Isolation of a low-molecular-weight growth inhibitory factor from hybridoma cell cultures.

A low-molecular-weight growth inhibitory factor was produced by hybridoma cells. The number of viable cells in hybridoma cell cultures reached a maximum of about 5 x 10(5) cells/ml when the inhibitory factor had accumulated to a critical level, after which the number of viable cells declined with a concomitant increase in the number of dead cells. The growth inhibitory factor was purified to apparent homogeneity by ultrafiltration, reverse-phase chromatography, passage through cation exchangers, and gel filtration. Analysis by reverse-phase chromatography and micellar electrokinetic chromatography using a capillary electrophoresis system indicated that the final inhibitory fraction was pure. The factor had a molecular weight of 500 or less, as judged by ultrafiltration, and its behavior upon ion-exchange chromatography indicated that it was uncharged. Its absorbance maximum at 263 nm indicated that it was not a peptide, but that it may have a conjugated system of carbon-carbon double bonds.

Detection of microbial-derived fatty acids in carious dentin by gas chromatography and gas chromatography-mass spectrometry.

The aim of the present study was to analyze the fatty acid content of carious and sound human dentin. Gas chromatography and gas chromatography-mass spectrometry revealed the presence of fatty acids of C10-C18 size in the carious dentin, whereas fatty acids of C16 size were present in minute amounts in three samples of the corresponding sound dentine controls. No fatty acids were detected in the other sound dentin control samples. The source of fatty acids was considered to be microorganisms invading the dentin during the progression of the caries lesion. The presence of bacterial fatty acids in carious dentin may serve as a marker for the pathological process and thus contribute to the understanding of the mechanisms involved.

Capillary zone electrophoresis as a new tool in the chemotaxonomy of oral treponemes.

The existing taxonomy of oral treponemes is not satisfactory. This is due to the fact that these strict anaerobic bacteria are not easily cultivated or differentiated. Therefore, new techniques that can contribute to improved cultivation, classification, and identification of these fastidious organisms should be welcomed. In the present study capillary zone electrophoresis (CZE) was used to distinguish oral treponemes by their metabolic product patterns generated in a liquid medium. To our knowledge this technique has not previously been used in bacterial chemotaxonomy. Reference strains of Treponema denticola, Treponema pectinovorum, Treponema vincentii, Treponema socranskii subspecies buccale and Treponema socranskii subspecies socranskii were cultured anaerobically in duplicate on different days in Pectin medium for 4 days at 37 degrees C under nitrogen atmosphere. Treponemal cells were harvested by centrifugation. Thereafter, their supernatants were filtered through 0.22-micron Millipore filters and subjected to CZE. The resulting electropherograms clearly distinguished T. denticola, T. pectinovorum and T. vincentii. Minor differences were detected between T. socranskii subspecies buccale and T. socranskii subspecies socranskii. Subspecies were clearly different from species. It seems that CZE of culture metabolites, which showed high resolution and good reproducibility, may be a valuable tool in the chemotaxonomy of oral treponemes, even at the subspecies level.

Multivariate chemosystematics demonstrate two groups of Actinobacillus actinomycetemcomitans strains.

Chemical analysis by us has indicated that Actinobacillus actinomycetemcomitans is not a homogeneous species. The present study used chemometric methods and a multitude of chemical characters to examine this further. Strains were characterized by cell sugar and fatty acid contents, lysis kinetics during EDTA and EDTA plus lysozyme exposure, methylene blue reduction, and API ZYM enzymatic assessment of whole cells and outer membrane vesicles/fragments. In total, 41 quantitative variables were analyzed from each of 9 strains and treated with principal component analysis and soft independent modeling of class analogy. These methods divided A. actinomycetemcomitans into 2 strain groups. One group contained ATCC 33384, ATCC 29522, FDC 2112 and FDC 2043; the other comprised ATCC 29524, ATCC 29523, FDC 2097, FDC 511 and FDC Y4. With an F-test, the groups (classes) of A. actinomycetemcomitans strains could be distinguished at 95% confidence limits. Both groups were distinct from members of the genera Haemophilus and Pasteurella (Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus influenzae, Pasteurella multocida and Pasteurella haemolytica).

Review of chemosystematics: multivariate approaches to oral bacteria and yeasts.

