Pulmonary Infection of Mice with Staphylococcus aureus

The survival of Staphylococcus aureus in the lungs of mice was studied under various conditions. Doses of 10⁷ to 10⁹ washed staphylococci were quantitatively introduced into the lungs after intratracheal inoculation in mice under either ether or sodium pentobarbital anesthesia. Mice were sacrificed at intervals, the lungs were excised and homogenized, and the cocci were enumerated by plate count. The 50% lethal dose was 6 × 10⁸ cocci per mouse, and mice died within 24 h but without proliferation of the inoculum. Mice given 10⁸ cocci intratracheally under pentobarbital anesthesia regularly survived and eliminated the organisms over a 48-h period. The use of ether anesthesia resulted in persistence of the inoculum for up to 48 h, but the organisms were then eliminated. Inability to proliferate did not appear to result from a lack of iron because pretreatment of the mice with ferric ammonium citrate or Imferon did not alter inoculum survival. Staphylococci inoculated intratracheally in mice infected with influenza virus 3 to 21 days previously showed no enhanced persistence or multiplication. Cocci preclumped with fibrinogen, inocula mixed with 10 times the number of Formalin-killed staphylococci, or inocula of the encapsulated Smith strain did not survive any better than conventional inocula, suggesting that phagocytosis might not be the sole mechanism for elimination. However, a sedimentable fraction from normal or infected lung homogenates proved either inhibitory or cidal for staphylococci in vitro.     

Chlamydial Infection in Inducible Nitric Oxide Synthase Knockout Mice

Type 1 CD4⁺-T-cell-mediated immunity is crucial for the resolution of chlamydial infection of the murine female genital tract. Previous studies demonstrating a correlation between CD4⁺-T-cell-mediated inhibition of chlamydial growth and gamma interferon (IFN-γ)-mediated induction of nitric oxide synthase suggested a potential role for the nitric oxide (NO) effector pathway in the clearance of Chlamydia from genital epithelial cells by the immune system. To clarify the role of this pathway, the growth levels of Chlamydia trachomatis organisms in normal (iNOS^+/+) mice and in genetically engineered mice lacking the inducible nitric oxide synthase (iNOS) gene (iNOS^−/− mice) were compared. There was no significant difference in the course of genital chlamydial infections in iNOS^+/+ and iNOS^−/− mice as determined by recovery of Chlamydia organisms shed from genital epithelial cells. Dissemination of Chlamydia to the spleen and lungs occurred to a greater extent in iNOS^−/− than in iNOS^+/+ mice, which correlated with a marginal increase in the susceptibility of macrophages from iNOS^−/− mice to chlamydial infection in vitro. However, infections were rapidly cleared from all affected tissues, with no clinical signs of disease. The finding of minimal dissemination in iNOS^−/− mice suggested that activation of the iNOS effector pathway was not the primary target of IFN-γ during CD4⁺-T-cell-mediated control of chlamydial growth in macrophages because previous reports demonstrated extensive and often fatal dissemination of Chlamydia in mice lacking IFN-γ. In summary, these results indicate that the iNOS effector pathway is not required for elimination of Chlamydia from epithelial cells lining the female genital tract of mice although it may contribute to the control of dissemination of C. trachomatis by infected macrophages.     

Pathogenic Role of Macrophages in Intradermal Infection of Methicillin-Resistant Staphylococcus aureus in Thermally Injured Mice

