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Background: Skin colonization or infection with Staphylococcus aureus is known to trigger aggravation of atopic dermatitis (AD). However, the exact mechanisms by which S. aureus can worsen AD are unknown., Objective: We investigated whether and how S. aureus-derived membrane vesicles (MVs) contribute to worsening of AD., Methods: Immunohistochemical and immunoelectron microscopic analyses were performed to detect staphylococcal protein A (SPA) in the epidermis of AD lesions. HaCaT cells were treated with S. aureus MVs and were analysed for the expression of cytokine genes. Immunopathology and cytokine gene profiles were analysed after topical application of S. aureus MVs to AD-like skin lesions in a mouse model., Results: The MV component SPA was detected in the keratinocytes as well as in the intercellular space of the epidermis of AD lesions colonized with S. aureus. Intact MVs from S. aureus delivered their components to keratinocytes and stimulated pro-inflammatory cytokine gene expression in vitro. A knock-down of Toll-like receptor 2 or nucleotide-binding oligomerization domain 2 using small interfering RNAs suppressed interleukin-8 gene expression. Topical application of intact S. aureus MVs to AD-like skin lesions in the mouse model induced massive infiltration of inflammatory cells and the resulting eczematous dermatitis. This inflammatory reaction was associated with a mixed Th1/Th2 immune response and enhanced expression of chemokine genes in AD-like skin lesions., Conclusions and Clinical Relevance: This study showed the importance of S. aureus MVs as a potent mediator for worsening of AD among many exogenous worsening factors of AD. Thus, S. aureus MVs may be regarded as one of the therapeutic targets for the management of AD aggravation., (© 2016 John Wiley & Sons Ltd.)

Staphylococcus aureus is a major human pathogen causing community- and hospital-acquired infections. Capsule production of S. aureus confers protection against host defence. There is a lack of information concerning the association of capsular polysaccharide (CP) expression and activity of the accessory gene regulator (agr) in clinical S. aureus isolates. Production of CP and agr expression were assessed in 195 S. aureus isolates from infected patients at a German University Hospital. Northern blot analysis revealed that S. aureus strains with a non-functional agr locus were more likely to be CP-negative than strains with a functional agr locus., (© 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.)

Non-duplicate methicillin-resistant Staphylococcus aureus (MRSA) isolates (n = 436), collected from four hospitals located in three Korean cities between 2001 and 2005, were investigated by SCCmec typing and multilocus sequence typing (MLST). Variations within SCCmec, especially type II, were detected in 165 (37.8%) isolates, and these variants were characterised using four different SCCmec typing methods. The predominant SCCmec type was a type II variant that differed from type II by the absence of a pUB110 insertion. MLST analysis showed that most of the isolates carrying SCCmec variants belonged to ST5.

Many bovine Staphylococcus aureus isolates from Argentina are nontypeable (NT), i.e., they do not produce serotype 5 or 8 capsular polysaccharides (CPs). Some of these NT strains have a deletion of the cap5(8) gene cluster mediated by a variant of IS257, now designated IScap. IScap showed 93% amino acid identity to S. aureus ORF49 but only 85% identity to IS431 from S. aureus N315 and 88% identity to an IS257-like element from bovine strain RF122. Thirty-six (53%) of 68 bovine isolates, drawn from a previously described S. aureus strain collection, carried some variant of IS257, including IScap. Of these 36 IS+ isolates, 6 were CP5+, 1 was CP8+, and 29 were NT. Forty-four of the 68 isolates were NT, and 24 of these 44 NT isolates (55%) exhibited IScap-mediated deletion of the cap5(8) gene cluster. IScap was not found among 20 human NT S. aureus isolates bearing the cap5HIJK genes, which suggests that IScap-mediated deletion of the capsule locus is restricted to bovine strains of S. aureus. We were unable to identify a precursor strain in which IScap flanked the cap5(8) capsule locus, nor were we able to select for deletion of the cap5(8) locus in vitro. Our results support the hypothesis that deletion of the cap5 locus occurred in the distant past and that the relative abundance of these NT strains may be a result of their ability to persist in subclinical mastitis infection in cows.

