Identification of mycobacterium tuberculosis complex species

Methods for using reporter mycobacteriophage (RM) and p-nitro-.alpha.-acetylamino-.beta.-hydroxy-propiophenone (NAP) to identify TB complex mycobacteria and distinguish these species from MOTT. RM-infected MOTT show little or no reduction in signal when treated with NAP. In contrast, TB complex mycobacteria infected with RM are distinguishable from RM-infected MOTT by a reduction in signal with NAP treatment.

FIELD OF THE INVENTION 
The invention relates to detection and identification of microorganisms, 
and in particular to detection and identification of microorganisms of the 
Mycobacterium tuberculosis (M.tb.) complex. This work was supported in 
part by grant No. CCR209585-01 from the Centers for Disease Control to 
John Chan. 
BACKGROUND OF THE INVENTION 
The Mycobacteria are a genus of bacteria which are acid-fast, non-motile, 
gram-positive rods. The genus comprises several species which include, but 
are not limited to, Mycobacterium africanum, M. avium, M. bovis, M. 
bovis-BCG, M. chelonae, M. fortuitum, M. gordonae, M. intracellulare, M. 
kansasii, M. microti, M. scrofulaceum, M. paratuberculosis and M. 
tuberculosis. Certain of these organisms are the causative agents of 
disease. For the first time since 1953, cases of mycobacterial infections 
are increasing in the United States. Of particular concern is 
tuberculosis, the etiological agent of which is M. tuberculosis. M. 
tuberculosis and other mycobacteria which are closely related to M.tb. (M. 
bovis, M. africanum, M. tuberculosis BCG and M. microti) are referred to 
as the TB complex mycobacteria. Many of these new cases of mycobacterial 
infection are related to the AIDS epidemic, which provides an immune 
compromised population which is particularly susceptible to infection by 
Mycobacteria. Mycobacterial infections other than tuberculosis are also 
increasing as a result of recent increases in the number of immune 
compromised patients. For example, Mycobacterium avium, Mycobacterium 
kansasii and other non-tuberculosis mycobacteria are found as 
opportunistic pathogens in patients infected with HIV as well as in in 
other immune compromised patients. 
In recent years there has also been an increase in the number of clinical 
isolates of tuberculosis which are resistant to at least one of the 
antibiotics normally used to treat the disease (e.g., isoniazid, rifampin 
or streptomycin). Multidrug-resistant tuberculosis strains have emerged in 
several countries, resulting in a corresponding increase in the number of 
fatalities in both immunocompetent and immunocompromised individuals. 
Because M.tb. grows very slowly (doubling time 20-24 hrs.), conventional 
methods for identifying this organism and determining drug susceptibility 
require 2-18 weeks. During that time, patients are often treated 
empirically with antibiotics which may be ineffective, as lack of any 
treatment allows the patient to remain infectious and puts the patient and 
patient contacts at risk. Such empirical treatment can also exacerbate the 
development of drug resistance. 
Conventional diagnosis of mycobacterial infections is dependent on 
acid-fast staining and cultivation of the organism, followed by 
biochemical and morphological assays to confirm the presence of 
mycobacteria and identify the species. These procedures are 
time-consuming, and a typical diagnosis using conventional culture methods 
can take as long as six weeks. Automated culturing systems such as the 
BACTEC.TM. system (Becton Dickinson Diagnostic Instrument Systems, Sparks, 
Md.) can decrease the time for detection of mycobacteria to one to two 
weeks. Once detected, culturing these slow-growing microorganisms in the 
presence of antibiotics to determine their drug susceptibility requires 
several additional weeks. There is still a need to reduce the time 
required for diagnosing mycobacterial infections and determining 
antibiotic susceptibility even further in order to allow prompt, informed 
treatment of M.tb. infections. 
The BACTEC TB System provides one means for determining whether or not a 
positive mycobacterial culture is the result of TB complex mycobacteria or 
mycobacteria other than tuberculosis (MOTT). This is important information 
for the initial diagnosis of tuberculosis, and shortens the time required 
for determining the species present in a positive mycobacterial culture. 
