Biological and Immunological Properties of Encapsulated Strains of Staphylococcus aureus from Human Sources

Of 875 strains of Staphylococcus aureus isolated from human source clinical specimens, 37 (4.2%) were encapsulated strains. These were all negative for clumping factor and could not be typed with bacteriophages or by serology. Twenty-one of these did not produce any hemolysins, 15 produced alpha hemolysin, 1 produced beta hemolysin, and 1 produced both beta and delta hemolysins. After one or two subcultures, 27 of the encapsulated strains converted to the compact variant form, all became positive for clumping factor, 12 became phage-typable, and 24 became sero-typable. In addition, 7 strains converted from negative to alpha hemolysin production. Comparison of phage- and sero-types did not reveal any relationships. Immunologically, mice challenged with heat-killed encapsulated strains were protected against a challenge infection with the Smith diffuse strain. Protective antibodies in rabbit anti-Smith diffuse strain antisera were removed by adsorption using the encapsulated organisms isolated in this study. The adsorbed sera no longer protected against challenge infection in mice with the Smith diffuse strain. From these results, it appears that the encapsulated strains isolated were immunologically and biologically similar to the classical Smith diffuse strain.     

Passive protection by human serum in mice infected with encapsulated Staphylococcus aureus.

The occurrence and nature of passive protective antibody in 100 samples of human serum was investigated in mice challenged with strains of Staphylococcus aureus capsular types A (Smith diffuse strain) and B (strain NS58D). Sixty of the sera passively protected mice against the capsular type-A strain, three against type B, and one against both types. Rabbit antisera against human IgG, IgA and IgM could remove the protective activity from a human serum of high potency, and the activity was also sensitive to 2-mercaptoethanol. Absorption with Smith surface antigen removed protective activity and reduced the concentration of IgG 7-fold, IgA 2.7 fold and of IgM 3-fold more than in a non-protective serum. Consequently, the protective activity of human serum is believed to be associated with antibodies to the S. aureus capsular antigen in the three immunoglobulin classes.     

Detection of Capsular Antigen Production in Unencapsulated Strains of Staphylococcus aureus

Of 91 compact-type strains of Staphylococcus aureus in regular serum soft agar (SSA), 82 converted diffuse-type growth in serum soft agar (pH adjusted to 6.0). With the addition of four different rabbit anticapsular sera (anti-type A, B, C, and D sera) in low pH (6.0) SSA, 21 strains of S. aureus showed compact-type colonial morphologies. Eleven, one, and one strains of S. aureus reacted singly with rabbit anticapsular sera types A, B, and C, respectively, and no strain reacted with rabbit anticapsular type D. Eight of the e strains reacted with both rabbit anticapsular sera types A and B. When the ability to absorb the converting activities of the antisera (changes of colonial morphologies of anticapsular sera in SSA) was quantitatively tested, 7- to 27-fold of these organisms were capable of absorbing the activities compared with the Smith diffuse organisms. These results suggest that even unencapsulated S. aureus strains are capable of producing capsular substance, although the capability is quantitatively different from strain to strain.     

Protective efficacy of protein A-specific antibody against bacteremic infection due to Staphylococcus aureus in an infant rat model.

Staphylococcal protein A (SpA) is a potent antiphagocytic component of the cell wall of most pathogenic Staphylococcus aureus strains. We studied the in vitro opsonophagocytic and in vivo protective activities of rabbit immunoglobulin G (IgG) antibody to purified SpA obtained from two unencapsulated S. aureus strains (Cowan I and 17A). Postimmune serum contained high titers of specific IgG to SpA, as measured by a modified enzyme-linked immunosorbent assay that blocked nonspecific binding of IgG to SpA. In vitro, both S. aureus strains were efficiently phagocytosed and killed by polymorphonuclear leukocytes in the presence of nonimmune sera and complement. With one strain (Cowan I), opsonophagocytosis was significantly enhanced in the presence of SpA antibody, but with the other strain (17A), killing was significantly decreased with immune serum. We then evaluated the potential protective benefit of SpA antibody in preventing S. aureus bacteremia in infant rats. Two-day-old rats received saline or various doses of SpA antiserum and were challenged subcutaneously 1 day later, but even the highest levels of antibody did not significantly reduce mortality, bacteremia or metastatic infection to lungs or liver (frequency or magnitude). This lack of protective efficacy was not related to a failure of SpA F(ab')2 to bind to cell surface-exposed epitopes, since F(ab')2 fragments prepared from hyperimmune serum bound avidly to the whole organism in an enzyme-linked immunosorbent assay.     

