Combination of capillary electrophoresis, PCR and physiological assays in differentiation of clinical strains of Staphylococcus aureus

Fast, sensitive and cheap determination of pathogenic bacteria is extremely important in many branches, for example biotechnology, quality control, analysis of samples and antimicrobial therapy. The development and application of analytical techniques in practice could provide new possibilities in this regard. The bacterial pathogen Staphylococcus aureus is responsible for a significant amount of human morbidity and mortality. Rapid and sensitive determination is therefore very important. In the present study, novel methods, based on capillary zone electrophoresis and (as confirmation of these results) molecular analysis of a part of the coag gene, were developed for identification and differentiation of three S. aureus strains. The electrophoretic measurements rely on the differential mobility of bacteria in the fused silica capillary under the direct current electric field. To perform coagulase gene typing, the repeated units encoding hypervariable regions of the S. aureus gene were amplified using the PCR technique followed by restriction enzyme digestion and analysis of restriction fragment length polymorphism patterns as well as sequencing. Finally, the results of electrophoretic measurements with molecular analysis were compared.     

Direct detection of nasal Staphylococcus aureus carriage via helicase-dependent isothermal amplification and chip hybridization

ABSTRACT: BACKGROUND: The bacterium Staphylococcus aureus constitutes one of the most important causes of nosocomial infections. One out of every three individuals naturally carries S. aureus in their anterior nares, and nasal carriage is associated with a significantly higher infection rate in hospital settings. Nasal carriage can be either persistent or intermittent, and it is the persistent carriers who, as a group, are at the highest risk of infection and who have the highest nasal S. aureus cell counts. Prophylactic decolonization of S. aureus from patients' noses is known to reduce the incidence of postsurgical infections, and there is a clear rationale for rapid identification of nasal S. aureus carriers among hospital patients. FINDINGS: A molecular diagnostic assay was developed which is based on helicase-dependent target amplification and amplicon detection by chip hybridization to a chip surface, producing a visible readout. Nasal swabs from 70 subjects were used to compare the molecular assay against culturing on "CHROMagar Staph aureus" agar plates. The overall relative sensitivity was 89%, and the relative specificity was 94%. The sensitivity rose to 100% when excluding low-count subjects (<100 S. aureus colony-forming units per swab). CONCLUSIONS: This molecular assay is much faster than direct culture and has sensitivity that is appropriate for identification of high-count (>100 S. aureus colony-forming units per swab) nasal S. aureus carriers who are at greatest risk for nosocomial infections.     

Molecular docking analysis of DNA ligase from Staphylococcus aureus with identical polysaccharides

Staphylococcus aureus has been recognized as an important human pathogen for more than 100 years. DNA ligase is the main protein responsible for the replication of S. aureus. DNA ligase was selected as successive target to control the replication mechanism. The antibacterial activity of polysaccharide is known. Therefore, it is of interest to study the activity of Polysaccharide analogues against DNA ligase in S. aureus using molecular docking analysis. We report ten analogues using scoring parameters with best two analogues as potential drug candidate for the combat of S. aureus infection.     

A simple procedure for isolation of cloning vectors and endogenous plasmids from viridans group streptococci and Staphylococcus aureus.

Isolation of plasmid DNA from viridans group streptococci is difficult, and preparations are often heavily contaminated with chromosomal DNA. We developed a simple protocol to isolate pure plasmid DNA for use in different molecular techniques, including automated sequencing. The protocol is also applicable for plasmid isolation from Staphylococcus aureus. In addition, the protocol allows isolation of pure endogenous plasmids from streptococci and S. aureus.     

Immune proteomics of Staphylococcus aureus

Immune proteomics is an increasingly powerful tool for the investigation of the adaptive immune response to natural encounters between micro-organisms and their hosts. The versatile species Staphylococcus aureus serves to illustrate how these techniques can be employed to appreciate the complexity and diversity of the host-pathogen interactions in unprecedented detail and completeness. Such knowledge is important for the development of effective vaccines as well as informative diagnostic and novel therapeutic tools. From high-resolution immune proteome studies, general rules underlying the human adaptive immune response to S. aureus colonization and infection are beginning to emerge against a background of extreme diversity: S. aureus carriers develop immune memory for their colonizing strain, but even non-carriers are frequently exposed to S. aureus, resulting in specific antibodies. During bacterial invasion, immune-competent individuals rapidly mount an antibody response to a large panel of S. aureus antigens. However, every patient starts from a personal baseline antibody profile reflecting his or her history of encounters with S. aureus.     

