The role of persistence and virulence factors in microecological changes in a humans

The patterns of persistence and virulence factors expression in the representatives of human microbial biocenosis depends on a complex of the environmental conditions: influence of microbes-symbionts, biotope peculiarities, properties of microorganisms located within eukaryotes. Interactions of symbionts in pairs "indigen-indigen" isolated from mucous membrane of tonsils in healthy persons, did not lead to changes in expression of pathogenic properties. Interinfluence in pairs "pathogen-indigen" and "indigen-indigen", isolated from patients with chronic tonsilitis were accompanied by an increase of anti-lysozyme, hemolytic and lecithovitellase activities. Migration of strains of non-enzymatized gram-negative bacteria (NEYNB) from nasal into tympanic cavity in experimental acute purulent otitis is connected with an earlier increase of their number in the nasal cavity and the expression of anti-lysozyme activity. In acute and chronic pyoderma, expression of ALA is more marked in bacteria from a perifocal damage in contrast to focal damage of normal skin. In conditions of interaction between erythrocytes and staphylococcal clones with different levels of expression of pathogenic factors, differences were observed in dynamics of hemolytic and anti-hemoglobin activities.     

Comparative effects of mono-, di-, tri-, and tetrasulfides derived from plants of the Allium family: redox cycling in vitro and hemolytic activity and Phase 2 enzyme induction in vivo

Epidemiological evidence indicates that a high dietary intake of plants of the Allium family, such as garlic and onions, decreases the risk of cancer in humans. It has been suggested that this effect is due to the ability of the aliphatic mono-, di-, tri-, and tetrasulfides derived from these vegetables to increase tissue activities of Phase 2 detoxification enzymes. In contrast, toxic effects have been recorded in domestic and farm animals after the consumption of garlic or onions, involving oxidative damage to erythrocytes and consequent hemolytic anemia. This effect again has been attributed to the aliphatic sulfides. In the present study, the ability of sulfides derived from garlic and onions to generate "active oxygen" species and cause oxidative damage to erythrocytes in vitro has been compared, together with their ability to cause hemolytic anemia and increase the activity of the Phase 2 enzymes quinone reductase (QR) and glutathione S-transferase (GST) in rats. Monosulfides were without significant effect on any parameter. Di-, tri-, and tetrasulfides generated hydrogen peroxide in the presence of GSH and hemoglobin and caused oxidative damage to erythrocytes in vitro. The activity decreased in the order of tetra- > tri- > disulfide, with the allyl compounds being more potent than the propyl. In vivo, both allyl and propyl tri- and tetrasulfides were powerful hemolytic agents. In contrast, only the allyl sulfides increased the activities of QR and GST; the propyl derivatives were completely without effect. Allyl and propyl tri- and tetrasulfides, thus, may contribute to the toxic effects of Allium vegetables, while only the allyl derivatives are effective in increasing tissue activities of cancer-protective enzymes.     

Conformation-specific antibodies to the alpha chain COOH terminus of hemoglobin A0.

An anti-hemoglobin antiserum obtained from a sheep immunized with human carboxyhemoglobin A0 demonstrated little difference in its reactivity with deoxy- or carboxyhemoglobin A0. However, a subpopulation of this antiserum isolated by synthetic peptide affinity chromatography clearly distinguished between these two hemoglobin species. This subpopulation, designated alpha(129-141) anti-hemoglobin antibodies, represents less than 1% of the total anti-hemoglobin antibodies. They are nonprecipitating by Ouchterlony analysis, and fluorescence-quenching studies demonstrate the interaction of a single antibody binding site per hemoglobin dimer. These antibodies bind preferentially to carboxyhemoglobin with a median affinity constant of 5 X 10(8) M-1 compared to binding to deoxyhemoglobin with a binding affinity of less than 1 X 10(8) M-1. Furthermore, the presence of these antibodies in stoichiometric amounts increases the oxygen affinity of hemoglobin, and thus antibody and oxygen binding to hemoglobin can be considered as a linked function.     

