Japan BCG Central Laboratory, 3-1-5 Matsuyama, Kiyose-shi, Tokyo 204-0022, Japan
Correspondence
Yukiko Fujita
y-fujita{at}bcg.gr.jp
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Since mycolic acids are very high molecular mass, branched-chain, 3-hydroxy fatty acids with 70 to 90 carbon atoms and possess a complicated alkyl chain with cyclopropane rings, methyl branches and a methoxy- or carbonyl group, it has been difficult to establish a strict relationship between molecular characteristics and immunopotentiating activities. Furthermore, these -branched-chain 3-hydroxy fatty acids are unstable at high temperatures and are not amenable to gas chromatographic analysis. Wax ester-mycolates are easily broken down by alkaline hydrolysis to form dicarboxymycolic acid and secondary alcohol (Miquel et al., 1963
; Toriyama et al., 1980
, 1982
). We intended to introduce a simpler, non-degradative technique for the molecular characterization of mycoloyl glycolipids, without hydrolysis or pyrolysis.
Previously, structural analysis of mycolic acids has mainly been performed by direct electron impact mass spectrometry (EI/MS) (Dubnau et al., 1997; Toubiana et al., 1979
), fast atom bombardment mass spectrometry (FAB/MS) (Fujiwara et al., 1999
; Watanabe et al., 2001
) or gas chromatography mass spectrometry (GC/MS) (Tomiyasu & Yano, 1984
; Toriyama et al., 1978
; Yano et al., 1978
) of mycolic acid ester derivatives or pyrolysis products (meromycolate). However, EI/MS and FAB/MS analysis were insufficient for molecular mass estimation of intact mycoloyl glycolipids such as TMM (molecular mass >1400 Da) and TDM (molecular mass >2600 Da), and GC/MS analysis is limited to shorter chain mycolic acids up to C50 to C60. Recently, Watanabe et al. (2001
, 2002)
described the separation and characterization of individual mycolic acids in representative mycobacteria and the determination of the location of functional groups in meromycolate chains in detail by FAB/MS and collision-induced dissociation mass spectrometry (CID/MS). Also, accurate molecular mass determination of mycolic acids was reported by highly sensitive matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (Laval et al., 2001
). However, molecular mass determination of intact mycoloyl glycolipids has not yet been performed successfully.
In the present study, we first applied MALDI-TOF mass spectrometry in a positive reflectron mode to molecular mass determination of intact mycoloyl glycolipid with molecular mass greater than 1400 Da. Prior to the complete analysis of TDM, we show the results of comparative MALDI-TOF mass analysis of intact TMM.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Growth conditions.
M. tuberculosis, M. bovis (including BCG strains), M. smegmatis, M. phlei and M. flavescens were grown at 37 °C on Sauton medium as surface pellicles until early stationary phase. Members of the M. aviumintracellulare group and M. kansasii were grown on Middlebrook 7H9 medium with shaking at 37 °C until early stationary phase according to their growth rate.
Extraction, isolation and purification of TMM.
Bacterial culture was centrifuged after autoclaving at 121 °C for 15 min. Lipids were extracted from heat-killed, packed cells with 20 vols chloroform/methanol (2 : 1, v/v) three times with grinding. The two phases were separated in a funnel. After the lower phase containing major glycolipid was collected, the solvent was evaporated off with a rotary evaporator. The total lipids were first separated by solvent fractionation and then each acetone-soluble or chloroform-soluble or tetrahydrofuran-soluble or -insoluble fraction obtained was further separated by thin-layer chromatography (TLC) on silica plates (Uniplate; Analtech) with the solvent system of chloroform/methanol/water (90 : 10 : 1, by vol.) or chloroform/methanol/acetone/acetic acid (90 : 10 : 6 : 1, by vol.). Glycolipid spots were visualized with a 9 M H2SO4 spray followed by charring at 200 °C for analytical purposes or with iodine vapour for a few minutes for preparative purposes. TMM was recovered from the plate immediately after the iodine colour had disappeared by passing through a small glass column with the solvent chloroform/methanol (2 : 1, v/v). Finally, TMM was purified until a single spot was obtained by repeating TLC.