There are several problems related to the classification and identification of bacterial and yeast species assigned to the genera Actinobacillus, Haemophilus, Pasteurella, Bacteroides, Prevotella, Porphyromonas, Campylobacter, Wolinella, Treponema, Candida, Torulopsis, and Saccharomyces, most of which belong to the resident oral microflora. The present review was written to demonstrate how multivariate analyses of data on cellular fatty acids, sugars, enzyme activities, and lysis kinetics during ethylenediaminetetraacetic acid (EDTA) and EDTA plus lysozyme treatment can be used to distinguish closely related species of these bacterial and yeast genera. With the exception of the Actinobacillus-Haemophilus-Pasteurella group, fatty acids were more discriminating than sugars. Enzymes from whole cells and outer membrane vesicles also contributed to taxonomic distinction. Apparently, chemosystematics, involving multivariate analyses, is a useful adjunct in oral microbial taxonomy.

Intra-injector methylation of free fatty acids from aerobically and anaerobically cultured Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus.

Free fatty acids from the type strains of anaerobically and aerobically broth-cultured Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus cells were Soxhlet-extracted with hexane. The fatty acids were identified and quantified by gas chromatography and gas chromatography-mass spectrometry after intra-injector derivation with trimethylanilinium hydroxide. This derivatization method, which we propose as suitable for routine use in clinical microbiology, is fast, accurate and sensitive, with low toxicity. Whereas the fatty acid content of A. actinomycetemcomitans was affected by the cultivation atmosphere, i.e. C16:1, decreased under aerobic growth and C16:0 increased, that of the closely related H. aphrophilus was more stable.

Application of multivariate analyses of enzymic data to classification of members of the Actinobacillus-Haemophilus-Pasteurella group.

Outer membrane vesicles and fragments from Actinobacillus actinomycetemcomitans, Actinobacillus lignieresii, Actinobacillus ureae, Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus influenzae, Haemophilus parainfluenzae, Pasteurella haemolytica, and Pasteurella multocida were isolated and examined semiquantitatively for 19 enzyme activities by using the API ZYM micromethod. The enzyme contents of vesicles and fragments were compared with the enzyme contents of whole cells of the same organisms. Enzymic data were analyzed by using principal-component analysis and soft independent modeling of class analogy. This technique allowed us to distinguish among the closely related organisms A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus. A. actinomycetemcomitans was divided into two groups of strains. A. lignieresii fell outside or on the border of the A. actinobacillus class. A. ureae, H. influenzae, H. parainfluenzae, P. haemolytica, and P. multocida fell outside the A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus classes.

Multivariate analyses of fatty acid data from whole-cell methanolysates of Prevotella, Bacteroides and Porphyromonas spp.

The genus Bacteroides contains a number of biochemically and physiologically heterogeneous groups of organisms and needs taxonomic revision. In this study cellular fatty acids from a number of Bacteroides spp. were identified and quantified using gas chromatography and gas chromatography-mass spectrometry. The chemical data were then subjected to principal components analysis. In B. fragilis, which is the type species of the genus Bacteroides, C3-OH-iso17 was the predominant fatty acid (38.0%) and Cante15 was present in higher amounts (32.7%) than Ciso15 (14.6%). B. fragilis thus differed from all the other species examined: Prevotella (Bacteroides) buccae, P. (B.) oralis, P. (B.) oris, P. (B.) disiens, P. (B.) veroralis, P. (B.) heparinolytica and Porphyromonas (Bacteroides) endodontalis. Principal components analysis also enabled the closely related P. buccae, P. oralis and P. oris to be differentiated.

Multivariate analyses of cellular fatty acids and carbohydrates of 1:2:1 and 2:4:2 spirochetes.

Delimitation of small-sized spirochetes of the oral cavity can be difficult. The endoflagella pattern has long served as the main criterion for taxonomic distinction. For approved species, e.g. Treponema denticola, several endoflagella patterns are observed, which indicates that this criterion is inadequate. The present study with GC and GC-MS used fatty acids and carbohydrates of whole-cell methanolysates to distinguish 1:2:1 and 2:4:2 subgingival spirochetes. Thirteen fatty acids: C12:0, C13:0, C14:0, Ciso-14:0, C2-OH-14:0, C15:0, Cante-15:0, C16:0, Ciso-16:0, C16:1, C18:0, C18:1, and C18:2, and three carbohydrates: rhamnose, glucose, and glucosamine, were detected. The carbohydrate contents did not differ between the two groups. While 1:2:1 spirochetes contained Ciso-14:0 and Cante-15:0 acid, 2:4:2 spirochetes did not. Also multivariate analyses of quantitative fatty acid data distinguished between these groups.

Multivariate analyses of cellular fatty acids in Bacteroides, Prevotella, Porphyromonas, Wolinella, and Campylobacter spp.

