Vol. 58, No. 5INFECTION AND IMMUNITY, May 1990, p. 1350-13540019-9567/90/051350-05$02.00/0Copyright © 1990, American Society for Microbiology

Antiphagocytic Effect of the Capsule of Staphylococcus simulansY. OHSHIMA,t F. SCHUMACHER-PERDREAU, G. PETERS, P. G. QUIE,t* AND G. PULVERER

Institute of Medical Microbiology and Hygiene, University of Cologne, Goldenfelsstrasse 19-21, 5000 Cologne 41,Federal Republic of Germany

Received 11 September 1989/Accepted 26 January 1990

An encapsulated strain of Staphylococcus simulans was observed to be more resistant to phagocytosis byhuman granulocytes than was a nonencapsulated strain. Phagocytosis of the encapsulated strain was enhancedby antisera to S. simulans, but opsonic activity of antisera was removed by absorption with S. simulans capsularmaterial. The encapsulated strain of S. simulans was also more invasive than the nonencapsulated S. simulansin vivo. More encapsulated than nonencapsulated S. simulans were found in heart blood when equal numbersof organisms were injected intraperitoneally into mice. Invasion of the bloodstreams of mice by encapsulatedS. simulans was prevented by passive immunization (rabbit antiserum). Thus, the capsule of S. simulansinhibited phagocytosis in vitro and contributed to virulence in vivo.

Coagulase-negative staphylococci (CNS) are ubiquitousinhabitants of human skin and mucous membranes. Thenovobiocin-resistant Staphylococcus saprophyticus groupcan cause urogenital infections in sexually active adults (20,33). The novobiocin-susceptible species of the Staphylococ-cus epidermidis group and Staphylococcus simulans arerecognized more and more as important opportunistic patho-gens in immunocompromised patients, e.g., premature neo-nates and leukopenic cancer patients, and in hospitalizedpatients after invasive procedures and with indwelling plas-tic devices (2, 4, 7, 10, 12, 15, 29, 36, 37). The strainsinvolved are especially characterized by their abilities toadhere to polymer surfaces and to produce a slime substance(8, 9, 24, 25).

It is further evident that specific strains are able to causesystemic infections, i.e., septicemia or endocarditis, in im-munologically normal hosts without foreign-body associa-tion (3, 28). These strains are reported to possess specialvirulence factors such as capsules; however, few studieshave been published on encapsulated CNS (1, 13, 14, 18).

Encapsulated strains of Staphylococcus aureus are moreresistant to phagocytosis than nonencapsulated strains (11,21, 34, 35). Similarly, encapsulated strains of S. epidermidishave been found to be resistant to phagocytosis (39).The present investigation was undertaken to examine the

role of capsule on phagocytosis and virulence of S. simulans.

MATERIALS AND METHODS

Bacteria. Encapsulated strain 698 of S. simulans wasisolated from an intra-abdominal abscess following appen-dectomy. The unencapsulated strain CCM 2742 of S. simu-lans that was used as a control is maintained in the straincollection of the Institute of Hygiene, University of Cologne.The two strains were identical in cell wall composition, andboth produced the following enzymes and toxins: protease,hemolysin, lipase, esterase, and DNase. Capsule is identifiedby the presence of a halo in India ink preparations (6). A halo

* Corresponding author.t Present address: School of Medicine, St. Marianna University,

2-16-1, Sugao, Miyamae-Ku, Kawasaki, Japan.t Present address: Department of Pediatrics, University of Min-

nesota Medical School, Box 483, Mayo Memorial Building, Minne-apolis, MN 55455.

was noted with S. simulans 698 but not with strain CCM2742.

Animals. Female mice weighing approximately 15 to 18 g(4 weeks old) were inoculated intraperitoneally with S.simulans. New Zealand rabbits from the Central Institute forExperimental Animals (Hanover, Federal Republic of Ger-many) were inoculated intravenously to raise antibodies toS. simulans.

S. simulans vaccine. S. simulans 698 was suspended to aconcentration of 108 CFU/ml and heat killed, and 0.5 ml ofthis suspension was injected into the marginal ear veins ofrabbits three times a week for 6 weeks. Ten days after thefinal inoculation, the rabbits were exsanguinated, and serawere separated and stored. Antibody titers were determinedby bacterial agglutination.

