Filter-based assay for Escherichia coli in aqueous samples using bacteriophage-based amplification.

This paper describes a method to detect the presence of bacteria in aqueous samples, based on the capture of bacteria on a syringe filter, and the infection of targeted bacterial species with a bacteriophage (phage). The use of phage as a reagent provides two opportunities for signal amplification: (i) the replication of phage inside a live bacterial host and (ii) the delivery and expression of the complementing gene that turns on enzymatic activity and produces a colored or fluorescent product. Here we demonstrate a phage-based amplification scheme with an M13KE phage that delivers a small peptide motif to an F(+), α-complementing strain of Escherichia coli K12, which expresses the ω-domain of ß-galactosidase (ß-gal). The result of this complementation-an active form of ß-gal-was detected colorimetrically, and the high level of expression of the ω-domain of ß-gal in the model K12 strains allowed us to detect, on average, five colony-forming units (CFUs) of this strain in 1 L of water with an overnight culture-based assay. We also detected 50 CFUs of the model K12 strain in 1 L of water (or 10 mL of orange juice, or 10 mL of skim milk) in less than 4 h with a solution-based assay with visual readout. The solution-based assay does not require specialized equipment or access to a laboratory, and is more rapid than existing tests that are suitable for use at the point of access. This method could potentially be extended to detect many different bacteria with bacteriophages that deliver genes encoding a full-length enzyme that is not natively expressed in the target bacteria.

Dual functional polyelectrolyte multilayer coatings for implants: permanent microbicidal base with controlled release of therapeutic agents.

Here we present a new bifunctional layer-by-layer (LbL) construct made by combining a permanent microbicidal polyelectrolyte multilayered (PEM) base film with a hydrolytically degradable PEM top film that offers controlled and localized delivery of therapeutics. Two degradable film architectures are presented: (1) bolus release of an antibiotic (gentamicin) to eradicate initial infection at the implant site, or (2) sustained delivery of an anti-inflammatory drug (diclofenac) to cope with inflammation at the site of implantation due to tissue injury. Each degradable film was built on top of a permanent base film that imparts the implantable device surface with microbicidal functionality that prevents the formation of biofilms. Controlled-delivery of gentamicin was demonstrated over hours and that of diclofenac over days. Both drugs retained their efficacy upon release. The permanent microbicidal base film was biocompatible with A549 epithelial cancer cells and MC3T3-E1 osteoprogenitor cells, while also preventing bacteria attachment from turbid media for the entire duration of the two weeks studied. The microbicidal base film retains its functionality after the biodegradable films have completely degraded. The versatility of these PEM films and their ability to prevent biofilm formation make them attractive as coatings for implantable devices.

Design of multi-drug release coatings targeting infection and inflammation.

Although infection and inflammation commonly coexist, there is a paucity of appropriate methods for concurrent localized delivery of antibiotics and non-steroidal anti-inflammatory drugs (NSAIDs) at appropriate concentrations and timescales. The commonly used therapeutic approach of systemic delivery of antibiotics and NSAIDs is associated with many complications, including rises in antibiotic resistant bacteria and severe gastrointestinal problems. As a potential solution, in this work we have assembled polymer multilayers to concurrently release therapeutic concentrations of an antibiotic, vancomycin, and an NSAID, diclofenac. Prior to film assembly, interactions between film components were thoroughly examined. Taking advantage of novel interactions found to exist between film components, several optimal film architectures were engineered using both dip and spray layer-by-layer assembly. A wide range of drug release profiles were obtained, with vancomycin delivery lasting from 4 hours to 2.3 days at final loadings of 13 to 30 µg/cm(2), while diclofenac released over 1.7 to 14 days at final loadings of 10 to 36 µg/cm(2). These drug release profiles have the potential to address a range of infection and inflammation requirements, from short term infection and inflammation eradication for trauma relief to infection prevention and long term inflammation mitigation from biomedical implants. Film-released vancomycin and diclofenac were found to be highly active against their respective targets in vitro, Staphylococcus aureus and cyclooxygenase. These films were also successfully applied to several medically relevant substrates, including intraocular lenses, bandages, and sutures, demonstrating potential for use as medical device coatings.