1.NTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY,July 1991, p. 390-394 Vol. 41, No. 3 0020-7713/91/03039O-05$02 .OO/O Copyright 0 1991, International Union of Microbiological Societies Distribution of a Novel Mycolic Acid in Species of the Genus Mycobacterium M. LUQUfN,' M. A. LANEELLE,, V. AUSINA,'" M. GARCIA BARCELO,' F. BELDA,' C. ALONS0,l AND G. PRATS' Departamento de Microbiologia, Hospital de la Sta. Cruz y San Pablo, Facultad de Medicina de la Universidad Autbnoma de Barcelona, Barcelona, Spain,' and Centre de Biochimie et Gkne'tique Cellulaires du CNRS et Universite' P. Sabatier, Toulouse, France2 We found that Mycobacterium porcinum ATCC 33776T (T = type strain) contains a new kind of mycolic acid with a methoxy group at the w-1 position. This mycolic acid was identified by comparing it with the previously described methoxymycolic acids. The patterns of mycolic acid methyl esters from 418 strains belonging to 44 species of mycobacteria were studied by using thin-layer chromatography. In addition to M. porcinum ATCC 33776T, representative strains of M. porcinum, Mycobacterium fortuitum, "Mycobacterium peregrinum," Mycobacterium senegalense, and a recently isolated Mycobacterium sp. contained appreciable amounts of the newly described mycolic acid. Mycolic acids are characteristic 2-branched, 3-hydroxy Mycobacterium aichiense, Mycobacterium chubuense, My- acids with very long chains (up to 90 carbon atoms) and are cobacterium obuense, and Mycobacterium rhodesiae were found only in mycobacteria, nocardiae, rhodococci, coryne- incubated at 30"C, and Mycobacterium thermoresistibile bacteria, and related taxa (1, 12, 14, 20). Differences in the strains were incubated at 42°C. structures of mycolic acids have proved to be valuable Analysis of mycolic acids. Saponification was performed by criteria in the classification and identification of members of treating wet organisms with a 5% (wt/vol) potassium hydrox- these taxa (1, 2, 6, 11, 15, 19-23, 28). Two types of ide solution in methanol-benzene (8:2, vol/vol) and heating nonoxygenated mycolates (with ethylenic double bounds or the mixture overnight under reflux. After acidification with cyclopropane rings in their longest chains [the mero-chains]) 20% sulfuric acid, fatty acids were extracted in diethyl ether and four types of mycolates with oxygenated functions in and methylated with diazomethane. To check for the pres- their mero-chains (methoxymycolates, ketomycolates, ep- ence of epoxymycolates, an acidic methanoly sis procedure oxymycolates, and wax ester mycolates) have been identi- (23) was performed. This treatment destroyed epoxydes and fied in the genus Mycobacterium (1-3, 5-9, 13, 18, 25, 26). transformed them into much more polar compounds. Recently, a fifth type of oxygenated mycolate was found in a The patterns of rnycolates were obtained on high-perfor- Mycobacterium sp. isolated from the environment (17). mance thin-layer chromatographic plates (10 by 10 cm; Structural analysis showed that this mycolate contained a HPTLC Alufolien Kieselgel 60 F254;Merck) by using either methoxy group at the 0-1 position (instead of the 0-17 or double development with petroleum ether-diethyl ether (85: 0-18 position, as in previously described methoxymycolates) 15, vol/vol) (solvent A) or dichloromethane (solvent B). The and two double bounds in the long mero-chain (instead of the positions of separated components were revealed by spray- one double bond found in the previously described myco- ing the plates with a 10% solution of molybdophosphoric lates with oxygenated groups). Thin-layer chromatography acid in ethanol, followed by charring. showed that the Mycobacteriurn porcinum type strain con- Purification of mycolates from M. porcinum. Crude meth- tains, in addition to a- and epoxymycolates (16), a more ylmycolates from the M. porcinum type strain were obtained polar component with the same chromatographic behavior by precipitating them in methanol. They were then frac- as the w-1 methoxymycolate mentioned above. In this study tioned on a Florisil column by eluting them with increasing we found that the structure of the most polar mycolic acid of concentrations (up to 20%) of diethyl ether in light petroleum the M. porcinum type strain is the same as the structure of ether (boiling point, 50°C). Final purification of mycolates the recently describe 0-1 methoxymycolate, and the patterns was performed by preparative thin-layer chromatography, of the mycolates of representative strains of 44 mycobacte- using CH2C12 as the solvent. Spots were detected by spray- rial species were investigated to assess the distribution of the ing the plates with rhodamine B (0.01% in 0.25 M phosphate novel 0-1 methoxymycolate. buffer). Mycolates were recovered from the