S. simulans capsular material. S. simulans 698 was incu-bated for 20 h at 37°C in chemically defined medium (30).After centrifugation (25,000 x g for 20 min), the bacterialpellet was suspended in 0.15 M phosphate-buffered saline(pH 7.2) and treated with sonic oscillation (Sonifier 13 12;Branson Sonic Power Co.) for 10 min at 4°C.

After centrifugation (20,000 x g for 20 min at 4°C), thesupernatant was filtered (0.45-,um-pore-size membrane filter;Millipore Corp., Bedford, Mass.), dialyzed with distilledwater for 48 h at 4°C, and lyophilized.

Bacterial clearance from peritoneal cavities and heart bloodof mice. S. simulans 698 and CCM 2742 were incubated onsheep blood agar for 20 h at 37°C, harvested, and washedtwice with sterile saline. Bacterial suspensions were ad-justed to an optical density of 1.2 at 580 nm by a model 2.5spectrophotometer (Beckman Instruments, Inc., Fullerton,Calif.). The number of viable bacteria was determined by thenumber of colonies in appropriate 10-fold dilutions of bacte-rial suspension inoculated on blood agar and incubatedovernight at 37°C. Bacterial suspension (0.5 ml) in salinecontaining 7.0 x 107 CFU of strain 698 per ml and 9.0 x 107CFU of strain CCM 2742 per ml was injected into theperitoneal cavities of mice (groups of 25). Five mice in eachgroup were killed at 2, 6, 12, 24, and 48 h after inoculation,and the number of CFU in the peritoneal cavity and heartblood was determined. A total of 0.5 ml of heart blood permouse was cultured, and peritoneal cavities were lavagedwith 3 ml of sterile saline, which was then cultured. Thecolonies were counted, and results were expressed as the

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ANTIPHAGOCYTIC EFFECT OF S. SIMULANS CAPSULE

total number of organisms in the peritoneal cavity and heartblood.Absorbed antisera. Rabbit antisera against encapsulated

strain 698 were absorbed with whole bacterial cells or withcapsular material. Rabbit antiserum (1 ml) was incubatedwith 5 mg of lyophilized heat-killed whole cells or capsularmaterial at 37°C for 1 h and at 4°C overnight. After incuba-tion, the sera were filtered (Millipore filter, 0.45-,um poresize). Sera were heat inactivated by being incubated at 56°Cfor 30 min.

Passive immunization with rabbit antiserum. Doses (0.5 ml)of rabbit antiserum or absorbed antiserum were injectedintraperitoneally into mice. Thirty minutes later, the micewere challenged with encapsulated strain 698 (0.5 ml, 5 x 107CFU/ml of saline). After 2 h, the mice were killed and thenumber of viable bacteria in peritoneal fluid and heart bloodwas determined as described above.

Preparation of PMNs. Human polymorphonuclear leuko-cytes (PMNs) were prepared for the bacterial killing assayby using the modified test of Van Furth et al. (31). Heparin-ized blood from a healthy volunteer was mixed with 6%(wt/vol) dextran T2000 (Pharmacia, Uppsala, Sweden; bloodto dextran ratio, 4:1 [vol/vol]) and incubated at 37°C for 20min. PMNs were collected from the leukocyte-enrichedplasma layer by centrifugation at 200 x g for 10 min.Erythrocytes were removed by hypotonic lysis with 0.2%(wt/vol) NaCI for 45 s followed by the restoration of isotox-icity by the addition of an equal amount of 1.6% (wt/vol)NaCl. After centrifugation at 200 x g for 10 min, the PMNswere washed with Hanks balanced salt solution (withoutphenol red; GIBCO Ltd., Glasgow, Scotland). PMNs weresuspended in RPMI 1640 (GIBCO) containing 10% (vol/vol)fetal calf serum at 106 cells per ml. This preparation con-tained 85% PMNs.

