Detection of mycoloylglycerol by thin-layer chromatography as a tool for the rapid inclusion of corynebacteria of clinical origin in the genus Corynebacterium

A chemotaxonomic study of some corynebacteria isolated from clinical samples revealed characteristic thin-layer chromatographic patterns for meso-diaminopimelic acid containing species included in the genera Corynebacterium, Dermabacter and Brevibacterium. Notably, a specific compound was consistently detected in mycolic acid containing species of the genus Corynebacterium. This compound was composed by glycerol and mycolic acids and structural analyses carried out by fast atom bombardment mass spectrometry in C. minutissimum confirmed its identification as mycoloylglycerol. The chain length of mycoloyl groups in this molecule ranged from 28 to 34 carbon atoms, being mono-, di- or triunsaturated. Detection of mycoloylglycerol by thin-layer chromatography may be thus useful for the rapid inclusion of a great variety of corynebacteria of clinical origin in the genus Corynebacterium in laboratories employing chromatographic techniques as an adjunct for the identification of these microorganisms.     

High-performance liquid chromatography analysis of mycolic acids as an aid in laboratory identification of Rhodococcus and Nocardia species.

High-performance liquid chromatography analysis of the p-bromophenacyl esters of mycolic acids from whole organisms gave chromatographic patterns that were useful in differentiation of Rhodococcus and Nocardia species. Rhodococcus equi, R. erythropolis, and R. rhodochrous contained more-polar mycolic acids and were easily separated from the less-polar mycolic acid-containing species of R. sputi, R. bronchialis, R. corallinus, R. rubropertinctus, and R. terrae. The less-polar mycolic acid-containing Rhodococcus species showed chromatographic patterns that partially overlapped (in elution times) the patterns of Nocardia asteroides, N. otitidiscaviarum, and N. brasiliensis, but the larger number of peaks in the last species made separation between the genera possible. Distinct chromatographic patterns were found for most species, except for R. equi strains that showed two different patterns. Strains of R. rubropertinctus and R. terrae appeared identical. N. asteroides and N. otitidiscaviarum showed similar mycolic acid patterns.     

Capillary gas chromatographic analysis of mycolic acid cleavage products, cellular fatty acids, and alcohols of Mycobacterium xenopi.

The fatty acids, alcohols, and mycolic acids of 26 strains of Mycobacterium xenopi were studied by capillary gas chromatography and thin-layer chromatography. All strains contained alpha-, keto-, and omega-carboxymycolates. The primary mycolic acid cleavage product was hexacosanoic acid. The fatty acid patterns and, especially, the presence of 2-docosanol are characteristic markers of M. xenopi.     

Analysis of mycolic acid cleavage products and cellular fatty acids of Mycobacterium species by capillary gas chromatography.

After growth and experimental conditions were established, the mycolic acid cleavage products, constituent fatty acids, and alcohols of representative strains of Mycobacterium tuberculosis, M. smegmatis, M. fortuitum complex, M. kansasii, M. gordonae, and M. avium complex were determined by capillary gas chromatography. Reproducible cleavage of mycolic acid methyl esters to tetracosanoic (24:0) or hexacosanoic (26:0) acid methyl esters was achieved by heating the sample in a high-temperature muffle furnace. The major constituent fatty acids in all species were hexadecanoic (16:0) and octadecenoic (18:1 omega 9-c, oleic) acids. With the exception of M. gordonae, 10-methyloctadecanoic acid was found in all species; moreover, M. gordonae was the only species tested which contained 2-methyltetradecanoic acid. M. kansasii was characterized by the presence of 2,4-dimethyltetradecanoic acid, M. avium complex by 2-eicosanol, and M. tuberculosis by 26:0 mycolic acid cleavage product. The mycolic acid cleavage product in the other five species tested was 24:0. Although a limited number of strains and species were tested, preliminary results indicate that this gas chromatographic method can be used to characterize mycobacterial cultures by their mycolic acid cleavage products and constituent fatty acid and alcohol content.     

Relationship among cell wall composition, stage of growth, and virulence of Nocardia asteroides GUH-2.

