Optimal data processing procedure for automatic bacterial identification by gas-liquid chromatography of cellular fatty acids.

Gas-liquid chromatography of cellular fatty acids was used in automatic identification of clinical bacterial isolates. The intraspecies variation in the occurrence of fatty acids and the variation in the relative gas-liquid chromatography peak areas of different fatty acids were evaluated and compared with the relative peak areas of these acids. A new chromatogram comparison method involving the use of an exponential function was developed to adjust to data variation optimally. This method was compared with several previously published methods of correlation analysis with data from representative clinical bacteriological isolates. The efficacies of the methods in separating different bacterial species into distinct clusters were compared. The new exponential function method was superior to the others both in its ability to separate species into different clusters and in giving a greater degree of identity to strains within a proper cluster. The results indicate that the gas-liquid chromatography of bacterial cellular fatty acids can be used effectively in the identification of clinically isolated bacteria. However, the usefulness of the analysis depends on the comparison method used and on its ability to cope with data variations.     

Determination of hippurate hydrolysis by gas-liquid chromatography.

A rapid gas-liquid chromatographic procedure was developed to determine hippurate hydrolysis by microorganisms. Bacterial cells were inoculated into 0.4 ml of 1% sodium hippurate and incubated for 2 h at 37 degrees C. Cells were removed by centrifugation, and the benzoate released by enzyme activity was converted to methyl benzoate and analyzed by gas-liquid chromatography. This procedure is sensitive, and its specificity provides a high degree of reliability for organisms with weak hippuricase activity.     

Identification of yeast species by fatty acid profiling as measured by gas-liquid chromatography

An improved rapid method for the identification of yeasts and yeast-like fungi from clinical sources which is based on fatty acid profiles obtained by gas-liquid chromatography (GLC) is described. The fatty acid profile database is based upon internal standardisation and using the relative retention times and the retention index of the analysed fatty acids. Differentiation between yeast species was achieved by the quantitative and qualitative comparison of measured fatty acid profiles with those in the database. A total of 1024 clinical isolates were analysed by GLC to test the validity of the database. 96.2% of all tested samples were identified correctly to the species level by the improved GLC method.     

Differentiation of Peptococcus and Peptostreptococcus by gas-liquid chromatography of cellular fatty acids and metabolic products

Gas-liquid chromatographic (GLC) profiles of cellular fatty acids and metabolic products were useful in identifying strains of Peptococcus saccharolyticus, Peptococcus asaccharolyticus, Peptostreptococcus anaerobius, Peptostreptococcus micros, and Streptococcus intermedius. The GLC results supported the recent taxonomic decision to transfer aerotolerant Peptostreptococcus species to the genus Streptococcus. Because inconsistencies in the results prevented our differentiating Peptococcus prevotii. Peptococcus magnus, and Peptococcus variabilis by GLC, additional strains will have to been examined. These GLC techniques are amenable to routine use; however, for interlaboratory results to be meaningful, the classification and nomenclature of the anaerobic gram-positive cocci should be standardized.     

Discriminant analysis of cellular fatty acids of Candida species, Torulopsis glabrata, and Cryptococcus neoformans determined by gas-liquid chromatography.

We used discriminant analysis of cellular fatty acid compositions determined by gas-liquid chromatography to differentiate yeastlike fungi (a total of 190 strains; including 37 Candida albicans strains, 21 Candida krusei strains, 13 Candida guilliermondii strains, 37 Candida tropicalis strains, 10 Candida pseudotropicalis strains, 24 Candida parapsilosis strains, 32 Torulopsis glabrata strains, and 16 Cryptococcus neoformans strains). Previous results with a standard strain of C. albicans indicated that reproducible fatty acid chromatograms can be obtained with cells grown in a medium of 2% Sabouraud glucose agar at 35 degrees C for between 48 and 72 h. These conditions were also maintained in cultures of the other organisms that we studied. The cellular fatty acid compositions of the organisms were determined quantitatively by gas-liquid chromatography and analyzed by discriminant analysis. The total correct identification expressed as relative peak percent was 95.8% (89.2% for C. albicans to 100% for C. krusei, C. guilliermondii, C. pseudotropicalis, T. glabrata, and C. neoformans). The total correct identification expressed as the common peak (palmitic acid) ratio was 94.7% (87.5% for C. parapsilosis to 100% for C. pseudotropicalis, T. glabrata, and C. neoformans). Both results suggest that cellular fatty acid compositions can be differentiated by this method.     