Analysis of mycolic acid methyl esters.
To confirm the possibility of direct analysis of intact molecular species composition of TMM by MALDI-TOF mass spectrometry, we also analysed mycolic acid methyl esters obtained from alkaline hydrolysis of TMM and other mycoloyl glycolipids. For this purpose, TMM, TDM and cell-wall-bound lipids were hydrolysed with 1·25 M NaOH in 90 % methanol at 70 °C for 1 h and the resultant mycolic acids were then extracted with n-hexane after acidification with HCl, followed by methylation with benzene/methanol/H2SO4 (10 : 20 : 1, by vol.) in a powerful fume cupboard under reduced pressure so as not to aspirate the vapour. Mycolic acid methyl esters from each lipid were fully separated into subclasses by TLC with the solvent system of benzene in the fume cupboard, considering the clear health hazards associated with benzene.
Sample preparation for mass spectrometry.
For MALDI-TOF mass analysis, TMM or mycolic acid methyl esters and 2,5-dihydroxybenzoic acid (2,5-DHB) as the matrix were dissolved in chloroform/methanol (2 : 1, v/v) at a concentration of 1 mg ml1 (TMM or methyl mycolate) or 10 mg ml1 (2,5-DHB). Aliquots of 5 µl of both samples and matrix were mixed and applied onto the sample plate as 1·5 µl droplets. The samples were then allowed to crystallize at room temperature.
Mass spectrometry analysis.
MALDI-TOF mass spectra (in the positive mode) were acquired on a Voyager-DE STR mass spectrometer (Applied Biosystems) with a pulse laser emitting at 337 nm. Samples were analysed in the reflectron mode with an accelerating voltage operating in positive ion mode of 20 kV. An external mass spectrum calibration was performed using calibration mixture 2 of the Sequazyme Peptide Mass Standards kit (Perseptive Biosystems), including known peptide standards in a mass range from 1290 to 5700 Da.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
MALDI-TOF mass spectrometry analysis of TMM from various mycobacterial species
Since natural mycolic acids occur generally as series of structurally related molecules differing from one another by two methylene units (28 a.m.u.), mycobacterial TMM generated complex mass spectra. Therefore, for the first approach in applying MALDI-TOF mass spectrometry to the analysis of TMM, we carefully took account of the calibration of pseudomolecular ions.
Fig. 1(a, b) shows positive MALDI-TOF mass spectra of TMM from two strains of M. tuberculosis. TMMs from M. tuberculosis H37Rv (Fig. 1a
) and Aoyama B (Fig. 1b
) showed similar pseudomolecular ion [M+Na]+ distributions ranging widely from m/z 1443 (nominal mass due to
-C75 TMM) to m/z 1725 (due to methoxy-C94 TMM). The characteristic feature is the biphasic distribution of pseudomolecular ions in this species, those due to
-mycoloyl TMM being in the lower mass range and those due to methoxy- or ketomycoloyl TMM in the higher mass range. A further characteristic is the occurrence of abundant odd-carbon-numbered (C77, C79, C81 and C83; in each case, the predominant member of each class is indicated in bold)
-mycoloyl TMM, which have not been reported so far in M. tuberculosis. Furthermore, these odd-carbon-numbered
-mycoloyl TMM showed monoenoic (or monocyclopropanoic) mycolate mass numbers, differing from even-carbon-numbered
-mycoloyl trehalose having dienoic (or dicyclopropanoic) mass numbers. This suggests that one of two cyclopropanation reactions occurs on the precursor destined to become dicyclopropyl
-mycolic acid at the level of TMM just before it is transferred to the cell wall, and not at the pre-
-meroacyl-S-acyl carrier protein level as previously considered (Takayama et al., 2005
), although the precursor double-bond position should be clarified in future. TDM mycolate also contained a small but significant amount of odd-carbon-numbered monoenoic
-mycolates. Methoxy- and ketomycoloyl TMM were also identified based on the analytical results of [M+Na]+ ions of the intact TMM. In these TMM subclasses, C85, C87, C88 and C89 methoxy- and C87 and C88 ketomycoloyl TMM predominated.
