Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, Spain1
Hospital Costa del Sol, Marbella, Málaga, Spain2
Hospital de la Santa Creu i Sant Pau, Barcelona, Spain3
Departamento de Genética y Microbiología, Facultad de Medicina, Universidad de Murcia, Aptdo. 4012, Campus Universitario de Espinardo, 30100 Murcia, Spain4
Author for correspondence: Pedro L. Valero-Guillén. Tel:+34 968 360953. Fax:+34 968 360950. e-mail: plvalero{at}um.es
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ABSTRACT |
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Keywords: mycolic acids, high-performance liquid chromatography, M. gordonae
Abbreviations: EI-MS, electron-impact MS; FAB-MS, fast-atom bombardment MS; Gen-Probe, Gen-Probe Rapid Diagnostic System; PRA, PCR-restriction length polymorphism analysis
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INTRODUCTION |
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Molecular-based methodologies for the rapid identification of M. gordonae isolates have revealed genetic variability within this species. In contrast to other mycobacteria, which show conservation of rDNA sequences at the species level, M. gordonae strains exhibit variation within a region of their rDNA; this region is a common target for diagnostic species-specific probes (Kirschner & Böttger, 1992 ). The microheterogeneity observed among strains of M. gordonae could be the reason for the hybridization problems reported by Walton & Valesco (1991)
when using the Gen-Probe Rapid Diagnostic System (Gen-Probe). Studies using other molecular-based identification methods, such as PCR-restriction length polymorphism analysis (PRA), have concluded that M. gordonae is the most heterogenous member of the genus Mycobacterium studied to date, generating several closely related subclusters upon analysis of strain data (Plikaytis et al., 1992
; Telenti et al. 1993
).
Mycolic acids are high-molecular-mass 3-hydroxy, 2-alkyl-branched fatty acids found in all Mycobacterium species. The different structural types of mycolic acids include the so-called -mycolates and the mycolates which have other oxygen functions (i.e. the keto-, methoxy-, dicarboxy-, epoxy- and
-1 methoxy-mycolates) in addition to the 3-hydroxy acid unit (Minnikin, 1982
; Minnikin et al., 1982
; Luquin et al., 1990
). The use of TLC to analyse mycolic acid methyl esters allowed the different structural types of mycolic acids to be separated. The mycolate patterns obtained upon separation of mycolic acid methyl esters by TLC have been widely used in the classification and identification of mycobacteria (Daffé et al., 1983
; Minnikin et al., 1984
; Luquin et al., 1991
; Valero-Guillén et al., 1985
). M. gordonae exhibits a TLC mycolate pattern characterized by the presence of
-, methoxy- and keto-mycolates. Analysis of mycolic acids by HPLC has permitted a large proportion of mycobacterial species to be identified by their unique and reproducible chromatographic patterns (Butler & Guthertz, 2001
). However, M. gordonae isolates have been shown to produce two different HPLC mycolic acid patterns a single-cluster pattern, which is the most common pattern, and a double-cluster pattern (Cage, 1992
; Butler et al., 1991
). To date, no studies have been done to correlate these patterns with differences in mycolate production between strains of M. gordonae or to identify the compounds responsible for the second cluster of peaks seen in the double-cluster pattern.
The aim of this work was to elucidate the lipidic compounds of M. gordonae that give rise to the HPLC-double-cluster pattern. We studied five clinical isolates of M. gordonae that produced the HPLC-double-cluster pattern; for comparative purposes, we also studied two reference strains of M. gordonae. The lipidic component responsible for the second peak in the double-cluster pattern was purified and identified by spectrometric methods as dicarboxy-mycolate, a mycolic acid that has not been described in M. gordonae until now.
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METHODS |
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Identification of the strains was done by using standard biochemical methods (Tsukamura, 1967 ) and Gen-Probe for M. gordonae (used according to the manufacturers instructions; Biomérieux). The strains were also typed by using PRA, as described by Telenti et al. (1993)
. Briefly, a region of DNA (439 bp) corresponding to the gene encoding Hsp65 was amplified by PCR and then digested with HaeIII or BstEIII; the fragments obtained were separated by agarose-gel electrophoresis and visualized under UV.
Extraction and analysis of fatty acids, mycolic acids and alcohols.
