Grantee Research Project Results

Final Report: A New Biosensor for Rapid Identification of Bacterial Pathogens

EPA Contract Number: 68D01016
Title: A New Biosensor for Rapid Identification of Bacterial Pathogens
Investigators: Tabacco, Mary Beth
Small Business: Echo Technologies Inc.
EPA Contact: Richards, April
Phase: I
Project Period: April 1, 2001 through September 1, 2001
Project Amount: $69,733
RFA: Small Business Innovation Research (SBIR) - Phase I (2001) RFA Text |  Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , SBIR - Monitoring , Small Business Innovation Research (SBIR)

Description:

There is an immediate need for rapid, portable analytical instruments to ensure the safety of natural and manmade water supplies, including source, recreational, treated, and distributed waters. Echo Technologies, Inc., has completed a Phase I SBIR project that demonstrated a new approach to the identification of bacteria in aqueous systems. The approach uses bacteriophage as the molecular recognition element. Bacteriophage are virus particles that generally attach to and infect a narrow range of host cells. This specificity has been demonstrated at both the species and strain level in the literature. The bacteriophage are optically encoded with fluorescent reporter molecules that allow for the detection and quantitation of the phage and the host/phage complex. Biosensors based on this molecular recognition offer a rapid, selective, and potentially very sensitive method to detect bacteria and bacterial pathogens in water reclamation systems. The approach could be used as an alternative, and possibly complement to, immunodiagnostic or nucleic acid-based detection.

Summary/Accomplishments (Outputs/Outcomes):

Feasibility of the concept was demonstrated by fabricating fluorescently labeled virus probes (FLVPs) to detect and identify bacteria for representative waterborne bacteria, including: Escherichia coli, Enterococcus faecalis, and Vibrio natriegens. The experiments conducted and key accomplishments are summarized as follows:

• Developed and tested methods for preparation of FLVPs for three genera of bacteria.

• Developed a method for direct-count enumeration of FLVPs.

• Demonstrated selectivity of phage and stained phage (FLVPs) for host bacteria using agar overlay.

• Demonstrated selectivity of FLVPs for host bacteria in solution.

• Measured attachment rate (< 1 minute).

• Demonstrated sensitive, quantitative optical response to target bacteria.

• Initial studies of matrix effects.

• Preliminary studies in mixed-cell populations.

• Fabricated FLVP biosensor and verified quantitative response.

Conclusions:

These cumulative results lay an excellent foundation for a Phase II development effort. The preparation of FLVPs has been made quite reproducible; the adhesion to host cells is very rapid; good selectivity was demonstrated in samples with host cells and mixtures; and the response from a solid-phase FLVP biosensor was demonstrated. Several experiments were conducted with a customized detection system to demonstrate the feasibility of making a small in-line instrument capable of high-sensitivity detection. In Phase II, a prototype rapid bacteria identification system will be designed, fabricated, and evaluated in the laboratory and in the field. The heart of the instrument will be an array of encoded FLVPs integrated with an array detection system. The system has biosensors for the identification of four bacteria at the genus or species level.

Application of the FLVP technology to solid-state optical sensing represents a new approach to the real-time detection of bacterial pathogens. This approach eliminates the need for culturing to identify pathogens, and is an important departure from immunoassay or DNA-based sensing concepts. The miniature probes are perfectly suited for incorporation into a sensor array for the simultaneous detection of many bacterial pathogens.

The proposed biosensor array is applicable to both military and civilian problems such as identifying terrorist threats, ensuring the safety of food supplies, agricultural engineering, and ensuring the purity of processing water in the biotechnology and semiconductor industries. The sensors also are ideally suited to routine monitoring of municipal water supplies for outbreaks of enteric pathogens.

Supplemental Keywords:

optical sensing, fluorescent bacteriophage assay, water pathogens., RFA, Scientific Discipline, Ecosystem Protection/Environmental Exposure & Risk, Water, Pathology, Chemistry, Recreational Water, Ecology, Biology, Engineering, Chemistry, & Physics, Microbiology, Monitoring/Modeling, Drinking Water, Environmental Engineering, Environmental Monitoring, biosensors, enterococci, Streptococci, microbial risk management, pathogens, escherichia, Bifidobacterium, rapid identification

SBIR Phase II:

A New Biosensor for Rapid Identification of Bacterial Pathogens  | Final Report