Device and method for detecting antibiotic inactivating enzymes

Abstract

The present invention provides a method of determining the antibiotic susceptibility of a microorganism comprising the following steps. First, a culture of the microorganism whose susceptibility is to be determined is admixed with an antibiotic to which susceptibility is to be assayed, and a permeabilizing agent for the microorganism present in a non-growth-inhibiting microorganism-permeabilizing effective amount to form an assay culture. Next, the assay culture is incubated under appropriate culture conditions and for a time sufficient to determine the susceptibility of the microorganism to the antibiotic. In another aspect, the present invention provides an improved method for antibiotic susceptibility testing of a microorganism in a culture by admixing the culture with an antibiotic to which susceptibility is to be assayed, and incubating the culture for a time sufficient to determine the susceptibility of the microorganism to the antibiotic, the improvement comprising admixing the culture with a permeabilizing agent for the microorganism present in a non-growth inhibiting microorganism-permeabilizing effective amount.

Description

[0001] This application claims priority of U.S. Provisional Application Serial No. 60 / 364,232 filed Mar. 13, 2002.[0002] Not applicable.[0003] Clinicians and veterinarians often select antibiotic therapies for infections on the basis of laboratory test results. The laboratory tests, known as antimicrobial or antibiotic susceptibility tests, determine the inhibitory activity of antibiotics against the microorganisms that cause infections. If the antibiotic susceptibility test indicates that an antibiotic is sufficiently potent to treat an infection, the microorganism causing the infection is reported to be "susceptible" to the antibiotic. If the test indicates a lack of sufficient antimicrobial potency for successful therapy, the microorganism is reported as "resistant" to the antibiotic. In some tests other categories of susceptibility may be reported, e.g. "moderate susceptibility" or "intermediate susceptibility."[0004] A problem with currently available antimicrobial susceptibili...

AI Technical Summary

Benefits of technology

[0045] It has been found that a permeabilizing agent can be used to detect antibiotic-resistant microorganisms that would otherwise register as falsely sensitive to a test antibiotic, by facilitating the liberation or release of antibiotic-inactivating factors. Surprisingly, the microorganism can grow in the presence of a permeabilizing agent that is normally lethal to a microorganism, or inhibits the growth of a microorganism.

[0077] As can be readily seen, the invention provides novel benefits and advantages over other antibiotic-sensitivity testing methods. The invention permits antibiotic-sensitivity testing to be performed, for example, on the surface of the agar, rather than the technically challenging aspect of some other methods that require the insertion of bacterial inoculum into a slit in the agar. Furthermore, the invention discloses the use of a permeabilizing agent that facilitates the release of antibiotic inactivating factors from microorganisms, without significantly inhibiting the growth of those microorganisms. Current antibiotic susceptibility testing techniques cannot enhance the release of such factors, leading to the possibility of false-positive results. Therefore, current antibiotic susceptibility testing techniques can lead a clinician to believe that a particular microorganism is sensitive to an antibiotic based on these in vitro results, only to find empirically that the antibiotic is ineffectual in vivo. The present invention provides more robust in vitro information that can detect microorganisms that are likely to be resistant to a particular antibiotic in vivo.

Problems solved by technology

If the antibiotic susceptibility test indicates that an antibiotic is sufficiently potent to treat an infection, the microorganism causing the infection is reported to be "susceptible" to the antibiotic.

A problem with currently available antimicrobial susceptibility tests is their failure to reliably predict the outcome of therapy.

Sometimes an antibiotic will fail to cure an infection even though the microorganism is susceptible to the antibiotic in the laboratory test.

That is, the current routine laboratory tests can be misleading and give an over-optimistic impression of the therapeutic potential of antibiotics.

These tests can therefore cause patients to be given ineffective treatments.

In serious infections, this inadequacy of current laboratory tests can have fatal consequences.

However one explanation is error arising from a deficiency in the antibiotic susceptibility test itself.

That deficiency is that current routine antibiotic susceptibility tests do not detect the antibiotic-inactivating potential of some microorganisms.

Such enzymes, which are not reliably detected in routine antibiotic susceptibility tests, may cause sufficient antibiotic inactivation at the site of an infection in a patient to cause a treatment failure.

The genes are typically encoded on large plasmids containing other antibiotic encoding resistance genes, leaving few therapeutic options.

However, this test is technically demanding and this has precluded its widespread adoption.

Multiplex PCR is also currently available as a research tool for detection of plasmid-mediated AmpC .beta.-lactamases, but is not yet available as a routine test for clinical labs.

The currently used antibiotic susceptibility tests, which measure only the antimicrobial activity of antibiotics and not the ability of the microorganisms to cause antibiotic inactivation, fail to take into account this very important determinant of the outcome of therapy.

This deficiency in the tests places clinicians at a disadvantage in selecting the most appropriate antibiotics for their infected patients.

Both disk diffusion and dilution methods are generally deficient in that they do not yield information about the ability of microorganisms to inactivate antibiotics.

These are complex tests to detect enzymatic inactivation of chloramphenicol and require special instruments capable of measuring the absorbance of light at specific wavelengths or of measuring radioactivity.

Such tests are not antibiotic susceptibility tests and their complexity is such that they are unsuitable for routine clinical microbiology laboratories.

They do not predict the potential for any other bacteria to resist these penicillins, and they do not predict the potential for any bacteria to be resistant to any of the other classes of .beta.-lactam antibiotics, such as cephalosporins, cephamycins, monobactams, monocarbams, penems or carbapenems.

The preincubation procedure thereby invalidates the interpretive tables that are necessary to determine antibiotic susceptibility or resistance.

This is a serious deficiency because it would be unethical to base therapy on this procedure which lacks validated interpretive criteria.

This is inconvenient and therefore a disadvantage.

Furthermore there are doubts about the validity of results obtained with this procedure.

It is therefore inconvenient and limited in scope.

Antibiotic inactivation results in a distortion or discontinuity in the usually circular inhibition zone.

There are several problems with the 3-dimensional test.

a. The procedure for making the slit in the agar for the 3-dimensional test is inconvenient and technically difficult to perform correctly.

b. Making the slit is potentially dangerous to laboratory staff because a scalpel blade contaminated with pathogenic bacteria is an infection hazard.

c. It is also technically difficult to accurately deliver the liquid 3-dimensional inoculum into the slit without overfilling the slit and possibly invalidating the test.

A variety of chemicals have been reported to disrupt or permeabilize microbial membranes, thereby increasing their permeability and causing loss of cellular contents.

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