Phagocytic killing of encapsulated and microencapsulated Staphylococcus aureus by human polymorphonuclear leukocytes.

Phagocytosis by human polymorphonuclear leukocytes (PMNs) is an important host defense against infections caused by Staphylococcus aureus. Using an in vitro assay, we compared the opsonic requirements for phagocytic killing of prototype strains of encapsulated (type 1) and microencapsulated (type 5 and type 8) S. aureus by human PMNs. More than 85% of broth-grown, logarithmic-phase type 5 and 8 S. aureus organisms were killed by PMNs incubated with fresh normal human, rabbit, or guinea pig serum with complement activity. Under similar conditions, the highly encapsulated type 1 strain was not killed. Both encapsulated and microencapsulated strains were opsonized for phagocytosis by heat-inactivated serum raised in rabbits to killed bacteria. Opsonization by homologous serum was required for phagocytosis of the type 1 strain. In contrast, microencapsulated type 5 and 8 S. aureus organisms were killed by heat-inactivated rabbit serum raised to type 5, type 8, or nonencapsulated isolates; this result suggested that antibodies to the capsule or to cell wall components other than the capsule could opsonize these organisms for phagocytosis. The specificity of the assay was confirmed with capsule type 5-specific monoclonal antibodies, which were opsonic only for the type 5 S. aureus isolate. These studies indicate that, unlike the highly encapsulated type 1 strain, broth-grown microencapsulated S. aureus strains do not resist opsonophagocytic killing in vitro by normal serum.     

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.     

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.     

Localization of the third component of complement on the cell wall of encapsulated Staphylococcus aureus M: implications for the mechanism of resistance to phagocytosis.

Encapsulated Staphylococcus aureus strains are more virulent than unencapsulated staphylococci, and this phenomenon has been associated with decreased opsonization of encapsulated bacteria by normal human serum. Peptidoglycan, a major cell wall component of S. aureus, has been shown to promote opsonization of this bacterial species by certain components of the serum complement system. However, when the processes of complement activation and opsonization of encapsulated staphylococci have been studied, it has been found that encapsulated bacteria activate complement efficiently and C3 is bacteria associated, yet these organisms are not efficiently phagocytized by human polymorphonuclear leukocytes. In this study, the hypothesis was tested that opsonically active molecules are not on the true external surface of encapsulated organisms but rather are cell wall associated and thus "hidden" from human polymorphonuclear leukocytes. By using immunoelectronmicroscopy, C3 was found to be localized on the cell wall of an encapsulated S. aureus strain after incubation of the organism in normal human serum. When shrinkage of the capsule was prevented by treatment with anticapsular antibody, the C3 was again shown to be cell wall associated and located beneath the bacterial capsule. These results suggest that encapsulated S. aureus may resist phagocytosis because opsonically active C3 molecules are not exposed at the true external surface of the bacterium.     

Gentamicin encapsulated within a biopolymer for the treatment of Staphylococcus aureus and Escherichia coli infected skin ulcers

Abstract

Skin wound infection requires carefully long-term treatment with an immense financial burden to healthcare systems worldwide. Various strategies such as drug delivery systems using polymer matrix from natural source have been used to enhance wound healing. Natural rubber latex (NRL) from Hevea brasiliensis has shown angiogenic and tissue repair properties. Gentamicin sulfate (GS) is a broad-spectrum antibiotic which inhibits the growth of a wide variety of microorganisms and, because of this, it has also been applied topically for treatment of local infections. The aim of this study was to develop a GS release system using NRL as matrix for Staphylococcus aureus and Escherichia coli infected skin ulcers treatment, without changing drug antibiotic properties. The matrix did not change the GS antimicrobial activity against S. aureus and E. coli strains. Moreover, the NRL-GS biomembrane did not exhibit hemolytic activity, being non-toxic to red blood cells. The eluates of NRL-GS biomembranes and GS solutions did not significantly reduce the survival of Caenorhabditis elegans worms for 24?h at any of the tested concentrations. Thus, these results emphasize that the NRL-GS biomembrane proved to be a promising biomaterial for future studies on the development of dressings for topical uses, inexpensive and practicable, keeping drug antibiotic properties against pathogens and to reduce the side effects.     

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.     