The clearance and organ distribution of virulent Nocardia asteroides GUH-2P and the avirulent mutant GUH-2AI at different stages of growth was determined after intravenous inoculation into BALB/c mice. The mutant differed significantly from the parent strain in its ability to survive and grow within the murine host. Since the mutant GUH-2AI had a very different colonial morphology compared with GUH-2P, it was believed that cell surface components might be affected by the mutation that resulted in the loss of virulence. Therefore, cell walls of both GUH-2P and GUH-2AI at different stages of growth were prepared and their composition determined. There were growth-stage-dependent changes in the composition of the cell walls that appeared to correlate with concurrent alterations in virulence; however, the overall chemical composition of the cell wall of the mutant (GUH-2AI) was not significantly different from that of the parent strain (GUH-2P). Both strains demonstrated significant modifications in fatty and mycolic acid composition at different stages of growth. Furthermore, the specific composition of C54 mycolic acids was very different in virulent log-phase cells compared with less-virulent stationary-phase cells, and the avirulent mutant lacked a C54:3 mycolate that was prominent in the virulent log-phase GUH-2P. Thus, C54:3 mycolic acid represented 2.5% of the cell wall (dry weight) in log-phase GUH-2P, but it was undetectable in the walls of GUH-2AI at the stationary phase of growth. These results suggest that certain mycolic acids are associated with virulence.     

Rapid method for the detection and identification of mycolic acids in aerobic actinomycetes and related bacteria.

A rapid method for the identification of lipids characteristic of the genera Corynebacterium, Mycobacterium, Nocardia, and the "rhodochrous group" has been developed. Modifications of previously described methods make this procedure suitable for use in the clinical laboratory. Thin-layer chromatography is used to demonstrate the presence of the lipid characteristic of Nocardia spp. (type A) in some corynebacteria, nocardias, and members of the "rhodochrous group." Precipitation in ether and ethanol is used to demonstrate the presence of mycobacterial mycolic acids. Since this procedure can be carried out in less than 2 days and the lipids are extracted from the same batch of cells grown for diaminopimelic acid and whole-cell sugar analyses, it can readily be added to the battery of tests performed in reference laboratories that deal with aerobic actinomycetes and related bacteria.     

Effect of mycobacterial lipids on surface properties of Curosurf: implications for lung surfactant dysfunction in tuberculosis

In this study, the effect of mycobacterial lipids on the surface activity of lung surfactant was evaluated. Mycolic acid and cord factor, the most abundant surface active lipids of the mycobacterial cell wall when combined with Curosurf led to alteration of its surface properties. Addition of graded amounts of mycolic acid increased the minimum surface tension of Curosurf monolayers from <10 mN/m to approximately 20-27 mN/m. Presence of mycolic acid also slowed the rate of Curosurf adsorption. Similarly, presence of cord factor increased the minimum surface tension achieved by Curosurf to approximately 16-27 mN/m. AFM imaging revealed presence of aggregates on addition of mycobacterial lipids to Curosurf monolayers. Results of this study show that mycolic acid and cord factor can biophysically inactivate porcine lung surfactant extract (Curosurf). This suggests that biophysical inhibition of lung surfactant is possible in vivo in pulmonary tuberculosis which could aggravate areas of alveolar atelectasis.     

Separation of Mycobacterium bovis BCG from Mycobacterium tuberculosis and Mycobacterium bovis by using high-performance liquid chromatography of mycolic acids.

Profile analysis of mycolic acid ester patterns of Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium bovis bacillus Calmette-Gúerin (BCG) using high-performance liquid chromatography indicated that separation of BCG from M. tuberculosis and M. bovis by elution and relative retention times is possible. Mycolic acid patterns of BCG eluted from the column 0.5 min before M. tuberculosis or M. bovis, resulting in relative retention times for two peaks not seen in the pattern of M. tuberculosis or M. bovis. Identification was confirmed by phage typing, which has been the standard procedure for confirmation of BCG strains. These results showed that high-performance liquid chromatographic analysis of mycolic acid esters can be used in the mycobacterial reference laboratory for separation of BCG from M. tuberculosis and M. bovis.     

Improved rapid identification of mycobacteria by combining solid-phase extraction with high-performance liquid chromatography analysis of BACTEC cultures.

