lar fatty acid composition. The cellular fatty acid composition of H. equigenitalis shows partial similarity to those of. Brucella canis and species of Moraxella and.
Vol. 15, No. 5
JOURNAL OF CLINICAL MICROBIOLOGY, May 1982, p. 791-794 0095-1137/82/050791-04$02.00/0
Cellular Fatty Acid Composition of Haemophilus equigenitalis CHIHIRO SUGIMOTO,* EIICHI MIYAGAWA, KENJI MITANI, MUNEO NAKAZAWA, AND YASURO ISAYAMA First Research Division, National Institute of Animal Health, Yatabe, Tsukuba, Ibaraki 305, Japan Received 8 October 1981/Accepted 18 December 1981
The cellular fatty acid composition of eight Haemophilus equigenitalis strains was determined by gas-liquid chromatography. All strains showed a grossly similar pattern characterized by large amounts of 18:1 and 16:0. The amounts of 16:1, 18:2, 18:0, 3-OH 14:0, 3-OH 16:0, and 3-OH 18:1 were relatively small. Haemophilus equigenitalis is the causative agent of contagious equine metritis. This venereal disease of horses, whose occurrence was first reported by Crowhurst (2) in 1977 in Ireland, rapidly spread over the world, to the European countries, the United States, Australia, and Japan (5, 15, 20, 21). Taylor et al. (23) proposed that this organism be classified within the genus Haemophilus on the basis of its DNA guanine-plus-cytosine contents (36.1 mol%) and its requirement of X factor as reported by Shreeve (19). Taylor et al. (23) and Sugimoto et al. (20), however, confirmed that this organism does not require X nor V factors and is positive in the porphyrin test (9). Consequently, the taxonomic position of H. equigenitalis is still questionable (24). This organism is a gram-negative, microaerophilic, and nonfermentative coccobacillus and is very unreactive in the conventional biochemical tests; it is positive only in the catalase, oxidase, and phosphatase tests (23). This characteristic makes taxonomic study of this organism difficult. The cellular fatty acid composition of bacterial cells has been successfully utilized in the identification and classification of bacteria (4, 8, 18). Braunthal et al. (1) and Jantzen et al. (7) studied the cellular fatty acid compositions of species of Haemophilus and revealed that they were grossly similar to those of species of Pasteurella and Actinobacillus. The present study was undertaken to determine the cellular fatty
acid composition of H. equigenitalis. MATERIALS AND METHODS Bacterial strains and culture conditions. Eight strains of H. equigenitalis, including a reference strain (NCTC 11184 [23]), were used. Six strains were isolated from horses in Japan (20), and one was isolated in the United States. The strains were cultivated on Eugon agar (BBL Microbiology Systems) supplemented with 5% Fildes pepsin-digested sheep blood (Fildes agar). To examine the effects of culture condition on the cellular fatty acid composition, 5% sheep blood
agar, 10o horse blood chocolate agar, and IsoVitaleX (BBL Microbiology Systems)-hemin agar prepared from Eugon agar as a basal medium were also used. Incubation was performed for 72 h at 37°C in the presence of 10%0 CO2. The bacteria were harvested and washed three times with saline before lyophilization. Chemical procedures and gas-liquid chromatography. Dried bacterial cells (about 20 mg) were methanolyzed by 5% HCI in methanol as previously described (13). Gas-liquid chromatography of fatty acid methyl esters was carried out on a Hewlett-Packard 5710A gas chromatograph equipped with a flexible fused silica capillary column (0.28 mm by 30 m) coated with OV101. Identification of fatty acids. Fatty acids were primarily identified by equivalent chain length calculated using a standard mixture of fatty acid methyl esters (11:0, 12:0, 13:0, 14:0, 15:0, 16:0, 17:0, 18:0, 19:0) as previously described (13). All structural elucidations were verified by a gas chromatograph-mass spectrometer-computer system (Hewlett-Packard 5992B) equipped with a glass capillary column (0.28 mm by 30 m) coated with OV-101.
