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Vol. 36, No. 3 INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, July 1986, p. 431434 0020-7713/86/030431-04$02.00/0 Copyright 0 1986, International Union of Microbiological Societies Phenotypic and Genotypic Comparisons among Strains of the Lobster Pathogen Aerococcus viridans and Other Marine Aerococcus viridans-Like Cocci R. WIIK,’* V. TORSVIK,’ AND E. EGIDIUS’ Institute of Marine Research, Directorate of Fisheries, 501 1 Bergen-Nordnes, and Department of Microbiology and Plant Physiology, University of Bergen, N-5014 Bergen- University,’ Norway ’ The lobster pathogen Aerococcus viridans and other gram-positive, marine A. viridans-like cocci were examined morphologically, biochemically, and genetically. Morphologically, the lobster pathogenic strains were unique in their tetrad-forming capacity. Because of intragroup fermentative variations among the lobster-pathogenic strains and values of deoxyribonucleic acid (DNA) base composition overlapping those of the other cocci, the lobster pathogens did not stand out as a separate group based on these data. According to the DNA-DNA hybridization studies, however, the lobster-pathogenic strains were very closely related (80 to 100% homology) and could be easily separated from the remaining strains. All the cocci had extraordinarily small genomes ranging from 0.57 X lo9 to 1.01 X lo9 daltons. The lobster pathogen Aerococcus viridans (8), originally “ G a r n u homari” (12), is a gram-positive tetracoccus which causes fatal septicemia (gaffkemia) in lobsters. There has been considerable disagreement about the taxonomic relationship of A . viridans and “ G . homari.” A . viridans strains initially included cocci isolated from air (22). Deibel and Niven ( 5 ) proposed to include both the air cocci and the lobster pathogens in the genus Pediococcus, although subsequent deoxyribonucleic acid (DNA) homology studies (18, 19) did not support a close relationship to this genus. Kelly and Evans (14) examined DNA-DNA homology among lobster-pathogenic strains and nonmarine strains of A . viridans and found strong evidence that both groups belong to the same species; this designation was introduced in the 8th edition of Bergey ’s Manual of Determinative Bacteriology (8). Kelly and Evans (14) also suggested that it might be desirable to separate the lobster-pathogenic strains from avirulent strains of A . viridans by considering the lobster pathogen a subspecies. In the present text we generally refer to strains of A , viridans originating from gaflkemic lobsters as A . viridans GL. The present study compared the morphology, physiology, and genetic features of A . viridans GL strains, isolated from two species of lobsters (Homarus gammarus L. and H . americanus H. Milne Edwards 1837), and of representative avirulent strains of A . viridans-like cocci of marine origin. Since most earlier genomic studies of A . viridans GL examined only isolates from the lobster H . americanus (14, 18, 19), the present study provides additional data on the biology and genetic characteristics, including DNA base composition, genome size, and ,DNA-DNA hybridization comparisons, among other lobster strains. MATERIALS AND METHODS Bacterial strains. A . viridans GL strains HI520, HI510, and SH21 were isolated from Norwegian H. gammarus; STA.14, STA.18, and Rabin were from H . americanus (J. E. Stewart, Fisheries and Oceans, Resource Branch, Nova Scotia, Canada); ATCC 10400 was from H . americanus (American Type Culture Collection, Rockville, Md.); * Corresponding author. NCMB 1120 was from British H . gammarus (National Collection of Marine Bacteria, Aberdeen, Scotland); and A . viridans strain NCTC 8251T (T indicates type strain) was from air (National Collection of Type Cultures, Colindale, England). A . viridans GL strain KB161 and the unidentified marine strains KB162, KB172, SH22, SH23, and SH31 were isolated from water or sediment in Norwegian lobster ponds. The marine isolates 37R and 88A were supposed to be avirulent strains of A . viridans (J. E. Stewart). Escherichia coli B (ATCC 11303) was used as a reference strain in the DNA studies. Morphological and biochemical characteristics. Capsule staining was done by the method of Collins (3). Bacterial cells were grown at 30°C for 24 h on nutrient agar (Oxoid Ltd., London, England) supplemented with 5% human blood. This temperature and incubation period were generally used throughout the study as standard growth conditions. The Hucker modification of Gram stain (4) was used on cells grown on brain heart infusion agar (BHIA) (Difco Laboratories, Detroit, Mich.). Cell morphology and motility were determined microscopically on cultures grown in tryptone soya broth (TSB) (Oxoid). Stability of tetrad formation was tested by ultrasonic treatment (MSE-Mullard Ultrasonic Disintegrator, MSE Scientific Instruments Ltd., Crawley, West Sussex, England) of 5-ml TSB cultures of A . viridans GL strains for 5 , 10, 20, 30, 60, and 120 s . Each culture was studied microscopically and counted with a counting chamber (Hawksley and Sons Ltd., Lansing, West Sussex, England) before and after treatment. Hemolysis was tested by growing the bacteria for 48 h on nutrient agar containing 5% defibrinated sheep blood. Catalase production was detected by transferring bacterial cells grown on BHIA to 1 drop of 3% H202solution. Acid production was measured by growing the organisms in a medium containing 0.65% glucose, 0.45% yeast extract (Oxoid), 1.5% tryptone (Oxoid), 0.64% NaCl, 0.25% phenyl ethyl alcohol, and 0.0008%bromcresol purple (wthol) (pH 7.4) (21). pH readings were taken after 24 h. The fermentative ability of the organisms was determined by the API 50 CHE system (API System SA, France). The bacteria were grown on tryptone soya agar (Oxoid) before transfer to the API 50 CHE medium. Readings were taken after 48 h. 431 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 14:31:49 432 INT. J . SYST.BACTERIOL. WIIK ET AL. Virulence tests. Bacterial strains were grown in TSB and diluted in 3% NaCl to concentrations from 6 x lo1 to 2.6 x lo4 CFU/ml; 0.5 ml of the suspension was injected into the lobsters, each of which weighed approximately 0.5 kg and had been shown to be free of the pathogen by the method of Stewart et al. (21) before being included in the experiment. In moribund or newly dead lobsters, gaffkemia was easily recognized by microscopic appearance of numerous grampositive tetrads in the hemolymph. DNA extraction and purification. Strains were grown in TSB for 17 to 30 h, and cells were harvested just before the stationary growth phase. E. coli B was cultivated in a medium containing 0.3% meat extract (Difco), 0.5% peptone (Oxoid), and 0.2% yeast extract (Oxoid) dissolved in distilled water (wt/vol) at 37°C for 15 h. The cells were harvested and lysed, and the nucleic acids were extracted and purified by the method of Marmur (16), with two modifications: immediately after adding sodium lauryl sulfate, the lysozymetreated cell mixtures became viscous and nearly crystal clear, and the prescribed incubation at 60°C was therefore omitted; and the DNA from the cocci was easily spooled onto a glass rod after mild shaking of the mixture by hand. Ratios of absorbance at 260 nm (A260)/A280 and A260/A230 were used to assess DNA purity. Guanine plus cytosine (G+C) content of DNA was determined by thermal denaturation (15), with 0 . 5 ~standard saline citrate (SSC) ( l x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) as the solvent and DNA at concentrations corresponding to initial A260 of 0.4 to 0.7, representing 20 to 35 pg of DNA per ml(l5). A Pye Unicam spectrophotometer connected to a microprocessor (Rockwell, AIM 65, MicroNor) was used in the DNA studies. Moles percent (mol%) G + C was calculated by the equation: mol% G+C = ( T , - 69.37 - 15.2 log 0.5)/0.41, where T,,, is melting temperature (10). Hyperchromicity was calculated as the percent increase in A260. Determination of M. The molecular weight of the bacterial genome (M) was determined based on initial renaturation rate of fragmented DNA, recorded spectrophotometrically (11). The