Comparative opsonic and protective activities of Staphylococcus aureus conjugate vaccines containing native or deacetylated Staphylococcal Poly-N-acetyl-beta-(1-6)-glucosamine

Staphylococcus aureus and Staphylococcus epidermidis both synthesize the surface polysaccharide poly-N-acetyl-beta-(1-6)-glucosamine (PNAG), which is produced in vitro with a high level (>90%) of the amino groups substituted by acetate. Here, we examined the role of the acetate substituents of PNAG in generating opsonic and protective antibodies. PNAG and a deacetylated form of the antigen (dPNAG; 15% acetylation) were conjugated to the carrier protein diphtheria toxoid (DT) and used to immunize animals. Mice responded in a dose-dependent fashion to both conjugate vaccines, with maximum antibody titers observed at the highest dose and 4 weeks after the last of three weekly immunizations. PNAG-DT and dPNAG-DT vaccines were also very immunogenic in rabbits. Antibodies raised to the conjugate vaccines in rabbits mediated the opsonic killing of various staphylococcal strains, but the specificity of the opsonic killing was primarily to dPNAG, as this antigen inhibited the killing of S. aureus strains by both PNAG- and dPNAG-specific antibodies. Passive immunization of mice with anti-dPNAG-DT rabbit sera showed significant levels of clearance of S. aureus from the blood (54 to 91%) compared to control mice immunized with normal rabbit sera, whereas PNAG-specific antibodies were ineffective at clearing S. aureus. Passive immunization of mice with a goat antiserum raised to the dPNAG-DT vaccine protected against a lethal dose of three different S. aureus strains. Overall, these data show that immunization of animals with a conjugate vaccine of dPNAG elicit antibodies that mediated opsonic killing and protected against S. aureus infection, including capsular polysaccharide types 5 and 8 and an untypable strain.     

Detection of encapsulation in Staphylococcus aureus by use of antiserum agar

We examined an antiserum agar method to study its reliability in screening Staphylococcus aureus strains for capsule production. The encapsulated S. aureus Smith diffuse strain was compared with its nonencapsulated variant, Smith compact, in CCY medium containing 0.5% NaCl and 5.0% Smith diffuse rabbit antiserum. A halo was visible surrounding colonies of the Smith diffuse strain but not the Smith compact strain. On this same medium, the protein A-producing Cowan I strain possessed a halo that was visible on photographs. Single high-salt medium is known to inhibit protein A production, halo formation by the strains was also compared in 7.5% NaCl medium. The halo surrounding the Cowan I strain was not present when the salt content of the medium was increased. In contrast, the halo surrounding the Smith diffuse strain persisted in the 7.5% NaCl medium. By use of this medium, the antiserum agar technique may be valuable for the identification of encapsulated staphylococci without appreciable interference from protein A.     

Fingerprinting methicillin-resistant Staphylococcus aureus by the immunoblot technique

A series of 133 isolates of methicillin-resistant Staphylococcus aureus was fingerprinted by the immunoblot technique. Extracts were prepared by lysostaphin degradation of overnight cultures and peptides were separated by SDS polyacrylamide gel electrophoresis. The peptides were transblotted on to nitrocellulose membranes and probed with (1) a hyperimmune rabbit serum raised against a methicillin-resistant S. aureus isolate, (2) a hyperimmune rabbit serum raised against an isolate of S. epidermidis, and (3) serum from a patient who had recovered from an infection with a methicillin-resistant S. aureus. This typing method confirmed the existence of an epidemic strain that accounted for 102 of the isolates. The remaining 31 isolates were grouped into a further seven types which correlated with the results of phage typing and antibiograms.     

Morphological examination of the glycocalyces of Staphylococcus aureus strains Wiley and Smith.

The glycocalyces of gram-positive bacteria have only been studied to a limited extent, with most studies being directed at the elucidation of capsules. With modern methods of electron microscopy, it has been shown that an extensive, diffuse polyanionic matrix surrounds Staphylococcus aureus cells of the Smith and Wiley strains, both in vivo and in modified staphylococcus 110 media. This slime layer was extracapsular in the case of the Smith strain, yet appeared to be the only layer peripheral to the teichoic acid in the Wiley strain. It is proposed that these glycocalyces serve a protective function and that their production is induced not only by excess nutrients in the growth medium but also by metabolic stress.     

Mouse protection assay for group B streptococcus type III.

