Immobilized phage proteins for specific detection of staphylococci.

Rapid, specific detection of pathogenic bacteria remains a major challenge in infectious disease diagnostics. Bacteriophages can show genus- or even species-level specificity and have been developed for biosensing purposes, but the possibility of using individual phage proteins for detection has not been fully explored. This work exploits the ability of specific phage proteins, the endolysins LysK and Φ11, and the bacteriocin lysostaphin, fixed on silicon wafers to bind staphylococci. The proteins show activity against eight tested clinical isolates of S. aureus and to S. epidermidis, but no binding to Escherichia coli and limited binding to Micrococcus. Binding was quantified by clearing assays in solution and by functionalization of silicon wafers followed by light microscopy. Bacterial binding densities on functionalized surfaces were ~3 cells/100 µm(2). The small size of the proteins makes the system robust and easy to handle, and the principle is generalizable to many different biosensor platforms, including label-free systems such as optical microresonators.

Solubilization and bio-conjugation of quantum dots and bacterial toxicity assays by growth curve and plate count.

Quantum dots (QDs) are fluorescent semiconductor nanoparticles with size-dependent emission spectra that can be excited by a broad choice of wavelengths. QDs have attracted a lot of interest for imaging, diagnostics, and therapy due to their bright, stable fluorescence. QDs can be conjugated to a variety of bio-active molecules for binding to bacteria and mammalian cells. QDs are also being widely investigated as cytotoxic agents for targeted killing of bacteria. The emergence of multiply-resistant bacterial strains is rapidly becoming a public health crisis, particularly in the case of Gram negative pathogens. Because of the well-known antimicrobial effect of certain nanomaterials, especially Ag, there are hundreds of studies examining the toxicity of nanoparticles to bacteria. Bacterial studies have been performed with other types of semiconductor nanoparticles as well, especially TiO(2), but also ZnO and others including CuO. Some comparisons of bacterial strains have been performed in these studies, usually comparing a Gram negative strain with a Gram positive. With all of these particles, mechanisms of toxicity are attributed to oxidation: either the photogeneration of reactive oxygen species (ROS) by the particles or the direct release of metal ions that can cause oxidative toxicity. Even with these materials, results of different studies vary greatly. In some studies the Gram positive test strain is reportedly more sensitive than the Gram negative; in others it is the opposite. These studies have been well reviewed. In all nanoparticle studies, particle composition, size, surface chemistry, sample aging/breakdown, and wavelength, power, and duration of light exposure can all dramatically affect the results. In addition, synthesis byproducts and solvents must be considered. High-throughput screening techniques are needed to be able to develop effective new nanomedicine agents. CdTe QDs have anti-microbial effects alone or in combination with antibiotics. In a previous study, we showed that coupling of antibiotics to CdTe can increase toxicity to bacteria but decrease toxicity to mammalian cells, due to decreased production of reactive oxygen species from the conjugates. Although it is unlikely that cadmium-containing compounds will be approved for use in humans, such preparations could be used for disinfection of surfaces or sterilization of water. In this protocol, we give a straightforward approach to solubilizing CdTe QDs with mercaptopropionic acid (MPA). The QDs are ready to use within an hour. We then demonstrate coupling to an antimicrobial agent. The second part of the protocol demonstrates a 96-well bacterial inhibition assay using the conjugated and unconjugated QDs. The optical density is read over many hours, permitting the effects of QD addition and light exposure to be evaluated immediately as well as after a recovery period. We also illustrate a colony count for quantifying bacterial survival.

Cytotoxicity of InP/ZnS quantum dots related to reactive oxygen species generation.

Indium phosphide (InP) quantum dots (QDs) have emerged as a presumably less hazardous alternative to cadmium-based particles, but their cytotoxicity has not been well examined. Although their constituent elements are of very low toxicity to cells in culture, they nonetheless exhibit phototoxicity related to generation of reactive oxygen species by excited electrons and/or holes interacting with water and molecular oxygen. Using spin-trap electron paramagnetic resonance (EPR) spectroscopy and reporter assays, we find a considerable amount of superoxide and a small amount of hydroxyl radical formed under visible illumination of biocompatible InP QDs with a single ZnS shell, comparable to what is seen with CdTe. A double thickness shell reduces the reactive oxygen species concentration approximately two-fold. Survival assays in five cell lines correspondingly indicate a distinct reduction in toxicity with the double-shell InP QDs. Toxicity varies significantly across cell lines according to the efficiency of uptake, being overall significantly less than what is seen with CdTe or CdSe/ZnS. This indicates that InP QDs are a useful alternative to cadmium-containing QDs, while remaining capable of electron-transfer processes that may be undesirable or which may be exploited for photosensitization applications.

Antimicrobial activity and cellular toxicity of nanoparticle-polymyxin B conjugates.

We investigate the antimicrobial activity and cytotoxicity to mammalian cells of conjugates of the peptide antibiotic polymyxin B (PMB) to Au nanoparticles and CdTe quantum dots. Au nanoparticles fully covered with PMB are identical in antimicrobial activity to the free drug alone, whereas partially-conjugated Au particles show decreased effectiveness in proportion to the concentration of Au. CdTe-PMB conjugates are more toxic to Escherichia coli than PMB alone, resulting in a flattening of the steep PMB dose-response curve. The effect is most pronounced at low concentrations of PMB, with a greater effect on the concentration required to reduce growth by half (IC50) than on the concentration needed to inhibit all growth (minimum inhibitory concentration, MIC). The Gram positive organism Staphylococcus aureus is resistant to both PMB and CdTe, showing minimal increased sensitivity when the two are conjugated. Measurement of reactive oxygen species (ROS) generation shows a significant reduction in photo-generated hydroxyl and superoxide radicals with CdTe-PMB as compared with bare CdTe. There is a corresponding reduction in toxicity of QD-PMB versus bare CdTe to mammalian cells, with nearly 100% survival in fibroblasts exposed to bactericidal concentrations of QD-PMB. The situation in bacteria is more complex: photoexcitation of the CdTe particles plays a small role in IC50 but has a significant effect on the MIC, suggesting that at least two different mechanisms are responsible for the antimicrobial action seen. These results show that it is possible to create antimicrobial agents using concentrations of CdTe quantum dots that do not harm mammalian cells.