Acinetobacter Radioresistens Sp. Nov. Isolated from Cotton and Soil

Home , Acinetobacter

INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1988, p. 209-211 Vol. 38, No. 2 0020-7713/88/020209-03$02.00/0 Copyright 0 1988, 1nt.ernationalUnion of Microbiological Societies

Acinetobacter radioresistens sp. nov. Isolated from Cotton and Soil YUKIMASA NISHIMURA,” TAKESHI INO, AND HIROSHI IIZUKA Institute of Applied Biology, Science University of Tokyo, Noda 278, Japan

A new species of Acinetubacter, Acinetubacter radioresistens, is proposed for three radiation-resistant Acinetobacter strains which were isolated from samples of cotton and soil. This species is phenotypically, genetically, and enzymatically distinguished from other Acinetubacter species. Strains of this species are gram-negative, oxidase-negative, nonsporeforming, nonmotile, nonfermentative, aerobic, pleomorphic coccobacilli and produce no acid from saccharides; they assimilated 16 of the 52 carbon sources which we examined. The radiation-resistantAcinetubacter strains had little deoxyribonucleic acid homology (15 to 44%) with other Acinetubacter strains and showed a different pattern from other Acinetubacter strains on electrophoretic analysis of enzymes. The guanine-plus-cytosine contents of the deoxyribonucleic acids are 44.1 to 44.8 mol%. The major cellular fatty acids are C,8:1, c16:1 and c16:0, and the ubiquinone system is Q-9. The type strain of this species is strain FO-1 (= IAM 13186).

Since Brisou and Prevot (2) in 1954 established the genus assays and of electrophoretic analyses of enzymes are sum- Acinetobacter by separating the nonmotile species from the marized in Table 2. We also present a phenotypic description genus “Achromobacter,” the taxonomy of Acinetobacter of the species. The three RRA strains which we studied are has been widely studied, and a number of named species deposited in the culture collection of the Institute of Applied have been proposed. However, only two species (Acineto- Microbiology (IAM; University of Tokyo, Tokyo, Japan) as bacter calcoaceticus and Acinetohacter lwofii) appear in the strains IAM 13MT(= FO-lT), IAM 13187 (= G82012), and Approved Lists of Bacterial Names (16), and only one IAM 13188 (= G82076). species (A. calcoaceticus) is described in Bergey ’s Manual The RRA strains were characterized by using the methods of Systematic Bacteriology (5). Recently, Bouvet and Gri- described below. Assimilation tests with carbon sources mont (1) described 12 hybridization groups and gave new were carried out by using a basal medium of Stanier et al. names to four of these genospecies (Acinetobacter bauman- (17), and the carbon sources were added at concentrations of nii, Acinetobacter haemolyticus, Acinetobacter johnsonii, 1 to 10 g/l,OOO ml of basal medium. Oxidation-fermentation and Acinetobacter junii). tests were performed by using the method of Hugh and We have isolated acinetobacters from samples of soil and Leifson (3). Cytochrome oxidase test strips (Eiken Chemical cotton. One of these organisms, strain FO-lT (T = type Co., Ltd., Tokyo, Japan) were used to detect the presence of strain), was isolated as a radiation-resistant organism from this enzyme. Penicillin