Susceptibility of erythrocytes from several animal species to Vibrio vulnificus hemolysin

The hemolytic activity of Vibrio vulnificus hemolysin (VVH) against erythrocytes from several animal species (sheep, horse, cow, rabbit, chicken) was investigated. VVH was active against erythrocytes from all species, but the amount of VVH causing 50% hemolysis under identical conditions (hemolytic susceptibility to VVH) differed. The degree of 125I-labeled VVH (125I-VVH) binding to each erythrocyte species correlated with the susceptibility of the cells to hemolysis. However, marked differences in the binding ability of 125I-VVH were not observed against liposomes constructed with lipids from each erythrocyte membrane. On the other hand, release of hemoglobin (Hb) differed for each of the erythrocyte species despite administration of approximately the same hemolytic VVH concentration to each species. Furthermore, under hypotonic conditions, the stability of each erythrocyte species varied markedly; the more susceptible the erythrocyte to VVH, the more unstable it was under such conditions. These results, therefore, suggest that the susceptibility of erythrocytes to VVH may be closely associated with the binding ability of VVH and erythrocyte membrane stability.     

In vivo hemolytic activity of group B streptococcus is dependent on erythrocyte-bacteria contact and independent of a carrier molecule

Experiments were performed to determine the interaction between the hemolysin of group B streptococcus (GBS) and sheep erythrocytes. Growing GBS were shown to possess a potent hemolysin at a very early stage of the growth cycle. After separation of the cells from the growth medium, all the hemolytic activity remained with the bacterial cells, and no activity could be detected in the growth medium. When fetal calf serum was added to the media, some soluble activity was detected. This activity, however was completely removed by ultracentrifugation, the hemolytic activity being found solely in the pellet. After the hemolysin had formed, no new protein synthesis was needed to cause hemolysis because the addition of chloramphenicol to cells caused no difference in their hemolytic potential. For proof that no short-lived, soluble factors are produced by the bacteria, bacteria and sheep erythrocytes were incubated in contiguous media, separated by a 0.22-m membrane. No hemolytic activity was detected on the erythrocyte side of the membrane, although high amounts of hemolysin could be extracted from the bacteria. Only when a detergent was added to the growth medium was hemolysis detected from the erythrocytes, showing that extracted hemolysin could indeed pass through the membrane. These results suggest that the hemolysin is attached to the surface of the cell and that contact is needed between the bacteria and erythrocyte to cause lysis. Where soluble activity was detected, it was connected to bacterial fragments.     

The hemolytic activity of six arachnid cationic peptides is affected by the phosphatidylcholine-to-sphingomyelin ratio in lipid bilayers

The hemolytic activity of six cationic amphipathic peptides (Oxki1, Oxki2, Pin1, Pin2, IsCT1 and IsCT2) from arachnids strongly depends on the source of red blood cells. The hemolytic activity of the amphipathic peptides was correlated to the phosphocholine-to-sphingomyelin ratio (PC/SM) content, the potency order of which on mammal erythrocytes ranked as follows Guinea pig>pig>sheep. The spider peptides, Oxki1 and Oxki2, prefer small unilamellar vesicles (SUV) composed of PC, but they could not disrupt SUVs made of SM only. Moreover, the membrane-disrupting activity of the scorpion peptide Pin1 was affected by increasing concentrations of SM. Only the scorpion hemolytic peptide Pin2 was able to disrupt SUVs composed merely of SM at high concentrations. Finally, the short scorpion peptides IsCT1 and IsCT2 seem to tolerate high concentrations of SM in the presence of PC for disruption of SUVs; however, the disrupting activities of IsCT1 and IsCT2 are much lower than that of the other four hemolytic peptides. The hemolytic activity caused by all six cationic peptides in mammalian erythrocytes was positively correlated to increases in temperature and increases in the concentration of benzyl alcohol, a membrane fluidizing agent. It was concluded that the hemolytic activity of the cationic peptides strongly depends on the PC/SM content of mammalian erythrocytes, in which cell membranes with a low PC/SM ratio (i.e., of low fluidity) were less disturbed than membranes with a high PC/SM ratio (i.e., of high fluidity).     