renaturation temperatures of DNA from the cocci and E. coli B were 64.5 and 72”C, respectively. The A260 of sheared DNA in the renaturation solution was 0.56 at 22”C, equivalent to a DNA concentration of 28 pglml (15). The molecular weight of the bacterial genome was calculated by the equation: M = (70.03 - 0.35 x mol% G+C) x 107/k’ (9), where k’ represents the reaction rate constant. DNA-DNA hybridization. Determination of the genetic relationship between two bacterial strains was based on initial renaturation rates of the DNA types and their mixture, recorded spectrophotometrically (6). The percentage of hybridized DNA was calculated by equation 19 of De Ley et al. (6). Two modifications of the methods of De Ley et al. (6) and Gillis et al. (10) were used: spectrophotometric instead of chemical determination of DNA concentration in the renaturation solution, and decrease of DNA concentration in the renaturation solution from a 80 to 28 Fg/ml (A260 = 0.56). As the value of k’ for E . coli B DNA is in agreement with the values given by Gillis et al. (9, lo), determining the DNA concentration spectrophotometrically instead of chemically and decreasing the concentration from 80 to 28 pg of DNA per ml seems justifiable. The genome size of E. coli B is also in agreement with figures given by Bak et al. (1) and Cairns (2). Huss et al. (13) recommended that a concentration of 30 to 40 pg of DNA per ml be used in the method developed by De Ley et al. (6). Four to six replicates per DNA type mixture were used, and reproducibility was obtained with an TABLE 1. Virulence of A . vividuns GL strains tested by lobster infection experiments Strain No. of lobsters infected ATCC 10400 2 HI520 2 SH21 2 KB161 HI510 1 1 CFU injected Water temp (“‘I 18.8 +18.2 +18.8 ? 18.2 k 18.7 2 18.3 2 18.0 2 12.0 2 1.2” 1.4 2.0 2.2 1.4 1.1 2.0 1.0 (2.17 2 (2.60 5 (1.31 ? (1.53 k (9.75 ? (3.50 & (5.60 2 (3.00 2 0.19’) 0.21) 0.05) 0.05) 0.79) 0.30) 0.41) 0.19) x 10’ x 103 X lo4 x 10’ x lo2 x lo2 x lo3 x 10’ Time to death (h) 7 7 5 5 7 11 Average deviation from the average temperature. Standard error (11). accuracy for change in A260of 2 5 x represents v’ 5 1.88 X lop5. (k0.25 mm), which RESULTS All bacterial strains examined were nonmotile, gram positive, alpha-hemolytic, catalase positive, and acid producing. For the A . viridans GL strains, the pH of the medium after growth, which stabilized after 24 h, varied from 5.5 to 5.8, and for the remaining strains, the pH ranged from 4.2 to 5.4. Only the A . viridans GL strains formed tetrads. The other strains were diplococci, except for SH23, which formed slightly ovoid cells in pairs and chains. Before ultrasonic treatment, A . viridans GL occurred in tetrads and in clusters of numerous cells. After 20 s of ultrasonic treatment, only tetrads could be seen under the microscope, and the number of cell units had increased 17 times, indicating that each cluster contained an average of 17 tetrads. After 120 s of treatment many of the tetrads had divided into diplococci, and the number of cell units had increased by a factor of 28. Only strains of A . viridans GL appeared to form capsules on blood agar. In the API 50 CH tests, A . viridans GL strains gave the same general reaction pattern, fermenting galactose, D-glucose, D-fructose, D-mannose, mannitol, N acetylglucosamine, amygdalin, arbutin, esculin, cellobiose, maltose, lactose, saccharose, trehalose, inulin, D-raffinose, P-gentibiose, gluconate, and giving a rather weak positive fermentation of ribose. The strains showed intragroup variation with respect to fermentation of sorbitol, a-methyl-Dglucoside, melibiose, D-turanose, and 5-ketogluconate. A . viridans NCTC 8251T differed from the lobster-derived strains in inability to ferment trehalose, inulin, D-raffinose, p-gentibiose, and gluconate, and by demonstrating only weak positive fermentat i o n of N - acet y lglu c o s ami n e , amygdalin, arbutin, and esculin. The avirulent A . viridans strains also presented some differences in fermentative patterns: strain 37R did not ferment ribose or inulin, and