Vol. 15, No. 3JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1982, p. 447-4550095-1137/82/030447-09$02.00/0

Thin-Layer Chromatography of Lipid Antigens as a Means ofIdentifying Nontuberculous Mycobacteria

PATRICK J. BRENNAN,' 2* MICHAEL HEIFETS,1 AND BETH P. ULLOM1Department of Clinical Laboratories, National Jewish Hospital and Research Center, Denver, Colorado

80206,1 and Department ofMicrobiology, Colorado State University, Fort Collins, Colorado 805232

Received 4 August 1981/Accepted 12 October 1981

The previously described thin-layer chromatography procedure (Brennan et al.,J. Clin. Microbiol. 8:374-379, 1978) for identifying serovars of the Mycobacteriumavium-M. intracellulare-M. scrofulaceum complex, based on their specific C-mycoside glycopeptidolipid antigens, has now been extended to all 31 knownserovars. Photographs of the characteristic pattern of the glycopeptidolipids arepresented. Although the procedure is fraught with the difficulties inherent tocomparative thin-layer chromatography, it is particularly suitable for the screen-ing of isolates which are not amenable to seroagglutination, such as those whichautoagglutinate or for which antisera are not presently available. In this way it waspossible to tell whether isolates were of the M. avium-M. intracellulare-M.scrofulaceum complex and whether or not they had previously been recognized.Knowledge of the chemical features of the typing antigens of nontuberculousmycobacteria other than M. avium-M. intracellulare-M. scrofulaceum strains alsoenables us to develop thin-layer chromatography systems for the identification ofM. kansasii, M. szulgai, members of the M. gordonae complex, M. terrae, M.xenopi, and M. gastri.

During the past 30 years it has become evidentthat tuberculosis-like diseases due to mycobac-teria other than tubercle bacilli occur more fre-quently than was earlier assumed. Based on theprevalence rate of acid-fast organisms other thantubercle bacilli in hospitalized patients, it isestimated that there may be up to 3,000 newcases per annum of nontuberculous mycobacter-ioses in the United States (5). Members of theMycobacterium avium-M. intracellulare-M.scrofulaceum (MAIS) complex are among themost common nontuberculous mycobacteria re-covered from humans (5). Aspects of the clinicalsignificance, bacteriology, and epidemiology ofnontuberculous mycobacteria have been recent-ly reviewed (18).The usefulness of serotyping in distinguishing

M. avium and M. intracellulare strains was firstemphasized by Schaefer (11, 12, 14), and hisseroagglutination procedure is now the primarymeans of identifying MAIS serovars and, in-deed, other nontuberculous mycobacteria. Thetest is applicable only to smooth colony-formingmycobacteria and is based on the presence ofspecies- or type-specific antigens. We have re-cently defined the structures of these antigensfrom some serovars (1-4). They are glycopepti-dolipids (GPLs), based on the well-known "C-mycosides" (2, 3) and have the generic structureshown in Fig. 1. Whereas the fattyacyl-peptidyl-monoglycosyl segment is prevelant in the anti-

gens from all serovars, the outer regions of theoligosaccharide segments are peculiar to eachserovar and, accordingly, confer both serologi-cal specificity and distinct chromatographic mo-bility (3). In the past, knowledge of these struc-tures allowed us to formulate chromatographicprocedures for identification of some serovars(4). In the present context, we have elaboratedon these procedures and extended them to all 31known serovars; furthermore, we have demon-strated the versatility of the procedure in classi-fying autoagglutinating and previously unrecog-nized isolates.

In the second part of this paper the approachhas been extended to other species of smoothcolony-forming mycobacteria. Seroagglutinationof a host of non-MAIS species is also based onamino acid-containing glycolipid antigens, butthese, unlike the C-mycosidic GPLs, are com-pletely labile to mild alkali treatment and aredistinguished by sugars other than 6-deoxyhex-oses. This information is herein utilized to differ-entiate between non-MAIS and MAIS orga-nisms and to identify certain non-MAIS species.