Interactions between peroxiredoxin 2, hemichrome and the erythrocyte membrane

Peroxiredoxin 2 (Prx2) is an abundant antioxidant protein in erythrocytes that protects against hemolytic anemia resulting from hemoglobin oxidation and Heinz body formation. A small fraction of Prx2 is bound to the cell membrane, but the mechanism and relevance of binding are not clear. We have investigated Prx2 interactions with the erythrocyte membrane and oxidized hemoglobin and whether these interactions are dependent on Prx2 redox state. Membrane binding of Prx2 in erythrocytes decreased when the cells were treated with H₂O₂, but studies with purified Prx2 and isolated ghosts showed that the interaction was independent of Prx2 redox state. Hemoglobin oxidation leads to the formation of hemichrome, a denatured form of the protein that binds to Band3 protein in the cell membrane as part of the senescence process and is a precursor of Heinz bodies. Hemichrome competed with Prx2 and decreased Prx2 binding to the membrane, potentially explaining the decreased binding in oxidant-exposed cells. The increased membrane binding of Prx2 seen with increasing intracellular calcium was less sensitive to H₂O₂ or hemichrome, suggesting an alternative mode of binding. Prx2 was also shown to exhibit chaperone-like activity by retarding the precipitation of pre-formed hemichrome. Our results suggest that Prx2, by restricting membrane binding of hemichrome, could impede Band3 clustering and exposure of senescence antigens. This mechanism, plus the observed chaperone activity for oxidized hemoglobin, may help protect against hemolytic anemia.     

Ligand spectrum of hemoglobin activity of methemoglobin-reductase and hemolytic resistance of erythrocytes during chronic exposure to nitrates]

It is found that nitrite-ions formed as a result of biotransformation during long term feeding of calves with sodium and potassium nitrates induce changes in some biochemical parameters of blood, including HS-glutathione content in erythrocytes, acid hemolytic resistance of erythrocytes, activity of NAD-dependent methemoglobin-reductase, correlation of ligand forms of hemoglobin and its total content. It is supposed that the observed changes are of an adaptational character and, as a whole, provide for the optimization of both quantitative and qualitative composition of population of erythroid cells at the expense of erythropoiesis intensification.     

New antibacterial peptide derived from bovine hemoglobin

Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields an intermediate peptide fraction exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis after separation by reversed-phase HPLC. From this fraction a pure peptide was isolated and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). This peptide correspond to the 107-136 fragment of the alpha chain of bovine hemoglobin. The minimum inhibitory concentrations (MIC) towards the four strains and its hemolytic activity towards bovine erythrocytes were determined. A MIC of 38 microM was reported against L. innocua and 76 microM for other various bacterial species. This peptide had no hemolytic activity up to 380 microM concentration.     

Evidence for the production of a novel proteinaceous hemolytic exotoxin by dinoflagellate Alexandrium taylori

We found that a whole cell suspension of Alexandrium taylori, which is toxic to Artemia, causes species-specific hemolysis against mammalian erythrocytes. Among the erythrocytes tested, rabbit and guinea-pig erythrocytes were highly sensitive, but human, sheep, and cattle erythrocytes were insensitive. The cell-free culture supernatant also showed potent hemolytic activity toward rabbit erythrocytes as seen in whole cell suspension. The hemolytic activity in the culture medium gradually increased with increase in cell number during exponential growth phase, and relatively high activity was maintained even after reaching the death phase. These results suggest that the hemolytic substance is actively released into the medium from A. taylori cells rather than simple leakage from ruptured or dead cells, and a part of them are steadily accumulated in the medium during the algal growth. Chemical characterization with ultrafiltration and trypsin-treatment suggested that the hemolytic substance released into the medium is protein-like compound with molecular weight more than 10,000 Da. The ammonium sulfate precipitated fraction obtained from the cell-free supernatant of A. taylori showed cytotoxic effect on HeLa cells as well as the hemolytic activity in a similar concentration range on a protein content basis. Our results suggest that A. taylori produces a novel proteinaceous hemolytic exotoxin.     

alpha-Hemolysin of E. coli]

The activity of alpha-hemolysin increased at the log growth phase in the culture of E. coli P678 Hly+ hemolytic strain; this activity diminished with the change into the stationary phase, and then fell sharply. Replacement of the culture medium in the stationary growth phase by fresh one led to restoration of the hemolytic activity of the culture. The culture fluid separated from the cells at the stationary growth phase produced an inhibitory action on the alpha-hemolysin Ca ions activated and stabilized the alpha-hemolysin. Sodium citrate and sucrose served as hemolysis inhibitors. The action of alpha-hemolysin was maximal against human erythrocytes at pH 6.5. Hemolytic activity was characterized in time by a distinct lag-phase and the phase of the greatest rate of reaction. The duration of the lag-phase and also the rate of hemolysis depended on the concentration of alpha-hemolysin (with the increase of the hemolysin concentration lag-phase was shortened and the reaction was accelerated). There proved to be a linear relationship between the amount of erythrocytes taken into the reaction and the rate of hemoglobin release, and also there was noted a temperature activation of the hemolytic reaction.     

