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J. Med. Microbiol. Vol.

51 (2002), 10971101 # 2002 Society for General Microbiology ISSN 0022-2615

MOLECULAR IDENTIFICATION

Development of a PCR assay specic for Peptostreptococcus anaerobius


M. P. RIGGIO and A. LENNON
Infection Research Group, University of Glasgow Dental School, 378 Sauchiehall Street, Glasgow G2 3JZ

Peptostreptococcus anaerobius is a gram-positive anaerobic coccus that is widely distributed in the normal human ora. The organism has also been implicated as a causative agent of several systemic infections, including endocarditis and infections of the genitourinary and gastrointestinal tracts. Its role in oral disease is less well dened, although it has been implicated in periodontal disease, gingivitis and root canal infections. Identication of P. anaerobius in clinical samples is currently reliant upon traditional culture and biochemical methods. The aim of this study was to develop a novel PCR assay for the detection of P. anaerobius and to attempt detection of this organism in oral samples. PCR primers specic for P. anaerobius DNA were developed by alignment of bacterial 16S ribosomal RNA gene sequences and selection of sequences specic at their 39 ends for P. anaerobius. When used in a PCR assay, positivity for P. anaerobius DNA was indicated by the amplication of a 943-bp product. The primers were shown to be specic for P. anaerobius DNA, as no PCR products were obtained when genomic DNA from a wide range of other Peptostreptococcus species and other oral bacteria were used as templates. The PCR assay was then applied to the detection of P. anaerobius DNA in subgingival plaque samples from adult periodontitis patients and pus aspirates from subjects with acute dento-alveolar abscesses. All of 60 subgingival plaque samples from 16 patients were negative for P. anaerobius DNA. None of the 43 pus samples analysed contained P. anaerobius DNA. These results suggest that P. anaerobius is not a major pathogen in adult periodontitis and dento-alveolar abscesses. The PCR assay is a more rapid, sensitive and specic alternative to culturebased methods for identication of P. anaerobius in clinical samples.

Introduction
Peptostreptococcus anaerobius is a gram-positive anaerobic coccus that is the type species of the genus Peptostreptococcus [1]. P. anaerobius is regarded as a member of the normal gastrointestinal ora, although it is uncertain as to whether it is a member of the normal oral and vaginal ora [2]. The organism is involved in polymicrobial infections and has been isolated from a wide variety of human clinical specimens. These include abscesses of the brain, ear, jaw, pleural cavity, pelvic, urogenital and abdominal regions [1], nasal septal abscesses [3], infected haemorrhoids [4], infections of the abdominal cavity [5] and of the female genitourinary tract [6]. P. anaerobius is also involved in
Received 31 Jan. 2002; revised version received 14 May 2002; accepted 19 May 2002. Corresponding author: Dr M. P. Riggio (e-mail: m.riggio@ dental.gla.ac.uk).

soft tissue infections, including those of the leg and external genitalia [7]. The isolation of P. anaerobius in pure culture from a case of endocarditis has been reported [8]. The involvement of P. anaerobius in oral infections such as periodontal disease is less well dened. P. anaerobius has been associated with gingivitis and periodontitis [9, 10], although an earlier study suggested that the organism was found in subgingival plaque at very low levels [11]. P. anaerobius is one of the species found most frequently in the root canals of teeth with periapical periodontitis [12] and has been isolated from a peritonsillar abscess [13]. Identication of P. anaerobius in clinical samples has traditionally been accomplished by a combination of microbiological culture and biochemical tests. These tests are based upon carbohydrate fermentation reactions and production of saccharolytic and proteolytic

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enzymes, and have been used with limited success in identifying P. anaerobius [2, 14, 15]. Volatile fatty acid (VFA) production has proved to be a useful identication method, as P. anaerobius is the only gram-positive anaerobic coccus to produce isocaproic acid as the major end-product of metabolism [2]. Although some of these traditional methods have proved useful in the identication of P. anaerobius, their routine use in a diagnostic laboratory is often hindered by the ambiguous nature of their results and by the fact that they are laborious and expensive to carry out. The purpose of this study was to develop a novel PCR assay for the specic detection of P. anaerobius DNA in clinical samples, that could be used as a more reliable, specic and rapid alternative to currently used detection methods. The present report describes the development of a PCR assay specic for P. anaerobius DNA and its application in the attempted detection of P. anaerobius in subgingival plaque samples from adult periodontitis patients and pus aspirates from subjects with acute dento-alveolar abscesses.