MATERIALS AND METHODSExtraction of lipid antigens and mild saponification.

Type (11) and untyped isolates were grown on 7H11agar medium as described before (4). The procedurepreviously developed for extraction of the specificlipids from serovars of the MAIS complex (4) was

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448 BRENNAN, HEIFETS, AND ULLOM

FattyAcyl-CO-NH-D-Phe-D-aThr--D-Ala-L-Alaninol-O-(3,4-di-O-Me-Rha)

0

[ 6-Deoxytalose-Rhamnose-(Sugar)x] -0-Acetyl

FIG. 1. Generic structure of mycobacterial GPLs.

extended to other mycobacteria without alteration.The native lipids so obtained were dried, redissolvedin distilled CHCl3, containing 0.005% 'butylated hy-droxytoluene, and stored at -20°C. Where indicated,lipids, in CHCl3-CH30H (2:1), were exposed to anequal volume of 0.2 M NaOH in CH30H at 37°C for 20min. CH30H-water (1:1) was then added to the reac-tion mixture, which was mixed in a Vortex mixer andcentrifuged. The CHCl3-rich bottom phase was thesource of the alkali-treated lipids. This crucial proce-dure of mild alkali treatment is identical to that alreadydescribed (4). Alkali-treated lipids could also be storedat -20°C in CHCl3 containing 0.005% butylated hy-droxytoluene.

Thin-layer chromatography (TLC). Homemade thin-layer plates (20 cm high and 10, 20, or 40 cm wide) ofSilica Gel H (Merck & Co., Inc., Rahway, N.J.) orcommercial plates from Analtech (Newark, Del.) wereused. Commercial plates gave "tighter" spots and arepreferred. The 40-cm-wide plates were poured byusing the standard Desaga device (15). Extracts of thenative or the alkali-treated lipids were applied as spots(20 pl per spot), usually 1 cm apart, using a hair dryerto conserve the spot size. Chromatography tanks linedwith Whatman 3MM chromatography paper were pre-equilibrated 1 h before use. The glass tank (19.5 by 12by 15 in. [ca. 50 by 31 by 38 cm]) marketed by SMIProducts (Emeryville, Calif.) is suitable for the 40-cm-wide plates. The four chromatographic solvents usedare labeled as before (4): I, CHCl3-CH30H-water,60:27:4; II, CHCl3-CH30H-water, 65:25:4; III, CHCl3-CH30H-water, 60:16:2; V, CHCl3-CH30H-water, 60:35:8.

Plates were sprayed with orcinol reagent (0.1% in40%o H2SO4) (4) and heated in an oven at 110°C for 7min. The C-mycoside GPLs produced a characteristicyellow-gold color due to the inherent 6-deoxyhexoses.The developed plates were photographed immediatelywith a Polaroid MP-3 Land camera.

RESULTS AND DISCUSSIONGeneral approach. The TLC procedure was

designed to supplement the more conventionalso-called biochemical tests (17) and seroagglu-tination. Above all, biochemical analyses arenecessary if only to aid in the choice of anappropriate chromatographic approach. For ex-ample, if preliminary data suggest a member ofthe MAIS complex, then the lipid extracts aretreated with alkali to destroy the nonspecificlipids and, by the process of deacetylation, tosimplify the pattern of specific C-mycosideGPLs (1). On the other hand, if biochemicalanalyses suggest, e.g., M. kansasii, M. szulgai,M. gordonae, M. terrae, M. xenopi, or M.gastri, then the lipid extracts remain untreated

and are chromatographed directly. Likewise, ifthe biochemical analyses are equivocal (i.e.,cannot differentiate between MAIS and non-MAIS), then the untreated lipids are chromato-graphed directly, and if vivid yellow-gold spotsare seen on spraying the TLC plates with orcin-ol, the lipids are then exposed to alkali andrechromatographed to identify the particularMAIS serovar. Alternatively, if alkali-stable yel-low-gold reacting spots are not evident on thechromatograms, then the organism must be ofthe non-MAIS grouping and treated accordingly(see below).C-mycosidic GPLs of the 31 MAIS serovars.