Identification of mycobacteria from BACTEC 12B cultures is achieved in 7 to 21 days by reverse-phase high-performance liquid chromatography (HPLC) using a UV spectrophotometer to detect nonpolar p-bromophenylacyl mycolic acid derivatives. However, cultures grown in BACTEC and other liquid media seldom contain sufficient mycolic acids to permit reliable identification under usual HPLC assay conditions, so the sample size must be increased. Unfortunately, samples prepared from cultures in liquid media such as BACTEC cultures also contain large amounts of extraneous polar and strongly nonpolar contaminants that interfere with the analysis and hasten deterioration of the HPLC column. The contaminants were removed from 10 samples simultaneously by solid-phase extraction (SPE), i.e., by passing the crude suspension containing the mycolic acid derivatives into disposable 500-mg tC18 SPE columns in place of the usual final filtration step used to prepare specimens for HPLC. Fifteen milliliters of 20% (vol/vol) dichloromethane in methanol was passed through the columns (< 3 ml/min) to wash through the undesired contaminants and bind the mycolic acid derivatives. The mycolates were quantitatively eluted in 3 ml of dichloromethane for analysis by HPLC. Treating a panel of 31 strains of frequently isolated mycobacteria by SPE reduced the content of contaminants by 89.3 to 99.9% without altering the chromatographic patterns compared with the same strains grown on conventional solid media and processed without SPE. Peak heights of mycolates prepared from BACTEC cultures were increased from < or = 6 to > or = 25 absorbance milliunits with SPE, sufficient for reliable interpretation by visual inspection of chromatograms obtained with a UV detector. Also, removal of the contaminants improved column longevity.     

Differentiation of Mycobacterium genavense and Mycobacterium simiae by automated mycolic acid analysis with high-performance liquid chromatography.

Mycobacterium genavense, a fastidious opportunist in patients with AIDS, cannot be identified by conventional biochemical methods. Computerized mycolic acid analysis by high-performance liquid chromatography offers an alternative that distinguishes the mycolic acid profile of M. genavense from those of all other organisms in the database developed at the Centers for Disease Control and Prevention.     

Evaluation of practical chromatographic procedures for identification of clinical isolates of mycobacteria.

After experimental conditions were established, 366 strains of mycobacteria belonging to 23 different species were studied for fatty acids, secondary alcohols, and mycolic acid cleavage products by capillary gas-liquid chromatography. Additionally, the mycolic acid pattern was studied by thin-layer chromatography. Capillary gas-liquid chromatography allowed direct identification of the following Mycobacterium spp.: M. kansasii, M. marinum, M. szulgai, M. xenopi, M. malmoense, and M. gordonae. The patterns of mycolic acid methyl esters recorded for the test strains of M. chelonae and M. agri may be of value in the identification of these species. Moreover, the combined use of the two chromatographic techniques provided precise identification of the M. tuberculosis complex, M. simiae, M. fallax, M. triviale, and M. chelonae-like organisms. A minimal set of biochemical tests is usually required to obtain identification to the species level when chromatographic procedures alone are not sufficient. Under the reported experimental conditions, thin-layer chromatography and capillary gas-liquid chromatography are rapid and very useful techniques for the identification of mycobacteria.     

Determination of drug susceptibility of Mycobacterium tuberculosis through mycolic acid analysis.

In the present work a rapid method to determine the susceptibility of Mycobacterium tuberculosis to isoniazid and streptomycin by determining levels of mycolic acids by high-performance liquid chromatography (HPLC) was developed. Mycobacterial growth kinetics in the presence and absence of antituberculosis drugs was characterized by evaluating the total area corresponding to mycolic acid peaks (TAMA). Results show a linear relationship between the logarithm of CFU per milliliter and TAMA and show that it is possible to detect growth inhibition of M. tuberculosis in the presence of isoniazid or streptomycin by using HPLC in 3 and 4 days, respectively.     

Gas-chromatographic analysis of mycolic acid cleavage products in mycobacteria.

