Fatty Acid and Mycolic Acid Composition of JK and D2 Corynebacteria

System. App!. Microbio!. 15, 336-339 (1992) © Gustav Fischer Verlag, StuttgartlNew York

Fatty Acid and Mycolic Acid Composition of JK and D2 Corynebacteria JACQUELINE CHEVALIER\ MARIE-THERESE POMMIER2, ANDREE CREMIEUX\ and GEORGES MICHEL2 1

2

Laboratoire de Microbiologie, Hygiene Microbienne, Immunologie, Faculte de Pharmacie, 13385 Marseille Cedex 5, France Laboratoire de Biochimie Microbienne, Universite Claude Bernard Lyon I, 69622 Villeurbanne Cedex, France

Received December 6, 1991

Summary The patterns of cellular fatty acids and mycolic acids of 15 strains of cutaneous corynebacteria, including 6 strains of the Corynebacterium JK group and 7 strains of the Corynebacterium D2 group, were examined. All strains had large amounts of saturated and monounsaturated straight chain fatty acids and only trace amounts of tuberculostearic acid. Iso and anteiso branched acids were not present. Their mycolic acids in the range from C26 to C36 were identified. JK, ANF and I strains differed in their patterns of mycolic acid composition from Corynebacterium D2 group. JK strains showed high levels of C34-C36 components and lower levels of C26-C28 mycolic acids while D2 strains demonstrated a high level of C26-C28 acids and no C34-C36 content.

Key words: JK group - D2 group - Corynebacterium - Skin bacteria - Fatty acid - Mycolic acid Taxonomy - Gas-liquid chromatography

Introduction Analysis of fatty acid content and mycolic acid composition is considered to be important in the identification and classification of saprophytic, opportunistic and pathogenic cutaneous coryneform bacteria that are difficult to identify using conventional methods. Several researchers (Athalye et ai., 1984; Athalye et ai., 1985; Mac Ginley et ai., 1985a) have examined the patterns of cellular fatty acids and corynemycolic acids of the diphterolds which are highly multiresistant to antibiotics, the JK group diphterolds, now known as Corynebacterium jeikeium Uackman et ai., 1987). For the other multi-resistant diphterold group, D2, called Corynebacterium urealyticum (Soriano et ai., 1986), which has been separated from the JK group on the basis of biochemical tests (Coyle et ai., 1985; Soriano et ai., 1985), the cellular lipid content, i. e., fatty acid pattern and corynomycolic acid profile has been reported only by Soriano et ai., 1988 and Herrera-Alcaraz et ai., 1990). In this study 13 strains of cutaneous corynebacteria including strains of both JK and D2 groups and 2 strains

belonging to ANF and I groups were examined by gas liquid chromatography (GLC). This work was carried out, not only to promote research in taxonomy but also to note any differences in cellular fatty acid and corynemycolic acid content that might be related to pathogenicity. Materials and Methods Strains and growth conditions. The studied strains, listed in Table 1 were checked using a biochemical micromethod (Api Coryne, Bio Merieux; data not shown) and tested for antimicrobial susceptibilities. One of the JK strains (21) and rwo of the D2 strains (71 and 83) showed a very limited pattern of resistance (Table 1). Strains were grown for 24 h on Tryptic Soy Agar (Difco) supplemented with Tween 80 (0.3%, w/v; Sigma) for investigation of fatty acids, and in shaken flasks of Tryptic Soy Broth (Difco), supplemented with 0.3%, w/v Tween 80 for mycolic acids study. Cells grown in shaken flasks were harvested by centrifugation, washed with distilled water and freeze-dried.