Determination of unchanged clonazepam in plasma by gas-liquid chromatography

A gas-liquid chromatography method for unchanged clonazepam determination in plasma using a nitrogen-phosphorus detector is presented. The drug is extracted from buffered plasma at pH 9.0 with benzen. The proposed method is simple, rapid and permits to determine clonazepam with a good precision (delta = 3.10 p. cent) and reproductibility (CV = 3.04 p. cent). The method allows the determination of other benzodiazepins. Plasmatics levels of inchanged clonazepam had been determined on some clinical cases.     

Differentiation between Candida dubliniensis and Candida albicans by fatty acid methyl ester analysis using gas-liquid chromatography

Candida dubliniensis is often found in mixed culture with C. albicans, but its recognition is hampered as the color of its colonies in primary culture on CHROMagar Candida varies. Furthermore, definite identification of C. dubliniensis is difficult to achieve, time-consuming, and expensive. Therefore, a method to discriminate between these two closely related yeast species by fatty acid methyl ester (FAME) analysis using gas-liquid chromatography (Sherlock Microbial Identification System [MIS]; MIDI, Inc., Newark, Del.) was developed. Although the chromatograms of these two species revealed no obvious differences when applying FAME analysis, a new library (CADLIB) was successfully created using Sherlock Library Generation Software (MIDI). The amount and frequency of FAME was analyzed using library training files (n = 10 for each species), preferentially those comprising reference strains. For testing the performance of the CADLIB, clinical isolates genetically assigned to the respective species (C. albicans, n = 32; C. dubliniensis, n = 28) were chromatographically analyzed. For each isolate tested, MIS computed a similarity index (SI) indicating a hierarchy of possible strain fits. When using the newly created library CADLIB, the SIs for C. albicans and C. dubliniensis ranged from 0.11 to 0.96 and 0.53 to 0. 93 (for all but one), respectively. Only three isolates of C. albicans (9.4%) were misidentified as C. dubliniensis, whereas all isolates of C. dubliniensis were correctly identified. Resulting differentiation accuracy was 90.6% for C. albicans and 100% for C. dubliniensis. Cluster analysis and principal component analysis of the resulting FAME profiles showed two clearly distinguishable clusters matching up with two assigned species for the strains tested. Thus, the created library proved to be well suited to discriminate between these two species.     

Subgrouping of Pseudomonas cepacia by cellular fatty acid composition.

The cellular fatty acid compositions were determined for 42 strains of Pseudomonas cepacia from five cystic fibrosis centers in North America. All isolates contained significant (20%) amounts of hexadecanoic (C16:0), and cis-9 hexadecenoic (C16:1 cis9) acids and an isomer of octadecenoic acid (C18:1). None had hydroxy acids containing fewer than 14 carbon atoms. The quantitative data from the fatty acid analysis were highly reproducible and provided a basis for numerical analysis. Five subgroups comprising all the strains were obtained by cluster analysis and further characterized by principal-component analysis. With minor exceptions, the predominant subgroup identified in each center was different from that identified in other centers and accounted for one-half of the isolates within each center. Cellular fatty acid composition is a useful adjunct to biochemical characterization for the identification of P. cepacia isolated from cystic fibrosis patients. Numerical analysis of the fatty acid data can separate P. cepacia into subgroups, which may provide useful epidemiologic information or a basis for further analysis by more complex techniques such as DNA probe analysis.     

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.     

Applications of cellular fatty acid analysis.