The five Staphylococcus aureus strains used in the manufacture of a commercially available bacterin were examined for capsular and surface polysaccharide serotypes. Double immunodiffusion assays of antigenic extracts of test and reference strains with monospecific typing sera to capsular serotypes 1, 2, 5, and 8 and to surface polysaccharide serotype 336 were performed to detect the specific reactivities and antigenic relationships of test samples. Antigenic extracts of two S. aureus strains reacted with antibodies to serotype 8, but not with antibodies to serotype 5, by producing specific precipitin lines. A third strain reacted with monospecific antibodies to serotype 5 and not with the antibodies to serotype 8. The extracts of two other strains failed to exhibit any detectable reaction with antiserum to serotypes 1, 2, 5, or 8. Antibodies to serotype 336, however, precipitated an identical, specific 336 antigen from the antigenic extracts of these two nontypeable strains. Thus, S. aureus bacterin includes one serotype 5, two serotype 8, and two serotype 336 strains, the three predominant serotypes responsible for bovine mastitis.

Complement-mediated opsonization of bacteria by C3 binding is an important component of the host innate immune system. Little information is available concerning the interaction between complement proteins and capsule type 5 and 8 Staphylococcus aureus strains, even though these isolates are responsible for approximately 70% of human staphylococcal infections. To investigate the importance of an intact complement pathway in an experimental staphylococcal infection, control and C3-depleted mice were challenged intravenously with 10(7) CFU of a serotype 5 S. aureus isolate. Whereas only 8% of the control mice succumbed to the infection, 64% of the complemented-depleted animals died. In vitro parameters of C3 binding to two heavily encapsulated (CP++) strains, three encapsulated (CP+) strains, and an isogenic capsule-negative (CP-) mutant were examined. The alternative pathway contributed 90% of C3 binding in 20% serum at 30 min, whereas it accounted for only 13% of C3 binding in 2% serum. Stationary-phase organisms bound only 10% as much C3 as mid-log-phase organisms; this was only in part due to capsule. When the S. aureus strains were cultivated on solid medium, the CP++ isolates bound 50% less C3 than CP+ strains; a CP+ strain bound 42% less C3 than the CP- mutant. Both C3b and iC3b fragments of C3 bound to S. aureus cells, and about one-third of the bound C3 was shed from the staphylococcal surface as iC3b, regardless of the CP phenotype of the strain. Thus, the phase of growth and presence of capsule are critical to opsonization.

Staphylococcus aureus is a medically important bacterial pathogen that is a common cause of superficial and deep-seated abscesses in humans. Most S. aureus isolates produce either a serotype 5 or 8 capsular polysaccharide (CP) that has been shown to enhance bacterial virulence. We investigated the role of S. aureus CPs in modulating abscess formation in an experimental animal model of intraabdominal infection. Structural studies of CP8 revealed that it has a zwitterionic charge motif conferred by the negatively charged carboxyl group of N-acetylmannosaminuronic acid and free amino groups available on partially N-acetylated fucosamine residues. We report that purified CP5 and CP8 facilitated intraabdominal abscess formation in animals when given i.p. with a sterile cecal contents adjuvant. Chemical modifications that neutralized the positively or negatively charged groups on CP8 abrogated its ability to provoke abscesses. Rats prophylactically treated with CP8 s.c. were protected against abscess formation induced by homologous or heterologous zwitterionic polysaccharides. Likewise, treatment with CP8 protected against challenge with viable S. aureus strains PS80 (a capsule type 8 strain) or COL (a methicillin-resistant capsule type 5 strain). Purified CP8 was a potent activator of rat and human CD4(+) T cells in vitro. When transferred to naive rats, these activated T cells modulated the development of intraabdominal abscess formation. These results provide a structure/function rationale for abscess formation by S. aureus and expand the sphere of encapsulated organisms that interact directly with T cells to regulate this host response to bacterial infection.

The Staphylococcus aureus serotype 5 capsular polysaccharide (CP5) has a repeating unit composed of (-->4)-3-O-acetyl-beta-D-ManNAcA-(1-->4)-alpha-L-FucNAc (1-->3)-beta-D-FucNAc-(1-->)(n). Sixteen chromosomal genes (cap5A through cap5P) are involved in the synthesis of CP5. We recently demonstrated that Cap5P, a 2-epimerase, catalyzes the conversion of UDP-N-acetyl glucosamine (UDP-GlcNAc) to UDP-N-acetylmannosamine (UDP-ManNAc). In this study, we show that UDP-ManNAc is oxidized to UDP-N-acetylmannosaminuronic acid (UDP-ManNAcA) by a UDP-ManNAc dehydrogenase encoded by S. aureus cap5O. We expressed Cap5O in Escherichia coli and purified the recombinant protein. The UDP-ManNAc dehydrogenase activity of purified Cap5O was assessed by incubating Cap5P and UDP-GlcNAc (to produce UDP-ManNAc), together with Cap5O, NAD(+), and a reducing agent. Enzymatic activity was quantitated indirectly by measuring the increase in absorbance at 340 nm resulting from NADH formation. The product of the reaction was confirmed as UDP-ManNAcA by gas chromatography-mass spectroscopy. A cap5O mutation, created by deletion of 727 bp in the 5' end of the gene, was introduced by allelic replacement into S. aureus Reynolds, rendering it CP5 negative. Mice inoculated intravenously or subcutaneously with the wild-type strain Reynolds had greater numbers of S. aureus recovered from their kidneys (P = 0.019) or their subcutaneous abscesses (P = 0.0018), respectively, than did animals inoculated with the cap5O mutant. The results of this study indicate that S. aureus cap5O is essential for capsule production and that capsule promotes staphylococcal virulence in mouse models of abscess formation.