Fatty and Mycolic Acid Contents of JK and D2 Corynebacteria

337

Table 1. Strains: Designation sources and antimicrobial susceptibilities CoryneStrain bacterium nO Group

JK

NAL PEF TRI

FUR NOV

R S R R R R

R S R R R R

R S R R R R

R S R R R R

S S S S S S

R S R R R R

R S R S S S

R R R R R R

R R R S R R

S S S S S S

11 12 16

isolated from patient, Nice, France isolated from patient, Nice, France ATCC 43044, American Type Culture Collection 26-1 isolated from healthy human skin, received from Dr. F. Soriano, Madrid, Spain isolated from healthy human skin, Marseilles, France isolated from healthy human skin, Marseilles, France isolated from healthy human skin, Marseilles, France

S R S

R R R

R R R

R R R

S S S

R R R

R S S

R R R

R R R

S S R

S

R

R

R

S

R

S

R

R

S

S

S

S

S

S

R

S

R

R

S

S

S

S

S

S

R

S

R

R

S

S

R

R

R

S

R

S

R

R

S

51

isolated from healthy human skin, Marseilles, France

S

S

S

S

S

R

S

R

R

S

333S

isolated from healthy human skin, Marseilles, France

S

S

S

S

S

R

S

R

R

S

71

331

I

ERY GEN AMP CEF PRI isolated from patient, Nice, France CIP 82451, CNCM Institut Pasteur, Paris isolated from drain, Lyon, France isolated from axilla, CDC, USA isolated from catheter, CDC, USA isolated from chest wound, CDC, USA

83

ANF

Antimicrobial susceptibility*

13 21 22 29 30 31

20 D2

Source

* Abbreviations: R = resistant; S = Susceptible; ERY = Erythromycin; GEN = Gentamycin; AMP = Ampicillin; CEF = Cefotaxin; PRI = Pristinamycin; NAL NOV = Novobiocin.

= Nalidixic acid; PEF = Pefloxacin; TRI = Trimethoprim-Sulfamethoxazole; FUR = Nitrofurantoin;

Extraction and analysis of fatty acids. Cells harvested from agar plates (about 40 mg, wet weight) were immediately saponified. The fatty acids were methylated, and methyl esters were extracted and examined as previously described (Miller, Hewlett-Packard gas chromatography application note 228-41; Hewlett-Packard Co., Palo Alto, CAl as reported by Olson et al., 1990. GLC analysis was performed by using a fused silica capillary column with high separation efficiency and resolution, and was automated using a HP 5898A gas chromatograph with the Microbial Identification System from Hewlett-Packard. Extraction and analysis of mycolic acids. Mycolic acid methyl esters were obtained after acid methanolysis of dried bacteria (Minnikin et al., 1980). Methyl mycolates were purified by preparative thin-layer chromatography (TLC) and converted to thtl corresponding trimethyl silyl (TMS) ethers. TMS ethers were analyzed by gas liquid chromatography (Chevalier et al., 1988).

Results and Discussion Fatty acid composition The profiles of fatty acids obtained from ]K, D2, ANF and I strains are presented in Table 2. Identified fatty acids were chiefly saturated or monounsaturated straight chain C12 to C18. No iso nor anteiso branched fatty acids were found, but 10 methyl octadecanoic acid (tuberculostearic acid) was detected in some D2 strains. Furthermore, low

levels of hydroxylated fatty acids were found in two strains. The major compounds were hexadecenolc (3.5%-20.1 %), hexadecanoic (10.9%-25.0%) and octadecenoic (55.3%-80.1 %) acids, which were always present. These results generally agree with previous reports (Minnikin et al., 1978; Collins et al., 1982b; Suzuki et al., 1982; Bousfield et al., 1983) in which C16: 0 and C18 : 1 were predominant (20.0% and 65.0% respectively). The high level of C18: 1 (average mean: 65.0%) noted in all strains may be attributed to Tween 80, which was used to increase the yield of the growth culture (Athalye et al., 1985). For the same reason the fatty acid C16 : 1 was often found at levels close to 15% to 20%, while, in other studies it reached a maximum of 10% (Minnikin et al., 1978; Collins et al., 1982a; Suzuki et al., 1982; Bousfield et al., 1983;, Soriano et al., 1988). Conversely, small quantities of palmitic (C16: 0) and stearic (C18 : 0) acids were obtained. In a previous study on the effect of Tween 80 on the fatty acid content, it was shown that C16: 0 and C18 : 0 acids decreased whereas the proportions of the monounsaturated homologous increased (Chevalier et al., 1987). In the case of tuberculostearic acid, present only in trace amounts in our D2 strains, Soriano et al. (1988) and Herrera-Alcaraz et al. (1990) reported measurable amounts ranging from 2.8% to 7.9% and from 2.0% to 20.0% respectively in strains grown on blood agar.

