Mild Staphylococcus aureus Skin Infection Improves the Course of Subsequent Endogenous S. aureus Bacteremia in Mice

Staphylococcus aureus carriers with S. aureus bacteremia may have a reduced mortality risk compared to non-carriers. A role for the immune system is suggested. Here, we study in mice the effect of mild S. aureus skin infection prior to endogenous or exogenous S. aureus bacteremia, and evaluate protection in relation to anti-staphylococcal antibody levels. Skin infections once or twice by a clinical S. aureus isolate (isolate P) or S. aureus strain 8325-4 were induced in mice free of S. aureus and anti-staphylococcal antibodies. Five weeks later, immunoglobulin G (IgG) levels in blood against 25 S. aureus antigens were determined, and LD50 or LD100 bacteremia caused by S. aureus isolate P was induced. S. aureus skin infections led to elevated levels of anti-staphylococcal IgG in blood. One skin infection improved the course of subsequent severe endogenous bacteremia only. A second skin infection further improved animal survival rate, which was associated with increased pre-bacteremia IgG levels against Efb, IsaA, LukD, LukE, Nuc, PrsA and WTA. In conclusion, S. aureus isolate P skin infection in mice reduces the severity of subsequent endogenous S. aureus bacteremia only. Although cellular immune effects cannot be rules out, anti-staphylococcal IgG against specified antigens may contribute to this effect.     

Increased Susceptibility of Humanized NSG Mice to Panton-Valentine Leukocidin and Staphylococcus aureus Skin Infection

Staphylococcus aureus is a leading cause of skin and soft-tissue infections worldwide. Mice are the most commonly used animals for modeling human staphylococcal infections. However a supra-physiologic S. aureus inoculum is required to establish gross murine skin pathology. Moreover, many staphylococcal factors, including Panton-Valentine leukocidin (PVL) elaborated by community-associated methicillin-resistant S. aureus (CA-MRSA), exhibit selective human tropism and cannot be adequately studied in mice. To overcome these deficiencies, we investigated S. aureus infection in non-obese diabetic (NOD)/severe combined immune deficiency (SCID)/IL2rγ^null (NSG) mice engrafted with human CD34⁺ umbilical cord blood cells. These “humanized” NSG mice require one to two log lower inoculum to induce consistent skin lesions compared with control mice, and exhibit larger cutaneous lesions upon infection with PVL⁺ versus isogenic PVL^- S. aureus. Neutrophils appear important for PVL pathology as adoptive transfer of human neutrophils alone to NSG mice was sufficient to induce dermonecrosis following challenge with PVL⁺ S. aureus but not PVL^- S. aureus. PMX53, a human C5aR inhibitor, blocked PVL-induced cellular cytotoxicity in vitro and reduced the size difference of lesions induced by the PVL⁺ and PVL^- S. aureus, but PMX53 also reduced recruitment of neutrophils and exacerbated the infection. Overall, our findings establish humanized mice as an important translational tool for the study of S. aureus infection and provide strong evidence that PVL is a human virulence factor.     

Active Immunization with an Octa-Valent Staphylococcus aureus Antigen Mixture in Models of S. aureus Bacteremia and Skin Infection in Mice

Proteomic studies with different Staphylococcus aureus isolates have shown that the cell surface-exposed and secreted proteins IsaA, LytM, Nuc, the propeptide of Atl (pro-Atl) and four phenol-soluble modulins α (PSMα) are invariantly produced by this pathogen. Therefore the present study was aimed at investigating whether these proteins can be used for active immunization against S. aureus infection in mouse models of bacteremia and skin infection. To this end, recombinant His-tagged fusions of IsaA, LytM, Nuc and pro-Atl were isolated from Lactococcus lactis or Escherichia coli, while the PSMα1-4 peptides were chemically synthesized. Importantly, patients colonized by S. aureus showed significant immunoglobulin G (IgG) responses against all eight antigens. BALB/cBYJ mice were immunized subcutaneously with a mixture of the antigens at day one (5 μg each), and boosted twice (25 μg of each antigen) with 28 days interval. This resulted in high IgG responses against all antigens although the response against pro-Atl was around one log lower compared to the other antigens. Compared to placebo-immunized mice, immunization with the octa-valent antigen mixture did not reduce the S. aureus isolate P load in blood, lungs, spleen, liver, and kidneys in a bacteremia model in which the animals were challenged for 14 days with a primary load of 3 × 10⁵ CFU. Discomfort scores and animal survival rates over 14 days did not differ between immunized mice and placebo-immunized mice upon bacteremia with S. aureus USA300 (6 × 10⁵ CFU). In addition, this immunization did not reduce the S. aureus isolate P load in mice with skin infection. These results show that the target antigens are immunogenic in both humans and mice, but in the used animal models do not result in protection against S. aureus infection.     