The genera Bacteroides, Wolinella, and Campylobacter contain several similar species that require taxonomic revision. Fatty acid profiles of whole bacterial cells have proven useful for taxonomy. In this study, cellular fatty acids from Bacteroides, Prevotella, Porphyromonas, Wolinella, and Campylobacter spp. were identified and quantitated by gas chromatography and gas chromatography-mass spectrometry, and the data were subjected to principal component analyses. Bacteroides fragilis, the type species of the genus Bacteroides, was distinct from the other organisms. While Bacteroides gracilis, Wolinella succinogenes, Wolinella curva, Wolinella recta, and Campylobacter fetus subsp. venerealis were close to each other, Prevotella (Bacteroides) buccae, Prevotella oralis, Prevotella oris, Prevotella disiens, Prevotella veroralis, Prevotella heparinolyticus, Porphyromonas (Bacteroides) endodontalis, and Bacteroides ureolyticus could be distinguished. B. fragilis was characterized by the presence of C3OH-i-1-, Ca-15, and Ci-15 and the absence of C12:0 and unsaturated fatty acids. For comparison, B. gracilis, B. ureolyticus, W. succinogenes, W. curva, W. recta, and Campylobacter fetus subsp. venerealis contained C12:0, C16:1, C18:1, and C3-OH-14 acids but lacked branched hydroxy and branched nonhydroxy acids. B. gracilis and B. ureolyticus are not "true" bacteroides.

Multivariate analyses of carbohydrate data from lipopolysaccharides of Actinobacillus (Haemophilus) actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus.

The taxonomic distinction between Actinobacillus (Haemophilus) actinomycetemcomitans and Haemophilus aphrophilus and the taxonomic distinction between H. aphrophilus and Haemophilus paraphrophilus have been questioned. This study was done to determine whether multivariate statistical analyses of carbohydrate data from lipopolysaccharides could be used to distinguish between these closely related species. Lipopolysaccharides were extracted with phenol-water and purified. Carbohydrates were assessed by using gas chromatography and gas chromatography-mass spectrometry after methanolysis and derivatization with trifluoroacetic acid anhydride. The lipopolysaccharides from all of the species contained rhamnose, fucose, galactose, glucose, L-glycero-D-mannoheptose, and glucosamine plus galactosamine, but in varying amounts. A. actinomycetemcomitans and H. paraphrophilus also contained D-glycero-D-mannoheptose, while H. aphrophilus did not. Sample- and variable-oriented principal-component analyses of the carbohydrate data clearly distinguished among A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus. Soft independent modelling of class analogy showed that no sample in the A. actinomycetemcomitans class fell within the 95% confidence limits of the H. aphrophilus class. H. paraphrophilus fell outside both classes.

Multivariate analyses of cellular carbohydrates and fatty acids of Candida albicans, Torulopsis glabrata, and Saccharomyces cerevisiae.

Quantitative data of major cellular carbohydrates distinguished Candida albicans or Torulopsis glabrata from Saccharomyces cerevisiae but not C. albicans from T. glabrata. Multivariate analyses of both carbohydrate and fatty acid variables (I. Brondz, I. Olsen, and M. Sjöström, J. Clin. Microbiol. 27:2815-2819, 1989), however, differentiated all three species.

Multivariate analysis of quantitative chemical and enzymic characterization data in classification of Actinobacillus, Haemophilus and Pasteurella spp.

Chemotaxonomic data for strains of Actinobacillus, Haemophilus and Pasteurella spp. were analysed using three multivariate statistical strategies: principal components, partial least squares discriminant, and soft independent modelling of class analogy. The species comprised Actinobacillus actinomycetemcomitans. Haemophilus aphrophilus, H. paraphrophilus, H. influenzae, Pasteurella multocida, P. haemolytica and P. ureae. Strains were characterized by cell sugar and fatty acid composition, lysis kinetics during EDTA and EDTA plus lysozyme treatment, and methylene blue reduction. In total 23 quantitative variables were compiled from chemotaxonomic analyses of 25 strains. A. actinomycetemcomitans and H. aphrophilus formed distinct classes which differed from those of H. paraphrophilus, H. influenzae and Pasteurella spp. All characterization variables, except those describing fatty acid content, contributed significantly to inter-species discrimination.

Gas chromatographic assessment of alcoholyzed fatty acids from yeasts: a new chemotaxonomic method.