Population biology of Gram-positive pathogens: high-risk clones for dissemination of antibiotic resistance

Infections caused by multiresistant Gram-positive bacteria represent a major health burden in the community as well as in hospitalized patients. Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium are well-known pathogens of hospitalized patients, frequently linked with resistance against multiple antibiotics, compromising effective therapy. Streptococcus pneumoniae and Streptococcus pyogenes are important pathogens in the community and S. aureus has recently emerged as an important community-acquired pathogen. Population genetic studies reveal that recombination prevails as a driving force of genetic diversity in E. faecium, E. faecalis, S. pneumoniae and S. pyogenes, and thus, these species are weakly clonal. Although recombination has a relatively modest role driving the genetic variation of the core genome of S. aureus, the horizontal acquisition of resistance and virulence genes plays a key role in the emergence of new clinically relevant clones in this species. In this review, we discuss the population genetics of E. faecium, E. faecalis, S. pneumoniae, S. pyogenes and S. aureus. Knowledge of the population structure of these pathogens is not only highly relevant for (molecular) epidemiological research but also for identifying the genetic variation that underlies changes in clinical behaviour, to improve our understanding of the pathogenic behaviour of particular clones and to identify novel targets for vaccines or immunotherapy.     

金黄色葡萄球菌荚膜多糖研究进展

金黄色葡萄球菌作为引起人和动物发病的一种主要的致病菌,已严重影响到人们的身体健康和畜牧业的发展。致病性金黄色葡萄球菌多数含有荚膜成分,并且这种荚膜成分与金黄色葡萄球菌的毒力和抗噬菌作用有关。主要从金黄色葡萄球菌荚膜多糖的5型和8型血清学分类、分子结构、影响因素、表达调控等方面简要介绍了目前国内外关于金黄色葡萄球菌荚膜多糖的研究进展。     

ADSRRS-fingerprinting and PCR MP techniques for studies of intraspecies genetic relatedness in Staphylococcus aureus

The present study was designed to evaluate the usefulness of two novel molecular typing methods, amplification of DNA fragments surrounding rare restriction sites (ADSRRS-fingerprinting) and the PCR melting profile (PCR MP), for Staphylococcus aureus strain differentiation. Thirty-seven S. aureus strains isolated from patients with a history of furunculosis were studied. The strains were identified by determining several phenotypic properties and were genotyped using three differentiation methods: macrorestriction analysis of the chromosomal DNA by pulsed-field gel electrophoresis (REA-PFGE), ADSRRS-fingerprinting, and PCR MP technique. In some cases the results obtained showed that the S. aureus isolated from the nose was identical to the one from the furuncle of the same patient. The same genotype was also identified for S. aureus strains isolated from two different members of a family with a history of recurrent furunculosis, although the active lesions were present in only one of them when the investigation was done. Results from strain genotyping illustrated that the recently developed ADSRRS-fingerprinting and PCR MP techniques are useful for studies of intraspecies genetic relatedness of S. aureus strains. They are as effective in discriminating closely related strains as the PFGE method, which is currently considered to be "the gold standard" for epidemiological studies.     

Hemoglobin promotes Staphylococcus aureus nasal colonization

Staphylococcus aureus nasal colonization is an important risk factor for community and nosocomial infection. Despite the importance of S. aureus to human health, molecular mechanisms and host factors influencing nasal colonization are not well understood. To identify host factors contributing to nasal colonization, we collected human nasal secretions and analyzed their ability to promote S. aureus surface colonization. Some individuals produced secretions possessing the ability to significantly promote S. aureus surface colonization. Nasal secretions pretreated with protease no longer promoted S. aureus surface colonization, suggesting the involvement of protein factors. The major protein components of secretions were identified and subsequent analysis revealed that hemoglobin possessed the ability to promote S. aureus surface colonization. Immunoprecipitation of hemoglobin from nasal secretions resulted in reduced S. aureus surface colonization. Furthermore, exogenously added hemoglobin significantly decreased the inoculum necessary for nasal colonization in a rodent model. Finally, we found that hemoglobin prevented expression of the agr quorum sensing system and that aberrant constitutive expression of the agr effector molecule, RNAIII, resulted in reduced nasal colonization of S. aureus. Collectively our results suggest that the presence of hemoglobin in nasal secretions contributes to S. aureus nasal colonization.     