Staphylococcus aureus and S. epidermidis are among the most common causes of nosocomial infection, and S. aureus is also of major concern to human health due to its occurrence in community-acquired infections. These staphylococcal species are also major pathogens for domesticated animals. We have previously identified poly-N-succinyl beta-1-6 glucosamine (PNSG) as the chemical form of the S. epidermidis capsular polysaccharide/adhesin (PS/A) which mediates adherence of coagulase-negative staphylococci (CoNS) to biomaterials, serves as the capsule for strains of CoNS that express PS/A, and is a target for protective antibodies. We have recently found that PNSG is made by S. aureus as well, where it is an environmentally regulated, in vivo-expressed surface polysaccharide and similarly serves as a target for protective immunity. Only a minority of fresh human clinical isolates of S. aureus elaborate PNSG in vitro but most could be induced to do so under specific in vitro growth conditions. However, by immunofluorescence microscopy, S. aureus cells in infected human sputa and lung elaborated PNSG. The ica genes, previously shown to encode proteins in CoNS that synthesize PNSG, were found by PCR in all S. aureus strains examined, and immunogenic and protective PNSG could be isolated from S. aureus. Active and passive immunization of mice with PNSG protected them against metastatic kidney infections after intravenous inoculation with eight phenotypically PNSG-negative S. aureus. Isolates recovered from kidneys expressed PNSG, but expression was lost with in vitro culture. Strong antibody responses to PNSG were elicited in S. aureus infected mice, and a PNSG-capsule was observed by electron microscopy on isolates directly plated from infected kidneys. PNSG represents a previously unidentified surface polysaccharide of S. aureus that is elaborated during human and animal infection and is a prominent target for protective antibodies.

The adherence of Staphylococcus aureus to human endothelial cells (EC) is probably an important step in the pathogenesis of systemic staphylococcal infections. We examined the influence of type 5 capsular polysaccharide (CP5) production, the global regulator agr, and the bacterial growth phase on S. aureus adherence to EC. Whereas S. aureus Newman showed maximal adherence to EC in the logarithmic phase of growth, an isogenic agr mutant showed maximal adherence in the stationary growth phase. S. aureus adherence to EC and CP5 expression were negatively correlated: a mutation in the agr locus diminished CP5 production and led to increased adherence. Likewise, induction of CP5 expression by addition of NaCl to the growth medium resulted in reduced staphylococcal adherence to EC. S. aureus Newman cells that adhered to EC did not express CP5. A Newman cap5O mutant was acapsular and showed significantly greater adherence to EC than the parental strain did (P<0.005). Complementation of the cap5O mutation in trans restored CP5 expression and reduced EC adherence to a level similar to that of the parental strain. The enhanced adherence shown by the cap5O mutant was similar in magnitude to that of the agr mutant or the cap5O agr double mutant. Cells of the cap5O mutant and cap5O agr double mutant harvested from stationary-phase cultures adhered significantly better than did cells harvested in the exponential growth phase. These data are consistent with the postexponential and agr-independent expression by S. aureus of at least one putative EC adhesin, whose binding domain may be masked by CP5.

Bovine mastitis caused by Staphylococcus aureus is responsible for major economic losses to the dairy industry, and more-effective therapeutic or preventive approaches are sorely needed. The predominance of staphylococcal capsular polysaccharide types 5 and 8 among human isolates from many sources is well documented, but there seems to be a greater variation in the distribution of capsular serotypes among isolates from cows. A total of 636 isolates of S. aureus from cases of bovine mastitis in Sweden, Denmark, Finland, Iceland, Ireland, and the United States were investigated for production of capsular polysaccharide types 5 and 8. Approximately half of all the European isolates tested were of serotype 8, although variation among countries and among isolates of clinical and subclinical origin was observed. Sweden had the highest frequency (87%) of serotypeable isolates, and Finland had the lowest (48%). Capsule types 5 and 8 accounted for only 42% of the U.S. isolates tested. A few isolates showed weak reactivity with CP5 antiserum in a colony blot assay, and an enzyme-linked immunosorbent assay inhibition method confirmed that the levels of capsule produced by these strains were <10% of those produced by control strains. Fifty isolates that failed to react with capsular antisera all possessed the genes for production of capsular polysaccharide type 5 or 8. These results underscore the variability in capsule production by bovine isolates of S. aureus from different geographic regions. This information is important for the rational design of a capsule-based vaccine to prevent S. aureus bovine mastitis.