Intradermal infection of methicillin-resistant Staphylococcus aureus (MRSA) in burned mice was pathogenically analyzed. An abscess was formed in normal mice intradermally infected with 10⁸ CFU/mouse of MRSA, and all of these mice survived after the infection; however, abscess formation was not demonstrated to occur in burned mice similarly exposed to the pathogen, and all of these mice died within 5 days of infection. In burned mice, MRSA infected at the burn site intradermal tissues spread quickly throughout the whole body, while in normal mice, the pathogen remained localized at the infection site. Macrophages (Mφ) isolated from the infection site tissues of normal mice produced interleukin-12 (IL-12) but not IL-10 and were characterized as M1Mφ. These M1Mφ were not isolated from the infection site tissues of burned mice. When normal-mouse infection site tissue Mφ were adoptively transferred to burned mice at the MRSA infection site, an abscess formed, and the infection did not develop into sepsis. In contrast, an abscess did not form and sepsis developed in normal mice that were inoculated with burned-mouse infection site tissue Mφ. These Mφ produced IL-10 but not IL-12 and were characterized as M2Mφ. These results indicate that abscess formation is a major mechanism of host resistance against intradermal MRSA infection. M1Mφ in the tissues surrounding the infection site play a pivotal role in abscess formation; however, the abscess is not formed in burned mice where M2Mφ predominate. M2Mφ have been described as inhibitor cells for Mφ conversion from resident Mφ to M1Mφ.Infection is the major cause of morbidity and mortality in severely burned patients (27, 33). Methicillin-resistant Staphylococcus aureus (MRSA) is known as a typical pathogen in such infections. Generally, healthy individuals are resistant against MRSA infection; however, severely burned patients with greatly suppressed immune responses are particularly susceptible to MRSA infection. In severely burned patients, MRSA was represented in 40% of the wounds, and 14% to 17% of all wounds became infected once they were colonized with MRSA (11, 14, 25). Therefore, intervention targeting the host''s antibacterial immune responses seems to be critical for successful regulation of MRSA infection.Recently, defects in antibacterial innate immunities have been demonstrated to occur in patients and animals following severe burn injuries (5). Innate immunity is the major host defense against early burn wound infection with MRSA (10), and classically activated macrophages (Mφ) (M1Mφ [interleukin-12-positive {IL-12⁺} IL-10⁻ Mφ]) have been identified as a major effector cell in host antimicrobial innate immunities (6, 19, 21). M1Mφ kill bacteria through the production of lysosomal enzymes, reactive oxygen intermediates, reactive nitrogen intermediates, and antimicrobial peptides (12, 21, 23). In general, resident Mφ (IL-12⁻ IL-10⁻ Mφ, isolatable from healthy donors) convert to M1Mφ following stimulation with invasive pathogens via pattern recognition receptors (19). Contrarily, a majority of thermally injured hosts are shown to be carriers of alternatively activated macrophages (M2Mφ [IL-12⁻ IL-10⁺ Mφ]) (15, 16, 32). M2Mφ have reduced abilities to kill bacteria, and soluble factors released from M2Mφ inhibit pathogen-stimulated macrophage conversion from resident Mφ to M1Mφ (15).Bacterial replication causes tissue destruction, and an abscess is commonly formed through this necrotizing process (18). An abscess, defined as a circumscribed collection of pus, is a very important host antibacterial defense against intradermal MRSA infection (8). The well-developed abscess has a wall or capsule of fibrous tissue separating it from the surrounding tissue. Histologically, the abscess lesions consist of accumulating leukocytes with live bacteria (26). In recent studies (7, 17, 26), abscess formation was caused by the accumulation of phagocytic Mφ to eliminate the pathogen. In this study, the influence of severe burn injury on abscess formation and bacterial growth at the infection site tissue was investigated for burned mice intradermally infected with MRSA. In the results, an abscess was not formed in burned mice, unlike in normal mice, after intradermal infection with MRSA. MRSA infected in burned mice at local intradermal tissues spread throughout the whole body quickly. In normal mice, IL-12-producing and IL-10-nonproducing Mφ (M1Mφ) were characterized as effector cells for abscess formation. After intradermal infection with MRSA, an abscess formed in burned mice that were inoculated with infection site tissue Mφ (M1Mφ). These results indicate that M1Mφ appearing in response to the MRSA infection are major effector cells in the host antibacterial defense. Through abscess formation, M1Mφ work to control the bacterial growth at the infection site and to inhibit the dissemination of the pathogen. M2Mφ inhibit Mφ conversion from resident Mφ to M1Mφ following bacterial stimulation. Therefore, an abscess is not formed in hosts where M2Mφ predominate, which results in the development of sepsis from local infections.     