Kinetics of erythrocyte lysis by snake venom cardiotoxins

Hemolysis of guinea pig erythrocytes by snake venom cardiotoxins was investigated with a semi-automatic method based on light-scattering changes of erythrocyte suspensions at 700 nm which are directly related to hemoglobin release. Small amounts of phospholipase-free cardiotoxin (<100 μg) could be conveniently and rapidly assayed with the high reproducibility in a recording spectrophotometer, and reliable kinetic data were accumulated.Cardiotoxins from two different genera (Hemachatus haemachates and Naja mossambica mossambica) displayed virtually identical hemolytic properties. Hemolysis increased linearly with time, in contrast with a sigmoidal pattern when phospholipase was present as an impurity. Low concentrations of Ca²⁺ (<1 mM) stimulated cardiotoxin action. A limiting plateau rate of hemolysis reached during concentration dependence experiments in which the level of either cardiotoxin or of erythrocytes was varied, suggested that the interaction of cardiotoxin with erythrocyte membranes is a saturation phenomenon only at a high ratio of cardiotoxin: erythrocytes. No hemolysis was observed with an homologous neurotoxin of S-methylated cardiotoxin, providing evidence for specificity. The linear Arrhenius plots obtained for the temperature dependence of cardiotoxin-induced hemolysis strengthened the conclusion that its action involves more than a detergent-like effect on membrane phospholipids.     

Simultaneous occurrence of hypercholesterolemia and hemolytic anemia in rats fed cholesterol diet

Cholesterol diet-induced hemolytic anemia in rats was described. When rats were fed a cholesterol diet for 11 weeks, serum cholesterol rapidly increased within the first week, and was maintained in 5-10 times higher levels throughout the study as compared to those of control rats. Erythrocyte count, hematocrit and hemoglobin concentration decreased from about 2 weeks of feeding. The spleen showed an increase of hemosiderin deposition from 6 weeks of feeding. The half life of erythrocytes labelled with 51Cr was shortened significantly at 6 weeks of feeding. These findings indicate that cholesterol diet can induce hemolytic anemia. Serum cholesterol and phospholipid were markedly increased, but in erythrocyte membrane, free cholesterol content was not persistently increased and phospholipid content was decreased. In hemorrheological studies, erythrocyte deformability and mechanical hemolysis tended to reduce. In conclusion, it was considered that as a result of reduced phospholipid content the erythrocytes of cholesterol-fed rats were decreased in its deformability and were captured more easily by the spleen. The profile of hemolytic anemia in cholesterol-fed rats was quite different from those reported in cholesterol-fed guinea pigs, rabbits and dogs.     

Factors influencing variable oxidative hemolysis of inbred mouse erythrocytes

The hemolysis of erythrocytes from certain inbred mouse strains (e.g., BALB/c) in response to hydrogen peroxide stress has been shown to be correlated with the type of hemoglobin beta chain (Kruckeberg, W.C., et al. (1987) Blood 70, 909-914). The characteristic hemolytic response of BALB/c red cells to oxidative stress resembles that of human red cells in that carbon monoxide and iron chelators inhibit hemolysis of both. Gross hemoglobin oxidation rates were similar in hemolytic (BALB/c) and nonhemolytic (C57BL/6) strains. The rate and degree of in vitro catalase inhibition by sodium azide was also the same for the two strains. Even in the presence of this catalase inhibitor the assayable hydrogen peroxide disappeared within seconds of its addition, yet hemolysis was not observed for about 15 min. The mechanism underlying this delay between hydrogen peroxide addition and disappearance and subsequent hemolysis is under investigation.     