Two to three loopfuls of bacteria (2030 mg wet wt of each strain) were collected from the surface of the Sautons agar plates. Fatty acids, mycolic acids and alcohols were liberated from each strain sample by saponification. These compounds were then extracted with diethyl ether. For each strain, an aliquot of the extract was treated with diazomethane to obtain the methyl esters of the fatty acids and mycolic acids (Daffé et al., 1983 ); in another aliquot of the extract the mycolic acids were transformed to p-bromophenacyl derivatives (Butler et al., 1991
).
The p-bromophenacyl derivatives of the mycolic acids were separated in a HPLC system (Waters Associates) equipped with an UV/visible detector. A reverse-phase C18 column (Nova-Pack 60A, 4 µm, 3·9x75 mm; Waters Associates) was used in the system; the mycolic acids were eluted using a linear gradient of methanol/chloroform [from 98:2 (v/v) to 30:70 (v/v)]. A high-molecular-mass standard (Ribi; ImmunoChem Research) was used as an internal standard, to assist in the identification of the peaks. Pattern-recognition software (PIROUETTE; Infometrix) was employed to evaluate the similarity of the HPLC chromatograms obtained, and was used in conjunction with a library containing the chromatograms of the most relevant Mycobacterium species and related bacteria (Glickman et al., 1994 ). Designation of HPLC peaks followed the arbitrary nomenclature used by several authors in the literature (Glickman et al., 1994
).
Mycolic acid methyl esters were studied by analytical one-dimensional TLC using silica gel 60 TLC plates (Merck). A triple development with a mixture of n-hexane/diethyl ether (85:15, v/v) was performed to separate the individual mycolates. The components separated by TLC were revealed as dark-blue spots by spraying the TLC plates with 10% (w/v) molybdophosphoric acid (Merck) in ethanol and heating them at 120 °C for 5 min. The methyl mycolates on the TLC plates were identified by comparing their positions on the plates with the mycolate patterns of reference strains (Luquin et al., 1991 ).
Fatty acid methyl esters, alcohols and methyl mycolate cleavage products were determined by GLC and GLC-MS as described previously (Luquin et al., 1991 ), employing a fused-silica capillary column (cross-linked methyl silicone, 15 mx0·25 mm, HP-1; Hewlett Packard) that was programmed from 175 to 300 °C at 8 °C min-1 and maintained at 300 °C for 15 min.
Purification and structural analysis of mycolates.
Crude methyl mycolates from M. gordonae strains ATCC 14470T, ATCC 35759 and CL-416C were obtained by precipitating their lipidic extracts with cold methanol (-20 °C, overnight). The samples were then centrifuged at 1500 g for 30 min at 4 °C. Unless indicated otherwise, the boiling point of the petroleum ether used in the following procedures was 6080 °C. For purification of the methyl mycolates, the precipitates were recovered, dissolved in the smallest possible volume of petroleum ether and applied to a silica gel 60 (Merck; particle size 0·0630·200 µm) column equilibrated in petroleum ether. Successive elutions, 35 bed-volumes each, were performed with petroleum ether followed by increasing concentrations of diethyl ether (5, 10, 20 and 100%) in petroleum ether. The eluates were separated by TLC, as described above. Dicarboxy-mycolates were obtained in the 20% (v/v) diethyl ether/petroleum ether fraction.
Purified mycolic acid methyl esters were identified by MS and NMR. Electron-impact MS (EI-MS) (70 eV) and fast-atom bombardment MS (FAB-MS) (8 eV) were performed in a VG AutoSpec (Fison) mass spectrometer. FAB-MS was carried out in the positive mode, employing m-nitrobenzyl alcohol as the matrix. Because FAB-MS seemed to produce (M+Na)+/z pseudomolecular ions, we doped the matrix with NaCl in some analyses. Dicarboxy-mycolates from strain CL-416C were also analysed by FAB-MS in the negative mode and by chemical ionization (methane; temperature of ionization, 180 °C) in the negative mode. For the latter method of analysis, a Thermoquest-Trace mass spectrometer (Thermo) with a temperature range of 60300 °C was used.
All methyl mycolates from strains ATCC 14470T, ATCC 35759 and CL-416C were analysed by 1H-NMR at 300 MHz in a Varian NMR spectrometer. Dicarboxy-mycolates from strains ATCC 35759 and CL-416C were also studied by 13C-NMR (75 MHz) in the same spectrometer. In all cases, the spectra were recorded in deuterochloroform [10 mg (ml sample)-1] at 25 °C.
