lated saliva was collected into a sterile glass tube. Plaque and saliva .... 79:269â272. 4. Douglas, C. W. I., J. Heath, K. K. Hampton, and F. E. Preston. 1993.
JOURNAL OF CLINICAL MICROBIOLOGY, May 1995, p. 1399–1401 0095-1137/95/$04.0010 Copyright q 1995, American Society for Microbiology
Vol. 33, No. 5
Identity of Streptococcal Blood Isolates and Oral Isolates from Two Patients with Infective Endocarditis ¨ GUTSCHIK,2 TOVE LARSEN,1 NILS-ERIK FIEHN,1* ERNO
AND
JETTE MARIE BANGSBORG1
Department of Oral Microbiology, School of Dentistry, Health Science Faculty, University of Copenhagen,1 and Department of Clinical Microbiology, Bispebjerg Hospital,2 Copenhagen, Denmark Received 11 October 1994/Returned for modification 10 December 1994/Accepted 10 February 1995
The purpose of this study was to isolate streptococcal strains from the oral cavities of streptococcal endocarditis patients and compare these strains biochemically and genetically with the corresponding streptococcal blood isolates. Total identity was observed between the blood and oral cavity isolates from the two patients studied.
phy showed vegetations on the septal leaflets of the aortic valve. Before antibiotic therapy was instituted in the two patients, samples of gingival plaque and saliva were obtained. Gingival plaque was sampled with a curette from sites with gingival inflammation and immediately transferred to 2 ml of sterile phosphate-buffered saline. Two to three milliliters of unstimulated saliva was collected into a sterile glass tube. Plaque and saliva samples and blood isolates were cultivated on mitis salivarius (M-S) agar (Difco Laboratories, Detroit, Mich.) at 378C under anaerobic conditions (70% N2, 20% H2, 10% CO2) for 2 days followed by 2 days of aerobic incubation. After 4 days, the colonies on the M-S agar plates were examined under a stereomicroscope (magnification, 310 to 340). Five to ten colonies from plaque and saliva with a colony morphology similar to that of the blood isolate on M-S agar were subcultivated on M-S agar until purity was obtained. All isolates including the blood isolate were Gram stained and characterized in accordance with the following criteria: catalase; fermentation of sorbitol, mannitol, salicin, inulin, and amygdalin; acetoin and H2O2 production; hydrolysis of arginine and esculin; dextran formation from sucrose; and activity of b-glucosaminidase, a-L-fucosidase, and acid and alkaline phosphatases (15). For patient 1, the blood isolate and a plaque isolate were identical, and according to the characteristics examined they were identified as Streptococcus mutans. The diagnosis of S. mutans was confirmed in the second blood sample. For patient 2, the blood isolate and a saliva isolate were identified as S. oralis or S. mitis. The diagnosis was verified in the three separate blood cultures. Blood isolates and corresponding plaque and saliva isolates were ribotyped. Furthermore, the following reference strains and clinical isolates from healthy subjects were included in the study and ribotyped: S. mutans ATCC 25175 (type strain); S. mutans 6715; S. mutans IB; S. oralis ATCC 35037 5 NCTC 11427 (type strain); S. mitis ATCC 33399 (type strain); S. mutans 40, 54, 70, 74, 80, 84, and 88 (8); S. oralis SK105 and SK109 (15); S. mitis SK145 (15); and S. oralis-S. mitis 53 (8). The ribotyping comprised the following steps: (i) DNA extraction by a modification of the method of Marmur (9, 18); (ii) digestion with restriction endonucleases BamHI, ClaI, EcoRI, HindIII, PstI, HaeIII, and Sau3A (Boehringer GmbH, Mannheim, Germany) in accordance with the manufacturer’s instructions; (iii) agarose gel electrophoresis (7); and (iv) Southern blotting and hybridization as described by Gerner-Smidt (9). Hybridization was carried out with a digoxigenin 11-dUTP
The incidence of infective endocarditis is about 50 cases per million citizens per year in western countries (11). Despite the apparently low number of cases, the disease must be regarded very seriously because of the high mortality of 30 to 50% (5, 14, 20). On the basis of cultivation of blood samples from endocarditis patients, up to 63% of the cases can be attributed to streptococci (1, 4, 16), and among those most frequently encountered are viridans streptococci, which are normal inhibitants of the oral cavity. Streptococcal blood isolates from endocarditis patients probably originate in the oral cavity, but identity between blood isolates and corresponding isolates from the oral cavity has not been shown. Therefore, the purpose of the present study was to isolate streptococcal strains from the oral cavities of streptococcal endocarditis patients and compare the strains biochemically and genetically with the corresponding streptococcal blood isolates. Two patients were included in the study. Both patients fullfilled the ‘‘Duke criteria’’ for definite diagnosis of infective endocarditis (6). Patient 1 was a 72-year-old male who had a persistent fever (38 to 398C) for 4 weeks, had no identifiable risk factors for endocarditis, and was