The mucin model for group B Streptococcus (GBS) type III was used to assay the protective effect of sera against a type III challenge in mice. Hyperimmune rabbit sera, prepared by the Lancefield method against the laboratory reference strain (SS620) and a clinical isolate (M732), protected against a lethal challenge with either strain of GBS type III. Absorption of the sera with either of these type III strains removed the protective effect. Neither normal rabbit sera nor heterologous antisera (anti-Ia, SS615) provided protection; however, protection was obtained with pooled human gamma globulin. Sera from adult volunteers were tested to assay protective levels in the mouse model. Human sera enhanced the mouse lethality of the clinical isolate, M732, but not the laboratory reference strain, SS620. Sera from adults vaccinated with type III polysaccharide of GBS were also tested. The murine-mucin-GBS model may be developed as a screening test to measure protective antibody levels in the pre- and postvaccine treatment period. The model may also be used to measure protective antibody in pooled human gamma globulin for use in the passive immunization of high-risk individuals.     

Passive immunization with antiserum to a nontoxic alpha-toxin mutant from Staphylococcus aureus is protective in a murine model.

A nonhemolytic, nonlethal variant of Staphylococcus aureus alpha-toxin constructed via oligonucleotide-directed mutagenesis and containing a single amino acid substitution (H-35 to L) was used to immunize a rabbit. The resulting antiserum was cross-reactive with wild-type alpha-toxin and neutralized its hemolytic activity in vitro. Passive immunization of mice with rabbit antiserum conferred protection against lethal challenge with wild-type alpha-toxin and against acute lethal challenge with a high-alpha-toxin -producing S. aureus strain. H35L alpha-toxin may be useful as a protective immunogen in S. aureus vaccine studies.     

Demonstration of Serologically Different Capsular Types Among Strains of Staphylococcus aureus by the Serum-Soft Agar Technique

Colonies of Staphylococcus aureus exhibiting diffuse-type growth in regular serum-soft agar containing 7.5% sodium chloride were isolated. After isolation, further identification of the encapsulated strains of S. aureus was performed. With this procedure, 19 encapsulated strains were obtained from 103 clinical specimens (18.4%). With these strains, three serologically distinct diffuse types of organisms were observed by the conversion of diffuse to compact type colonial morphology in serum-soft agar containing specific antidiffuse sera. Capsule-inhibiting activity of antisera was adsorbable with homologous encapsulated organisms and not adsorbed with either heterologous encapsulated organisms nor the derived compact variant, suggesting a specific activity for the antispecific capsular antibody. Fourteen strains were similar to the Smith diffuse-type strain, four strains were the same as NS58D, and one was identical to NS41D. These were provisionally designated as capsule types A, B, and C, respectively.     

Protective Opsonic Activity of Antibodies against Fibronectin-Binding Proteins (FnBPs) of Staphylococcus aureus

In this report, opsonic activity of hyperimmune rabbit IgG against fibronectin-binding proteins (gal-FnBP A and ZZ-FnBP B) of Staphylococcus aureus is described. Moreover, the action of IgG purified from serum of rabbits immunized with 'combined vaccine' (fibronectin-binding protein A + collagen-binding protein 4-α-toxoid) is shown. The opsonic activity has been studied in an in vitro phagocytosis assay as well as in vivo . Mice which had been infected inlraperitoncally with S. aureus strain Cowan l pretreated (opsonized in vitro ) with specific anti-FnBPs IgG were able to eliminate the staphylococci from the peritoneal cavity and liver more rapidly than controls. Also, clearance from the bloodstream of intravenously injected 5. aureus Cowan l as well as S. aureus U320, opsonized with IgG anti-FnBPs or anti-FnBP+CnBP +α-toxoid. was more effective than observed in control groups. In other in vivo experiments it was shown that mice passively immunized with hyperimmune IgG anti-FnBP (one or two doses, intravenously) before challenge with S. aureus Cov/an l eliminated the bacteria belter than controls injected only with preimmune IgG.     

Influence of Staphylococcus aureus antibody on experimental endocarditis in rabbits.

