Characterization of passage-selected vancomycin-resistant Staphylococcus aureus strains of diverse parental backgrounds.

A series of 12 Staphylococcus aureus strains of various genetic backgrounds, methicillin resistance levels, and autolytic activities were subjected to selection for the glycopeptide-intermediate S. aureus (GISA) susceptibility phenotype on increasing concentrations of vancomycin. Six strains acquired the phenotype rapidly, two did so slowly, and four failed to do so. The vancomycin MICs for the GISA strains ranged from 4 to 16 microg/ml, were stable to 20 nonselective passages, and expressed resistance homogeneously. Neither ease of acquisition of the GISA phenotype nor the MIC attained correlated with methicillin resistance hetero- versus homogeneity or autolytic deficiency or sufficiency. Oxacillin MICs were generally unchanged between parent and GISA strains, although the mec members of both isogenic methicillin-susceptible and methicillin-resistant pairs acquired the GISA phenotype more rapidly and to higher MICs than did their susceptible counterparts. Transmission electron microscopy revealed that the GISA strains appeared normal in the absence of vancomycin but had thickened and diffuse cell walls when grown with vancomycin at one-half the MIC. Common features among GISAs were reduced doubling times, decreased lysostaphin susceptibilities, and reduced whole-cell and zymographic autolytic activities in the absence of vancomycin. This, with surface hydrophobicity differences, indicated that even in the absence of vancomycin the GISA cell walls differed from those of the parents. Autolytic activities were further reduced by the inclusion of vancomycin in whole-cell and zymographic studies. The six least vancomycin-susceptible GISA strains exhibited an increased capacity to remove vancomycin from the medium versus their parent lines. This study suggests that while some elements of the GISA phenotype are strain specific, many are common to the phenotype although their expression is influenced by genetic background. GISA strains with similar glycopeptide MICs may express individual components of the phenotype to different extents.

Unique morphogenesis in Saccharomyces cerevisiae strain GS1731.

During the lag and early exponential phase of growth, 50-60% of budded cells of Saccharomyces cerevisiae strain GS1731 were multiply budded. During subsequent culture growth, the frequency of multiply budded cells decreased until by stationary phase multiply budded cells were rare. Data from renewed growth of a culture after hydroxyurea treatment indicated that GS1731 mother cells could assemble up to three pre-bud sites and begin bud growth and development in each. Light and scanning electron microscopy showed two or three very small buds emerging simultaneously on a mother cell and either reaching full size at the same time or enlarging sequentially. Immunofluorescence studies revealed that these multiply budded cells had multiple bundles of cytoplasmic microtubules. DAPI staining of nuclei revealed that some of the unbudded mother cells were multinucleate and completed cytokinesis giving rise to normal daughter cells.

Characterization of an NaCl-sensitive Staphylococcus aureus mutant and rescue of the NaCl-sensitive phenotype by glycine betaine but not by other compatible solutes.

To further study mechanisms of coping with osmotic stress-low water activity, mutants of Staphylococcus aureus with transposon Tn917-lacZ-induced NaCl sensitivity were selected for impaired ability to grow on solid defined medium containing 2 M NaCl. Southern hybridization experiments showed that NaCl-sensitive mutants had a single copy of the transposon inserted into a DNA fragment of the same size in each mutant. These NaCl-sensitive mutants had an extremely long lag phase (60 to 70 h) in defined medium containing 2.5 M NaCl. The osmoprotectants glycine betaine and choline (which is oxidized to glycine betaine) dramatically shortened the lag phase, whereas L-proline and proline betaine, which are effective osmoprotectants for the wild type, were ineffective. Electron microscopic observations of the NaCl-sensitive mutant under NaCl stress conditions revealed large, pseudomulticellular cells similar to those observed previously in the wild type under the same conditions. Glycine betaine, but not L-proline, corrected the morphological abnormalities. Studies of the uptake of L-[14C]proline and [14C]glycine betaine upon osmotic upshock revealed that the mutant was not defective in the uptake of either osmoprotectant. Comparison of pool K+, amino acid, and glycine betaine levels under NaCl stress conditions in the mutant and the wild type revealed no striking differences. Glycine betaine appears to have additional beneficial effects on NaCl-stressed cells beyond those of other osmoprotectants. The NaCl stress protein responses of the wild type and the NaCl-sensitive mutant were characterized and compared by labeling with L-[35 S]methionine and two-dimensional gel electrophoresis. The synthesis of 10 proteins increased in the wild type in response to NaCl stress, whereas the synthesis of these 10 proteins plus 2 others increased in response to NaCl stress in the NaCl-sensitive mutant. Five proteins, three of which were NaCl stress proteins, were produced in elevated amounts in the NaCl-sensitive mutant under unstressed conditions compared to the wild type. The presence of glycine betaine during NaCl stress decreased the production of three NaCl stress proteins in the mutant versus one in the wild type.

