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Description  |
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BACKGROUND OF THE INVENTION
1. Field of the Invention:
The present invention relates to a method for rapidly determining the
effectiveness of antimicrobial agents against bacteria. More particularly,
the invention relates to a method for rapidly determining the
effectiveness of antimicrobial agents against bacteria by measuring the
amount of adenosine triphosphate (ATI) isolated from the bacteria
remaining after treatment with the antimicrobial agent.
2. Description of the Prior Art:
A rapid and routine procedure for the determination of the effectiveness of
antimicrobial agents against various bacteria is very frequently of vital
importance, particularly for the rapid measurement of the effectiveness of
antimicrobial agents against bacteria in urine. Present techniques for the
determination of microbial susceptibility generally require overnight
incubation of an infecting organism after its isolation.
Thus, generally a minimum of 48 hours is required to test the effectiveness
of antimicrobial agents after a specimen is received in the laboratory.
The most commonly used conventional techniques for the determination of
microbial susceptibility to antimicrobial agents include agar diffusion
(Kirby-Bauer technique), broth dilution and agar dilution. A long-standing
need continues to exist for a rapid means for determining microbial
sensitivity to antimicrobial agents to avoid the inclusion of
inappropriate, unnecessary, and often toxic agents in the therapeutic
regimen of an infected person.
The conventional broth dilution method (MIC-Broth Dilution) of determining
microbial sensitivity to antimicrobial agents involves visual detection of
the least amount of antimicrobial agent required to cause complete
inhibition of bacterial growth in a culture medium. In the technique,
two-fold dilutions of antibiotics are made in a suitable culture medium. A
control tube containing the culture medium, but no antibiotic is also
included for each organism tested. The organism is then allowed to grow to
a logarithmic or early stationary phase of growth in the medium, and then
diluted to a solution containing 10.sup.4 to 10.sup.5 viable units per
milliliter. A quantity of the cultured medium is then placed in each tube
of a series containing varying amounts of an antibiotic, and the tubes are
allowed to incubate at 37.degree. C for 16 to 20 hours. Thereafter, the
end point of the test is determined visually, as mentioned above. A
disadvantage of this technique besides the relatively very long time
required to complete the test, is that care must be taken to recognize
that slight amounts of turbidity present may be caused by the inoculum
itself and not by the growth of the organism.
The Kirby-Bauer method involves an agar diffusion technique for the
measurement of microbial susceptibilities. In the test an inoculum of an
organism, which is an overnight culture, is prepared by diluting the
inoculum in trypticase soy broth to such a concentration that a dense, but
not confluent growth is observed. The culture turbidity is adjusted to a
concentration which conforms to a turbidity standard. The standard is
formed by mixing 0.5 ml of 0.048 M barium chloride (1.175% w/v Ba Cl
.sup.. 2H.sub.2 O) with 99.5 ml of 0.36 NH.sub.2 SO.sub.4 (1% w/v). A
cotton swab is then soaked in the diluted culture, and an agar plate is
then streaked in four directions to obtain an even and thorough
distribution of the organism on the plate. The treated plates are dried
for 15 minutes at 37.degree. C. Thereafter, antibiotic discs are applied
to the surface of the agar and pressed into place. No more than 12 discs
are used per plate to prevent overlapping of zones. The plates are then
allowed to stand at room temperature for 30 minutes and incubated at
37.degree. C for 18 to 20 hours. The end point is shown by a clearly
defined zone around the antibiotic disc where no growth has occurred. The
zone is measured and compared with the given sensitivity or resistance of
the antibiotic. Only a zone having a diameter greater than 6.0 mm is
significant because the antibiotic discs are 6.0 mm in diameter. The chief
disadvantage of the diffusion technique is the long period of time
required to obtain the microbial sensitivity results.
SUMMARY OF THE INVENTION
Accordingly, one object of the present invention is to provide a rapid
method for determining the sensitivity of bacterial organisms to
antimicrobial agents.
Another object of the invention is to provide a method of determining the
sensitivity of bacterial organisms to antimicrobial agents by utilizing a
bioluminescent reaction involving bacterial adenosine triphosphate (ATP)
and a mixture of luciferase and luciferin.