Vaccine Protection of Leukopenic Mice against Staphylococcus aureus Bloodstream Infection

The risk for Staphylococcus aureus bloodstream infection (BSI) is increased in immunocompromised individuals, including patients with hematologic malignancy and/or chemotherapy. Due to the emergence of antibiotic-resistant strains, designated methicillin-resistant S. aureus (MRSA), staphylococcal BSI in cancer patients is associated with high mortality; however, neither a protective vaccine nor pathogen-specific immunotherapy is currently available. Here, we modeled staphylococcal BSI in leukopenic CD-1 mice that had been treated with cyclophosphamide, a drug for leukemia and lymphoma patients. Cyclophosphamide-treated mice were highly sensitive to S. aureus BSI and developed infectious lesions lacking immune cell infiltrates. Virulence factors of S. aureus that are key for disease establishment in immunocompetent hosts—α-hemolysin (Hla), iron-regulated surface determinants (IsdA and IsdB), coagulase (Coa), and von Willebrand factor binding protein (vWbp)—are dispensable for the pathogenesis of BSI in leukopenic mice. In contrast, sortase A mutants, which cannot assemble surface proteins, display delayed time to death and increased survival in this model. A vaccine with four surface antigens (ClfA, FnBPB, SdrD, and SpA_KKAA), which was identified by genetic vaccinology using sortase A mutants, raised antigen-specific immune responses that protected leukopenic mice against staphylococcal BSI.     

Biofilm Matrix Exoproteins Induce a Protective Immune Response against Staphylococcus aureus Biofilm Infection

The Staphylococcus aureus biofilm mode of growth is associated with several chronic infections that are very difficult to treat due to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the formation of S. aureus biofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response against S. aureus infections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and protein-based biofilm matrices produced by two clinical S. aureus strains. Remarkably, results showed that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and killing of S. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside a biofilm and on the surrounding tissue, using an in vivo model of mesh-associated biofilm infection. Furthermore, immunized mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle. Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine against S. aureus biofilm-associated infections.     

Development of Tetracycline-Resistant Staphylococcus aureus in Gnotobiotic Mice

Germ-free mice were colonized with a pigmented, tetracycline-sensitive strain of Staphylococcus aureus and maintained in flexible plastic isolators. Treatment of the gnotobiotic mice with oral tetracycline (20 mg/ml) resulted in the development of staphylococci resistant to tetracycline (5 mug/ml or higher). Resistant staphylococci did not appear in feces until several days after exposure of mice to the antibiotic and persisted for as long as specimens were collected (64 days subsequently). Resistance developed after a single exposure of gnotobiotes to antibiotic. Resistant staphylococci were present in the intestinal tracts of mice at counts of 10(3) per g of contents, whereas sensitive organisms coexisted at counts of 10(5) to 10(11) per g. Resistant staphylococci were isolated only from treated mice and not from untreated mice in adjacent cages. Initial colonization of germ-free mice with sensitive staphylococci interfered with subsequent colonization by resistant staphylococci and provided an example of bacterial interference. Resistance to tetracycline was not associated with resistance to chloramphenicol, penicillin, ampicillin, erythromycin, streptomycin, or kanamycin. Hydrolysis of gelatin was the only biochemical characteristic in which isolates varied but was not correlated with resistance to tetracycline or pigmentation of colonies. A nonpigmented, gray variant of S. aureus appeared in all specimens after colonization with the original, pigmented strain. Only the nonpigmented strain was obtained from gnotobiotes colonized with the nonpigmented variant. Contact between bacteria and antibiotic in the intestinal tract of gnotobiotes was considered to be essential for the development of tetracycline-resistant staphylococci.     