To evaluate the potential protective benefit of antibody to whole cells of Staphylococcal aureus for the prevention of endocarditis, the rabbit endocarditis model was used. Methicillin-sensitive (17A) and methicillin-resistant (173) S. aureus strains were evaluated in rabbits with or without indwelling intracardiac catheters. All immunized rabbits developed significant homologous agglutinating antibody titers (the mean reciprocal titers were 15,300 to strain 17A and 1,150 to strain 173). After challenge, virtually no significant differences were observed between immunized and unimmunized animals with respect to (i) incidence of endocarditis, (ii) concentration of bacteria in infected vegetations, (iii) incidence of metastatic renal abscesses, or (iv) concentrations of bacteria in infected kidneys. The clearance of homologous S. aureus strains from blood cultures was similar for immunized and unimmunized animals at 10 to 90 min after intravenous challenge. In vivo adherence of homologous S. aureus strains to aortic valves and vegetations was similar in immunized and unimmunized animals when evaluated at 30 and 90 min postchallenge. Even without catheterization, the incidence of bacteremia and renal abscesses was the same in immunized and unimmunized rabbits. Whole-cell-induced S. aureus antibody did not prevent or modify any stage in the development of endocarditis in rabbits.     

Antibodies to capsular polysaccharides are not protective against experimental Staphylococcus aureus endocarditis.

The protective efficacy of antibodies to the Staphylococcus aureus capsular polysaccharide was examined in a rat model of catheter-induced endocarditis. Capsular antibodies were induced either by active immunization with killed S. aureus or by passive immunization with hyperimmune rabbit antiserum to S. aureus. Control rats were injected with phosphate-buffered saline or passively immunized with normal rabbit serum or rabbit antiserum to a nonencapsulated strain. Animals with indwelling catheters were challenged intravenously with 5 x 10(4) to 4 x 10(6) CFU of the homologous S. aureus strain (capsular serotype 5 strain Reynolds or serotype 1 strain SA1 mucoid). Both immunized and control rats developed S. aureus endocarditis. The numbers of S. aureus cells recovered from the blood and aortic valve vegetations of immunized rats were similar to those of control rats, indicating that capsule-specific antibodies were not protective. To determine whether the presence of an indwelling catheter interfered with antibody-mediated protection against S. aureus endocarditis, catheters were removed 2 h after insertion in additional groups of rats. An inoculum of 10(8) CFU of strain Reynolds was needed to provoke endocarditis in rats catheterized for 2 h, compared with 5 x 10(4) CFU for rats with indwelling catheters. Passively transferred capsular antibodies were not protective since both immunized and nonimmunized animals developed endocarditis, and quantitative cultures of blood and valvular vegetations revealed no differences between immunized and control animals. The findings of this study indicate that antibodies to the capsular polysaccharide are not protective in the rat model of experimental S. aureus endocarditis.     

Facile penetration of the Staphylococcus aureus capsule by lysostaphin.

The ability of macromolecules to cross the capsular layer of encapsulated microorganisms and interact with their cell walls is important in considerations of the mechanisms of resistance to phagocytosis and of antigen masking in such strains. Lysostaphin was employed as a probe of the penetrability of the Staphylococcus aureus capsule. The rates of lysostaphin-induced lysis of encapsulated and unencapsulated S. aureus strains were compared. Encapsulated S. aureus strains M and Smith diffuse were lysed by lysostaphin at the same rate as their respective unencapsulated counterpart strains M variant and Smith compact. Growth of the M strain in a medium designed to enhance capsule production did not delay the onset or decrease the rate of lysis of the strain compared with organisms grown in normal medium. Cations did not selectively decrease the rate of lysis of the encapsulated strain, but inhibited the lysis of both the M and M variant strains. Peptidoglycan, the presumed lysostaphin target, isolated from both M and M variant strains was digested by lysostaphin at very similar rates. In contrast to whole cells, cations stimulated the rate of lysostaphin digestion of peptidoglycan. It is concluded that the fraction of lysostaphin active in cell lysis, believed to be a glycylglycine endopeptidase with a molecular weight of about 25,000, passes freely through the capsular layer to its target in the staphylococcal cell wall.     

Antiserum agar method for identification of Smith type exopolysaccharides in clinical isolates of Staphylococcus aureus.

We used an antiserum agar method to identify clinical Staphylococcus aureus strains producing an exopolysaccharide antigenically identical to the S. aureus Smith diffuse strain. S. aureus blood isolates were obtained from 137 patients, and three additional isolates were obtained from bone debridement. The 140 patients were clinically divided into the following groups: endocarditis (7 patients); pneumonia, empyema, or both (33 patients); intravascular device (34 patients); superficial or wound infection or both (35 patients); deep tissue infections (18 patients); and 6, unknown bacteremias (13 patients). Ninety (64.3%) of the total 140 S. aureus isolates were found to produce precipitin halos on the antiserum agar. The percentage was greatest in the isolates from the endocarditis group (100%) and least in deep tissue infections (55.5%). The presence of clinical S. aureus strains producing exopolysaccharides antigenically identical to the Smith diffuse strain exopolysaccharide appears to be a common phenomenon.     

