Genetic Heterogeneity of Porphyromonas (Bacteroides) gingivalis by Genomic DNA Fingerprinting B.G. LOOS, D. MAYRAND', R.J. GENCO, and D.P. DICKINSON2 Department of Oral Biology, School of Dental Medicine, State University of New York at Buffalo, New York 14214; and 'Groupe de Recherche en Ecologie Buccale, Departement de Biochimie, FacultW des Sciences et de Genie, Universitd Laval, Quebec City, Quebec, Canada GJK 7P4

This study describes the use of total genomic DNA fingerprinting with the use of restriction endonucleases to characterize clinical isolates of Porphyromonas girgivalis (Bacteroides gingivalis) obtained from patients with periodontitis or with rootcanal infections. The majority of independent isolates had a unique DNA fingerprint, indicating extensive genetic heterogeneity within this species. Twenty-nine distinct DNA fingerprints were found among the 33 isolates investigated. This is in contrast to biotyping and serotyping, where only one type and three types, respectively, have been reported. The observed heterogeneity indicates that DNA fingerprinting is a sensitive measure of genetic dissimilarity between P. gingivalis isolates and is able to characterize individual isolates. These results have ecological implications, indicating that there is considerable natural diversity in the global population of P. gingivalis, and that there are likely to be relatively large numbers of genetically distinct clonal lines. Furthermore, DNA fingerprinting is a sensitive and powerful tool for longitudinal and cross-sectional epidemiological studies. This technique provides far greater discrimination between isolates than either biotyping or serotyping, and will be most helpful in, for example, the analysis of distribution of clonal lines within one periodontal patient, or the analysis of the transmission to and turnover of strain populations within a patient population, since the probability of two strains with the same DNA fingerprint being found by chance is small. J Dent Res 69(8):1488-1493, August, 1990

Introduction. Gram-negative anaerobic micro-organisms comprise a significant proportion of the oral microflora. Several such species of bacteria have been implicated as pathogenic and causative agents for periodontitis (Moore et al., 1982; Tanner et al., 1984; Slots et al., 1986; Dzink et al., 1988). Periodontal lesions can be chronic (quiescent), with periods of disease activity (Goodson et al., 1982; Haffajee et al., 1983). In a recent study, Dzink et al. (1988) compared the predominant cultivable Gram-negative microflora from active periodontal lesions with the flora of quiescent sites. Porphyromonas gingivalis-formerly Bacteroides gingivalis (Shah and Collins, 1988)-Bacteroides intennedius, Fusobacterium nucleatum, Bacteroides forsythus, and Wolinella recta were the predominant species found to be significantly elevated in the active sites, compared with quiescent sites. P. gingivalis has been implicated as a periodontal pathogen