RESULTS The cellular fatty acid composition of H. equigenitalis (NCTC 11184) cells cultivated under various culture conditions were compared. There was slight difference in the cellular fatty acid composition among cells cultivated on four different media (Table 1). The cells cultivated on blood agar contained a larger amount of 18:0 and a smaller amount of 18:1 than those cultivated on the other media. A minor component, 18:2, was not detected in the cells cultivated on IsoVitaleX-hemin agar. The cellular fatty acid composition of H. equigenitalis, however, was thought not to be critically influenced by the culture media. Fildes agar was used throughout the following experiments because it gave gopd growth of all strains tested. There was no significant difference between the cellular fatty acid compositions of 48-h-old cells and 72-h-old cells cultivated on Fildes agar. The structures of all fatty acids detected were verified by gas chromatograph-mass spectrome791
792
SUGIMOTO ET AL.
TABLE 1.
J. CLIN. MICROBIOL.
Cellular fatty acid composition of H. equigenitalis (NCTC 11184) cells cultivated under various culture conditions % of total acids
ECLa
72 hb
Fatty acid
FAC 1.72 3.61 1.70 29.4 0.28 0.84
CA
BA
IHA
48 hb, FA
13.97 14:0 0.53 0.75 1.23 0.85 15.42 3-OH 14:0 3.71 4.59 3.81 4.48 15.75 16:1 1.00 1.09 1.42 1.03 16.00 16:0 28.1 29.8 32.8 27.6 17.43 3-OH 16:0 0.42 0.31 0.25 0.33 17.58 18:2 0.20 0.33 NDd 0.67 17.76 18:1 55.3 41.2 52.5 50.9 54.8 17.99 18:0 4.02 12.8 6.22 5.03 6.03 19.16 3-OH 18:1 1.66 1.16 1.73 1.38 1.96 a ECL, Equivalent chain length. b Culture age. C Media: FA, Fildes agar; BA, 5% sheep blood agar; CA, 10%o horse blood chocolate agar; IHA, IsoVitaleXhemin agar; Eugonagar (BBL Microbiology Systems) was used as a basal medium. d ND, Not detected.
103
100
3-OH 14:0
I-%
43
~
1Z
0
.*_
50 ._ .-*
1~
I
~
50
AU II
100
1.I..
III
i
150
M-50 M-18 208 240 200
250
m/e
100 0
In
C
50
C
GP
m/e FIG. 1. Mass spectra of two 3-hydroxy fatty acid methyl esters.
300
FATTY ACIDS OF H. EQUIGENITALIS
VOL. 15, 1982
793
TABLE 2. Cellular fatty acid composition of H. equigenitalis (seven strains) ECLa
Fatty acid
Kentucky-188b NDC
12:0 11.97 13.04 13:0 ND 14:0 0.74 13.97 3-OH 14:0 6.51 15.42 15.75 16:1 0.89 16:0 35.0 16.00 17.43 3-OH 16:0 0.58 0.79 18:2 17.58 18:1 45.0 17.76 6.03 17.99 18:0 19.16 3-OH 18:1 2.98 a ECL, Equivalent chain length. b Strain. C ND, not detected. d Tr, Less than 0.50%°.
