anaerobic jar equipped with a BBL Campy-Pak apparatus. (Becton Dickinson and Co.) or in a candle jar (strains LPKS. 31 and LPKS 32 only), and anaerobically ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1991, p. 3037-3039
Vol. 57, No. 10
0099-2240/91/103037-03$02.00/0 Copyright © 1991, American Society for Microbiology
Phenotypic Characterization of C02-Requiring Strains of Streptococcus bovis from Koalas R. OSAWA1* AND L. I. SLY2 Veterinary Service and Research, Lone Pine Koala Sanctuary, Fig Tree Pocket,' and Department of Microbiology,
University of Queensland, St. Lucia,2 Brisbane, Queensland, Australia Received 16 April 1991/Accepted 29 July 1991
We examined phenotypic characteristics of six mannitol-fermenting strains of Streptococcus bovis, including two unusual C02-requiring strains isolated from koala feces. These strains did not grow in air, but grew in air supplemented with CO2 and under reduced oxygen conditions. All six strains had the same biochemical characteristics, except that the C02-requiring strains did not produce ,D-N-acetylglucosaminidase.
colonial morphology, cellular morphology, Gram staining, and catalase and hemolytic activities of the strains were also determined. Growth and clear zone formation on tannintreated brain heart infusion agar were observed in order to determine tannin-protein complex degradation, as described previously (14, 15). The strains were all catalase negative, gram-positive cocci which produced smooth, white, alpha-hemolytic colonies on Columbia blood agar and larger white flat colonies with surrounding clear zones on tannin-treated brain heart infusion agar. The anaerobiosis and biochemical characteristics of the strains are summarized in Table 1. Strains LPKS 1, LPKS 6, UQM 3549, and UQM 3552 grew on Columbia blood agar aerobically and anaerobically, whereas strains LPKS 31 and LPKS 32 did not grow in the presence of 100% air but grew under anaerobic or reduced oxygen conditions in a candle jar or in a jar equipped with a Campy-Pak apparatus. This observation suggested that strains LPKS 31 and LPKS 32 are aerotolerant. However, further testing showed that these strains had a requirement for CO2 and grew well in air supplemented with CO2. Most anaerobic atmospheres currently used include CO2 at concentrations ranging from 3 to 15%. Our observations demonstrate the need to include a C02-air control to accurately interpret requirements for anaerobic growth. In addition, the possibility of the presence of C02-requiring strains must be taken into account when the total streptococci in koala feces are determined. A CO2 requirement has been determined for some strains of other species of streptococci. For example, the inability of some strains of "Streptococcus milleri" to grow in air alone has been misinterpreted as a dependence on anaerobic conditions (1, 6) which could be overcome by CO2 supplementation of an aerobic atmosphere. Strains of S. bovis normally grow aerobically. However, Latham et al. (11) described the isolation of anaerobic strains from rumina and ceca of cows and calves. The growth of these strains was enhanced by the addition of CO2, and after repeated subculturing in air containing 10% CO2 these strains grew on plates incubated aerobically. In contrast, we did not observe any tendency for the koala isolates to grow in air alone after repeated subculturing. Devriese et al. (4) described Streptococcus cecorum as a species which includes strains that are similar to S. bovis strains obtained from the ceca of chickens, the growth of which is enhanced by, but not dependent on, the addition of CO2. However, S. cecorum is only distantly related to S.