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RESULTS |
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Mycolate chromatographic patterns
Upon HPLC analysis of their mycolates, the five clinical strains of M. gordonae and strain ATCC 35759 showed an HPLC-double-cluster pattern (Cage, 1992 ) (Fig. 1a
). TLC analysis of the mycolates revealed that the six strains contained
-, methoxy-, keto-mycolates and additional components, such as secondary alcohols and dicarboxy-mycolates (Fig. 1b
). M. gordonae ATCC 14470T showed the most common HPLC-single-cluster pattern (Cage, 1992
) (data not shown); TLC revealed this strain to contain only
-, methoxy- and keto-mycolates in its cell wall (Fig. 1b
; lane 4). HPLC of the p-bromophenacyl derivatives of the total mycolic acids from M. gordonae CL-416C are shown in Fig. 1(a)
. As further demonstrated by HPLC (data not shown), dicarboxy-mycolates purified from strains ATCC 35759 and CL-416C and transformed to their p-bromophenacyl derivatives eluted as three peaks, which corresponded exactly to peaks A1, A4 and A5 in Fig. 1(a)
. Other components detected in the chromatogram, peaks B2B8 (Glickman et al., 1994
), corresponded to a mixture of keto-, methoxy- and
-mycolates (Fig. 1a
).
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Structures of the dicarboxy-mycolates of M. gordonae
In the 1H-NMR spectrum of strain ATCC 35759 (Fig. 2), major resonances were detected between 0·1 and 3·7 p.p.m. A multiplet centred at 0·15 p.p.m. (3H) and another one at 0·47 p.p.m. (1H) were attributed to a trans-1,2-disubstituted cyclopropane ring (Draper et al., 1982
; Watanabe et al., 1999
). A signal at 0·67 p.p.m. (Fig. 2
) was assigned to a CHCH3 adjacent to the cyclopropane ring (Watanabe et al., 1999
); the protons of the methyl branch resonated as a doublet at 0·90 p.p.m., and overlapped a signal of a terminal methyl at 0·88 p.p.m. (6H in total) (Draper et al., 1982
). Singlets at 3·66 and 3·70 p.p.m. corresponded to two carbomethoxy groups, and the triplet at 2·29 p.p.m. was assigned to a CH2 attached to a carbonyl group. The protons of other CH2 groups resonated between 1·2 and 1·6 p.p.m. Finally, the signal at 2·42 p.p.m. was due to the proton of C-2 of the molecule. Minor resonances situated at -0·3 and 0·73 p.p.m. (Fig. 2
) were due to the presence of low amounts of a compound with a cis-1,2-disubstituted cyclopropane ring (Watanabe et al., 1999
).
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These data indicate that the additional component detected by TLC and HPLC in the M. gordonae clinical strains and in strain ATTC 35759 can be identified as a dicarboxy-mycolate (dimethyl ester form) that contains predominantly a trans-1,2-disubstituted cyclopropane ring with a methyl branch adjacent to it (Fig. 3; Table 1
).
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In the region of the EI-MS trace attributed to meroaldehydes (Fig. 3), a major fragment at m/z 601 was accompanied by another one at m/z 632; other fragments were present at lower intensities. A formula for dicarboxy-mycolates was not satisfactory when m/z 601 was considered. However, negative-mode FAB-MS (data not shown) revealed very low intensities for pseudomolecular ions, (M-H)-1/z, but important fragments at (M-15)/z, which were tentatively assigned to the loss of CH3 from a carbomethoxy group. The elimination of a methyl group to give a negatively charged molecule (a carboxylate ion) could apparently take place in the meroaldehyde part of the structure, because intense fragments at m/z 381 and 353 (similar intensities) were clearly related to methyl esters derived from a breakdown between C-2 and C-3 of the molecule. Thus, an intense fragment at m/z 617 was detected by negative-mode FAB-MS and was considered to be related to a meroaldehyde at m/z 632 seen in the EI-MS analysis. Finally, by chemical ionization an important fragment (relative abundance 90%) was detected at m/z 631 (data not shown). Thus, it was concluded that the major meroaldehyde produced by the compound under study was actually m/z 632. Moreover, this fragment satisfied the formula given in Fig. 3
, when an n2+n3 value equal to 34 was considered. Then, the meroaldehyde at m/z 632 fragments to m/z 601 due to loss of OCH3 (Fig. 3a
), because of the existence of the second ester group at the
end. Similarly, an m/z 573 (Fig. 3a
) was derived from a meroaldehyde at m/z 604 (very low intensity) (n2+n3 equal to 32).