Mycolic acids were detected in both reference strains and clinical isolates of mycobacteria using gas chromatography of fatty acid methyl esters prepared by acid methanolysis. The methyl esters were extracted with hexane, concentrated, and analyzed with a gas chromatograph by using two different injector temperatures. When the samples were analyzed at high injector temperatures of 300 to 350 degrees C, characteristic thermal cleavage products from mycolic acids, C22:0, C24:0, or C26:0 fatty acid methyl esters, were detected. When analyzed at injector temperatures of 235 degrees C or lower, the mycolic acids were heat stable and the characteristic methyl ester cleavage products were not observed.     

Mycolic acid analysis for clinical identification of Mycobacterium avium and related mycobacteria.

We examined the mycolic acid composition of 133 strains belonging to MAIS complex (Mycobacterium avium-M. intracellulare-M. scrofulaceum) and MAIS intermediate strains and the related species M. asiaticum, M. malmoense, M. shimoidei, and M. simiae. The analysis revealed that about 10% of the strains identified as M. avium-M. intracellulare complex by conventional cultural and biochemical tests were in fact M. simiae strains according to their mycolate composition. Of 25 strains previously studied by the International Working Group on Mycobacterial Taxonomy, 2 (MAIS intermediate and M. asiaticum) presented patterns incompatible with the clusters to which they had been assigned. M. malmoense and both M. simiae serovars shared the same pattern with alpha-, alpha'-, and ketomycolates. We describe here the method used to identify the mycolic acid profiles in detail. We found it to be highly reproducible and convenient for use in mycobacterial reference laboratories.     

Mitogenic effects of bacterial cell walls, their fragments, and related synthetic compounds on thymocytes and splenocytes of guinea pigs.

Stimulation of [(3)H]thymidine incorporation of thymocytes and splenocytes from guinea pigs by various bacterial cell walls and their peptidoglycans, by enzymatic digests, and by synthetic muramyl dipeptides was studied as an indication of mitogenic activity. Cell wall and peptidoglycan preparations, isolated from 19 strains belonging to 18 different species, definitely increased [(3)H]thymidine incorporation of thymocytes as well as splenocytes, regardless of mycolic acid contents as a non-peptidoglycan component. Both the cell walls from Nocardia corynebacteriodes (containing mycolic acids) and those from Streptomyces gardneri (lacking mycolic acids) showed far stronger mitogenic activities on splenocytes than other cell walls (stimulation index, 25 to 30). Furthermore, water-soluble enzymatic digests, notably the endopeptidase digests, which generally were greater in degree of polymerization of peptidoglycan subunits than the glycosidase digests obtained from representative cell walls, were found to have as distinct a stimulating activity on splenocytes as the original cell walls. In contrast, solubilization of the cell walls by enzymes, irrespective of endopeptidases or glycosidases, was accompanied by disappearance of the mitogenic activity on thymocytes. On the other hand, studies with synthetic 6-O-acyl-MurNAc-l-Ala-d-isoGln preparations (6-O-acyl-MDPs) revealed that 6-O-stearoyl-MDP and 6-O-(2-tetradecylhexadecanoyl)-MDP, unlike MDP, had distinct mitogenic activity on thymocytes, whereas their activity on splenocytes was rather weaker than MDP itself. The findings presented here suggest that MDP is the minimal structure for the mitogenic activities of bacterial cell walls on guinea pig splenocytes, but that MDP, though distinctively active by itself, requires a polymerized form to exert effectively its inherent stimulating activities on splenocytes. On the other hand, on thymocytes, MDP, unless it takes a particular form or has appropriate additive groups, cannot exert its mitogenic activities.     

Direct identification of Mycobacterium species in BACTEC 7H12B medium by high-performance liquid chromatography.

Primary cultures of mycobacteria grown in BACTEC 7H12B medium (Becton Dickinson and Co., Paramus, N.J.), with and without the addition of oleic acid-albumin-dextrose-catalase (OADC) enrichment (Becton Dickinson and Co., Cockeysville, Md.), were analyzed for their mycolic acid patterns by high-performance liquid chromatography. Of the 126 isolates grown in medium to which OADC was added, 117 (93%) were successfully identified to the species level. The time to identification of Mycobacterium tuberculosis (n = 65) averaged 19 days, and the average time was 21 days for nontuberculosis mycobacteria (n = 52) from initial specimen processing. None of the 10 isolates cultured without OADC were identified. The mycolic acid patterns were considered reliable for identification if the height of the tallest peak in the chromatogram was at least 50% of the internal standard peak height.     