Genetic Diversity and Ecological Success of Staphylococcus aureus Strains Colonizing Humans

The genetic determinants and phenotypic traits which make a Staphylococcus aureus strain a successful colonizer are largely unknown. The genetic diversity and population structure of 133 S. aureus isolates from healthy, generally risk-free adult carriers were investigated using four different typing methods: multilocus sequence typing (MLST), amplified fragment length polymorphism analysis (AFLP), double-locus sequence typing (DLST), and spa typing were compared. Carriage isolates displayed great genetic diversity which could only be revealed fully by DLST. Results of AFLP and MLST were highly concordant in the delineation of genotypic clusters of closely related isolates, roughly equivalent to clonal complexes. spa typing and DLST provided considerably less phylogenetic information. The resolution of spa typing was similar to that of AFLP and inferior to that of DLST. AFLP proved to be the most universal method, combining a phylogeny-building capacity similar to that of MLST with a much higher resolution. However, it had a lower reproducibility than sequencing-based MLST, DLST, and spa typing. We found two cases of methicillin-resistant S. aureus colonization, both of which were most likely associated with employment at a health service. Of 21 genotypic clusters detected, 2 were most prevalent: cluster 45 and cluster 30 each colonized 24% of the carrier population. The number of bacteria found in nasal samples varied significantly among the clusters, but the most prevalent clusters were not particularly numerous in the nasal samples. We did not find much evidence that genotypic clusters were associated with different carrier characteristics, such as age, sex, medical conditions, or antibiotic use. This may provide empirical support for the idea that genetic clusters in bacteria are maintained in the absence of adaptation to different niches. Alternatively, carrier characteristics other than those evaluated here or factors other than human hosts may exert selective pressure maintaining genotypic clusters.     

Association of C-reactive Protein and Circulating Leukocytes with Resistance to Staphylococcus aureus Infection in Endotoxin-treated Mice and Rabbits

The response of rabbits and mice to treatment with Escherichia coli endotoxin, as measured by C-reactive protein (CRP) and leukocyte levels, and resistance to Staphylococcus aureus infection was studied to evaluate the significance of these responses and their associations. In both species, there was an initial leukopenia without early recovery of normal lymphocyte levels. This was followed by an increase in polymorphonuclear leukocytes and a return to near the normal range. The CRP level was slightly altered during the stage of decreased resistance and increased throughout the remainder of the period of observation. The resistance level was decreased initially, recovered to normal levels, and continued to increase. The changes in CRP and resistance levels were closely associated. It would appear that this association between CRP and resistance, the antibacterial activity of CRP, and its action on the polysaccharides obtained from bacterial cell walls are evidence for the participation of CRP in nonspecific resistance to infection.     

Regulatory Adaptation of Staphylococcus aureus during Nasal Colonization of Humans

The nasopharynx is the main ecological niche of the human pathogen Staphylococcus aureus. Although colonization of the nares is asymptomatic, nasal carriage is a known risk factor for endogenous staphylococcal infection. We quantified S. aureus mRNA levels in nose swabs of persistent carriers to gain insight into the regulatory adaptation of the bacterium to the nasal environment. We could elucidate a general response of the pathogen to the surrounding milieu independent of the strain background or the human host. Colonizing bacteria preferentially express molecules necessary for tissue adherence or immune-evasion whereas toxins are down regulated. From the analysis of regulatory loci we found evidence for a predominate role of the essential two-component system WalKR of S. aureus. The results suggest that during persistent colonization the bacteria are metabolically active with a high cell surface turnover. The increased understanding of bacterial factors that maintain the colonization state can open new therapeutic options to control nasal carriage and subsequent infections.     