(reviewed by Mayrand and Holt, 1988; Slots and Listgarten, 1988; van Winkelhoff et al., 1988). It is isolated in high numbers from periodontal lesions, and its proportion of the microflora seems to be increased in heavily inflamed subgingival lesions (White and Mayrand, 1981; Zambon et al., 1981; Tanner et al., 1984; Loesche et al., 1985; Slots et al., 1986; Dzink et al., 1988). P. gingivalis is rarely found in subgingival sites in periodontally healthy humans or in patients with gingivitis (Christersson et al., 1989). Consistent with these observations, periodontal patients demonstrate elevated serum antibody levels to P. gingivalis (Mouton et al., 1981; Tew et al., 1985; Ebersole et al., 1986). Furthermore, P. gingivalis can sometimes be isolated from endodontic infections (van Winkelhoff et al., 1985; Haapasalo et al., 1986). Our understanding of the role that P. gingivalis plays in periodontal disease and in root-canal infections would be furthered if individual isolates could be tracked through the oral ecological niches in both health and disease, and if their transmission from individual to individual could be followed. Ecological and epidemiological studies of P. gingivalis are hampered by the difficulty of identification of individual isolates. Currently, P. gingivalis isolates are characterized by biotyping or by serotyping. Only two biotypes, based on catalase activity, have been identified (Laliberte and Mayrand, 1983; Parent et al., 1986), and just three serotypes have been reported (Fisher et al., 1987). Therefore, the probability of any two unrelated isolates having identical biotypes and serotypes is high. Since genetic diversity among isolates is a reflection of sequence differences in their chromosomes, total genomic DNA fingerprinting of P. gingivalis isolates with the use of restriction endonucleases could provide a sensitive method for characterization of isolates. This relatively new technique, sometimes referred to as restriction endonuclease analysis (REA), is now widely used for characterization of individual isolates and for investigation of the epidemiology of bacteria and yeast, and appears to be a powerful tool in the study of microbial transmission (Langenberg et al., 1986; Scherer and Stevens, 1987; Wren and Tabaqchali, 1987; Dickinson et al., 1988; Grothues et al., 1988; Caufield and Walker, 1989; Denning et al., 1989; Kulkarni et al., 1989; Loos et al., 1989b; and reviewed by Wachsmuth, 1986). The present study was undertaken to fingerprint individual isolates of P. gingivalis by inspection for restriction site polymorphisms. We found extensive heterogeneity among the isolates of P. gingivalis studied. Furthermore, we tested the stability and reproducibility of DNA fingerprinting after multiple laboratory transfers, and we were able to track laboratory strains.

Materials and methods. Received for publication December 15, 1989 Accepted for publication March 27, 1990 2Present address: Department of Biological Chemistry, University of Texas Health Science Center, Dental Branch, Houston, Texas 77025 This work was supported by USPHS grants DE 08240, DE 04898, and DE 07034 from the National Institute of Dental Research and by the Medical Research Council of Canada. 1488

Isolates and growth conditions. -Thirty-three isolates of Porphyromonas gingivalis, generously supplied from different geographical areas, were included in this study. Origins of the isolates and clinical diagnoses are summarized in Table 1. Isolates were obtained from individual dental patients either with periodontitis or with infected root canals. Isolates were

