Molecular typing of actinomyces pyogenes isolates

Zbl. Bakt. 281,174-182 (1994) © Gustav Fischer Verlag, Stuttgart· Jena . New York

Molecular Typing of Actinomyces pyogenes Isolates VERONIQUE GUERIN-FAUBLEEl, DOMINIQUE DECORET 2 , ANGELI KODJ0 1 , BRUNO TILL yl, ARNAUD CARLOTTI 3 , JEAN FRENEY4, and ODILE BARSOTTI 2 Laboratoire de Microbiologie et Immunologie, Ecole Nationale Veterinaire de Lyon, BP 83, 69280 Marcy l'etoile, France 2 Laboratoire de Microbiologie et Immunologie, Faculte d'Odontologie, rue Guillaume Paradin, 69372 Lyon cedex 08, France 3 Laboratoire de Mycologie Fondamentale et Appliquee aux Biotechnologies Industrielles, Faculte de Pharmacie, 8 avenue Rockfeller, 69373 Lyon cedex 08, France 4 Departement de Recherche en Bacteriologie Clinique, Faculte de Medecine Alexis Carrel, 69376 Lyon cedex 08, France 1

Received July 26, 1993 . Revision received November 8, 1993 . Accepted March 21, 1994

Summary The molecular characterization of 28 clinical Actinomyces pyogenes strains was attempted. SDS-PAGE protein profiles did not allow to distinguish isolates. Restriction endonuclease analysis of total DNA gave the finest differentiation between strains but the profiles were difficult to read. Ribotypes after DNA digestion by Bst Ell or Sma I have a high discriminatory power and are more helpful epidemiological markers. No relationship could be demonstrated between molecular types and clinical sources. Introduction

Actinomyces pyogenes is an opportunistic pathogen which is commonly isolated, as a primary or secondary invader, from a variety of pyogenic diseases mainly in cattle, sheep, goats and pigs, but also in various other domestic animals and in humans. It is responsible for bronchopneumonia, endometritis, abortions, abscesses, septicaemia, arthritis as well as summer mastitits in cattle. Its distribution seems to be worldwide and it is responsible for heavy economic losses. The transfer of Corynebacterium pyogenes to the genus Actinomyces was proposed on the basis of its chemical and physiological characteristics by Reddy et al. (17) and was confirmed by Collins and Jones (4). This species is very homogeneous and no suitable markers are available for the typing of A. pyogenes strains. Numerical phenetic analysis by Roberts (18) showed that all the strains were linked at high similarity values and no biotype could be reported by Hoi Sorensen (14) nor by Tainaka et al. (21). In preliminary studies (12),

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examination in our laboratory of 103 field isolates for morphological, cultural and biochemical properties by standard tests and by Api 50 CH trays was unable to demonstrate any heterogeneity between strains except for 3 atypical non-proteolytic (13) or less proteolytic strains. Antimicrobial susceptibility patterns are of limited value in epidemiological investigations since most of the strains are either sensitive to numerous antibiotics or resistant to tetracycline and streptomycin (11). Other phenotypic markers have been investigated on a few isolates: serovars have been described by Tainaka et al. (21) and a bacteriocin fingerprinting procedure has been proposed by Uimmler (16) but further studies are necessary before they can be used routinely. It has been the purpose of this study to attempt molecular characterization by 3 techniques (electrophoretic protein profiles, restriction endonuclease analysis of total DNA, ribotyping) of A. pyogenes isolates associated with different types of infections and from different animal species in order to evaluate their value as a tool in epidemiological studies.

Materials and Methods Bacterial strains. A total of 28 strains of A. pyogenes were used in this study. Most of them had been obtained from cattle. They were collected from clinical specimens, in France, between 1986 and 1990. Eight of them were isolated from the respiratory tract, 2 from uterine discharges, 2 from milk, 2 from arthritis, 1 from an aborted foetus and the remaining 2 from pus and cerebrospinal fluid. Four strains originated from sheep, 2 from goats, 1 from a rabbit and 3 from pigs including the type strain ATCC 19411 (103129T, Collection de l'Institut Pasteur, Paris, France). A human strain isolated from the blood and foot of a diabetic patient was included (7).