Yet another object of the invention is to provide a method of measuring the
sensitivity of microbial organisms present in urine to various
antimicrobial agents.
Briefly, these objects and other objects of the invention, as hereinafter
will become more readily apparent, can be attained by a method for
determining the sensitivity of bacteria to antimicrobial agents by
measurement of an ATP index by culturing a bacterium in a growth medium;
assaying the amount of bacterial adenosine triphosphate in a sample of
said cultured bacterium by measuring the amount of luminescent light
emitted when said bacterial adenosine triphosphate is reacted with a
luciferase-luciferin mixture; subjecting said sample of said cultured
bacterium to an antibiotic agent; and assaying the amount of bacterial
adenosine triphosphate after treatment with said antibiotic by measuring
the luminescent light resulting from said reaction; whereby the ATP index
is determined from the values obtained from said assay procedures.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Adenosine triphosphate (hereinafter referred to as ATP) is a component of
all cellular matter, thereby making it an excellent indicator of the
presence of various life forms. A very sensitive test for the presence of
ATP is an enzymatic biolumine scent assay. The test is accomplished by
reacting ATP with the enzyme luciferase and luciferin in the presence of a
divalent metal ion such as magnesium or manganese.
It has previously been shown that the concentration of ATP derived from
several bacteria strains from pure cultures can be closely correlated to a
bacterial count. This correlation is described by E. W. Chappelle and G.
V. Levin in "Use of the Firefly Bioluminescent Reaction for Rapid
Detection and Counting of Bacteria" as described in "Biochemical
Medicine", 2, 41-52 (June, 1968), and the article is incorporated herein
by reference. By using this technique on a number of bacterial species, an
average ATP content of a bacterial cell has been established as about 3
.times. 10.sup..sup.-10 .mu.g, i.e., from 0.28 .times. 10.sup..sup.-10
.mu.g to 8.9 .times. 10.sup..sup.-10 .mu. g.
In the adaption of this technique for the detection of bacteria in urine, a
method first had to be developed to separate non-bacterial sources of ATP
from the bacteria. Typical non-bacterial sources of ATP in urine include
free soluble ATP and the ATP present in red and white blood cells. The
separation process involves treating a urine sample with a non-ionic
detergent which ruptures any red and white blood cells present, thus
releasing ATP into solution in a freely soluble form. The freed
non-bacterial ATP is then hydrolyzed with an ATP hydrolyzing enzyme, i.e.,
an ATPase, and thereafter the ATPase is denatured by heating or by some
chemical means.
Bacterial ATP can now be released after removal of non-bacterial ATP by
rupturing the bacterial cells by the addition of acid. Subsequently, the
solution is neutralized, and the pH and ionic strength of the urine sample
is adjusted by a buffer for optimum luciferase activity. The solution is
then treated with a luciferin-luciferase mixture, and if ATP is present,
light is emitted from the ensuing bioluminescent reaction and is detected
and recorded by an appropriate apparatus. The following is believed to be
the reaction sequence by which light is emitted by the reaction of ATP
with luciferase-luciferin. This is the same reaction which occurs when
fireflies emit their characteristic light.
______________________________________
##STR1##
##STR2##
E = luciferase
LH.sub.2 =
reduced luciferin
ATP = adenosine triphosphate
AMP = adenosine monophosphate
PP = pyrophosphate
T = thiazolinone
h.nu. =
Light at 550 nm
______________________________________
If all of the components of the above reaction are maintained in a
concentration in excess of the ATP, the emission of light is directly
proportional to the amount of ATP introduced. This allows a direct
correlation between the magnitude of light emission and ATP concentration,
when a treated urine sample is combined with the luciferase-luciferin
mixture and when a standard curve is prepared. This detection mechanism
was adapted for an accurate technique of determining bacterial levels in
urine as described in U.S. Pat. No. 3,745,090 and is herein incorporated
by reference.