Streptococcus bovis is commonly found in the normal gastrointestinal floras of many animals (2, 3, 8, 16) and also is often isolated in clinical situations, such as cases of human bacteremia (9, 10, 13), endocarditis (7, 17), and bovine mastitis and udder infections (5, 12). Although S. bovis is usually considered to be a facultative anaerobe (6), Latham et al. (11) reported that this species includes strains which are strictly anaerobic when they are first isolated. Recently, Osawa (14) and Osawa and Mitsuoka (15) investigated the phenotypic characteristics of facultatively anaerobic strains of S. bovis obtained from various sources. We isolated apparently anaerobic strains from the feces of two healthy koalas (Phascolarctos cinereus (Goldfuss), and in this study we characterized these strains. Six strains of S. bovis, including two that are not able to grow in air, were obtained from our collections at the Lone Pine Koala Sanctuary (strains LPKS 1, LPKS 6, LPKS 31, and LPKS 32) and the Department of Microbiology, University of Queensland, Queensland, Brisbane, Australia (strains UQM 3549 and UQM 3552). The sources of the strains are indicated in Table 1. Cultures of these strains, which were stored as lyophiles, were revived and subcultured anaerobically at least three times on plates containing Columbia blood agar (Oxoid, Ltd., Basingstoke, Hampshire, United Kingdom) in an atmosphere enriched with 8 to 10% CO2 by using Bio-bags (Becton Dickinson and Co., Cockeysville, Md.) before use. The oxygen requirements of the strains were determined by incubating inoculated Columbia blood agar plates aerobically in the air, microaerophilically (5 to 15% C02) in an anaerobic jar equipped with a BBL Campy-Pak apparatus (Becton Dickinson and Co.) or in a candle jar (strains LPKS 31 and LPKS 32 only), and anaerobically in an anaerobic jar equipped with a BBL GasPak apparatus (Becton Dickinson and Co.) and in Bio-bags (Beckton Dickinson and Co.) at 37°C for up to 5 days. The ability to grow in an atmosphere containing 3 to 12% CO2 in air was determined by enriching the air in a jar with CO2 generated by using a BBL CO2 GasPak apparatus (Becton Dickinson and Co.). The biochemical characteristics of the strains were determined by using the following three commercially available identification kits: API 20 STREP (API System, Montalieu, Vercieu, France), ATB 32A (API System), and RapID ANA II system (Innovative Diagnostic Systems, Inc., Atlanta, Ga.). The *
Corresponding author. 3037
3038
APPL. ENVIRON. MICROBIOL.
NOTES TABLE 1. Phenotypic characteristics of six S. bovis strainsa Strain LPKS 1
Strain LPKS 6
Strain
LPKS 31
Strain LPKS 32
Strain
Characteristic
UQM 3549
UQM 3552
Gram stain Catalase Beta-hemolysis Growth and clear zone formation on T-TBHIAb Oxygen tolerance under the following growth conditions: Aerobic Microaerophilic Anaerobic CO2 requirement Voges-Proskauer reaction' Hippurate hydrolysis' Esculin hydrolysisc Production of: Pyrrolidonylarylamidasec ot-Galactosidase'
+
+
+
+
+
+
+
+
+
+
+
+
+ + +
+ + +
+ + +
+ + +
+ + +
+
+ + + +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Alkaline phosphatasec Leucine arylamidasec Arginine dihydrolase'
+
+
+
+
+
+
P-N-Acetylglucosaminidased
-
-
+
+
+
+
+
+
+
+
+
+
+ +
+ +
+ +
+ +
+ +
+ +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
.Strain Characteristc
P-Glucuronidase'
P-Galactosidase'
Acid produced from': Ribose L-Arabinose Mannitol Sorbitol Lactose Trehalose Inulin Raffinose Starch Glycogen
' Strains LPKS 31, LPKS 32, LPKS 1, and LPKS 6 were isolated from koala feces. Strains UQM 3549 (= NCDO 2135) and UQM 3552 (= NCDO 2631) were obtained from the National Collection of Dairy Organisms, Reading, United Kingdom. b T-TBHIA, tannin-treated brain heart infusion agar. Determined by using API 20 STREP tests. d Determined by using ATB 32A and RapID ANA II tests.