Protective Immunity against Recurrent Staphylococcus aureus Skin Infection Requires Antibody and Interleukin-17A

Although many microbial infections elicit an adaptive immune response that can protect against reinfection, it is generally thought that Staphylococcus aureus infections fail to generate protective immunity despite detectable T and B cell responses. No vaccine is yet proven to prevent S. aureus infections in humans, and efforts to develop one have been hampered by a lack of animal models in which protective immunity occurs. Our results describe a novel mouse model of protective immunity against recurrent infection, in which S. aureus skin and soft tissue infection (SSTI) strongly protected against secondary SSTI in BALB/c mice but much less so in C57BL/6 mice. This protection was dependent on antibody, because adoptive transfer of immune BALB/c serum or purified antibody into either BALB/c or C57BL/6 mice resulted in smaller skin lesions. We also identified an antibody-independent mechanism, because B cell-deficient mice were partially protected against secondary S. aureus SSTI and adoptive transfer of T cells from immune BALB/c mice resulted in smaller lesions upon primary infection. Furthermore, neutralization of interleukin-17A (IL-17A) abolished T cell-mediated protection in BALB/c mice, whereas neutralization of gamma interferon (IFN-γ) enhanced protection in C57BL/6 mice. Therefore, protective immunity against recurrent S. aureus SSTI was advanced by antibody and the Th17/IL-17A pathway and prevented by the Th1/IFN-γ pathway, suggesting that targeting both cell-mediated and humoral immunity might optimally protect against secondary S. aureus SSTI. These findings also highlight the importance of the mouse genetic background in the development of protective immunity against S. aureus SSTI.     

Role of Interleukin-17A in Cell-Mediated Protection against Staphylococcus aureus Infection in Mice Immunized with the Fibrinogen-Binding Domain of Clumping Factor A

Clumping factor A (ClfA) is a fibrinogen-binding cell wall-attached protein and an important virulence factor of Staphylococcus aureus. Previous studies reported that an immunization with the fibrinogen-binding domain of ClfA (ClfA_40-559) protected animals against S. aureus infection. It was reported that some cytokines are involved in the pathogenesis of staphylococcal diseases and in host defense against S. aureus infection. However, the role of cytokines in the protective effect of ClfA_40-559 as a vaccine has not been elucidated. In this study, we demonstrated that the spleen cells of ClfA_40-559-immunized mice produced a large amount of interleukin-17A (IL-17A). The protective effect of immunization was exerted in wild-type mice but not in IL-17A-deficient mice. IL-17A mRNA expression was increased in the spleens and kidneys of immunized mice after infection. CXCL2 and CCL2 mRNA expression was increased in the spleens and kidneys, respectively. Consistent with upregulation of the mRNA expression, neutrophils infiltrated into the spleens extensively and macrophage infiltration was observed in the kidneys of immunized mice. These results suggest that immunization with ClfA_40-559 induces the IL-17A-producing cells and that IL-17-mediated cellular immunity is involved in the protective effect induced by immunization with ClfA_40-559 against S. aureus infection.Staphylococcus aureus has been known as a major nosocomial pathogen and to cause a variety of infections, ranging from superficial infections to more life-threatening diseases (18). Recently, infections caused by community-acquired methicillin-resistant S. aureus have been reported to be increasing in healthy children and young adults (40). Most of clinically isolated strains show antibiotic resistance, leaving few options for effective antimicrobial therapy. Effective treatment for and prevention strategies against S. aureus infection are urgently needed.Various staphylococcal virulence factors have been identified as targets for novel therapeutics, including microbial surface components recognizing adhesive matrix molecule proteins (MSCRAMM) located on the surface of S. aureus. Clumping factor A (ClfA) is a fibrinogen-binding MSCRAMM and is expressed by virtually all S. aureus strains (15). ClfA is composed of 933 amino acids, and the structure of ClfA at the molecular level has been clarified (20, 21). Residues 40 to 559 compose region A, which contains the fibrinogen-binding domain. The biological roles of ClfA were also clarified in previous studies. ClfA promotes clumping of S. aureus in plasma and adherence of bacterial cells to blood clots and to plasma-conditioned biomaterials (21, 24, 36). These studies indicate that ClfA must be an important virulence factor of S. aureus in causing infections.The potential of ClfA as a vaccine target has been shown. Mice vaccinated with a recombinant region A of ClfA (ClfA_40-559) induced specific antibody production, and animals were protected against septic arthritis (15). In addition to active immunization, passive immunization with antibodies for ClfA_40-559 also protected animals (2, 6, 15, 38). Aurexis (tefibazumab), a humanized monoclonal antibody, was shown to enhance opsonophagocytosis and to protect animals against infective endocarditis caused by S. aureus (2, 6).Pathogen-activated antigen-presenting cells induce activation and differentiation of naïve CD4⁺ T cells into T helper (Th) cells, and the resultant cells play an important role in immune responses by exertion of a variety of effector functions. Gamma interferon (IFN-γ) produced by Th1 cells is involved in immune response to combat intracellular pathogens. Cytokines produced by Th2 cells are essential for the generation of antibodies to eliminate extracellular pathogens (32). Interleukin-17 (IL-17) is produced mainly by Th17 cells (7, 30). IL-17 regulates CXC and CC chemokine expression and several proinflammatory cytokines (4, 29, 30). Recent studies showed that IL-17 plays an important role in cell-mediated host defense, especially against extracellular bacterial infections (4, 29). The roles of specific cell-mediated responses in pathogenesis of staphylococcal diseases and host defense against S. aureus infection were previously reported. Delayed hypersensitivity to staphylococcal antigens developed in mice after repeated staphylococcal infections (3). S. aureus capsular polysaccharide activates T cells and induces production of IFN-γ, which potentiates pathogenesis of S. aureus (22). It was also reported that various T cell populations activated by vaccination are involved in adaptive host defense against S. aureus infection. Gómez et al. reported that the ratio of IFN-γ-producing CD4⁺ and CD8⁺ T cells increased in mice immunized with an S. aureus mutant via an intramammary route and that IFN-γ plays an important role in the eradication of intracellular staphylococci (5). Lin et al. showed that vaccination with the recombinant N terminus of the candidal Als3p adhesin induced CD4⁺ lymphocyte-derived IL-17A, which is necessary for vaccine efficacy against S. aureus and Candida albicans (16).In this study, we investigated the role of cytokines in the protective effect against S. aureus systemic infection in mice immunized with ClfA_40-559. We demonstrate that immunization with ClfA_40-559 induces IL-17A-producing cells and that IL-17A plays a role in the protective effect against systemic S. aureus infection in ClfA_40-559-immunized mice.     