The BACTEC identification scheme relies on a combination of three tests, 
namely, morphology on smear, growth characteristics and the NAP 
(p-nitro-.alpha.-acetylamino-.beta.-hydroxy-propiophenone) TB 
differentiation test. NAP is an intermediate compound in the synthesis of 
chloramphenicol which markedly inhibits the growth mycobacteria belonging 
to the TB complex. MOTT show little or no growth inhibition, and any 
slight inhibition of growth is usually temporary. The mechanism of action 
of NAP on TB complex mycobacteria is not known, nor is the reason for its 
TB complex-specificity. When cultured in the presence of NAP, TB complex 
organisms show sharply reduced evolution of CO.sub.2, whereas MOTT 
continue to grow with increasing CO.sub.2 production. The BACTEC TB System 
measures CO.sub.2 evolution, as a "growth index" (GI) by monitoring 
production of 14.sub.C -labeled CO.sub.2 in cultures containing 14.sub.C 
-labeled palmitate. Once a positive culture is obtained, speciation by 
determining growth (CO.sub.2 production) in the presence of NAP generally 
requires an additional 4-6 days. 
Luciferase is useful as a biological reporter or signal generating molecule 
because it catalyzes the reaction of luciferin with adenosine triphosphate 
(ATP), resulting in the production of light. Sensitive light-detection 
systems are available to detect and measure light (luminescence) generated 
by this reaction. Luciferase has been used for many years in the standard 
assay for measuring ATP. The cDNA coding for firefly luciferase (FFluc) 
has been cloned, which has allowed its use as a direct reporter molecule 
in a variety of transformed and transfected cells. In mycobacteria, FFluc 
has been inserted into the genomes of mycobacteriophage and into plasmids 
as a reporter gene for use in antibiotic susceptibility testing as an in 
vivo measure of cell viability after exposure to antibiotics. W. R. 
Jacobs, et al. (1993) Science 260:819 and WO 93/16172. Inhibition of 
culture growth results in reduced or absent light production from the 
cloned luciferase gene. This effect has been attributed to reduced amounts 
of ATP (required for the luciferase reaction) in antibiotic-sensitive 
cells, which exhibit reduced metabolic activity in the presence of an 
anti-TB antibiotic. 
.beta.-galactosidase is an enzyme which cleaves lactose into glucose and 
galactose. Other substrates for this enzyme are also known. X-gal 
(5-bromo-4-chloro-3-indolyl-.beta.-D-galactoside) and chlorophenol 
red-.beta.-D-galactopyranoside are colorimetric substrates for 
.beta.-galactosidase. Enzymatic cleavage of X-gal produces a reaction 
product which is blue in color. Enzymatic cleavage of chlorophenol 
red-.beta.-D-galactopyranoside produces a reaction product which is yellow 
to red in color. Methyl umbelliferyl-.beta.-D-galactopyranoside is a 
fluorometric substrate for .beta.-galactosidase which produces a 
fluorescent signal when enzymatically cleaved. This ability to produce a 
signal makes .beta.-galactosidase useful as a reporter molecule in 
conjunction with colorimetric or fluorometric the enzymatic substrates, 
and these signal generating systems have been used in a variety of 
biological assays. Like FFluc, the bacterial gene which encodes 
.beta.-galactosidase (LacZ) has been cloned and used as a reporter gene in 
recombinant organisms in both inducible and constitutive expression 
systems. 
As used herein, the term "reporter gene" refers to a gene which can be 
expressed to produce a gene product which directly or through further 
reaction generates a detectable signal. This signal can be used to detect 
or identify cells carrying the gene, either on a plasmid or inserted into 
the genome of the cell. Examples of reporter genes are the gene encoding 
firefly luciferase (resulting in a luminescent signal upon reaction with 
luciferin) and the gene encoding .beta.-galactosidase (resulting in a 
colored or fluorescent signal upon reaction with appropriate enzyme 
substrates). A mycobacteriophage carrying a reporter gene is referred to 
herein as a "reporter mycobacteriophage" or "RM." Mycobacteriophage 
carrying a luciferase reporter gene are referred to as "luciferase 
reporter mycobacteriphage" or "LRM." Mycobacteriphage carrying a 
.beta.-galactosidase reporter gene are referred to as 
".beta.-galactosidase reporter mycobateriphage" or ".beta.-GRM." 