Morphological, cultural and biochemical properties have been reported (12); all isolates had the biochemical profile characteristic of A. pyogenes except one bovine non-proteolytic strain (strain C). Bacterial cells were obtained by aerobic culture on Brain Heart Infusion broth (Difco, Detroit, USA) for 2 days at 3rC, and harvested by centrifugation at 6000 g for 15 min. Electrophoretic protein profiles. Polyacrylamide gel electrophoresis of whole-cell proteins in the presence of sodium dodecyl sulphate (SDS-PAGE) was performed using a discontinuous buffer system as specified by Barsotti et al. (1). Proteins were extracted by shaking bacterial cells in PBS buffer containing 0.5% Triton X-100 (Prolabo, Paris) and 0.02 M EDTA (Merck, Darmstadt, Germany) (2 mUg of washed bacteria) in the presence of glass beads. Each profile was compared with every other profile by the Dice coefficient (6). Total DNA Restriction Fragment Length Polymorphism. Total DNA extract was prepared using a modification of the method of Barsotti et al. (3), lysis of the cells being obtained by the simultaneous action for 2.5 h at 37°C of lysozyme (Sigma, St. Louis, USA) (20 mg/g of wet cells) and achromopeptidase (Wako, Neuss, Germany) (10 mg/g) and further treatment for 2 h at 64°C with SDS (0.25%) and proteinase K (Sigma) (20 mg/g). Restriction digestion of DNA samples was carried out using Bst Ell, Pvu II or Sma I endonucleases (Boehringer-Mannheim, Mannheim, Germany) (5 U/!-lg of DNA) according to the manufacturer's instructions. DNA fragments were then separated electrophoretically and stained according to conditions described elsewhere (2). rRNA gene restriction patterns. For the analysis of rRNA gene restriction patterns, DNA was cleaved and restriction fragments were separated in the same way. Southern transfer to a Hybond-N Plus membrane (Amersham International, England), prehybridization and hybridization were conducted as previously described (9). A universal probe consisting of ribosomal 16 + 23 S RNA from Escherichia coli labelled with acetylaminofluorene (AAF) 12 Zbl. Bakt. 28112

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(Eurogentec, Seraing, Belgium) was used. The DNA fragment size marker Raoul I was hybridized with AAF-labelled DNA from plasmid pBR322 (Eurogentec). rDNNrRNA and DNNDNA duplexes were detected by an immunological technique according to Crimont et al. (8) using monoclonal anti-AAF IgG and alkaline phosphatase-labelled anti-mouse IgG antibodies supplied in the AAF kit (Eurogentec). The hybridized fragment sizes were calculated from migration distances by repeated side-ta-side comparisons with size marker Raoul I using the "RNA pattern", a computer program written by P. A. D. Crimont (Institut Pasteur, Paris) using the algorithm described by Schaffer and Sederoff (20).

Results Electrophoretic protein profiles

Representative whole-cell protein patterns are shown in Figure 1. Depending on the strains, 35 to 50 bands were identified. The overall protein profiles were visually similar to numerous common bands, but a number of substantial differences occurred between individual samples. Nevertheless, all the Dice coefficients were above 70%, 302/392 being superior to 80%, which, when the reproducibility of the technique was taken into account, did not allow us to differentiate strains according to their electrophoretic protein profiles.

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Fig. 1. Whole-cell protein patterns of Actinomyces pyogenes isolates in SDS-polyacrylamide gel electrophoresis. Lanes: 1, caprine strain, H; 2, 3, 4, 5, ovine strains, E, 0, F, G; 6, reference strain ATCC19411; 7, bovine strain, C; 8, molecular weight standards in kD.

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Total DNA Restriction Fragment Length Polymorphism

As previously reported (2), the enzymes showing the best resolution of the larger molecular weight fragments for restriction endonuclease analysis of DNA of Actinomyces species were Bst Ell, Pvu II, and Sma I; however, with A. pyogenes, digestion with Sma I was not complete. With the technique described, DNA digest patterns were reproducible (2). Complex but well-resolved patterns of about 50 fragments with a molecular size ranging from 49 kbp to 0.75 kbp were obtained (Figure 2). Pvu II, Bst Ell as well as Sma I digested fragments gave numerous bands that were common to all isolates. However, many fine strain variations were observed particularly in the high molecular

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Fig. 2. Restriction endonuclease analysis profiles of Pvu II digested DNA of Actinomyces pyogenes isolates. Lanes: 1,2,3,4,5,6, 7, 8, 10, 12, bovine strains, Q, K, U, 6, N, M, P, Z, Y, W; 9, human strain, 2; 11, ovine strain, G; 13, molecular weight marker Raoul I (Appligene) in kbp.

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mass range of the gels and every strain exhibited a unique pattern except for 2 isolates 6 and M (Figure 2, lanes 4 and 6, respectively) that had the same restriction profiles as the 3 endonucleases. These latter strains were isolated from calf nasal swabs on the same date and in the same area, and were most probably related.

