An Electron Microscopic Study of Antagonism between Cephalexin and Erythromycin in Staphylococcus aureus

This study concerns investigations at the cellular level of antagonism between cephalexin (CEX) and erythromycin (EM) with the aid of electron microscopes and a liquid scintillation counter. Exposure of Staphylococcus aureus 209-P to CEX and EM in combination was found to result in a marked antagonism between the two antibiotics in their effects on the growth of the organism. Observations under a scanning electron microscope revealed lysed cells in the presence of CEX alone but almost no lysis in the presence of a combination of CEX and EM. Observations under a transmission electron microscope, on the other hand, disclosed that nearly all of the cells exposed to 20 μg/ml of CEX were transformed into protoplasts with their morphological changes being most marked after 4 hr of exposure. When 1 μg/ml of EM was allowed to act alone, this exposure resulted in thickening of the cell walls. The combined use of CEX and EM, however, resulted in neither thickening of the cell walls as in the presence of EM alone nor in the formation of protoplasts as in the presence of CEX alone but merely produced the swelling of separating walls. Cellular uptake of ¹⁴C-L -lysine and N-acetylglucosamine-1-¹⁴C into the cell wall fraction and the protein fraction was affected by CEX and EM, respectively, when used alone or in combination.     

Cell Wall Synthesis in Staphylococcus aureus in the Presence of Protein Synthesis Inhibitory Agents

Electron-microscopic and biochemical studies on morphological changes in Staphylococcus aureus following exposure to protein synthesis inhibitory agents such as lincomycin (LCM), clindamycin (CLM), erythromycin (EM), and spiramycin (SP) are presented in this paper. It was demonstrated that bacterial cell walls became extremely thickened usually with the formation of multilayers, when exposed to each of the above-mentioned antibiotics. Furthermore, electron density of the cytoplasm was higher in those cells exposed to drugs than in intact control cells. Incorporations of ¹⁴C-labeled l-lysine into the cell-wall fraction and the protein fraction were measured for biochemical elucidation of these phenomena. Labeled lysine was selectively incorporated into the cell-wall fraction when the test organism was exposed to the respective antibiotics. Uptake at 15 min after exposure was about twice as large as that of intact control cells. SP and CLM inhibited protein synthesis while they stimulated cell-wall synthesis. The evidences for thickening of, and formation of multilayers in the bacterial cell walls following exposure to drugs were closely related to the stimulating action of these antibiotics on the cell-wall synthesizing system. Morphology of resistant clinical isolates following such antibiotic exposure was also investigated using two staphylococcal strains, one resistant to EM alone and the other completely cross-resistant to all the macrolides.     

THE LABELING OF CULTURED CELLS OF ACER WITH [14C]PROLINE AND ITS SIGNIFICANCE

The distribution of the radioactivity from [¹⁴C]proline that is bound in cultured cells of Acer has been determined by electron microscope autoradiography. In this way proline may be related to the cell wall as a morphological entity rather than as a fraction in a biochemical separation of a heterogeneous crop of cells. The cells in culture may vary greatly. Some are active growing, turgid cells, with thin protoplasts tightly pressed against their walls; in others the protoplasts may spontaneously withdraw from the wall; in still others the protoplasts disorganize, and walls thicken and become sculptured as the cells differentiate and even senesce. Different culturing practices may affect the status of the cells, and this, in turn, affects the distribution of radioactivity from proline in the cells. Cells which are actively growing, turgid, and nucleated have the highest grain density in their protoplasts and nuclei; as the protoplasts of such cells withdraw from their walls, they retain the bulk of the radioactivity. On the other hand, in cells which have thickened walls and sparse protoplast contents, the radioactivity is accumulated in their walls. A high content of proline and hydroxyproline-rich protein is, therefore, not a necessary or invariable feature of the cell walls of cultured Acer cells but depends on the state of development of these cells.     

Synthesis of peptidoglycan in the form of soluble glycan chains by growing protoplasts (autoplasts) of Streptococcus faecalis.

Protoplasts (autoplasts) of Streptococcus faecalis were produced by the action of native autolytic N-acetylmuramidase in the absence of added peptidoglycan hydrolases and were grown in osmotically stabilized medium containing L-[3H]lysine and D-[14C]alanine. To reduce the level of muralytic hydrolysis of glycan chains during growth, heat-inactivated cell walls were added to the medium to bind autolytic enzyme, and tetracycline (1 mug/ml) was added to inhibit further enzyme synthesis. Under these conditions, protoplasts synthesized newly labeled peptidoglycan in the form of soluble, infrequently peptide cross-linked glycan chains which were released into the supernatant medium. These relatively large glycan chains were not transferred to exogenously added cell walls.     

Distribution and effects of 2,4,5-trichlorophenoxyacetic acid in cells of Bacillus megaterium.

Cell death in a resting population of an asporogenous Bacillus megaterium was accelerated by ambient concentrations of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) equal to or greater than 10 mug/ml or 5 mug/mg of cells (dry weight), but only after prolonged exposure. Conversely, populations of growing cells were not markedly influenced even at 100 mug/ml. Effects on cell respiration were not manifest until the ambient concentration reached 1,000 mug of 2,4,5-T/ml, or 500 mug/mg. Cells of B. megaterium did, however, accumulate 2,4,5-T passively to a level approximately twofold above the ambient concentration. Most of the accumulated compound was easily washed from the cells, but, of the firmly bound herbicide, about 0.5 mug/mg of cells (dry weight), nearly 60% by weight, was localized in the protoplast membrane. The foregoing results, obtained with a purified preparation of 2,4,5-T were also elicited by 2,4,5-T analytical standards. The extracted contaminants did not produce the results alone nor did they influence the results when present in combination with 2,4,5-T.     

Protoplast induction in Chlorella pyrenoidosa

Chlorella pyrenoidosa (UTEX 1230) cells in late log phase of growth were induced to form viable protoplasts by enzymatic digestion only when incubated in 2-deoxy-d-glucose (2DG) for 24 h. The combination of hemicellulase (4% w/v), Cellulysin (4% w/v), and glucuronidase (5% v/v) with 0.8 M mannitol and 8 mM CaCl₂ in modified Bristol's solution, was most effective for obtaining viable protoplasts as determined by light and electron microscopy, and vital staining with primuline (0.01% w/v). Resistance of cell walls to extensive extraction (acetolysis), and infrared analysis indicated that sporopollenin is a component of the cell wall. Transmission electron miscroscopy of acetolysed cell walls also allowed visualization of the laminate nature of the wall. This is the first report of successful induction of protoplasts from algae which contain sporopollenin in their cell walls.     

Mechanism of Action of the Fungicide Thiabendazole, 2-(4′-Thiazolyl) Benzimidazole

Thiabendazole, 2-(4'-thiazolyl) benzimidazole (TBZ) inhibited the growth of Penicillium atrovenetum at 8 to 10 mug/ml. Oxygen consumption with exogenous glucose was inhibited at 20 mug/ml, but endogenous respiration required more than 100 mug/ml. TBZ inhibited completely the following systems of isolated heart or fungus mitochondria: reduced nicotinamide adenine dinucleotide oxidase, succinic oxidase, reduced nicotinamide adenine dinucleotide-cytochrome c reductase, and succinic-cytochrome c reductase at concentrations of 10, 167, 10, and 0.5 mug/ml, respectively. Cytochrome c oxidase was not inhibited. Antimycin A and sodium azide caused the usual inhibition patterns for both fungus and heart terminal electron transport systems. In the presence of antimycin, the fungicide inhibited completely succinate-dichloro-phenolindophenol reductase and succinate-2, 2-di-p-nitrophenyl-(3, 3-dimethoxy-4, 4-biphenylene-5, 5-diphenylditetrazolium)-reductase at 2 and 4 mug of TBZ per ml, respectively. Coenzyme Q reductase required 15 mug/ml. TBZ reduced the uptake by P. atrovenetum of glucose and amino acids and decreased the synthesis of various cell components. At 120 mug/ml, the incorporation of labeled carbon from amino acids-U-(14)C was decreased: lipid, 73%; nucleic acids, 80%; protein, 80%; and a residual fraction, 89%. TBZ did not inhibit peptide synthesis in a cell-free protein-synthesizing system from Rhizoctonia solani. Probably the primary site of inhibition is the terminal electron transport system and other effects are secondary.     

Antimicrobial action of silver nitrate

Silver nitrate 3 mug/ml prevented the separation into two daughter cells of sensitive dividing cells of Pseudomonas aeruginosa growing in nutrient broth plus the chemical. Cell size of sensitive cells was increased and the cytoplasmic contents, cytoplasmic membrane and external cell envelope structures were all abnormal. P. aeruginosa cells grown in the presence of silver nitrate 9 mug/ml showed all these changes to a marked degree except inhibition of cell division was not observed. Silver nitrate (1.5 mug/ml) in distilled water inactivated bacteriophage T2 particles as determined by their infectivity to Escherichia coli B cultures. Lysozyme (50 mug/ml) reduced, and sodium chloride (0.9%) blocked this activity.     

Lytic Action of β(1-3)-Glucanase on Yeast Cells

Candida utilis, Saccharomyces cerevisiae, S. fragilis, Pichia polymorpha, and Hansenula anomala yeast cells, harvested in the early logarithmic phase, were attacked with purified beta(1-3)-glucanase from Micromonospora chalcea, which resulted in the liberation of protoplasts. The treated cells were observed under the electron microscope before the protoplasts were liberated. Differences in the cell walls of the enzyme-treated and untreated cells were observed. The action of the glucanase was also tested against isolated walls of C. utilis. The enzyme attacked the S. cerevisiae cell wall in a uniform manner. The attack on S. fragilis was located in certain zones of the cell wall, where breakage occurred and through which the protoplast emerged. On the other three yeasts, an intermediate attack was observed, not as definitely located as in S. fragilis, yet less uniformly than in S. cerevisiae.     

Effect of pyrimethamine and sulfadiazine on the fine structure and multiplication of Toxoplasma gondii in cell cultures.

Rhesus monkey kidney cell cultures were inoculated with Toxoplasma gondii organisms obtained from peritoneal fluid of mice infected with the RH strain. Pyrimethamine and sulfadiazine were added either singly or in combination to the cultures 4 hr after inoculation. Twenty-four hours later the effect of the drugs on the parasites were studied by light and electron microscopy. Pyrimethamine (1.0 mug/ml) inhibited multiplication of the parasites and caused striking morphological changes. Organisms were rounded and often had a fragmented nucleus. Division was inhibited as indicated by abnormal daughter membrane formation during endodyogeny. No effect was evident in sulfadiazine-treated parasites when concentrations up to 50 mug/ml were used. However, combination of ineffective levels of pyrimethamine (0.1 mug/ml) and sulfadiazine (0.5 mug/ml) produced effects similar to those seen at a higher concentration of pyrimethamine indicating a synergistic action of the 2 drugs.     

Fine structure of isolated mesophyll protoplasts of tobacco

Summary Protoplasts of palisade cells isolated enzymatically from mature leaves of tobacco were studied with the electron microscope. A cell wall was completely absent, and the chloroplasts contained large inclusion bodies which were believed to be a crystalline form of fraction I protein. The fine structure of the protoplasts was otherwise that of healthy mesophyll cells, indicating that they are in a good physiological state. Some protoplasts were multinucleate as a result of fusion during the isolation process.     

Structure and association of wall fibrils produced by regenerating tobacco protoplasts

A study has been made of the wall fibrils produced by tobacco protoplasts, using scanning electron microscopy in conjunction with negative staining. It has been shown that the fibres seen in scanning electron microscopy correspond to aggregates of microfibrils. These aggregates are only visible where they are lifted clear of the protoplast surface. Negative staining of fixed protoplasts shows that the aggregation of microfibrils into the fibres visible in scanning electron microscopy is probably produced by air-drying. Gentle disruption of microfibrils produces both random broken fragments and bundles of short pieces of fibrillar material about 60 nm in length. This material is present in undisrupted young walls, but not in undisrupted older walls. The microfibrils in young walls seem much more fragile and liable to breakage than those in older walls. These results are discussed in terms of the interpretation of scanning electron microscope images and the mechanism of cellulose microfibril formation by higher plants.Abbreviations SEM Scanning electron microscopy     

Further evidence for a partially folded intermediate in penicillinase secretion by Bacillus licheniformis.

Protoplasis of Bacillus licheniformis 749/C (a mutant constitutive for penicillinase production) continued to synthesize and release penicillinase in hypertonic growth medium in the presence of trypsin and chymotrypsin at 25 mug each per ml. When the protoplasts were stripped of about half of their membrane-bound penicillinase by pretreatment at pH 9.5 or with a higher level of trypsin, penicillinase activity no longer increased in the presence of the proteases. This effect was immediately eliminated after addition of soybean trypsin inhibitor. These proteases do not significantly inhibit general protein synthesis. Stripped protoplasts of strain 749/C and of uninduced strain 749 (unable to synthesize penicillinase) were incubated with 50 mug of chymotrypsin per ml, and the supernatent fluids were examined immunochemically for peptides derived from the penicillinase chain. Such fargments were found only with the protoplasts capable of synthesizing penicillinase (strain 749/C). The direct detection of the products of protease degradation of a susceptible form of penicillinase provides strong evidence that, in stripped protoplasts of B. licheniformis 749/C, penicillinase synthesis continues in the presence of trypsin or chymotrypsin and that, in these modified membranes, the protease is able to act on an early form of the enzyme that has not yet attained the protease-resistant conformation characteristic of the membrane-bound and exopenicillinases. This finding is discussed in terms of the current models of penicillinase secretion.     

Tobramycin (Nebramycin Factor 6): In Vitro Activity Against Pseudomonas aeruginosa

Tobramycin (factor 6 of the nebramycin complex) is a new aminoglycoside antibiotic isolated from Streptomyces tenebrarius which is active against S. aureus, Enterobacteriaceae, and Pseudomonas aeruginosa. Susceptibility to tobramycin of 96 strains of P. aeruginosa, including 45 recent isolates from blood, was studied by using agar and broth dilution methods. The minimum inhibitory concentration (MIC) for 83 of 96 strains was 3.12 mug/ml or lower in Mueller Hinton agar; MIC values were two to eight times lower in Mueller Hinton broth tests. Agar dilution MIC values were generally lower than those obtained in parallel tests with gentamicin. Killing curves obtained from serial sampling of broth cultures showed a 100- to 10,000-fold decline in viability of log-phase organisms within 30 min of exposure to the drug. Two-dimensional agar dilution tests with carbenicillin and tobramycin with 79 strains showed additive or synergistic effects; no antagonism was documented. Seventy-eight of 79 strains were inhibited by a combination of 50 mug of carbenicillin per ml and 1.56 mug of tobramycin per ml, blood levels which seem attainable in man. Tobramycin appears to be a potent, rapidly bactericidal antibiotic against P. aeruginosa and merits clinical evaluation.     

Growth of Streptococcus mutans protoplasts is not inhibited by penicillin.

A method is described in which cells of Streptococcus mutans BHT can be converted to spherical, osmotically fragile protoplasts. Exponential-phase cells were suspended in a solution containing 0.5 M melezitose, and their cell walls were hydrolyzed with mutanolysin (M-1 enzyme). When the resultant protoplasts were incubated in a chemically defined growth medium containing 0.5 M NH4Cl, the protoplast suspensions increased in turbidity, protein, ribonucleic acid, and deoxyribonucleic acid in a balanced fashion. In the presence of benzylpenicillin (5 microgram/ml), balanced growth of protoplasts was indistinguishable from untreated controls. This absence of inhibition of protoplast growth in the presence of benzylpenicillin was apparently not due to inactivation of the antibiotic. When exponential-phase cells of S. mutans BHT were first exposed to 5 microgram of benzyl-penicillin per ml for 1 h and then converted to protoplasts, these protoplasts were also able to grow in chemically defined, osmotically stabilized medium. The ability of wall-free protoplasts to grow and to synthesize ribonucleic acid and protein in the presence of a relatively high concentration of benzylpenicillin contrasts with the previously reported rapid inhibition of ribonucleic acid and protein synthesis in intact streptococci. These data suggest that this secondary inhibition of ribonucleic acid and protein synthesis in whole cells is due to factors involved with the continued assembly of an intact, insoluble cell wall rather than with earlier stages of peptidoglycan synthesis.     

Steroid Lysis of Protoplasts and Effects of Stabilizers and Steroid Antagonists

Six synthetic antimicrobial steroids were examined for indications of their mechanism of action. Dequadin acetate, cetyl pyridinium chloride (CPC), and sodium deoxycholate were studied for comparison. Aerated cells of Sarcina lutea were washed, suspended in 1.06 M sucrose, and converted to protoplasts with 20 mug/ml of lysozyme. Lysis was measured optically at 650 mmu as a decrease in optical density. Screening tests with 50 mug/ml of each compound showed five steroids and CPC to be lytic. Protoplasts were strongly protected from lysis by pretreatment with 0.001 to 0.004 M spermine tetrahydrochloride. Other polyamines, such as spermidine phosphate, were less protective, and putrescine was ineffective. Uranyl nitrate (5 x 10(-4) M) rapidly agglutinated protoplasts and protected them from rupture by the lytic agents. Similar studies with 0.001 to 0.004 M Mg(++) showed varying degrees of protection, which, in most cases, was only temporary. Steroidal lysis did not appear to be related to chelation, since ethylenediaminetetraacetate did not cause lysis alone and antagonized some lytic compounds. Lecithin, Tween 80, Tween 20, and Span 20 at 0.05% exhibited certain effects on protoplast stability. Span 20 strongly prevented lysis by steroids. Tween 20 alone quickly caused protoplast rupture. Lecithin and Tween 80, which also caused lysis alone, interfered with lytic steroids and CPC. The test compounds were both inhibitory and lethal to cells of Sarcina lutea. The results suggest that direct action on cell membranes may be chiefly responsible for the antimicrobial properties of the steroids.     

Nutrition and metabolism of marine bacteria. XVI. Formation of protoplasts, spheroplasts, and related forms from a gram-negative marine bacterium

When cells of a marine pseudomonad were washed and suspended in 0.5 m sucrose, they retained their rod shape, but thin sections, when examined in an electron microscope, revealed that the outer layer of the cell wall had separated a considerable distance from the cytoplasmic membrane. Treatment of such cells with lysozyme alone produced no obvious change, but treatment with ethylenediaminetetraacetic acid (EDTA) alone caused the outer wall to disappear. A combination of EDTA and lysozyme resulted in the rapid formation of spheres essentially free from hexosamine and indistinguishable from protoplasts of gram-positive bacteria. When cells were washed with 0.5 m NaCl and then suspended in 0.5 m sucrose, they also retained their rod shape, but in this case the outer layer separated from the cells completely and could be recovered from the suspending medium. Such cells were converted to protoplasts by the action of lysozyme alone. Cells washed and finally suspended in 0.5 m NaCl, when treated with EDTA and lysozyme, slowly became spherical. Thin sections revealed typical spheroplasts of gram-negative bacteria in which the outer wall remained intact. Protoplasts took up alpha-aminoisobutyric acid by a Na(+)-dependent process.     

Antimicrobial Actions of Hexachlorophene: Lysis and Fixation of Bacterial Protoplasts

Hexachlorophene was found to be both a lytic and a fixative agent for protoplasts isolated from Bacillus megaterium. Concentrations of 50 to 100 mug of drug per mg of original cell dry weight were required to lyse 4.4 x 10(9) protoplasts (2 mg of original cell dry weight). At higher drug concentrations, protoplasts became fixed against osmotic stress and reduced in sensitivity to disruption by n-butanol. Lower drug concentrations caused proportionate lysis in the protoplast population. Intact cells lost the ability to become plasmolyzed at these same hexachlorophene concentrations. Nonplasmolyzed, drug-treated cells were resistant to the action of lysozyme, whereas plasmolyzed, drug-treated cells were sensitive. But the sensitivity of isolated cell walls to lysozyme digestion was not markedly altered by hexachlorophene treatment. These effects appeared to be secondary in the killing of cells by hexachlorophene because they occurred at concentrations higher than the minimum lethal concentration.     

Ultrastructural and biochemical aspects of cell wall reconstitution in recalcitrant (grapevine) and regenerating (tobacco) leaf protoplasts

Summary To identify possible reasons that may contribute to recalcitrance in plant protoplasts, the time course of new cell wall deposition was studied by scanning electron microscopy in protoplasts of a recalcitrant species, the grapevine. Results showed that microfibrils were developed after 2 days of culture, that complete cell wall formation occurred on Day 6 to 7 of protoplast culture, and its ultrastructural appearance was identical to that of grapevine leaf-derived callus cells. In addition, a comparative study was undertaken on [U-¹⁴C]glucose uptake and incorporation in ethanol-soluble, cellulosic, and noncellulosic polysaccharide fractions in protoplasts of grapevine and of a readily regenerating species, tobacco, during culture. There was a significantly higher [U-¹⁴C]glucose uptake by tobacco than by grapevine protoplasts. The label distribution in the ethanol-soluble, cellulosic, and noncellulosic fractions of newly synthesized cell walls differed quantitatively between the two species. In particular, the labeled glucose incorporated in the noncellulosic cell wall fraction was threefold greater in tobacco than in grapevine protoplasts. Differences were also revealed in the monosaccharide composition of this fraction between the two species. Addition of dimethyl sulfoxide to the culture medium resulted in a dramatic increase in [U-¹⁴C]glucose uptake by grapevine protoplasts, whereas it exhibited a limited effect in tobacco protoplasts. It showed no effect on the ultrastructural characteristics of new cell wall nor on the incorporation rate of labeled glucose in the cellulosic and noncellulosic cell wall fractions.     

Inactivation of bovine herpesvirus-1 and bovine viral diarrhea virus in association with preimplantation bovine embryos using photosensitive agents

Hematoporphyrin (HP), hematoporphyrin derivative (HPD), and thiopyronine (TP) are photosensitive agents (PSA) that have a germicidal effect when they are activated by light: helium neon laser (He Ne ) light (HP, HPD), white light (HP, HPD), and yellow-green light (TP). Experiments were conducted with appropriate controls to determine the effect of photosensitive agents a) for inactivating bovine herpesvirus-1 (BHV-1; titre 10(6) TCID(50) /ml) and bovine viral diarrhea virus (BVDV; titre 10(6) TCID(50) /ml); b) for disinfecting Day-7, zona pellucida-intact (ZP-I) bovine embryos that had been exposed to BHV-1 (titre 10(6) TCID(50) /ml) or BVDV (titre 10(6) TCID(50) /ml); and c) on the in vitro development of embryos. Exposure to HP, HPD and TP followed by light irradiation inactivated BHV-1 and BVDV. Embryos exposed to BHV-I were disinfected by HP or HPD (5 mug/ml) in combination with He Ne light, or by HP or HPD (10 mug/ml) in combination with white light. Embryos exposed to BVDV were disinfected by HPD (5 and 10 mug/ml) followed by He Ne or white light irradiation. Exposure of embryos to light alone or to light and HP or HPD had no detrimental effect on their in vitro development; however, exposure of embryos to TP (5 mug/ml) followed by irradiation caused embryonic degeneration. Exposure of embryos to 5 mug of HPD followed by He Ne light, or 10 mug/ml of HP or HPD, followed by white light, is simple methods of disinfecting them of BHV-I and BVDV.