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TABLE 1

CLINICAL CONDITIONS AND SAMPLE SITES, GEOGRAPHIC LOCATIONS, AND SOURCES OF ISOLATES OF P. gingivalis Siteb City and Country Diagnosisa Sourcec Isolate sinus tract chron. inf. root canal Herweijer, J.A. Buffalo, NY 4ZR6-13 Zambon, J.J. Buffalo, NY periodontitis (IDDM) subgingival 9-14K-1 Wolff, L. severe periodontitis Minneapolis, MN subgingival 17-5 Quebec City, Canada Mayrand, D. adv. adult periodontitis subgingival 19A4 severe periodontitis Wolff, L. Minneapolis, MN subgingival 34-4 Carlsson, J. chronic periodontitis Umea, Sweden subgingival 295-1 SUNYAB collection Boston, MA 381 loc. chron. periodontitis subgingival Edwardsson, S. Malm6, Sweden 817H periodontitis subgingival SUNYAB collection unknown subgingival 2561 Buffalo, NY Pima Indian Res., AZ Zambon, J.J. periodontitis (NIDDM) subgingival A7A1-28 van Winkelhoff, A.J. Buffalo, NY AJW1 periodontitis subgingival van Winkelhoff, A.J. periodontitis Buffalo, NY subgingival AJW2 van Winkelhoff, A.J. Buffalo, NY periodontitis subgingival AJW4 van Winkelhoff, A.J. AJW5 periodontitis Buffalo, NY subgingival ATCC ATCC 33277T severe periodontitis Bowden, G.H.W. Winnipeg, Canada BH 18/10 subgingival Pima Indian Res., AZ periodontitis (NIDDM) Loos, B. G. CLN16-6-4 subgingival Pima Indian Res., AZ Loos, B.G. CLN17-6-1 periodontitis (NIDDM) subgingival acute inf. root canal root canal Herweijer, J.A. EM-3 Buffalo, NY Suido, H. adult periodontitis Japan subgingival ESO-75 adult periodontitis Suido, H. ESO-127 Japan subgingival Zambon, J.J. periodontitis Buffalo, NY FAY19M-1 subgingival abscess van Winkelhoff, A.J. periodontitis Amsterdam, The Netherlands HG445 van Winkelhoff, A.J. periodontitis Amsterdam, The Netherlands subgingival HG564 van Winkelhoff, A.J. periodontitis HG565 Amsterdam, The Netherlands subgingival root canal inf. root canal Sundqvist, G. Umea, Sweden JBB-c Ann Arbor, MI Loesche, W. periodontitis (IDDM) subgingival JKG 6 periodontitis Dahlkn, G. Kenya, Africa OMG 406 subgingival Dahl6n, G. periodontitis Kenya, Africa OMG 434 subgingival Quebec City, Canada adv. adult periodontitis Mayrand, D. RB22D-1 subgingival periodontitis Zambon, J.J. Buffalo, NY THUR28BM-2 subgingival unknown SUNYAB collection unknown clin. specimen W50 SUNYAB collection unknown unknown clin. specimen W83 aIsolates were obtained from patients having either (severe) periodontitis or (localized) chronic periodontitis or (advanced) adult periodontitis, or chronic or acute infected root canals. IDDM: patient was suffering from insulin-dependent diabetes mellitus. NIDDM: patient was suffering from non-insulindependent diabetes mellitus. bP. gingivalis was isolated from samples from either subgingival periodontal pockets or endodontic fistulae or root canals or dental abscesses. Strains W50 and W83 are "clinical specimens". cStrains were obtained either commercially from ATCC (American Type Culture Collection) or from colleagues mentioned in this Table or from the SUNYAB collection (strain collection from the Department of Oral Biology, School of Dental Medicine, State University of New York at Buffalo, NY).

speciated as P. gingivalis by the investigators who supplied the isolates. In our laboratory, the isolates were inspected for purity; for production of non-fluorescent, black-pigmented colonies on blood agar plates (Slots and Reynolds, 1982); and for their demonstration of a characteristic API ZYM profile for P. gingivalis (Slots, 1981; Laughon et al., 1982). Until this point, individual characterization for the majority of these isolates has been based on biotyping and serotyping, and these results are presented in Table 2. All isolates were biotype 1 (catalase-negative), as determined by us with the method described by Syed (1980), and their serotypes were either a, b, or c, as determined by Fisher et al. (1987). The isolates were revived from frozen or lyophilized stocks and maintained on blood agar plates (Trypticase Soy Agar, BBL Microbiological Systems, Becton Dickinson & Co, Cockeysville, MD) enriched with 5% defibrinated sheep blood, hemin (5 pLg/mL), and Vitamin K1 (1 pg/mL), and incubated anaerobically at 370C in an anaerobic chamber (Forma Scientific, Marietta, OH). All isolates were tested for resistance to streptomycin by inclusion of 2000 [xg/mL of this antibiotic in the enriched blood agar plates. Preparation of genomic DNA.-Total genomic DNA was isolated according to a modification of the procedure of Wilson (1987). Briefly, cells were scraped with a sterile cotton swab from a lawn on one plate grown for two to four days before