The host range of mycobacteriophage varies greatly, with some capable of 
infecting only a single species. Certain mycobacteriophage (e.g., TM4 or 
phAE40) have been characterized as preferentially infecting species of the 
TB complex, whereas others (e.g., L5) have a very broad range of 
mycobacterial hosts. A reporter mycobacteriophage constructed in TM4 or 
phAE40 would therefore be expected to be useful for specific 
identification of TB complex organisms, as primarily TB complex species 
should be infected and produce a signal. However, in practice, these 
mycobacteriophage are not perfectly species-specific, infecting and 
producing high levels of signal in certain MOTT species as well. This 
results in false-positives which are unacceptable for clinical detection 
and identification of TB complex mycobacteria. The present invention not 
only meets the need for a more rapid method for detection, identification 
and antibiotic susceptibility testing of TB complex organisms, it solves 
the problem of identifying false-positives and provides more accurate 
identification of TB complex organisms using a reporter mycobacteriophage. 
SUMMARY OF THE INVENTION 
The present invention uses luciferase reporter mycobacteriophage or 
.beta.-galactosidase reporter mycobacteriphage and NAP to identify TB 
complex organisms and distinguish them from MOTT. As the mechanism of TB 
complex growth inhibition by NAP is not known, it was not known prior to 
the present invention whether NAP inhibition of bacterial growth would 
affect the production of light by LRM or affect the production of a 
reaction product by .beta.-GRM. That is, it was not known prior to the 
present invention whether or not exposure to NAP would result in reduced 
production of light by a luciferase reporter mycobacteriophage and, if so, 
whether or not reduced light production could be used to distinguish TB 
complex organisms from MOTT. It was also unknown whether or not exposure 
to NAP would result in a reduction in .beta.-galactosidase reaction 
products produced by .beta.-galactosidase reporter mycobacteriophage and, 
if so, whether or not this response could be used to distinguish these 
species of mycobacteria. It has now been discovered that use of the 
luciferase reporter gene or the .beta.-galactosidase reporter gene in 
place of CO.sub.2 measurements in the conventional NAP identification 
system allows TB Complex vs. MOTT species identification in 48 hours or 
less. In many cases identification can be made in as little as 1-3 hours. 
The successful assays performed with these two reporter genes suggest that 
many of the reporter genes known in the art may be substituted in the 
RM/NAP assays of the invention with similar results. 
Further, in conjunction with antibiotic susceptibility testing, the RM/NAP 
identification system the invention allows more information to be obtained 
from a determination of antibiotic susceptibility. Antibiotic resistant 
MOTT and antibiotic resistant TB complex organisms infected with RM both 
produce a positive signal when cultured in the presence of the antibiotic. 
However, a positive signal (e.g., luminescence from luciferin or a colored 
reaction product from a colorimetric substrate for .beta.-galactosidase) 
which is due to an antibiotic-resistant TB complex organism is 
substantially reduced upon exposure to NAP, whereas antibiotic-resistant 
MOTT continue to produce a signal when treated with NAP.

DETAILED DESCRIPTION OF THE INVENTION 
The invention provides methods for clinical identification or antimicrobial 
susceptibility testing of M. tuberculosis and other TB complex organisms 
using a reporter mycobacteriophage assay and the differential effect of 
NAP on the growth of mycobacteria species. The inventive methods not only 
allow rapid identification of TB complex organisms, they distinguish 
between a false-positive signal arising from MOTT infected by the reporter 
mycobacteriophage and a true positive signal arising from phage-infected 
TB complex organisms. The methods may also be adapted to determine 
antibiotic susceptibility, distinguishing between a positive signal 
arising from an antibiotic-resistant TB complex organism and a positive 
signal arising from antibiotic resistant MOTT. 
Applicants have for the first time demonstrated that NAP treatment can be 
used in a reporter phage assay to distinguish between TB complex organisms 
and MOTT. The mechanism of TB-complex-specific growth inhibition by NAP is 
not well understood, although it was previously known that growth 
inhibition was accompanied by reduced CO.sub.2 evolution by cultures. Of 
significance to the present invention, however, is that it was not 
previously known whether or not NAP treatment would also selectively 
inhibit luminescence in TB complex organisms in an LRM assay, as several 
mechanisms of NAP-induced growth inhibition which would not affect the 
intracellular level of ATP could be hypothesized (e.g., cell surface 
effects, inhibition of transcription or translation factors, etc.). 
