A major determinant in enhanced antibiotic resistance of hospital-acquired (i.e. nosocomial) infections, and implant-device associated infections is the embedding of bacteria in extracellular polymers (EPS). This proposal seeks to fabricate novel nanoparticles capable of killing bacteria in biofilms via antibiotics, quorum sensing quenchers, and quorum sensing disruptors conjugated to the nanoparticles. The mutlifunctionalized nanoparticle strategy of penetrating, analyzing and destroying highly resistant bacteria embedded in a protective EPS, is conceptually simple, but involves complex chemistry using CLICK chemistry for efficient synthesis and RAFT methodology for massive functionalization on each nanoparticle. Broader Impact A strength of this proposal is the multidisciplinary linkage of nanochemistry with biofilm microbiology research and its applicability to a range of biofilm problems. The applicant proposes a 4-phased outreach plan, which includes outreach activities in elementary school, development of an instructional video for middle school, multiple lectures in the two graduate-level course and recruitment of minorities to the research group.
Over the past several decades there has been an emerging crisis, involving the increasing prevalence of antibiotic-resistance among bacteria that cause human and animal infections. This issue, although health-related, addresses the fundamental biology of bacteria and how they resist antibiotics. A basic research project was conducted to investigate the potential capabilities of nanoparticles as antibiotic delivery vehicles (ADVs) against infection causing bacteria. Nanoparticles are ultra-small particles, approximately 1-100 nanometers (nm) in diameter. Owing to their very small sizes, nanoparticles often possess unique physical and/or chemical properties. We hypothesized that antibiotics can be carried as small, concentrated packets by nanoparticles, and upon delivery to a bacterial cell, they can release the antibiotics as a concentrated ‘pulse’, perhaps overwhelming the bacterium’s resistance mechanisms. This formed the basis of our research. In this work, silica nanoparticles, approximately 15 nm in diameter, were engineered with either surface carboxylic acid groups, or having elongated RAFT molecules with attched carboxyl groups that extended from the surfaces of the nanoparticles. The purpose of the carboxyl groups was to bind and carry the common antibiotic penicillin-G. The latter RAFT design potentially allowed several thousand antibiotic molecules to be attached to a single nanoparticle. The densities of surface carboxyl groups, and on RAFT molecules, were deliberately varied in order to bind differing amounts of penicillin-G. The silica nanoparticles were also labeled with a fluorescent tag to permit efficient detection and quantification by standard fluorometry and confocal scanning laser microscopy (CSLM). Finally, iron oxide clusters were synthesized inside of the silica shell of the nanoparticle in order to impart magnetic responses and retrieval (using magnets) of the nanoparticles during various stages of the experiments. The results characterizing the densities of carboxyl groups on nanoparticle surfaces confirmed that very high densities of antibiotic molecules per nanoparticle could be achieved, and therefore provided the potential for the delivery of large numbers of antibiotic molecules to a single bacterial cell. In using standardized assays, called Kirby Bauer assays, to test the ability of the antibiotic-nanoparticles to kill bacteria, the plate assays revealed that they were able to kill bacteria with high efficiency. By using Penicillin-G (PenG) attached to 15 nm diameter nanoparticles, the minimum inhibitory concentration (MIC) was less than 5 ug PenG, compared with no-nanoparticle controls whose MIC concentrations varied between 20 – 60 ug PenG, depending on the bacterial strain. Several strains of Level-2 bacterial pathogens were tested including Escherichia coli, Psuedomonas aeruginosa, Klebsiella pneumoniae, Bacillus subtilus, and Staphylococcus aureus. We then tested the PenG-nanoparticles against bacteria that were specifically resistant to the antibiotic PenG, including several stains of MRSA (i.e. methicillin-resistant Staphylococcus aureus). Our results showed that the PenG-nanoparticles were highly effective in killing MRSA strains at low concentrations (MIC were less than 5 ug/mL). The results confirmed the effectiveness of the nanoparticle delivery system using antibiotics. Many disease-causing (i.e. pathogenic) bacteria are currently resistant to the antibiotic penicillin-G. Our study showed that the antimicrobial activities of commonly-used antibiotics, such as penicillin, can be made more-powerful in their antimicrobial effects, when carried by nanoparticles. When Penicillin was delivered to penicillin-resistant bacteria, the nanoparticle-based delivery system was highly-effective in killing the bacteria. In more practical terms, the results of this study offer the likely possibility that nanoparticles can be used to overcome antibiotic-resistant bacteria using common antibiotics that may have been previously considered ineffective against resistant bacterial strains. Several connections were established to promote science, especially regarding nanoparticle research, with local high schools, middle schools and primary school. Such presentations were well-received. During the course of this study two Ph.D. students were supported; one of which has already received his degree. Additionally two undergraduate students worked on the project, and are currently in graduate school studying the sciences.
