Probiotic disruption of quorum sensing reduces virulence and increases cefoxitin sensitivity in methicillin-resistant Staphylococcus aureus.

Therapies which target quorum sensing (QS) systems that regulate virulence in methicillin-resistant Staphylococcus aureus (MRSA) are a promising alternative to antibiotics. QS systems play a crucial in the regulation of MRSA antibiotic resistance, exotoxin production, antioxidant protection and immune cell evasion, and are therefore attractive therapeutic targets to reduce the virulence of a pathogen. In the present work the the effects of bioactive peptides isolated from two strains of lactic acid bacteria were tested against antibiotic resistance, carotenoid production, resistance to oxidative killing and biofilm structure in two clinical MRSA isolates. The results obtained from fractional-inhibitory concentration assays with bulk and semi-purified bioactive molecules showed a significant synergistic effect increasing cefoxitin mediated killing of MRSA. This was coupled to a six-fold decrease of the major membrane pigment staphyloxanthin, and a 99% increase in susceptibility to oxidative stress mediated killing. Real-time quantitative PCR analysis of the QS-genes agrA and luxS, showed differential expression between MRSA strains, and a significant downregulation of the hemolysin gene hla. Light microscopy and scanning electron microscopy revealed alteration in biofilm formation and clustering behavior. These results demonstrate that bioactive metabolites may be effectively applied in tandem with beta-lactam antibiotics to sensitize MRSA to cefoxitin. Moreover, these results shown that several key QS-controlled virulence mechanisms are diminished by probiotic metabolites.

Enzyme-Linked Immunosorbent Assay (ELISA).

Enzyme-linked immunosorbent assay (ELISA) is one of the most specific and straightforward assays for detecting biomolecules in research and clinics. With advances in analytical methods, ELISA assay has been constantly optimized to improve its sensitivity, and different types of ELISA are now available to detect various biomarkers. This chapter provides an overall summary of the basic principle of ELISA, discusses different components of ELISA assay, and clearly outline protocols for different types of ELISA assays, including direct, indirect, sandwich, competitive, and nanoparticle-based ELISA.

Size and macromolecule stabilizer-dependent performance of gold colloids in immuno-PCR.

Gold nanoparticles (GNPs) are well-documented for their size and surface chemistry-dependent electronic and optical properties that are extensively utilized to develop highly sensitive immunoassays. GNP-based immuno-polymerase chain reaction (immuno-PCR) is especially interesting due to the facile loading of biomolecules on the surface of GNP probes and has been utilized to develop analyte-specific assays. In this study, the role of size and surface chemistry of GNPs is explored in detail to develop a highly sensitive and reproducible immuno-PCR assay for specific detection of biotinylated analytes. Our results indicate that smaller-sized gold nanoparticles outperform the larger ones in terms of their sensitivity in immuno-PCR assay and show superior loading of proteins and oligonucleotides on the surface of nanoparticles. Furthermore, the role of different macromolecular stabilizers (such as polyethylene glycol (PEG), bovine serum albumin (BSA), and PEGylated BSA) was compared to optimize the loading of biomolecules and to improve the signal-to-noise ratio of GNP probes. mPEG-BSA-functionalized GNP probes of 15 nm were found to be highly sensitive at low concentrations of analytes and significantly (~ 30 fold) improve the limit of detection of analytes in comparison with ELISA assay.

Applications of gold nanoparticles in ELISA, PCR, and immuno-PCR assays: A review.

Development of state-of-the-art assays for sensitive and specific detection of disease biomarkers has received significant interest for early detection and prevention of various diseases. Enzyme Linked Immunosorbent assays (ELISA) and Polymerase Chain Reaction (PCR) are two examples of proteins and nucleic acid detection assays respectively, which have been widely used for the sensitive detection of target analytes in biological fluids. Recently, immuno-PCR has emerged as a sensitive detection method, where high specificity of sandwich ELISA assays is combined with high sensitivity of PCR for trace detection of biomarkers. However, inherent disadvantages of immuno-PCR assays limit their application as rapid and sensitive detection method in clinical settings. With advances in nanomaterials, nanoparticles-based immunoassays have been widely used to improve the sensitivity and simplicity of traditional immunoassays. Owing to facile synthesis, surface functionalization, and superior optical and electronic properties, gold nanoparticles have been at the forefront of sensing and detection technologies and have been extensively studied to improve the efficacies of immunoassays. This review provides a brief history of immuno-PCR assays and specifically focuses on the role of gold nanoparticles to improve the sensitivity and specificity of ELISA, PCR and immuno-PCR assays.