Whether or not NAP would inhibit processes necessary for the production of 
colored or fluorescent reaction products in the .beta.-GRM assay was also 
unknown. It has now been discovered that mycobacteria cultured in a 
suitable liquid media can be treated with NAP in an amount sufficient to 
inhibit growth of TB complex mycobacteria, and detected in an LRM assay 
such as the assay described by W. R. Jacobs, et al. and in WO 93/16172, 
supra, to distinguish TB complex from MOTT organisms by reduction in 
luminescence. Alternatively, NAP inhibition of growth of TB complex 
organisms may be detected as reduced production of colored or fluorescent 
reaction products in a .beta.-GRM assay. 
The following discussion employs LRM as an example. However, it is equally 
applicable to .beta.-GRM as well as other RM carrying other reporter 
genes. In the conventional LRM assay, mycobacterial cells are infected 
with the LRM and begin to synthesize luciferase by expression of the 
cloned gene carried by the reporter mycobacteriophage. Upon addition of 
luciferin, infected cells emit a signal (luminescence), whereas uninfected 
cells do not. Identification of the species of mycobacteria should 
therefore be possible by selection of a RM with the desired host range. 
However, the RM's currently available are not perfectly specific for the 
TB complex organisms. Although they do not infect all MOTT, many MOTT 
species produce high luminescence signals due to infection with "TB 
complex-specific" LRM such as those derived from TM4 and phAE40 . For 
example, luciferase reporter mycobacteriophage phAE42-12 is a mutant of 
phAE40 isolated for increased luminescence signal and deposited with the 
American Type Culture Collection (Rockville, Md.) on Feb. 7, 1995 as 
Accession Number ATCC 97046. phAE40 is a mutant derived from TM4, selected 
for broadened range of infection of TB complex organisms. Like its parent 
phage, phAE42-12 efficiently infects M. flavescens, M. smegmatis, M. 
intracellulare and M. chelonae and gives high luminescence signals. 
phAE42-12 generally does not infect M. fortuitum; M. gordonae, M. kansasii 
and M. xenopi, but certain strains of these species have been found which 
are inefficiently infected. Similarly, although many strains of M. avium 
do not support infection or are inefficiently infected, some strains have 
been found which are efficiently infected. Temperature may also affect the 
infection capability of mycobacteriophage. For example, phAE42-12 will 
infect M. fortuitum and M. gordonae at temperatures greater than about 
37.degree. C. 
Treatment with NAP can reduce the luminescence signal 5-10 fold or more in 
TB complex organisms with little or no effect on the high levels of 
luminescence signal from LRM-infected MOTT, regardless of whether the 
infection is due to lack of infection specificity or is induced by higher 
temperatures. This differential effect has now been used to determine 
whether or not a positive signal in a reporter mycobacteriophage assay for 
TB complex organisms is indicative of a TB complex organism or MOTT. The 
invention therefore increases the utility of reporter phage constructions, 
as perfect infection specificity for the TB complex is no longer required 
for a useful clinical diagnostic test. Sometimes a minor reduction in the 
luminescence signal is seen initially in LRM infected MOTT treated with 
NAP, however, this reduction is less than 5-fold and the organisms often 
recover. Therefore, a substantial reduction in signal (defined as a 
reduction in signal of about 5-fold or more) is indicative of TB complex 
mycobacteria. 
A typical assay for distinguishing species of mycobacteria according to the 
invention is performed as follows. Log phase cultures of the mycobacteria 
to be identified are exposed to NAP under conditions appropriate for 
continued growth of the culture. For mycobacteria, growth conditions are 
typically a liquid medium conventionally used for growth of mycobacteria 
(e.g., Lowenstein Jensen media, 7H9 or BACTEC liquid media) and an 
incubation temperature of about 37.degree. C. The culture is exposed to an 
amount of NAP sufficient to inhibit growth of TB complex organisms if they 
are present. A NAP concentration of 0.5-100 .mu.g/ml is typically 
sufficient to inhibit growth of TB complex mycobacteria. The culture may 
be infected with the RM either simultaneously with exposure to NAP, or the 
culture may be incubated for a period of time in the presence of NAP prior 
to infection with the RM. Typically, cultures are incubated in the 
presence of NAP for about 1-48 hours to ensure that growth of TB complex 
mycobacteria, if present, is inhibited. However, as illustrated in Example 
2, the period of exposure to NAP in many cases may be as short as 1-3 
hours. It is only necessary that the exposure to NAP be long enough to 
result in a detectable reduction in luminescence or .beta.-galactosidase 
reaction product in TB complex mycobacteria in the RM assay. If the 
culture medium contains a detergent, the cells must be pelleted, washed 
and resuspended in a medium which does not contain a detergent prior to 
mycobacteriophage infection. The RM are then added to the culture (with 
NAP if the cells were not previously exposed) and incubated at about 
35.degree.-50.degree. C. for a period of time to allow phage infection. 