strain 88A was not able to catabolize ribose, inulin, gluconate, mannitol, or amygdalin. In addition, the other marine, A . viridans-like cocci displayed fermentative patterns that differed from those of A . viridans G L isolates: strains KB162 and KB172 both failed to ferment inulin, amygdalin, arbutin, esculin, cellobiose, trehalose, P-gentibiose, or gluconate; strain SH22 did not ferment mannitol, trehalose, or gluconate, but D-lyxose, D-fucose, and L-fucose; strain SH23 did not ferment galactose, lactose, trehalose, inulin, D-raffinose, or gluconate; and strain SH31 failed to ferment ribose, galactose, P-gentibiose, or gluconate. The limited number of lobsters available excluded virulence tests on all bacterial strains in this study. Of those Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 14:31:49 VOL.36, 1986 LOBSTER PATHOGEN A . VZRIDANS TABLE 2. DNA base composition and genome size of various A . viriduns and A . viriduns-like strains G+CQ Mol% Hyperchromicity (%) kt M (109 daltons)b E . coli B 52.2 34.0 0.192 2.70 A . viriduns GL HI520 HI510 KB161 SH21 Rabin STA. 14 STA.18 ATCC 10400 NCMB 1120 38.8 38.8 38.8 39.8 38.3 38.8 39.8 37.6 39.0 34.1 35.9 27.4 36.9 28.7 28.4 33.8 30.8 26.3 0.804 0.804 0.877 0.765 0.785 0.804 0.731 0.824 0.70 0.70 0.64 0.73 0.71 0.70 0.77 0.69 A . viriduns (avirulent) 37R 88A 39.3 39.8 30.4 35.4 0.692 0.987 Strain Unidentified marine isolates ( A . viriduns-like) KB162 KB172 SH22 SH23 SH31 34.7 31.5 34.4 39.2 34.0 39.8 40.0 37.3 37.1 39.5 0.81 0.63 0.984 0.565 0.57 1.01 0.897 0.63 The values of DNA base composition (mol% G + C) and hyperchromicity were determined by thermal denaturation in 0.5 x SSC. M was determined from the DNA renaturation rate, measured spectrophotometrically; k', apparent renaturation rate constant. ~~ ~~~ ~~~~~~~~ ~ 433 TABLE 3. DNA-DNA hybridization among various A . viridans GL isolates and other A . viriduns and A . viridans-like strains Strains used for recipient DNA A . viriduns GL HI520 HI510 KB161 SH21 Rabin STA. 14 STA.18 ATCC 10400 A . viriduns (avirulent) 37R 88A Unidentified marine isolates ( A , viriduns-like) KB172 SH22 SH31 Donor DNA" prepared from: HI520 100 100 87. 86 93 96 80 84 KB161 SH21 86 100 86 86 100 56 48 38 26 1 33 31 a Percent hybridized DNA, determined spectrophotometrically, from initial renaturation rates. Values are normalized to 100% for homologous combinations. ~ a examined, only strain ATCC 10400 was found to be avirulent (Table 1). The A26dA280ratio of DNA ranged from 1.8 to 2.0, with a mean value of 1.8. The A26dA230 ratio ranged from 1.6 to 1.8 with a mean of 1.7 for the A . viriduns G L strains plus 37R and 88A, and from 1.9 to 2.2 with a mean of 2.1 for the unidentified strains and for E. coli B. The G+C values of DNA are given in Table 2. By melting the DNAs several times, an accuracy of k0.2 mol% G + C could be estimated. For E . coli B DNA, G+C was 52.2 mol%, which is in accordance with previously found G + C values (10; K. Salte, Ph.D thesis, University of Bergen, Norway, 1981). DNA from A . viriduns ATCC 10400 had G + C of 37.6 mol%, which is in accordance with previous results (14, 18, 19). In addition to the absorbance ratios, the hyperchromicity values indicated pure DNA (lo), as did the regularity of thermal melting profiles. Molecular weights of the bacterial genomes are given in Table 2. The mean 2 standard deviation for A . viriduns G L is (0.71 +. 0.04) x lo9. The DNA-DNA hybridizations showed a high degree of relatedness (80 to 100% hybridized DNA) among the strains of A . viriduns GL; the relatedness between A . viriduns GL and the remaining strains varied from 1 to 56% (Table 3). At similarities below 25 to 30%, hybridized DNA values calculated by equation 19 of De Ley et al. (6) are less reliable and give only semiquantitative values (6, 13). DISCUSSION The nonmarine strain A . viriduns NCTC 8251, which was originally described by Williams et al. (22), deviated from the lobster-pathogenic group in five fermentative tests, and by giving a sparse, granular suspension of growth in TSB. Because of variation in