Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2000-02-17
2002-10-29
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S004000
Reexamination Certificate
active
06472166
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field
The present invention relates to methods for determining the effects of a growth-altering agent on one or more microorganisms within a biological sample in general, and to methods for determining the effects of a growth-altering agent on individual microorganism colonies within a biological sample in particular.
2. Background Information
Effective patient treatment often requires an identification of microorganisms within a biological sample and a determination of the sensitivity of those microorganisms to growth-altering agents. Historically, biological samples have been taken and applied to or added to microbiologic growth media (called “cultures”), which were then examined and tested primarily on a macroscopic basis. In most conventional tests, a suitable growth medium is inoculated with a patient's sample and subsequently incubated until there is visible evidence of microorganism growth. Most organisms require an incubation period of at least 18 to 24 hours to form visible colonies. The individual colonies start as a single, or a small cluster of microscopic cells or viable units (collectively referred to as colony-forming units or CFU's) contained within the inoculum. After an initial lag period during which time the organism acclimates itself to its new environment and experiences little or no growth, the viable microorganisms settle into exponential growth: one cell will give rise to two cells in one generation, eight cells in three generations, sixty-four cells in six generation, and so forth until a visible colony is created.
If the inoculum contains a plurality of different microorganisms, each organism type will form its own characteristic colony, which may or may not be distinguishable from another. For most purposes, however, it is desirable to have only a single species of organism present within the organism growth. For example, if one wishes to test a biological sample for sensitivity to a particular antibiotic and the sample and subsequent culture contain multiple organism species, it may not be possible to determine the sensitivity of individual organism species within the culture to the antibiotic. To determine the sensitivity of individual organism species, it is necessary to make a “pure” culture (i.e., one that contains a single species of microorganism) by incubating the initial sample inoculum on a first solid growth medium and removing a single colony, or a group of identical colonies, from a first growth medium and plating it onto a second solid growth medium or forming a suspension if a liquid culture is used. A person of skill in the art will recognize the process is time consuming and generally requires a skilled technician.
In those instances where it is desirable to know the effectiveness of a growth-altering agent (e.g., antibiotics, growth promoting agents, nutrients, antiseptics, etc.) on an organism, prior art practice generally dictates the use of one of the following evaluative methods. In one method, a growth-altering agent is applied to a region of a solid inoculated growth medium prior to incubation and the organism growth in the applied region is evaluated or compared against organism growth in a region where growth-altering agent was not applied. A Kirby-Bauer plate test is an example of this type of macroscopic method. The Kirby-Bauer method includes incubating a growth medium until confluent growth forms over the growth medium. A region of growth medium bearing an effective growth-altering agent (antimicrobial) diffused out from a disk will not contain organism growth if the antimicrobial is effective in suppressing the organism. The size of the growth-free zone surrounding the disk is then compared to a reference to determine whether the organism is susceptible to the growth-altering agent in a clinically useful concentration. A second evaluative method involves adding a known amount of the growth-altering agent to a liquid medium that is inoculated with the organism to be tested. Turbidity testing is an example of this type of macroscopic method. A turbidity test measures the “cloudiness” of a liquid sample to determine the organism content of the sample. An increase in the turbidity of the sample indicates an increase in the organism content within the sample. A third evaluative method involves observing the effect organism growth has on a colored reagent that responds to one or more constituents or metabolic products of the growing organism. The information available from any of these macroscopic evaluation methods is, generally speaking, also macroscopic in nature; e.g., the growth-altering agent applied in a particular concentration either has or does not have an effect on the growth of the organism(s). Little or no additional information is available regarding, for example, the mechanism of death, whether the organism experienced septum formation, or any statistical information vis-a-vis the population of organisms within the culture.
One of the problems with the above macroscopic methods is test error that results from waiting until a visible layer or an acceptable concentration of organism develops. Organism colonies growing on or within a growth medium compete for food and as a result may be growth inhibited because of competition rather than because of a growth-altering agent. Those same organisms can also affect each other by their excretions and metabolic by-products. A more accurate analysis of the effect of a growth-altering agent on a particular microbe would be possible if such interference did not occur.
Another problem with macroscopic evaluation of an organism is the time required to produce meaningful results. As noted above, it is typically necessary to incubate an organism culture anywhere from 18 to 24 hours to produce a growth adequate for macroscopic evaluation (e.g., if the organism replicates every 20 to 60 minutes, there should be at least 20 generations of the organism). Practically speaking, however, generating a culture and analyzing it using conventional methods takes at least 48 hours because of handling, evaluation, etc. Because the rate of microbial growth is so rapid and the time for testing so great, patients suspected of having a microbial infection are often initially treated with a wide-spectrum antibiotic prior to the identification of the actual organism and its sensitivity. A more targeted treatment can be administered after the test data is received. A person of skill in the art will recognize, however, that wide spectrum antibiotics having the utility to provide more expeditious treatment are not favored over the more targeted treatments available with specific information. In fact, a wide spectrum antibiotic can be considerably more expensive and have more adverse side effects than a more targeted drug. There is also considerable concern today that the overuse of wide spectrum antibiotics might promote the development of antibiotic resistance within the organisms, consequently limiting their effectiveness.
In recognition of the problems associated with the time it takes to perform the above described macroscopic tests, a number of methods for rapidly determining antibiotic susceptibility have been proposed, including methods that examine individual organisms. These methods utilize the fact that susceptible bacteria may change their shape, size, or internal chemistry (or some combination thereof) when exposed to an antibiotic Some types of bacteria, however, do not detectably react to an antibiotic until after the propagation of the first few generations. Tests that only consider organisms in their first few generations, therefore, cannot provide useful information in every case and are considered to be ineffective unless the behavior of the organism is known in advance. An example of an analysis for a specific microorganism is proposed by Ledley (U.S. Pat. No. 5,922,282) for the determination of antibiotic susceptibility for mycobacterium tuberculosis (MTB). In the Ledley method, the DNA of individual organisms are altered by the
Levine Robert A.
Wardlaw Stephen C.
Leary Louise N.
McCormick Paulding & Huber LLP
Wardlaw Partners LP
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