Mycolic acids analysis from various species mycobacterium by high pressure liquid chromatography (HPLC)

HPLC is the most useful method to analyze various species of mycobacteria by using mycolic acids. The purpose was to prepare a library containing chromatographic patterns of mycolic acids derived from reference species Mycobacterium, which had been cultivated in standard conditions. 28 reference strains (27 ones from American Type Culture Collection and one cultivated from the vaccine M. bovis BCG) were used. The analysis of mycolic acids involved chromatographic separation of their bromophenacyl derivatives according to Centers for Disease Control recommendation. Mycolic acids profiles formed by HLPC were reproducible for all reference species in this study. Standard deviation of relative retention time of every peak did not exceed 2.5%. The species included into M. tuberculosis complex beyond M. bovis BCG shared the same mycolic acids pattern. HPLC is the only mean to distinguish M. tuberculosis from M. bovis BCG. The other studied stains had species specific patterns which differed from M. tuberculosis complex and M. bovis BCG. The prepared library comprising 28 reference elution profiles of mycolic acids from known mycobacteria species can be applied in diagnostic procedure of tuberculosis and mycobacteriosis.     

Identification of major slowly growing pathogenic mycobacteria and Mycobacterium gordonae by high-performance liquid chromatography of their mycolic acids.

A rapid, reverse-phase high-performance liquid chromatography method was used to detect rho-bromophenacyl mycolic acid ester patterns for strains of four major pathogenic Mycobacterium species and for the most commonly encountered saprophytic species, Mycobacterium gordonae. Mycobacteria in low numbers (2.5 X 10(6) CFU) were detected and identified to the species level. Standard chromatographic patterns characteristic of each species were established. Simple pattern recognition enabled rapid identification of M. tuberculosis, M. kansasii, M. avium, M. intracellulare, and M. gordonae.     

Isolation of an unusual mycobacterium from an AIDS patient.

A mycobacterium isolated from a clinical sample of an AIDS patient was identified as Mycobacterium interjectum by direct 16S rRNA sequence determination. High-performance liquid chromatography, however, revealed a mycolic acid pattern which was different from the one shared by the previously analyzed strains of this species.     

Rapid, standardized method for determination of Mycobacterium tuberculosis drug susceptibility by use of mycolic acid analysis

Multidrug-resistant (MDR) Mycobacterium tuberculosis and extrensively drug-resistant (XDR) M. tuberculosis are emerging public health threats whose threats are compounded by the fact that current techniques for testing the susceptibility of M. tuberculosis require several days to weeks to complete. We investigated the use of high-performance liquid chromatography (HPLC)-based quantitation of mycolic acids as a means of rapidly determining drug resistance and susceptibility in M. tuberculosis. Standard susceptibility testing and determination of the MICs of drug-susceptible (n = 26) and drug-resistant M. tuberculosis strains, including MDR M. tuberculosis strains (n = 34), were performed by using the Bactec radiometric growth system as the reference method. The HPLC-based susceptibilities of the current first-line drugs, isoniazid (INH), rifampin (RIF), ethambutol (EMB), and pyrazinamide (PZA), were determined. The vials were incubated for 72 h, and aliquots were removed for HPLC analysis by using the Sherlock mycobacterial identification system. HPLC quantitation of total mycolic acid peaks (TMAPs) was performed with treated and untreated cultures. At 72 h, the levels of agreement of the HPLC method with the reference method were 99.5% for INH, EMB, and PZA and 98.7% for RIF. The inter- and intra-assay reproducibilities varied by drug, with an average precision of 13.4%. In summary, quantitation of TMAPs is a rapid, sensitive, and accurate method for antibiotic susceptibility testing of all first-line drugs currently used against M. tuberculosis and offers the potential of providing susceptibility testing results within hours, rather than days or weeks, for clinical M. tuberculosis isolates.