The present invention resides in the adaptation of the bioluminescent
reaction of luciferase-luciferin with ATP for the rapid determination of
microbial sensitivity to antimicrobial agents. The method of the invention
is effective for determining the sensitivity of bacteria isolated from
various body fluids, particularly from urine. The measurement of adenosine
triphosphate for determining microbial sensitivities to antibiotics was
selected because (1) the substance is present in all viable
microorganisms; (2) the concentration of ATP in bacteria is directly
related to fundamental processes of oxidative phosphorylation and
respiration; (3) although none of the commonly used antimicrobial agents
act directly on oxidative phosphorylation and respiration of the
organisms, it is evident that the antimicrobial agents may have profound,
indirect consequences on the ATP content of bacteria and thus their rate
of growth; and (4) the measurement of ATP by the luciferase
bioluminescence reaction is precise, reproducible and compatible with
automation.
In the present process of measuring microbial sensitivity, a sample of a
body fluid such as urine, lymph fluid, plasma, blood, spinal fluid,
saliva, mucus, ascites and the like is cultured. Normally, from 0.01 to
1.0 ml of a body fluid specimen is cultured. The amount of specimen to be
cultured is not a critical factor. Many types of conventional culture
media can be used with the nutrients either in agar or a liquid medium.
(See Bailey and Scott, Diagnostic Microbiology, Third Edition, C. V. Mosby
Co., 1970). The selection of a particular culture medium is a matter of
choice and is not a critical factor. Culturing of the specimen is normally
accomplished from 8 - 96 hours at temperatures ranging from 20.degree. to
45.degree. C. Each individual bacterial colony produced which is
recognized as being different is isolated, and then placed in a separate
culture broth, again for about 16 to 48 hours. The method by which the
bacterial colony is isolated is not critical and can be accomplished by
such techniques as a metal loop or by a differential media. With the
differential media technique, for instance, gram-positive bacteria are
mixed with gram-negative bacteria. The mixed bacteria are grown in a
phenylalanine agar medium to inhibit the gram-negative bacteria and to
enhance the growth of the gram-positive bacteria.
A fresh sample of bacterial culture is then diluted in a broth to obtain a
uniform inoculum of about 10.sup.4 to 10.sup.8 CFU/ml, preferably 10.sup.6
CFU/ml. (The abbreviation "CFU" refers herein to colony forming units.)
Each broth culture is then preincubated at 20.degree. to 45.degree. C to
initiate growth of the bacterial strain.
The antibiotic solutions used to treat the cultured bacteria are formulated
by diluting an antibiotic solution to an appropriate concentration, which
depends only on the useful range of each antibiotic, with water or a
saline solution. Usually, the concentration of antibiotic in solution
ranges from 0.1 - 100 .mu. gm/ml. The salt concentration of the saline
solution can assume any value that is not detrimental to the bacteria.
Normally, however, the saline solutions are about 0.9% solutions. Suitable
antibiotics used in the present process include ampicillin trihydrate,
tetracycline hydrochloride, potassium penicillin G, sodium cephalothin,
erythromycin base, sodium colistimethate, nitrofurantoin, sodium
nafcillin, sodium ampicillin, nalidixic acid, chloramphenicol, disodium
carbenicillin, gentamicin sulfate, clindamycin phosphate, and
sulfisoxazole. Other antibiotics can also be used. The only requirement is
that the antibiotic used should give a good correlation.
At this point in the procedure a sample of the broth culture is placed into
a receptacle containing the particular antibiotic to be tested. The amount
of antibiotic used relative to the culture varies as a function of the
particular antibiotic used. Normally, from about 0.1 ml to 10 ml of a
culture specimen is added to the antibiotic solution. Simultaneously, a
sample of broth culture is added to two separate receptacles containing a
0% to 2% saline solution. This forms duplicate control samples. The amount
of saline solution added to each control sample equals the volume of the
antibiotic added to the sample with same. One of the control samples is
assayed by the ATP-luciferase technique to establish a bacterial count at
time zero. Thereafter, the other control sample and the test sample with
the antibiotic are incubated for 0.5 to 10 hours at 20.degree. to
45.degree. C. This incubation temperature range will allow for comparison
for growth at body temperature. In the final step of the procedure, the
later control sample and the test sample with the antibiotic are assayed,
again by the ATP-luciferase technique, to determine the sensitivity of the
particular bacterium used to the particular antibiotic under test. It
should be clearly understood that many types of bacteria may be
simultaneously tested with many types of antibiotics.
