Cellular fatty acid composition of Streptococcus mutans and related streptococci.

The cellular fatty acid composition of 18 strains of Streptococcus mutans and 17 isolates of related streptococci were examined by gas-liquid chromatography. The cultures could be divided into two distinct groups on the basis of their fatty acids. The first group, which included S mutans and S salivarius, contained palmitic (16:0), octadecenoic (18:1), stearic (18:0), eicosenoic (20:1), and eicosanoic (20:0) acids. The presence of the two 20-carbon fatty acids distinguished S mutans and S salivarius from all of the other species. Two of the S mutans cultures were further distinguished by the presence of 19-carbon and 21-carbon cyclopropane acids. The second group included S sanguis, S mitis, S uberis, and a culture of Lancefield group C streptococci. The percentages of the major acids (16:0, 18:1, and 18:0) found in these species were essentially identical. A culture of Lancefield group E streptococci contained large amounts of 16:1 and 18:1 and small amounts of two unidentified acids which were not present in any of the other cultures.

Effects of cage density on fear-related behavioral response and activity of layers.

Two experiments were conducted to study the effects of cage density on fear-related behavioral response and activity of White Leghorn layers. In Experiment 1, 20-wk old pullets were placed into 30.5 x 50.8 cm cages at the rate of one, two, three, and four birds per cage for Treatments 1, 2, 3, and 4, respectively. In Experiment 2, birds were placed into cages (30.5 x 50.8 cm) at the rate of one, two, three, four, three, two, and three birds per cage for Treatments 1, 2, 3, 4, 5, 6, and 7, respectively, at 20 wk of age. At 28 wk of age, one bird was removed from each cage in Treatment 5, and one bird was added to each cage in Treatments 6 and 7. Fear-related behavioral response of the birds, as determined by the duration of induced tonic immobility (TI), and bird activity, as determined by the head movement (HM) count, were measured at 52 wk of age in Experiment 1, and at 29, 36, and 52 wk of age in Experiment 2. Increasing cage density had no effect on fear-related response of the birds in both experiments. Cage density effect on bird activity was not consistent. In Experiment 1, HM count was not affected by cage density, whereas in Experiment 2, pullets in the multiple-bird cage had a lower (P < or = .05) HM count (three-period average) than the birds housed individually. There was no correlation between the duration of induced TI and HM count in both experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of population density on layer performance.

Four experiments were conducted to study the effects of population density on layer performance. in each experiment, 20-wk--old White leghorn pullets were housed in 30.5 x 50.8 cm cages at the rate of one, two, three, and four birds per cage for Treatments 1, 2, 3, and 4, respectively. in Experiment 4, there were three additional treatments: for Treatments 5, 6, and 7, birds were placed into identical cages (30.5 x 50.8 cm) at the rate of three, two, and three birds per cage, respectively, at 20 wk of age. At 28 wk of age, one bird was removed from each cage in Treatment 5, and one bird was added to each cage in Treatments 6 and 7. In the first three experiments, all eggs that were collected for 2 consecutive d every 8 wk starting at 28 wk of age were used to measure egg traits. In Experiment 4, egg traits were determined once at 52 week of age. Birds at the highest population density had the lowest percentage hen-day egg production and had one of the poorest feed efficiencies. Egg production was negatively correlated (P < or = .01, Experiments 1, 2, 3; P < or = .05, Experiment 4) with population density. Feed required to produce a dozen eggs was positively correlated (P < or = .01, Experiments 1, 2; P < or = .05, Experiment 3) with population density in three out of the four experiments. The addition or removal of a cage mate to or from a multiple-bird cage in Experiment 4 did not (P > .05) affect egg production or feed efficiency. Final BW, mortality, and egg weight were not (P > .05) affected by population density. Only in one out of the four experiments was feed consumption (Experiment 1), eggshell thickness (Experiment 2), or albumen height (Experiment 3 lowered (P < or = .05) by having more than one bird per cage. This study showed that increasing population density decreased laying performance of the birds.

Gas-liquid chromatography as an analytical tool in microbiology.

The use of gas-liquid chromatography (GLC) in clinical and diagnostic bacteriology laboratories has increased significantly in recent years. GLC analysis of metabolic products from bacterial growth and chemical components of bacterial cells has provided useful information for rapid detection and identification of several bacterial groups or species. The use of short-chain acid products and cellular fatty acid composition for identifying and classifying Pseudomonas species and other medically important gram-negative non-fermentative bacteria is illustrated. Application of the flexible, fused, silica-glass, capillary column for increased resolution of bacterial fatty acids is also discussed.