Role of Nitric Oxide in Ovulation, Meiotic Maturation of Oocytes, and Implantation in Mice

Nitric oxide synthase blockers inhibited meiotic maturation of oocytes, decreased the count of oocytes during hormonal stimulation of superovulation, and increased embryonic death in experimental females mated with intact males.     

Proteomic Identification of saeRS-Dependent Targets Critical for Protective Humoral Immunity against Staphylococcus aureus Skin Infection

Recurrent Staphylococcus aureus skin and soft tissue infections (SSTIs) are common despite detectable antibody responses, leading to the belief that the immune response elicited by these infections is not protective. We recently reported that S. aureus USA300 SSTI elicits antibodies that protect against recurrent SSTI in BALB/c but not C57BL/6 mice, and in this study, we aimed to uncover the specificity of the protective antibodies. Using a proteomic approach, we found that S. aureus SSTI elicited broad polyclonal antibody responses in both BALB/c and C57BL/6 mice and identified 10 S. aureus antigens against which antibody levels were significantly higher in immune BALB/c serum. Four of the 10 antigens identified are regulated by the saeRS operon, suggesting a dominant role for saeRS in protection. Indeed, infection with USA300Δsae failed to protect against secondary SSTI with USA300, despite eliciting a strong polyclonal antibody response against antigens whose expression is not regulated by saeRS. Moreover, the antibody repertoire after infection with USA300Δsae lacked antibodies specific for 10 saeRS-regulated antigens, suggesting that all or a subset of these antigens are necessary to elicit protective immunity. Infection with USA300Δhla elicited modest protection against secondary SSTI, and complementation of USA300Δsae with hla restored protection but incompletely. Together, these findings support a role for both Hla and other saeRS-regulated antigens in eliciting protection and suggest that host differences in immune responses to saeRS-regulated antigens may determine whether S. aureus infection elicits protective or nonprotective immunity against recurrent infection.     