Increased cell size and shortened peptidoglycan interpeptide bridge of NaCl-stressed Staphylococcus aureus and their reversal by glycine betaine.

Staphylococcus aureus cells grown in a defined medium under conditions of high ionic stress (2.5 M NaCl) were significantly larger than cells grown under unstressed conditions, even though the cells grew much more slowly under stressed conditions. Analysis of the structure of peptidoglycan from stressed cells showed a shorter interpeptide bridge than in peptidoglycan from unstressed cells. Glycine betaine inclusion in the high-NaCl medium resulted in cells with sizes and interpeptide bridges similar to those of cells grown under unstressed conditions.

Autolysis-defective mutant of Staphylococcus aureus: pathological considerations, genetic mapping, and electron microscopic studies.

In an earlier report, we had described the isolation and characterization of autolysis-defective mutants of Staphylococcus aureus (N. Mani, P. Tobin, and R.K. Jayaswal, J. Bacteriol. 175:1493-1499, 1993). In the study reported here, an autolysis-defective mutant showed attenuated virulence in a rat model of experimental endocarditis, supporting the role of autolysins in pathogenicity. Transmission electron micrographs of the mutant cells revealed a rough outermost surface as compared with the parent strain, ISP2018. Treatment of mutant cells with lysozyme, proteases, and lipase failed to alter this rough appearance. Physical and genetic data locate the site of mutation between the omega 1100 and ilv loci on the S. aureus chromosome.

Characterization of cell surface material removed by water and NaCl washing of Paracoccus denitrificans grown under conditions of divalent cation deficiency and sufficiency.

Paracoccus denitrificans grown on complex medium deficient in Mg2+ and Ca2+ are rendered lysozyme susceptible by washing with NaCl, whereas cells grown in a succinate-salts medium (Mg2+ and Ca2+ sufficient) or complex medium supplemented with Mg(2+)+Ca2+ are not. The material released by water washing of cells grown on complex medium and complex medium supplemented with Mg2+ and Ca2+ was characterized by a high protein content. There was a high lipid: protein ratio and an appreciable amount of 3-deoxyoctulosonic acid in the material released by NaCl washing of cells grown under all conditions, indicating release of outer membrane material. The lipid ornithine: lipid phosphorous ratios of NaCl wash from cells grown on complex medium and complex medium supplemented with Mg2+ and Ca2+ were 0.54 and 0.34, respectively. Although NaCl washing removed outer membrane material from cells grown under all conditions, only divalent cation deficient cells were rendered lysozyme susceptible. This might be explained by the increased outer membrane ornithine-containing lipid to phospholipid ratio in these cells yielding a more permeable outer membrane.

Effect of dietary restriction on the age-dependent changes in the expression of antioxidant enzymes in rat liver.

The effects of aging and dietary restriction on the expression of several enzymes (superoxide dismutase, catalase and glutathione peroxidase) that are involved in free radical detoxification were studied in liver tissue from male Fischer F344 rats. The expression (i.e., activities and mRNA levels) of superoxide dismutase (Cu-Zn) and catalase decreased with age in liver. Dietary restriction (40% restriction of energy intake) increased the activities of superoxide dismutase (24 to 38%) and catalase (64 to 75%) in liver at 21 and 28 mo of age. Glutathione peroxidase activity in liver of diet-restricted rats was significantly higher (37%) at 28 mo of age than that of rats fed ad libitum. The age-related changes in the relative levels of mRNA for superoxide dismutase, catalase and glutathione peroxidase paralleled the changes in the activities of these enzymes in rats fed ad libitum or rats fed the restricted diet. Thus, the changes in the activities of superoxide dismutase, catalase and glutathione peroxidase with age and dietary restriction appear to arise from changes in the levels of mRNAs coding for these enzymes. Free radical damage, as measured by thiobarbituric acid-reactive material and lipofuscin accumulation, was lower in diet-restricted rats than in rats fed ad libitum.

Effects of ingested gossypol on the ultrastructure of rat ovarian follicles.

Ultrastructural changes were observed in the oocytes and granulosa cells of the ovarian follicles of sexually mature female rats treated daily for 60 days with gossypol acetic acid at a dosage known to induce sterility in male rats (20 mg/kg body weight, orally). The changes included an increased lysosomal population in the ooplasm and a reduced, poorly developed smooth endoplasmic reticulum in the granulosa cells. The latter changes suggest a suppression of the secretory activity of the granulosa cells.

Dietary restriction retards age-related decrease in cell population of rat corneal endothelium.

The surface areas of corneal endothelial cells from 12- and 18-month-old male Fischer 344 rats fed ad libitum or a calorie-restricted diet were compared. The rats fed the restricted diet in both age groups showed a statistically significant reduction in the mean cell area of the corneal endothelium. The data indicate that dietary restriction retarded the age-related endothelial cell loss and the subsequent enlargement that takes place to compensate for cell loss. This is the first report to suggest that dietary restriction retards age-related cell loss.

Lipoprotein as a regulator of starch synthesizing enzyme activity.

Amylose precipitating factor, a lipoprotein, functions as a regulator of in vitro activity of glycogen/starch phosphorylase and of A/UDPglucose glucosyltransferase. The results suggest that this lipoprotein could act to stimulate the in vivo production by phosphorylase of long, linear glucans (amylose) from the short chain precursors. The lipoprotein also appears to switch A/UDPglucose glucosyltransferase from the elongation of branched glucan molecules (amylopectin and glycogen) to the elongation of linear glucans (amylose).

Age-related changes in the corneal endothelium of the mouse.

The endothelial cells of the C57BL/6J mouse cornea show a progressive age-related increase in cell area, and an increasing departure from the initial hexagonal form. Mean endothelial surface areas were 304, 386, 458, 499, and 653 micron2 for mice of ages 1, 4, 11, 22, and 27 months, respectively. Increasing cell areas are interpreted to represent a spreading and thinning of surviving cells to compensate for cell loss. These age-related changes correspond to those previously observed in the rat, although the rat endothelial cells tend to have smaller cell areas and show less variation at each age studied.

Effect of brief vascular perfusion on the ultrastructure and activity of mitochondria isolated from the rat heart.

Mitochondria isolated from heart tissue after a 1-min perfusion with Hanks medium were found to have significantly lower rates of State-3 respiration and respiratory control ratios compared to mitochondria isolated from non-perfused hearts. Examination of the mitochondrial preparations by electron microscopy revealed that a large proportion of the mitochondria isolated from perfused heart tissue were swollen and broken compared to mitochondria from non-perfused hearts.

Prevention by pinealectomy of short-photoperiod-induced ultrastructural changes in the hamster Harderian gland.

Harderian gland ultrastructure was examined at biweeky intervals for 16 weeks after either pinealectomy or sham-operation of male and female hamsters kept in 1L:23D and 14L:10D photoperiods. Female characteristics such as membrane formations and extensive smooth endoplasmic reticulum appeared in the males kept under short photoperiod conditions. Similarly, male characteristics such as the tubular clusters appeared in the glands of females kept in the short photoperiod. When male and female hamsters were pinealectomized and exposed to the short photoperiod, the glands in each sex retained the characteristics typical of that sex. Atrophy of the testes and uteri was also observed in the sham-operated hamsters maintained under short photoperiods, but not in the pinealectomized animals. The changes observed in the Harderian glands and reproductive organs of hamsters kept in the short photoperiod were not observed in the hamsters maintained in the long photoperiod. The photoperiod-induced ultrastructural changes in the hamster Harderian glands are most likely due to changes in hormonal levels brought about by the regression of reproductive organs and can be prevented by pinealectomy.

Sexual dimorphism in the Harderian gland proteins of the golden hamster.

SDS polyacrylamide gel electrophoresis of male and female hamster Harderian gland homogenates has shown a clear-cut sexual dimorphism. At least three major proteins present in the male gland are missing from the female gland. Two of the above are associated with the tubular clusters of the male gland while the third seems to be a structural component.

