Experimental investigation of preparations for the immunotherapy of staphylococcal infections

A comparative experimental study was carried out of antigenic staphylococcal preparations developed in the USSR and Czechoslovakia for the immune therapy of chronic staphylococcal infection. The efficiency of the preparations was unequivocally confirmed using the rabbit staphylococcal sepsis model. The immunogenicity of tested strains was shown not to always correlate with their virulence. Preparations obtained by means of aqueous extraction from mildly virulent immunogenic strains exhibited greater protective activity than those prepared from highly virulent strains. The PCA phenomenon did not differ significantly provided the tested preparations were administered at doses which ensured equal protective action.     

Sensitivity to polyvalent therapeutic staphylococcal bacteriophage as a supplementary criterion of staphylococcal pathogenicity]

It is recommended to use the capacity of pathogenic staphylococci to be lysed by polyvalent therapeutic staphylococcal bacteriophage in the capacity of an additional simple and accessible criterion of staphylococcus pathogenicity. Of 147 strains of the pathogenic plasmacoagulating staphylococci 101 were lysed by the phage and of 166 nonplasmocoagulating nonpathogenic strains--only 6. This test correlated with the other signs of staphylococcus (lecithinase and hemolytic activity). The simplicity and sufficient specificity of this test permits to use it in any practical laboratory. Polyvalent diagnostic phage can be used on the basis of therapeutic bacteriophage by its additional adaptation to the pathogenic strains of staphylococcus.     

Prevention and immunotherapy of staphylococcal infections with bacterial vaccines

Pathogenesis of staphylococcal infection both local and systemic is associated with many pathogenicity factors, which in foreign literature are called virulence factors of Staphylococcus aureus, which were studied as potential candidates for vaccine development. Much difficulties are related to use of known experimental models, which virtually do not allow to determine direct appropriate effect by survival of animals, as well as to data about absence of correlation between increase of antibody titers in animals and protective effect of studied preparations. Despite the importance of the problem of prevalence and severity of staphylococcal infection and intensive research in order to determine protective components able to protect from infection caused by S. aureus, there are no licensed prophylactic preparations with proven efficacy.     

Transformation of Bacillus subtilis by crude lysates containing staphylococcal plasmid DNA

Crude lysates from staphylococcal strains, containing DNA, were capable of transforming Bacillus subtilis at a rate of 1.68 X 10(-10) - 20.6 X 10(-10) depending on the marker according to which the transformers were selected. In a new host, plasmids showed the same behavior pattern as in the staphylococcus but their spontaneous loss was in all the cases recorded significantly more often.     

Immunocorrecting and protective effect of proteolytic enzymes on antibody formation in mice in staphylococcal infection and antibiotic treatment

The impact of proteolytic enzymes on the humoral immune response, survival rate and mean survival time of mice, infected with S. aureus culture and receiving antibiotics was studied. Infection with staphylococcal suppressed the formation of antibodies to sheep red blood cells. Ampicillin made this immunosuppression even more pronounced, while gentamicin produced practically no effect on the degree of immunosuppression in the infected animals. Proteolytic enzymes terrilytin and terridecase exhibited immunocorrecting properties when used in combination with antibiotics. Terridecase, the immobilized form of the enzyme proved to have the highest activity. In experimental generalized staphylococcal infection all preparations under study produced a protective effect. The maximum effect was noted after the use of ampicillin in combination with terridecase.     

Effect of synthetic muramyl dipeptide derivatives on staphylococcal infection in mice

The work deals with the results of experimental evaluation of the influence of some new modified derivatives of muramyldipeptide (MDP) on the course of staphylococcal infection in mice. The preparations under study were found to produce rapid elimination of bacteria from kidneys and the increase of phagocytic activity of blood macrophages in animals. At the same time MDP and its derivatives stimulated natural killer cells whose activity was inhibited during infection. The dependence between the structure of these compounds and their protective action in staphylococcal infection, as well as the increase of the natural immunity characteristics of the body was followed.     

Protective efficacy of combined administration of the multicomponent vaccine and the immunomodulator myelopid in experimental infections in mice

The influence of myelopid (MP) on the protective activity of polycomponent vaccine VP-4 prepared from the antigens of opportunistic bacteria was studied on experimental infections of mice, caused by Klebsiella pneumoniae, Salmonella typhimurium and Staphylococcus aureus. In staphylococcal and Klebsiella infections the joint administration of vaccine VP-4 and MP produced more pronounced protective effect than each of these preparations, introduced alone. The protective action of vaccine VP-4 was specially enforced by MP in cases of local staphylococcal infection. Recommendations on the joint use of two or more immunomodulating agents are possible only on the basis of the experimental substantiation of their effect in definite infections.     