Th1-Th17 Cells Mediate Protective Adaptive Immunity against Staphylococcus aureus and Candida albicans Infection in Mice

We sought to define protective mechanisms of immunity to Staphylococcus aureus and Candida albicans bloodstream infections in mice immunized with the recombinant N-terminus of Als3p (rAls3p-N) vaccine plus aluminum hydroxide (Al(OH₃) adjuvant, or adjuvant controls. Deficiency of IFN-γ but not IL-17A enhanced susceptibility of control mice to both infections. However, vaccine-induced protective immunity against both infections required CD4+ T-cell-derived IFN-γ and IL-17A, and functional phagocytic effectors. Vaccination primed Th1, Th17, and Th1/17 lymphocytes, which produced pro-inflammatory cytokines that enhanced phagocytic killing of both organisms. Vaccinated, infected mice had increased IFN-γ, IL-17, and KC, increased neutrophil influx, and decreased organism burden in tissues. In summary, rAls3p-N vaccination induced a Th1/Th17 response, resulting in recruitment and activation of phagocytes at sites of infection, and more effective clearance of S. aureus and C. albicans from tissues. Thus, vaccine-mediated adaptive immunity can protect against both infections by targeting microbes for destruction by innate effectors.     

The Effects of Continuous In Vivo Administration of Nisin on Staphylococcus aureus Infection and Immune Response in Mice

Mice were intraperitoneally infected with 2 × 10⁸ cfu Staphylococcus aureus Xen 36 and treated with 2,130 AU (arbitrary units) nisin (equivalent to 27.7 μg pure nisin), a class Ia lantibiotic, over 7 days. The metabolic activity of S. aureus Xen 36, concluded from changes in cell bioluminescence, declined for the first 3.5 h, but increased over the next 24 h and remained at this level for the remainder of the 7-day trial. Similar results were obtained with heat-inactivated (25 min at 121 °C) nisin, suggesting that the decline in metabolic activity of S. aureus Xen 36 cannot be attributed to the bacteriostatic activity of nisin. The decline in lymphocyte numbers in infected mice was of smaller magnitude after treatment with active nisin compared to inactive nisin, suggesting that active nisin limited the apoptosis of lymphocytes. The drastic increase in neutrophil versus lymphocyte (N:L) ratio observed in the presence of active nisin suggested that the decline in metabolic activity of S. aureus Xen 36 was due to an immune response triggered by the infection. Nisin, active or inactive, stimulated the activity of cytokines interleukin-6, interleukin-10 and tumour necrosis factor. However, the overall immune response triggered by both forms of nisin was too minute to trigger an abnormally high antigenic immune reaction.     

金黄色葡萄球菌致小鼠慢性输卵管炎性不孕模型制作

目的通过金黄色葡萄球菌直接感染小鼠输卵管,建立炎症致不孕的动物模型。方法用1×109/mL的金黄色葡萄球菌接种小鼠,制作慢性输卵管炎症模型,观察输卵管病理炎性改变以及小鼠的受孕情况。结果造模术75 d后,模型组小鼠受孕率、输卵管通畅率显著低于对照组（P〈0.001）。模型组肉眼观察输卵管有不同程度积水积脓、僵硬,输卵管与周围组织均有不同程度的粘连;病理学观察输卵管管腔被异物肉芽组织完全阻塞,全层均见大量慢性炎细胞浸润。结论输卵管内接种浓度1×109/mL的金黄色葡萄球菌可以成功建立小鼠输卵管炎性不孕模型。     

Monoclonal Antibody Targeting Staphylococcus aureus Surface Protein A (SasA) Protect Against Staphylococcus aureus Sepsis and Peritonitis in Mice

Epidemic methicillin-resistant Staphylococcus aureus (MRSA) imposes an increasing impact on public health. Due to multi-antibiotics resistance in MRSA strains, there is an urgent need to develop novel therapeutics such as effective monoclonal antibodies (mAbs) against MRSA infections. Staphylococcus aureus surface protein A (SasA), a large surface-located protein (~240 kDa), is one of MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) and a potential target for immunotherapeutic approaches against S. aureus infections. In the present study, we analyzed the sequence of SasA with bioinformatics tools and generated a protective monoclonal antibody (2H7) targeting the conserved domain of SasA. 2H7 was shown to recognize wild-type S. aureus and promote opsonophagocytic killing of S. aureus. In both sepsis and peritoneal infection models, prophylactic administration of 2H7 improved the survival of BALB/c mice challenged by S. aureus strain USA300 and ST239 (prevalent MRSA clones in North America and Asian countries, respectively) and enhanced bacterial clearance in kidneys. Additionally, 2H7 prophylaxis prevented the formation of intraperitoneal abscess in a murine model of peritoneal infection and therapeutic administration of 2H7 showed protective efficacy in a murine sepsis model. Our results presented here provide supporting evidences that an anti-SasA mAb might be a potential component in an antibody-based immunotherapeutic treatment of MRSA infections.     