EQ-51
% of total acids EQ-56 EQ-59
0.55 ND
Trd Tr
ND ND
1.74 3.95 1.96 30.2 Tr 0.98 53.6 3.66 1.41
2.01 5.99 2.17 31.6 0.59 1.10 45.4 3.15 3.39
1.55 3.46 1.87 35.4 Tr 0.81 47.6 5.22 1.95
ter spectra. Mass spectra of two 3-hydroxy fatty acid methyl esters are shown in Fig. 1. The peak at mle 103 which characterizes 3-hydroxy fatty acid methyl esters (13, 17) was detected in both of the fatty acids, and the base peak was found in the spectra of 3-OH 14:0. Molecular ion peak (M) and peaks at mle (M-18) and (M-50) were detected in 3-OH 18:1. Peaks at mle (M-18) and (M-50) were detected in 3-OH 14:0. All eight strains of H. equigenitalis exhibited a grossly similar fatty acid composition characterized by large amounts of 18:1 and 16:0 (Tables 1 and 2). Relatively small amounts of 14:0, 16:1, 18:2, and 18:0 were also detected. 3-Hydroxy fatty acids, 3-OH 14:0, 3-OH 16:0, and 3-OH 18:1 also existed, but they amounted to less than 10% of total fatty acids. Branched chain fatty acid was not detected. DISCUSSION The cellular fatty acid compositions of eight H. equigenitalis strains tested in the present study were very similar, although two of these strains. were collected from geographically separate regions. Consequently, the cellular fatty acid composition well characterizes this organism whose characterization by the conventional biochemical tests is difficult because of its extreme unreactiveness. The cellular fatty acid composition is known to be influenced by composition of culture medium, culture age of cells, temperature of incubation, and other culture conditions (4). The cellular fatty acid composition of H. equigenitalis, however, was relatively stable even if the culture conditions had changed. This fact makes it possible to compare the results in our study with those in other species. The cellular fatty acid composition was char-
EQ-64
EQ-70
ND Tr 1.60 4.04 1.90 34.1 Tr 0.89 51.4 3.58 1.75
Tr Tr
EQ-Chiba 0.64 Tr
1.40 3.81 1.85 34.8 Tr 0.56 47.4 8.09 1.43
1.74 4.81 1.92 28.7 Tr 0.84 52.4 5.32 1.75
acterized by large amounts of 18:1 and 16:0 and relatively small amounts of 14:0, 16:1, 18:2, and 18:0. Amounts of 3-OH hydroxy fatty acids were not so large, but the existence of 3-OH 18:1, which had not been detected in other species thus far studied, was characteristic. The cellular fatty acid composition of H. equigenitalis is clearly distinct from those of true members of the genus Haemophilus. Species of Haemophilus contain large amounts of C16 fatty acids (16:1, 16:0) and small amounts of C18 fatty acids (18:2, 18:1, 18:0) as reported by Jantzen et al. (7). Species of Haemophilus showed a cellular fatty acid pattern indistinguishable from those of Pasteurella and Actinobacillus species, which also share a number of phenotypic characters with Haemophilus species and whose DNA guanine-plus-cytosine contents are in the same range as the genus Haemophilus (10, 12, 24). H. equigenitalis, however, seems to be clearly separated from Actinobacillus-Haemophilus-Pasteurella group in its cellular fatty acid composition. The cellular fatty acid composition of H. equigenitalis shows partial similarity to those of Brucella canis and species of Moraxella and Acinetobacter in its abundance of 18:1 (3, 8, 11, 16). These species contain 18:1 as a major fatty acid, but their amounts of C16 fatty acids are not so large as those of species of Haemophilus. The cellular fatty acid composition of H. equigenitalis is distinctively different from those of other gram-negative bacteria so far analyzed, including species of Francisella, Legionella, and Brucella, except Brucella canis (3, 6, 14, 22). Further studies of H. equigenitalis based on the cellular fatty acid composition is now proceeding in our laboratory to clarify the taxonomic position of this organism.
794
SUGIMOTO ET AL.