Bacterial killing assay. Bacterial killing activity of PMNswas measured by the techniques of Metcalf et al. (22)modified as follows. S. simulans 698 and CCM 2742 wereincubated on sheep blood agar plates at 37°C for 20 h,harvested, and washed twice with sterile saline. The amountof each strain was adjusted to approximately 5 x 107CFU/ml. Both S. simulans strains were opsonized by incu-bation at 37°C for 30 min in RPMI 1640 containing 10%(vol/vol) normal pooled human serum, rabbit antiserum, orabsorbed rabbit antiserum. After opsonization, bacteriawere washed twice with sterile phosphate-buffered salineand resuspended in RPMI 1640 containing 10% (vol/vol) fetalcalf serum to a concentration of approximately 107 CFU/ml.Bacterial suspension (0.1 ml; 107 or 106 CFU/ml) was addedto 1.0 ml ofPMN suspension (106 cells per ml) and incubatedat 37°C for 60 or 120 min with gentle shaking. After incuba-tion, 0.1 ml of each reaction mixture was added to 9.9 ml ofdistilled water, mixed vigorously and incubated at roomtemperature for 20 min. The number of viable bacteria wasdetermined.

RESULTS

Bacterial clearance of S. simulans from peritoneal cavitiesand heart blood of mice. A large capsule was detected on S.simulans 698 (Fig. 1). Mice were inoculated intraperitoneallywith the strains, and the kinetics of clearance of bacteriafrom the peritoneal cavity was determined at 2-h intervals.The number of viable bacteria in the peritoneal cavity wasunchanged during the first 6 h in mice inoculated withencapsulated strain 698, but more than 95% of strain 2742was already cleared in 2 h (Fig. 2A).

FIG. 1. Capsular detection on the outer surface of S. simulans698 with India ink stain. Clear halo of capsule noted.

When heart blood of mice inoculated intraperitoneally wascultured, more colonies of S. simulans 698 than of thenonencapsulated strain of S. simulans were found. A 4-logdifference between the two strains was noted after 2 h.Although strain 698 persisted for longer than 12 h, strain2742 was cleared from the blood within 12 h (Fig. 2B).However, none of the mice infected with either strain died.

After passive immunization of mice with rabbit antiserum(intraperitoneal application) against encapsulated strain 698,a 99% reduction in organisms in the peritoneal fluid wasnoted, and only a very small number of organisms wererecovered from the blood (Fig. 3A). The protective effect ofantisera was removed by absorption of the antisera withheat-killed whole cells of S. simulans 698 (Fig. 3B). Absorp-tion with S. simulans capsular material reduced the protec-tive effect to some extent but was not statistically significant(Fig. 3C).

Phagocytosis and killing of S. simulans by human PMNs.Phagocytosis and killing of S. simulans 698 and CCM 2742were examined in vitro by using human PMNs and pooledhuman serum for opsonization. More than 80% of nonencap-sulated S. simulans CCM 2742 were phagocytized and killedwithin 2 h by phagocytic mixtures containing PMNs (Fig.4A). Little phagocytosis or killing of encapsulated strain 698was observed during 2 h of incubation with PMNs, evenwhen the ratio of PMNs to bacteria was increased from 1:1to 10:1 (Fig. 4B).However, encapsulated S. simulans 698 was rapidly

phagocytized and killed by PMNs after opsonization withrabbit antiserum against S. simulans capsule (-Fig. 5A). Morethan 90% of the inoculated bacteria were killed by PMNswithin 1 h. Opsonic activity of the rabbit antiserum wasremoved to a great extent by absorption of the antiserumwith heat-killed whole cells (Fig. SB) or with capsularmaterial prepared from encapsulated strain 698 (Fig. SC).

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FIG. 2. Clearance of nonencapsulated strain CCM 2742 andencapsulated strain 698 of S. simulans in peritoneal fluid (A) andheart blood (B) of mice. Results are shown as averages of viablenumber of bacteria from each group of five mice at 2-h intervals.

DISCUSSIONCNS are recognized as significant nosocomial pathogens

despite being normal inhabitants of human skin and mucosalmembranes (2, 4, 7, 14, 19, 29, 37). The presence of slime isstrongly correlated with these CNS infections (8, 9, 24, 25).Several investigators have reported that some CNS strainsisolated from clinical specimens produce capsular material(1, 13, 14, 17, 18, 40). These encapsulated strains of CNSshow higher resistance to phagocytosis than do unencapsu-lated strains (39). Additionally, the capsular substances fromS. aureus and S. epidermidis contribute to increased viru-lence in experimental infections with these microorganisms(11, 18, 21, 39).

