Mycobacterial species-specific reporter mycobacteriophages

Details Mycobacterial species-specific reporter mycobacteriophages Mycobacterial species-specific reporter mycobacteriophages

1996-08-29

2001-10-09

Ketter, James (Department: 1805)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

Reexamination Certificate

active

06300061

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to mycobacterial species-specific reporter mycobacteriophages (reporter mycobacteriophages), methods of making such reporter mycobacteriophages, and the use of such reporter mycobacteriophages, for example, to rapidly diagnose mycobacterial infection and to assess drug susceptibilities of mycobacterial strains in clinical samples. Specifically, this invention relates to the use of mycobacterial species-specific luciferase reporter mycobacteriophages to diagnose tuberculosis and to assess the drug susceptibilities of the various strains of
Mycobacterium tuberculosis
(
M. tuberculosis
).
To produce the mycobacterial species-specific reporter mycobacteriophages of the invention, transcriptional promoters and reporter genes are introduced into the genomes of mycobacterial species-specific mycobacteriophages. These reporter genes may be the genes for luciferase or the &bgr;-galactosidase gene, and provide the DNA which encodes the production of a gene product.
The reporter mycobacteriophages may be used for diagnosing mycobacterial infections by incubating same with samples which may contain the specific mycobacteria of interest. If the mycobacteria of interest is present, then the reporter mycobacteriophages introduce the recombinant nucleic acids which encode expression of the gene product into the mycobacteria of interest, and the mycobacteria then express the gene product. The expressed reporter gene product may be detected by a suitable assay, for example, through the detection of photons or the conversion of an easily assayable chemical reaction. The presence of such gene product indicates that the sample contains the mycobacteria of interest, and hence the mycobacterial species-specific reporter mycobacteriophages may be used to detect and thereby diagnose the specific mycobacterial infection.
Since signals may not be generated by cells which are not metabolically active in the presence of antibiotics, the mycobacteria species-specific reporter mycobacteriophages of this invention may be used to assess the drug susceptibilities of various strains of mycobacteria. If antibiotic drugs are added to the sample containing the reporter mycobacteriophages and the gene product is detected, the mycobacteria is metabolically active and hence resistant to the antibiotic drug.
BACKGROUND OF THE INVENTION
In 1990, there was a 10% increase in the incidence of tuberculosis in the United States. In addition, there has been an increase in the appearance of clinical isolates of tuberculosis that are resistant to antibiotics used to treat the disease. This problem is exacerbated by the length of time that is currently needed both to diagnose tuberculosis, and to determine the drug susceptibilities of various strains of
M. tuberculosis
. As a result, patients with
M. tuberculosis
may remain infectious for long periods of time without being treated, or may be treated with a drug to which the bacterial strain is resistant. Therefore, a need has arisen in the field for a method of diagnosis of
M. tuberculosis
(and other mycobacterial infections) which is rapid, sensitive and specific, which method is also capable of assessing the drug susceptibilities of the various strains of
M. tuberculosis
and other mycobacterial strains. It is critical that a mycobacterial strain be assessed for drug resistance rapidly because a patient infected with a strain of
M. tuberculosis
or another mycobacteria must be treated immediately with the particular antibiotic drug(s) to which the strain is not resistant, and not with antibiotic drug(s) to which the strain is resistant, or the patient may die.
Currently, the most rapid test available for the diagnosis of
M. tuberculosis
is the staining of sputum samples for acid-fast bacilli, which is a tedious procedure, and which procedure has low sensitivity. Alternative methods for diagnosis require cultivation of the bacilli for approximately two to six weeks followed by classification of the cultured organism. Typical diagnostic tools include biochemical tests, analysis of mycolic acids and serotyping. All of these tests are time-consuming. More recently, the use of oligonucleotide probes and Polymerase Chain Reaction have been suggested for the identification of
M. tuberculosis
species. Although these methods may be useful approaches, their uses in a clinical setting have not yet been determined. Further, these methods do not distinguish between live and dead organisms, and are therefore of limited use in the determination of drug sensitivities of clinical isolates.
In addition,
Mycobacterium avium
(
M. avium
) is a mycobacteria which is often found in immunosuppressed patients. This mycobacteria is typically disseminated throughout the bodies of immunosuppressed patients, such as AIDS patients, and causes
M. avium
infection. Because this mycobacteria often causes death in immunosuppressed patients, it is necessary to be able to diagnose and assess the drug susceptibilities of the various strains of
M. avium.
It is therefore an object of this invention to construct broad mycobacterial host range and mycobacterial species-specific reporter mycobacteriophages.
It is another object of this invention to provide mycobacterial species-specific reporter mycobacteriophages which may be used to rapidly diagnose mycobacterial infections.
It is still another object of this invention to provide mycobacterial species-specific reporter mycobacteriophages which may be used to rapidly assess the drug susceptibilities of different strains of mycobacteria in clinical samples.
It is yet another object of this invention to provide mycobacterial species-specific reporter mycobacteriophages wherein the reporter genes are luciferase genes, which mycobacterial species-specific reporter mycobacteriophages may be used to rapidly diagnose mycobacterial infections and to rapidly assess the drug susceptibilities of various strains of mycobacteria.
It is a further object of this invention to provide mycobacterial species-specific luciferase gene reporter mycobacteriophages which may be used to rapidly diagnose tuberculosis and assess the drug susceptibilities of the various strains of
M. tuberculosis.
SUMMARY OF THE INVENTION
This invention relates to broad host range and mycobacterial species-specific reporter mycobacteriophages, (reporter mycobacteriophages), methods of producing such reporter mycobacteriophages, and the use of such reporter mycobacteriophages to rapidly diagnose mycobacterial infection, such as
M. tuberculosis
, and to distinguish which strains of the mycobacteria are drug-resistant.
To produce these reporter mycobacteriophages, reporter genes and transcriptional promoters are introduced into the genomes of mycobacterial species-specific mycobacteriophages. The promoter and reporter gene-containing mycobacteriophages (reporter mycobacteriophages) are then incubated with a clinical sample which may contain the mycobacteria of interest, such as
M. tuberculosis
. The reporter mycobacteriophages are specific for the mycobacteria which is sought to be detected. The reporter mycobacteriophages efficiently introduce the recombinant nucleic acids which encode the expression of the reporter gene's gene product into the mycobacteria of interest, and the mycobacteria then express the gene product. A substrate or other means capable of allowing for the detection of the gene product is then added to the sample. If the gene product or the signal generated by the gene product is detected, the presence of the infectious mycobacteria is known, thereby diagnosing the disease.
To assess drug susceptibility of mycobacteria, drugs such as antibiotics may be added to a sample containing the reporter mycobacteriophages of this invention. If the mycobacteria are susceptible to a drug after exposure to the drug, the mycobacteria will be killed. However, drug-resistant mycobacteria will continue to be metabolically active in the presence of the drug, and will continue to express the detectable gene product of the reporter genes. P

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