The assay technique is accomplished by first placing a culture sample in a
sterile receptacle suitable for centrifuging. Usually, a minimum of about
0.1 ml to a maximum amount of about the capacity of the receptacle or
about 15 ml, preferably 10 ml, of the culture sample is placed in the
receptacle. Scrupulous procedures must be followed to avoid contamination
by extraneous ATP sources. The sample containers must be thoroughly
cleaned, for instance, by acid washing, because residual ATP readily
clings to glass surfaces. Further, the deionized, distilled water should
be used in preparing all reagents. Many commercial detergents will
interefere with the assay so that all glassware used must be scrupulously
free of residue.
The test solutions are now ready for the removal of all non-microbial
sources of ATP which are present in the original specimen. This is
accomplished by treating the test solutions containing the cultured
organism with an ATPase. An ATPase hydrolyzing solution is formed by
mixing a calcium salt, a surfactant and ATPase. The function of the
surfactant is to rupture all non-bacterial cells in solution and free all
non-bacterial adenosine triphosphate into solution. Any convenient means
can be used to rupture the non-bacterial cell as long as the bacterial
cells are not detrimentally affected. A convenient manner of rupturing the
non-bacterial cells is to reduce the surface tension of the non-bacterial
cells.
It has been found that certain surfactant compounds, which effectively
lower the surface tension of aqueous solution, also readily rupture
non-bacterial cells. Suitable surfactants include cationic surfactants,
anionic surfactants and nonionic surfactants such as octyl phenoxy
polyethoxyethanols (Triton-X100). Other types of surfactants include
triterpenoid saponins, steroid saponins, sulfosuccinates such as dioctyl
sodium sulfosuccinates, various glycosides and some polyoxyethylene
derivatives of fatty acid partial esters of sorbitol anhydrides. The
concentration of surfactant in solution varies and can be any amount which
achieves a surface tension of about 20 - 40 dynes /cm at 25.degree. C in
H.sub.2 O.
The concentration of ATPase in the hydrolyzing solution varies. The only
requirement is that the minimum amount used should promote hydrolysis of 1
.mu. mole of ATP in about 15 minutes. Suitable sources of ATPase for the
ATPase solution include potato apyrase, insect apyrase, muscle ATPase,
liver ATPase, and the like. More preferred as a hydrolyzing agent is
potato apyrase since it is more completely characterized, easily purified,
easily obtained and yields excellent results. In this hydrolyzing
solution, any convenient calcium salt can be used as long as the anion
does not have an inhibitory effect such as calcium chloride, calcium
bromide, calcium iodide, calcium sulfate and the like, such that a calcium
ion concentration ranging from 5 .times. 10.sup..sup.-4 M to 5 .times.
10.sup..sup.-3 M is achieved. The presence of calcium ion is essential as
a catalyst because it is a required cofactor for apyrase.
After the ATPase solution is added to the bacteria-containing solution, the
sample is allowed to stand at ambient temperature for sufficient time to
permit complete hydrolysis, preferably from 1 to 25 minutes, more
preferably from 5 to about 15 minutes.
After removal of all non-microbial sources of ATP, the hydrolyzing ATPase
enzyme solution is denatured or destroyed and simultaneously the bacterial
cells are ruptured which releases the ATP in the cells. This is usually
accomplished by physical or chemical means. Suitable chemical agents
include acids such as nitric acid, phosphoric acid, sulfuric acid,
perchloric acid, hydrochloric acid, and the like; organic acids, such as
formic acid, acetic acid, and the like; bases such as alkali metal and
alkaline earth metal hydroxides and carbonates, organic bases such as
pyridine; organic solvents such as acetone, methylene chloride, chloroform
and the like. The important feature is that rupture of the bacterial cell
walls does not occur and destroy bacterial ATP. Physical means of
denaturing include heat and short wavelength radiation. It has been found
that heating the test solution for a brief period, i.e., about 1 to 15
minutes, preferably about 5 to 12 minutes, from 60.degree. to 100.degree.