fermentative ability, both among nonmarine strains of A . viriduns (22) and lobster-pathogenic strains, fermentative testing does not clearly differentiate between these two groups. A prominent phenotypic characteristic of the A . viriduns G L strains was their tetrad-forming capacity, not found in other strains in the present study. Nonmarine strains of A . viriduns may also form packets of but according to Williams et al. (22) they normally four (3, occur in pairs or irregular clusters. Serological grouping of virulent and avirulent strains of A . viriduns indicates that virulent strains possess a special antigen absent from avirulent strains (20). Steenbergen et al. (20) emphasize, however, that the presence or absence of the virulence antigen is not in itself justification for dividing the organisms into two taxonomic groups. By injection in lobster, strain ATCC 10400 appeared to be avirulent. Earlier, this strain was shown to be pathogenic to both H . gammurus (7) and H . americunus (12). This loss of virulence was not reflected in morphologic, biochemical, or genetic changes, when compared to corresponding characteristics of virulent strains. The G + C values of DNA obtained for the A . viriduns GL strains overlap those previously found for nonmarine strains of A . viriduns (14, 18, 19). Our DNA-DNA hybridization results showed a very close relationship among the lobsterpathogenic strains. In a previous DNA-DNA hybridization study, a comparatively high degree of homology between lobster-pathogenic and nonmarine A . viriduns was observed (14). In contrast to the very high degree of homology among the lobster-pathogenic strains, however, the nonmarine aerococci showed a rather large intragroup variation. According to our DNA-DNA hybridization results, the strains 37R, 88A, KB172, SH22, and SH31 hardly belong to the species A . viridans, represented by the lobsterpathogenic group. The different homology values between reference strain HI 520 and each of the unidentified strains may reflect a rather distant relationship among the unidentified strains. The API 50 CH tests, however, did not give a Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 14:31:49 434 INT. J. SYST.BACTERIOL. WIIK ET AL. satisfactory separation of the bacterial groups or strains. As an example, strain 37R deviated from the lobster pathogenic group, which in itself varied with respect to five traits, solely in its inability to ferment ribose and inulin, while the avirulent A. viridans strain NCTC 8251 deviated from the lobster pathogens in five different fermentative traits. The fermentative variations indicate, however, that these strains probably belong to several different groups. Nor did determination of DNA base composition give a clear differentiation between these strains, exhibiting G + C values ranging from 37.1 to 40.0 mol%. According to Schultes and Evans (19), A. viridans species represents G + C values ranging from 37.0 to 40.2 mol%. The A. viridans GL strains examined here have remarkably small genomes, ranging from 0.57 X lo9 to 1.01 X lo9 daltons. Organisms in the class Mollicutes (mycoplasmas) which represent the smallest genomes previously recorded for procaryotes, have genome sizes of 0.5 x lo9 1.0 X lo9 daltons (17). Bak et al. (1)determined bacterial genome sizes by DNA-DNA renaturation studies and found molecular weights ranging from 1.0 X lo9 to 7.0 x lo9; selected members of the family Micrococcaceae had genome sizes from 1.12 x lo9 to 2.82 x lo9 daltons, and those in the genus Streptococcus had genome sizes from 1.20 x lo9 to 1.37 x lo9 daltons. Haemophilus influenzae and Neisseria catarrhalis had the smallest genomes (1.01 x lo9 and 1.04 x lo9 daltons, respectively), and Pseudomonas aeruginosa had the largest (6.96 x lo9 daltons). Based on DNA-DNA renaturation rates, Gillis and De Ley (9) determined the genome sizes of 40 different bacterial strains ranging from 1.40 X lo9 to 3.99 x lo9 daltons, the extremes represented by Nitrosomonas sp. and Serratia marcescens, respectively. The very small genomes obtained for the bacteria in the present study show that genomes smaller than 1.0 x lo9 daltons also exist among procaryotes other than the mycoplasmas . ACKNOWLEDGMENTS We thank J. Goksgyr? K. Salte, and K. A. Hoff for their support during this study, and A. S . Lyssand for her secretarial assistance. LITERATURE CITED Bak, A. L., C. Christiansen, and A. Stenderup. 