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Fig. 3. Normalized graph showing migration patterns of rRNA gene restriction fragments of Actinomyces pyogenes strains after DNA digestion with Sma I.

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rRNA gene restriction patterns

Figures 3 and 4 display a normalized representation of rRNA gene restriction patterns of 27 clinical isolates of A. pyogenes and the type strain. Comparison of the number and size of fragments led us to describe the ribotypes when they differed by at least one band. Size of rRNA gene restriction fragments

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Fig. 4. Normalized graph showing migration patterns of rRNA gene restriction fragments of Actinomyces pyogenes strains after DNA digestion with Bst Ell.

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Only one fragment hybridized with the universal probe when DNA was cleaved with Pvu II. In contrast, ribotyping revealed some heterogeneity after DNA digestion with Sma I or Bst Ell. Sma I digests (Figure 3) generated 3 to 5 bands ranging from 1.6 to 16 kbp. Two bands of about ].6 and 13 kbp were present in all isolates. Eleven ribotypes were observed; 13 isolates exhibited pattern characterized by 3 fragments (1.6, 4.6, and 13 kbp). When the DNA was digested with Bst Ell (Figure 4), more bands (2 to 10) ranging from 1.9 to 19.5 kbp were observed and 19 ribotypes could be described, each made up of 1 to 3 strains. All the strains except isolate 4 (obtained from a culture collection) harboured a fragment of approximatively 2.7 kbp. After Sma I as well as Bst Ell digestion, no correlation could be found between ribotypes and either clinical source, geographical origin and date of isolation, or antibiotic resistance pattern (data not shown). The two strains with identical restriction endonuclease analysis profiles had ribotypes that were similar when DNA was cleaved with Bst Ell but different after DNA digestion with Sma I. Discussion Comparison of the restriction endonuclease analysis profiles showed a genetic polymorphism within the species A. pyogenes confirmed by the ribotyping studies. To our knowledge, this is the first report of the molecular characterization of A. pyogenes isolates. Nevertheless, the preliminary description of serotypes (19, 21) implied a genetic diversity. These results suggest that the molecular typing of A. pyogenes strains is a potential tool for epidemiological investigations. For this purpose, electrophoretic protein patterns are not a helpful marker. With regard to the genus Actinomyces, they seem to be more useful for species identification (see for example reference 5) than for type description. Restriction endonuclease analysis of total DNA gave the most accurate differentiation between isolates but was of limited value since the profiles were not easy to read because of the large number of bands. Restriction enzymes with rare cleavage sites must be used with pulse-field gel electrophoresis to visualize the large fragments generated, but this technique has never been applied to the genus Actinomyces. The hybridization of Sma I or Bst Ell digests with a universal rRNA probe reduced the number of visible fragments thereby making comparisons easier, with a higher level of discrimination. The determination of ribosomal ribonucleic acid gene restriction patterns was proposed in 1986 as a general tool for bacterial strain fingerprinting (9). The use of non-isotopic labelled probes (8) made ribotyping easier. This technique could not only be employed in taxonomy (22) and identification (10) but also in epidemiology (15) depending on the degree of intra species genomic homogeneity. A. pyogenes ribotypes, as defined by us, are useful epidemiological markers. However, the choice of the restriction endonucleases may be critical to divergence patterns, and the finest differentiation between isolates required the use of at least two enzymes instead of only one. Molecular techniques (restriction endonuclease analysis as well as rRNA gene restriction patterns) are complementary and each of them can reveal individual differences.

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These results need to be expanded to evaluate the usefulness of molecular characterization in the investigation of A. pyogenes infections. No relationship has yet been demonstrated between molecular types and clinical sources. Acknowledgments. We thank]. L. Martel (Laboratoire de Pathologie Bovine, CNEVA, Lyon, France) who collected the bovine strains from clinical specimens and Professor Y. Peloux (H6pital de la Timone, Marseille, France) for generously supplying the human strain.