Livestock-associated Staphylococcus aureus

Staphylococcus aureus has long been associated with livestock. Livestock can be carriers of S. aureus, but can also become infected. The best-known infection is bovine mastitis. The discovery of methicillin-resistant S. aureus belonging to sequence type (ST)398 boosted interest in livestock-associated S. aureus. ST398 is pandemic. Whole genome sequencing and other genetic analyses have shown that livestock-associated strains are distinct from human-derived strains. However, there is also an exchange of strains between the reservoirs. Livestock-associated and human-associated strains share virulence factors, but have also distinct virulence factors that appear to be important in host adaptation. Exchange of genes encoding these virulence factors between strains may expand the host range and thereby threaten public health. Vaccination of animals may be a solution to this problem, but new avenues for vaccination need to be explored, because no vaccine is currently available.     

Staphylococcus aureus bacteremia

Staphylococcus aureus bacteremia (SAB) is still associated with a high mortality, and knowledge on risk factors and the clinical and the therapeutic aspects of SAB is still limited. This thesis focuses on the clinical aspects of SAB and its metastatic infections. In a study of all patients with bacteremia in Copenhagen County October 1992 through April 1993 (study I) we emphasized previous findings, that S. aureus is one of the most frequent pathogens in bacteremia, and in a case control study also in Copenhagen County 1994-95 (study II) we demonstrated, that not only an inserted central venous catheter and nasal S. aureus carriage but also hyponatremia and anemia are important risk factors for hospital-acquired SAB (study II). Studies on the treatment of SAB have pointed out, that the eradication of a primary is important, but there are only limited clinical studies dealing with antibiotic treatment. By logistic regression analysis, we were able to demonstrate that focus eradication is essential, but also that treatment with dicloxacillin 1 g x 4 or 2 g x 3 are superior to 1 g x 3 (studie III), indicating that the time for serum concentration above the Minimal Inhibitory Concentration (MIC) for the bacteria plays a role in the outcome of SAB treatment. S. aureus osteomyelitis secondary to SAB is frequently observed. No other countries, however, have a centralized registration, which make it possible to evaluate a large number of these patients. Since 1960, The Staphylococcal Laboratory, Statens Serum Institut in Copenhagen, has registrated selected clinical informations from nearly all patients with positive blood cultures of S. aureus. Based on this registration, we were able to show an increased number of S. aureus osteomyelitis among older patients and a decreased number of S. aureus osteomyelitis of femur and tibia among younger infants in the period 1980-90 (study IV). By reviewing the records of a large number of patients with vertebral S. aureus osteomyelitis, we could evaluate important aspects in the diagnosis and treatment of these patients (study V). We illustrated, that symptoms and laboratory findings were relatively unspecific, and CT-scanning or bone scintigraphy were absolutely necessary for the diagnosis (study V). The relatively high number of patients in the study allowed us to evaluate different treatment regimens, and we found, that treatment with penicillinase-stable penicillins four grams daily for at least eight weeks was necessary (study V). S. aureus meningitis is relatively uncommon and most often a neurosurgical infection based on the presence of a catheter. Meningitis secondary to SAB is relatively rare. The nationwide registration on Statens Serum Institut enabled us to study a large number of patients with special emphasis on clinical, outcome and treatment (study VI). We found, that these patients often were older people with chronic underlying diseases, the infection developed as a community-acquired infection, and the patients also had an unknown focus of infection. Furthermore, these patients often had other secondary manifestations such as endocarditis or osteomyelitis and an extremely high mortality (study VI). Finally, I believe that our studies will contribute to reduce the incidence of SAB and improve the diagnosis and treatment of SAB in the future.     

Staphylococcus aureus on the hair

Staphylococcus aureus was found to be present on the hair of about 10% of people having no hospital contact and on that of about 50% of patients with skin diseases.     

Unusual properties of Staphylococcus aureus strains of the new epidemic phage type 95

Three hundred and seventy-one penicillin-resistant Staphylococcus aureus strains of phage type 95 isolated in the years 1977-1983 were investigated. They had characteristic resistance patterns to cadmium (Cd), arsenate (As) and mercury (Hg). Most were susceptible to all three heavy metals; 25 strains were resistant to As only and one to Cd and As. The susceptible strains had a uniform medium level of penicillinase production, whereas the As-resistant strains produced large amounts of penicillinase. In most of the strains, penicillin resistance was located on a very unstable penicillinase plasmid. The combination of rare properties found in Danish type-95 strains seems to point to the spread of one or two clones. Co-reactions with other phage groups or complexes and results of lysogenisation experiments suggest that the Danish type-95 strains are derived from strains of the 52, 52A, 80, 81 complex.     