C is effective to denature the hydrolyzing enzyme.
It is easily seen that an entire range of compounds and procedures may be
used to effect the inactivation or denaturation of the hydrolyzing enzyme,
requiring only that they do, in fact, destroy the activity thereof without
appreciably affecting the bacterial cells or the bioluminescent reaction
used to determine the bacterial ATP. Any compound used which is toxic to
the luciferase-ATP reaction would ultimately have to be removed or
neutralized.
The denaturation of the hydrolyzing enzyme is generally carried out at an
elevated temperature, preferably 95.degree. C, and ambient pressure;
however, if it should be more convenient or necessary to carry this out at
elevated pressures, there is no apparent reason for not doing so.
After bacterial ATP has been released in solution, the test solution is
diluted with water to raise the pH of the same to about 1.8 - 2.9,
preferably 2.1, if an acid is used to denature the ATPase, and then
vortexed to mix the solution. Thereafter, the test solution is treated
with a solution containing a luciferase-luciferin mixture, and the light
which is emitted is measured in a luminescence photometer in order to
measure the ATP index. The pH of the final solution containing bacterial
ATP and luciferase is that which is sufficient for the bioluminescent
reaction to occur, usually from 6.5 to 8.5, preferably 7.4 to 8.0, most
preferably 7.75. A buffer is present in the solution to adjust the ionic
strength as well as keep the pH within the appropriate range. The amount
of buffer added is within the range of about 0.05 ml to about 1.0 ml at a
concentration of about 2 to 2.5 M. Suitable buffers include TES buffer
[N-tris (hydroxymethyl)methyl-2-amino-sulfuric acid]; phosphate buffers,
arsenate buffers; Tris buffers, e.g., tris (hydroxymethyl)aminomethane;
arsine buffers; glycylglycine buffer; and
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid buffer. Also present in
the luciferase-luciferin mixture can be any soluble manganese or magnesium
salt. Any manganese or magnesium salt containing an anion which has an
inhibitory effect should not be used. Usually, the mixture contains from
0.1 mg - 100 mg/ml of luciferin, preferably 0.7 M luciferin. It is readily
apparent that the receptacle which contains the control bacterial sample
will emit the greatest amount of luminescent light because no antibiotic
has been present in the control sample to diminish the amount of bacteria
present. The antibiotic containing solutions will emit lesser amounts of
luminescent light if there is an adverse influence of the antibiotic on
the growth of the bacteria. The bioluminescent readings are normally
conducted at room temperature in a photometer and the ATP index is
calculated according to the following formula:
##EQU1##
wherein B.sub.t represents the light reading obtained for a solution of
cultured broth which is treated with an antibiotic and then allowed to
incubate, A.sub.t represents the light reading obtained for a solution of
cultured broth (control sample) which is allowed to incubate without the
presence of antibiotic, and A.sub.o represents the light reading obtained
for a solution of cultured broth (control sample) at time zero which is
not incubated nor treated with antibiotic. The interpretation of the
results obtained by the light measurements is as follows: Those samples of
antibiotic treated cultures having an ATP index >+ 0.05 are deemed as
resistant bacterial samples, while those samples of antibiotic treated
cultures having an ATP index .ltoreq.+ 0.05 are deemed as sensitive
bacterial samples.
An alternative method of calculating the ATP index is to use the following
logarithmic relationship:
##EQU2##
This formula was used to obtain the data in the examples. According to the
logarithmic formula, antibiotic treated cultures having an ATP index value
>+ 0.25 are deemed as resistant bacterial samples, while those samples of
antibiotic treated cultures having an ATP index .ltoreq.+ 0.25 are deemed
as sensitive bacterial samples. However, in determining the ATP index for
a certain antibiotic-bacteria combination, it is recommended that the
logarithmic method should not be used. Rather, the non-logarithmic formula
is the method of choice and should be used to determine all ATP index
values.