admitted to our hospital for further diagnostic investigation. On admission, the patient presented no signs of skin or mucosal embolic phenomena but complained of pain in the left kidney region. The temperature was 38.18C, and sinus rhythm with no murmur was recognized by stethoscopy. Two sets of blood cultures were drawn with a 4-h interval, and cultures yielded positive growth of nonhemolytic streptococci after 48 h of incubation. A systolic murmur was recognized 2 days after admission, and transthoracic echocardiography showed large (3 by 3 mm) vegetations on the mitral valve and mitral insufficiency with valvular regurgitation. Patient 2 was a 72-year-old female who had a persistent fever (38 to 398C) for 5 weeks and no history of cardiac disease except a myocardial infarction 2 years earlier. On admission, the patient was found to be chronically ill with a temperature of 39.58C, sinus tachycardia, and a systolic murmur with a maximum in the second right intercostal space but presented no signs of skin or mucosal phenomena. Three set of blood cultures were drawn, the last two more than 12 h apart. All three cultures yielded positive growth of nonhemolytic streptococci after 48 h of incubation. Transthoracic echocardiogra-
* Corresponding author. Mailing address: Department of Oral Microbiology, School of Dentistry, Nørre Alle´ 20, 2200 Copenhagen N, Denmark. Phone: 45 35326660. Fax: 45 35326664. 1399
1400
NOTES
J. CLIN. MICROBIOL.
FIG. 1. Ribotypes of blood and plaque isolates from patient 1 and for three S. mutans reference strains after digestion of chromosomal DNAs with EcoRI, PstI, or HindIII.
FIG. 2. Ribotypes of blood and saliva isolates from patient 2 and for the S. oralis and S. mitis type strains after digestion of chromosomal DNAs with EcoRI, PstI, or HindIII.
VOL. 33, 1995
copy DNA probe of 16S and 23S rRNAs from Escherichia coli (Boehringer). For each restriction enzyme, the ribotyping was repeated twice. The band patterns for the isolates examined differed between endonucleases, but the two pairs of blood and plaquesaliva isolates always showed identical patterns with each of the different enzymes. Figure 1 shows the ribotypes of blood and plaque isolates from patient 1 and three S. mutans reference strains after digestion with EcoRI, PstI, or HindIII. The blood and plaque isolate pairs are identical to each other but different from the reference strains. The S. mutans reference strains also differ from each other, except for strains ATCC 25175 and 6715 after digestion with PstI, where the band patterns seem identical. The observations after digestion with BamHI, ClaI, HaeIII, and Sau3A were similar to the above-mentioned findings. The ribotypes of seven clinical isolates of S. mutans after digestion with each of the seven endonucleases examined were different. For patient 2, comparisons similar to those for patient 1 were made. Figure 2 shows ribotypes of a blood isolate, a saliva isolate, and the type strains of S. oralis and S. mitis after digestion with EcoRI, PstI, or HindIII. For each enzyme, the band patterns for the two isolates were identical to each other but different from those of the type strains. Similar results were observed when the other four endonucleases were used. The ribotypes of four clinical isolates of S. oralis and S. mitis after digestion with each of the endonucleases examined were different. In the present study, samples from the oral cavity were obtained immediately after it was confirmed that endocarditis was caused by nonhemolytic streptococci, presumably by a viridans streptococcus. Isolation of the viridans streptococci from M-S agar was carried out on the basis of colony morphology identity between the blood isolate and single colonies of samples from the oral cavity. For the two patients examined, total identity between the streptococcal blood isolate and an oral isolate was observed on the basis of both conventional microbiological methods and ribotyping. Ribotyping has proved useful in distinguishing between different strains of many species (3, 10, 12, 13, 17, 21). This is the first report which describes a direct comparison between blood isolates and corresponding oral cavity isolates in endocarditis patients. The identity between the corresponding pairs of isolates was verified by digestion of chromosomal DNA with seven different endonucleases. Previous investigation has demonstrated a high discriminatory power of ribotyping with three or more restriction enzymes and established the value of this method as an epidemiological tool (2). The reference strains and clinical isolates of the relevant streptococcal species from other, healthy individuals showed different ribotypes after digestion of their DNAs with the same endonucleases; this documents the genetic diversity within S. mutans and S. oralis-S. mitis, which has previously been observed for mutans streptococci (19), and strengthens the point of view that the streptococcal species isolated in blood and identified in the two endocarditis cases originated in the oral cavity.