金黄色葡萄球菌生物膜形成机制研究进展

金黄色葡萄球菌是医院和社区获得性感染最常见的病原菌之一,而且可形成生物膜,从而导致生物膜相关疾病的产生。患者一旦发生金黄色葡萄球菌生物膜感染,难以彻底治愈。深入研究金黄色葡萄球菌生物膜形成的分子机制和调控网络,对寻找有效的防治、治疗药物和手段具有重要意义。我们就金黄色葡萄球菌生物膜形成过程和调控机制的研究近况做一综述。     

Inflammasome activation and IL-1β/IL-18 processing are influenced by distinct pathways in microglia

Microglia are important innate immune effectors against invading CNS pathogens, such as Staphylococcus aureus (S. aureus), a common etiological agent of brain abscesses typified by widespread inflammation and necrosis. The NLRP3 inflammasome is a protein complex involved in IL-1β and IL-18 processing following exposure to both pathogen- and danger-associated molecular patterns. Although previous studies from our laboratory have established that IL-1β is a major cytokine product of S. aureus-activated microglia and is pivotal for eliciting protective anti-bacterial immunity during brain abscess development, the molecular machinery responsible for cytokine release remains to be determined. Therefore, the functional role of the NLRP3 inflammasome and its adaptor protein apoptosis-associated speck-like protein (ASC) in eliciting IL-1β and IL-18 release was examined in primary microglia. Interestingly, we found that IL-1β, but not IL-18 production, was significantly attenuated in both NLRP3 and ASC knockout microglia following exposure to live S. aureus. NLRP3 inflammasome activation was partially dependent on autocrine/paracrine ATP release and α- and γ-hemolysins produced by live bacteria. A cathepsin B inhibitor attenuated IL-β release from NLRP3 and ASC knockout microglia, demonstrating the existence of alternative inflammasome-independent mechanisms for IL-1β processing. In contrast, microglial IL-18 secretion occurred independently of cathepsin B and inflammasome action. Collectively, these results demonstrate that microglial IL-1β processing is regulated by multiple pathways and diverges from mechanisms utilized for IL-18 cleavage. Understanding the molecular events that regulate IL-1β production is important for modulating this potent proinflammatory cytokine during CNS disease.     

Detection of enterotoxin and toxic shock syndrome toxin 1 genes in Staphylococcus, with emphasis on coagulase-negative staphylococci

The detection of staphylococcal enterotoxins is decisive for the confirmation of an outbreak and for the determination of the enterotoxigenicity of strains. Since the recognition of their antigenicity, a large number of serological methods for the detection of enterotoxins in food and culture media have been proposed. Since immunological methods require detectable amounts of toxin, molecular biology techniques represent important tools in the microbiology laboratory. In the present study, polymerase chain reaction (PCR) was used to identify genes responsible for the production of enterotoxins and toxic shock syndrome toxin 1 (TSST-1) in S. aureus and coagulase-negative staphylococci (CNS) isolated from patients and the results were compared with those obtained by the reverse passive latex agglutination (RPLA) assay. PCR detection of toxin genes revealed a higher percentage of toxigenic S. aureus strains (46.7%) than the RPLA method (38.3%). Analysis of the toxigenic profile of CNS strains showed that 26.7% of the isolates produced some type of toxin, and one or more toxin-specific genes were detected in 40% of the isolates. These results suggests the need for further studies in order to better characterize the pathogenic potential of CNS and indicate that attention should be paid to the toxigenic capacity of this group of microorganisms.     

Staphylococcus aureus: a blemish on skin immunity

Staphylococcus aureus is the leading cause of human skin infections. In this issue of Cell Host & Microbe, new research probes how a change in surface hydrophobicity mediated by a single S. aureus protein renders the pathogen resistant to key molecular effectors of skin innate immunity, including cationic antimicrobial peptides and fatty acid constituents of sebum. Novel treatment strategies for S. aureus infection may lie in supplementing the very same innate defense molecules to therapeutic levels.     