Identification of Achromobacter species by cellular fatty acids and by production of keto acids.

The cellular fatty acid composition and metabolic products of 12 reference strains of Achromobacter sp. and A. xylosoxidans were determined by gas-liquid chromatography (GLC). Results showed that the two Achromobacter groups are strikingly different and can be readily distinguished on the basis of cellular fatty acids and the short-chain acids produced by Achromobacter sp. The major cellular fatty acids of Achromobacter sp. were octadecenoic (18:1) and a 19-carbon cyclopropanoic (19:0 delta) acid, whereas hexadecanoic (16:0) and a 17-carbon cyclopropanoic (17:0 delta) acid were principal components of the lipids of A. xylosoxidans. Hydroxy acids were not found in strains of Achromobacter sp. but comprised approximately 20% of the cellular fatty acids of A. xylosoxidans. In addition, Achromobacter sp. produced relatively large amounts of 2-ketoisocaproic acid, which was detected in only trace amounts from strains of A. xylosoxidans. The data show that GLC tests provide additional criteria for differentiating groups which are very closely related when evaluated with conventional tests. The GLC tests can be readily adapted in the clinical laboratory because they are rapid, highly reproducible, relatively inexpensive, and simple to perform.

Production of p-hydroxyhydrocinnamic acid from tyrosine by Peptostreptococcus anaerobius.

Peptostreptococcus anaerobius was found to metabolize tyrosine to p-hydroxyhydrocinnamic acid [3-(p-hydroxyphenyl)propionic acid]. This acid was detected in spent growth media by gas-liquid chromatography, and its identity was confirmed by mass spectrometry.

Cellular fatty acid compositions and isoprenoid quinone contents of 23 Legionella species.

The cellular fatty acid compositions and ubiquinone contents of 182 Legionella strains representing 23 species were determined by capillary gas-liquid chromatography and reverse-phase high-performance liquid chromatography, respectively. Except for the type strain of Legionella erythra (ATCC 35303T), all Legionella species contained large (40 to 90%) amounts of branched-chain fatty acids and only trace to small (less than 0.5 to 5%) amounts of ester-linked hydroxy acids. The 23 species were placed in three major fatty acid groups on the basis of differences in the relative amounts of 14-methylpentadecanoic (Ci16:0), hexadecanoic (C16:1), and 12-methyltetradecanoic (Ca15:0) acids. All Legionella species contained ubiquinones with 9 to 14 isoprene units in the side chains and were divided into five different ubiquinone groups. The species were further differentiated into 16 groups on the basis of qualitative and quantitative differences in their fatty acid compositions and ubiquinone contents. Both of these chemical characteristics can be used to distinguish Legionella species from other gram-negative bacteria and rapidly and accurately identify suspected isolates before serologic and other tests are done.

Fatty acid composition of Propionibacterium propionicum (Arachnia propionica).

The cellular fatty acids of Arachnia propionica are largely 13-methyltetradecanoic acid (Ci15:0) and 12-methyltetradecanoic acid (Ca15:0); thus, the fatty acid pattern of this organism resembles the pattern found in propionibacteria. This finding supports the transfer of members of the genus Arachnia to the genus Propionibacterium.

Use of gas chromatography for detecting ornithine and lysine decarboxylase activity in bacteria.

A gas-liquid chromatography (GLC) procedure for the detection of L-ornithine and L-lysine decarboxylase (EC 4.1.1.17 and EC 4.1.1.18, respectively) activities of bacteria was developed and evaluated against Møller's method, a conventional biochemical test. Cultures were incubated for 2 to 4 h in a simple growth medium and tested by GLC for putrescine and cadaverine, the direct decarboxylation products of ornithine and lysine, respectively. Results obtained with various Enterobacteriaceae, pseudomonads, and vibrios showed that the GLC procedure was superior to the conventional test; clear, well-defined results were obtained within 3 to 5 h, even with cultures which gave weak, delayed, or variable reactions by Møller's method. This GLC procedure for the determination of decarboxylase reactions would be useful in microbiological laboratories for culture identification and for various other enzymatic studies.

Short-chain acids of Pseudomonas species encountered in clinical specimens.