bovis as determined by DNA-DNA homology experiments and may be distinguished by positive reactions for ribose, melezitose, ,-glucuronidase, and alkaline phosphatase. In this study aerobic strains and C02-requiring strains were found to have the same biochemical characteristics, as determined by API 20 STREP tests, and were identified as S. bovis biotype I (mannitol fermenting). On the other hand, subsequent tests in which we used ATB 32A and RapID ANA II tests on the six strains of S. bovis revealed that these strains share the same biochemical characteristics, except that C02-requiring strains LPKS 31 and LPKS 32 lack P-N-acetylglucosaminidase. Studies with more strains are needed to determine whether the observed absence of this enzyme is specific to the C02-requiring strains of S. bovis or is a variable characteristic within the species. We thank D. Grassick of the Department of Microbiology, University of Queensland, for expert technical assistance. REFERENCES 1. Ball, L. C., and M. T. Parker. 1979. The cultural and biochemical characters of Streptococcus milleri strains isolated from human sources. J. Hyg. 82:63-78. 2. Bauchop, T. 1971. Stomach microbiology of primates. Annu. Rev. Microbiol. 25:429-435. 3. Dehority, B. A., and J. A. Grubb. 1977. Characterisation of the predominant bacteria occurring in the rumen of goats (Capra
hircus). Appi. Environ. Microbiol. 33:1030-1036. 4. Devriese, L. A., G. N. Dutta, J. A. E. Farrow, A. Van De Kerckhove, and B. A. Phillips. 1983. Streptococcus cecorum, a new species isolated from chickens. Int. J. Syst. Bacteriol. 33:772-776. 5. Garvie, E. I., and A. J. Bramely. 1979. Streptococcus bovis-an approach to its classification and its importance as a cause of bovine mastitis. J. Appl. Bacteriol. 46:557-566. 6. Hardie, J. M. 1986. Anaerobic streptococci, p. 1066-1071. In P. H. A. Sneath, N. S. Mair, M. E. Sharp, and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 2. The Williams & Wilkins Co., Baltimore. 7. Hoppes, W. L., and P. I. Lerner. 1974. Nonenterococcal group D streptococcal endocarditis caused by Streptococcus bovis. Ann. Intern. Med. 81:588-593. 8. Hungate, R. E. 1966. The rumen and its microbes. Academic Press, Inc., London. 9. Keil, P., and K. Skadhauge. 1973. Studies on mannitol fermenting strains of Streptococcus bovis. Acta Pathol. Microbiol. Scand. Sect. B 81:10-14. 10. Klein, R. S., M. T. Catalano, S. C. Edberg, J. I. Casey, and N. H. Steigbigel. 1979. Streptococcus bovis septicemia and carcinoma of the colon. Ann. Intern. Med. 91:560-562. 11. Latham, M. J., M. E. Sharpe, and N. Weiss. 1979. Anaerobic cocci from the bovine alimentary tract, the amino acids of their cell wall peptidoglycans and those of various species of anaerobic Streptococcus. J. Appl. Bacteriol. 47:209-221. 12. McDonald, T. J., and J. S. McDonald. 1976. Streptococci isolated from bovine intramammary infections. Am. J. Vet. Res.
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37:377-381. 13. Murray, H. W., and R. B. Roberts. 1978. Streptococcus bovis bacteremia and underlying gastrointestinal disease. Arch. Intern. Med. 138:1097-1099. 14. Osawa, R. 1990. Formation of a clear zone on tannin-treated brain heart infusion agar by a Streptococcus sp. isolated from feces of koalas. Appl. Environ. Microbiol. 56:829-831. 15. Osawa, R., and T. Mitsuoka. 1990. Selective medium for enu-
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meration of tannin-protein-complex-degrading Streptococcus spp. in feces of koalas. Appl. Environ. Microbiol. 56:3609-3611. 16. Raibaud, P., M. Caulet, J. V. Galpin, and G. Mocquot. 1961. Studies on the bacterial flora of the alimentary tract of pigs. Il. Streptococci: selective enumeration and differentiation of the dominant group. J. Appl. Bacteriol. 24:285-306. 17. Watanakunakorn, C. 1974. Streptococcus bovis endocarditis. Am. J. Med. 56:256-260.
