INTRODUCTION

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The Microbial Identification System (MIS; Microbial ID, Newark, Del.) is a well-established fully automated gas chromatography (GC) analytical system which identifies bacteria and fungi based on their unique fatty acid profiles (6, 9, 14). The identification of mycobacteria by using the MIS standard library (M17H10) requires well-defined conditions of growth and sample preparation. Whole-cell fatty acid methyl esters (FAMEs) between 9 and 20 carbons in length are also used for the identification of mycobacteria, but the result of library search is often not discriminative enough for clinical application, i.e., in many cases the system does not allow discrimination between different species. Mycobacterial mycolic acid cleavage products (MACPs) with chain lengths of C₂₂ to C₂₆ are not released by the MIS sample preparation (8). The MACP pattern, however, is often highly significant for the identification of mycobacteria (4, 5, 7).

Trimethylsulfonium hydroxide (TMSH) converts fatty acids bound in biomolecules such as phospholipids and/or glycerides into the corresponding FAMEs. The transesterification can be performed at room temperature in a fast, single-step reaction. Secondary alcohols and MACPs are also released from mycobacteria under these conditions. The samples arising from TMSH treatment can be injected directly into the GC (12).

This paper describes the identification of mycobacteria by using the MIS library search with a TMSH-modified MIS sample preparation technique. The results of the conventional standard library search are compared with those of the TMSH-assisted library search.

	MATERIALS AND METHODS

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Chromatographic conditions. Specimens were processed on a Hewlett-Packard (Avondale, Pa.) GC system that included a model 5890 Series II GC equipped with a split injector and a flame ionization detector, a model 6890 automatic sampler, a Vectra XU 5/90C computer, a model 3365 Series II ChemStation (Rev A.03.34), and a fused-silica column (25 m by 0.2 mm by 0.33 µm; 5% phenylmethyl silicone, Ultra 2, HP 19091B-102). For MACP analysis, the column temperature program was increased from 260 to 310°C (starting at 170°C and increasing by 5°C min¹, with a final time of 3 min at 310°C) and two integration events were modified (Baseline All Valleys ON, 20.000 min; Integrator OFF, 30.000 min). All other parameters of chromatography were the following, as recommended in the operational manual of the MIS (8): carrier gas, hydrogen; sample volume, 2 µl; split ratio, 1:100; injector temperature, 250°C; detector temperature, 300°C.

Bacteria and growth conditions. The examined reference strains were Mycobacterium avium (DSM 43216), M. chelonae (DSM 43804), M. fortuitum (ATCC 6841, DSM 43271, DSM 43075), M. gastri (ATCC 15754), M. gordonae (ATCC 14470), M. intracellulare (DSM 43223), M. kansasii (ATCC 12478), M. malmoense (ATCC 29571), M. marinum (ATCC 927), M. scrofulaceum (ATCC 19981), M. simiae (ATCC 25275), M. smegmatis (DSM 43756, DSM 43061, DSM 43078), M. szulgai (NCTC 10831), M. terrae (DSM 43227), and M. tuberculosis (ATCC 27294), as well as clinical isolates (Table 2). The clinical isolates were identified by conventional biochemical tests, including tests of growth conditions, pigment, catalase, aryl sulfatase, phosphatase, niacin, and amides. Cultures were grown on Middlebrook agar (Difco 0627-01-2) plus Middlebrook OADC enrichment (Difco 0722-64-0) in 5 to 10% CO₂ at 35°C as described by MIDI (8).

Sample preparation. Sample preparation was done as described in the MIDI operating manual for the MIS (8): each sample was saponified (with sodium hydroxide in methanol), methylated (with hydrochloric acid in methanol), extracted (with hexane in methyl tert-butyl ether), and cleaned by base wash (with sodium hydroxide). After that step in the MIDI protocol, mycolic acids were removed by means of anhydrous sodium sulfate. This is very important to keep the chromatographic system clean; otherwise, the long-chain mycolic acids might lead to disturbances of later GC runs. For MACP analysis, the last step was replaced by adding 10 µl of 0.2 M methanolic TMSH (Macherey-Nagel, Düren, Germany).

	RESULTS

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Library search by using TMSH-modified sample preparation. One hundred ninety-nine strains of mycobacteria, including 180 clinical isolates (Table 2) and 19 reference strains, were prepared in accordance with the MIS sample preparation protocol and MIS sample preparation combined with the TMSH technique. The named species in the MIS library report were usually identical for both methods of sample preparation. In particular, the similarity indices showed no relevant difference. This means that MIS sample preparation combined with the TMSH technique allows the use of the MIS library search without modification.

Identification of MACPs. Identification of MACPs was done by comparing the chromatographic data resulting from the present study with fatty acid profiles reported in the literature (5, 7, 12). All FAME patterns, including those of MACPs and fatty alcohols, show excellent agreement with data in the literature. Table 1 shows the ECL values for important MACPs, long-chain FAMEs, and an alcohol. The ECL values were calculated by MIS after different samples were run. The ECL_MIS value is the interpolated ECL value determined by using the conventional MIDI calibration mixture 1 (C₉ to C₂₀). The ECL_MISplus value is the ECL value determined by using the supplemented MIDI calibration mixture 1 (C₉ to C₂₆). The calculated ECL values are the mean values of several (at least six) GC runs done on three independent chromatographic MISs. There was no significant difference between the ECL values of these systems, i.e., there was no mismatch between different runs and all peaks were identified by the systems.