Staphylococcus aureus is an important cause of bovine mastitis worldwide, and effective preventive or therapeutic modalities are lacking. Although most human S. aureus isolates produce capsular polysaccharides (CPs), few reports have described the prevalence of capsules on bovine isolates. This information is important for the rational design of a vaccine for the prevention of staphylococcal mastitis. We serotyped 195 S. aureus strains isolated between 1989 and 1997 from the milk of mastitic cows in Argentina. Only 14 (7.1%) of the strains were serotype 5, and all were recovered between 1989 and 1992. Thirteen serotype 8 strains were identified, and 12 of these were isolated between 1991 and 1994. The remaining 168 isolates were nonreactive (NR) with CP serotype 5 (CP5)- or CP8-specific antibodies. Hybridization studies performed with genomic DNA from eight NR strains revealed that only three of them carried the capsule genes. Pulsed-field gel electrophoresis (PFGE) performed with 127 of the 195 S. aureus isolates revealed that most (86%) strains belonged to one of four major PFGE groups. Although 8 of 14 CP5 isolates showed a common PFGE pattern (arbitrarily defined as A1), 31 other A1 isolates from the same time period (1989 to 1992) were not CP5 positive. In contrast, only nine PFGE type B3 isolates were recovered between 1990 and 1994, and eight of these were positive for CP8 (P < 0.0003). The results of this study underscore the variability in capsule expression by S. aureus strains isolated from different geographical regions and cast doubt on the roles of CP5 and CP8 in the pathogenesis and immunoprophylaxis of bovine mastitis in Argentina.

The recent identification of glycopeptide intermediate-resistant Staphylococcus aureus (GISA) clinical isolates has provided an opportunity to assess the stability of the glycopeptide resistance phenotype by nonselective serial passage and to evaluate reversion-associated cell surface changes. Three GISA isolates from the United States (MIC of vancomycin = 8 microg/ml) and two from Japan (MICs of vancomycin = 8 and 2 microg/ml) were passaged daily on nutrient agar with or without vancomycin supplementation. After 15 days of passage on nonselective medium, vancomycin- and teicoplanin-susceptible revertants were obtained from each GISA isolate as determined by broth dilution MIC. Revertant isolates were compared with parent isolates for changes in vancomycin heteroresistance, capsule production, hemolysis phenotype, coagulase activity, and lysostaphin susceptibility. Several revertants lost the subpopulations with intermediate vancomycin resistance, whereas two revertants maintained them. Furthermore, although all of the parent GISA isolates produced capsule type 5 (CP5), all but one revertant tested no longer produced CP5. In contrast, passage on medium containing vancomycin yielded isolates that were still intermediately resistant to vancomycin, had no decrease in the MIC of teicoplanin, and produced detectable CP5. No consistent changes in the revertants in hemolysis phenotype, lysostaphin susceptibility, or coagulase activities were discerned. These data indicate that the vancomycin resistance phenotype is unstable in clinical GISA isolates. Reversion of the vancomycin resistance phenotype might explain the difficulty in isolating vancomycin-resistant clinical isolates from the blood of patients who fail vancomycin therapy and, possibly, may account for some of the difficulties in identifying GISA isolates in the clinical laboratory.

Staphylococcus aureus nasal carriage is a risk factor for infection in humans, particularly in the hospital environment. Attenuation of carriage has proven effective in reducing the prevalence of infection in some high-risk groups. To study staphylococcal factors that influence nasal colonization, a mouse model of S. aureus nasal colonization was developed. Mice were inoculated intranasally with S. aureus Reynolds, and nasal carriage was evaluated by quantitating cultures of the nasal tissues from mice sacrificed at various time points after inoculation. The majority of mice inoculated with 10(8) CFU of S. aureus maintained nasal carriage for at least 20 days. Nasal colonization rates were similar for inbred (BALB/c and C57BL/6) and outbred (ICR) mice. Colonization was not affected by mouse passage of strain Reynolds. Lower inoculum doses (<10(7) CFU) resulted in reduced colonization after 7 days. However, mice given streptomycin in their drinking water developed long-term carriage of S. aureus, and they were colonized with inocula as low as 10(5) CFU. Nasal colonization was also established with two other S. aureus strains (one strain each of human and murine origins). S. aureus recovered from the nares of experimentally colonized mice expressed high levels of capsule, and the ability of a capsule-defective mutant to persist in the nares was reduced in comparison to that of the parent strain. This nasal colonization model should prove useful for studies of factors that mediate S. aureus colonization and for assessment of targets for antimicrobial intervention or vaccine development.