obvious black-pigmentation was observable. The bacterial cells were re-suspended in 3 mL TE (10 mmol/L Tris-HCI, 1 mmol/ L EDTA [pH 8.0]). Two units of RNAse A (Boehringer Mannheim Biochemicals, Indianapolis, IN) were added (final concentration, 19 Lg/mL). Cells were then lysed with sodium dodecyl sulfate (0.5% final concentration). Proteinase K (Bethesda Research Laboratories, Life Technologies Inc., Gaithersburg, MD), to give a final concentration of 100 pg/mL, was added, and the mixture was incubated at 370C for one h. Cell-wall debris was precipitated by the addition of denatured protein and polysaccharides, 500 pL of 5 mol/L NaCl, followed by 400 pL of a mixture of 10% CTAB (hexadecyltrimethyl ammonium bromide) in 0.7 mol/L NaCl, to give approximate final concentrations of 1% CTAB and 0.7 mol/L NaCl. This mixture was incubated at 65TC for ten min. The DNA was purified by one extraction with TE-saturated chloroform-isoamyl alcohol, followed by an extraction with TEsaturated phenol-chloroform-isoamyl alcohol. The nucleic acid was recovered by precipitation with isopropyl alcohol, washed in 70% alcohol, dried, and dissolved in 200 puL TE. The DNA preparations for each isolate of P. gingivalis were stored in sealed microtubes at 40C until further use. The DNA yield was quantitated by measurement of the absorbance at 260 nm (Maniatis et al., 1982). We typically extracted from 200 to 800 pg of chromosomal DNA from one plate of cells. The

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integrity of the DNA, as well as the presence of any extrachromosomal elements, was assessed by gel electrophoresis on a 0.7% agarose gel with TAE buffer (0.04 mol/L Tris-acetate, 1 mmol/L EDTA) containing 0.5 Aig/mL ethidium bromide (Maniatis et al., 1982). Restriction digests. -Restriction endonucleases with six base, non-redundant recognition sequences, BamHl, Cla!, EcoRI, PstI, PvuII, and SacI were tested [under the conditions recommended by the manufacturer (Bethesda Research Laboratories, Life Technologies Inc., Gaithersburg, MD)] for their ability to digest the genomic DNA preparations. The total reaction volume of the reaction mixture was 25 pLL, and it contained 2-4pg DNA. The resulting digests were inspected on a 0.7% agarose gel with TAE buffer. Gel electrophoresis was performed in 12 x 20 x 0.7-cm gels (gel box from Blair Craft Scientific Inc., Huntington Station, NY) for six h at 4 V/cm. The agarose gel and running buffer both contained 0.5 Fg/mL ethidium bromide, and electrophoresis was followed by photography (under UV illumination) with a Polaroid camera. Commercially obtained restriction digests with HindIII and EcoRI lambda phage DNA were used as size markers (Boehringer Mannheim Biochemicals, Indianapolis, IN). TABLE 2

RESULTS AND SUMMARY FOR BIOTYPING, SEROTYPING, DNA FINGERPRINTING, AND RESISTANCE FOR STREPTOMYCIN TESTING FOR ISOLATES OF P. gingivalis Isolate Clonal Streptomycind Biotypea Serotypeb Typec 2561 a 1 1 R a ATCC 33277T 1 1 R a 1 381 1 R a 34-4 1 2 a 817H 1 3 a CLN17-6-1 1 4 ESO-75 a 1 5 ESO-127 a 1 6 JKG 6 1 b 7 THUR28BM-2 1 b 8 W50 1 b 9 W83 b 1 9 4ZR6-13 1 c 10 9-14K-1 1 c 11 17-5 1 c 12 AJW2 1 c 13 AJW4 1 c 14 OMG 434 1 c 15 A7A1-28 1 c 16 nd CLN16-6-4 1 16 19A4 nd 1 17 295-1 1 nd 18 AJW1 nd 1 19 AJW5 1 nd 20 BH 18/10 1 nd 21 EM-3

1

nd

22

-

FAY19M-1 1 nd 23 1 HG445 nd 24 HG564 1 nd 25 HG565 1 nd 26 JBB-c 1 nd 27 1 OMG 406 nd 28 RB22D-1 nd 1 29 aBiotyping based on catalase activity; biotype 1 means negative for catalase. bSerotyping data from Fisher et al. (1987): isolates were either serotype a, b, or c. For several isolates the serotype was not determined (nd). cClonal types based on unique DNA fingerprints as performed in this study (assigned numbers are arbitrary). dIsolates were either resistant (R) or sensitive (-) to 2000 pig/mL streptomycin in media.