|
Differing from M. tuberculosis and M. bovis, the M. aviumintracellulare group showed a unique pseudomolecular ion distribution due to the existence of wax ester-mycoloyl TMM, as shown in Fig. 2(a, b). In the lower mass range, substantial amounts of ions due to even-carbon-numbered dienoic
-mycoloyl TMM with C78 to C84 mycolate centred at C80 were found. However, in the higher mass range, a large amount of ions due to wax ester-mycoloyl TMM with odd- (and even-) carbon-numbered mycolate with C82 to C89 centred at C85 and ions due to ketomycoloyl TMM with the same carbon-numbered mycolate were demonstrated. A slight, but significant difference between M. aviumintracellulare serotypes 4 and 16 was observed in the relative amounts of keto- and wax ester-mycoloyl TMM.
|
Tables 1 and 2 summarize the results of comparative studies of TMM molecular species from 11 strains of slowly and rapidly growing mycobacteria by MALDI-TOF mass analysis. Total numbers of carbons and double bonds (or cyclopropane rings) of major species of mycoloyl trehalose were determined by [M+Na]+ ions of
-, methoxy-, keto- and wax ester-mycoloyl TMM.
|
|
|
|
Wax ester-mycolic acids are known to be a characteristic component in the M. aviumintracellulare group, M. phlei and other rapidly growing photochromogenic mycobacteria (Miquel et al., 1963). Owing to the alkali-instability of the intramolecular ester bond, intact molecule analysis of wax ester-mycolates has not been successful so far. Instead, analysis of dicarboxy mycolic acid and secondary alcohols derived from alkaline hydrolysis products of wax ester-mycolates has usually been performed (Toriyama et al., 1980
, 1982
). In the present investigation, we report the first results of molecular characterization of intact wax ester-mycoloyl TMM by MALDI-TOF mass spectrometry without any degradation or hydrolysis procedure. In the M. aviumintracellulare group, distinctive [M+Na]+ ions due to C83, C85 and C87 wax ester-mycoloyl TMM centred at C85 were clearly demonstrated at m/z 1586, 1614 and 1642 in the higher mass range, while, in M. phlei and M. flavescens, [M+Na]+ ions due to C78, C79, C80, C81, C82 and C83 wax ester-mycoloyl TMM were observed at m/z 1516, 1530, 1544, 1558, 1572 and 1586, respectively, in the lower mass range. Interestingly, as expected, numbers of carbons of keto- and wax ester-mycoloyl TMM were almost identical, reflecting the metabolic precursorproduct relationship between the former and the latter, since the wax ester-mycolates are synthesized directly from ketomycolates by a biological BaeyerVilliger type oxidation system (Toriyama et al., 1982
).
The MALDI-TOF mass spectra of intact TMM extracted from each mycobacterium therefore give a characteristic fingerprint of the species (or subspecies) useful for chemical taxonomy or species identification and, furthermore, such molecular characterization may be especially important for the structureimmunomodulating activity relationship of cell-wall mycoloyl glycolipids for the host defence system.