The role of nasal carriage in Staphylococcus aureus burn wound colonization

Thermal injury destroys the physical skin barrier that normally prevents invasion of microorganisms. This and concomitant depression of local and systemic host cellular and humoral immune responses are important factors that contribute to colonization and infection of the burn wound. One of the most common burn wound pathogens is Staphylococcus aureus. Staphylococcus aureus is both a human commensal and a frequent cause of infections leading to mild to life-threatening diseases. Despite a variety of infection control measures, for example patient cohorting and contact precaution at burn centres, S. aureus is still frequently encountered in burn wounds. Colonization with S. aureus has been associated with delayed wound healing, increased need for surgical interventions, and prolonged length of stay at burn centres. In this minireview, we focus on S. aureus nasal carriage in relation to S. aureus burn wound colonization and subsequent infection, and its impact on strategies for infection control.     

Comparative study of Staphylococcus aureus isolated from lesional and non-lesional skin of atopic dermatitis patients

The skin of patients with atopic dermatitis (AD) is often colonized by Staphylococcus aureus, and superantigenic exotoxins produced by the organism are thought to be an important precipitating factor of AD. However, there are few reports comparing the characteristics of S. aureus isolated from the lesional and non-lesional skin of identical AD patients. In this study, therefore, we examined whether the presence of superantigen-producing S. aureus correlates with the formation of eczematous lesion of AD patients. The detection rate of S. aureus on the lesional skin of AD patients was higher than on the non-lesional skin of AD patients. Furthermore, the bacterial cell count of S. aureus on the lesional skin of AD patients was also significantly higher than that of the non-lesional skin of AD patients. However, there was no significant difference between the detection rate of superantigenic exotoxin-producing S. aureus on the lesional and nonlesional skin of AD patients. These results suggest that the number of S. aureus present is more important in the formation of eczematous lesion of AD patients than the presence of superantigenic exotoxin-producing S. aureus strains per se.     

Characterization of a new cytotoxin that contributes to Staphylococcus aureus pathogenesis

Staphylococcus aureus is an important pathogen that continues to be a significant global health threat because of the prevalence of methicillin-resistant S. aureus strains (MRSA). The pathogenesis of this organism is partly attributed to the production of a large repertoire of cytotoxins that target and kill innate immune cells, which provide the first line of defence against S. aureus infection. Here we demonstrate that leukocidin A/B (LukAB) is required and sufficient for the ability of S. aureus, including MRSA, to kill human neutrophils, macrophages and dendritic cells. LukAB targets the plasma membrane of host cells resulting in cellular swelling and subsequent cell death. We found that S. aureus lacking lukAB are severely impaired in their ability to kill phagocytes during bacteria-phagocyte interaction, which in turn renders the lukAB-negative staphylococci more susceptible to killing by neutrophils. Notably, we show that lukAB is expressed in vivo within abscesses in a murine infection model and that it contributes significantly to pathogenesis of MRSA in an animal host. Collectively, these results extend our understanding of how S. aureus avoids phagocyte-mediated clearance, and underscore LukAB as an important factor that contributes to staphylococcal pathogenesis.     

Staphylococcus aureus isolated from Kawasaki disease patients hyper-releases extracellular protein A.

S. aureus isolates from patients with Kawasaki disease (KD) release high levels of extracellular protein A (SpA), as compared to S. aureus in other diseases. The molecular weight of this released protein A is about 70 kDa. Extracellular KD SpA purified by affinity chromatography possessed the same amino acid sequence at the NH2-terminal IgG binding region and the same antigenic specificity as recombinant and cell-wall-bound SpA preparations. The size of DNA fragments containing the spa gene from S. aureus KD strains was 160-165 kb. All of these DNA fragments contained the igb portion encoding the IgG-binding region of KD SpA. Significantly higher molecular size of the SpA molecules hyper-released in the stationary-phase culture and the lack of production of other exo-proteins allow us to speculate that S. aureus isolated from patients with KD have mutations occurring in the agr locus.     

金黄色葡萄球菌非编码小RNA的研究进展

金黄色葡萄球菌(Staphylococcus aureus,S.aureus)是困扰全球公共卫生及人类健康的重要病原菌,其引起的各种临床感染与该菌表达的多种毒力因子密切相关,而这些毒力因子表达受到调节性因子的精确调控,在细菌致病机制中发挥着核心作用。非编码小RNA(Small non-coding RNA,s RNA)是基因表达的一类重要调节因子,可使细菌对环境因素做出反应,调节其应激适应性及毒力因子表达。但到目前为止,仅少数金黄色葡萄球菌s RNA的生物学功能得到阐述。本文将针对这些调节性s RNA的研究进展作一综述。