Regulated Antisense RNA Eliminates Alpha-Toxin Virulence in Staphylococcus aureus Infection

The ability to selectively disrupt gene function remains a critical element in elucidating information regarding gene essentiality for bacterial growth and/or pathogenesis. In this study, we adapted a tet regulatory expression system for use in Staphylococcus aureus, with the goal of downregulating gene expression via induction of antisense RNA. We demonstrate that this system exhibits a 50- to 100-fold dose-dependent level of induction in bacterial cells grown in culture (i.e., in vitro) and also functions in mice (i.e., in vivo) following oral administration of inducer. To determine whether induced antisense RNA could interfere with chromosomally derived gene expression, we cloned a fragment of the S. aureus alpha-toxin gene (hla) in antisense orientation downstream of the tet promoter system and introduced the construct into S. aureus. Induced antisense hla RNA downregulated chromosomally derived hla gene expression in vitro approximately 14-fold. Similarly, induction of hla antisense RNA in vivo dramatically reduced alpha-toxin expression in two different murine models of S. aureus infection. Most importantly, this reduction completely eliminated the lethality of the infection. These results indicate that the tet regulatory system functions efficiently in S. aureus and induced antisense RNA can effectively downregulate chromosomal gene expression both in vitro and in vivo.     

Whole-Genome Sequence of Livestock-Associated ST398 Methicillin-Resistant Staphylococcus aureus Isolated from Humans in Canada

Despite reports of high colonization rates of ST398 livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) among pigs and pig farmers, the incidence of LA-MRSA infection in the general population in Canada appears to be rare in comparison to that in some European countries. In this study, the complete genome sequence of a Canadian representative LA-MRSA isolate (08BA02176) from a human postoperative surgical site infection was acquired and compared to the sequenced genome of an LA-MRSA isolate (S0385) from Europe to identify genetic traits that may explain differences in the success of these particular strains in some locales.     

Staphylococcus aureus Leukocidins Target Endothelial DARC to Cause Lethality in Mice

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Characterization of the Humoral Immune Response during Staphylococcus aureus Bacteremia and Global Gene Expression by Staphylococcus aureus in Human Blood

Attempts to develop an efficient anti-staphylococcal vaccine in humans have so far been unsuccessful. Therefore, more knowledge of the antigens that are expressed by Staphylococcus aureus in human blood and induce an immune response in patients is required. In this study we further characterize the serial levels of IgG and IgA antibodies against 56 staphylococcal antigens in multiple serum samples of 21 patients with a S. aureus bacteremia, compare peak IgG levels between patients and 30 non-infected controls, and analyze the expression of 3626 genes by two genetically distinct isolates in human blood. The serum antibody levels were measured using a bead-based flow cytometry technique (xMAP®, Luminex corporation). Gene expression levels were analyzed using a microarray (BµG@s microarray). The initial levels and time taken to reach peak IgG and IgA antibody levels were heterogeneous in bacteremia patients. The antigen SA0688 was associated with the highest median initial-to-peak antibody fold-increase for IgG (5.05-fold) and the second highest increase for IgA (2.07-fold). Peak IgG levels against 27 antigens, including the antigen SA0688, were significantly elevated in bacteremia patients versus controls (P≤0.05). Expression of diverse genes, including SA0688, was ubiquitously high in both isolates at all time points during incubation in blood. However, only a limited number of genes were specifically up- or downregulated in both isolates when cultured in blood, compared to the start of incubation in blood or during incubation in BHI broth. In conclusion, most staphylococcal antigens tested in this study, including many known virulence factors, do not induce uniform increases in the antibody levels in bacteremia patients. In addition, the expression of these antigens by S. aureus is not significantly altered by incubation in human blood over time. One immunogenic and ubiquitously expressed antigen is the putative iron-regulated ABC transporter SA0688.     

Staphylococcus aureus Bloodstream Infection and Endocarditis - A Prospective Cohort Study

