Aug 21, 1989 - paper was published by Holzer et al. (2), who ... Holzer, G., T. F. Bourne, and W. Bertsch. 1988. ... Marcel Dekker, Inc., New York. 4. Kossa, W.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1990, p. 1717-1724
Vol. 56, No. 6
0099-2240/90/061717-08$02.00/0 Copyright C) 1990, American Society for Microbiology
Pyrolytic Methylation-Gas Chromatography of Whole Bacterial Cells for Rapid Profiling of Cellular Fatty Acids JACEK P. DWORZANSKI,t LUC BERWALD,t AND HENK L. C. MEUZELAAR* Center for Micro Analysis and Reaction Chemistry, University of Utah, Salt Lake City, Utah 84112 Received 21 August 1989/Accepted 7 March 1990
A novel, on-line derivatization technique has been developed which enables generation of fatty acid methyl ester (FAME) proffles from microorganisms by gas chromatography-mass spectrometry without the need for laborious and time-consuming sample preparation. Microgram amounts of bacterial cells are directly applied to a thin ferromagnetic filament and covered with a single drop of methanolic solution of tetramethylammonium hydroxide. After air drying, the ifiament is inserted into a special gas chromatograph inlet equipped with a high-frequency coil, thus enabling rapid inductive heating of the ferromagnetic filament. This so-called Curie-point heating technique is shown to produce patterns of bacterial FAMEs which are qualitatively and quantitatively nearly identical to those obtained from extracts of methylated lipids prepared by conventional sample pretreatment methods. Relatively minor differences involve the loss of hydroxy-substituted fatty acids by the pyrolytic approach as well as strongly enhanced signals of FAMEs derived from mycolic acids. This type of pyrolysis enables on-line derivatization and thermal extraction of volatile derivatives for analysis, whereas the residual components remain on a disposable probe (ferromagnetic wire) of a pyrolytic device. The reduced sample size (micrograms instead of milligrams) and the lack of sample preparation requirements open up the possibility of rapid microbiological identification of single colonies (thus overcoming the need for timeconsuming subculturing) as well as analysis of FAME proffles directly from complex environmental samples.
Classification and identification of microorganisms based on rapid and specific methods for determining the chemical composition have proved to be a valuable approach in microbiology (1). Especially, the combination of modern chromatographic methods such as gas chromatography (GC) and, more recently, liquid chromatography with mass spectrometry (MS) techniques has led to the identification of a large number of chemical constituents showing a degree of
specificity suitable for chemotaxonomic and/or diagnostic purposes (7). To make full use of the potentially high speed and specificity offered by these chemical techniques, much research has been devoted to the development of improved methods for sample preparation prior to chromatographic analysis. Because only volatile components are appropriate for GC analysis, two different methodological approaches capable of converting nonvolatile components to volatile products were widely studied during the past decade. The first approach is based on thermal degradation (pyrolysis) of whole microorganisms or their components (3), whereas the second one uses wet chemical methods involving saponification, extraction, and initial separation or derivatization or both of bacterial constituents prior to GC analysis. The application of pyrolysis to the volatilization of normally nonvolatile constituents of cells is based on thermal cleavage (in an inert atmosphere) to smaller or modified substances which are suitable for GC, MS, or combined GC-MS analysis. Pyrolytic methods provide the required speed in sample preparation, because the only step involves placing the bacterial sample on a pyrolytic probe. However,
* Corresponding author. t Permanent address: Silesian Medical Academy, Sosnowiec,
Poland. t Permanent address: Rijksinstituut voor Millieuhygiene, Bilthoven, The Netherlands.
Volksgezondheid
en
compared with the wet chemical techniques, pyrolytic methods appear to lag behind with regard to overall specificity. Chemical methods of sample preparation enable determination of highly specific substances, e.g., fatty acids derived from bacterial cell walls, but generally require laborious and long procedures which seriously limit response time as well as analytical throughput. These considerations prompted us to investigate the possibility of developing a novel analytical approach which combines the speed of pyrolytic methods with the specificity offered by chemical derivatization procedures. Injection port methylation during analysis of blood serum extracts by GC is a known phenomenon. Wong et al. (13) observed that injection of a solution of phenobarbital mixed with a solution of lecithin generates the formation of Nmethyl-phenobarbital and methyl palmitate. The known ability of quarternary alkyl amines to alkylate acidic compounds (4) suggested to us that substances such as choline might be responsible for some of these observations. Hence, we decided to add small amounts of a quaternary amine to bacterial samples coated on a pyrolysis filament such as is used in so-called Curie-point pyrolysis. Addition of tetramethylammonium hydroxide (TMAH) to a pyrolysis filament coated with bacterial lipids or whole bacterial cells and subsequent pyrolysis of that mixture revealed strong fatty acid methyl ester (FAME) signals, whereas the free fatty acid signals observed in typical pyrolysis experiments (11) appeared to be low or absent. The above observations have formed the basis for a series of studies aimed at the development of on-line pyrolytic derivatization techniques for cells and tissues. Preliminary results of on-line pyrolysis GC-MS experiments with a selected group of gram-negative and gram-positive bacteria are reported here. Mechanistic aspects of the observed phenomena will be reported in a separate paper. (The work reported here was presented in part at the 36th 1717
1718
ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco, Calif., June 1988.)