Peritoneal Fluid

m Heart Blood

Nonimmunized Preimmunized with(control ) A B c

FIG. 3. Clearance of encapsulated S. simulans 698 from perito-neal fluid and heart blood of mice. Each group of five mice waspassively immunized with rabbit antiserum (A), rabbit antiserumabsorbed with whole cells (B), or rabbit antiserum absorbed withcapsule (C). Nonimmunized mice were used as a control. Resultsare shown as the means ± the standard deviations of three experi-ments.

We have compared an encapsulated and an unencapsu-lated S. simulans strain identical in cell wall compositionsand enzymatic properties. Our experimental results suggestthat the surface capsule of S. simulans had a major anti-phagocytic effect. The capsule appeared to inhibit interac-tion of bacterial surfaces with opsonins such as complementand antibacterial antibodies which promote phagocytosisand killing of bacteria by phagocytic cells. The role of thecapsule, therefore, is similar in this CNS species and in S.aureus and other bacterial species (1, 35, 38).

Cell wall peptidoglycan of S. aureus plays a key role inresistance to phagocytosis, but opsonization of the cell wallby complement activated via the alternative pathway pro-vides a natural host defense mechanism (27, 32, 35). How-ever, strains of S. aureus which are encapsulated are notopsonized by complement in serum without specific antibod-ies (26, 35). Therefore, specific antibodies against capsularantigens are required for effective opsonization of encapsu-lated S. aureus, and results from this study suggest that theyare necessary for opsonization of encapsulated S. simulansas well.

After intraperitoneal inoculation of mice with encapsu-lated S. simulans, rapid invasion and persistent colonizationof the bloodstream was observed. Large numbers of organ-isms also persisted in the peritoneal cavity. Despite this,none of the infected mice developed disease or died. Afterpassive immunization with rabbit antiserum containing anti-capsular antibodies, however, no differences between en-capsulated and nonencapsulated strains of S. simulans were

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ANTIPHAGOCYTIC EFFECT OF S. SIMULANS CAPSULE

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FIG. 4. Bacterial killing activity of human PMNs against nonen-capsulated strain CCM 2742 (A) and encapsulated strain 698 (B) ofS. simulans. After preopsonization with normal pooled humanserum, bacteria were incubated with PMNs at 1:1 and 10:1 PMN/bacteria ratios. Symbols: 0, without PMNs; 0, with PMNs. Resultsare shown as the means + the standard deviations of three experi-ments.

observed, suggesting that anticapsular antibodies are op-

sonic, allow rapid clearing of intraperitoneal organisms, andprevent bloodstream invasion. Specific antibodies againstcapsular material, therefore, appear to be necessary for hostdefense against encapsulated strains of CNS.Opsonic binding of the C3 molecule of complement to

bacterial surfaces appears to be inhibited by the presence ofa capsule (18, 34, 38). Heat inactivation of the S. simulansantiserum (56°C, 30 min) does not diminish its effectivenessas an opsonin for encapsulated S. simulans, suggesting that

D without PMNs

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FIG. 5. Bacterial killing activity of PMNs against encapsulatedstrain 698 of S. simulans preopsonized with rabbit antiserum (A),antiserum absorbed with heat-killed whole cells (B), or antiserumabsorbed with capsule (C). Bacteria were incubated with PMNs at a1:1 ratio. Nonopsonized bacteria were used as a control. Results areshown as the means + the standard deviations of three experiments.

these organisms are effectively opsonized by specific anti-bodies and that enhanced opsonization by complement can-not be detected (5, 16). Absorption of these antisera with S.simulans capsular substance decreased opsonic activity,providing strong evidence that it was anticapsular antibodieswhich provide effective opsonin for encapsulated S. simu-lans, promoting rapid uptake and killing by human PMNs.

Bacterial cell wall peptidoglycan of encapsulated S. sim-ulans may bind complement on the cell wall surface, aspreviously demonstrated with S. aureus (26). However,opsonic or ligand activity, i.e., binding of bacteria to phago-cytic cell surface, is blocked by the capsule (5, 16). Recently,we demonstrated that the S. simulans capsule also acts as abarrier for the interaction of a polyvalent staphylococcalbacteriophage with its receptor in the S. simulans cell wall(23).The presence of capsule on the surface of S. simulans

appeared to play a role in resistance to phagocytosis; how-ever, this property may be associated with other virulencefactors in vivo.

ACKNOWLEDGMENT

Y. Ohshima is deeply grateful for a fellowship from the Alexandervon Humboldt Foundation.

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