Isolation and characterization of light-dependent hemolytic cytotoxin from harmful red tide phytoplankton Chattonella marina

Chattonella marina (C. marina), a raphidophycean flagellate, is a causative organism of red tide, and highly toxic to fish. In this study, we found that the cell-free methanol extract prepared from this flagellate exhibited potent hemolytic activity against rabbit erythrocytes. Interestingly, the hemolytic activity of the extract was absolutely light-dependent, and no hemolytic activity was detected in the dark even at very high concentration. Gel filtration chromatography of the methanol extract on a column of Sephadex LH-20 revealed that the extract contained hemagglutinin as well as hemolytic agents, and the substances responsible for these activities were separately eluted. These results suggest that the hemagglutinating and hemolytic activities were derived from distinct compounds. The hemolytic fraction obtained after gel filtration (F4) caused marked inhibition of the growth of C. marina itself and other species of phytoplanktons. Furthermore, F4 showed a potent cytotoxicity toward various mammalian cultured cell lines including human tumor cells (HeLa cells) in a dose-dependent manner. The cytotoxicity was also light-dependent, and no cytotoxic effect was exhibited in any cell lines tested in the dark. After further purification procedures via preparative thin-layer chromatography and subsequent HPLC, a major hemolytic agent was obtained as highly purified form. Since the methanol extracts prepared from other raphidophycean flagellates such as Heterosigma akashiwo, Olisthodiscus luteus, and Fibrocapsa japonica showed light-dependent hemolytic activity toward rabbit erythrocytes, it was suggested that the light-dependent hemolytic agents commonly exist at least in these raphidophycean flagellates.     

HEMOLYTIC ACTIVITY OF HETEROCAPSA CIRCULARISQUAMA (DINOPHYCEAE) AND ITS POSSIBLE INVOLVEMENT IN SHELLFISH TOXICITY

Heterocapsa circularisquama Horiguchi is lethal to shellfish, particularly bivalves such as pearl oysters ( Pinctada fucata Gould). No detrimental effects of this flagellate on fish have been observed thus far. In this study, we found that H. circularisquama causes mammalian erythrocytes to lyse. Among the erythrocytes tested, rabbit erythrocytes showed the highest susceptibility, whereas erythrocytes from cattle, sheep, and human were relatively insensitive. Heterocapsa triquetra Stein, which is morphologically similar to H. circularisquama but not toxic to bivalves, showed no hemolytic activity toward rabbit erythrocytes. Culture supernatant or ultrasonic-ruptured cells of H. circularisquama showed only weak hemolytic activity. Hemolytic activity was found in the ethanol extract of H. circularisquama cells, suggesting that the hemolytic agents may be more stable in ethanol than in aqueous solution. Both an intact flagellate cell suspension and the ethanol extract caused morphological changes and eventual collapse of unfertilized eggs of Pacific oyster. Furthermore, the ethanol extract was lethal to the microzooplankton rotifer Brachionus plicatilis Müller, which is highly sensitive to H. circularisquama. Our results suggest that a hemolytic toxin produced by H. circularisquama may be one of the causative agents responsible for the shellfish toxicity.     

Hemolysis of human erythrocytes induced by tamoxifen is related to disruption of membrane structure

Tamoxifen (TAM), the antiestrogenic drug most widely prescribed in the chemotherapy of breast cancer, induces changes in normal discoid shape of erythrocytes and hemolytic anemia. This work evaluates the effects of TAM on isolated human erythrocytes, attempting to identify the underlying mechanisms on TAM-induced hemolytic anemia and the involvement of biomembranes in its cytostatic action mechanisms. TAM induces hemolysis of erythrocytes as a function of concentration. The extension of hemolysis is variable with erythrocyte samples, but 12.5 microM TAM induces total hemolysis of all tested suspensions. Despite inducing extensive erythrocyte lysis, TAM does not shift the osmotic fragility curves of erythrocytes. The hemolytic effect of TAM is prevented by low concentrations of alpha-tocopherol (alpha-T) and alpha-tocopherol acetate (alpha-TAc) (inactivated functional hydroxyl) indicating that TAM-induced hemolysis is not related to oxidative membrane damage. This was further evidenced by absence of oxygen consumption and hemoglobin oxidation both determined in parallel with TAM-induced hemolysis. Furthermore, it was observed that TAM inhibits the peroxidation of human erythrocytes induced by AAPH, thus ruling out TAM-induced cell oxidative stress. Hemolysis caused by TAM was not preceded by the leakage of K(+) from the cells, also excluding a colloid-osmotic type mechanism of hemolysis, according to the effects on osmotic fragility curves. However, TAM induces release of peripheral proteins of membrane-cytoskeleton and cytosol proteins essentially bound to band 3. Either alpha-T or alpha-TAc increases membrane packing and prevents TAM partition into model membranes. These effects suggest that the protection from hemolysis by tocopherols is related to a decreased TAM incorporation in condensed membranes and the structural damage of the erythrocyte membrane is consequently avoided. Therefore, TAM-induced hemolysis results from a structural perturbation of red cell membrane, leading to changes in the framework of the erythrocyte membrane and its cytoskeleton caused by its high partition in the membrane. These defects explain the abnormal erythrocyte shape and decreased mechanical stability promoted by TAM, resulting in hemolytic anemia. Additionally, since membrane leakage is a final stage of cytotoxicity, the disruption of the structural characteristics of biomembranes by TAM may contribute to the multiple mechanisms of its anticancer action.     