Typically, about 1-5 hours is allowed for infection, however, the time for 
infection can be routinely optimized and adjusted for a particular RM/NAP 
assay system. The luminescence of LRM-infected cells is then determined 
upon addition of luciferin, measuring light emission in a luminometer. If 
a .beta.-GRM is used, the colorimetric or fluorometric substrate is added 
and incubated with the .beta.-GRM infected cells for a period of time 
sufficient for the enzymatic reaction to occur (typically several hours or 
overnight). The amount of the reaction product is then determined 
qualitatively or quantitatively. Colored reaction products may be 
detected, for example, visually or by optical density. Fluorescence may be 
detected, for example, visually or by instrumentation. 
The above assay for distinguishing species of mycobacteria may be included 
in protocols for antibiotic susceptibility testing (AST) of mycobacteria 
to determine whether or not an antibiotic-resistant strain is an 
antibiotic-resistant TB complex mycobacterium or antibiotic-resistant 
MOTT. This allows identification of the mycobacterium at essentially the 
same time as its antibiotic susceptibility is determined. For example, a 
clinical specimen suspected of containing a species of mycobacteria may be 
cultured and then tested in parallel in the NAP RM assay of the invention 
and in an RM assay for antibiotic susceptibility, for example the LRM 
assay described by Jacobs, et al., supra. After culturing, several 
subcultures are prepared: 1) a subculture containing 0.5-100 .mu./ml NAP, 
2) at least one subculture containing an antibiotic (e.g., streptomycin, 
isoniazid, rifampicin or ethambutol), and 3) a control subculture 
containing neither antibiotic or NAP. After incubation of the subcultures 
for a period of time to allow NAP and the antibiotics to take effect, the 
control, NAP and antibiotic subcultures are infected with an RM as 
described above. Typically, the subcultures are infected about 48 hours 
after subculturing so that the NAP result and the antibiotic sensitivity 
result are available at approximately the same time. The luminescence of 
LRM-infected cells or reaction product produced by .beta.-GRM infected 
cells is then determined as described above, and the signal in the NAP and 
antibiotic subcultures is compared to the uninhibited control. If signal 
is substantially reduced or absent in the NAP subculture, the 
mycobacterium present in the clinical sample is a member of the TB 
complex. If the RM signal is also reduced or absent in one or more of the 
antibiotic subcultures, that TB complex mycobacterium is also sensitive to 
the antibiotic present in the subculture or subcultures in which the 
signal is reduced or absent. If the RM signal is comparable to the control 
in the NAP subculture and reduced or absent in one or more of the 
antibiotic subcultures, the mycobacterium is a MOTT which is sensitive to 
the antibiotic present in the subculture or subcultures in which the 
signal is reduced or absent. Antibiotic-resistant TB complex mycobacteria 
produce levels of signal comparable to the control in the subculture(s) 
containing the antibiotic(s) to which they are resistant, but signal would 
be reduced or absent in the NAP subculture. Antibiotic-resistant MOTT 
produce levels of RM signal comparable to the control in the subculture(s) 
containing the antibiotic(s) to which they are resistant and in the NAP 
subculture. Of course, a microorganism may be resistant to one 
concentration of an antibiotic but sensitive at higher concentrations. The 
level of resistance or sensitivity may be determined by adjusting the 
concentration of antibiotic in the subcultures in the methods of the 
invention. 