NOTES
1401
We thank laboratory technicians Birgit Schro ¨der Nielsen and Trine Lemvigh for excellent technical assistance. REFERENCES 1. Bayliss, R., C. Clarke, C. M. Oaklay, W. Somerville, A. G. W. Whitfield, and S. E. J. Young. 1983. The microbiology and pathogenesis of infective endocarditis. Br. Heart J. 50:513–519. 2. Blanc, D. S., H. H. Siegrist, R. Sahli, and P. Francioli. 1993. Ribotyping of Pseudomonas aeruginosa: discriminatory power and usefulness as a tool for epidemiological studies. J. Clin. Microbiol. 31:71–77. 3. Bowman, R. A., G. L. O’Neill, and T. V. Riley. 1991. Non-radioactive restriction fragment length polymorphism (RFLP) typing of Clostridium difficile. FEMS Microbiol. Lett. 79:269–272. 4. Douglas, C. W. I., J. Heath, K. K. Hampton, and F. E. Preston. 1993. Identity of viridans streptococci isolated from cases of infective endocarditis. J. Med. Microbiol. 39:179–182. 5. Douglas, I. L., and C. G. Cobbs. 1992. Prosthetic valve endocarditis, p. 375–396. In D. Kaye (ed.), Infective endocarditis, 2nd ed. Raven Press, Ltd., New York. 6. Durack, D. T., A. S. Lukes, and D. K. Bright. 1994. New criteria for diagnosis of infective endocarditis: utilization of specific findings. Duke Endocarditis Service. Am. J. Med. 96:200–209. 7. Fiehn, N.-E., J. M. Bangsborg, and H. Colding. 1995. Ribotyping on smallsized spirochetes isolated from subgingival plaque. Oral Microbiol. Immunol. 10:13–18. 8. Fiehn, N.-E., and D. Moe. 1984. Sucrase and maltase activities in supragingival dental plaque in humans of streptococcal, actinomyces, and lactobacilli species. Scand. J. Dent. Res. 92:97–108. 9. Gerner-Smidt, P. 1992. Ribotyping of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex. J. Clin. Microbiol. 30:2680–2685. 10. Goering, R. V., and T. D. Duensing. 1990. Rapid field inversion gel electrophoresis in combination with an rRNA gene probe in the epidemiological evaluation of staphylococci. J. Clin. Microbiol. 28:426–429. 11. Gutschik, E., and S. Lippert. 1990. Dental procedures and endocarditis prophylaxis: experiences from 108 dental practices. Scand. J. Dent. Res. 98:144–148. 12. Hadorn, K., W. Lentz, F. H. Kayser, I. Shalit, and C. Krasemann. 1990. Use of a ribosomal RNA gene probe for epidemiological study of methicillin and ciprofloxacin resistant Staphylococcus aureus. Eur. J. Clin. Microbiol. Infect. Dis. 9:649–653. 13. Irino, K., F. Grimont, I. Casin, P. A. D. Grimont, and The Brazilian Purpuric Fever Study Group. 1988. rRNA gene restriction patterns of Haemophilus influenzae biogroup aegyptius strains associated with Brazilian purpuric fever. J. Clin. Microbiol. 26:1535–1538. 14. Janatuinen, M. J., E. A. Va ¨ndttinen, V. Rantakokko, et al. 1991. Prosthetic valve endocarditis. Scand. J. Thorac. Cardiovasc. Surg. 25:127–132. 15. Kilian, M., L. Mikkelsen, and J. Henrichsen. 1989. Taxonomic study of viridans streptococci: description of Streptococcus gordonii sp. nov. and emended descriptions of Streptococcus sanguis (White and Niven 1946), Streptococcus oralis (Bridge and Sneath 1982), and Streptococcus mitis (Andrews and Harder 1906). Int. J. Syst. Bacteriol. 39:471–484. 16. Kjerulf, A., M. Tvede, and N. Ho ¨iby. 1993. Crossed immunoelectrophoresis used for bacteriological diagnosis in patients with endocarditis. APMIS 101: 746–752. 17. Lawson, A. P., S. E. Gharbia, H. N. Shah, and D. R. Clark. 1989. Recognition of Fusobacterium nucleatum subgroups Fn-1, Fn-2, and Fn-3 by ribosomal RNA gene restriction patterns. FEMS Microbiol. Lett. 65:41–46. 18. Marmur, J. 1961. A method for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol. 3:208–217. 19. Saarela, M., S. Alaluusua, T. Takei, and S. Asikainen. 1993. Genetic diversity within isolates of mutans streptococci recognized by an rRNA gene probe. J. Clin. Microbiol. 31:584–587. 20. Strate, M., and J. S. Nielsen. 1987. Infective endocarditis. A retrospective analysis of 34 cases. Ugeskr.Læg. 149:3031–3033. 21. Yogev, D., D. Halachmi, G. E. Kenny, and S. Razin. 1988. Distinction of species and strains of mycoplasmas (Mollicutes) by genomic DNA fingerprints with an rRNA gene probe. J. Clin. Microbiol. 26:1198–1201.