Conclusions
Mycoloyl glycolipids such as TDM, TMM and arabinogalactan mycolate are extremely characteristic surface molecules in the mycobacterial cell wall. They play crucial roles not only in characterizing the mycobacterial surface structure, but also in affecting host immune responses as antigen, immunopotentiator or virulence factor at the site of infection. The present paper describes, for the first time, that direct MALDI-TOF mass spectrometry of TMM, the most simple mycoloyl glycolipid, can give precise information of the intact mycolic acid molecule without a degradation process. This technique is particularly promising for the analysis of more complicated mycoloyl glycolipids such as TDM or arabinogalactan polymycolate of the cell-wall skeleton, since it seems likely that the mycolic acid composition differs distinctively according to the mycobacterial species, growth conditions such as growth temperature and each glycolipid class, although analysis of TDM appears more laborious. Furthermore, the fast and easy separation of samples for MALDI-TOF mass spectrometry seems to be suitable for fingerprinting mycoloyl glycolipids from a given mycobacterial strain. It may also provide more information on the mechanism of biosynthesis of mycoloyl glycolipids in each mycobacterial species in the context of selective utilization of mycolic acid molecular species.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barry, C. E., III, Lee, R. E., Mdluli, K., Sampson, A. E., Schroeder, B. G., Slayden, R. A. & Yuan, Y. (1998). Mycolic acids: structure, biosynthesis and physiological functions. Prog Lipid Res 37, 143179.[CrossRef][Medline]
Beckman, E. M., Porcelli, S. A., Morita, C. T., Behar, S. M., Furlong, S. T. & Brenner, M. B. (1994). Recognition of a lipid antigen by CD1-restricted + T cells. Nature 372, 691694.[CrossRef][Medline]
Bekierkunst, A. (1968). Acute granulomatous response produced in mice by trehalose-6,6-dimycolate. J Bacteriol 96, 958961.[Medline]
Bekierkunst, A., Levij, I. S. & Yarkoni, E. (1971a). Suppression of urethan-induced lung adenomas in mice treated with trehalose-6,6-dimycolate (cord factor) and living bacillus Calmette Guerin. Science 174, 12401242.[Medline]
Bekierkunst, A., Yarkoni, E., Flechner, I., Morecki, S., Vilkas, E. & Lederer, E. (1971b). Immune response to sheep red blood cells in mice pretreated with mycobacterial fractions. Infect Immun 4, 256263.[Medline]
Bloch, H. (1950). Studies on the virulence of tubercle bacilli; the relationship of the physiological state of the organisms to their pathogenicity. J Exp Med 92, 507526.
Daffé, M., Lanéelle, M. A., Asselineau, C., Lévy-Frébault, V. & David, H. (1983). Taxonomic value of mycobacterial fatty acids: proposal for a method of analysis. Ann Microbiol 134B, 241256 (in French).
Dubnau, E., Lanéelle, M. A., Soares, S., Benichou, A., Vaz, T., Prome, D., Prome, J. C., Daffé, M. & Quemard, A. (1997). Mycobacterium bovis BCG genes involved in the biosynthesis of cyclopropyl keto- and hydroxy-mycolic acids. Mol Microbiol 23, 313322.[CrossRef][Medline]
Dubnau, E., Chan, J., Raynaud, C., Mohan, V. P., Lanéelle, M. A., Yu, K., Quemard, A., Smith, I. & Daffé, M. (2000). Oxygenated mycolic acids are necessary for virulence of Mycobacterium tuberculosis in mice. Mol Microbiol 36, 630637.[CrossRef][Medline]
Enomoto, K., Oka, S., Fujiwara, N., Okamoto, T., Okuda, Y., Maekura, R., Kuroki, T. & Yano, I. (1998). Rapid serodiagnosis of Mycobacterium avium-intracellulare complex infection by ELISA with cord factor (trehalose 6, 6'-dimycolate), and serotyping using the glycopeptidolipid antigen. Microbiol Immunol 42, 689696.[Medline]
Fujiwara, N., Pan, J., Enomoto, K., Terano, Y., Honda, T. & Yano, I. (1999). Production and partial characterization of anti-cord factor (trehalose-6,6'-dimycolate) IgG antibody in rabbits recognizing mycolic acid subclasses of Mycobacterium tuberculosis or Mycobacterium avium. FEMS Immunol Med Microbiol 24, 141149.[CrossRef][Medline]
Glickman, M. S., Cox, J. S. & Jacobs, W. R., Jr (2000). A novel mycolic acid cyclopropane synthetase is required for cording, persistence, and virulence of Mycobacterium tuberculosis. Mol Cell 5, 717727.[CrossRef][Medline]
Laval, F., Lanéelle, M. A., Deon, C., Monsarrat, B. & Daffé, M. (2001). Accurate molecular mass determination of mycolic acids by MALDI-TOF mass spectrometry. Anal Chem 73, 45374544.[CrossRef][Medline]
Matsunaga, I., Oka, S., Inoue, T. & Yano, I. (1990). Mycolyl glycolipids stimulate macrophages to release a chemotactic factor. FEMS Microbiol Lett 55, 4953.[Medline]
Matsunaga, I., Oka, S., Fujiwara, N. & Yano, I. (1996). Relationship between induction of macrophage chemotactic factors and formation of granulomas caused by mycoloyl glycolipids from Rhodococcus ruber (Nocardia rubra). J Biochem 120, 663670.[Abstract]
Minnikin, D. E. (1982). Lipids: complex lipids, their chemistry, biosynthesis and roles. Part I. Physiology of the mycobacteria. In The Biology of the Mycobacteria, pp. 95184. Edited by C. Ratledge & J. Stanford. London: Academic Press.