The serotype 5 capsule gene cluster of Staphylococcus aureus comprises 16 genes (cap5A through cap5P), but little is known about how the putative gene products function in capsule biosynthesis. We propose that the N-acetylmannosaminuronic acid (ManNAcA) component of the S. aureus serotype 5 capsular polysaccharide (CP5) is synthesized from a UDP-N-acetylglucosamine (UDP-GlcNAc) precursor that is epimerized to UDP-N-acetylmannosamine (UDP-ManNAc) and then oxidized to UDP-ManNAcA. We report the purification and biochemical characterization of a recombinant UDP-GlcNAc 2-epimerase encoded by S. aureus cap5P. Purified Cap5P converted approximately 10% of UDP-GlcNAc to UDP-ManNAc as detected by gas chromatography-mass spectrometry. The epimerization of UDP-GlcNAc to UDP-ManNAc occurred over a wide pH range and was unaffected by divalent cations. Surprisingly, CP5 expression in S. aureus was unaffected by insertional inactivation of cap5P. Sequence homology searches of the public S. aureus genomic databases revealed the presence of another putative UDP-GlcNAc 2-epimerase on the S. aureus chromosome that showed 61% identity to Cap5P. Redundancy of UDP-GlcNAc 2-epimerase function in S. aureus was demonstrated by cloning the cap5P homologue from strain Newman and complementing an Escherichia coli rffE mutant defective in UDP-GlcNAc 2-epimerase activity. Our results confirm the putative function of the S. aureus cap5P gene product and demonstrate the presence of a second gene on the staphylococcal chromosome with a similar function.

Vaccines based on preferential expression of bacterial antigens during human infection have not been described. Staphylococcus aureus synthesized poly-N-succinyl beta-1-6 glucosamine (PNSG) as a surface polysaccharide during human and animal infection, but few strains expressed PNSG in vitro. All S. aureus strains examined carried genes for PNSG synthesis. Immunization protected mice against kidney infections and death from strains that produced little PNSG in vitro. Nonimmune infected animals made antibody to PNSG, but serial in vitro cultures of kidney isolates yielded mostly cells that did not produce PNSG. PNSG is a candidate for use in a vaccine to protect against S. aureus infection.

Controversy persists over the role that the capsular polysaccharide plays in the pathogenesis of Staphylococcus aureus infections. To address this issue, we compared the mouse virulence of S. aureus Reynolds and capsule-defective mutant strains cultivated under conditions of high or low capsule expression. Strain Reynolds cells cultivated on Columbia salt agar plates expressed approximately 100-fold more type 5 capsular polysaccharide than did cells cultivated in Columbia salt broth. The relative virulence of strain Reynolds and its capsule-defective mutants after growth on either solid or liquid medium was examined in mice challenged intraperitoneally or intravenously. The results indicated that agar-grown Reynolds cells were cleared from the bloodstream of mice less readily than broth-grown Reynolds cells. When the parental and mutant strains were cultivated on solid medium, strain Reynolds sustained a higher level of bacteremia than did the capsular mutants. We performed in vitro opsonophagocytic killing assays to determine whether staphylococcal virulence for mice correlated with resistance to phagocytosis. S. aureus Reynolds cultivated on solid medium was susceptible to phagocytic killing only in the presence of specific capsular antibodies and complement. Strain Reynolds grown in broth showed opsonic requirements for phagocytic killing that were similar to those of the capsular mutants (grown in broth or on agar); i.e., the bacteria were opsonized for phagocytosis by nonimmune serum with complement activity. These studies indicate that optimal expression of capsule enhances bacterial virulence in the mouse model of bacteremia, probably by rendering the organisms resistant to opsonophagocytic killing by leukocytes.