Results High-molecular-weight genomic DNA was obtained from all isolates of P. gingivalis, and no isolates showed the presence of extra-chromosomal DNA. The restriction endonucleases BamHI, Cial, PstI, PvuII, and Sac! digested the total genomic DNA preparations to completion and produced acceptable DNA fingerprints. Incomplete digestion was obtained with EcoRI. We continued to use the relatively inexpensive enzyme PstI as the primary restriction endonuclease for genomic fingerprinting of isolates in this study. We also used ClaI and PvuII for some additional digests (see below). For most isolates, marked differences in the DNA fingerprint of each isolate were observed by visual inspection of the gels. Fig. 1 shows an example of PstI digestion of ten independent isolates of P. gingivalis. Each isolate shows a different DNA fingerprint, most easily seen by a comparison of restriction patterns in adjacent lanes. So that otherwise-similar DNA fingerprints were not scored as different because of artifacts associated with fingerprints seen on different regions of the gel, or on different gels, pairwise inspection of all 33 isolates was performed. Results from all possible permutations indicated that there were 29 different DNA fingerprints among the 33 isolates examined (Table 2). Three sets of isolates gave indistinguishable DNA fingerprints with endonuclease PstI (Fig. 2), and they were: (i) W50 and W83. These isolates are commonly used laboratory strains for in vitro experiments studying physiological and pathological properties of P. gingivalis. Both are clinical specimens of unknown origin. (ii) CLN16-6-4 and A7A1-28. These isolates were recovered from different patients in the same Pima Indian Reservation in Arizona on two separate occasions at least two years apart. (iii) 2561, ATCC 33277T, and 381. Strain 2561 is a laboratory isolate of P. gingivalis and subsequently became the parent strain when it was submitted to the American Type Culture Collection (ATCC) as the type strain (ATCC 33277T) (Coykendall et al., 1980). Strain 381 is a commonly used laboratory strain. In one study, a mutant (strain 381-R) that showed resistance to 2000 [Lg/mL streptomycin was isolated (Slots and Gibbons, 1978). Each of these sets of isolates was therefore also tested with two additional restriction enzymes, Clal and PvuII, but no differences in their respective DNA fingerprints were found, indicating either close genetic similarity or identity of these sets of isolates (data not shown). To test further the apparent genetic similarity among strains 2561, 381, and ATCC 33277T, we determined their resistance to streptomycin. Both the laboratory progenitor strain 2561 and the type strain ATCC 33277r, as well as strain 381, showed resistance to 2000 ug/mL streptomycin in the media. All of the other isolates used in this study were sensitive to this level of antibiotic (Table 2). Finally, we investigated the stability and reproducibility of the restriction digests for two isolates after repeated transfers in the laboratory. Isolates W83 and CLN16-6-4 were both transferred every week for one year. With three restriction enzymes (PstI, Clal, and PvuII) used again, the respective DNA fingerprints from these manipulated bacteria were compared with DNA fingerprints produced from DNA preparations at the beginning of transferring. No differences were observed

(data not shown).

Discussion. It has been established that global bacterial populations are not composed of an infinite number of different isolates. Rather,