Previously we presented diagrams of the charac-teristic deacetylated GPL antigens from 17 ofthe 31 known serovars (4). Figure 2 is a photo-graph of an actual thin-layer plate of the GPLsfrom all 31 serovars. Solvent II is our "univer-sal" solvent since it is applicable to a greaterarray of serovars than is any other known sol-vent. It and solvent V are particularly useful forthose serovars with slow-moving GPLs (e.g.,serovars 8, 9, 10, 11, 19, 21, 25, 26, 27, and 42).It is not entirely suitable for those with fast-moving GPLs (e.g., serovars 1, 2, 3, 4, 6, 7, 12,13, 14, 15, 16, 17, 20, and 28). However, themajority of these can be resolved in solvent I orIII.

In general, each serovar is distinguished by itsown unique complement of one, two, or threeGPLs. There are exceptions, the most obviousbeing serovars 10 and 11, both of which yield asimilar array of lipids. Other apparent excep-tions are serovars 2, 4, 6, 14, and 20, also 15 and23, and still others which are obvious from Fig. 2and 3. The similarities in the mobilities of theGPLs from these organisms make their unequiv-ocal identification by TLC particularly difficult.Among certain combinations of serovars there isa distinct reciprocity between chromatographicmobility and cross-agglutination, which is notsurprising since the GPLs are the typing anti-gens (2, 3). For example, Schaefer (14) reportedthat serovar 10 (formerly serotype III) is coag-glutinated by serovar 11 (formerly serotype Illa)sera. Also, serovars 1 and 2 coagglutinate, as do12 and 13, 8 and 21, 14 and 15, and 16 and 17.Schaefer noted yet other examples of cross-agglutination; e.g., strains of serovar 9 are agglu-tinated by serovar 3 sera. Interestingly, there isevidence for the presence in serovar 3 of a GPL

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TLC OF MYCOBACTERIAL LIPID ANTIGENS 449

.wSolvent Front

44t 44

i 1 i i I I i I i

. S S

*S S

a0 0 0 *

-... Origin!;,. Ir, 1 1 ',, 1 .! i'- ',< .S .'-,- 23 4 4- . x-? 2 42 -'

FIG. 2. TLC in CHCl3-CH30H-water (65:25:4) (solvent II) of the alkali-treated lipid extracts from allserovars of the MAIS complex. The nonspecific apolar C-mycosides (reference 3) are congregated at the solventfront. The major characteristic (identifying) polar GPLs are marked with daggers. Some of the lipids ran in askewed fashion (e.g., 17) and are to the right of the origin. The plate was sprayed with orcinol-H2SO4, to whichthe spots gave a bright yellow-gold color.

41

.1!.r:i '1-'.!% ' 7 -e

I 0

4

4

4

4

14 15 16 20 23

FIG. 3. TLC in CHCl3-CH30H-water (60:16:2; sol-vent III) of the alkali-treated lipid extracts from sero-vars 14, 15, 16, 20, and 23 of the MAIS complex. Otherconditions are as in the legend to Fig. 2.