resistance was tested by incubating cotton sterilized by gamma radiation (7) and was considered the organisms in a medium with a definite quantity of to be a separate species from A. calcoaceticus on the basis of potassium benzylpenicillin added to heart infusion broth phenotypic characteristics (11). We also reported the differ- (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan). Nitrate ential characteristics of the outer membrane in a comparison reduction was determined by using both succinate-nitrate A. calcoaceticus of strain FO-lT and type strain IAM 12087 medium and Casitone-nitrate medium. Methods in general (= ATCC 23055) (10). Furthermore, we grouped our collection of acinetobacters, both these isolates and authentic use were employed in all of the other common tests. The strains, on the basis of deoxyribonucleic acid (DNA)-DNA incubation temperature used in all tests was 30°C. hybridization (13) and electrophoretic patterns of enzymes The cellular fatty acids and the ubiquinones were analyzed (Y. Nishimura, H. Kanzaki, and H. Iizuka, J. Basic Micro- by methods described previously (14, 15). The ubiquinones biol., in press). These studies showed that two isolates were identified by reversed-phase high-performance liquid (G82012 and G82076) from soils belong to the same group as chromatography (column: LiChrospher 100 RP-18,5 pm; E. strain FO-lT; these two isolates were also resistant to Merck AG, Darmstadt, Federal Republic of Germany) by gamma rays (13). The radiation susceptibilities reported comparing their retention times with those of standard previously (6, 7, 13) for the three radiation-resistant Acine- ubiquinones. The cells used for these analyses were obtained tobacter (RRA) strains and the reference Acinetobacter from a culture shaken at 30°C for ca. 24 h; heart infusion strains are shown in Table 1. The RRA strains showed D,, broth was used for the fatty acid analyses, and Trypto-Soya values which were ca. 19 to 34 times that of the type strain broth (Nissui) was used for the ubiquinone analyses. of A. calcoaceticus, measured under air equilibrium in 0.067 The RRA strains are gram-negative, cytochrome oxidase- M phosphate buffer, and their survival curves showed no negative, catalase-positive, nonfermentative, nonsporeform- shoulders. ing, nonmotile, aerobic, pleomorphic coccobacilli. These RRA strains have been described by Thornley and Glauert strains could assimilate 16 of the 52 carbon sources which we (18) and Welch and Maxcy (19) but these authors reported no examined and were unable to oxidize 15 of the saccharides detailed taxonomic characteristics. tested. Previously (ll), we reported that strain FO-lT pro- In this paper, we propose that the RRA strains typify a duced acids from arabinose, xylose, rhamnose, galactose, new species of the genus Acinetobacter, Acinetobacter glucose, and mannose, but we have now found that strain radioresistens. The results of our previous DNA homology FO-lT cannot produce acids from any saccharide. This is considered the result of a mutation during cultivation or preservation of the strain. The RRA strains reduced nitrate -* Corresponding author. to nitrite in succinate-nitrate medium but not in Casitone-