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the populations are composed of a limited number of clonal lines, with independent isolates of any one clonal line being genetically identical to each other (0rskov and 0rskov, 1983; Ochman and Selander, 1984; Selander et al., 1987). In general, each independent isolate of P. gingivalis we examined in the present study had a unique DNA fingerprint. This result indicates that there is considerable genetic diversity in the worldwide population of P. gingivalis, and that therefore there is likely to be a relatively large number of genetically distinct clonal lines. All isolates in this study were biotype 1 (Table 2), in accordance with previous studies, which reported this property for human isolates (Laliberte and Mayrand, 1983; Parent et al., 1986). Only three serotypes have been found for a large collection of P. gingivalis isolates (Fisher et al., 1987). These traditional techniques are therefore likely to have only limited application for assessment of the extent of heterogeneity of strains and for ecological and epidemiological studies of P. gingivalis. Other potential techniques to characterize individual isolates include whole-cell protein profiles on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subtyping based on outer membrane protein (OMP) profiles or lipopolysaccharide (LPS) profiles. These techniques, as well as biotyping and serotyping, may have limited resolution, since they rely on a small number of phenotypic variables, and therefore the same biotype, serotype, OMP, or LPS pattern could be detected in two otherwise genotypically unrelated isolates. Also, phase variation in the expression of certain phenotypic characteristics or serologically important antigens may complicate the interpretation of results obtained with one of the above-described techniques. For example, differences in SDS-PAGE membrane profiles were observed when the same strains were grown under iron limitation (Barua et al., 1989) or hemin limitation (Fisher and Zambon, 1989). To estimate further the amount of genetic diversity and to classify the different isolates into a genetic framework, we have started to use multilocus enzyme electrophoresis (MEE)

Fig. 1-DNA fingerprints (PstI digests) of ten independent isolates of Porphyromonas gingivalis. Each isolate shows a different DNA fingerprint, most easily seen by comparison of a lane with two adjacent lanes. Lane 1, 381; lane 2, OMG 434; lane 3, CLN17-6-1; lane 4, ESO-75; lane 5, HG564; lane 6, HG565; lane 7, 34-4; lane 8, 9-14K-1; lane 9, RB22D-1; lane 10, BH 18/10. Size (kb): Lambda phage DNA size markers in kilobases (kb).

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(Selander et al., 1986, 1987). Preliminary results confirm the existence of extensive genetic diversity within the species of P. gingivalis and have allowed us to investigate the extent of genetic relationships among independent isolates (Loos et al., 1989a). Therefore, most of the observed differences among the P. gingivalis DNA fingerprints as seen in the present study are probably due to natural diversity in DNA sequence in the population of this species. The observed genetic heterogeneity among isolates of P. gingivalis shows similarity to findings for other bacterial species, for example, mutans streptococci (Caufield and Walker, 1989; Kulkarni et al., 1989), Campylobacter pyloris (Langenberg et al., 1986), and non-typable Haemophilus influenzae (Loos et al., 1989b). One caution in the application of DNA fingerprinting, as used in this study, is that it is a sensitive technique for detection of differences between isolates. Identical restriction patterns on their own indicate, but do not prove, strain identity in any single case. In the present study, any differences observed in the DNA fingerprints of two isolates in adjacent lanes on the same gel were considered to indicate different isolates, regardless of the molecular origin of the restriction site polymorphisms. Genomic fingerprints of isolates were always compared by the running of samples in adjacent lanes, followed by visual inspection. Differences were always easily noticeable (Kulkarni et al., 1989). However, differences in DNA restriction fingerprints due to the presence of plasmids are not likely, since none of the isolates showed the presence of extra-chromosomal DNA in the present study. This finding extends previous findings on the lack of plasmids in P. gingivalis isolates (Dickinson et al., 1987; Sako et al., 1988). DNA fingerprinting as performed in the present study appears to be a sensitive and powerful tool for investigation of the ecology of P. gingivalis populations, as well as for lon-

Fig. 2-DNA fingerprints (PstI digests) of three sets of isolates of Porphyromonas gingivalis. Indistinguishable DNA fingerprints were observed for lane 1, W50, and lane 2, W83; for lane 3, CLN16-6-4, and lane 4, A7A1-28; and for lane 5, 2561, lane 6, ATCC 33277T, and lane 7, 381. But each set was different from the others. Each of these sets of isolates was also digested with the restriction enzymes ClaI and PvuII, but no differences in their respective DNA fingerprints were found (data not shown). Size (kb): Lambda phage DNA size markers in kilobases (kb).

