CAMP-reaction among skin isolates obtained from a dog with an acute squamous eczema.

The primary culture of a clinical specimen obtained from a dog with an acute squamous eczema revealed 3 different bacterial cultures. Two of these cultures, a beta-hemolytic Staphylococcus aureus and a group B streptococcal culture, demonstrated synergistic hemolytic activities on this primary culture plate. The group B streptococcus had the serotype surface antigens Ib/c, protein antigen c in its c beta component.

Synergistic hemolytic reactions between staphylococci and Micrococcus lylae.

The primary culture of a clinical specimen obtained from a dog with an acute squamous eczema revealed three different bacterial species which demonstrated synergistic hemolytic activities on sheep blood agar plates. The three cultures were identified as beta-hemolytic Staphylococcus intermedius, as a coagulase-negative staphylococcal species, producing a delta-like hemolysin and as non-hemolytic Micrococcus lylae. The coagulase-negative staphylococcal species as well as M. lylae produced synergistically with beta-hemolytic S. intermedius zones of complete hemolysis. The occurrence of three different synergistically active bacterial species from one clinical specimen might be of clinical significance.

Inhibition of staphylococcal alpha-hemolysis by monoclonal antibodies against oligomer 12 S alpha-toxin.

Oligomer 12 S alpha-toxin as well as 3 S alpha-toxoid of Staphylococcus aureus induced the formation of monoclonal antibodies (mabs). Mabs against the 12 S alpha-toxin could be demonstrated in 31 and those against 3 S alpha-toxoid in 18 of 120 hybrid cell colonies. Each of these mab-preparations reacted with 12 S, 3 S alpha-toxin and 3 S alpha-toxoid. The reactions were more pronounced with the homologous than the heterologous toxin preparations. Mabs against 12 S alpha-toxin inhibited the hemolytic effects of native 3 S alpha-toxin as well or better than the respective polyclonal antisera.

Purification of oligomeric staphylococcal alpha-toxin by affinity chromatography on digitonin-sepharose.

An effective concentration of alpha-toxin from Staphylococcus aureus Wood 46, directly from the culture supernatant, could be achieved by adsorption on digitonin-sepharose and elution with 3 mol/l sodium thiocyanate (NaSCN). The toxin was further purified by gelchromatography. The purified product yielded 1 single protein band upon SDS-polyacrylamide electrophoresis. It was nonhemolytic, but reacted with anti-alpha-toxin under complement fixation. Dialysis against 0.14 mol/l NaCl with hydrophobic amino acids partially reactivated the alpha-hemolytic activity of the toxin. Ultracentrifugal analysis yielded sedimentation coefficients for the purified toxin of approximately 3,7 S when dissolved in 3 mol/l NaSCN and of about 12 S after dialysis against 0.14 mol/l NaCl (Table 1). The spontaneous oligomerization of the alpha-toxin during dialysis against 0.14 mol/l NaCl possibly resulted from a change in configuration induced by its adsorption to digitonin-sepharose.

[Clumping-factor-reactions of Staphylococcus aureus of different origin in plasma- and fibrinogen-preparations(author's transl)]. / "Clumping-Factor"-Reaktionen von Staphylococcus aureus verschiedener Herkunft in Plasma- und Fibrinogenpräparationen.

The clumping-factor (CF) test in microtiter-plates proved suitable for the preliminary identification of Staphylococcus aureus, provided a "susceptible" plasma- or fibrinogen-preparation had been applied (Table 1). Thus, all of 100 S. aureus-cultures from humans gave strongly positive CF-reactions with plasma from humans, rabbits, pigs, cattle, and dogs as well as with fibrinogen-solutions from humans and cattle. Equally, all of 100 S. aureus-cultures from cattle clumped in plasma from pigs, cattle and humans. All of the 50 S. aureus-cultures from dogs were CF-positive in plasma from dogs, humans and horses. Only part of the CF-positive S. aureus-culture reacted with plasma from sheep and goats.

[Lipase and phospholipase C from Staphylococcus aureus of different origin. I. Determination and occurrence (author's transl)]. / Lipase und Phospholipase C von Staphylococcus aureus verschiedener Herkunft.I. Nachweis und Vorkommen.

Lipase and phospholipase C from Staphylococcus aureus of different origin were demonstrated qualitatively by agar diffusion on tributyrin- and lecithin agar. On test media with either 0,3% Na-azide or 0,3% KCN lipase-activity was not inhibited, phospholipase C, on the other hand, completely blocked (Table 1, Fig. 2). In this manner a tentative differentiation was possible between lipase and phospholipase C. For the quantitative determination of lipase the hydrolysis of p-nitrophenyl palmitate proved to be most useful (Fig. 1). S. aureus-cultures of human origin produced more often and more actively lipase and phospholipase C than those from cattle (Table 2).

[Lipase and phospholipase from Staphylococcus aureus of different origin. II. Purification and characterization (author's transl)]. / Lipase und Phospholipase C von Staphylococcus aureus verschiedener Herkunft. II. Reinigung und Charakterisierung.

Lipase and phospholipase C from Staphylococcus aureus could be isolated by gel filtration on Sephacryl S 200 (Fig. 1a, b) and completely separated by refiltration under the same conditions. Isoelectric focusing gave maximal enzyme-activities for lipase at pH 8.6 and 9.5 and for phospholipase C at pH 7.4 (Fig. 2). Thin-layer chromatography revealed that the reaction products in lecithin agar of the phospholipase C-preparations from S. aureus and Bacillus cereus were identical (Table 1).