Minnikin, D. E., Minnikin, S. M., Parlett, J. H., Goodfellow, M. & Magnusson, M. (1984a). Mycolic acid patterns of some species of Mycobacterium. Arch Microbiol 139, 225231.[CrossRef][Medline]
Minnikin, D. E., Parlett, J. H., Magnusson, M., Ridell, M. & Lind, A. (1984b). Mycolic acid patterns of representatives of Mycobacterium bovis BCG. J Gen Microbiol 130, 27332736.[Medline]
Miquel, A. M., Ginsburg, H. & Asselineau, J. (1963). Composition of waxes C and D from Mycobacterium avium. Bull Soc Chim Biol 45, 715730 (in French).[Medline]
Moody, D. B., Reinhold, B. B., Guy, M. R. & 9 other authors (1997). Structural requirements for glycolipid antigen recognition by CD1b-restricted T cells. Science 278, 283286.
Moody, D. B., Reinhold, B. B., Reinhold, V. N., Besra, G. S. & Porcelli, S. A. (1999). Uptake and processing of glycosylated mycolates for presentation to CD1b-restricted T cells. Immunol Lett 65, 8591.[CrossRef][Medline]
Niazi, K., Chiu, M., Mendoza, R. & 8 other authors (2001). The A' and F' pockets of human CD1b are both required for optimal presentation of lipid antigens to T cells. J Immunol 166, 25622570.
Noll, H., Bloch, H., Asselineau, J. & Lederer, E. (1956). The chemical structure of the cord factor of Mycobacterium tuberculosis. Biochim Biophys Acta 20, 299309.[CrossRef][Medline]
Orbach-Arbouys, S., Tenu, J. P. & Petit, J. F. (1983). Enhancement of in vitro and in vivo antitumor activity by cord factor (6-6'-dimycolate of trehalose) administered suspended in saline. Int Arch Allergy Appl Immunol 71, 6773.[Medline]
Ozeki, Y., Kaneda, K., Fujiwara, N., Morimoto, M., Oka, S. & Yano, I. (1997). In vivo induction of apoptosis in the thymus by administration of mycobacterial cord factor (trehalose 6,6'-dimycolate). Infect Immun 65, 17931799.[Abstract]
Pan, J., Fujiwara, N., Oka, S., Maekura, R., Ogura, T. & Yano, I. (1999). Anti-cord factor (trehalose 6,6' dimycolate) IgG antibody in tuberculosis patients recognizes mycolic acid subclasses. Microbiol Immunol 43, 863869.[Medline]
Rastogi, N., Legrand, E. & Sola, C. (2001). The mycobacteria: an introduction to nomenclature and pathogenesis. Rev Sci Tech 20, 2154.[Medline]
Ribi, E., Granger, D. L., Milner, K. C., Yamamoto, K., Strain, S. M., Parker, R., Smith, R. W., Brehmer, W. & Azuma, I. (1982). Induction of resistance to tuberculosis in mice with defined components of mycobacteria and with some unrelated materials. Immunology 46, 297305.[Medline]
Ryll, R., Kumazawa, Y. & Yano, I. (2001). Immunological properties of trehalose dimycolate (cord factor) and other mycolic acid-containing glycolipids a review. Microbiol Immunol 45, 801811.[Medline]
Silva, C. L., Ekizlerian, S. M. & Fazioli, R. A. (1985). Role of cord factor in the modulation of infection caused by mycobacteria. Am J Pathol 118, 238247.[Abstract]
Sueoka, E., Nishiwaki, S., Okabe, S., Iida, N., Suganuma, M., Yano, I., Aoki, K. & Fujiki, H. (1995). Activation of protein kinase C by mycobacterial cord factor, trehalose 6-monomycolate, resulting in tumor necrosis factor-alpha release in mouse lung tissues. Jpn J Cancer Res 86, 749755.[Medline]
Takayama, K., Wang, C. & Besra, G. S. (2005). Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis. Clin Microbiol Rev 18, 81101.