Effect of some antibiotics on the resistance of white mice to staphylococcal toxin]

Penicillin and streptomycin in experimental staphylococcal intoxication had different effects on the disease depending on the period of their use. Penicillin injections prior to administration of the toxin increased significantly the resistance of the animals to it, while with the use of streptomycin there was only a tendency to its increase. The use of both antibiotics against the background of the intoxication had a reverse effect: there was a tendency to a decreased animal resistance to a greater extent with the use of streptomycin. The use of tetracycline hydrochloride had no effect on the animal resistance to the staphylococcal toxin.     

Biomaterial-associated staphylococcal peritoneal infections in a neutropaenic mouse model

Abstract Adhesion of staphylococcal cells to polyethylene with end point-attached heparin was quantified by bioluminescence. Staphylococcus epidermidis 3380 and the slime-producing S. epidermidis RP12 adhered to the highest extent, and S. lugdunensis 2342 to the least extent. Preincubation of the polymer with dialysis fluid reduced adhesion of S. epidermidis 3380 and RP12 but enhanced that of S. aureus , and preadsorption of the surface with fibronectin decreased subsequent adhesion of S. epidermidis and S. haemolyticus strains. When staphylococci were grown in the presence of a biomaterial their ability to activate peritoneal cells was decreased. The bactericidal activity was impaired, whereas ingestion of opsonized coagulase-negative staphylococci (CNS) strains was unaffected. With S. epidermidis RP12 the presence of biomaterial did not influence either phagocytosis or bactericidal effect of peritoneal cells. After intra-peritoneal challenge with staphylococcal strains, the organ uptake of S. aureus Cowan 1 was increased in normal mice whereas immunosuppressed mice died. CNS strains increased mainly in the peritoneal cavity of immunosuppressed mice. The uptake of bacteria in liver and kidneys was increased with S. epidermidis 3380, S. lugdunensis 2343 and S. schleiferi 667-88. Generally, CNS strains persisted in the peritoneal cavity of both normal and immunosuppressed mice. These data indicate that host defense mechanisms, mainly polymorphonuclear neutrophils, fail to eliminate CNS infections in the peritoneum, and that initial adhesion to an implanted biomaterial may be of lesser importance in the peritoneal cavity than in e.g. catheter-associated infections. There are strain-specific virulence factors of bacteria, and slime producing strains evade the host defense mechanisms more efficiently than non-slime producing strains.     

Factors influencing transduction of genetic determinants of penicillinase activity and pathogenicity in staphylococcus aureua. II. Antiphage activity of acridine derivatives]

Acridine dyes examined earlier (acrichine, acridine orange, proflavine and rivanol) and newly-synthesized preparations (acridines No. No. 37--40) were studied in the capacity of nonspecific agents influencing the lytic cycle in development of staphylococcus phages. Acrichine and acridine No. 37 failed to prevent lysis of the indicator staphylococcus cultures (strains 16/160 and 8325) by bacteriophages; proflavine, rivanol, acridines No. No. 39--40 produced a marked inhibitory effect; acridine orange and acridine No. 38 inhibited the staphylococcus lysis completely. Some preparations could be used to investigate the transduction phenomenon.     

Opsonic effect of fibronectin on staphylococcal phagocytosis by human polymorphonuclear leukocytes: its relative inefficiency in post-phagocytic metabolic activities and in intracellular killing

The binding of 125I-labeled human plasma fibronectin (FN) to two strains of live Staphylococcus aureus (S. aureus) (a coagulase-positive Cowan I and a coagulase-negative Newman D2C) and the opsonic effect of FN on phagocytosis of these bacteria by human polymorphonuclear leukocytes (PMN) have been studied. 125I-FN bound to a similar extent in both staphylococcal strains. The 125I-FN-binding was significantly inhibited by human fibrinogen as well as unlabeled FN. The FN-binding was also reduced markedly by trypsinization of these bacteria, but the extent of its decrease did not correlate with their tryptic susceptibility of protein A and clumping factor. FN enhanced the uptake of these bacteria by PMN. However, its binding had no effect on superoxide anion (O2-) generation. The FN-binding definitely stimulated staphylococcal ingestion and intracellular killing by PMN, but the extent of such promotion was dissimilar between these two strains of bacteria. These results suggest that post-phagocytic metabolic activities as well as intracellular killing of these Staphylococci may also be greatly influenced by FN-unrelated factors as are other bacteria having no FN-receptors.     