Spontaneous lytic activity against heterologous erythrocytes in cotton rat (Sigmodon hispidus) serum

Although innate immunity has been well studied in laboratory animal models, no such documentation exists for wild species possessing a diversity of physiological adaptations to their environment. We examined the blood sera of 188 hispid cotton rats (Sigmodon hispidus) for naturally occurring hemolytic activity against heterologous erythrocytes. Ninety-two percent of the blood sera samples from cotton rats lysed sheep erythrocytes. All sera tested against chicken erythrocytes showed hemolytic activity, while only 44% of the same sera could lyse bovine erythrocytes. No hemolytic activity was present in cotton rat sera against erythrocytes from other rodent species (Eastern woodrat, Neotoma floridana, and pine vole, Microtus pinetorum). Hemolytic activity was heat labile and appeared to be mediated through the classical complement pathway. The protective nature of this hemolytic factor is unclear but it is probably directed at a more relevant molecule. These data, along with other reports of naturally occurring target specific serum factors in the cotton rat, may reflect the importance of innate protective mechanisms to small mammal populations.     

Studies on the factors affecting the growth and hemolytic activity of <Emphasis Type="Italic">Anabaena variabilis</Emphasis>

The hemolytic activity of the cell-free culture supernatant of Anabaena variabilis OL S1 was investigated using the hemolysis of rabbit erythrocytes as an assay. The culture medium of A. variabilis started to exhibit hemolytic activity at the late exponential growth phase, and maximized at the stationary phase. The hemolytic toxin is heat-stable and can be extracted in dichloromethane. The hemolytic activities under different temperature, light intensity and pH showed a high correlation with the cell densities (r=0.965, 0.951, 0.865, respectively), and the optimum condition is 28~30°C, pH 7.5~8.0, light intensity 120 μmol photons m⁻²s⁻¹. The addition of 10~20 μg mL⁻¹ chloramphenicol, an inhibitor of protein synthesis, exhibited no marked suppression on the hemolytic activity. The supplement of 1~20 μg mL⁻¹ glycerol increased the hemolytic activity significantly, suggesting that synthesis of hemolysin was dependent on carbohydrate and lipid metabolism. The spectrum of erythrocyte sensitivity to the hemolysin indicated that rabbit erythrocytes were more sensitive to the hemolysin than were rat and human erythrocytes. Goldfish and cat erythrocytes were, however, insensitive to the hemolytic toxin of A. variabilis.     

Hemolytic activity of Serratia marcescens

A cell-bound hemolytic activity was found in several strains of Serratia marcescens. One Serratia cell per ten erythrocytes was sufficient to cause complete lysis of human erythrocytes within 2 h in the liquid assay. The hemolytic activity resided in the membrane fraction and could be inactivated by incubating cells with proteases. The hemolytic activity was greatly enhanced in actively metabolizing Serratia cells and was partially controlled by the iron supply. Hemolysis was accompanied by degradation of erythrocyte membrane proteins (band 3 and 6, glycophorin) and was independent of the blood group. The exoprotease secreted by S. marcescens in large amounts was not involved in hemolysis. Comparison with various hemolytic strains of Escherichia coli showed that hemolysis of erythrocytes was more pronounced with S. marcescens than with E. coli. In contrast to hemolysis by E. coli, lysis of erythrocytes by S. marcescens was not enhanced by Ca²⁺ ions.Dedicated to Professor Dr. Gerhart Drews on the occasion of his 60th birthday