More than ever, new technology is having an impact on the tools of clinical microbiologists. The analysis of cellular fatty acids by gas-liquid chromatography (GLC) has become markedly more practical with the advent of the fused-silica capillary column, computer-controlled chromatography and data analysis, simplified sample preparation, and a commercially available GLC system dedicated to microbiological applications. Experience with applications in diagnostic microbiology ranges from substantial success in work with mycobacteria, legionellae, and nonfermentative gram-negative bacilli to minimal involvement with fungi and other nonbacterial agents. GLC is a good alternative to other means for the identification of mycobacteria or legionellae because it is rapid, specific, and independent of other specialized testing, e.g., DNA hybridization. Nonfermenters show features in their cellular fatty acid content that are useful in identifying species and, in some cases, subspecies. Less frequently encountered nonfermenters, including those belonging to unclassified groups, can ideally be characterized by GLC. Information is just beginning to materialize on the usefulness of cellular fatty acids for the identification of gram-positive bacteria and anaerobes, despite the traditional role of GLC in detecting metabolic products as an aid to identification of anaerobes. When species identification of coagulase-negative staphylococci is called for, GLC may offer an alternative to biochemical testing. Methods for direct analysis of clinical material have been developed, but in practical and economic terms they are not yet ready for use in the clinical laboratory. Direct analysis holds promise for detecting markers of infection due to an uncultivable agent or in clinical specimens that presently require cultures and prolonged incubation to yield an etiologic agent.     

Determination of blood urea by gas-liquid chromatography. A comparison with two other methods.

An original method for the quantitative determination of plasmatic urea by gas-liquid chromatography (GLC) is described. It is based upon the transformation of urea into urethane by alcoholic deamination in a warm and strictly anhydrous medium. Compared with the two other usual methods (enzymatic and colorimetric), the GLC technique is extremely reliable and specific, and can easily be adapted to biological fluids.     

Detection of anaerobic wound infection by analysis of pus swabs for volatile fatty acids by gas-liquid chromatography.

Swabs were able to absorb enough extractable volatile fatty acids from broth cultures of anaerobic organisms for detection and analysis by gas-liquid chromatography (GLC). Similarly, volatile fatty acids were often detected in swabs dipped into liquid pus. Fifty-three liquid pus specimens were then investigated fully to determine if GLC analysis of swab samples gave the same result as microbial culture of the specimens and GLC analysis of the liquid pus. Anaerobic bacteria failed to grow from 36 and volatile fatty acids were not extracted from swabs of 31 of these pus samples but were extracted from swabs of five. Anaerobic bacteria were isolated from 17 of the specimens, and in 15, volatile fatty acids were also detected in the swab samples; in two, volatile fatty acids were absent from both swab samples and liquid pus. In this study, results by culture and GLC analysis of swabs were similar in 87% of specimens.     

Purification of fatty acid methyl esters by high-performance liquid chromatography.

The determination by gas chromatography (GC) of fatty acid methyl esters (FAMEs) prepared from complex biological samples is subject to interference from cholesterol. During sample injection on the GC system of FAMEs prepared from tissues that contain cholesterol, we observed a major contaminant that co-eluted with docosahexaenoic acid (DHA, 22:6n-3). To address this problem, FAMEs were purified on an amino-phase high-performance liquid chromatography (HPLC) column using a hexane-isopropanol gradient. The HPLC retention times for both the FAME fraction and cholesterol were stable and reproducible when the amino column was used for sample purification. The purified extracts were analyzed by GC without artifacts or impurity peaks after 50 analytical runs. The method described here will be useful for measurement of 22:6n-3 and other fatty acids important for studies of nutrition or pathology.     

Rapid diagnosis of anaerobic infections by gas-liquid chromatography.

It was postulated that the short chain fatty acids (SCFA) produced by anaerobic bacteria might serve as microbial markers in purulent material. Eighteen pus specimens from various sources were analysed by gas-liquid chromatography (GLC), and the SCFA detected were compared with the microorganisms isolated by conventional methods. It was found that the detection of propionic, isobutyric, butyric, or isovaleric acids by direct GLC of pus specimens is strong evidence for anaerobic infection but not specific for Bacteroides fragilis. It was also shown that the presence of succinic acid in pus specimens does not necessarily indicate infection by anaerobes. It can be concluded that direct GLC of purulent material provides a rapid and reliable presumptive method for the differentiation between anaerobic and aerobic infections.     

Differentiation of Campylobacter and Campylobacter-like organisms by cellular fatty acid composition.