The short-chain acids of 36 strains of Pseudomonas grown on Trypticase soy agar were determined by gas-liquid chromatography. Distinct acid profiles were observed for each of the eight species tested. Propionic, isobutyric, and isovaleric acids were the principal acids detected in media extracts of P. maltophilia, P. cepacia, P. pseudoalcaligenes, P. diminuta, and P. vesiculare. The presence and relative amounts of the isobutyric and isovaleric acids clearly distinguished P. maltophilia, P. pseudoalcaligenes, and P. cepacia from other species. P. diminuta could be distinguished from P. vesiculare by the production of glutaric acid; P. testosteroni was the only species tested which produced relatively large amounts of phenylacetic acid.

Production of glutaric acid: a useful criterion for differentiating Pseudomonas diminuta from Pseudomonas vesiculare.

A gas-liquid chromatographic procedure was used to determine short-chain acids produced by Pseudomonas diminuta and P. vesiculare after growth on Trypticase soy agar. Each of nine strains of P. diminuta produced glutaric acid, whereas none of the strains of P. vesiculare produced this acid.

Detection by gas chromatography of 3-deoxy-D-manno-2-octulosonic acid and L-glycero-D-manno-heptose in whole cells of Neisseria elongata.

Lipopolysaccharide components 3-deoxy-D-manno-2-octulosonic acid and L-glycero-D-manno-heptose were detected in hydrolysates from whole cells of Neisseria elongata by gas-liquid chromatography. Cells from a single plate were hydrolyzed, and carbohydrate components were converted to aldononitrile and O-methyloxime acetate derivatives for subsequent analyses by gas-liquid chromatography. 3-Deoxy-D-manno-2-octulosonic acid was well separated from other cell components as the O-methyloxime acetate derivative. With both derivatives, L-glycero-D-manno-heptose was readily identified by their different retention times. The procedure requires only a relatively small number of cells, and detection is accomplished without prior isolation of the lipopolysaccharide.

Identification of some uncommon monounsaturated fatty acids of bacteria.

Location of the double-bond position of monounsaturated fatty acids of various bacteria was accomplished with combined gas chromatography-mass spectrometry analysis of dimethyl disulfide (DMDS) derivatives. The monoenoic fatty acids from whole cells were converted to methyl esters and then to DMDS adducts and analyzed by capillary gas chromatography-mass spectrometry. The mass spectra of DMDS adducts gave an easily recognizable molecular ion and two major diagnostic ions attributable to fragmentation between the two CH3S groups located at the original site of unsaturation. Twenty-one relatively novel monoenoic fatty acids were identified among the bacteria studied. All Flavobacterium species contained i17:1 omega 8c, Bacillus alvei contained i16:1 omega 11c and i17:1 omega 12c, and Psychrobacter immobilis contained 12:1 omega 9c. Resolution of cis and trans isomers with capillary gas chromatography and subsequent mass spectrometry permitted positive identification of 16:1 omega 7c and 16:1 omega 7t in Arcobacter (Campylobacter) cryaerophila and 16:1 omega 9c and 16:1 omega 9t in Aerococcus viridans.

Identification of nutritional components in trypticase responsible for recovery of Escherichia coli injured by freezing.

Moss, C. Wayne (North Carolina State University, Raleigh), and M. L. Speck. Identification of nutritional components in Trypticase responsible for recovery of Escherichia coli injured by freezing. J. Bacteriol. 91:1098-1104. 1966.-Freezing and storage of Escherichia coli at -20 C resulted in nonlethal or "metabolic" injury to a proportion of the surviving population. The injury was manifested as an increased nutritional requirement after freezing. Injured cells could not grow on a minimal agar medium, but could develop on Trypticase Soy Agar. The percentage of injured survivors varied among strains, but was little affected by altering the freezing menstruum. Trypticase was found to be the component in Trypticase Soy Agar responsible for the recovery of injured cells, and contained five closely related peptides that possessed most of the biological activity. Isolation of the peptides was accomplished by Sephadex gel chromatography, paper chromatography, and high-voltage paper electrophoresis. Hydrolysis of the peptides destroyed the ability to restore injured cells.

Release of biologically active peptides from Escherichia coli at subzero temperatures.