The effect of NAP on TB complex mycobacteria can be detected in the RM 
assay with 1-3 hours exposure to NAP, whereas longer exposure to the 
antibiotic (e.g., 48 hours) may be required to detect sensitivity or 
resistance. For this reason, it may be useful to first determine whether 
or not a clinical specimen contains TB complex mycobacteria using the NAP 
RM assay and subsequently determine antibiotic sensitivity if TB complex 
mycobacteria are found to be present. In this way, a clinician will know 
within a few hours whether or not clinically relevant TB complex 
mycobacteria are present in a specimen. 
The invention improves RM assays by simplifying the interpretation of assay 
results for identification of mycobacterial species and significantly 
shortening the time required to obtain these results. The conventional 
BACTEC NAP identification system is not begun until the culture reaches GI 
50, and then requires an additional 4-6 days to complete. Results may be 
obtained using the inventive methods in approximately the same amount of 
time as in probe-based hybridization systems for identification of 
mycobacteria (often 3 hours or less). In addition, antibiotic 
susceptibility testing (AST) using the RM/NAP methods of the invention 
provides a time savings of about 5-8 days (starting from BACTEC cultures) 
as compared to conventional BACTEC AST protocols. The conventional BACTEC 
AST is begun when the culture reaches GI 500 and then requires an 
additional 5-6 days to complete. Conventional AST on solid media requires 
even longer time periods than conventional BACTEC AST to obtain a result. 
In contrast, AST performed with the RM assay of the invention begins at GI 
&lt;500 (typically GI less than about 250) and can be completed in 1-2 
additional days. 
EXAMPLE 1 
Representative TB complex organisms (M. bovis bcg and M. tuberculosis 
strain 201) and various mycobacteria other than tuberculosis were 
differentiated using LRM detection with and without NAP treatment. The 
growth of the test mycobacteria on Lowenstein Jensen slants was 
standardized by inoculation into BACTEC liquid media, subculturing when 
necessary to obtain log phase cultures at moderate growth index (typically 
GI 100-300). BACTEC culture bottles containing 5 .mu.g/ml NAP were 
prepared by addition of 0.1 ml of 200 .mu.g/ml NAP to each 4.0 ml bottle. 
The organism to be tested was subcultured into BACTEC culture bottles with 
or without NAP by addition of 0.5 ml of the liquid culture to each bottle. 
Subculture bottles containing NAP were incubated at 37.degree. C. for an 
additional 24-48 hours, monitoring GI daily. The luciferase phage assay 
was then performed using phage phAE42-12, comparing the luminescence 
signals obtained from infections of NAP treated organisms with untreated 
organisms, as follows. 
After the NAP treatment period, media were removed and the organisms in 
each culture bottle were washed. Cells were pelleted by centrifugation at 
1500 xg for 15 minutes at room temperature and resuspended in room 
temperature 7H9 medium (Difco) containing 0.2% glycerol and 10% ADC (5% 
albumin/2% dextrose/145 mM sodium chloride). The cells were washed a 
second time as before, resuspending the cells in a final volume of 350 
.mu.gl 7H9. To initiate infection, phage phAE42-12 were added in a volume 
of 7.5 .mu.l to give 4.times.10.sup.9 pfu/ml and incubated at 37.degree. 
C. for 3 hours. The luminescence signal was measured after 3 hours of 
infection by transferring 100 .mu.l of phage-infected cells to a 
luminometer cuvette and adding 100 .mu.l of 1 mM luciferin in 0.1 M sodium 
citrate pH 4.5. Light emission was measured immediately after addition of 
these reagents, integrating the signal for 15 seconds. Luminescence was 
measured as the ratio of luminescence signal to background luminescence in 
uninfected cultures. A ratio of signal to background of 2 or greater is 
considered positive for light production by the LRM. 
To assess the effect of temperature on the assay, similar experiments were 
performed with phage infection at 47.degree. C. For several MOTT the 
signal was higher for infections at 47.degree. C. as compared to 
infections at 37.degree. C. NAP treatment had little effect on the 
luminescence signal generated by these organisms, regardless of the 
temperature of phage infection. However, the signal from untreated TB 
complex organisms was greatly reduced with infection at 47.degree. C. as 
compared to untreated TB complex organisms infected at 37.degree. C. 
