrates the AP-PCR analysis of A. actinomycetemcomitans isolates from both study subjects. The banding patterns in lanes 2 and 3 for the male patient were identical to the banding patterns in lanes 6 and 7 for the female patient, using the two PCR primers. The AP-PCR analysis for P. gingivalis showed different nucleotide banding patterns for strains obtained from the twins.

Discussion

The present AP-PCR analysis showed that two fraternal twins harbored identical isolates of A. actinomycetemcomitans. Both individuals had probably received the A. actinomycetemcomitans strain from the same source, most likely one of the parents, and had probably been infected by this bacterium for more than 26 years.

This may be the first report to lend support to the notion of decades of longevity of an oral A. actinomycetemcomitans colonization. Asikainen and colleagues19 presented microbiological evidence of at least two years' A. actinomycetemcomitans survival in teenagers. Previously, Preus and colleagues9 suggested that one predominant genotype of A. actinomycetemcomitans could persist for 13 years in the subgingival plaque of a patient with established periodontal disease. The inability of mechanical therapy to completely eliminate A. actinomycetemcomitans from the periodontal pocket, and the organism's colonization of buccal mucosa and tongue dorsum contribute to a prolonged period of colonization.6,13,15 Although this report cannot indisputably determine the origin of the A. actinomycetemcomitans, reports by Preus and colleagues9 and Asikainen and colleagues10 suggest a vertical intrafamilial transmission of the organism, most likely occurring from the mother during childhood. It is also of interest that A. actinomycetemcomitans seems to have undergone a multitude of generations over many years within the periodontal pocket of each individual without changing the AP-PCR profile.

The possibility of a transmission of A. actinomycetemcomitans during recent adult life of our study patients was not supported by these twins' social history. Since 1968, when the male twin was drafted for military service, these individuals had rarely had contact with each other until recently. Clinical and radiographic evidence of each patient's periodontal disease status indicates a history of periodontal breakdown beginning earlier in life, perhaps in adolescence. The pattern of vertical and horizontal periodontal destruction is consistent with the diagnosis of post-juvenile periodontitis and an involvement of A. actinomycetemcomitans, and possibly of P. gingivalis in adolescence and early childhood. Also, Preus and colleagues9 provided evidence for the improbability of a transmission of A. actinomycetemcomitans between adults who already have an established periodontal A. actinomycetemcomitans infection. These data, therefore, support the notion of a prolonged A. actinomycetemcomitans colonization in some individuals. If so, it seems that the dentist cannot rely upon the host defense to clear the organism but may have to perform active intervention directed against the species. In periodontitis patients for whom antimicrobial therapy directed against A. actinomycetemcomitans is indicated, a systemic antibiotic therapy of amoxicillin-metronidazole (250 mg/TID/eight days of each drug) combined with thorough subgingival scaling and root planing constitutes an effective regime.20

Conclusion

This paper reports a case of fraternal twins who have been infected with an identical strain of A. actinomycetemcomitans and who may have harbored the bacterium in subgingival plaque for more than 26 years. A prolonged subgingival colonization of A. actinomycetemcomitans may contribute to severe periodontitis, as implied in several previous studies. The combination of subgingival A. actinomycetemcomitans and P. gingivalis may have been significant in the periodontal destruction observed in the twins studied.

Authors

Peter A. Russo, DDS, is a clinical assistant professor at the University of Southern California School of Dentistry.
Hessam Nowzari, DDS, is director of advanced periodontics at the USC School of Dentistry.
Jorgen Slots, DDS, PhD, is chairperson of periodontology and associate dean for research at the USC School of Dentistry.

References

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To request a printed copy of this article, please contact/Peter A. Russo, DDS, 18800 Main St., Suite 201, Huntington Beach, CA 92648.