gels by eluting them with diethyl ether, and rhodamine B was removed by MATERIALS AND METHODS passing the preparation through a short Florisil column (0.5 by 2 cm). The sources of the 418 strains selected for this study are Instrumentation. Gas chromatography was performed by shown in Table 1. All of the strains were identified by using a Perkin-Elmer model 8310B gas chromatograph conventional methods (27, 29, 30). For isolation of lipid equipped with a fused-silica wide-bore column (12 m by 0.53 components, bacteria were cultivated on plates of Middle- mm [inside diameter]) with BP1 as the stationary phase. The brook 7H10 agar and incubated at 37°C in 5% CO, for 21 column temperature was programmed to increase at a rate of days, except as noted below. Strains of Mycobacterium 3"C/min from 140 to 260°C. The injector and detector tem- fallax, Mycobacterium marinum, Mycobacterium gadium, peratures were set at 260 and 290"C, respectively. For pyrolytic gas chromatography the injector temperature was set at 400°C. * Corresponding author. Nuclear resonance spectra were obtained with either a 390 VOL. 41, 1991 NOVEL MYCOLIC ACID IN MYCOBACTERZUM SPP. 391 TABLE 1. List of strains studied ~ ~ ~~ No. of No. of strains Total no. Taxon Reference strain(s) clinical from other of strains isolatesu sources examined Mycobacterium agri ATCC 27406T, CIP 141320002 2 Mycobacterium aichiense ATCC 27280T 1 Mycobacterium aurum ATCC 23366T 3 4 Mycobacterium avium ATCC 25291T 4b 5 Mycobacterium bovis ATCC 19210T 9 10 Mycobacterium chelonae subsp. ATCC 19977T 1 4 6 abscessus Mycobacterium chelonae subsp. NCTC 946T, ATCC 35752, TMC 1542 12 13 28 chelonae Mycobacterium chitae ATCC 19627T, CIP 141160002, CIP 141160003 3 Mycobacterium chubuense ATCC 27278T 1 Mycobacterium duvalii NCTC 358T, CIP 141180001 2 Mycobacterium fallax CIP 141390005T,CIP 141390011, CIP 141390013, CIP 5 10 141390016, CIP 141390023 Mycobacterium farcinogenes NCTC 109TjT, CIP 141100001 1 3 Mycobacterium flavescens ATCC 14474= 3 4 Mycobacterium fortuitum ATCC 6841T, NCTC 8573, TMC 1530 29 10 42 Mycobacterium gadium ATCC 27726T 1 Mycobacterium gastri ATCC 15754T, CIP 140340003, CIP 140340005, CIP 2 6 140340006 Mycobacterium gilvum NCTC 10742T 1 Mycobacterium gordonae ATCC 14470T 27 6 34 Mycobacterium intracellulare ATCC 13950T, TMC 1403 33b 35 Mycobacterium kansasii ATCC 12478= 51 52 Mycobacterium komossense ATCC 33013T 1 Mycobacterium malmoense ATCC 29571T 1 Mycobacterium marinum ATCC 927T 2 3 Mycobacterium moriokaense ATCC 43059T, CIP 141470002 2 Mycobacterium neoaurum ATCC 25795= 1 Mycobacterium obuense ATCC 27023T 1 Mycobacterium parafortuitum ATCC 19686T 1 L'Mycobacteriumperegrinum" TMC 1545, TMC 1547, NCTC 8573, Grange 487 3 19 26 Mycobacterium phlei ATCC 11758T, CIP 141300001 2 Mycobacteriurn porcinum ATCC 33776T, CIP 141460002, CIP 141460003 3 Mycobacterium rhodesiae ATCC 27024T 1 Mycobacterium scrofulaceum ATCC 19981T 18 19 Mycobacteriurn senegalense NCTC 10956T, CIP 141350001 2 Mycobacterium shimoidei ATCC 27962T 1 Mycobacterium simiae ATCC 25275T 3 4 Mycobacterium smegmatis ATCC 19420T, ATCC 14468, TMC 1515, CIP 141330001, 7 CIP 141330004, CIP 141330005, CIP 141330006 Mycobacterium szulgai NCTC 10831T 1 2 Mycobacterium terrae ATCC 157ST 5 6 Mycobacterium thermoresistibile ATCC 19527T, CIP 14131001 2 Mycobacterium triviale CIP 140330001T,CIP 140330002, CIP 140330003 3 Mycobacterium tuberculosis ATCC 27294T, ATCC 25584 44 46 Mycobacterium vaccae ATCC 15483T 1 Mycibacterium xenopi ATCC 19276T 26 27 Mycobacterium sp." 2 4 6 Clinical isolates included strains isolated from human patients. The other sources were animals and several environmental sources. A total of 418 strains were included in this study. These strains were identified by using standard cultural and biochemical properties as M. avium-M.intrucellulure. Rapidly growing nonphotochromogenic mycobacteria belonging to a new taxon. 200-MHz Brucker instrument or a 300-MHz Brucker instru- graphic behavior as known epoxymycolates in both solvent ment by using deuterochloroform as the solvent. A and solvent B (Fig. l), and the corresponding spot disappeared when acid methanolysis was used (23). The RESULTS infrared spectrum of this mycolate was consistent with the presence of a trans-epoxide ring (900 cm-l). The most polar Structural analysis of the more polar component of the M. oxygenated compound was stable under acidic and alkaline porcinum type strain. The mycolate chromatographic pattern conditions, and its Rf values were similar to those of the of M. porcinum ATCC 33776T (T = type strain) revealed the newly described w-1 methoxymycolate with both solvent presence of nonoxygenated mycolates and two types of systems (17); we found that the migration of this compound more polar oxygenated mycolates. One of these