with mobility similar to one of these in serovar 9(Fig. 2). Hence, several of the cases of cross-agglutination reported by Schaefer may be ex-plained by either shared GPL antigens or unusu-ally close similarities in the composition of themajor specific lipid antigens.TLC of C-mycoside GPLs as a means of recog-

nizing classified and unclassified MAIS serovars.Under the aegis of the U.S.-Japan CooperativeMedical Science Program (sponsored by theNational Institutes of Health), we serve as areference laboratory for the identification of"atypical" mycobacterial isolates submitted forstudy and classification. The primary means ofidentification is seroagglutination (11, 12, 14).However, only between 40 and 70% of isolatesare amenable to this test. For example, of 511cultures recently submitted to us, 213 weresuccessfully typed, 109 autoagglutinated, and189 were either non-agglutinating or multiplyagglutinating. These non-agglutinating and mul-tiply agglutinating isolates are the prime targetsfor TLC examination.The TLC approach for handling nontubercu-

lous mycobacterial isolates which are thought tobe MAIS organisms and which produce a nega-tive or an equivocal response to the seroagglu-tination test is as follows. The alkali-treatedlipids from 10 to 12 such isolates are chromato-graphed in solvent II against similarly derivedlipids from 25 to 30 typed serovars on 40-cm-wide plates and sprayed with orcinol. Alkali-stable yellow-gold spots (due to the 6-deoxyhex-

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450 BRENNAN, HEIFETS, AND ULLOM

oses inherent to the C-mycoside GPLs) arehighly indicative of a MAIS strain. If, on theother hand, no characteristic lipid spots areevident on the TLC plates, then the isolate isprobably of a non-MAIS specification, and theapproach described later, using the untreatednative lipids, is implemented.The chromatograms in Fig. 4A to D contain

alkali-treated extracts from non-agglutinatingisolates. Recognized MAIS serovars are giventheir Arabic numerals. Isolates which failed theseroagglutination test are represented by alpha-betical rudiments and, where alphabeticals runout, by lowercase Roman numerals. Seldom isthere perfect reproducibility between differentchromatograms despite running them in thesame solvent, same tank, and similar climaticconditions. For example, regard serovar 4 inFig. 4B and D, serovar 7 in Fig. 4C and D,serovar 8 in Fig. 4A, B, and D, and serovar 15 inFig. 5B and D. Also, the absolute Rf values formany of the serovars do not agree with previousvalues (4). Hence, each chromatogram must beexamined in its own right, and a comparisonbetween chromatograms is not generally ad-vised. This problem can be partially overcomeby applying a common standard, such as methylorange, to all chromatograms. There can also beinconsistencies in the relative mobilities of someGPLs when run in fast solvents (e.g., serovars 2and 4 are similar in Fig. 2 but differ in Fig. 5).However, this irregularity disappears when asolvent is used which is more compatible withthe polarity of the particular GPL (e.g., Fig. 3).Yet another difficulty with this system is thatsome products become degraded on prolongedstorage and develop extra spots (e.g., serovar 14in Fig. 4A). In general, it is the most polar,slower moving, major spots which are character-istic of each serovar and which are the basis ofcomparison. It is important to realize this whenexamining chromatograms such as these in Fig.4 because, due to unequal application of lipids atthe origin, the lesser apolar spots may not beobvious and patterns may therefore look dissim-ilar in the upper regions.

Despite these problems, meaningful conclu-sions can be made from such chromatography,particularly if sufficient numbers of extractsfrom known organisms are included on eachplate and if unknowns are rechromatographed,preferably in other solvents, and closer to themore likely candidates. The results of the chro-matography shown in Fig. 4 followed by otherchromatograms are summarized as follows: notMAIS-c, f, h, j, n, o, p, r, t, u, I, II, V, VI, VII,XI, XII, XIII, XIV, XVIII, XIX; MAIS strainsstill under examination-a, b, i, X; MAIS strainstentatively identified-g, m, q, s, VIII, IX, XV,XVI; MAIS strains unclassified-d, e, k, w, III,