1970. Bacterial genome sizes determined by DNA renaturation studies. J. Gen. Microbiol. 64:377-380. Cairns, J. 1963. The chromosome of Escherichia coli. Cold Spring Harbor Symp. Quant. Biol. 28:4346. Collins, C. H. 1964. Microbiological methods, p. 84. Butterworth & Co., Ltd., London. Conn, H. J., J. W. Bartholornew, and M. W. Jennisson. 1954. Leaflet IV, Staining methods, p. 7. In Committee on Bacteriological Technic of the Society of American Bacteriologists (ed.), Manual of methods for pure culture study of bacteria. Biotech Publications, New Y ork. 5. Deibel, R. H., and C. F. Niven, Jr. 1960. Comparative study of G a m u homari, Aerococcus viridans, tetrad-forming cocci from meat curing brines, and the genus Pediococcus. J. Bacte1301. 79:175-180. 6. De Ley, J., H. Cattoir, and A. Reynaerts. 1970. The quantitative measurement of DNA hybridization from renaturation rates. Eur. J. Biochem. 12:133-142. 7. Egidius, E. 1972. On the internal bacterial flora of the European lobster (Homarus vulgaris) and its susceptibility of gaffkemia. Aquaculture 1:193-197. 8. Evans, J. B. 1974. Genus IV. Aerococcus Williams, Hirch and Cowan 1953, 475, p. 515-516. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey’s manual of determinative bacteriology, 8th ed. The Williams & Wilkins Co., Baltimore. 9. Gillis, M., and J. De Ley. 1975. Determination of the molecular complexity of double-stranded phage genome DNA from initial renaturation rates: the effect of DNA base composition. J. Mol. Biol. 98:447-464. 10. Gillis, M., J. De Ley, and M. De Cleene. 1970. The determination of molecular weight of bacterial genome DNA from renaturation rates. Eur. J. Biochem. 12:143-153. 11. Heuch, I. 1982. Syllabus of the subject M 152: applied statistics. Institute of Mathematics, University of Bergen, Norway. 12. Hitchner, E. R., and S. F. Snieszko. 1947. A study of a microorganism causing a bacterial disease of lobsters. J. Bacteriol. 5458. 13. HUSS,V. A. R., H. Festl, and K. H. Schleiter. 1983. Studies on the spectrophotometric determination of DNA hybridization from renaturation rates. Syst. Appl. Microbiol. 4:18&192. 14. Kelly, K. F., and J. B. Evans. 1974. Deoxyribonucleic acid homology among strains of the lobster pathogen “ G a m a homari” and Aerococcus viridans. J . Gen. Microbiol. 81:257-260. 15. Mandel, M., and J. Marrnur. 1968. Use of ultraviolet absorbance-temperature profile for determining the guanine plus cytosine content of DNA. Methods Enzymol. 12:195-206. 16. Marmur, J. 1963. A procedure for the isolation of deoxyribonucleic acid from microorganisms. Methods Enzymol. 6: 726-738. 17. Razin, S., and E. A. Freundt. 1984. Class I. Mollicutes Edward and Freundt 1967, 267*=, p. 740-741. In N. R. Krieg and J. G. Holt (ed.), Bergey’s manual of systematic bacteriology, Vol. 1. The Williams & Wilkins Co., Baltimore. 18. Schultes, L. M. 1969. Deoxyribonucleic acid homologies among aerococci and related bacteria. University Microfilms International, Ann Arbor, Mich. 19. Schultes, L. M., and J. B. Evans. 1971. Deoxyribonucleic acid homology of Aerococcus viridans. Int. J. Syst. Bacteriol. 21:207-209. 20. Steenbergen, J. F., H. S. Kirnball, D. A. Low, H. C. Schapiro, and L. N. Phelps. 1977. Serological grouping of virulent and avirulent strains of the lobster pathogen Aerococcus viridans. J. Gen. Microbiol. 99:425430. 21. Stewart, J. E., J. W. Cornick, and D. I. Spears. 1966. Incidence of G a m a homari in natural lobster (Homarus americanus) populations of the Atlantic region of Canada. J. Fish. Res. Board Can. 23:1325-1330. 22. Williams, R. E. O., A. Hirch, and S. T. Cowan. 1953. Aerococcus, a new bacterial genus. J. Gen. Microbiol. 8: 475430. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 18 Jun 2017 14:31:49