Effect of lincomycin and staphylococcal vaccine on the course of experimental staphylococcal sepsis

Therapeutic efficacy of lincomycin used alone and in combination with inactivated staphylococcal vaccine and the effect of these agents on synthesis of antibodies and their content in blood serum were investigated. Lincomycin was shown to inhibit septic processes in the host. After its administration the number of the pathogens in the blood and organs markedly decreased. At the same time, lincomycin lowered antibody synthesis in the lymphoid organs and the content of alpha-antitoxins in blood serum. The use of lincomycin in combination with inactivated staphylococcal vaccine promoted an increase in the number of the antibody forming cells in the spleen and lymph nodes and the content of the antibodies to the staphylococcal alpha-toxin in blood serum of the animals with staphylococcal sepsis.     

Production of monoclonal antibodies to staphylococcal enterotoxin A.

Spleen cells from mice immunized with staphylococcal enterotoxin A were successfully fused with NS-1 mouse myeloma cells. Two of the four clones studied produced monoclonal antibodies to staphylococcal enterotoxin A in growth medium which showed titers of greater than 10(6) to 10(7) when tested by the indirect enzyme-linked immunosorbent assay. These monoclonal antibodies showed reactivity with enterotoxins A and E in the enzyme-linked immunosorbent assay. However, the reactivity was higher with enterotoxin A than with enterotoxin E. Nanogram quantities of crude staphylococcus enterotoxin A from Staphylococcus aureus growth were detected by the monoclonal antibodies in electroimmunoblots via autoradiography.     

Production of a monovalent staphylococcal enterotoxic antiserum type A and its use for typing staphylococcal enterotoxins

A monovalent specific staphylococcal antiserum, type A, was obtained by means of the isolated and purified preparation of type A staphylococcal enterotoxin. This antiserum was proved to be identical to antiserum of the same type, manufactured by Serva Feinbiochemica GmbH & Co. (West Germany). The titer of the newly obtained antiserum in the precipitation test was 1 : 16, and its use allowed one to detect enterotoxin of the above-mentioned type at a concentration of 0.004 mg/ml. The study of 320 staphylococcal strains with the use of this antiserum revealed that 25 strains (7.8%) produced type A enterotoxin.     

Heat stability and species range of purified staphylococcal alpha-toxin

Cooper, Louis Z. (New England Medical Center Hospital, Boston, Mass.), Morton A. Madoff, and Louis Weinstein. Heat stability and species range of purified staphylococcal alpha-toxin. J. Bacteriol. 91:1686-1692. 1966.-Heating of high-titer purified staphylococcal alpha-toxin at 60 and 80 C resulted in a double-sloped curve of inactivation of the hemolytic effect on rabbit erythrocytes. Early inactivation was less at the lower temperature, but activity persisted for a longer time at 80 C. Toxin inactivated at 60 C showed renewed activity when heated briefly at 80 C. A precipitate which formed during heating of alpha-toxin at 60 or 80 C yielded hemolytic activity when resuspended and heated at 80 but not at 60 C. Supernatant fluid of heat-precipitated toxin was heat-labile and did not regain activity when heated at 80 C. The results indicate that the "paradoxical effect" of heating of staphylococcal alpha-toxin is not due to a thermolabile inhibitor, but results from alteration of the toxin molecule to a heat-stable active form. Demonstration of renewed activity by 80 C heating of purified toxin requires potent toxin preparations and brief heating periods. Hemolysis of erythrocytes of several animal species by purified alpha-toxin was generally similar to that produced by impure toxin. Rabbit cells were most susceptible. Human and horse erythrocytes hemolyzed to less than 0.1% of the extent of rabbit cells. Blood cells of other species were intermediate in their response to the lytic effect of alpha-toxin.     

Mechanism of the protective effect of sympathetic denervation on the development of experimental staphylococcal sialadenitis

Membrane potentials (MP) of different cell types of submaxillary glands were studied on rats with intact innervation and after preliminary sympathectomy at varying stages of experimental staphylococcal sialadenitis (2 and 24 h). Two hours and especially 24 hours after priming the rats with intact innervation manifested an abrupt fall in the MP in acinar and ductal cells. Two hours after administration of staphylococcus toxin the MP of salivary gland cells in sympathectomized rats did not differ from normal. Following 24 hours there were far less changes in the MP as compared to those in poisoned rats with intact innervation of the salivary glands.     