An alternative chemotaxonomic method to methanolysis was developed for gas chromatographic assessment of fatty acids in whole yeast cells. Clinical and reference strains of the medically important yeasts Candida albicans, Torulopsis glabrata, and Saccharomyces cerevisiae were cultured for 48 h at 26 degrees C. Cellular lysis and transesterification were then performed with ethanol, propanol, butanol, or methanol. The relative recovery rates for cellular fatty acids, including the volatile acids C10:0 and C12:0, were similar after alcoholysis with ethanol, propanol, or butanol, while methanolysis gave lower recoveries of volatile fatty acids. Thus, after ethanolysis, the recovery of C10:0 acid (0.1, 1, and 10%) from a defined matrix (lyophilized Actinobacillus actinomycetemcomitans cells) varied from 97 to 102%, while the recovery of C10:0 after methanolysis varied from 49 to 75%. This indicated that with the frequently used methanolysis technique, there is a considerable loss of volatile fatty acids. These acids may be used as marker molecules for taxonomic differentiation between yeasts.

Chemical differences in lipopolysaccharides from Actinobacillus (Haemophilus) actinomycetemcomitans and Haemophilus aphrophilus: clues to differences in periodontopathogenic potential and taxonomic distinction.

While Actinobacillus actinomycetemcomitans has been associated with rapidly progressive periodontal destruction in man, the closely related Haemophilus aphrophilus has not been related to periodontal disease. This may be due to differences in composition and structure of the lipopolysaccharides (LPS) of these dental-plaque bacteria, since LPS probably exerts a series of detrimental effects on the periodontium. LPS was prepared by the phenol-water procedure from the type strains of A. actinomycetemcomitans and H. aphrophilus, purified by hexane extraction and ultracentrifugation, and analyzed with gas chromatography and gas chromatography-mass spectrometry. While the lipid content of LPS from A. actinomycetemcomitans constituted 35.4%, it was only 18.4% in H. aphrophilus: 3-hydroxytetradecanoic and tetradecanoic acids were 21.1 and 14.3% in A. actinomycetemcomitans and 10.9 and 7.5% in H. aphrophilus. There were qualitative and quantitative differences in the polysaccharide portions of their LPS. A actinomycetemcomitans contained both D-glycero-D-mannoheptose and L-glycero-D-mannoheptose (7.8 and 11.3%); H. aphrophilus contained only L-glycero-D-mannoheptose (17.4%). The rhamnose, fucose, galactose, glucose, and glucosamine/galactosamine contents in A. actinomycetemcomitans were 2.6, 5.2, 10.1, 22.4, and 5.2%, respectively; in H. aphrophilus, they were 2.1, 2.6, 19.4, 36.4, and 3.7%. Chemical differences in LPS from A. actinomycetemcomitans and H. aphrophilus may contribute to the divergence in periodontopathogenic potential of these organisms and help taxonomic differentiation.

Chemotaxonomy of selected species of the Actinobacillus-Haemophilus-Pasteurella group by means of gas chromatography, gas chromatography-mass spectrometry and bioenzymatic methods.

Instrumental analytical and bioenzymatic methods were used to differentiate between species of the Actinobacillus-Haemophilus-Pasteurella group. Long-chain fatty acids were analysed directly with gas chromatography (GC) without derivatization. GC of trifluoroacetylated whole-cell methanolysates was a rapid method for differentiation. Cellular sugars were more suitable for differentiation than fatty acids. D-Glycero-D-mannoheptose, the major localization of which was lipopolysaccharide, distinguished H. aphrophilus from A. actinomycetemcomitans, H. paraphrophilus, H. influenzae type b, P. haemolytica, P. multocida, and P. ureae. GC of single colonies, which is a new chemotaxonomic method, was preferable to GC of liquid-grown cells. Lysozyme-and EDTA-induced bacteriolysis and reduction of methylene blue by cellular hydrogenase served as additional criteria for differentiation.

Microbial chemotaxonomy. Chromatography, electrophoresis and relevant profiling techniques.

This review deals with the chemistry of marker substances used in microbial classification and identification, their isolation and purification and their biomedical application. A critical evaluation of current methods is also included. The information presented is partly based on personal experience, partly derived from more than 300 publications in this rapidly expanding field of science. Much has been done to improve the recognition of microorganisms by GC, but there are also a series of other techniques available that can assist in bacterial classification and identification. Some of these techniques have been made available to the clinical microbiologist through commercial systems, e.g., assessment of bacterial fatty acids. A fingerprint library has been developed by Hewlett-Packard for the analysis of fatty acids from approximately 6000 different bacteria. Other chemotaxonomic methods require great personal expertise and advanced equipment. Efforts should therefore be made to adapt and simplify such methods for application in the routine clinical laboratory. Chemical markers will probably have a great impact on future microbial taxonomy, particularly in cases where conventional methods fail to give satisfactory classifications. In order to make taxonomy more objective, there seems to be a need for screening of chemical markers in bacterial species and for compiling chemotaxonomic fingerprints in clinical manuals.