Regulatory Role of Nitric Oxide in Cutaneous Inflammation

Inflammation - Nitric oxide (NO), a signaling molecule, regulates biological functions in multiple organs/tissues, including the epidermis, where it impacts permeability barrier homeostasis, wound...     

Regulatory Role of Nitric Oxide in Neutrophil Apoptosis

Apoptosis of blood neutrophils from healthy donors was studied under conditions of cell culturing with different concentrations of H₂O₂, selective NO synthase inhibitor, and inductor of NO synthesis (L-arginine). In vitro incubation of neutrophilic leukocytes with 5 mM H₂O₂ led to activation of the apoptotic program in neutrophils, which was seen from increased content of Bax protein in the cells and increased number of apoptotic cells in the culture. Increased content of annexin-positive cells after incubation of neutrophil culture with NO synthase inhibitor suggests involvement of NO in the regulation of neutrophil apoptosis under conditions of oxidative stress, while L-arginine prevented H₂O₂-induced programmed cell death. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 146, No. 12, pp. 646–650, December, 2008     

Role of monocytes in experimental Staphylococcus aureus endocarditis

In the pathogenesis of bacterial endocarditis (BE), the clotting system plays a cardinal role in the formation and maintenance of the endocardial vegetations. The extrinsic pathway is involved in the activation of the coagulation pathway with tissue factor (TF) as the key protein. Staphylococcus aureus is a frequently isolated bacterium from patients with BE. We therefore investigated whether S. aureus can induce TF activity (TFA) on fibrin-adherent monocytes, used as an in vitro model of BE. We also assessed in vivo in rabbits with catheter induced vegetations, the effect of S. aureus infection on vegetational TFA. In vitro experiments showed that adherent S. aureus induced TFA on fibrin-adherent monocytes which was optimal at a bacterium/monocyte ratio of 1 to 1. Monocyte damage occurred when this ratio exceeded 4 to 1 (visually) or 6 to 1 (propidium iodide influx) Consequently, TFA decreased. In vivo S. aureus led to very high bacterial numbers in the vegetations and a significant increase of their weight. However, TFA of infected vegetations was the same as of sterile ones. This may be due to the high bacteria to monocyte ratio as well as bacterium-induced monocyte damage. Teicoplanin treatment of infected rabbits reduced bacterial numbers in the blood and in the vegetations. Two-day treatment resulted in an increase of vegetational TFA, but after four-day treatment vegetational TFA dropped, most probably due to a suboptimal bacterium/monocyte ratio. S. aureus endocarditis in etoposide (Vepesid)-treated rabbits, leading to a selective monocytopenia, caused a rapid death of the animals. In these rabbits no vegetations were found at all. We conclude that, like Streptococcus sanguis and Staphylococcus epidermidis, S. aureus is able to induce TFA in fibrin-adherent blood monocytes. In addition, monocytes have a protective effect during the course of S. aureus endocarditis.     

Effect of Diuresis on Staphylococcus aureus Kidney Infections in Mice

In the Cornett strain of mice, water diuresis did not prevent hematogenous production of pyelonephritis by Staphylococcus aureus. Increased fluid intake did not affect the numbers of organisms deposited in the kidneys or the rate of growth during the first 4 hr after inoculation. Drinking the glucose solution did not enhance bacterial proliferation within the renal parenchyma. Subcutaneous injection of saline to supplement for interruption of drinking after inoculation reduced the numbers of organisms recovered in the kidneys but not sufficiently to prevent production of pyelonephritis. Incorporating penicillin as a marker indicated that fluids administered by subcutaneous injections were rapidly delivered to the kidneys. Combining diuresis with treatment did not influence the rapidity of delivery of antimicrobial to the kidneys or the length of time that it was present in the renal homogenate.     

