Bacterial phospholipid analysis by fast atom bombardment mass spectrometry.

The structural analysis of naturally occurring bacterial phospholipids in mixtures by fast atom bombardment (FAB) mass spectrometry are reported. The bacterial strains examined included several genera of actinomycetes, two strains of Escherichia coli, and one strain each of Proteus mirabilis and Pseudomonas aeruginosa. FAB mass spectrometry proved to be a useful tool for the structural identification of phospholipids in mixtures and provided stable pseudo-molecular ions and characteristic fragment ions which permitted the identification of phosphatidylethanolamine and phosphatidyl choline. Information regarding the chain length of the fatty acids, their degree of unsaturation in the chains and the presence of hydroxyl groups was also obtained. The results obtained by FAB mass spectrometry were supported by high-resolution mass spectral data, tandem mass spectrometric studies and FAB mass spectrometry of components which had been separated and partially purified by thin-layer chromatography. Each organism displayed a highly characteristic phospholipid profile suggesting the possible use of FAB mass spectrometry as a method for rapid bacterial detection and identification.

Pitted keratolysis. The role of Micrococcus sedentarius.

Though pitted keratolysis of the foot is generally viewed to be caused by bacteria, there is confusion regarding the identity of the causative organism. Species of Corynebacterium, Actinomyces, Dermatophilus, and Micrococcus have been proposed by various investigators. We have studied eight cases of pitted keratolysis and have cultured an organism identified as Micrococcus sedentarius on the basis of colonial morphology, micromorphology, biochemical reactions, and chemical analysis of whole-cell components. Furthermore, pitted keratolysis was produced experimentally in a human volunteer by applying M sedentarius under an occlusive dressing onto the surface of the heel.

Characterization of pathogenic bacteria by automated headspace concentration-gas chromatography.

Automated headspace concentration-gas chromatography (AHC-GC) was used to profile the volatile metabolites produced by Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. Bacterial cultures were incubated in trypticase soy broth and examined at 24 h. The profiles were consistent for each genus examined and variation observed among the different strains of each species was chiefly quantitative. The volatiles were identified by concurrent headspace concentration-gas chromatography-mass spectrometry and consisted mainly of isobutanol, isopentanol, isopentyl acetate, 1-undecene and methyl ketones. There were sufficient differences in the profiles in the 4-6 min elution period to distinguish P. aeruginosa and S. aureus from each other and from the other two bacteria. P. mirabilis and K. pneumoniae typically showed three intense peaks which corresponded to isobutanol, isopentyl acetate and isopentanol. The determination of volatiles by AHC-GC is sensitive, rapid and offers a possible alternative for automatic detection and characterization of pathogenic bacteria.

Analysis of cellular components, biochemical reactions, and habitat of human cutaneous lipophilic diphtheroids.

The cutaneous distribution of lipophilic diphtheroids was determined in normal human volunteers. The organisms were found to be plentiful in moist regions (scalp, nares, axilla, groin, and toe web) and scarce in dry and purely oily regions. The lipid requirement, cellular fatty acids, mycolic acid and cell wall diaminopimelic acid content of these lipophilic diphtheroids was compared to those of strains of Corynebacterium bovis, C. xerosis, C. diphtheriae, and C. minutissimum. Only lipophilic diphtheroids and C. bovis strains were found to have a strict lipid requirement. Lipophilic diphtheroids were found to have meso-diaminopimelic acid and corynemycolic acid in their cell walls, consistent with membership in the genus Corynebacterium. Lipophilic diphtheroids were also found to comprise a homogeneous group which was distinct from the speciated strains on the basis of cellular fatty acids and mycolic acids.

Volatiles of Pseudomonas aeruginosa and related species by automated headspace concentration--gas chromatography.

The volatile metabolites of three strains of Pseudomonas aeruginosa and one strain each of Pseudomonas cepacia, Pseudomonas maltophilia, Pseudomonas fluorescens, and Pseudomonas putida were analyzed using an automated headspace concentrator incorporating a gas chromatograph. The procedure does not require sample preparation and automates the entire analytical sequence to yield reproducible profiles of volatile constituents. Gas chromatographic profiles of the volatile metabolites of each species were obtained using a 20-min concentration period and two fused silica capillary columns of different polarities. The production of headspace metabolites from trypticase soy broth was studied in relationship to culture incubation time and initial cell concentration. The volatiles identified after 24 h incubation consisted of 1-butanol, isopentanol, toluene, 1-undecene, 2-butanone, 2-heptanone, 2-nonanone, and 2-undecanone. Sufficient amounts of specific metabolites were produced after 5 h incubation to provide information of possible diagnostic value. In particular, all P. aeruginosa strains produced a distinctive series of 1-undecene and methyl ketones after 5 h incubation of media inoculated to provide 2 X 10(6) cells/mL. The results indicate that when growth and analytical conditions are held constant, P. aeruginosa and related species produce characteristic profiles of headspace metabolites. Since conventional bacteriological tests require 24 h or more for the identification of these pseudomonads, automated volatile analysis could provide an alternative means for the rapid detection of these bacteria.