The Staphylococcus aureus serotype 5 capsular polysaccharide (CP5) has a trisaccharide repeating unit of (-->4)-3-O-Ac-beta-D-ManNAcAp-(1-->4)-alpha-L-FucNAcp-(1-->3 )-beta-D-FucNAcp-(1-->). Tn918 mutagenesis of strain Reynolds yielded a mutant that produced wild-type levels of O-deacetylated CP5. The site and orientation of the single transposon insertion in mutant JL232 were determined by analysis of Southern blots and amplification of DNA flanking the transposon. DNA sequencing revealed that Tn918 was inserted within an open reading frame of 627 bp. The predicted amino acid sequence encodes a protein of approximately 26 kDa with homology to members of the NodL-LacA-CysE family of bacterial acetyltransferases. Southern blot analysis showed that genes similar to cap5H were present only in strains of S. aureus belonging to capsular serotypes 2, 4 and 5. In an in vitro assay, the parental strain was more resistant to opsonophagocytic killing than the mutant strain. In a mouse model of staphylococcal infection, the parental strain was able to seed the bloodstream from the peritoneal cavity and colonize the kidneys more efficiently than the O-deacetylated mutant. When cap5H was provided to the mutant in trans, it fully restored CP5 O-acetylation. The virulence of the complemented mutant strain closely approximated that of the parental strain.

The Staphylococcus aureus cap5P and cap5O genes of the type 5 capsule biosynthetic locus restore enterobacterial common-antigen expression to Escherichia coli mutants defective in rffE and rffD gene expression, respectively. Cap5P and Cap5O likely function as UDP-GlcNAc 2-epimerase and UDP-ManNAc dehydrogenase enzymes, respectively, in the synthesis of the capsule precursor UDP-ManNAcA.

Staphylococcus aureus arthritis is a rapidly progressive and highly erosive disease of the joints in which both host and bacterial factors are of pathogenic importance. One potential bacterial virulence factor is the ability to express a polysaccharide capsule (CP). Among 11 reported capsular serotypes, CP type 5 (CP5) and CP8 comprise 80 to 85% of all clinical blood isolates. The aim of this study was to assess the role of CP5 as a virulence factor in staphylococcal septicemia and septic arthritis with a recently established murine model of hematogenously spread S. aureus arthritis. NMRI mice were inoculated intravenously with S. aureus strains isogenic for expression of CP5, and clinical, bacteriological, serological, and histopathological progression of disease was studied. Inoculation of 7 x 10(6) CFU of S. aureus per mouse induced 55% mortality in the group inoculated with the CP-expressing bacteria, compared to 18% in the group inoculated with CP- mutants. A lower dose of inoculum (3 x 10[6] per mouse) did not give rise to mortality in mice inoculated with CP mutant strains, whereas 18% of the mice inoculated with the CP5-expressing S. aureus died. Importantly, mice inoculated with S. aureus expressing CP5 had a significantly higher frequency of arthritis and a more severe form of the disease. In vitro assays suggested that macrophages were not able to phagocytize CP5+ staphylococci as efficiently as they were CP5- strains. In addition, once phagocytized, CP5+ bacteria were less efficiently killed than CP- mutants. In summary, CP5 leads to a higher frequency of arthritis and a more severe course of the disease. This seems to be related to the effects of the downregulatory properties of CP on the ingestion and intracellular killing capacity of phagocytes. Our results clearly indicate that the expression of CP5 is a determinant of the virulence of S. aureus in arthritis and septicemia.

Staphylococcus aureus is an important bacterial pathogen with multiple virulence factors. The prevalence of antibiotic resistance among clinical isolates means that new vaccine strategies to prevent staphylococcal infections are needed. An ideal vaccine would induce antibodies to prevent bacterial adherence, promote opsonophagocytic killing by leukocytes and neutralize toxic secreted proteins.

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.

Capsules are produced by over 90% of Staphylococcus aureus strains, and approximately 25% of clinical isolates express type 5 capsular polysaccharide (CP5). We mutagenized the type 5 strain Reynolds with Tn918 to target genes involved in CP5 expression. From a capsule-deficient mutant, we cloned into a cosmid vector an approximately 26-kb EcoRI fragment containing the transposon insertion. In the absence of tetracycline selection, Tn918 was spontaneously excised, thereby resulting in a plasmid containing 9.4 kb of S. aureus DNA flanking the Tn918 insertion site. The 9.4-kb DNA fragment was used to screen a cosmid library prepared from the wild-type strain. Positive colonies were identified by colony hybridization, and a restriction map of one clone (pJCL19 with an approximately 34-kb insert) carrying the putative capsule gene region was constructed. Fragments of pJCL19 were used to probe genomic DNA digests from S. aureus strains of different capsular serotypes. Fragments on the ends of the cloned DNA hybridized to fragments of similar sizes in most of the strains examined. Blots hybridized to two fragments flanking the central region of the cloned DNA showed restriction fragment length polymorphism. A centrally located DNA fragment hybridized only to DNA from capsular types 2, 4, and 5. DNA from pJCL19 was subcloned to a shuttle vector for complementation studies. A 6.2-kb EcoRI-ClaI fragment complemented CP5 expression in a capsule-negative mutant derived by mutagenesis with ethyl methanesulfonate. These experiments provide the necessary groundwork for identifying genes involved in CP5 expression by S. aureus.