The luminescence signal from TB complex organisms was substantially reduced 
by more than 1 log by pretreating with 5 .mu.g/ml NAP for 24-48 hours. For 
MOTT the signal was essentially unchanged by NAP pretreatment. The results 
are shown in the following Table: 
______________________________________ 
NAP Exposure 
Luminescence 
Mycobacterium 
NAP 5 .mu.g/ml 
(hrs.) (Signal/Background 
______________________________________ 
TB COMPLEX 
TB201 - 24 27 
+ 3.9 
- 48 32 
+ 1 
BCG - 48 5650 
+ 4 
BCG - 48 120 
+ 0.9 
BCG - 48 100 
+ 1.1 
BCG - 48 138 
+ 1.2 
BCG - 48 169 
+ 1.3 
BCG - 48 378 
+ 2.4 
MOTT 
xenopi - 24 1 
+ 1 
- 48 1.9 
+ 1.1 
kansasii - 24 1.3 
+ 1.7 
- 48 2.4 
+ 2.1 
avium 1546 - 24 1.3 
+ 1.4 
- 48 5.6 
+ 4.2 
flavescens - 24 13 
+ 12 
- 48 51 
+ 33 
smegmatis &gt;37.degree. C. 
- 24 580 
+ 1132 
- 48 135 
+ 403 
fortuitum &gt;37.degree. C. 
- 24 57 
+ 96 
- 48 221 
+ 184 
gordonae &gt;37.degree. C. 
- 24 42 
+ 50 
- 48 146 
+ 164 
chelonae &gt;37.degree. C. 
- 24 1.2 
+ 1.1 
- 48 1.24 
+ 1.25 
gastri #2978 
- 48 9.9 
+ 10 
gastri #2977 
- 48 2.1 
+ 2.0 
gastri #2973 
- 48 5.3 
+ 3.3 
fortuitum 37.degree. C. 
- 48 1.1 
+ 1.1 
gordonae 37.degree. C. 
- 48 1.0 
+ 0.9 
smegmatis 37.degree. C. 
- 48 67 
+ 34 
chelonae 37.degree. C. 
- 48 1.9 
+ 2.6 
intracellulare 
- 48 37 
(Edgar B.) + 27 
intracellulare 
- 48 13 
(P-54) + 16 
______________________________________ 
As expected, M. fortuitum, M. gordonae, M. avium, M. kansasii and M. xenopi 
showed little or no infection by the LRM. Any low levels of luminescence 
detected in these species were essentially unchanged by treatment with 
NAP. MOTT strains which showed significant levels of infection by LRM were 
M. flavescens, M. smegmatis, M. chelonae, M. intracellulare, and M. 
fortuitum and M. gordonae at higher temperatures. The luminescence 
produced by these organisms was also essentially unaffected by treatment 
with NAP. In contrast, the luminescence signals of M. tuberculosis and M. 
tuberculosis BCG were high in the absence of NAP treatment but were 
substantially reduced to near or below 2 (an essentially negative 
signal/background) by treatment with NAP. 
EXAMPLE 2 
M. bovis BCG and phage phAE42-12 were used to measure the kinetics of the 
NAP effect on detection of TB complex organisms using LRM, comparing 
treatment with 5 .mu.g/ml and 10 .mu.g/ml NAP. Seven day roller bottle 
cultures of BCG were grown in 7H9 media containing 0.2% glycerol, 10% ADC 
and 0.01% TWEEN-80. The cells were washed twice in the same medium but 
without TWEEN-80 as described in Example 1. The cells were then diluted to 
a density of 10.sup.7 cells/ml for phage infection. In some samples NAP 
was added at the same time as the phage for 3 hours. In other samples NAP 
was added before or after phage to obtain treatment times varying from 0-5 
hours. All luminescence assays were performed as in Example 1 at 3 hours 
post infection. The results are shown in FIG. 1. 
Three hours of NAP treatment, obtained by simultaneous addition of NAP and 
phage phAE42-12, was sufficient to reduce the luminescence signal more 
than 10 fold. However, the time course of reduction in luminescence shown 
in FIG. 1 indicates that the reduction in signal would be substantial and 
easily detectable in as little as 1 hour (about a 5-fold reduction). This 
rapid effect of NAP indicates that the method of the invention provides an 
identification test which can identify TB complex organisms in three hours 
or less with a simple workflow. This is in contrast to prior art methods 
in which the effect of NAP on culture growth is followed by monitoring 
CO.sub.2 production, requiring 4-6 days for species identification. 