338

J. Chevalier, M.-T. Pommier, A. Cremieux, and G. Michel

Table 2. Fatty acid profiles of corynebacteria (% of total) Strains

JK

Carbon length

D2

Corynebacterium 13

21

22

29

30

ANF

Corynebacterium 31

11

12

16

20

71

83

331

CoryneCorynebacterium bacterium

51

5.9 3.3

C12 :0 C12:030H C 15 : 0 C 16 : 1 C 16 : 0 C 17 : 1 C17:020H C 18 : 1 C 18 :0

5.5 16.7 11.4

15.7 19.4

17.0 25.0

5.9 17.6 16.7

63.0 3.4

64.9

58.0

60.8

333S

7.8 16.1 15.1

20.1 21.5

18.5 19.2

61.0

58.4

7.0 55.3 tr

ClOMe18:0

5.4 11.2

18.6 16.3

15.2 17.8 4.9

16.8 14.4

17.4 14.0

18.9 11.0

3.5 10.9

17.9 13.9

80.1 3.3 tr

65.1

62.1

68.8

68.6

70.1

59.0

tr

78.2 7.4

tr

tr

(tr): trace amounts Figures are the means of 3 determinations.

The fatty acid profiles of Corynebacterium 02 and JK groups can be distinguished from those of Corynebacterium bovis (Athalye et a1., 1985) and Corynebacterium tuberculostearicum associated with leprosy lesions (Brown et a1., 1984). Significant amounts of tuberculostearic acid (20.0% to 40.0%) were found in two strains of Corynebacterium bovis examined by Athalye et a1. (1985) and seven strains studied by Mac Ginley et a1. (1985b).

acids were in the range from C26 to C36; the C30 and C32 components were always present. The profile of the JK, ANF, and I groups differed from that of the 02 group. The first three groups can be separated from the 02 group by the presence of high levels of C34-C36 compounds (mean from 29.0% to 53.0%). Furthermore, for the JK, ANF and I groups, the major corynemycolic acids were C30-C36 whereas, in the 02 group, they were C28-C32. These results are in general agreement with those published by Athalye et a1. (1984) relative to JK corynebacteria, by Mac Ginley et a1. (1985a) for JK corynebacteria and some lipophilic diphteroids and by Herrera-Alcaraz et a1. (1990) corresponding to 02 corynebacteria. All strains examined had high levels of C30-C32 mycolic acids, which ranged from 41.4% to 68.3%. These strains were clearly distinct from Corynebacterium bovis, characterized by a small quantity of C30-C32 derivates and a predominance of C22-C29 compounds (Collins et a1., 1982a; Athalye et a1., 1984; Mac Ginley et a1., 1985a,

Corynemycolic acid composition Thin layer chromatography analysis of the acid methanolysates obtained from all strains showed the presence of hydroxylated fatty acid methyl esters. They had the same mobility (Rf = 0.40) as that of corynemycolic methyl esters isolated from corynebacteria (Minnikin and Goodfellow, 1976; Minnikin et a1., 1980; Soriano et a1., 1988). The methyl corynemycolates were quantified by GLC analysis of the TMS ether derivatives (Table 3). Mycolic Table 3. Content of mycolic acid (% of total) of strains

Strains

JK

Number of Carbon atoms 13 C26 C28 C 30 C 32

C34 C36

D2

Corynebacterium

10.0 19.0 42.0 17.0 12.0

21

22

10.0 35.8 20.8 33.4

tr 11.0 17.4 38.3 17.0 15.1

29

30

13.0 44.0 22.0 21.0

7.5 4.7 13 .7 13.4 43.0 34.6 18.1 18.5 20.5 26.0

(tr): trace amounts Figures are the means of 3 determinations.