IV, XVII. The isolates which did not yield anycharacteristic alkali-stable lipid spots (Fig. 4A,B, C, and D), and therefore are not of the MAISspecification, are given first. Some of these weresubsequently identified by the procedures de-scribed below for non-MAIS isolates. Of theMAIS isolates which were tentatively identified,g and IX were recognized as either serovar 14 or20 by further chromatography in solvent III.Isolate m appeared to be serovar 18; q, serovar23 (see also Fig. 5A); s, serovar 16; VIII, serovar7 (see also Fig. SA); and XV and XVI, serovar 6,14, or 20. The evidence in Fig. 4 for some ofthese designations is not always entirely con-vincing due to the absence of some of the minorspots in the photographs, the distance betweencomparable spots, and the lack of reproducibili-ty between these 40-cm chromatograms, espe-cially for serovars with fast-moving spots. Nev-ertheless, when the less polar solvent III wasused, we had little doubt about the assignments.The characteristic GPLs from isolates d, e, k,

w, lII, IV, and XVII (Fig. 4) did not correspondexactly to those from any of the known serovarswhen a number of solvents were used. Rechro-matography of d, e, k, III, IV, and XVII (Fig.SA) indicated that d, e, and k, which wereobtained from one patient at National JewishHospital, are identical. Also, XVII, III, and IV,which arose from three different patients inanother hospital, apparently are identical anddifferent from d, e, and k. Indeed, XVII, III, andIV are clearly the same (Fig. 5B), but the evi-dence that they differ from d, e, and k is notcompletely convincing. These chromatogramscertainly help to emphasize the difficulties in-volved in making such decisions. Nevertheless,this is the only reasonable approach presentlyavailable to use when dealing with untypableisolates from patients or other sources.Thus, the results provide indications for the

existence of one or two additional members ofthe MAIS complex. Indeed, from an examina-tion of like plates for the many seroagglutina-tion-fail isolates examined by us, we are nowconvinced that the MAIS serocomplex extendswell beyond its present 31-member limit, a con-viction shared by others (D. J. Dawson andP. A. Jenkins, personal communications). How-ever, substantially more evidence is required toprove this point. Of particular necessity is thepreparation of antisera to new isolates and itstesting against standard strains of all knownserovars to ensure specificity, and also the rec-ognition of isolates with the same characteristicGPLs from other sources. Our purpose here isnot so much to determine the infrasubspecificstatus of the MAIS complex, but to demonstratethe usage of serology combined with chromatog-raphy of the GPL antigens for the identification

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TLC OF MYCOBACTERIAL LIPID ANTIGENS 451

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FIG. 4. TLC in solvent II of the lipids from some known MAIS serovars against those from seroagglutination-fail isolates. Again, some of the lipids ran in a skewed direction to the right. For instance, in (C) the two center

spots in the center of the plate correspond to III and IV, not to IV and V.

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452 BRENNAN, HEIFETS, AND ULLOM

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FIG. 5. Rechromatography of the lipid extracts from "unclassified" MAIS serovars against extracts fromother isolates and classified serovars. Chromatogram A was run in solvent I and chromatogram B was run insolvent II.

and classification of MAIS organisms.Identification of non-MAIS species by TLC of

their specific lipids. In general, the hallmark ofnon-MAIS species is that their characterizinglipids are all alkali labile. Accordingly, chroma-tography of the alkali-stable lipid extracts willshow nothing worthwhile (f. non-MAIS isolatesgiven above with the patterns of the correspond-ing isolates in Fig. 4). Certainly this rule appliesto M. kansasii, M. szulgai, M. gordonae, M.xenopi, M. terrae, M. gastri, and M. fortuitum.Therefore, to identify any of these species,whole untreated lipid extracts must be applied tothin-layer plates. Because many nonspecific al-kali-labile lipids, such as neutral and phospho-glycerides, are present in these extracts, thepatterns are not as unambiguous nor, due to theabsence of the yellow-gold color, as striking asthose from the alkali-treated extracts of MAISstrains.The general requirement and procedure for