;. . . .: APPL. ENVIRON. MICROBIOL.
DWORZANSKI ET AL.
carrier gas--
pyrolysi MATERIALS AND METHODS Bacterial cultures. Strains of Escherichia coli and Bacillus subtilis used in our study were obtained from Sigma Chemical Co., St. Louis, Mo., in the form of freeze-dried or frozen cells, respectively. Samples of Mycobacterium tuberculosis were obtained from the Rijksinstituut voor Volksgezondheid and Milieuhygiene, Bilthoven, The Netherlands, where the clinical isolates were cultured and identified by conventional microbiological procedures before being shipped to the University of Utah Center for Micro Analysis and Reaction Chemistry in the form of freeze-dried cells. Sample preparation. Bacterial cells were prepared for fatty acid analysis by two different methods: (i) saponificationmethylation-extraction (SME), and (ii) pyrolytic methylation with TMAH. The SME procedure we used is based on the method described by Moss (6) with modifications introduced by Miller and Berger (L. T. Miller and T. Berger, application note 228-41, Hewlett-Packard Co., 1985). The whole procedure was carried out in glass test tubes sealed with polytetrafluoroethylene-lined screw caps. Cells were placed in test tubes and saponified for 30 min at 100°C with methanolic sodium hydroxide (3 N in 50% methanol). Methyl esters of the fatty acid sodium soaps were prepared by adding 3 N HCl in 40% aqueous methanol and heating the mixture at 80 ± 1C for 10 min. After rapid cooling of the mixture, FAME components were extracted with a mixture of diethyl ether and hexane (1:1, vol/vol) under continuous rotation of the test tubes. This was followed by removal of the aqueous methanolic phase with a Pasteur pipette and washing the extract with a mildly basic solution of sodium hydroxide in deionized water. The organic layer was removed and analyzed immediately or stored for a short time at -20°C in the dark in a polytetrafluoroethylene-lined vial. The pyrolytic methylation procedure is based on the direct application of whole bacterial cells onto a ferromagnetic pyrolytic wire. To obtain known amounts of the bacteria on the wires, samples of bacterial cells were suspended in methanol (1 mg/ml) and dispersed by immersion in a weak ultrasonic bath. Aliquots, 5 ,ul, of each suspension were placed on the tips of ferromagnetic wires (diameter, 0.5 mm), and the solvent was allowed to evaporate at room temperature. In other experiments, the wires were simply touched to bacterial colonies on agar plates or to suspensions, lipid extracts, and bacterial pellets obtained after centrifugation of bacterial growth media. Immediately after the wires were coated with bacteria, a 5-,ul drop of 0.1 M TMAH in methanol was placed on each wire and the solvent was allowed to evaporate at room temperature. After drying, the wires were withdrawn into borosilicate glass tubes and inserted into the Curie-point pyrolyzer (Fig. 1). Curie-point pyrolysis. Pyrolytic derivatization was carried out by means of a newly developed microvolume Curie-point reactor (Fig. 1) described elsewhere (H. L. C. Meuzelaar, W. H. McClennen, and Y. Yun, Proc. 35th Am. Soc. Mass Spectr. Conf., p. 1142-1143, 1987) in more detail. Ferromagnetic wires with Curie-point temperatures of 358, 510, or 610°C were chosen for the various experiments. A homebuilt high-frequency power supply (0.5 MHz, 100-W maximum) was used to achieve a 1.0- to 2.0-s temperature rise time. Total heating time was 4 s. GC-MS. FAME mixtures were separated on a fused silica
filament:
to IITD
..
sample-4 hf3 hieated zone
C} C200
----
it
no split
Inlet restrictor and vent
_capillary
GC~G :column:
oven
FIG. 1. Schematic representation of the Curie-point pyrolysis GC-MS technique. ITD, Ion trap detector; hf, high frequency.