Moss, C. Wayne (North Carolina State University, Raleigh), and M. L. Speck. Release of biologically active peptides from Escherichia coli at subzero temperatures. J. Bacteriol. 91:1105-1111. 1966.-Freezing and storage of Escherichia coli at -20 C in phosphate buffer resulted in loss of cell viability and a pronounced leakage of cellular material which had maximal absorption at 260 mmu. Greater loss in cell viability occurred when cells were frozen in distilled water, but only small amounts of 260 mmu absorbing material were detected. Unfrozen cells stored at 2 and 22 C in each menstruum showed little loss in viability, but cells in phosphate buffer released significant amounts of material during storage. Leakage material from cells in phosphate buffer contained greater amounts of ribonucleic acid and amino acids than did material from cells in distilled water. Leakage material from frozen cells contained protein in the form of peptides of relatively small molecular weight; this was not observed for unfrozen cells. These compounds protected a dilute cell suspension from the lethal effects of freezing, and also possessed biological activity for the recovery of cells which had been "injured" by freezing. Direct cell counts indicated that the material released was not a result of cell lysis.

Characterization of clostridia by gas chromatography differentiation of species by trimethylsilyl derivatives of whole-cell hydrolysates.

Trimethylsilyl (TMS) derivatives prepared from whole-cell hydrolysates of 36 strains, representing 10 species of Clostridium were examined by gas-liquid chromatography (GLC). The TMS profile of each species contained a group of peaks which characterized the species. Variation among strains within a species was much lower than variation between species. Some of the closely related clostridia could be differentiated by comparing their TMS profiles. Strains of Clostridium botulinum were distinguished from C. sporogenes on the basis of the ratio of two GLC peaks which corresponded to arabinose and glucose. A peak with a retention time identical to that of mannose was present in all C. bifermentans strains but was absent in those of C. sordellii.

Characterization of clostridia by gas chromatography. I. Differentiation of species by cellular fatty acids.

Fatty acids of 41 strains representing 13 species of Clostridium were extracted directly from whole cells and examined as methyl esters by gas-liquid chromatography. Both visual and quantitative comparisons of the resulting chromatograms for the presence and relative amounts of large major peaks allowed rapid differentiation of C. perfringens, C. sporogenes, and C. bifermentans from each other and from 10 other species. Each of the three former species possessed a different characteristic fatty acid methyl ester profile that was exhibited by all strains tested within the respective species. Culture age and growth media influenced the relative proportions of certain of the acids, but such differences did not limit species differentiation.

Degradation of bacterial cyclopropane acids with boron trihalide reagents.

The cellular fatty acid compositions of Pseudomonas diminuta UC 501 and Streptococcus mutans OMZ-61 were compared in samples processed by a saponification-methylation procedure (method S) and in samples processed by a transesterification procedure (method T). All samples were heated in an inert atmosphere of nitrogen. The major acids found in samples of P. diminuta treated by method S included 16:0 (palmitic), 18:1 (octadecenoic) and 19 cyc (19 carbon cyclopropane acid). Those found in samples of S. mutans treated by the same method included 16:0, 18:1. 18:0 (stearic), 20:1 (eicosenoic), 20:0 (eicosanoic), 19 cyc and 21 cyc. When method T was used to process samples of both cultures, the cyc acids were degraded and artifacts were produced. Transesterification with boron trihalide reagents is therefore not recommended for routine analysis of bacterial fatty acids.

Gas chromatography-mass spectrometry of some biologically important short chain acid butyl esters.

Mass spectra of n-butyl esters of selected biologically important short chain fatty acids were obtained by using the technique of gas-liquid chromatography-mass spectrometry. The results indicate that simple cleavage is responsible for the primary fragmentation of the molecules. The mass spectral data are considered to be advantageous for the identification of unknown short chain acids.

Cellular fatty acids and metabolic products of Pseudomonas species obtained from clinical specimens.

The cellular fatty acid composition of 112 reference strains and clinical isolates of Pseudomonas species was determined by gas-liquid chromatography (GLC). The presence and relative amounts of cyclopropane, hydroxy, and branched-chain fatty acids were distinguishing features of these strains. Determination of short-chain fatty acids extracted from spent growth media provided an additional means for identifying some strains. Our results show that clinical isolates of pseudomonads can be divided into eight distinct GLC groups. The procedures were especially useful for distinguishing glucose-nonoxidizing pseudomonads, which are difficult to identify by conventional criteria. Since the GLC procedures are simple, rapid, and highly reproducible, they are useful in diagnostic laboratories that process large numbers of cultures. Coupled with selected conventional tests, the analysis of short-chain and cellular fatty acids can be very useful for rapid screening of clinical isolates of Pseudomonas species.