ObjectivesTo update the epidemiology of S. aureus bloodstream infection (SAB) in a high-income country and its link with infective endocarditis (IE).MethodsAll consecutive adult patients with incident SAB (n = 2008) were prospectively enrolled between 2009 and 2011 in 8 university hospitals in France.ResultsSAB was nosocomial in 54%, non-nosocomial healthcare related in 18% and community-acquired in 26%. Methicillin resistance was present in 19% of isolates. SAB Incidence of nosocomial SAB was 0.159/1000 patients-days of hospitalization (95% confidence interval [CI] 0.111-0.219). A deep focus of infection was detected in 37%, the two most frequent were IE (11%) and pneumonia (8%). The higher rates of IE were observed in injecting drug users (IE: 38%) and patients with prosthetic (IE: 33%) or native valve disease (IE: 20%) but 40% of IE occurred in patients without heart disease nor injecting drug use. IE was more frequent in case of community-acquired (IE: 21%, adjusted odds-ratio (aOR) = 2.9, CI = 2.0-4.3) or non-nosocomial healthcare-related SAB (IE: 12%, aOR = 2.3, CI = 1.4-3.5). S. aureus meningitis (IE: 59%), persistent bacteremia at 48 hours (IE: 25%) and C-reactive protein > 190 mg/L (IE: 15%) were also independently associated with IE. Criteria for severe sepsis or septic shock were met in 30% of SAB without IE (overall in hospital mortality rate 24%) and in 51% of IE (overall in hospital mortality rate 35%).ConclusionSAB is still a severe disease, mostly related to healthcare in a high-income country. IE is the most frequent complication and occurs frequently in patients without known predisposing conditions.     

金黄色葡萄球菌CHIPS基因克隆及原核表达

趋化抑制蛋白(Chemotaxis Inhibitory Protein ofStaphylococcus aureus,CHIPS)是由金黄色葡萄球菌分泌到菌体外的一种蛋白。在感染早期,它能特异性地与中性粒细胞和单核细胞上的C5a受体(C5aR)和fMLP受体(FPR)结合,从而阻止中性粒细胞和单核细胞对C5a和fMLP的结合作用,导致对病原吞噬作用的延迟。人们可以利用CHIPS对C5a-C5aR的阻止作用来研制治疗由C5a诱发的炎症性疾病的药物。将CHIPS作为免疫原防治疾病也将成为新的研究课题。实验成功构建了CHIPS蛋白的原核表达系统,为CHIPS免疫原性研究及蛋白的其他功能研究奠定了基础。     

金黄色葡萄球菌甲氧西林耐药辅助基因femB的克隆和原核表达

金黄色葡萄球菌femB基因与甲氧西林高水平耐药密切相关,可能成为开发抗MRSA药物的新靶位.以金葡菌基因组DNA为模板,PCR扩增femB全长基因,所得片段与pGM-T载体连接并转化感受态大肠杆菌DH5α,阳性克隆以PCR、双酶切及测序鉴定.将鉴定正确的目的片段定向克隆到pGEX-4T-1表达载体中,转化至大肠杆菌BL21后经IPTG诱导表达GST/FemB融合蛋白;采用SDS-PAGE及Western blot对融合蛋白进行鉴定.结果显示,重组质粒在宿主菌中获得了高效表达,融合蛋白相对分子质量为75 kD,该融合蛋白可与抗GST-tag抗体特异结合;表明femB基因的原核表达系统构建成功,为进一步研究其生物学功能奠定了基础.     

Protective Role of Surfactant Protein D in Ocular Staphylococcus aureus Infection

Staphylococcus aureus is one of the most common pathogens causing keratitis. Surfactant protein D (SP-D) plays a critical role in host defense and innate immunity. In order to investigate the role of SP-D in ocular S. aureus infection, the eyes of wild-type (WT) and SP-D knockout (SP-D KO) C57BL/6 mice were infected with S. aureus (10⁷ CFU/eye) in the presence and absence of cysteine protease inhibitor(E₆₄).Bacterial counts in the ocular surface were examined 3, 6, 12, 24 hrs after infection. Bacterial phagocytosis by neutrophils and bacterial invasion in ocular epithelial cells were evaluated quantitatively. S. aureus-induced ocular injury was determined with corneal fluorescein staining. The results demonstrated that SP-D is expressed in ocular surface epithelium and the lacrimal gland; WT mice had increased clearance of S. aureus from the ocular surface (p<0.05) and reduced ocular injury compared with SP-D KO mice. The protective effects of SP-D include increased bacterial phagocytosis by neutrophils (p<0.05) and decreased bacterial invasion into epithelial cells (p<0.05) in WT mice compared to in SP-D KO mice. In the presence of inhibitor (E₆₄), WT mice showed enhanced bacterial clearance (p<0.05) and reduced ocular injury compared to absent E₆₄ while SP-D KO mice did not. Collectively, we concluded that SP-D protects the ocular surface from S. aureus infection but cysteine protease impairs SP-D function in this murine model, and that cysteine protease inhibitor may be a potential therapeutic agent in S. aureus keratitis.