The examples which follow show the reproducability of the ATP index results
of the following bacterial cultures which are representative of the types
of bacteria to which the present method is applicable. Suitable bacteria
include Escherichia coli, Staphylococcus aureus, Klebsiella aeogenes,
Enterobacter cloacae, Serratea marcescens, Proteus vulgaris, Pseudomona
aerugenosa, Proteus mirabilis, Providencia stuartii, Staphylococcus
epidermidis and Enterococcus.
The invention is further illustrated by the following examples in which all
parts and percentages are by weight unless otherwise indicated. These
examples are illustrative of results obtained with one embodiment of the
invention and are provided to teach those skilled in the art how to
practice the invention and to represent one mode contemplated for carrying
out the invention.
PREPARATION OF BACTERIAL STRAINS
Bacterial strains were maintained on trypticase Soy Agar slants at
4.degree. C. At monthly intervals, fresh broth cultures were streaked onto
5% sheep's blood agar and esoin methylene blue agar to confirm the purity
of the sample. Five similar colonies from the blood agar were inoculated
into fresh broth from which new agar slants were prepared. Agar diffusion
sensitivity tests were done periodically on the bacterial strains to
confirm the stability of their susceptibility patterns.
PREPARATION OF STOCK ANTIBIOTIC SOLUTIONS
Stock solutions of the antibiotics shown in Tables 1 and 3 were prepared by
mixing each antibiotic into a volume of sterile, distilled water to a
concentration of 1280 .mu. g/ml and each solution was dispensed into
sterile, capped polypropylene tubes (12 .times. 75 mm) using a Cornwall
syringe pipette having a 0.22 micron Millepore filter. In the preparation
of antibiotic solutions of sulfisoxazole, erythromycin base, nitrofuranoin
and nalidixic acid, special solvents of 10% sodium hydroxide, ethanol,
dimethylformamide and 1 N sodium hydroxide were used. The solutions were
frozen at -20.degree. C for at most two weeks and were used within one
hour of being thawed.
The antibiotics studied and the concentrations used to determine the ATP
index relative to a specific bacterium are shown in Table 1. The
concentration of antibiotic selected was determined empirically by
measuring the effect of the mean inhibitory concentration (MIC)
breakpoints generally quoted for the agar diffusion method on a spectrum
of known organisms. The concentrations shown in Tables 1 - 4 fall
somewhere in between the MIC breakpoints.
PREPARATION OF A BODY FLUID SAMPLE FOR ATP INDEX MEASUREMENT
A fresh sample of an overnight broth culture of a urine specimen which was
cultured on a blood agar plate was diluted 1000-fold in Trypticase Soy
Broth at 37.degree. C, whereby an inoculum of approximately 10.sup.6
colony forming units (CFU/ml) was obtained. The inoculum was preincubated
at 37.degree. C for 30 minutes and 4.5 ml of the incubated inoculum was
placed in two tubes, one of which contained 0.5 ml of sterile 0.9% sodium
chloride (control sample) and the other contained 0.5 ml of an antibiotic
solution in 0.9% sodium chloride to yield the final concentration shown in
Tables 1 - 4. (Tables 1 - 4 also show the specific antibiotics tested). A
one-half ml quantity of the control sample was immediately assayed for ATP
(A.sub.o) as described specifically below. Both the control sample and the
antibiotic sample were incubated at 37.degree. C for 2.5 hours. After
incubation, 0.5 ml samples from each tube were assayed for ATP (A.sub.t
and B.sub.t values).
ATP ASSAY FOR THE DETERMINATION OF THE ATP INDEX
Preparation of ATPase Hydrolysis Solution:
To a solution of 0.3 M calcium chloride containing 0.6% Triton X-100 kept
frozen at -20.degree. C in aliquots suitable for a day's work was added 5
mg of ATPase (Apyrase, Grade 1, Sigma) per milliliter immediately before
an assay and gently mixed by inversion.