The value of n3 was deduced by taking into account the presence of a very intense fragment at m/z 292 in theEI-MS analysis (Fig. 3a), which was derived from a breakdown between the cyclopropane ring and the carbon attached to the methyl branch and a further rearrangement. This fragment should indicate that for m/z 632, n3 is equal to 16 and n2 is equal to 18, and suggested that the major dicarboxy-mycolate from M. gordonae contains 63 carbon atoms, with an alkyl chain at C-2 of 20 carbon atoms (producing a C22:0 methyl ester). For a meroaldehyde at m/z 604 (n2=16, n3=16), a compound with a molecular mass of 986 is also obtained assuming an alkyl chain of 22 carbon atoms at C-2 (producing a C24:0 methyl ester), thus justifying a more intense peak at m/z 936 (M+-50) (see above and Fig. 3
) and at (M+Na)+/z 1009 (data not shown).
The two remaining homologous compounds seen in the EI-MS analysis can also be formulated in a similar way. Thus, a C65 dicarboxy-mycolate with a molecular mass of 1014 is composed combining the m/z 632 meroaldehyde and the m/z 382 methyl ester; and a C61 dicarboxy-mycolate (molecular mass 958) is composed by combining m/z 604 (a meroaldehyde) and m/z 354 (a methyl ester).
Both 1H-NMR (Fig. 2) and MS [EI-MS (Fig. 3
) and FAB-MS (data not shown)] predicted the presence of minor amounts of dicarboxy-mycolates with a cis-1,2-disubstituted-cyclopropane ring in the strains producing a HPLC-double-cluster pattern. The major meroaldehyde for this series was detected at m/z 646 (fragment at m/z 615; Fig. 3a
), and the chain length varied between C60 and C64 according to the MS: pseudomolecular ions, (M+Na)+/z, were situated at 967 (C60), 995 (C62, probably the predominant pseudomolecular ion) and 1023 (C64) (data not shown).
Structures of the other mycolates of M. gordonae
The structures of the remaining mycolates (-, methoxy-and keto-mycolates) of strains ATCC 35759, CL-416C and ATCC 14470T were established by 1H-NMR and MS (EI-MS and positive-mode FAB-MS); the results of these analyses are presented in Table 1
.
-Mycolates from M. gordonae strains ATCC 35759 and CL-416C contained mainly two cis-1,2-disubstituted cyclopropane rings (their protons were located at -0·32, 0·59 and 0·64 p.p.m.), and even chain lengths of C72C84 (major series at C76 and C78 for CL-416C, and at C78 and C80 for ATCC 35759). Major meroaldehyde ions (m/z 740, 768 and 796) and their fragments (m/z 279, 307, 487, 515 and 543), together with methyl ester fragments at m/z 354 and 382, permitted the tentative structures in Table 1
to be proposed (Draper et al., 1982
). Moreover, other compounds probably containing one cis double bond (resonance at 5·34 p.p.m.) and one cis-1,2-disubstituted cyclopropane ring were also predicted in the two aforementioned strains; both EI-MS and FAB-MS signalled chain lengths of C73C77. The ratio of CH=CH protons to 1,2-disubstituted cyclopropane ring protons was 21 and 22·7 for strains ATCC 35759 and CL-416C, respectively, implying that these compounds could represent approximately 20% of the total amount of
-mycolates present in these strains. Finally, M. gordonae ATCC 14470T contained
-mycolates with only two cis-1,2-disubstituted cyclopropane rings, which ranged from C74 to C80 (major compounds C76 and C78).