identification of an agglutination-fail non-MAISisolate are as follows. First, no distinct lipidsshould be evident in the system described inFigure 4 (i.e., TLC after alkali treatment ofwhole lipid extracts). If this criterion is fulfilled,then whole lipid extract is applied to a TLC plateand chromatographed in solvent II beside ex-tracts from the above-listed group of non-MAISspecies. The color reaction is observed, and anidentification may be made at this stage, particu-larly if previous analyses, such as biochemicalreactions (17), are taken into account. Finally,the extract is rechromatographed along with thatfrom the most likely candidate and compared asdescribed below. In our experience, between 40

and 50% of non-MAIS seroagglutination-fail iso-lates can be identified in this fashion.Chromatographic properties of the lipid anti-

gens ofM. kansasii and M. szulgai. When variousextracted fractions from M. kansasii and M.szulgai were tested for serological activityagainst rabbit antisera to the homologous orga-nisms, it was found that only lipid extracts wereactive (Brennan and Ullom, unpublished data).Subsequently, it was shown that the pure activelipids were oligosaccharides acylated with fattyacids and amino acids and that serological activi-ty and structural integrity were destroyed byalkali. Location of these characteristic lipids onthin-layer plates provides a facile means foridentifying M. kansasii. Figure 6 is a chromato-gram of whole lipid extracts from several differ-ent isolates of M. kansasii. The characteristicserologically active lipids, three in number, areindicated with arrows. These produce a verytypical dark olive-green color which is due to thepresence of xylose and glucose in the lipids(Brennan and Ullom, unpublished data). Szulgaet al. (16) have already drawn attention to thecharacteristic array of lipids produced by M.kansasii. When a direct comparison of the pat-tern obtained with their solvent system wasmade with that shown in Fig. 6, it became clearthat they also were demarcating the lipids re-sponsible for specific agglutination of M. kansa-sii.The recognition of M. szulgai as a distinct

species is credited to a unique pattern of lipidswhich Marks, Jenkins, and co-workers could notreconcile with that of any known species (8, 10).The TLC profile of the characteristic lipids of

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TLC OF MYCOBACTERIAL LIPID ANTIGENS 453

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FIG 6. TLC in solvent II of the intact lipids from anassortment of isolates which had been characterized asM. kansasii by serological and biochemical means.These produced a characteristic dark olive-green colorwith orcinol-H2SO4.

seven randomly selected isolates of M. szulgaiare shown in Fig. 7. The patterns from five of theisolates are identical and very characteristic.The reaction of the lipids to the spray is mostaccurately described as a grey-brown hue. Ofthe six isolates, one (no. 1) presented an unusualpicture. The implications of this "aberrant"pattern for an organism which in all other re-

spects corresponds to M. szulgai is as yet un-known. However, there is a precedent for thistype of observation. Szulga et al. (16) describedtwo types of patterns for isolates identified as M.kansasii. Their "type 1" pattern was found inthe majority of strains isolated from humans,whereas the second type of pattern occurredonly once among 20 isolates. In the case of M.szulgai, the aberrant pattern was found once

among 15 strains which had all been serotyped

FIG. 7. TLC in solvent II of the intact lipids froman assortment of isolates which had been character-ized as M. szulgai by serological and biochemicalmeans. These give a grey-brown color on sprayingwith orcinol-H2SO4.

as M. szulgai. On a few occasions we havepreviously encountered the type of patternshown for isolate 5 in which there were traces ofthe normal array of lipids on the original plates.We now believe that this isolate had almostcompletely converted to "rough" morphology,with the consequent loss of most of its lipidantigens.