Clinical study of the efficacy of staphylococcal anatoxin in relation to the phage group to which the staphylococcal strains--the causative agents of infection--belong]

In comparing the bacteriophage group reference of the strains of pathogenic staphylococci isolated in case of postoperative complications from children given staphylococcus toxoid for prophylactic purpose and from control group it was found that prophylactic vaccinations of staphylococcus toxoid created the most intense immunity against staphylococci of the I bacteriophage group. There was found no significant association between the efficacy of the therapy and bacteriophage reference of staphylococci--the causative agents of the infection.     

医院感染葡萄球菌菌种变迁与耐药性近况

目的：了解近9年来医院感染葡萄球菌菌种的变迁与近3年来葡萄球菌药状况。方法：1993年1月至2001年12月我院传染病科等13科室住院病人的各种标本采用血琼脂培养,所分离的葡萄球菌采用美国DADE公司生产的MICROSCAN WALKAY-40全自动微生物分析仪鉴定到种及其亚种。药敏试验药物有青霉素、苯唑西林、氨苄西林、哌拉西林、阿莫西林／克拉维酸（阿莫仙）、头孢唑啉、头孢噻肟、头孢哌酮／舒巴坦（舒普深）、庆大霉素、复方新诺明、环丙沙星、氧氟沙星、利福平、万古霉素、红霉素、林可霉素、四环素、伊米配能／西司他丁（泰能）共18种。采用液体稀释法测定每株葡萄球菌对受试药物的最小抑菌浓度（MIC）,操作按说明书进行。质控菌ATCC25923。依据新近NCCLS标准判读结果。结果：1993年至1998年分离的葡萄球菌中,金黄色葡萄球菌（金葡萄）占71.43%,凝固酶阴性葡萄球菌（CNS）占28.57%,包括表皮葡萄球菌（表葡菌）、腐生葡萄球菌、里昂葡萄球菌和头状葡萄球菌4种。1999年至2001年分离的424株葡萄球菌中,金葡菌仅占29.01%,CNS增至13种,占70.995,以表葡菌、溶血葡萄球菌、松鼠葡萄球菌为主。近3年来分离的各种葡萄球菌甲氧西林耐药率在73.03%-100%之间,除对舒普深、复方新诺明、利福平和万古霉素较敏感外,对其余抗菌药物的耐药率均超过60％,以金葡菌、溶血葡萄球菌、松鼠葡萄球菌的耐药率为最高。MRS耐药率普遍高于MSS,且均呈多重耐药。5.42%(23/424)菌株万古霉素MIC＞16mg/L,除1株为MSCNS外,其余22株均为MRS。结论：3年来医院感染葡萄球菌菌种构成比发生了显著变化,以CNS为主。对抗菌药物呈多重耐药,部分菌株对万古霉素敏感性降低,应予警惕。     

Diagnostic utilization of hemolytically active exosubstances of certain gram-positive bacteria. I. Detection of staphylococcal hemolysins with prepurified preparations of staphylococcal beta-toxin and CAMP-factor of Streptococcus agalactiae.

The authors have modified the one-plate method for the detection of staphylococcal hemolysins. They recommend to use in this method a prepurified form of staphylococcal beta-toxin and of streptococcal CAMP-factor instead of the exclusively beta-tonin-producing strain of Staphylococcus aureus and instead of the intensively CAMP-test positive Streptococcus agalactiae strain, respectively. The authors determined concurrently staphylococcal hemolysins, using a three-plate method in which alpha-antitoxin was employed, to ensure a better evidence of alpha-toxin. A total of 494 staphylococcal strains were examined by both methods. Of this number, 446 Staphylococcus aureus strains were of diverse host origin and 48 were coagulase-negative staphylococcal strains. On the basis of the various hemolytically active staphylococcal toxins, the authors recommend the suggested modification of the one-plate method for their routine detection.     

Effect of terrilytin on the activity of nonspecific resistance factors during immunization with staphylococcal anatoxin and staphylococcal infection

Terrilytin and immobilized terrilytin enhance the activity and intensity of phagocytosis and increase the concentration of lysozyme in nonimmunized animals. Both preparations increase the production of antibodies to staphylococcal alpha-hemolysin, the titers of beta-lysins, the activity and intensity of the phagocytosis of bacterial cells by peripheral blood leukocytes in animals immunized with staphylococcal toxoid and challenged with live staphylococcal culture. In healthy animals terrilytin and immobilized terrilytin induce an increase in total proteolytic activity and in the activity of alpha-1-antitrypsin and alpha-2-macroglobulin, decreased as the result of staphylococcal infection.