lysates were prepared by adding 3 l of achromopeptidase (20 U/ l in 10 mM Tris-HCl, 1 mM EDTA, pH 7.0) to 100 l of plaque. Samples were incubated at 568C for 30 min, boiled for 5 min and stored at 708C until required. A 50- l volume of each pus sample was diluted 10100-fold in PCR diluent (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 1 mM EDTA); 30 l of SDS 10% and 3 l of proteinase K (10 mg/ml) were added to 300 l of diluted pus and incubated at 558C for 3 h. Lysed samples were extracted twice with an equal volume of phenol:chloroform (1:1) and once with an equal volume of chloroform. DNA was precipitated by adding 0.1 volume of 3 M sodium acetate, pH 5.3, and 2 volumes of ethanol 100%, followed by mixing and incubation at 708C for 30 min. Precipitated DNA was recovered by centrifugation and the pellet was resuspended in 100 l of sterile molecular biology grade water.

Selection of PCR primers


Primers for use in the PCR assay were derived from the 16S rRNA gene sequence of P. anaerobius ATCC 27337. The 16S rRNA gene sequences of Peptostreptococcus species and several other oral bacteria were aligned with Version 8 of the University of Wisconsin Genetics Computer Group sequence analysis program package [16]. Two primers that demonstrated sequence specicity for P. anaerobius at their 39 ends were selected from unique regions of the P. anaerobius 16S rRNA gene sequence. The primer sequences were: 59CGT CTW ATT TNA TGC TTG CA-39 (Pan-1; base position 6281), where N A C G T and W A T, and 59-AGC CCC GAA GGG AAG GTG TG-39 (Pan-2; base position 1004985), which give an expected amplication product of 943 bp.

Materials and methods Bacterial culture and genomic DNA extraction


P. anaerobius ATCC 27337 was cultured on Fastidious Anaerobe Agar (Life Technologies, Paisley) supplemented with debrinated horse blood 7.5% v/v and incubated at 378C for 45 days in an anaerobic chamber in an atmosphere of N2 85%, CO2 10% and H2 5%. Bacteria were harvested from the plates and genomic DNA was extracted with the Puregene DNA Isolation Kit (Novara Flowgen, Ashby de la Zouch).

Sample details
Subgingival plaque was obtained from patients with untreated chronic inammatory adult periodontitis who had been newly referred to Glasgow Dental Hospital. Criteria for inclusion in the study were the presence of at least three periodontal pockets with a depth of at least 5 mm and bleeding on probing, together with no history of antibiotic treatment during the preceding 6 months. In total, 60 subgingival plaque samples from 16 patients were analysed. The mean pocket depth of analysed samples was 7.2 mm (range 510 mm); the age range of the patients was 2958 years (mean age 42.1 years). One pus sample from each of 16 subjects with acute dento-alveolar abscess was analysed; their age range was 3254 years (mean 40.8 years).

PCR
All PCR reactions were performed in a total volume of 50 l and the PCR reaction mixture was essentially as described previously [17]. Each PCR reaction mixture comprised either 5 l of lysed plaque sample/pus extract or 1 l of bacterial genomic DNA and either 45 or 49 l of reaction mixture containing MgCl2 at the optimum concentration of 1.5 mM. The PCR cycling conditions comprised an initial denaturation step of 948C for 5 min, followed by 35 cycles of denaturation at 948C for 1 min, primer annealing at 608C for 1 min and extension at 728C for 1.5 min, and nally an extension step at 728C for 10 min. Stringent anti-contamination procedures were employed when performing PCR, as described previously [17]. Negative and positive controls were included with each batch of samples being analysed. The positive control was a standard PCR reaction mixture containing 10 ng of P. anaerobius genomic DNA instead of sample; the

Sample collection and preparation


Subgingival plaque samples were collected with a sterile curette into sterile tubes containing 0.5 ml of freshly prepared Fastidious Anaerobe Broth (Bioconnections, Leeds). Samples were mixed for 30 s and

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negative control contained sterile water instead of sample. PCR products were visualised by electrophoresis on agarose 2% gels as described previously [17].