A multiplex assay for the quantification of antibody responses in Staphylococcus aureus infections in mice

Staphylococcus aureus causes a variety of infections. Knowledge about the physiological role of most S. aureus antigens in colonization and infection is only limited. This can be studied by measuring antigen-specific antibody responses. In this study, we optimized the multiplex microsphere bead-based flow cytometry technique for mouse serum samples. We analysed immunoglobulin G (IgG) levels directed against 26 S. aureus proteins in a single small-volume mouse serum sample. We assessed possible cross reactivity. Furthermore, we analysed serum samples from mice with different types of S. aureus infections caused by different S. aureus strains. The results show that cross reactivity between proteins on microspheres and serum antibodies towards other proteins was limited. We found that lung-infected mice had a higher and broader IgG response than skin-infected mice. Clearly, the site of infection influences the IgG profile. Next, we compared sera from mice with intravenously-induced bacteraemia caused by different S. aureus strains. We showed different IgG responses depending on the causing S. aureus strain. It is concluded that the bead-based multiplex S. aureus antibody assay can be successfully applied to determine the immunogenicity of different S. aureus proteins in relation to the site of infection and the S. aureus strain causing the infection.     

Virulence and immunity of Staphylococcus aureus BB and certain deficient mutants.

Coagulase-negative and deoxyribonuclease-negative mutants were isolated from Staphylococcus aureus BB by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. Comparison of virulence (50% lethal dose) to mice of these six mutant strains and S. aureus BB was determined by both intravenous and intraperitoneal routes. The ratios of the 50% lethal dose of coagulase-negative mutants to that of the parental strain S. aureus BB ranged from 201 to 403 for intravenous infection and 30.7 to 52.7 for intraperitoneal infection. The virulence of deoxyribonuclease-negative mutants was essentially the same as that of S. aureus BB. When mice were immunized subcutaneously with live S. aureus BB or its deoxyribonuclease-negative mutants, the resulting protection against the intravenous challenge of S. aureus BB was remarkable. The ratios of the 50% lethal dose for the mice that were immunized by these strains to that for the untreated mice extended from 40.1 to 60.6 for intravenous infection and 6.61 to 11.5 for the intraperitoneal route. However, no effect against S. aureus BB challenge was shown in the mice that were immunized with coagulase-negative mutants.     

Binding of human immunoglobulin G to protein A in encapsulated Staphylococcus aureus.

Recent studies of the mechanism of resistance to phagocytosis in encapsulated Staphylococcus aureus have suggested that the capsule is readily penetrated by high-molecular-weight proteins such as antibodies and complement components. S. aureus strains contain a cell wall protein, protein A, that reacts with the Fc portion of immunoglobulins. The binding of immunoglobulin G (IgG) to encapsulated and unencapsulated S. aureus strains has been studied to assess the penetrability of the S. aureus capsule by IgG. Encapsulated S. aureus strains M and Smith diffuse bound large amounts of human IgG which were comparable to amounts bound by the unencapsulated strains Cowan I, M variant, and Smith compact. Trypsin treatment of bacteria reduced their ability to bind IgG. Bound IgG was not removed by extensive washing of bacteria with buffer. A non-protein A-containing, coagulase-negative, encapsulated staphylococcal strain did not bind IgG. These observations suggest that IgG is binding to cell wall protein A in encapsulated S. aureus. No differences in the rates of IgG binding by encapsulated and unencapsulated S. aureus strains were observed. It is concluded that the S. aureus capsule is freely permeable to IgG. This is of importance in considerations of the mechanisms of resistance to phagocytosis and antigen masking in encapsulated microorganisms.     

Chemical and biological properties of extracellular slime produced by Staphylococcus aureus grown in high-carbohydrate, high-salt medium.

Slime material produced by three strains of Staphylococcus aureus grown in the high-carbohydrate, high-salt modified 110 medium contained ribitol teichoic acid and, in two of the three strains, a basic protein reacting with antisera to S. aureus whole cells and cell walls. The basic protein differed chemically and serologically from cell wall mucopeptide and protein A. Substances resembling the capsular antigen of the Smith diffuse strain of S. aureus were not detected, nor were any other uronic acid-containing components. When cell walls, slime material, and teichoic acid were injected intradermally into cows, only cell walls produced a skin reaction.