Identification of strains. Most of the 19 reference strains were identified by MIS alone. M. avium (DSM 43216) and M. intracellulare (DSM 43223) were recognized as M. avium-intracellulare group; M. fortuitum (DSM 43271) was not distinguished from M. chelonae. M. chelonae (DSM 43804) was not distinguished from M. asiaticum. After MIS-plus-TMSH sample preparation, M. asiaticum could be excluded since the MACP C_26:0 was not found.

View larger version (12K): [in this window] [in a new window]   FIG. 1.   FAME pattern of M. tuberculosis resulting from the use of conventional MIS sample preparation (A) and MIS sample preparation combined with the TMSH technique (B).

View larger version (14K): [in this window] [in a new window]   FIG. 2.   FAME and MACP profile of M. szulgai resulting from the use of MIS sample preparation combined with the TMSH technique.

Reproducibility of FAME, alcohol, and MACP analysis. When the same strain is cultured under standardized conditions and analyzed repeatedly, there is a good reproducibility of the FAME, alcohol, and MACP profiles both qualitatively and quantitatively; similarity indices and blotting dendrograms based on Euclidian distance and principal-component analysis (8) of chromatographic patterns were used for comparison. Samples of the same strain were linked at a Euclidian distance of 2.5 or less (8).

	DISCUSSION

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The identification of mycobacteria by microbiological culture techniques is difficult to perform and requires up to several weeks. The identification by gene probes is highly specific, but in contrast to chromatographic methods, this method of identification is very expensive and the characterization of one single unknown species requires a series of many gene probes.

Cellular fatty acid compositions are widely used as a basis for the characterization of bacteria. The identification is done by one single chromatographic run, which usually requires less than half an hour. The MIS uses quantitative analyses of fatty acid profiles for reliable identification of many bacteria to the subspecies level. MIS sample preparation is simple and allows the identification of FAMEs and secondary alcohols but not the identification of the MACPs, a characteristic component of the mycobacterial cell wall. Thus, a reliable identification of mycobacteria must take into consideration the quantitative analyses of these molecules (7, 12).

TMSH (3, 10-13) reproducibly converts fatty acids bound in biomolecules such as phospholipids and/or glycerides into the corresponding FAMEs. The transesterification can be performed at room temperature in a fast, single-step reaction. MACPs and secondary alcohols are also released from mycobacteria under these conditions. The profiles of the chromatograms match well those obtained from other sample preparation techniques that need approximately 16 h to prepare MACPs (7).

The simple modification of sample preparation of applying 1 drop of TMSH reagent produces a pattern of FAMEs, alcohols, and MACPs in one single GC run. The results of the conventional MIS library search are not disturbed by the use of TMSH. The identification of long-chain fatty alcohols and MACPs is performed by MIS after ECL values are written into the peak library. The MACP patterns for mycobacteria presented in this study are consistent with those previously reported by others (7). These patterns enable one to differentiate between strains with similar indices of identification or identification of species outside the normal MIS library. Examples of this resolving power are found in differentiating M. avium, M. celatum, and M. xenopi by the MIS system. These species (data not shown) are characterized by similar FAME profiles in the region between C₁₀ and C₂₀ fatty acids (2). The dilemma is that there is no entry for M. celatum in the MIS library and the MIS library search often does not allow reliable discrimination between M. xenopi and M. avium. By using MACP and long-chain fatty alcohol patterns, differentiation of these three species is simple. M. xenopi is characterized by high amounts of C_22:0 alcohol and C_26:0, M. celatum contains high amounts of C_24:0 and C_26:0 fatty acids and lacks C_22:0 alcohol. M. avium contains high amounts of C_24:0; C_22:0 alcohol and C_26:0 are not found. M. celatum cannot be identified by conventional microbiological testing. Phenotypically, M. celatum is similar to M. avium, but genetic probes for M. avium do not react with it, suggesting that a specific probe is required for its identification (1). In contrast, GC of FAMEs with the enhanced ability to analyze mycolic acid cleavage fragments is less time-consuming and less expensive than DNA methods and can readily differentiate these three species.

The method described herein is helpful for users of the MIS since no significant change in sample preparation is necessary. A conventional library search can be done since the FAME pattern of MIS sample preparation is not changed by adding TMSH. The TMSH modification of sample preparation is not time-consuming since the reagents act immediately at room temperature and probes can be introduced directly into the GC. In contrast to conventional MIS sample preparation of mycobacteria, that of the chromatographic system described in this report is not disturbed by any residues of mycolic acids. So, FAME patterns of other bacteria can be investigated directly after running TMSH-treated probes.

Library generation software of MIS enables the creation of a new library for identification of mycobacteria based on TMSH-assisted sample preparation by using qualitative and quantitative profiles of FAMEs, alcohols, and MACPs. As long as such a library tool does not exist, the combination of a conventional library search report and an observation of a typical MACP pattern is an excellent tool for the identification of mycobacteria.