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gitudinal and cross-sectional epidemiological studies. For example, transmission and turnover of strain populations within a patient population could be examined, since the probability of finding two strains with the same DNA fingerprint is small. The application of DNA fingerprinting in the epidemiology of mutans streptococci (Kulkarni et al., 1989) and of P. gingivalis in a pilot study (Loos et al., 1989c) has recently been described. DNA fingerprinting may be of limited value in studies for the investigation of other micro-organisms with worldwide populations consisting of only a few predominant clonal lines, for example, Haemophilus influenza type b (Musser et al., 1985; Allan et al., 1987). Furthermore, DNA fingerprinting of Actinobacillus actinomycetemcomitans, a species implicated in the etiology of localized juvenile periodontitis, exhibited limited genetic diversity (Zambon et al., 1990). For this species, only three clonal types have been found among more than 100 human isolates, therefore limiting the usefulness of this technique for ecological and epidemiological purposes. During the course of our investigations, we found three sets of isolates with indistinguishable DNA fingerprints. In one, the isolates CLN16-6-4 and A7A1-28 were recovered from different patients in the Gila Indian Community in Arizona. The strains were isolated on two separate occasions over two years apart. This may indicate that the local diversity of the P. gingivalis population is limited, perhaps because this is a homogeneous isolated tribe of Native Americans living on a reservation. The two other sets are frequently used laboratory strains. Strains W50 and W83, which are "clinical specimens" of unknown origin, were indistinguishable in their DNA fingerprints with three different endonucleases tested. These results indicate that these isolates are genetically similar and may have been recovered from the same patient. The other set of laboratory strains with indistinguishable restriction patterns comprised strain 2561, its descendent strain ATCC 33277T (Coykendall et al., 1980), which we obtained commercially from the ATCC, and strain 381. Strain 381 was originally isolated by Dr. S.S. Socransky from subgingival plaque, and a streptomycin-resistant mutant, 381-R, was used in an in vivo attachment experiment (Slots and Gibbons, 1978). In addition to having identical DNA fingerprints, all three of these strains were streptomycin-resistant (2000 pig/mL incorporated in enriched blood-agar plates). All other isolates in the present study were sensitive to this level of streptomycin (Table 2). Furthermore, each of these three strains was serotype a (Table 2). Taken together, these data strongly suggest that both strain 2561 and ATCC 33277T are in fact strain 381-R. Therefore, the type strain of P. gingivalis may be a mutant laboratory strain with an unnatural streptomycin resistance, and this strain may not be a true representative of the species P. gingivalis associated with human periodontitis. We have deposited a new strain with ATCC and suggest its use for the replacement of the current type strain (ATCC 33277T). The chosen strain, RB22D-1 (ATCC 49417), originates from a human periodontal lesion and is representative of what the species should be: It is an anaerobic, non-motile, Gram-negative short rod that requires hemin for growth. It is collagenolytic and proteolytic, hemagglutinates red blood cells, and produces phenylacetic acid. The strain has been shown to be infective by itself in a guinea pig model (Grenier and Mayrand, 1987). In light of the results with laboratory strains, it seems prudent that during the exchange of strains among different laboratories, no new labels or new names be attached to received strains, to minimize confusion as to the origin of strains, and to avoid duplication of in vitro experiments with genetically identical strains.