209 210 NOTES INT. J. SYST.BACTERIOL.

TABLE 1. Radiation susceptibilities of the RRA and TABLE 2. Comparison of the DNA homology groups reference Acinetobacter strains and clusters based on electrophoretic analysis of enzymes of Acinetobacter strains D,, value Strain (krads)" DNA homology Cluster and subcluster Corresponding group" in enzyme pattern" species FO-lT 220' G82012 204' Z-la-c A. calcoaceticus G82076 125' 2-4 A. lwofii Acinetobacter anitratum S5" 26' 2-2 A. johnsonii A. cakoaceticus IAM 12087T (= ATCC 230ST) 6.5' Z-lb A. haemolyticus A. calcoaceticus IAM 12088 (= ATCC 19606) 12' 2-3 A. radioresistens

a Measured under air equilibrium in 0.067 M phosphate buffer. '' Data from reference 13. Data from reference 7. ' Data from Nishimura et al. (in press). Data from reference 13. Isolated and identified as a flavin nucleotide-forming organism by Nishi- mura and Iizuka (8, 9). Data from reference 6. Description of Acinetobacter radioresistens sp. nov. Acine- f Qata from reference 4. tobacter radioresistens (ra.di.o.re.sis'tens. L. n. radius ray, beam; L. part. adj. resistens resisting; M.L. part. adj. radioresistens ray resisting, because of high resistance to nitrate medium. Other characters are described below for gamma-ray irradiation). Cells are rods 0.6 to 0.7 by 0.8 to 1.0 the new species. pm. Pleomorphic. A plump form is 0.8 to 0.9 by 1.2 to 1.8 The qajor fatty acids of the RRA strains were ClgZ1,C16:1, pm; elongate cells (15 pm or more long) occur in young and C16:o (Table 3) and were similar to those of A. calcoace- cultures; and coccoid cells (0.6 to 0.9 pm in diameter) occur ticus (14). The major ubiquinone of the RRA strains was Q-9 in old cultures. The cells occur singly or in pairs. Nonspore- (Table 3); the ubiquinone system was the ubiquinone system forming, noncapsulated, nonmotile, non-acid fast, gram neg- of the genus Acinetobacter (12). ative. As mentioned above, Bouvet and Grimont (1) proposed Colonies on nutrient agar are smooth, entire, convex, four new genospecies. We have found that the levels of DNA glistening, opaque, and yellowish white to pale yellow. homology between the RRA strains and reference strains of A pellicle is formed on the surface of nutrient broth. A. calcoaceticus, A. lwofii, and three genospecies of Bouvet Metabolism is strictly respiratory. Catalase is produced. and Grimont (A. baumannii, A. haemolyticus, and A. john- Cytochrome oxidase is not produced. Nitrate is reduced to sonii) are low (15 to 44%). A. baumannii type strain ATCC nitrite in succinate-nitrate medium but not in Casitone- 19606 proposed by Bouvet and Grimont was assigned to nitrate medium. Indole and hydrogen sulfide are not pro- group 1 together with A. calcoaceticus type strain IAM duced. Tween 80 is hydrolyzed. Lysine and ornithine decar- 12087 (= ATCC 23055) in our DNA-DNA hybridization boxydase, phenylalanine deaminase, arginine dihydrolase, experiments. Although the relationship between the RRA gelatinase, and urease are not produced. Hemolysis on strains and the A. junii strains of Bouvet and Grimont has sheep blood agar is negative. Growth occurs in medium not been examined by DNA-DNA hybridization, the level of containing 5% NaCI. Good growth occurs at 27 to 31°C. DNA homology between them may be expected to be low Growth occurs at 42°C. L-Arabinose, D-ribose, D-xylose, because of the differing DNA base ratios of these organisms. D-galactose, D-glUCOSe, D-fructose, D-mannose, L-rham- In the electrophoretic analysis of enzymes, the RRA strains nose, D-glucosamine, D-cellobiose, lactose, maltose, melibi- also produced a different pattern (2-3 in Table 2) than other ose, sucrose, and raffinose are not oxidized and not assimi- Acinetobacter strains. As shown in Table 4, the RRA strains lated. Acetate, fumarate, DL-lactate, L-malate, malonate, were differentiated from A. calcoaceticus by a lack of succinate, hexadecane, heptadecane, octadecane, eicosane, assimilation of citrate and L-arabinose and by no acid ethanol, n-butanol, L-alanine, L-glutamate, L-leucine, and production from saccharides; and they were differentiated L-proline are assimilated. Citrate, gluconate, decane, nona- from A. lwofii by assimilation of malonate and L-leucine and decane, methanol, n-propanol, isopropanol, ethanolamine, by growth at 42°C. p-alanine, DL-phenylalanine, L-histidine, L-isoleucine, DL- The conclusion drawn from these results was that the norleucine, DL-serine, L-threonine, dulcitol, inositol, manni- RRA strains are genotypically and phenotypically distinct tol, and sorbitol are not assimilated. from other Acinetobacter species. Consequently, the RRA Highly resistant to gamma-ray irradiation (DIo value, 125 strains were considered to be a new species of the genus to 220 krads under air equilibrium in 0.067 M phosphate Acinetobacter, and the name Acinetobacter radioresistens is buffer). proposed for these isolates because of their high level of Resistant to benzylpenicillin (100 IU). radiation resistance. The DNA base compositions range from 44.1 to 44.8 mol%

~~ ~~ FO-lT 3.1 trh tr 18.1 24.9 tr tr tr 50.8 2.5 7.7 85.8 6.5 G82012 2.6 tr tr 20.8 22.8 tr tr tr 51.6 1.7 11.2 83.6 5.2 G82076 3.2 tr tr 20.5 24.1 tr tr tr 47.5 3.4 10.8 83.8 5.4 The number preceding the colon indicates the number of carbon atoms; the number after the colon indicates the degree of unsaturation. tr, Trace (fatty acids occurring in amounts less than l%j. VOL.38, 1988 NOTES 211

TABLE 4. Differential characteristics among the RRA strains and the valid Acinetobacter species