Effects of Intrarenal Inoculation of Staphylococcus aureus on Mice

The extreme susceptibility of the mouse kidney to infection with Staphylococcus aureus was confirmed by direct intrarenal (i.r.) inoculation with this organism. By applying Poisson's distribution formula to the results from infection of mice with small inocula (as few as one coccus or less per kidney), it was estimated that even three organisms could multiply in situ to produce abscess lesions, if the organisms were inoculated directly into the kidneys. This susceptibility of the mouse kidney for staphylococcal infection was not uniformly manifested with every strain, but correlated well with the virulence found through intravenous infection of the strains tested. The i.r. inoculation method by which infection of mice was easily established, using a small inoculum of S. aureus, similar to the amount suspected to occur in natural infections of man, is applicable for the analysis of the mechanisms of staphylococcal infections and any resulting immunity in man. The present paper describes the details and some experimental results obtained by our method.     

Manipulation of Autophagy in Phagocytes Facilitates Staphylococcus aureus Bloodstream Infection

The capacity for intracellular survival within phagocytes is likely a critical factor facilitating the dissemination of Staphylococcus aureus in the host. To date, the majority of work on S. aureus-phagocyte interactions has focused on neutrophils and, to a lesser extent, macrophages, yet we understand little about the role played by dendritic cells (DCs) in the direct killing of this bacterium. Using bone marrow-derived DCs (BMDCs), we demonstrate for the first time that DCs can effectively kill S. aureus but that certain strains of S. aureus have the capacity to evade DC (and macrophage) killing by manipulation of autophagic pathways. Strains with high levels of Agr activity were capable of causing autophagosome accumulation, were not killed by BMDCs, and subsequently escaped from the phagocyte, exerting significant cytotoxic effects. Conversely, strains that exhibited low levels of Agr activity failed to accumulate autophagosomes and were killed by BMDCs. Inhibition of the autophagic pathway by treatment with 3-methyladenine restored the bactericidal effects of BMDCs. Using an in vivo model of systemic infection, we demonstrated that the ability of S. aureus strains to evade phagocytic cell killing and to survive temporarily within phagocytes correlated with persistence in the periphery and that this effect is critically Agr dependent. Taken together, our data suggest that strains of S. aureus exhibiting high levels of Agr activity are capable of blocking autophagic flux, leading to the accumulation of autophagosomes. Within these autophagosomes, the bacteria are protected from phagocytic killing, thus providing an intracellular survival niche within professional phagocytes, which ultimately facilitates dissemination.     

Magnetic Nanoparticle Targeted Hyperthermia of Cutaneous Staphylococcus aureus Infection

The incidence of wound infections that do not adequately respond to standard-of-care antimicrobial treatment has been increasing. To address this challenge, a novel antimicrobial magnetic thermotherapy platform has been developed in which a high-amplitude, high-frequency, alternating magnetic field is used to rapidly heat magnetic nanoparticles that are bound to Staphylococcus aureus (S. aureus). The antimicrobial efficacy of this platform was evaluated in the treatment of both an in vitro culture model of S. aureus biofilm and a mouse model of cutaneous S. aureus infection. We demonstrated that an antibody-targeted magnetic nanoparticle bound to S. aureus was effective at thermally inactivating S. aureus and achieving accelerated wound healing without causing tissue injury.     

Community-Acquired Infection Caused by Small-Colony Variant of Staphylococcus aureus

Staphylococcus aureus and other Gram negative bacteria produce small colony variants (SCV) which usually emerge after exposure to antimicrobials. They cause repeated infections, treatment failures and often pass unnoticed during cultures due to unusual appearance and incomplete incubation. This infectious disease grand round highlights a similar clinical case with atypical history and appearance of a SCV of S. aureus and why prolonged incubation is necessary for aspirates from patients with recurrent infections like abscesses.     