The methoxy-mycolates of strains ATCC 35759, CL-416C and ATCC 14470T were identical (Table 1). They were clearly identified by 1H-NMR due to characteristic overlapping signals at 0·85 (a methyl branch adjacent to the methoxy group of the meroaldehyde chain), 0·88 (a terminal methyl group) and 0·90 p.p.m. (a methyl branch adjacent to a 1,2-disubstituted cyclopropane ring), and also by signals at 2·95 (CHOCH3) and 3·33 p.p.m. (OCH3). Methoxy-mycolates containing cis- (-0·32, 0·59 and 0·64 p.p.m.) or trans- (0·15 and 0·47 p.p.m.) 1,2-disubstituted cyclopropane series of C73C87 were detected, with series of C81 and C82 predominant for strains ATCC 35759 and ATCC 14470T, and series of C81 and C79 predominant for strain CL-416C. The ratio of cis to trans was approximately 1:1, and given the structure of methoxy-mycolates of other mycobacteria (Minnikin, 1982
; Watanabe et al., 2001
), it seems that the odd series can be attributed to cis compounds and the even series can be attributed to trans compounds. Meroaldehydes from methoxy-mycolates were not found in EI-MS due, probably, to the loss of 31 mass units (OCH3), which resulted in a series of m/z ions ranging from 769 to 867 (separated by intervals of 14 mass units) that predicted meroaldehydes at m/z 800898. Other fragments characteristic of meroaldehydes were located at m/z 297 (intensity 72%), 307 (intensity 19%) and 321 (intensity 16%); considering the m/z 354 and 382 for methyl esters, the tentative structure presented in Table 1
can be postulated for the major compounds found in the strains studied.
Keto-mycolates (Table 1) were characterized by resonances at 0·90 (a methyl branch adjacent to a 1,2-disubstituted cyclopropane ring), 1·05 (a methyl branch adjacent to the keto group in the meroaldehyde chain) and 2·32 p.p.m. (CH2CO). The major series varied from C79 to C85 and had one trans-1,2-disubstituted cyclopropane ring in the structure (resonances at 0·15 and 0·47 p.p.m.). C81 and C83 were the predominant series for strains ATCC 14470T and ATCC 35759; C81 and C79 were the predominant series for strain CL-416C. FAB-MS also predicted very minor series of mycolates with a chain length of C78C80 in the three aforementioned strains, probably containing a cis-1,2-disubstituted cyclopropane ring, as suggested by 1H-NMR (data not shown). The tentative structure of the major keto-mycolate series is given in Table 1
and was based on the presence of important fragments at m/z 293, 491, 547 and 601 (derived from meroaldehydes), and at m/z 354 and 382 (methyl esters).
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DISCUSSION |
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The elucidation of the structures of mycolic acids uses a variety of techniques (Minnikin, 1982 ), the most widely used of which are EI-MS and NMR. Recently, new techniques, such as matrix-assisted laser desorption ionization/time-of-flight MS (MALDI/TOF-MS) (Laval et al., 2001
; Watanabe et al., 2001
) and FAB-MS (Barry et al., 1998
), have been applied to the elucidation of these structures. In this study, FAB-MS data complemented the data obtained by EI-MS, giving more precise information on the molecular mass of the different structural types of mycolic acids present in M. gordonae. This information is not always evident when using EI-MS, as there is a tendency for several meroaldehydes (e.g. methoxy and dicarboxy) to break down further due to loss of OCH3.
Using HPLC, Cage (1992) demonstrated two different mycolic acid patterns for M. gordonae a single-cluster pattern and a double-cluster pattern. In the present study, we have demonstrated that the second cluster of peaks present in the double-cluster pattern actually corresponds to dicarboxy-mycolates, and that these compounds essentially contain a trans-1,2-disubstituted cyclopropane ring, although a minor series with a cis-1,2-disubstituted cyclopropane has also been detected. As reported by Lanéelle & Lanéelle (1970)
, dicarboxy-mycolates and secondary alcohols (2-eicosanol and 2-octadecanol) are naturally combined in the cell wall to form wax-ester mycolates. These compounds are widely distributed in mycobacteria (Barry et al., 1998
; Luquin et al., 1991
; Minnikin et al., 1984
, 1985a
), but only a limited number of studies have dealt with their structural elucidation. Thus, the dicarboxy-mycolates of Mycobacterium phlei (see Minnikin, 1982
) are mixtures of unsaturated and cyclopropyl homologues, and differ from those found in M. gordonae, in which only cyclopropyl derivatives were detected.