Tomiyasu, I. & Yano, I. (1984). Separation and analysis of novel polyunsaturated mycolic acids from a psychrophilic, acid-fast bacterium, Gordona aurantiaca. Eur J Biochem 139, 173180.[Abstract]
Toriyama, S., Yano, I., Masui, M., Kusunose, M. & Kusunose, E. (1978). Separation of C5060 and C7080 mycolic acid molecular species and their changes by growth temperatures in Mycobacterium phlei. FEBS Lett 95, 111115.[CrossRef][Medline]
Toriyama, S., Yano, I., Masui, M., Kusunose, E., Kusunose, M. & Akimori, N. (1980). Regulation of cell wall mycolic acid biosynthesis in acid-fast bacteria. I. Temperature-induced changes in mycolic acid molecular species and related compounds in Mycobacterium phlei. J Biochem 88, 211221.[Abstract]
Toriyama, S., Imaizumi, S., Tomiyasu, I., Masui, M. & Yano, I. (1982). Incorporation of 18O into long-chain, secondary alcohols derived from ester mycolic acids in Mycobacterium phlei. Biochim Biophys Acta 712, 427429.
Toubiana, R., Berlan, J., Sato, H. & Strain, M. (1979). Three types of mycolic acid from Mycobacterium tuberculosis Brevanne: implications for structure-function relationships in pathogenesis. J Bacteriol 139, 205211.[Medline]
Walker, R. W., Prome, J. C. & Lacave, C. S. (1973). Biosynthesis of mycolic acids. Formation of a C32 beta-keto ester from palmitic acid in a cell-free system of Corynebacterium diphtheriae. Biochim Biophys Acta 326, 5262.[Medline]
Watanabe, R., Yoo, Y. C., Hata, K. & 8 other authors (1999). Inhibitory effect of trehalose dimycolate (TDM) and its stereoisometric derivatives, trehalose dicorynomycolates (TDCMs), with low toxicity on lung metastasis of tumour cells in mice. Vaccine 17, 14841492.[CrossRef][Medline]
Watanabe, M., Aoyagi, Y., Ridell, M. & Minnikin, D. E. (2001). Separation and characterization of individual mycolic acids in representative mycobacteria. Microbiology 147, 18251837.[Medline]
Watanabe, M., Aoyagi, Y., Mitome, H., Fujita, T., Naoki, H., Ridell, M. & Minnikin, D. E. (2002). Location of functional groups in mycobacterial meromycolate chains; the recognition of new structural principles in mycolic acids. Microbiology 148, 18811902.[Medline]
Yamagami, H., Matsumoto, T., Fujiwara, N., Arakawa, T., Kaneda, K., Yano, I. & Kobayashi, K. (2001). Trehalose 6,6'-dimycolate (cord factor) of Mycobacterium tuberculosis induces foreign-body- and hypersensitivity-type granulomas in mice. Infect Immun 69, 810815.
Yano, I., Kageyama, K., Ohno, Y., Masui, M., Kusunose, E., Kusunose, M. & Akimori, N. (1978). Separation and analysis of molecular species of mycolic acids in Nocardia and related taxa by gas chromatography mass spectrometry. Biomed Mass Spectrom 5, 1424.[Medline]
Yarkoni, E. & Bekierkunst, A. (1976). Nonspecific resistance against infection with Salmonella typhi and Salmonella typhimurium induced in mice by cord factor (trehalose-6,6'-dimycolate) and its analogues. Infect Immun 14, 11251129.[Medline]
Yarkoni, E. & Rapp, H. J. (1977). Granuloma formation in lungs of mice after intravenous administration of emulsified trehalose-6,6'-dimycolate (cord factor): reaction intensity depends on size distribution of the oil droplets. Infect Immun 18, 552554.[Medline]
Received 26 November 2004;
revised 28 January 2005;
accepted 28 January 2005.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |