Bactericidal Substances Research Articles

Identification of a component of crystalline egg albumin bactericidal for thermophilic aerobic sporeformers.

During an investigation of the effect of basic and acidic proteins on the growth of thermophilic aerobic sporeformers, crystalline egg albumin was found to be strongly bactericidal. This finding was uncharacteristic of acidic proteins. The bactericidal fraction was heat sensitive and separated from the non-bactericidal albumin fraction during gel filtration on Sephadex G-75. Cells of Micrococcus lysodeikticus and Bacillus stearothermophilus were lysed rapidly by the bactericidal component, leading to its tentative identification as lysozyme. The bactericidal substance possessed an electrophoretic mobility on polyacrylamide gel containing sodium dodecyl sulfate identical to that of crystalline egg white lysozyme. Users of crystalline egg albumin are cautioned that commerical preparations may be contaminated with lysozyme. Destruction of the thermophilic aerobes by lysozyme should be considered when performing counts on egg products.

Bactericidal substance produced by Haemophilus influenzae b.

During bacteriophage studies on Haemophilus influenzer, it was observed that encapsulated type b and unencapsulated Rb strains released a bactericidal substance acitve against types a, c, d, e, and f H. influenzae, non-typable H. influenzae strains, other Haemophilus species, and certain members of the Enterobacteriaceae. The bactericidal activity was assayed by a plaque test utilizing an Rd strain as an indicator lawn and was also demonstrated in mixed broth cultures of a producer strain and an indicator strain. Immediately lysis of sensitive bacteria by the factor was not evident. The factor is sensitive to trypsin but resistant to deoxyribonuclease, treatment with 2-mercaptoethanol, lipase, alpha-amylase, and heating in a 100 degrees C water bath for 20 min. The activity is not dependent upon increased Ca2+ or Mg2+ concentration as is necessary for HP1C1 and S2 phage propagation. The bactericidal factor is not pelleted by high-speed centrifugation at 150,000 X g for 6 h. Treatment with ultraviolet light or mitomycin C does not result in observable phage, phage-like particles, or increased bactericidal activity. T-HE BACTERICIDAL FACTOR IS NOT A TYPICAL SMALL MOLECULAR WEIGHT "COLICIN-LIKE" BACTERiocin in that it is not inducible, has a wider range of activity, and does not kill by "single-hit" kinetics. On preliminary characterization, it is a thermostable protein toxic to certain bacterial strains.

Identification of a Component of Crystalline Egg Albumin Bactericidal for Thermophilic Aerobic Sporeformers

During an investigation of the effect of basic and acidic proteins on the growth of thermophilic aerobic sporeformers, crystalline egg albumin was found to be strongly bactericidal. This finding was uncharacteristic of acidic proteins. The bactericidal fraction was heat sensitive and separated from the non-bactericidal albumin fraction during gel filtration on Sephadex G-75. Cells of Micrococcus lysodeikticus and Bacillus stearothermophilus were lysed rapidly by the bactericidal component, leading to its tentative identification as lysozyme. The bactericidal substance possessed an electrophoretic mobility on polyacrylamide gel containing sodium dodecyl sulfate identical to that of crystalline egg white lysozyme. Users of crystalline egg albumin are cautioned that commerical preparations may be contaminated with lysozyme. Destruction of the thermophilic aerobes by lysozyme should be considered when performing counts on egg products.

Nature of bacteriocins

The bacteriocins comprise a large, heterogeneous group of bactericidal substances produced by bacteria from widely differing species. The best known bacteriocins are the colicins. Some aspects of research on colicins are discussed especially their chemical nature and mode of action, and their genetic determinants.

Myeloperoxidase-mediated iodination by granulocytes. Intracellular site of operation and some regulating factors.

The intracellular site of operation of the myeloperoxidase-H(2)O(2)-halide antibacterial system of granulocytes has been determined by utilizing measurements of the fixation of iodide to trichloracetic acid (TCA) precipitates of subcellular fractions, including intact phagocytic vesicles. Na(125)I was added to suspensions of guinea pig granulocytes in Krebs-Ringer phosphate buffer, and they were then permitted to phagocytize different particles. Phagocytic vesicles were formed by allowing cells to ingest a paraffin oil emulsion (POE) and collected by flotation on sucrose after homogenization. Measurement of (125)I bound to TCA precipitates of the different fractions and the homogenates disclosed that the lysosome-rich fraction obtained by centrifugation from control (nonphagocytizing) cells accounted for a mean 93.1% of the total cellular activity. With phagocytosis of POE, TCA-precipitable iodination increased two- to sevenfold, and the lysosomal contribution fell to a mean 36.9% of the total. The appearance of activity within phagocytic vesicles accounted for almost the entire increase seen with phagocytosis (a mean 75.7%), and iodide was bound within these structures with high specific activity. More iodide was taken up by cells than fixed, regardless of iodide concentration, and was distributed widely throughout the cell rather than selectively trapped within the vesicles. The amount of iodide taken up and fixed varied considerably with the phagocytic particle employed. Yeast particles were found to stimulate iodination to a far greater degree than the ingestion of POE or latex. Such observations are consistent with the concept that the ingested particle is a major recipient of the iodination process. Measurements of metabolic activities related to the formation and utilization of peroxide by cells phagocytizing different particles were made and correlated with iodination. The findings suggest that mechanisms must exist within granulocytes to collect or perhaps even synthesize H(2)O(2) within phagocytic vesicles to serve as substrate for myeloperoxidase. The simultaneous stimulation of other metabolic pathways for peroxide disposal and its release into the medium by phagocytizing cells is consistent with the high diffusability of this important bactericidal substance.

Studies on a bactericidal substance produced by group A streptococci.

Summary: During bacteriophage studies of group A streptococci, some strains produced an inhibitory substance active against other group A strains. The inhibitory material was not found in broth cultures and could only be extracted from cultures grown in semi-solid agar; its activity was assayed by the inhibition of growth and killing of a sensitive group A strain, 12203. Viable counts showed that 2 h exposure to the factor at 37°C produced greater than 90% inhibition and more than 60% killing of the treated culture. Turbidimetric measurements indicated no gross lysis of the sensitive culture and a dose-response curve demonstrated that approximately two bactericidal factor molecules were sufficient to inactivate a colony-forming unit of strain 12203. The inhibitory factor was of low molecular weight as judged by its diffusibility; it was concentrated by lyophilization and partially purified by gel filtration on G-10 Sephadex. The factor was resistant to proteolytic enzymes, nucleases and lipase as well as reducing and oxidizing conditions. Activity was lost at 60°C, but the factor was stable at ---70°C. The bactericidal substance was stable within the pH range 2 to 12. These properties, as well as the wide range of activity of the bactericidal factor on several Gram-positive and -negative organisms, suggest that it is an inhibitory substance other than a bacteriocin.

Production of bactericidal concentrations of hydrogen peroxide by Streptococcus sanguis

Streptococcus sanguis possess a bacteriocin-like activity against related bacteria and Gram-positive filamentous and diphtheroidal organisms in vitro. This activity was associated with the alpha-haemolysis produced by Strep. sanguis. During aerobic growth conditions, Strep. sanguis liberates hydrogen peroxide. Reduced oxygen tension and the enzymes catalase and peroxidase abolish bacteriocin-like activity, alphahaemolysis and peroxide production. Bacteriocin-like activity of Strep. sanguis is due to liberated peroxide and is identical with alpha-haemolysis. No other haemolytic or bactericidal substances are demonstrated. Variability in sensitivity to the inhibitory action by Strep. sanguis is related to catalase activity of sensitive strains. The potential for a bactericidal activity in vivo by Strep. sanguis due to secretion of hydrogen peroxide is reviewed. Since Strep. sanguis is the dominant organism in early dental plaque, the role of peroxide is discussed in maintaining a balance of the indigenous dental plaque flora.

Distribution of lysosomal enzymes, cationic proteins, and bactericidal substances in subcellular fractions of human polymorphonuclear leukocytes.

Separation of homogenates of human polymorphonuclear leukocytes (PMN) into different fractions by sedimentation in centrifugal fields that ranged from 126 x g to 50,000 x g resulted in a differential distribution of the lysosomal enzymes. Peroxidase, lysozyme, beta-glucuronidase, and acid phosphatase activity were separated from each other. This demonstrates that the lysosomes of human PMN comprise at least three and possibly four physically and chemically different cytoplasmic particles. Proteins which are more cationic than lysozyme and which may be analogous to cationic lysosomal protein of rabbit PMN were associated with lysozyme and beta-glucuronidase rich granules. Antibacterial activity was present in four of the five cell fractions which this work produced. These results are significant because they differ from those obtained with rabbits and because they directly influence future experimental design and interpretation, in attempts to analyze antibacterial, scavenging, and inflammatory capacities of human PMN. Since lysosomes differ physically, biochemically, and morphologically, they may well differ with respect to their function in the PMN.

Metabolic Event Involved in the Bactericidal Activity of Normal Mouse Macrophages

The intracellular bactericidal activity of normal mouse peritoneal macrophages requires a viable, actively metabolizing cell. The killing mechanism is apparently dependent upon an intact respiratory electron chain since inhibition of activity is achieved by anaerobiosis, cyanide, antimycin A, or amytal. By way of contrast, inhibitors of oxidative phosphorylation, glycolysis, the Krebs citric acid cycle, and the phosphogluconate oxidative pathway are without influence on this antibacterial activity. Various dyes and electron acceptors with selected reduction-oxidation values also inhibit killing. Although the bactericidal substance was not identified, the data are consistent with the hypothesis that the primary agent is hydrogen peroxide.

Comparative toxicity of various ozonized olefins to bacteria suspended in air

Air containing olefin vapour was treated with known amounts of ozone simulating natural concentrations. The bactericidal effect of the mixture was tested using microthreads sprayed with washed cultures of Escherichia coli var. communis or Micrococcus albus, aerosol strain. With 20 different olefins a wide range of activity was found, those in which the double bond formed part of a ring being the most bactericidal; petrol vapour was about as active as the average open-chain olefin. The two organisms behaved similarly at the experimental relative humidity of 80%. The estimated amount of bactericidal substance present was only about one hundredth of that required to give the same kill with a ;conventional' air disinfectant; a simple physical explanation is proposed for this enhanced effect.

Bactericidal substance from Staphylococcus aureus. Biological properties.

A bactericidal substance previously isolated from phage type 71 Slaphylococcus aureus has been further identified and characterized. Staphylococci belonging to phage type 71 produce the substance in higher titers than staphylococci lysed by other phages in group II in addition to phage 71. Other staphylococci do not produce the bactericidal substance. The bactericidal substance shares several of the properties of bacteriocins but differs from this group of antibiotic substances in some respects. A combination of ammonium sulfate fractionation and gel filtration on a Sephadex G-100 column resulted in considerable degree of purification of the bactericidal substance. The substance is a previously unrecognized product of S. aureus and is distinct from other extracellular products of this organism.

Effect of Bactericidal Substance from Staphylococcus aureus on Group A Streptococci II. Structural Alterations

Ultrastructural alterations brought about by treatment of a beta-hemolytic streptococcus with a bactericidal substance from Staphylococcus aureus are described and illustrated. The substance causes an early condensation of nucleoid deoxyribonucleic acid (DNA) and a partial loss of ribosomes. These changes are followed by a dissolution of the cell contents resulting in bacterial "ghosts" composed of empty cell wall and capsule. These morphological findings correlate with known biochemical effects of the bactericidal substance on ribonucleic acid degradation and cessation of DNA and protein synthesis.

Effect of Bactericidal Substance from Staphylococcus aureus on Group A Streptococci I. Biochemical Alterations

A bactericidal substance isolated from phage type 71 staphylococci has been studied relative to its mechanism of action on streptococcal cells. The substance exerts its effect best at 37 C. No cell lysis occurs as evidenced by lack of alteration in optical density of susceptible cell suspensions. The bactericidal substance results in immediate cessation of protein and deoxyribonucleic acid synthesis as well as degradation of newly and previously formed ribonucleic acid (RNA). The action on RNA seems to be independent of protein synthesis.

Demonstration of a bactericidal substance against beta-hemolytic streptococci in supernatant fluids of staphylococcal cultures.

Staphylococcal skin isolates belonging to phage type 71 were found to produce a bactericidal substance against some streptococci, pneumococci, and corynebacteria. Fifteen strains of group A streptococci belonging to 13 different M types, group C streptococci, and group D streptococci were uniformly inhibited on solid media and in broth by membrane-filtered supernatant fluids of the staphylococcal broth cultures. Inhibition of group G streptococci and other staphyloccoci was variable, and no inhibition of group B streptococci or of a variety of gram-negative rods was demonstrable. A quantitative variation observed to exist among susceptible organisms was a function of the inoculum size of the inhibited strains. The bactericidal substance could be detected best from 24 to 48 hr after inoculation of the staphylococci in tryptic soy broth or in a dialysate of tryptic soy broth. Little or no bactericidal activity was noted when the organisms were grown in several other liquid media. The bactericidal substance was nondialyzable and could be precipitated with ammonium sulfate. It was heat-stable and its activity was not altered within a pH range of 4.0 to 8.5. Pronase and three times crystallized trypsin totally abolished its activity. The concentrated ammonium sulfate precipitate could be fractionated on a Sephadex G-100 column into several peaks, with the bactericidal activity localized to a single peak.

Bactericidal substances in cytoplasm of leucocytes. An attempt to separate lysozyme by gel filtration.

A substance with a bactericidal effect onEscherichia coli was isolated from the cytoplasm of polymorphonuclear leucocytes. It consists of a mixture of different active substances and therefore a gel filtration using s Sephadex G-100 column was used to separate lysozyme from other active substances. The extract was separated by this procedure into five peaks, out of which peak I. possesed a significant bactericidal activity, peak II. and III. had a weak one, whereas the lysozyme activity was present in peak III., IV. and V.

Bactericidal factors in haemolymph from normal and immune wax moth larvae, Galleria mellonella

Haemolymph bactericidal factors were investigated by gel filtration. Sera from normal and immunized Galleria mellonella larvae contain a bactericidal substance with common properties. Immune serum also contains an active substance not found in normal serum. The nature and significance of the two bactericidal factors is discussed.

Neutralization of beta-lysin and cationic bactericidal agents by acidic phospholipid.

Acidic phospholipids neutralized the antistaphylococcal activity of rabbit, human, and rat serum and purified rabbit beta-lysin as well. Several tissue bactericidal factors and cationic antibiotics were also neutralized. The neutralizing ability of all anionic lipids tested, together with the lack of this ability for the electrostatically neutral phosphatidylcholine, suggested that neutralization resulted from formation of electrostatic complexes between negative charges on the lipids and positive charges on the bactericidal substances. The neutralization of these host defense systems by phospholipids can explain the ability of phospholipids to increase bacterial virulence.

Paper chromatographic analysis by a new test method of bactericidal substances in plant extracts and possible relations of these substances to plant growth inhibitors

1. Es wird eine neue besonders empfindliche Testmethode zum Nachweis baktericider Stoffe in Papier-Chromatogrammen beschrieben. Die Chromatogrammabschnitte werden mit den Testkeimen beimpft, darauf 1 Tag in einer feuchten Kammer aufbewahrt und dann auf Nahragar ausgelegt. Bei Anwesenheit einer baktericiden Substanz bleibt die Bakterienentwicklung an den betreffenden Chromatogrammabschnitten aus oder verzogert sich (Abb.3 und 5). Benutzt man Serratia marcescens als Testkeim, so zeigt sich die Wirkung auch an der ausbleibenden oder verspateten Rosafarbung der Chromatogrammabschnitte (Abb.2). 2. Atherische Extrakte der Knospen von Acer pseudoplatanus sowie der oberirdischen Teile junger Pflanzen von Pisum sativum und Helianthus annuuswurden mit Isopropanol/Wasser/Ammoniak (80:15:5) chromatographiert. Die Papier-Chromatogramme wurden mittels der neuen Methode auf bactericide Stoffe gegenuber Serratia marcescens und Pseudomonas fluorescens, teilweise auch gegenuber Aerobacter aerogenes, getestet. 3. In allen genannten Arten wurden baktericide Stoffe gefunden, vor allem am Start (Acer, Helianthus), und bei Rf 0,6–0,7 (Acer, Pisum). Zusatzlich traten mitunter auch weitere Stoffe auf (Abb. 1, 4, 7und 8). Die baktericiden Stoffe wirkten auf Serratia und Pseudomonas meist im wesentlichen gleich oder doch ahnlich; Aerobacter war in unserem Test weniger empfindlich. 4. Die Chromatogramme enthalten auch wachstumshemmende Stoffe, die durch den Avena-Zylindertest und den Lepidium-Keimungstest nachgewiesen wurden. Diese Hemmstoffe befinden sich vorzugsweise bei Rf 0,6–0,7 in allen drei Arten. Hierbei handelt es sich um den weitverbreiteten sogenannten Inhibitor β; diese Chromatogrammfraktion enthalt nach Literaturangaben fur Acer (Cornforth u. Mitarb., 1965) sowie Pisum (Dorffling, 1967) als physiologisch entscheidenden Stoff d-Abszisin II (Addicott u. Mitarb., 1964). 5. Der baktericide Stoff von Acer und Pisum mit dem Rf 0,6–0,7 und der wachstumshemmende Stoff mit dem gleichen Rf-Wert sind trotz weiterer Ubereinstimmungen (Saurecharakter; eine gewisse Parallelitat im Vorkommen beider Stoffe) nicht identisch, denn synthetisches d,l-Abszisin zeigte in physiologischen Konzentrationen keine Baktericidie. — Die Frage nach einer chemischen Verwandtschaft beider Stoffe sowie weiterer baktericider und wachstumshemmender Stoffe miteinander mus zunachst offenbleiben.

MONOCYTIN, A PROTECTING SUBSTANCE PRODUCED BY MURINE MONOCYTES.

SummarySeveral bactericidal substances were isolated from the supernatant of murine monocytes incubated for 2 hours at 37°C in Eagle's medium in vitro with addition of 5. typhi (Ty2). Smaller quantities of the same substances were liberated from monocytes incubated without addition of bacteria. An in vivo protecting substance has also been isolated. This substance protected mice if injected at least 4 hours before the bacterial inoculum, and the protection lasted for about 10 days, but when administered simultaneously with bacteria or after onset of infection no protection could be achieved. The substance turned out to be a basic protein, labile to acid treatment and trypsin and stable to heating at 100°C for one hour.

The Effect of Different Media on Viability, Metabolism and Phagocytic Activity of White Cells. Production of a Bactericidal Substance in the Course of Phagocytosis in vitro

The Effect of Different Media on Viability, Metabolism and Phagocytic Activity of White Cells. Production of a Bactericidal Substance in the Course of Phagocytosis in vitro