Type 8 capsular polysaccharide (CP8) is widely prevalent among clinical isolates of Staphylococcus aureus, but the role that the capsule plays in the pathogenesis of staphylococcal infections is unclear. This study was performed to identify growth conditions that would optimize the production of CP8 and to determine whether enhanced CP8 expression would influence staphylococcal virulence. S. aureus Becker grown in a chemically defined broth medium with < 1 microM ferric nitrate produced up to eightfold more CP8 per milligram of biomass than did bacteria cultivated in the same medium containing 20 microM ferric nitrate. The bacteria produced > 350-fold more cell-associated CP8 per milligram of biomass when grown on the surface of Columbia agar than when grown in Columbia broth. Most of the CP8 produced by broth-grown cells was secreted into the culture medium. S. aureus cultivated on the surface of nitrocellulose membranes floating on Columbia broth produced levels of CP8 similar to those produced by cells grown on Columbia agar. Similarly, bacteria harvested from endocardial vegetations of rabbits infected with S. aureus produced high levels of CP8. These results indicate that staphylococci grown on surfaces, both in vitro and in vivo, produce larger quantities of cell-associated CP8 than those grown in liquid cultures. However, no differences were observed in the 50% lethal dose for mice of strain Becker grown on solid medium (high levels of capsule expression) or in liquid medium (low levels of capsule expression).

The virulence of the Staphylococcus aureus strains that differed only in capsule expression was compared in a rat model of catheter-induced experimental endocarditis. The ID50 of all the strains was low (less than 3 x 10(3) cfu of S. aureus), suggesting that this model may be more sensitive than other animal models to differences in bacterial virulence. Compared with the wild-type strains that expressed type 5 or type 8 capsular polysaccharides, mutant strains devoid of capsule had significantly lower ID50 values. In contrast, a mutant that produced scant amounts of the type 5 polysaccharide had an ID50 similar to that of the parental type 5 isolate. As the bacterial inoculum was increased, each of the S. aureus strains reached final concentrations of 10(10)-10(11) cfu/g of vegetation; however, the nonencapsulated mutants colonized the left-sided vegetations at lower inocula than did the wild-type strains. This study indicates that microcapsule expression attenuates bacterial virulence in a rat model of catheter-induced endocarditis.

Most clinical isolates of Staphylococcus aureus produce microcapsules (uronic acid-containing extracellular polysaccharides) that are detectable by serologic methods but are not visible by negative staining. Among the 11 reported serotypes, capsule types 5 and 8 comprise approximately 75% of all isolates. Transposon mutagenesis was performed on S. aureus to create mutants altered in capsule expression. Tn918 was introduced into the capsule type 5 strain Reynolds by filter mating, and a capsule-deficient transconjugate, JL236, was isolated. The wild-type strain was transformed with JL236 chromosomal DNA to confirm that transfer of the appropriate-size chromosomal fragment containing Tn918 generated a capsule-deficient transformant. Strain Reynolds was mutagenized with ethyl methanesulfonate to obtain a capsule-negative mutant (strain JL240). Capsular phenotypes were determined by colony immunoblots, antibody adsorption experiments, and transmission electron microscopy. The virulences of the parental and mutant strains in mice were compared. The 50% lethal doses for strains Reynolds, JL236, and JL240 were similar (10(8.59), 10(8.98), and 10(8.93) CFU, respectively). Animals injected intraperitoneally with either wild-type or mutant strains had comparable levels of bacteremia at 3 and 24 h after challenge. Quantitative cultures of blood and kidneys from animals challenged intravenously with sublethal doses of the S. aureus strains also showed no differences in bacterial clearance or renal abscess formation. These studies indicate that the type 5 S. aureus microcapsule does not promote bacterial virulence in the animal models tested.

A colony immunoblot method was developed for serotyping the capsular polysaccharides expressed by Staphylococcus aureus isolates. The method was rapid and specific and was performed with either polyclonal or monoclonal antibodies specific for each of the capsule types. S. aureus isolates were obtained from patients with toxic shock syndrome (TSS) or other staphylococcal infections and from asymptomatic women with vaginal colonization. Among the vaginal isolates of S. aureus, expression of the type 8 capsule was significantly (P less than 0.001) more frequent among strains that produced TSS toxin 1 (TSST-1) than it was among TSST-1-negative strains. In contrast, the frequency of type 8 capsule expression was similar among both TSST-1-positive and -negative strains of S. aureus from patients with nonvaginal TSS. When all vaginal and nonvaginal isolates were compared, TSST-1-negative S. aureus strains were equally distributed among the type 5 and 8 and nontypeable capsule groups, whereas TSST-1-positive strains were predominantly capsule type 8.