RRA A. ccrlcoaceticus Iwofii Characteristic A. (3 strains)" (12 strains)" (3 strains)" Assimilation of Citrate - h + Malonate + 91 L-Arabinose - 75 L-Leucine + 91 Acid from: L-Arabinose + D-Ribose + D-xylose + D-Galactose + D-Glucose + D-Mannose + L-Rhamnose + D-Ghcosamine + Lactose 91 Growth at 42°C + 66 Major fatty acids c18:lv c16:1, c16:0 c18:13 c16:0? 'lh:lc c16:1, c18:1, clh:,d Guanine-plus-cytosine content of DNA (mol%) 44.144.8'' 39.042 .o' 45.845.9' Strains listed in reference 13. f,All strains positive; -, all strains negative. The numbers are percentages of positive strains. ' Data from reference 14. Data from reference 12. Data from reference 13. guanine plus cytosine (as determined by the thermal melting tance of an Acinetobucter species. J. Gen. Appl. Microbiol. temperature of the DNA). 25401406. The major cellular fatty acids are the even-numbered 8. Nishimura, Y., and H. Iizuka. 1972. Microbiological studies on straight-chain acids Clgtl, C16t1,and Clbt0. petroleum and natural gas. XII. Bacterial formation of the combined form of riboflavin from hydrocarbons. J. Agric. The ubiquinone system is Q-9. Chem. SOC.Jpn. 46:639-644. The type strain is strain FO-1 (= IAM 13186), which was 9. Nishimura, Y., and H. Iizuka. 1976. Microbiological studies on isolated from cotton. Two strains, G82012 (= IAM 13187) petroleum and natural gas. XIII. Identification of the flavin and G82076 (= IAM 13188), were isolated from soils. nucleotide-forming bacteria. J. Gen. Appl. Microbiol. 22:79-84. 10. Nishimura, Y., T. Ino, and H. Iizuka. 1986. Isolation and We thank E. Kairiyama of the National Commission of Atomic characterization of the outer membrane of Acinetobacter sp. Energy of Argentina for supplying strain FO-lT. FO-1. J. Gen. Appl. Microbiol. 32:177-184. This work was supported by a Grant-in-Aid for Scientific Re- 11. Nishimura, Y., E. Kairiyama, M. Shimadzu, and H. Iizuka. search from the Ministry of Education, Science and Culture of 1981. Characterization of radiation-resistant Acinetobacter. Z. Japan. Allg. Mikrobiol. 21:125-130. 12. Nishimura, Y., K. Kanbe, and H. Iizuka. 1986. Taxonomic studies of aerobic coccobacilli from seawater. J. Gen. Appl. LITERATURE CITED Microbiol. 32:l-11. 1. Bouvet, P. J. M., and P. A. D. Grimont. 1986. Taxonomy of the 13. Nishimura, Y., K. Kano, T. Ino, H. Iizuka, Y. Kosako, and T. genus Acinetobacter with the recognition of Acinetobacter Kaneko. 1987. Deoxyribonucleic acid relationship among the baumannii sp. nov., Acinetobacter haemolyticus sp. nov., Ac- radiation-resistant Acinetobacter and other Acinetobacter. J. inetobacter johnsonii sp. nov., and Acinetobacter junii sp. nov. Gen. Appl. Microbiol. 33:371-376. and emended descriptions of Acinetobacter calcoaceticus and 14. Nishimura, Y., H. Yamamoto, and H. Iizuka. 1979. Taxonomical Acinetobacter lwofii. Int. J. Syst. Bacteriol. 36:22&240. studies of Acinetobacter species-cellular fatty acid composi- 2. Brisou, J., and A. R. Prevot. 1954. Etudes de systematique tion. Z. Allg. Mikrobiol. 21:125-130. bacterienne. X. Revision des especes rCunies dans le genere 15. Nishimura, Y., S. Yamanaka, and H. Iizuka. 1983. Ubiquinone, Achromobacter. Ann. Inst. Pasteur (Paris) 86:722-728. fatty acid, polar lipid and DNA base composition in fseudomo- 3. Hugh, R., and E. Leifson. 1953. The taxonomic significance of nus radiora. J. Gen. Appl. Microbiol. 29:421-427. fermentative versus oxidative metabolism of carbohydrates by 16. Skerman, V. B. D., V. McGowan, and P. H. A. Sneath (ed.). various gram negative bacteria. J. Bacteriol. 66:24-26. 1980. Approved lists of bacterial names. Int. J. Syst. Bacteriol. 4. Ito, H., T. Sato, and H. Iizuka. 1976. Study of the intermediate 30:225420. type of Moraxella and Acinetobacter occurring in radurized 17. Stanier, R. Y., N. J. Palleroni, and M. Doudoroff. 1966. The vinna sausages. Agric. Biol. Chem. 40S67-873. aerobic pseudomonads: a taxonomic study. J. Gen. Microbiol. 5. Juni, E. 1984. Genus 111. Acinetobacter, p. 303-307. In N. R. 43: 159-271. Krieg and J. G. Holt (ed.), Bergey's manual of systematic 18. Thornley, M. J., and A. M. Glauert. 1968. Fine structure and bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore. radiation resistance in Acinetobacter: studies on a resistant 6. Kairiyama, E., H. G. Lescano, Y. Nishimura, and H. Iizuka. strain. J. Cell Sci. 3:273-294. 1980. Radio-resistance of a gram-negative bacterium. Z. Allg. 19. Welch, A. B., and R. B. Maxcy. 1975. Characterization of Mikrobiol. 20:149-152. radiation-resistant vegetative bacteria in beef. Appl. Microbiol. 7. Kairiyama, E., Y. Nishimura, and H. Iizuka. 1979. Radioresis- 30~242-250.