Acknowledgment. We thank our colleagues for their generous supply of isolates

(see Table 1). REFERENCES ALLAN, I.; LOEB, M.R.; and MOXON, E.R. (1987): Limited Genetic Diversity of Haemophilus influenzae (type b), Microb Pathog 2: 139-145. BARUA, P.K.; DYER, D.W.; and NEIDERS, M.E. (1989): Response of Bacteroides gingivalis to Iron Limitation, J Dent Res 68:358, Abst. No. 1415. CAUFIELD, P.W. and WALKER, T.M. (1989): Genetic Diversity within Streptococcus mutans Evident from Chromosomal DNA Restriction Fragment Polymorphisms, J Clin Microbiol 27:274278. CHRISTERSSON, L.A.; ZAMBON, J.J.; DUNFORD, R.G.; GROSSI, S.G.; and GENCO, R.J. (1989): Specific Subgingival Bacteria and Diagnosis of Gingivitis and Periodontitis, J Dent Res 68:1633-1639. COYKENDALL, A.L.; KACZMAREK, F.S.; and SLOTS, J. (1980): Genetic Heterogeneity in Bacteroides asaccharolyticus (Holdeman and Moore 1970) (Finegold and Barnes 1977) (Approved list, 1980) and Proposal of Bacteroides gingivalis sp. nov. and Bacteroides macacae (Slots and Genco) comb. nov., Int J Syst Bacteriol 30:559564. DENNING, D.W.; BAKER, C.J.; TROUP, N.J.; and TOMPKINS, L.S. (1989): Restriction Endonuclease Analysis of Human and Bovine Group B Streptococci for Epidemiological Study, J Clin Microbiol 27:1352-1356. DICKINSON, D.P.; RIVOLI, P.S.; BREWSTER, J.M.; and GENCO, R.J. (1987): Plasmids in Black-pigmented Members of the Genus Bacteroides, J Dent Res 66:223, Abst. No. 933.

DICKINSON, D.P.; LOOS, B.G.; DRYJA, D.M.; and BERNSTEIN, J.M. (1988): Restriction Fragment Mapping of Branhamella catarrhalis: A New Tool to Study the Epidemiology of this Middle Ear Pathogen, J Infect Dis 158:205-208. DZINK, J.L.; SOCRANSKY, S.S.; and HAFFAJEE, A.D. (1988): The Predominant Cultivable Microbiota of Active and Inactive Lesions of Destructive Periodontal Diseases, J Clin Periodontol 15:316-323. EBERSOLE, J.L.; TAUBMAN, M.A.; SMITH, D.J.; and FREY, D.E. (1986): Human Immune Responses to Oral Microorganisms: Patterns of Systemic Antibody Levels to Bacteroides Species, Infect Immun 51:507-513. FISHER, J.G.; ZAMBON, J.J.; and GENCO, R.J. (1987): Identification of Serogroup-specific Antigens among Bacteroides gingivalis Components, J Dent Res 66:222, Abst. No. 927. FISHER, J.G. and ZAMBON, J.J. (1989): The Effect of Hemin Concentration on the Outer Membrane of Bacteroides gingivalis, J Dent Res 68:358, Abst. No. 1414. GOODSON, J.M.; TANNER, A.C.R.; HAFFAJEE, A.D.; SORBERGER, G.C.; and SOCRANSKY, S.S. (1982): Patterns of Progression and Regression of Advanced Destructive Periodontal Disease, J Clin Periodontol 9:427-481. GRENIER, D. and MAYRAND, D. (1987): Selected Characteristics of Pathogenic and Nonpathogenic Strains of Bacteroides gingivalis, J Clin Microbiol 25:738-740. GROTHUES, D.; KOOPMANN, U.; VON DER HARDT, H.; and TUMMLER, B. (1988): Genome Fingerprinting of Pseudomonas aeruginosa Indicates Colonization of Cystic Fibrosis Siblings with Closely Related Strains, J Clin Microbiol 26:1973-1977. HAAPASALO, M.; RANTA, H.; RANTA, K.; and SHAH, H. (1986): Black-pigmented Bacteroides spp. in Human Apical Periodontitis, Infect Immun 53:149-153. HAFFAJEE, A.D.; SOCRANSKY, S.S.; and GOODSON, J.M. (1983): Comparison of Different Data Analyses for Detecting Changes in Attachment Levels, J Clin Periodontol 10:513-521. KULKARNI, G.V.; CHAN, K.H.; and SANDHAM, H.J. (1989): An Investigation into the Use of Restriction Endonuclease Analysis

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