The cellular fatty acid compositions of 368 strains of Campylobacter species or Campylobacter-like organisms were determined by gas-liquid chromatography. Most of the strains (339) were placed in one of three groups based on differences in fatty acid profiles. Group A contained Campylobacter jejuni (97%) and most C. coli (83%) strains and was characterized by the presence of a 19-carbon cyclopropane fatty acid (19:0 cyc) and 3-hydroxytetradecanoic acid (3-OH-14:0). Group B included all C. laridis and some C. coli (17%) strains; its profile was similar to that of group A, except that 19:0 cyc was absent. Group C contained C. fetus subsp. fetus and C. fetus subsp. veneralis and was characterized by the presence of 3-OH-14:0 and 3-hydroxyhexadecanoic acid (3-OH-16:0) and the absence of 19:0 cyc. Twenty-nine isolates were placed in four additional groups. Group D included the type strain of "C. cinaedi" and 14 other isolates, which were differentiated by the presence of dodecanoic acid (12:0), 3-hydroxydodecanoic acid (3-OH-12:0), and 3-OH-16:0 and the absence of hexadecenoic acid (16:1) and 3-OH-14:0. Group E contained the type strain of "C. fennelliae" and two additional isolates, which were differentiated by the presence of a 16-carbon aldehyde and a 16-carbon dimethylacetyl and the absence of 16:1. Group F included the type strain and one reference strain of C. cryaerophila and six human isolates whose phenotypic characteristics were similar to those of this species; this group was distinguished by the presence of two isomers of 16:1, tetradecenoic acid (14:1), and 3-OH-14:0. Group G included three stains of C. pyloridis and was characterized by the presence of 19:0 cyc, 3-OH-16:0, and 3-hydroxyoctadecanoic acid (3-OH-18:0) and by the absence of 16:1 and 3-OH-14:0.     

Characterization of CDC group DF-3 by cellular fatty acid analysis.

Fourteen strains of Centers for Disease Control group DF-3 bacteria were examined for cellular fatty acid composition to evaluate their chemical relatedness to known bacterial species and groups. The fatty acids were liberated from whole cells by base hydrolysis, methylated, and analyzed by capillary gas-liquid chromatography. All group DF-3 strains possessed a distinct fatty acid profile which was characterized by large amounts (24%) of 12-methyltetradecanoate (a-C15:0), moderate amounts of saturated iso-branched-chain acids (i-C14:0 and i-C15:0), and small to moderate amounts of both branched- and straight-chain hydroxy acids (3-OH C15:0, i-3-OH C16:0, 3-OH C16:0, and i-3-OH C17:0). This fatty acid profile was unique as compared with the profiles of other bacteria we have previously tested but was most similar to the profiles of Capnocytophaga species.     

Differentiation of Brucella ovis from Brucella abortus by gas-liquid chromatographic analysis of cellular fatty acids

The cellular fatty acid composition of Brucella ovis and Brucella abortus strains was determined by gas-liquid chromatography. Both species were characterized by the presence of fatty acids 16:0, 17:0, 17:0 cyclopropane, 18:0, 18:1, and 19:0 cyclopropane; B. ovis also contained some 15:0. There were differences in the relative proportions of the fatty acids present, and it was possible to differentiate B. ovis from B. abortus on the basis of the absence of 15:0, lower concentrations of 17:0 and 18:1, and higher concentrations of 19:0 cyclopropane in B. abortus. The data indicate that analysis of cellular fatty acid composition by gas-liquid chromatography can be used for the identification of B. ovis and its differentiation from B. abortus.     

Gas-liquid chromatography of cellular fatty acids for identification of staphylococci.

A commercially available, computer-assisted microbial identification system (MIS) employs gas-liquid chromatographic analyses of bacterial fatty acids. The MIS was used to identify 470 isolates of Staphylococcus species. The accuracy of the MIS was compared with the accuracies of conventional methods. There was a complete agreement between the MIS and conventional methods in the identification of 413 (87.8%) strains. For 36 of 45 misidentified strains, the correct identification was listed by the MIS as a choice but not as the first choice. Twelve strains could not be matched. All strains of Staphylococcus cohnii, S. epidermidis, S. intermedius, S. lugdunensis, S. schleiferi, S. sciuri, S. simulans, and S. xylosus were correctly identified. Two species, S. hominis and S. saprophyticus, accounted for 52.6% (30 of 57) of the misidentifications. Seventy-eight organisms were retested. Identification of 73 organisms remained unchanged, and for five organisms, the second choice became first and vice versa. The overall performance of the MIS is acceptable, and the system can be used as an alternate identification method for staphylococci.