LITERATURE CITED 1. Braunthal, S. D., S. C. Holt, A. C. R. Tanner, and S. S. Socransky. 1980. Cellular fatty acid composition of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus. J. Clin. Microbiol. 11:625-630. 2. Crowhurst, R. C. 1977. Genital infection in mares. Vet. Rec. 100:476. 3. Dees, S. B., D. G. Hollis, R. E. Weaver, and C. W. Moss. 1981. Cellular fatty acids of Brucella canis and Brucella suis. J. Clin. Microbiol. 14:111-112. 4. Drucker, D. B. 1976. Gas-liquid chromatographic chemotaxonomy, p. 51-125. In J. R. Norris and D. W. Ribbons (ed.), Methods in microbiology, vol. 9. Academic Press, Inc., London. 5. Huges, K. L., J. D. Bryden, and F. MacDonald. 1978. Contagious equine metritis. Aust. Vet. J. 54:101. 6. Jantzen, E., B. P. Berdal, and T. Omland. 1979. Cellular fatty acid composition of Francisella tularensis. J. Clin. Microbiol. 10:928-930. 7. Jantzen, E., B. P. Berdal, and T. Omland. 1980. Cellular fatty acid composition of Haemophilus species, Pasteurella multocida, Actinobacillus actinomycetemcomitans and Haemophilus vaginalis (Corynebacterium vaginale). Acta Pathol. Microbiol. Scand. Sect. B 88:89-93. 8. Jantzen, E., K. Bryn, T. Bergan, and K. Bovre. 1974. Gas chromatography of bacterial whole cell methanolysates. V. Fatty acid composition of Neisseriae and Moraxellae. Acta Pathol. Microbiol. Scand. Sect. B 82:767-779. 9. Killian, M. 1974. A rapid method for the differentiation of Haemophilus strains; the porphyrin test. Acta Pathol. Microbiol. Scand. Sect. B 82:835-842. 10. Kilian, M. 1976. A taxonomic study of the genus Haemophilus, with the proposal of a new species. J. Gen. Microbiol. 93:9-62. 11. Lambert, M. A., D. G. Hoilis, C. W. Moss, R. E. Weaver, and M. L. Thomas. 1971. Cellular fatty acids of nonpathogenic Neisseria. Can. J. Microbiol. 17:1491-1502. 12. Mannheim, W., S. Pohl, and R. Hollander. 1980. On the taxonomy of Actinobacillus, Haemophilus and Pasteurella: DNA base composition, respiratory quinones and biochemical reactions of representative collection cul-
J. CLIN. MICROBIOL. tures. Zentralbl. Bakteriol. Parasitenkd. Infectionskr. Hyg. Abt. I Orig. Reihe A 246:512-540. 13. Miyagawa, E., and T. Suto. 1980. Cellular fatty acid
14. 15. 16. 17. 18. 19.
20.
21. 22.
23.
24.
composition in Bacteroides oralis and Bacteroides ruminicola. J. Gen. Appl. Microbiol. 26:331-343. Moss, C. W., R. E. Weaver, S. B. Dees, and W. B. Cherry. 1977. Cellular fatty acid composition of isolates from Legionnaires disease. J. Clin. Microbiol. 6:140-143. Mumme, J., and L. Ahlswede. 1979. Isolation of Haemophilus equigenitalis from cervical swabs of halfbred mares. Dtsch. Tierarztl. Wochenschr. 86:257-259. Nishimura, Y., H. Yamamoto, and H. lizuka. 1979. Taxonomical studies of Acinetobacter species-cellular fatty acid composition. Z. Allg. Mikrobiol. 19:307-308. Ryhage, R., and E. Stenhagen. 1960. Mass spectrometric studies. VI. Methyl esters of normal chain oxo-, hydroxy-, methoxy- and epoxy-acids. Ark. Kemi 15:545-574. Shaw, N. 1974. Lipid composition as a guide to the classification of bacteria. Adv. Appl. Microbiol. 17:63-68. Shreeve, J. E. 1978. Preliminary observations on X factor requirement of the bacterium responsible for CEM. Vet. Rec. 102:20. Sugimoto, C., Y. Isayama, M. Kashiwazaki, T. Fujikura, and K. Mitani. 1980. Detection of Haemophilus equigenitalis, the causal agent of contagious equine metritis, in Japan. Natl. Inst. Anim. Health Q. 20:118-119. Swerczek, T. W. 1978. The first occurrence of contagious equine metritis in the United States. J. Am. Vet. Med. Assoc. 173:405-407. Tanaka, S., T. Suto, Y. Isayama, R. Azuma, and Y. Hatakeyama. 1977. Chemo-taxonomical studies on fatty acids of Brucella species. Ann. Sclavo 19:67-82. Taylor, C. E. D., R. 0. Rosenthal, D. F. J. Brown, E. S. P. Lapage, L. R. Hill, and R. M. Legros. 1978. The causative organism of contagious equine metritis 1977: proposal for a new species to be known as Haemophilus equigenitalis. Equine Vet. J. 10:136-144. Zinnemann, K. 1980. Newer knowledge in classification, taxonomy and pathogenicity of species in the genus Haemophilus: a critical review. Zentralbl. Bakteriol. Parasitenkd. Infectionskr. Hyg. Abt. I Orig. Reihe A 247:248-258.