. References 1. Barsotti, 0., C. Com by, J. J. Morrier, G. Benay, D. Decoret, R. Guinet and J. Dumont: Improved SDS-Page method for protein profiles analysis of Actinomyces species. J. BioI. Buccale 16 (1988) 219-224 2. Barsotti, 0., J. J. Morrier, D. Decoret, G. Benay, and J. P. Rocca: An investigation into the use of restriction endonuclease analysis for the study of transmission of Actinomyces. J. Clin. Periodontol. 20 (1993) 436-442 3. Barsotti, 0., F. Renaud, ]. Freney, G. Benay, D. Decoret, and J. Dumont: Rapid isolation of DNA from Actinomyces. Ann. Inst. Pasteur/Microbiol. 138 (1987) 529-536 4. Collins, M. D. and D. Jones: Reclassification of Corynebacterium pyogenes (Glage) in the genus Actinomyces, as Actinomyces pyogenes comb. nov. J. Gen. Microbiol. 128 (1982) 901-903 5. Dent, V. E. and R. A. D. Williams: Actinomyces howellii, a new species from the dental plaque of dairy cattle. Int. J. Syst. Bacteriol. 34 (1984) 316-320 6. Dice, L. R.: Measures of the amount of ecological association between species. Ecology 26 (1945) 297-302 7. Drancourt, M., O. Oules, V. Bouche, and Y. Peloux: Two cases of Actinomyces pyogenes infection in humans. Eur. J. Clin. Microbiol. Infect. Dis. 12 (1993) 55-56 8. Grimont, F., D. Chevrier, P. A. D. Grimont, M. Lefevre, and J.-L. Guesdon: Acetylamino fluorene-labelled ribosomal RNA for use in molecular epidemiology and taxonomy. Res. Microbiol. 140 (1989) 447-454 9. Grimont, F. and P. A. D. Grimont: Ribosomal ribonucleic acid gene restriction patterns as potential taxonomic tools. Ann. Inst. Pasteur/Microbiol. 137 B (1986) 165-175 10. Grimont, F., M. Lefevre, E. Ageron, and P. A. D. Grimont: rRNA gene restriction patterns of Legionella species: a molecular identification system. Res. Microbiol. 140 (1989) 615-626 11. Guerin-Faubtee, V., J. P. Flandrois, E. Broye, F. Tupin, and Y. Richard: Actinomyces pyogenes: Susceptibility of 103 clinical animal isolates to 22 antimicrobial agents. Vet. Res. 24 (1993) 251-259 12. Guerin-Faublee, V., S. Karray, B. Tilly, and Y. Richard: Actinomyces pyogenes: etude bacteriologique conventionnelle et sur galeries Api de 103 souches isolees chez les ruminants. Ann. Rech. Yet. 23 (1992) 151-160 13. Hartwigk, von H.: Atypische Merkmale von Corynebakterium (C.) pyogenes beim Rind. Dtsch. tieriirztl. Wschr. 68 (1961) 38-41 14. Hoi Sorensen, G.: Corynebacterium pyogenes. A biochemical and serological study. Acta Vet. Scand. 15 (1974) 544-554 15. Irino, K., F. Grimont, T. Casin, P. A. D. Grimont, and The brazilian purpuric fever study group: rRNA gene restriction patterns of Haemophilus in(luenzae biogroup Aegyptius strains associated with Brazilian purpuric fever. J. Clin. Microbiol. 26 (1988) 1535-1538 16. Liimmler, C.: Typing of Actinomyces pyogenes by its production and susceptibility to bacteriocin-like inhibitors. Zbl. Bakt. 273 (1990) 173-178

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17. Reddy, C. A., C. P. Cornell, and A. M. Fraga: Transfer of Corynebacterium pyogenes (Glage) Eberson to the genus Actinomyces as Actinomyces pyogenes (Glage) comb. nov. Int. J. Syst. Bact. 32 (1982) 419-429 18. Roberts, R. J.: A numerical taxonomic study of 100 isolates of Corynebacterium pyogenes. J. Gen. Microbiol. 53 (1968) 299-303 19. Ryff, J. F. and]. O. Browne: Corynebacterium pyogenes - A cultural and serological study. Am. J. Vet. Res. 15 (1954) 617-621 20. Schaffer, H. E. and R. R. Sederoff: Improved estimation of DNA fragment length from agarose gels. Analyt. Biochem. 115 (1981) 113-122 21. Tainaka, M., T. Kume, S. Takeuchi, S. Nishio, and H. Saito: Studies on the biological and serological properties of Corynebacterium pyogenes. Kitasato Arch. Exp. Med. 56 (1983) 105-117 22. Verger, J. M., F. Grimont, P. A. D. Grimont, and M. Grayon: Taxonomy of the genus Brucella. Ann. Inst. Pasteur/Microbiol. 138 (1987) 235-238 Veronique Guerin-Faublee, Service de Pathologie generale, Microbiologie et Immunologie, BP 83, F-69280 Marcy I'etoile, France, Tel (33) 78872592, Fax (33) 78872594