ANF

Corynebacterium 31

11

12

16

20

71

83

331

41.0 34.0 25.0

48.0 31.0 21.0

11.2 35.9 27.6 25.3

14.1 44.5 20.4 21.0

tr 34.7 32.3 32.0

31.7 37.7 30.6

9.0 36.0 24.9 31.1

CoryneCorynebacterium bacterium

51

333S

4.5 12.7 45.3 20.4 17.1

6.0 9.7 32.7 22.9 28.7

Fatty and Mycolic Acid Contents of JK and D2 Corynebacteria

1985b). Our strains appeared similar in mycolic acid composition to Corynebacterium diphteriae, Corynebacterium xerosis and Corynebacterium minutissimum (Collins et aI., 1982a; Athalye et aI., 1984; Mac Ginley et aI., 1985a; 1985b). In the JK group the antibiotic-susceptible strain 21 could be distinguished from the other strains by the presence of a high level (33.4%) of C36 corynemycolic acid. In the D2 group both sensitive strains, 71 and 83, demonstrated the lower level of C26-C28 mycolic acid. Because of their behavior in biochemical identification and their corynemycolic acid profiles, these three antibiotic-susceptible strains have been maintained in the JK and D2 groups. Our findings show that D2 group Corynebacterium may be distinguished from other groups on the basis of corynemycolic acid contents. On the contrary, JK group Corynebacterium showed' a very high similarity with cutaneous strains classified in ANF and I groups, which may be differentiated by their biochemical reactions and their antimicrobial susceptibility. References Athalye, M., Noble, W. c., Mallet, A. 1., Minnikin, D. E.: Gas chromatography-mass spectrometry of mycolic acids as a tool in the identification of medically important coryneform bacteria. J. Gen. Microbiol. 130, 513-519 (1984) Athalye, M., Noble, W. c., Minnikin, D. E.: Analysis of cellular fatty acids by gas chromatography as a tool in the identification of medically important coryneform bacteria. J. Appl. Bact. 58, 507-512 (1985) Bousfield, I. J., Smith, G. L., Dando, T. R., Hobbs, G.: Numerical analysis of total fatty acid profiles in the identification of coryneform, nocardioform and some other bacteria. J. Gen. Microbiol. 129,375-394 (1983) Brown, S., Laneelle, M. A., Asselineau, J., Barksdale, L.: Description of Corynebacterium tuberculostearicum sp. nov., a leprosy-derived Corynebacterium. Ann. Inst. Pasteur Microbiol. 135,251-267 (1984) Chevalier, J., Pommier, M. T., Cremieux, A.: Role du Tween 80 utilise dans la culture des corynebacteries cutanees (group JK) sur la composition en acides gras cellulaires. Ann. Inst. Pasteur Microbiol. 138, 427-437 (1987) Chevalier, J., Pommier, M. T., Cremieux, A., Michel, G.: Influence of Tween 80 on the mycolic acid composition of three cutaneous corynebacteria. J. Gen. Microbiol. 134,2457-2461 (1988) Collins, M. D., Goodfellow, M., Minnikin, D. E.: A survey of the structure of mycolic acids in Corynebacterium and related taxa. J. Gen. Microbiol. 128, 129-149 (1982a) Collins, M. D., Goodfellow, M., Minnikin, D. E.: Fatty acid composition of some mycolic acid containing coryneform bacteria. J. Gen. Microbiol. 128, 2503-2509 (1982b)