In this study we report the isolation and purification of the capsular polysaccharide elaborated by Staphylococcus aureus SA1 mucoid. The capsule was isolated from bacterial extracts and culture supernatants by a series of ethanol precipitations and enzyme digestions, followed by ion-exchange chromatography. Teichoic acid contamination was eliminated by oxidation with sodium metaperiodate, and the final product eluted in the void volume of a Sephacryl S-300 column. The purified capsular polysaccharide was analyzed by gas-liquid chromatography-mass spectroscopy, 13C and 1H nuclear magnetic resonance, amino acid analysis, immunelectrophoresis, and numerous biochemical assays. The major constituents of the capsule were 2-acetamido-2-deoxy-alpha-galacturonic acid (4-O linked), 2-acetamido-2-deoxy-alpha-fucose (3-O linked), and taurine. The polysaccharide also contained O-acetyl groups which were removed by mild alkaline hydrolysis. Serologically and biochemically, the capsule from strain SA1 mucoid appeared very similar to that produced by strain M. Purified capsular polysaccharide was immunogenic in both rabbits and mice. The optimal immunizing dose in mice was 0.1 microgram of purified capsular polysaccharide administered intraperitoneally. SA1 mucoid resisted opsonophagocytic killing by human leukocytes and complement. However, antibodies raised to the purified capsular polysaccharide neutralized the antiphagocytic effect of the capsule.

In this study, we determined that immunization with capsular polysaccharide from Staphylococcus aureus could protect mice against nonlethal infections induced by encapsulated staphylococci. We immunized mice with either formalin-killed bacteria or purified capsular polysaccharide (PCP) and challenged them with one of three related S. aureus strains that varied in capsule size. Quantitative cultures of blood and kidney from the animals were performed to evaluate protection. Immunization with whole bacteria protected mice against infection with the homologous strain. Mice immunized with PCP were protected when challenged intravenously with either a highly encapsulated S. aureus strain or a microencapsulated mutant but not with an unencapsulated mutant. Protection correlated with capsular antibody levels in the immunized animals. Immunity to staphylococcal infection could be passively transferred to naive animals by using immune serum. These experiments suggest that the S. aureus capsular polysaccharide merits further study as a potential vaccine candidate for preventing staphylococcal infection.

A mouse model of abscess formation is described in which toxic shock syndrome toxin 1 (TSST-1) is produced by Staphylococcus aureus in vivo. Mice injected intravenously with S. aureus developed renal abscesses within 4-7 days. Kidneys excised from infected mice were cultured quantitatively, and extracts from the kidneys were assayed for TSST-1 with use of a competitive enzyme-linked immunosorbent assay. Animals with less than 10(7) S. aureus/g of kidney had less than 6 ng of TSST-1/mL of extract. Toxin levels ranged from less than 6 ng/mL to 271 ng/mL in kidney extracts from mice with greater than 10(7) S. aureus/g of kidney. Urine from infected mice also contained measurable levels of TSST-1 (range, less than 6-728 ng/mL). Mice developed serum antibodies to TSST-1 by 2 weeks after challenge. Serum samples collected 5-7 days after bacterial challenge did not show biochemical changes typical of a toxic shock-like illness in these animals.

We used three related strains of Staphylococcus aureus to determine whether capsule size influenced bacterial virulence. Strain SA1 mucoid elaborated a large capsule demonstrable by transmission electron microscopy (TEM). Nonmucoid isolates were derived from strain SA1 mucoid by Tn551 insertional mutagenesis. By TEM, strain JL24 produced a "microcapsule," whereas strain JL25 was unencapsulated. Strain SA1 mucoid had a 50% lethal dose for mice greater than 3,000-fold lower than that of strains JL24 and JL25. Quantitative cultures of blood and kidney from animals challenged intravenously revealed that strain SA1 mucoid was cleared less readily from the bloodstream and kidneys than the nonmucoid mutants. In an in vitro assay, only strain SA1 mucoid demonstrated antibody-dependent, complement-mediated opsonophagocytosis by human leukocytes. Strains JL24 and JL25 were opsonized for phagocytosis by complement alone. Thus a highly encapsulated strain of S. aureus was more virulent in mice than two related nonmucoid strains. The microencapsulated mutant was not more virulent than the unencapsulated mutant.