Age-dependent changes in the cellularity and ultrastructure of the spleen of Fischer F344 rats.

The age-related changes in the cellularity (cells/gram of tissue) of the spleens and thymuses of Fischer F344 male rats were determined. A decline in the weight of the thymus with age was observed as previously reported by others. The decline was most drastic between 4 and 20 months of age. The spleen, however, increased in weight with age. The increase was almost linear between 4 and 30 months of age. Yet when the number of cells recovered from each organ as a function of age was determined, a decrease for both the thymus and the spleen was observed with increasing age. It was surprising to find that fewer cells were recovered from the spleens of old animals even though the weight of the spleen of the old animals was greater than the spleens from the younger animals. The ultrastructure of the splenic white pulp of rats ranging from 4 to 30 months of age was studied to determine the possible cause for the age-related decrease in cellularity of the spleen. The white pulp of the 4-month-old rats contained a large number of small lymphocytes, and the number of cells was found to decrease with increasing age. The 30-month-old animals had less than 20% the number of lymphocytes in the white pulp as the 4-month-old animals, and the white pulp exhibited an increased number of reticular cells and macrophages with enlarged cytoplasm. The decreased cellularity and increased structural disturbance might be significant in the age-related decline of spleen lymphocyte functions.

Methicillin-resistant septal peptidoglycan synthesis in a methicillin-resistant Staphylococcus aureus strain.

In a methicillin-resistant Staphylococcus aureus strain, electron micrographs showed that cell wall septa continued to be formed in the presence of methicillin, although they became distorted and enlarged. The results indicated that peripheral cell wall synthesis was inhibited. It is concluded that a methicillin-resistant mode of septal peptidoglycan synthesis is an important determinant of methicillin resistance.

Influence of short photoperiods on the ultrastructure of hamster Harderian glands.

A sexual dimorphism of the hamster Harderian gland at the ultrastructural level, has been reported. Castration of males and the administration of testosterone to females are known to alter the male type gland to the female type and vice versa. In this paper we present data that exposure to a short photoperiod (1L:23D) can induce similar structural alterations between the 4th and 10th week of exposure. Changes in the male included reduction in the number of tubular clusters and large vacuoles, while in the female the number of membrane formations decreased with an accompanying appearance of tubular clusters.

Age-related changes in the corneal endothelium of the rat.

The endothelial cells of the rat cornea show a progressive decline in numbers and an increase in pleomorphism from age 6 months to 30 months. These age-related changes correspond closely to those reported in the human cornea from approximately age 20 to age 70 years. The corneal endothelium may be found useful as an index of physiological aging in these and other mammals.

delta-Aminolevulinate synthase activity in the Harderian glands of male and female golden hamsters.

1. delta-Aminolevulinate (A1A) synthase activity was determined in the Harderian glands of male and female Golden Hamsters. 2. The enzyme activity was higher in the female gland than in the male gland. 3. The data suggest that the higher enzyme activity is, at least partly, related to the higher porphyrin content in the female gland. 4. This enzyme activity has not been reported in the male gland previously.

Effect of testosterone on the female hamster Harderian gland pigmentation and ultrastructure.

Distinct differences occur in the pigmentation and ultrastructural features of the Harderian glands in male and female hamsters. The results of a study on the effect of testosterone on the fine structure of the female Harderian glands are presented here. Glands from three groups of hamsters were examined at intervals up to 49 days: (1) testosterone injected, receiving 2 mg testosterone propionate in 0.1 ml sesame oil per day; (2) sham-injected, receiving 0.1 ml sesame oil per day; (3) untreated controls. Testosterone injections caused a reduction in the number of dark-brown pigment granules in the acinar cells starting on the 6th day, whereas clusters of tubules, typical of adult male glands, appeared on the 4th day and increased in number thereafter. Lamellar structures, normally present in the female gland, decreased in testosterone treated specimens. These changes reversed after cessation of testosterone treatment. It is concluded that exogenous testosterone administered to female hamsters modifies the pigmentation and ultrastructure of their Harderian glands towards the male type and that this is a reversable phenomenon. There also appears to be an inverse relationship between the presence of tubular clusters in the acinar cells, and the degree of pigmentation.