Effects of sodium saccharin on the activity of trypsin, chymotrypsin, and amylase and upon bacteria in small intestinal contents of rats.

Rats fed a diet containing 2.5% sodium saccharin (NaSacc) displayed a rapid (24-36 hr) increase in tryptic and chymotryptic activity in the lower half of the small intestine and the cecum compared with control animals. Cecal pH of rats fed NaSacc was lower than controls. The effect of NaSacc on enzymatic activity of intestinal contents and on indigenous bacterial microflora was studied further in vitro. Intestinal contents incubated anaerobically with or without NaSacc revealed that the presence of NaSacc led to higher tryptic and chymotryptic activity and higher final pH. Changes in pH do not appear, however, to be important for the increased proteolytic activity induced by NaSacc since autodigestion of trypsin and chymotrypsin in filter-sterilized samples was only slightly affected by pH during in vitro incubation. Amylolytic activity, on the other hand, was stabilized by higher pH values. Saccharin stabilized chymotryptic and led to almost complete loss of amylolytic activity during incubation of filter-sterilized samples maintained at adjusted pH values. The amount of reducing sugars remaining in the NaSacc-containing contents from either cecum (in vivo) or from in vitro incubation of unsterilized small intestinal samples was greater than controls not containing NaSacc. The growth of six bacterial strains isolated from small intestinal contents and incubated in laboratory media was inhibited by NaSacc. Extracellular proteolytic activity from bacterial sources was undetectable after incubation of intestinal bacteria in laboratory media. The present results suggest that the effect of NaSacc upon digestive enzyme composition in the small intestine of rats is not mediated through a direct physiological effect of NaSacc on pancreatic exocrine secretion. It is hypothesized that an inhibition of enzymatic activity by NaSacc in the small intestine and the bacteriostatic effect of NaSacc on bacteria may be responsible for the increased proteolytic activity observed in vivo in the cecum following the feeding of a NaSacc-containing diet to rats.

Perineal scent gland of wild and domestic cavies: bacterial activity and urine as sources of biologically significant odors.

Several studies were conducted to evaluate the source of biologically relevant odorous substances in male wild cavy (Cavia aperea ) perineal gland secretions. In the first study, using a habituation procedure, male wild cavies distinguished between the urine of two individuals after exposure to the perineal secretions of one of the individuals. However, these animals did not distinguish between the perineal secretions of two individuals after exposure to the urine of one of the individuals. These results suggest that urine is a component of cavy perineal gland secretions as normally found in the perineal sac. Other studies were designed to evaluate the possible role of bacteria in producing biologically relevant odors from cavy perineal gland secretions. Microbiological analyses of secretion that had accumulated in the perineal sacs ("dirty") or been squeezed directly from the glands ("clean") of wild and domestic (C. porcellus) cavies indicated large numbers of bacteria inhabited these secretions. In behavioral studies using two-choice preference tests, male wild cavies spent more time investigating conspecific dirty perineal gland secretions than clean secretions. Clean secretions, however, which had been incubated for 48 hr at 37 degrees C were preferred by these cavies over clean secretions which had been frozen at -60 degrees C during the same time period. In a final experiment it was found that wild cavies preferred conspecific clean secretion that had been sterilized and incubated with bacteria over uninoculated sterilized secretion. Together, these results indicate that urine and bacteria are responsible for components of biologically significant odors of cavy perineal scent marks.

Death of Pseudomonas aeruginosa in soil.

When incubated in natural (nonsterilized) soil, Pseudomonas aeruginosa died initially at a rate which approximated the rate for starvation of a pure culture in buffer. Predation by other soil microbes or phage did not appear to be involved, and pyocyanin either was not produced or was ineffective. The initial rate of death was followed by a second, considerably slower rate. Cells initially added in low numbers to soil also underwent biphasic death as above. Slow drying of the soil caused a period of rapid soil death of P. aeruginosa, but this then slowed to give residual numbers and a death rate similar to the second death rate noted for soil not allowed to dry. The cells in the dry soil had not changed genetically to a desiccation-resistant form. Pseudomonas aeruginosa died out completely in a relatively short time when the soil was first quickly dried to a water content similar to that obtained initially through slow drying and then further allowed to dry slowly. These observations appear to point to a dormant form, in some ways resembling a cyst, for P. aeruginosa in soil.