Lack of a Major Role of Staphylococcus aureus Panton-Valentine Leukocidin in Lower Respiratory Tract Infection in Nonhuman Primates

Panton-Valentine leukocidin (PVL) is a two-component cytolytic toxin epidemiologically linked to community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections, including serious invasive infections caused by the epidemic clone referred to as strain USA300. Although PVL has long been known to be a S. aureus virulence molecule in vitro, the relative contribution of this leukotoxin to invasive CA-MRSA infections such as pneumonia remains controversial. We developed a nonhuman primate model of CA-MRSA pneumonia and used it to test the hypothesis that PVL contributes to lower respiratory tract infections caused by S. aureus strain USA300. The lower respiratory tract disease observed in this monkey model mimicked the clinical and pathological features of early mild to moderate S. aureus pneumonia in humans, including fine-structure histopathology. In this experiment using a large sample of monkeys and multiple time points of examination, no involvement of PVL in virulence could be detected. Compared with the wild-type parental USA300 strain, the isogenic PVL deletion-mutant strain caused equivalent lower respiratory tract pathology. We conclude that PVL does not contribute to lower respiratory tract infection in this nonhuman primate model of human CA-MRSA pneumonia.Staphylococcus aureus is the most abundant cause of serious bacterial infections in the United States.^1–4 In addition, the organism acquires antibiotic resistance readily and methicillin-resistant S. aureus (MRSA) strains have been a major concern in health care facilities for decades.^3–7 The pathogen causes a wide spectrum of infections, ranging from mild skin problems to fatal invasive diseases.¹ The mortality rate associated with invasive MRSA infections is approximately 20%, the majority of which are health care– or hospital-associated.¹ Inasmuch as health care–associated S. aureus infections occur in individuals with predisposing risk factors, the health status of the patient plays a prominent role. In contrast, community-associated MRSA (CA-MRSA) strains generally cause infections in otherwise healthy individuals.CA-MRSA emerged unexpectedly in the 1990s and is now epidemic in the United States.^2,8,9 The prototype CA-MRSA strain in the United States, known as pulsed-field type USA400 (MW2), caused fatal pneumonia in children in the Midwest.^10,11 Whole-genome sequencing of a USA400 strain revealed the presence of a novel methicillin-resistance element SCCmecIV and lukS-PV and lukF-PV (lukS/F-PV) genes encoding a two-component cytolytic toxin known as Panton-Valentine leukocidin (PVL).¹² Although strains of USA400 remain a significant cause of CA-MRSA infections, especially in Canada,^13,14 they have been nearly replaced in the United States by the pulsed-field type USA300 epidemic clone (USA300).^9,15,16 Notably, SCCmecIV and PVL genes are also present in the genome of USA300,¹⁷ and thus, they are strongly linked by epidemiology to CA-MRSA infections in the United States. The great majority of USA300 infections involve skin and soft tissue, accounting for 75% to 95% of CA-MRSA infections.^8,16,18,19 However, this pathogen also has the ability to cause severe invasive disease, including cases of fatal pneumonia.^20,21Several lines of evidence suggest that PVL may be an important virulence factor in the pathogenesis of S. aureus pneumonia. Lina and colleagues first described an association of PVL with S. aureus strains that cause primary community-acquired pneumonia, some of which were fatal cases of necrotizing pneumonia.²² Subsequently, PVL-producing S. aureus, mostly methicillin-susceptible S. aureus strains, were reported to be associated with necrotizing pneumonia in young immunocompetent individuals.²³ Recent reports also document that PVL-positive strains of USA300 can cause necrotizing pneumonia, albeit these infections are infrequent and often associated with influenza or parainfluenza virus coinfection.²⁴Despite the epidemiological linkage, there is currently little direct experimental evidence that PVL is a primary virulence factor for CA-MRSA pneumonia. One study suggested that PVL promotes S. aureus pneumonia in a mouse infection model.²⁵ However, subsequent work by several investigators using wild-type USA300 and USA400 and isogenic PVL deletion mutant strains failed to support the idea that PVL contributes to experimental CA-MRSA pneumonia in mice or rats.^26–29Elucidating whether PVL has a role in pneumonia in humans is critical because the leukotoxin is widely considered to be a possible determining factor in CA-MRSA pneumonia, and therefore, it has become a target for antistaphylococcal therapeutics and vaccines.³⁰ We developed a nonhuman primate model of S. aureus lower respiratory tract infection and used it to test the hypothesis that USA300 wild-type and isogenic PVL mutant strains differ in their ability to cause CA-MRSA pneumonia.