Results Specicity and sensitivity of the P . anaerobius PCR assay


After 35 cycles of amplication, the lower limit of detection of P. anaerobius was c. 50 bacterial cells (data not shown). The specicity of the PCR assay was conrmed by performing PCR with 10 ng of genomic DNA from each of the bacterial species selected for use as controls. Only the use of P. anaerobius DNA as a template resulted in a PCR product of the expected size (Fig. 1, lane 2).

Bacterial species used as PCR controls


Bacterial species used as controls for PCR were the Peptostreptococcus species P. anaerobius ATCC 27337, P. magnus NCTC 11804, P. micros ATCC 33270, P. asaccharolyticus NCTC 11461, P. prevotii NCTC 11806, P. tetradius ATCC 35098, P. productus NCTC 11829 and P. indolicus NCTC 11088. The following oral bacteria were also used as PCR controls: Prevotella intermedia ATCC 25611, Porphyromonas gingivalis ATCC 33277, Bacteroides forsythus ATCC 43037, Helicobacter pylori ATCC 43504, Actinobacillus actinomycetemcomitans ATCC 33384, Fusobacterium nucleatum ATCC 25586, Eikenella corrodens ATCC 23834, Streptococcus mutans ATCC 25175, Staphylococcus aureus ATCC 12600 and Actinomyces naeslundii ATCC 12104.

PCR analysis of clinical specimens


The P. anaerobius PCR assay was used to determine the prevalence of this species in subgingival plaque samples from patients with adult periodontal disease and in pus aspirates obtained from subjects with acute dento-alveolar abscesses. P. anaerobius DNA was not detected by PCR in any of 60 subgingival plaques and 43 pus samples analysed. Sample inhibition of PCR was discounted because the spiking of selected plaque

Fig. 1. Specicity of the P. anaerobius PCR assay. Electrophoresis on an agarose 2% gel of PCR products obtained with primer pair Pan-1 and Pan-2 and genomic DNA from various bacteria as template: P. magnus NCTC 11804 (lane 1); P. anaerobius ATCC 27337 (2); P . micros ATCC 33270 (3); P. asaccharolyticus NCTC 11461 (4); P. prevotii NCTC 11806 (5); P. tetradius ATCC 35098 (6); P. productus NCTC 11829 (7); P. indolicus NCTC 11088 (8); Pr. intermedia ATCC 25611 (9); Por. gingivalis ATCC 33277 (10); B. forsythus ATCC 43037 (11); H. pylori ATCC 43504 (12); A. actinomycetemcomitans ATCC 33384 (13); F. nucleatum ATCC 25586 (14); Eik. corrodens ATCC 23834 (15); Str. mutans ATCC 25175 (16); S. aureus ATCC 12600 (17); Actinomyces naeslundii ATCC 12104 (18); 100-bp DNA ladder (19); negative control (20). PCR positivity is indicated by the presence of a 943bp product.

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and pus samples with 1000 whole cells of P. anaerobius yielded a PCR product of the expected size (data not shown).

