Technology for License...

Method for Rapid Microbial ID and AST

Traditional bacteriology systems for antibiotic susceptability testing (AST) generally fall into two categories; those like the Kirby-Bauer system seen here which looks at the growth of a confluent layer of organisms after overnight incubation, or more "rapid" systems which look at metabolic or redox changes produced by the organisms over the course of about six to eight hours. Both methods are subject to artifacts, in some part because the any heterogeneous organisms in the population may be swamped by their neighbors' metabolic activity. Over the past few decades there have been a number of attempts to rapidly determine the resistance of a microbe by looking at changes to single organisms when exposed to an antibiotic. This technique works well for a few antibiotics but is not generally applicable.

Our Technology

Our technology was developed to provide a microbial ID/AST, and specifically, an ID and a MIC for a number of antibiotcs on a simple, inexpensive platform. In operation, the technologist loads a suspension of organisms from a primary isolate into a small cassette, which is then placed into the instrument. The cassette is periodically examined by the reader until all of the results have been collected, at which time the results are presented. In (typically) two hours, a report is printed giving the microbial ID and the exact MIC for any desired number of antibiotics. Accompanying this report is an analysis of the required antibiotic dosage and total costs of each treatment option for the particular patient.

The features and benefits of the technology are:

Features:
• Small physical size of instrument
• Self-contained, sealed test units
• Rapid results (30 min to two hours, typical)
• Multiple MICs per test
• Combined growth, enzymatic and immunologic ID available
• Quantitation of colony growth rate vs growth-altering agents

Benefits:
• Bench-top complete bacteriology system
• Low volume of hazardous disposables
• Rapid reporting of optimum antibiotics
• Kinetic analysis provides "results when ready"
• Reduction in laboratory and hospital costs
• Enables rapid drug discovery applications
• Potential bio-weapon exposure screening

Theory of Operation

In contrast to all previous methods, our technology employs advances in image capture and recognition algorithms to digitally characterize the development of individual colony forming units (CFUs) over a few early generations. A suspension of organisms in a nutrient broth is placed into the sample cassette, which holds a thin-film hydrogel substrate where there is at least one control area where the organisms can freely grow and one or more areas containing growth-altering agents, such as antibiotics. The broth is absorbed into the hydrogel, which plates a layer of organisms on the surface. The system is relatively tolerant of variations in organism concentration and can work directly with urine samples or CSF. The active areas of the cassette are imaged, and any optical discontinuities are digitally mapped as shown above. These include dust, defects in the substrate and the CFUs. Also present on the surface of the substrate are small, randomly dispersed optical reference particles of approximately 5 microns, which can be seen as the larger dark object in the first picture. These allow focusing of the image (since the CFUs themselves may be almost invisible), and they allow accurate frame registration in the later images.

At intervals of from five to 30 minutes, the active areas are re-imaged and reanalyzed as shown here after 90 minutes of incubation. Areas which increase in size are interpreted as CFUs, and other areas are considered artifact. The reference particles are used to provide exact localization of the microcolony without the need for precisely re-positioning the cassette. Data is gathered at least until growth is confirmed in the control area. The rate of growth in the control area is compared with that in the area containing antibiotics to determine susceptability. The calculation of relative colony growth versus antibiotic concentration provides a level of information unavailable by any other technique. A video clip showing growth of micro-colonies and their digitization over the course of three hours can be seen here.

How the MIC is Obtained

An exact MIC is obtained for all antibiotics. An inert optical marker is admixed with each antibiotic so that there is a known ratio of marker for each unit of antibiotic. In separate areas of the substrate, the mixture of each antibiotic and marker is deposited on the hydrogel (by screen printing, jet printing, etc) so as to form an approximate a gradient from zero to some point over the presumed MIC. When the suspension of organisms hydrates the substrate, local diffusion completes the gradient. During the measurement process, the signal from the marker is taken along with the microcolony growth information, which allows the calculation of the antibiotic concentration at any given point on the gradient. Thus, microcolony growth versus antibiotic concentration can be plotted to determine the MIC. Note that because the antibiotic concentration is calculated during the measurement process, the initial deposition of the antibiotic mixture does not have to be particularly accurate, which simplifies the manufacturing process. A video of the growth of organisms on a control area and an area containing an antibiotic can be seen here.

This technique can be used with any growth-altering agent to provide information on growth rate versus agent concentration. This may be important for drug discovery or mutagen analysis.

Microbial Identification

On a separate section of the substrate are deposited a series of biochemical, immunologic and nutrient markers which are also imaged by the reader, measuring changes due to growth, metabolism, enzymatic cleavage of a substrate or attachment of an immunologic marker. The combination of markers plus the antibiotic sensitivity pattern identifies the organism.

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