339

Coyle, M. B., Hollis, D. G., Groman, N. B.: Corynebacterium spp. and other coryneforms microorganisms, pp. 193-204. In: Manual of Clinical Microbiology (E. H. Lennette, A. Ballows, W. J. Hausler, H. J. Shadomy, eds.), 4th ed. WashingtonlDC, American Society for Microbiology 1985 Herrera-Alcaraz, E. A., Vallero-Guillen, P. L., Martin-Luengo, F., Soriano, F.: Taxonomic implications of the chemical analysis of the D2 group of corynebacteria. FEMS Microbiol. Lett. 72,341-344 (1990) Jackman, P. J. H., Pitcher, D. G., Pelczynska, S., Borman, P.: Classification of corynebacteria associated with endocarditis (group JK) as Corynebacterium jeikeium sp. nov. System. Appl. Microbiol. 9, 83-90 (1987) Mac Ginley, K. J., Labows, J. N., Zehman, J. M., Nordstrom, K. M., Webster, G. F., Leyden, J. J.: Pathogenic JK group corynebacteria and their similarity to human cutaneous lipophilic diptherolds. J. Infect. Disea. 152, 801-806 (1985a) Mac Ginley, K. J., Labows, J. N., Zehman, J. M., Nordstrom, K. M., Webster, G. F., Leyden, J. J.: Analysis of cellular components, biochemical reactions, and habitat of human cutaneous lipophilic diptherolds. J. Invest. Dermatol. 85, 374-377 (1985b) Minnikin, D. E., Goodfellow, M., Collins, M. D.: Lipid composition in the classification and identification of coryneform bacteria, pp. 85-160. In: Coryneform bacteria (1. J. Bousfield, A. G. Callely, eds.). London, Academic Press 1978 Minnikin, D. E., Goodfellow, M.: Lipid composition in the classification and the identification of no cardiae and related taxa, pp. 160-219. In: The Biology of the Nocardiae (M. Goodfellow, G. H. Brownell, J. A. Serrano, eds.). London, Academic Press 1976 Minnikin, D. E., Hutchinson, I. G., Caldicott, A. B., Goodfellow, M.: Thin layer chromatography of methanolysates of mycolic acid containing bacteria. J. Chromatogr. 188, 221-233 (1980) Olson, W. P., Groves, M. J., Klegerman, E.: Identifying bacterial contaminants in a pharmaceutical manufacturing facility by gas chromatographic fatty acid analysis. Pharm. Technol. 14, 32-36 (1990) Soriano, F., Martin Luengo, F., Valero, P. L., Ponte, c., Santamaria, M., Roblas, F. R.: Caracterizacion e identificacion de Corynebacterium grupo D2 aislados de muestras clinicas. Enf. Microbiol. Clin. 6, 235-238 (1988) Soriano, F., Ponte, c., Santamaria, M., Aguado, J. M., Wilhelmi, I., Vela, R., Cifuentes, L.: Corynebacterium group D2 as a cause of alkaline encrusted cystitis: report of four cases and characterization of the organisms. J. Clin. Microbiol. 21, 788-792 (1985) Soriano, F., Ponte, c., Santamaria, M., Castilla, c., Roblas, R. F.: In vitro and in vivo study of stone formation by Corynebacterium group D2 (Corynebacterium urealyticum). J. Clin. Microbiol. 23, 691-694 (1986) Suzuki, K., Kawaguchi, A., Saito, K., Okuda, S., Komagata, K.: Taxonomic significance of the position of double bonds of unsaturated fatty acids in corynebacteria. J. Gen. Appl. MicrobioI. 28, 409-416 (1982)

Dr. Jacqueline Chevalier, Laboratoire de Microbiologie, Hygiene Microbienne, Immunologie, Faculte de Pharmacie, 27 Blvd. Jean Moulin, F-13385 Marseille Cedex 5, France

23 System. Appl. Microbiol. Vol. 15/3