Discussion
The aims of this study were to develop a novel PCR assay specic for P. anaerobius and to use this assay to determine the incidence of this organism in oral specimens. Conventional methods for identication of P. anaerobius and other members of the Peptostreptococcus genus are reliant upon bacterial culture coupled with biochemical identication of clinical isolates. P. anaerobius is commonly identied on the basis of colony morphology and Grams staining characteristics, followed by biochemical analyses. The Rapid ID 32A system, which analyses bacterial sugar fermentation characteristics and enzymic activities, has been used for identication of P. anaerobius [18]. This method is perhaps of more value for identication of P. anaerobius than other members of the Peptostreptococcus genus, because P. anaerobius is more saccharolytic than other species in this genus. The RapIDANA II panel of tests identies bacteria on the basis of their capacity to hydrolyse amino acid and phosphate substrates. However, this method has proved to be unreliable for identication of P. anaerobius, as in one study 37% of P. anaerobius isolates were misidentied [14]. Detection of VFA end-products of metabolism by gas-liquid chromatography has also been used to identify Peptostreptococcus spp. [2, 15, 19]. This has proved to be a reliable method for identication of P. anaerobius, as it is the only Peptostreptococcus species to produce a major terminal peak of isocaproic acid. These conventional methods for identication of P. anaerobius have undoubtedly been of considerable use. However, because of the emergence of phenotypically variable strains with altered biochemical characteristics, ambiguous results are often obtained with such methods. Other considerations are the lengthy processes involved and the high overall costs of performing such tests. PCR offers an alternative method of identication that is simpler, cheaper and quicker than conventional methods. Furthermore, as PCR can detect phenotypically variable strains unequivocally, it can be considered as a more specic and reliable method for bacterial identication. In the present study, a PCR assay for the specic detection of P. anaerobius was developed and evaluated. The assay was shown to be specic for P. anaerobius, as no other bacterial species tested which included the most closely related Peptostreptococcus spp. were detected with the PCR primers used. When the PCR assay was used to attempt detection of P. anaerobius in subgingival plaque samples from adult periodontitis patients and pus aspirates from dento-alveolar abscesses, no clinical

specimens examined were found to harbour the organism. This is in contrast to previous ndings for P. micros, which was detected in 19 (28%) of 60 subgingival plaque samples and 20 (71%) of 28 pus samples analysed by PCR [20]. This is not an unexpected nding, because the role of P. anaerobius in oral infections is less well dened than that of P. micros, which has been well documented as an oral pathogen [2]. Very few previous studies have attempted to detect P. anaerobius in oral specimens. The tenuous nature of the link between P. anaerobius and periodontal disease is further documented by the immunological ndings of another study. With a gingival explant culture system, Hall et al. [21] evaluated the reactivity of local antibodies produced by juvenile periodontitis tissue. Only 2 of 73 gingival explant culture supernatant uids demonstrated reactivity to P. anaerobius, indicating that this bacterium does not play a major role in periodontal disease. No clinical samples were shown to be PCR-positive for P. anaerobius in the present study. However, conrmation of PCR product identity in any future studies could readily be done by digestion of PCR products with a combination of the restriction endonucleases RsaI, MnlI and HinfI, which give a distinct restriction prole for P. anaerobius DNA. The classication of members of the genus Peptostreptococcus has generated much discussion. 16S rRNA sequence analysis has demonstrated that P. anaerobius is distinct from other anaerobic grampositive micro-organisms [22, 23]. Several studies have suggested that other members of the genus Peptostreptococcus should be removed from this genus and reclassied. On the basis of 16S rRNA sequence data, it has been proposed that P. productus be placed in the genus Ruminococcus [24]. It has also been proposed that, on the basis of biochemical analysis and 16S rRNA sequence data, P. magnus be reclassied in the new genus Finegoldia as Fin. magna, and that P. micros be reclassied in the new genus Micromonas as M. micros [25]. It was recently proposed that P. anaerobius should be the only remaining member of the genus Peptostreptococcus [26]. In that study, phylogenetic analysis of 16S rRNA sequence data demonstrated that Eubacterium brachy and Clostridium mayombeii were most closely related to P. anaerobius, although it was advocated that these species should not be placed in the same genus as P. anaerobius. The primers specic for P. anaerobius used in the present study are very divergent in sequence at their 39 ends from the corresponding region of the 16S rRNA genes of E. brachy and C. mayombeii, and as such would not be capable of detecting these closely related species. In conclusion, a novel PCR assay for the specic detection of P. anaerobius has been developed. This PCR assay was used to assess the prevalence of P. anaerobius in subgingival plaque from adult perio-

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dontitis patients and in pus aspirates from subjects with acute dento-alveolar abscesses. P. anaerobius was not detected in any of the clinical specimens analysed, which conrms previous suggestions that its association with the oral cavity is tenuous. Nonetheless, this PCR assay provides a rapid, specic and sensitive alternative to conventional methods for identication of P. anaerobius in clinical specimens.

14.

15. 16. 17. 18.

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