Taking into account the presence of 2-octadecanol and 2-eicosanol in the M. gordonae strains containing dicarboxy-mycolates, the chain length of wax-ester mycolates from M. gordonae should range from C81 to C85 for the principal components, which is similar to the chain lengths detected for the principal keto-mycolates. Like the keto-mycolates, the major series of the wax-ester mycolates contain a trans-1,2-disubstituted cyclopropane ring, thus supporting the hypothesis that keto-and wax-ester mycolates are biosynthetically related (Minnikin, 1982 ; Barry et al., 1998
).
Partial structural analyses of -, methoxy- and keto-mycolates from M. gordonae have been reported previously (Daffé et al., 1981
; Minnikin et al., 1985b
). However, for the first time, our work has established the nature of the double bonds and the cyclopropyl rings in this species. Thus,
-mycolates from M. gordonae are mostly of type-1, as defined by Watanabe et al. (2001)
i.e. they contain two cyclopropyl rings with no double bonds. Most slow-growing mycobacterial species examined to date (Minnikin, 1982
; Watanabe et al., 2001
) contain predominantly this type of
-mycolate, with a cis to trans ratio of 1:0 (Watanabe et al., 2001
), as in the case of M. gordonae, although minor series of trans-cyclopropyl
-mycolates have been found in Mycobacterium kansasii and in the Mycobacterium avium complex (Watanabe et al., 2001
). However, other types of
-mycolates have been defined in several mycobacterial species (Watanabe et al., 2001
), but only the so-called type-3
-mycolate (one cis-cyclopropyl ring plus one cis double bond) seems to be present in the M. gordonae strains containing dicarboxy-mycolates (this study). This type of
-mycolate has also been characterized in several strains of Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium microti and in strains of the M. avium complex (Watanabe et al., 2001
). The aforementioned species also contain a variety of methoxy-mycolates, the major ones being of the type methoxy-mycolate-1 (Watanabe et al., 2001
) (one series with a cis-cyclopropyl ring and one series with a trans-cyclopropyl ring), a characteristic shared by Mycobacterium leprae (Draper et al., 1982
) and, according to our results, by M. gordonae. As for its
-mycolates, M. gordonae seems to lack trans double bonds or additional cyclopropyl rings in the meroaldehyde chain and, contrary to other mycobacteria (Watanabe et al., 2001
), it lacks methoxy-mycolates with cis double bonds. Among the keto-mycolates of M. gordonae, we only detected the keto-mycolate-1 (Watanabe et al., 2001
), generally with a trans-cyclopropyl ring. Hence, the keto-mycolates of M. gordonae resemble those of species such as M. kansasii and M. avium, and differ from those of the M. tuberculosis complex, where, in general, the cis to trans ratio is more balanced (Watanabe et al., 2001
).
According to the results cited above, it seems that a high proportion of the mycolic acids of M. gordonae have trans cyclopropanation, but like other mycobacteria (Barry et al., 1998 ; Minnikin, 1982
; Watanabe et al., 2001
) M. gordonae still maintains a cis configuration in the cyclopropyls of its
-mycolates. An appropriate ratio in the cis to trans geometry of both double bonds and cyclopropyls seems to have physiological significance for the bacterial cell wall, but its true relevance remains to be elucidated (Barry et al., 1998
).
The M. gordonae clinical strains studied here exhibit PRA patterns III and V, which are only present in 12% of M. gordonae isolates (Telenti et al., 1993 ). The mycolic acid pattern exhibited by these clinical isolates is also uncommon in members of the genus Mycobacterium, as the presence of
-, methoxy-, keto- and dicarboxy-mycolates has only been reported in two additional species of the genus, Mycobacterium komossense and Mycobacterium bohemicum (Minnikin et al., 1985a
; Torkko et al., 2001
).
We conclude that the variability in the mycolic acid patterns of strains of M. gordonae, as detected by HPLC and TLC, exists because of the presence of additional components (dicarboxy-mycolates) in some strains of this species. These additional components give rise to the HPLC-double-cluster pattern. Hence, our findings reiterate the heterogeneity of M. gordonae reported previously by several authors (Kirschner & Böttger, 1992 ; Plikaytis et al., 1992
; Telenti et al., 1993
; Walton & Valesco, 1991
).
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ACKNOWLEDGEMENTS |
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Received 16 April 2002;
revised 11 July 2002;
accepted 12 July 2002.