Release of coagulase from staphylococci.

Immediately after inoculation with coagulase-positive staphylococci the coagulase-activity increased significantly in various culture media. The increase was much higher than the calculated coagulase-activity added with the inoculum (table 1). It appears that this release offers a possibility for the efficient production of coagulase prior to purification.

[Measurement of alpha 2-macroglobulin in human sera by quantitative inhibition of an acid staphylococcal protease (author's transl)]. / Messung von alpha 2-Makroglobulin in Humanseren durch quantitative Hemmung einer sauren Staphylokokken-Protease.

Concentrations of alpha 2-macroglobulin could be determined in the sera of 215 blood donors and 94 patients with various internal diseases by quantitative inhibition of an acid protease from Staphylococcus aureus, M 135 (fig. 1, 2). The determinations agreed closely with those obtained by immunodiffusion (tab. 1, fig. 3). However, the alpha 2-macroglobulin-measurements by the protease method required only 4 h and 40 microliter serum. This procedure would also be suitable for the determination of alpha 2-macroglobulin in sera from experimental and domestic animals.

[Improved method for the demonstration of protein A of Staphylococcus aureus (author's transl)]. / Verbessertes Verfahren zum Nachweis von Protein A bei Staphylococcus aureus.

Protein A (PA) could be extracted completely from Staphylococcus aureus by treatment with concentrated formic acid. This led to the development of a semi-quantitative determination of PA by hemagglutination (fig. 1). The treatment with formic acid yielded PA more effectively than the commonly used extraction by boiling (table 1). It could be conducted directly on a loopfull of staphylococci obtained from blood agar. It required no additional cultivation in a fluid medium. Most suitable for the hemagglutination was a commercial preparation of Rh-positive human erythrocytes, blood group O, loaded with Rh-antibodies from humans. This relatively stable preparation had also a higher susceptibility for PA in the slide-test and served for a better detection of PA-positive staphylococci (table 2).

[Effects of serums and plasmas of various species on a protease of Staphylococcus aureus. Human alpha2-macroglobulin as an inhibitor (author's transl)]. / Wirkungen von Seren und Plasmen verschiedener Spezies auf eine Protease von Staphylococcus aureus. Humanes alpha2-Makroglobulin als Inhibitor.

Serums and plasmas from various animals and man inhibited 85-100% of the activity of a purified protease from Staphylococcus aureus, strain M 135 (table 1). The inhibitory effects of corresponding serums and plasmas were approximately equal. The inhibition in human serum was caused by alpha2-macroglobulin. On the other hand, alpha1-antitrypsin proved to be ineffective (table 2). The complex alpha2-macroglobulin-protease could be separated by vertical polyacrylamide discelectrophoresis. Immunelectrophoretic analysis revealed no changes by the staphylococcal protease neither on alpha2-macroglobulin, nor on other human serum proteins. Only fibrinogen was split into at least 2 components (fig. 1).

[Use of a microtiter-system for the determination of the antibiotic susceptibility of staphylococci with tetrazolium salts (author's transl)]. / Mikrotitrierverfahren zur Bestimmung der antibiotischen Empfindlichkeit bei Staphylokokken mit Tetrazoliumsalzen.

Antibiograms of staphylococci were determined in a microtiter-system (fig. 1) within 4 h. The reactions could be clearly demonstrated by the use of tetrazolium salts. The procedure proved to be particularly useful for quantitative studies.

[Purification of protease from staphylococcus aureus (author's transl)]. / Reinigung einer Protease von Staphylococcus aureus.

380 (80%) of 475 Staphylococcus aureus cultures isolated from humans, cattle and dogs were proteolytically active either on casein or gelatin or both (table 1). Protease-activity could also be demonstrated in experimental body-cavities of rabbits (fig. 1). The enzyme-activity was estimated with azocasein. Protease from S. aureus, M 135 precipitated from the culture supernatant with ammonium sulfate at 65% saturation (table 2). It was purified by 2 filtrations on Ultrogel AcA 44 (fig. 2,3) and subsequent isoelectric focusing between pH 3.5-7.0 (fig. 4). The purified protease yielded only 1 line in the SDS-polyacrylamidegel-electrophoresis, in the gelatin-polyacrylamidegel-electrophoresis and in the double immuno-diffusion test (fig. 5). Its isoelectric point was at pH 4.6, and its highest proteolytic activity between pH 7.5-8.3. The molecular weight was estimated by SDS-polyacrylamidegel-electrophoresis to be near 29.000. The protease-activity was completely inhibited in the presence of EDTA, partially inhibited by Cu2+ and Zn2+ and increased by Mn2+ (table 3).

["Clumping factor"-reactions using staphylococci after their extraction with guanidiniumchloride (author's transl)]. / "Clumping Factor"-Reaktionen mit Guanidiniumchloridextrahierten Staphylokokken

Staphylococci of strain K 807 (ATCC: 31243) have much "clumping factor" (CF) on their surface. Extraction of the staphylococci with 6 M guanidinium chloride, removed all soluble substances, including coagulase, without reduction in CF-activity. The extracted staphylococci proved to be most suitable for the quantitative determination of fibrinogen and fibrin degradation products in a microtiter procedure (fig. 2). The CF-test with staphylococci of strain K 807 was more sensitive compared with the hitherto used strain Newman D2C (table 1). After staining with "Astrazonrot (BBL)" no loss of CF occurred. With the stained staphylococci the CF-reactions became more distinctly visible and gave sharp endpoints.