capillary column (12 m by 0.22-mm inside diameter) coated with 0.15-,um BP-1 (Perkin-Elmer bonded phase), using a Perkin-Elmer model 8500 GC. The column exit was inserted via a heated transfer line into the vacuum system of a model 700 Finnigan MAT ion trap detector MS system, controlled by an IBM PC-XT compatible computer. Thus, the conventional open splitter inlet of the ion trap detector system was bypassed, and the column exit was brought directly to within the ion trap detector electrode region. Column temperature was programmed from 50 to 140°C at a rate of 30°C/min and then to 310°C at a rate of 15°C/min. High-purity helium was used as a carrier gas, with a linear velocity of 50 cm/s, corresponding to a flow rate of 1.1 ml/min. Samples were split 5:1 to 100:1. RESULTS AND DISCUSSION Comparison of the reconstructed ion current chromatogram of the lipid extract of B. subtilis prepared by means of the conventional SME technique and injected into the capillary GC column by Curie-point flash vaporization (Fig. 2a) with the chromatographic profile obtained by the application of the pyrolytic methylation technique to B. subtilis cells (Fig. 2b) shows a remarkable degree of similarity in spite of the marked differences in sample preparation methods. Whereas the conventional method required 10 to 15 mg of sample and took 2 h, the pyrolytic method used only 5 p,g (wet weight) of bacterial cells, less than a single colony of average size, and required no sample preparation beyond the subsequent application and drying of a single drop of methanolic TMAH solution onto the pyrolysis filament. Moreover, Fig. 2b suggests that the pyrolytic methylation yields are very high in spite of the very short reaction time. Although quantitative studies, e.g., involving labeled reference compounds, need to be performed to obtain precise yield estimates, the high degree of similarity between Fig. 2a and b seems to indicate closely similar yields. This can be
VOL. 56, 1990
PROFILING FATTY ACIDS BY PYROLYTIC METHYLATION
1719
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FIG. 2. Normalized ion current profiles of B. subtilis ATCC 6633. (a) Total ion chromatogram of the extract obtained by the SME procedure; (b) total ion chromatogram of pyrolytic products of whole cells obtained by the TMAH procedure; (c) reconstructed mass chromatogram (m/z 69 to 74) from the total ion current chromatogram shown in panel b. Peak designation: Number before the colon refers to the number of carbon atoms of fatty acid and the number after the colon is the number of double bonds; i, iso; ai, anteiso; c, cyclcopropane acid; n, normal (unbranched) acid.
argued as follows: since it is unlikely that the reaction rates for all lipids are the same, identical product profiles can only be expected if the conversion percentages are comparable, e.g., nearing completion. Close examination of Fig. 2b, however, reveals a somewhat elevated and noisy looking base line in comparison
with Fig. 2a. Here, the high discrimination power afforded by the GC-MS method used proves its value, as can be seen in the reconstructed chromatogram obtained by adding only fragment ion signals in the m/z 69 to 74 range. This mass range contains highly characteristic fragment ion signals for nearly all of the FAME compounds present but is relatively
1720
APPL. ENVIRON. MICROBIOL.
DWORZANSKI ET AL. -6 n 100%
a) E.
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FIG. 3. Normalized ion current profiles of E. coli W (ATCC 9647). (a) Extract obtained by the SME procedure; (b) SME extract pyrolyzed in the presence of TMAH; (c) pyrolytic products of whole cells obtained by the TMAH procedures. For peak designation, see legend to Fig. 2.
free of background interference. Moreover, it should be pointed out that the noisy looking base line is in reality composed of a large number of overlapping peaks, most of which represent pyrolytic products of other cellular components. Many of these components can be separated and identified on the basis of selected characteristic ion signals. As demonstrated in previous work (8), pyrolytic products of
cellular components such as polysaccharides, proteins, or nucleic acids can provide important chemotaxonomic information. In this preliminary study, however, we decided to concentrate entirely on lipid components in general and FAME signals in particular. In addition to the above-discussed characteristics of the pyrolytic methylation procedure, some marked disadvan-
VOL. 56, 1990
PROFILING FATTY ACIDS BY PYROLYTIC METHYLATION 18:1
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(I) FIG. 4. Normalized ion current profiles of M. tuberculosis H3820. (a) Extract obtained by the SME procedure; (b) SME extract pyrolyzed in the presence of TMAH; (c) pyrolytic products of whole cells obtained by the TMAH procedure. 10-Me-18, 10-Methyloctadecanoic acid (tuberculostearic acid). For remaining peak designations, see legend to Fig. 2. TEIME
tages and advantages are illustrated in Fig. 3 and 4, respectively. Figure 3 shows the loss of hydroxy-substituted C14 FAME, apparently through degradation during the pyrolytic procedure (cf. Fig. 3a, b, and c). Although thus far we have compared only 20 bacterial strains, representing approximately 15 different species, the loss of hydroxy-substituted FAMEs appears to be a generalized phenomenon. This observation prompted us to take also a closer look at the fate
of the unsaturated FAMEs, especially in view of literature reports describing pyrolytic degradation of this type of compounds (12). Table 1 compares the signal ratios of unsaturated versus saturated species and demonstrates the absence of obvious shifts which might be ascribed to pyrolytic degradation of unsaturated FAMEs. In contrast to the E. coli patterns in Fig. 3, Fig. 4 reveals that M. tuberculosis profiles obtained by the pyrolytic meth-
1722
APPL. ENVIRON. MICROBIOL.
DWORZANSKI ET AL. 0 16:0
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