EXAMPLE 3 
The methods of the invention were used to distinguish between a TB complex 
species (M. bovis BCG) and a MOTT species (M. smegmatis) in about three 
hours. Seven day roller bottle cultures of these species were grown in 7H9 
media containing 0.2% glycerol, 10% ADC and 0.01% TWEEN-80. The cells were 
washed twice in the same medium but without TWEEN-80 as described in 
Example 1. The cells were then diluted to a density of 10.sup.7 /ml for 
phage infection. NAP (5 .mu.g/ml) was added at the same time as phage. 
Luminescence assays were performed as in Example 1 at 3 hours post 
infection. The results are shown in FIG. 2. Luminescence in M. smegmatis 
was essentially unchanged in the presence of NAP, however, luminescence in 
M. bovis BCG was reduced about 14-fold, providing a clear distinction 
between the TB complex species and the MOTT species. 
EXAMPLE 4 
The FFluc gene of phAE40 (expressed from a heat shock protein "hsp" 
promoter) was removed and replaced with the .beta.-galactosidase gene 
(LacZ) for use as a .beta.-GRM in the NAP assay. This phage was identified 
as phAE40-LACZ. Colorimetric systems such as this are useful for 
situations in which instrumentation, such as a luminometer, is not readily 
available. Seven day roller bottle cultures of M. bovis BCG and overnight 
log phase cultures of M. smegmatis in 7H9 with 10% ADC and 0.01% TWEEN-80 
were centrifuged to pellet the cells and washed twice with 7H9 medium. 
Assays were set up for each species at 10.sup.8 cells/ml, 10.sup.7 
cells/ml and 10.sup.6 cells/mi. These samples were infected with 
phAE40-LACZ at 2.times.10.sup.10 pfu/ml, simultaneously adding 5 .mu.g/ml 
or 10 .mu.g/ml NAP. Infection was allowed to proceed for 2 hours at 
37.degree. C. Uninfected controls and infected controls without NAP 
treatment were also included in the analysis. 
Following infection, X-Gal was added to a final concentration of 0.02% and 
the samples were incubated overnight to allow color development. The 
intensity of the blue color produced was evaluated visually and by reading 
absorbance at 620 nm (A.sub.620). Inhibition of color development in the 
BCG samples was generally not as complete as inhibition of luminescence in 
the LRM assay. However, it was possible to differentiate BCG from MOTT 
visually, particularly at lower cell concentrations (10.sup.6 cells/ml), 
where an estimated 4-5-fold reduction in signal was observed after 
treatment with either 5 .mu.g/ml or 10 .mu.ml NAP. As cell concentration 
increased, the difference in color production between the two species 
became less distinct. The variable effect of NAP on culture growth 
depending on cell density ("inoculum effect") has been previously observed 
in other NAP assay systems, and is not believed to be due to any feature 
of this particular assay. Absorbance readings were also more variable than 
those obtained in the LRM assay, but the signal/background ratios were 
generally lower for BCG treated with NAP than for M. smegmatis treated 
with NAP. 
The above experiment was repeated, substituting chlorophenol 
red-.beta.-D-galactopyranoside for X-gal as the colorimetric enzyme 
substrate. Similar results were obtained, however, reduction in the 
reaction product could be detected in several hours. This substrate may 
therefore provide a more sensitive detection system than X-gal. It is 
expected that the .beta.-GRM assay system cab be improved by further 
optimization of parameters such as the time of substrate addition, and the 
time and dose for NAP treatment. It is also expected that other 
colorimetric or fluorometric substrates for .beta.-galactosidase may be 
routinely used in this assay to distinguish TB complex organisms from 
MOTT. 
.beta.-galactosidase is much more stable in the cell than luciferase, and 
it was therefore unexpected that a reduction in the amount of reaction 
product could be detected in this system within several hours of exposure 
to NAP. Further, the .beta.-galactosidase signal generating system does 
not require ATP for signal production as luciferase does. It was therefore 
uncertain whether NAP treatment would result in a reduction in 
.beta.-galactosidase reaction product at all. The discovery that two 
reporter genes with such different enzymatic mechanisms can be used in the 
RM assay of the invention suggests that the effect of NAP on growth of TB 
complex mycobacteria may affect multiple biochemical processes specific to 
TB complex organisms.