Exotoxins of Staphylococcus aureus

This article reviews the literature regarding the structure and function of two types of exotoxins expressed by Staphylococcus aureus, pyrogenic toxin superantigens (PTSAgs) and hemolysins. The molecular basis of PTSAg toxicity is presented in the context of two diseases known to be caused by these exotoxins: toxic shock syndrome and staphylococcal food poisoning. The family of staphylococcal PTSAgs presently includes toxic shock syndrome toxin-1 (TSST-1) and most of the staphylococcal enterotoxins (SEs) (SEA, SEB, SEC, SED, SEE, SEG, and SEH). As the name implies, the PTSAgs are multifunctional proteins that invariably exhibit lethal activity, pyrogenicity, superantigenicity, and the capacity to induce lethal hypersensitivity to endotoxin. Other properties exhibited by one or more staphylococcal PTSAgs include emetic activity (SEs) and penetration across mucosal barriers (TSST-1). A detailed review of the molecular mechanisms underlying the toxicity of the staphylococcal hemolysins is also presented.     

Possible common biological and immunological properties for detecting encapsulated strains of Staphylococcus epidermidis.

Twenty strains of capsular type II Staphylococcus epidermidis, determined by the method of Ichiman, were obtained from clinical specimens. Among them, 5, 5, and 10 strains were 4+, 3+, and 2+ in the intensities of their reactions against fluorescent antibody, respectively. Strains exhibiting 4+ and 3+ intensities were mouse virulent and phage nontypable, while 2+ strains were mouse avirulent and phage typable. When three strains randomly selected from each of the mouse-virulent and mouse-avirulent strains were compared in terms of their cell volume indices, all mouse-virulent strains had significantly higher indices (average, 1.86 times) than the mouse-avirulent strains. With intraperitoneal injection of the strains into mice, strains with higher cell volume indices resisted ingestion by peritoneal cells, while strains with low cell volume indices were sensitive to phagocytosis. When the capacity to absorb a definite amount of passive protective activity in rabbit antiserum prepared with capsular type II strains was compared among these strains, 10 to 20 mg of mouse-virulent strains was capable of completely absorbing the passive protective activity, whereas more than 80 mg of the cells was required for similar absorption by mouse-avirulent strains. In ultra-thin sections of three mouse-virulent strains stained with ferritin-conjugated rabbit antiserum, well-defined capsules were detected around cell walls; however, no capsule was seen around the walls of three mouse-avirulent strains.     

Emergence of vancomycin resistance in Staphylococcus aureus. Glycopeptide-Intermediate Staphylococcus aureus Working Group

BACKGROUND: Since the emergence of methicillin-resistant Staphylococcus aureus, the glycopeptide vancomycin has been the only uniformly effective treatment for staphylococcal infections. In 1997, two infections due to S. aureus with reduced susceptibility to vancomycin were identified in the United States. METHODS: We investigated the two patients with infections due to S. aureus with intermediate resistance to glycopeptides, as defined by a minimal inhibitory concentration of vancomycin of 8 to 16 microg per milliliter. To assess the carriage and transmission of these strains of S. aureus, we cultured samples from the patients and their contacts and evaluated the isolates. RESULTS: The first patient was a 59-year-old man in Michigan with diabetes mellitus and chronic renal failure. Peritonitis due to S. aureus with intermediate resistance to glycopeptides developed after 18 weeks of vancomycin treatment for recurrent methicillin-resistant S. aureus peritonitis associated with dialysis. The removal of the peritoneal catheter plus treatment with rifampin and trimethoprim-sulfamethoxazole eradicated the infection. The second patient was a 66-year-old man with diabetes in New Jersey. A bloodstream infection due to S. aureus with intermediate resistance to glycopeptides developed after 18 weeks of vancomycin treatment for recurrent methicillin-resistant S. aureus bacteremia. This infection was eradicated with vancomycin, gentamicin, and rifampin. Both patients died. The glycopeptide-intermediate S. aureus isolates differed by two bands on pulsed-field gel electrophoresis. On electron microscopy, the isolates from the infected patients had thicker extracellular matrixes than control methicillin-resistant S. aureus isolates. No carriage was documented among 177 contacts of the two patients. CONCLUSIONS: The emergence of S. aureus with intermediate resistance to glycopeptides emphasizes the importance of the prudent use of antibiotics, the laboratory capacity to identify resistant strains, and the use of infection-control precautions to prevent transmission.     

Frequency and some properties of clinical isolates of methicillin-resistant Staphylococcus aureus.

Of 420 Staphylococcus aureus isolates, 3.1% were methicillin resistant. Most of the 13 isolates were from the flora of hospitalized patients. The organisms were also resistant to nafcillin and cephalothin. They shared many of the properties with methicillin-resistant staphylococci accumulated from other sources except for the lack of lysozyme-like activity.