Characteristic lipid patterns for the M. gor-donae complex. Goslee et al. (7) recognizedseven distinct agglutination types of M. gor-donae, a group of saprophytic scotochromo-genic mycobacteria widely scattered in nature.TLC of the lipid extracts from all seven serovarsis shown in Fig. 8. Although the characteristiclipids differ widely in polarity, they all respond-ed to the orcinol spray with a representativeyellow-green color, suggesting a basic structural

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454 BRENNAN, HEIFETS, AND ULLOM

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the M. gordonae serocomplex. All gave a yellow-green color with the orcinol spray.

similarity. Serovar 5 consistently gave a veryfaint pattern. Jenkins et al. (8) have alreadyshown characteristic lipid patterns for twostrains with cultural resemblence to M. gor-donae. Since their solvents differ from thoseused in Fig. 8, it is not possible to determinewhich of the seven serovars was examined. Ourapproach of combined serology and chemicalanalysis has not yet been extended to these lipidproducts from the M. gordonae complex. How-ever, since all are alkali labile, they are presum-ably structurally related to the acylated oligosac-charides of M. kansasii.

Characteristic lipids of M. gastri, M. terrae, M.flavescens, and M. xenopi. In none of these caseshas serological activity yet been definitely corre-lated with species-specific lipids. Because of theinordinate simplicity and similarity of the pat-terns from M. terrae, M. flavescens, and M.xenopi in solvent V (Fig. 9), we are cautiousabout the use of TLC for identification of thesespecies. Nevertheless, a reasonable differentia-

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FIG. 9. TLC in CHC13-CH30H-water (60:35:8; sol-vent V) of the intact lipids from M. szulgai, M. gastri,M. terrae, M. flavescens, and M. xenopi. Methylorange is included as an internal marker.

tion between M. terrae and M. xenopi can beachieved with solvent III, in which M. terraeshows a characteristic lipid, absent in M. xen-

opi, with an Rmethyl orange value of 3.5. On theother hand, M. gastri produces a unique patternwith three major lipids, each responding to theorcinol spray with a characteristic green color.Hence, the system described in Fig. 9 is usefulfor identification of M. gastri, whereas a changein the composition of the solvent to that ofsolvent III will further distinguish between sus-pected M. terrae and M. xenopi. Further im-provement of the TLC systems for identifyingthese species is required and will undoubtablyemerge once more is known about the chemicalcomposition of their characteristic lipids.

ACKNOWLEDGMENTS

This investigation was funded by Public Health Servicecontract AI-02079 from the U.S.-Japan Medical SciencesCooperative Program, administered by the National Instituteof Allergy and Infectious Diseases.We thank J. K. McClatchy and M. B. Goren for their

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TLC OF MYCOBACTERIAL LIPID ANTIGENS 455

continuing interest in this work. We also thank Anna Tsang forproviding the isolates and their histories and Ilga Drupa for theseroagglutination analyses.

LITERATURE CITED

1. Barrow, W. W., B. P. Ullom, and P. J. Brennan. 1980.Peptidoglycolipid nature of the superficial cell wall sheathof smooth-colony-forming mycobacteria. J. Bacteriol.144:814-822.

2. Brennan, P. J., and M. B. Goren. 1979. Structural studieson the type-specific antigens and lipids of the Mycobacte-rium avium-Mycobacterium intracellulare-Mycobacte-rium scrofulaceum serocomplex. J. Biol. Chem.254:4205-4211.

3. Brennan, P. J., H Mayer, G. 0. Aspinall, and J. E. NamShin. 1981. Structures of the glycopeptidolipid antigensfrom serovars in the Mycobacterium avium/Mycobacte-rium intracellularelMycobacterium scrofulaceum sero-complex. Eur. J. Biochem. 115:7-15.

4. Brennan, P. J., M. Souhrada, B. Ullom, J. K. McClatchy,and M. B. Goren. 1978. Identification of atypical myco-bacteria by thin-layer chromatography of their surfaceantigens. J. Clin. Microbiol. 8:374-379.

5. Davidson, P. T. 1979. The management of disease withatypical mycobacteria. Clin. Notes Respir. Dis. 18:3-13.

6. Goren, M. B., and P. J. Brennan. 1979. Mycobacteriallipids: chemistry and biologic activities, p. 64-194. InG. P. Youmans (ed.), Tuberculosis. W. B. Saunders Co.,Philadelphia.

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