Preparation of Luciferase-Luciferin Mixture:
The contents of a vial of luciferase (Luminescence Biometer Reagent Kit,
DuPont Instruments) was reconstituted with 1.5 ml of 0.2 M TRIS (Trizma
base, Sigma) containing 0.01 M magnesium sulfate at a pH of 8.4. This
solution was also kept frozen in suitable aliquot sizes. After complete
solution of the mixture, 0.1 ml of the reagent was dispensed into reaction
cuvettes.
The actual assays were conducted as follows. A 0.5 ml sample of a broth
culture was placed in a sterile polypropylene tube. To the culture was
added 0.1 ml of the ATPase hydrolysis solution, which was then vortexed
and allowed to stand for 15 minutes. By this procedure, all non-bacterial
ATP in solution was denatured or destroyed. Thereafter, 0.1 ml of 1.5 N
HNO.sub.3 was added to the denatured broth, and the tube was vortexed and
allowed to stand for 5 minutes. This procedure released ATP from the
bacterial cells present in the culture. The acid solution was then diluted
with 4.3 ml of sterile, deionized water (Travenol). Thereafter, 0.1 ml of
the diluted broth was withdrawn into a disposable tuberculin syringe and
then injected into a working sample of the luciferase-luciferin mixture.
Luminescent readings were then taken in a Luminescence Biometer, DuPont
Instruments, which was standardized to 1.0 .times. 10.sup.8 femtograms
(fg) ATP/ml of the original sample by adding 0.05 ml of a freshly thawed
ATP standard (0.1 .mu. g/ml) to the final reaction volume of a blank tube,
i.e., a sterile broth. The ATP index of each culture-antibiotic solution
tested was then calculated by the formula shown above.
KIRBY-BAUER AGAR DIFFUSION SENSITIVITY TEST
Agar diffusion sensitivity testing was conducted by the tentative standards
recommended by the National Committee for Clinical Laboratory Standards
Subcommittee on Antimicrobial Susceptibility Testing.
MIC-BROTH DILUTION TEST
This assay procedure was accomplished in a trypticase Soy Broth according
to the method proposed by the International Collaborative Study on
Antibiotic Susceptibility Testing.
The data in Tables 1 - 3 show the ATP indexes for each antibiotic-bacterium
combination investigated. Also shown are the results for the same
bacterium-antibiotic combinations as tested for sensitivity by the
conventional Kirby-Bauer agar diffusion technique and the conventional MIC
tube dilution techniques. The number of disagreements between the results
of the present process and the Kirby-Bauer technique are summarized in
Table 4.
TABLE 1
__________________________________________________________________________
COMPARISON OF RESULTS OBTAINED BY ATP INDEX, AGAR DIFFUSION
(KIRBY-BAUER),
AND TUBE DILUTION MIC
MICROORGANISM
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus epidermidis
ATCC 25923 S-187 05995
MIC MIC MIC
ANTIBIOTIC
K-B (.mu.g/
ATP K-B (.mu.g/
ATP K-B (.mu.g/
ATP
(.mu.g/ml)
(mm.)
mg) INDEX
INTERPR.
(mm.)
ml) INDEX
INTERPR.
(mm.)
ml) INDEX
INTERPR.
__________________________________________________________________________
PENICILLIN
32.5
<0.125
-0.86
S ND ND ND ND 21 1.0 -0.46
S
G (8) (0.15) -0.70
S 14.5
ND +0.53
R
AMPICILLIN
31 <0.125
-0.17
S ND ND ND ND 25 0.5 -0.02
S
(8)
NAFCILLIN
19.5
ND -0.79
S 19.5
ND -0.39
S ND ND ND ND
(0.6)
CARBENICILLIN
ND ND ND ND ND ND ND ND ND ND ND ND
(128)
CEPHALOTHIN
32.5
<0.125
-0.38
S ND ND ND ND 35 0.25
-0.24
S
(16)
TETRACYCLINE
25.5
0.5 -0.13
S ND ND ND ND 0 >128
+1.01
R
(6)
ERYTHRO- 28 0.25
0.00
S ND ND ND ND 29.5
0.25
+0.08
S
MYCIN (4)
CLINDAMYCIN
26 <0.25
-0.26
S ND ND ND ND 29 <0.125
-0.01
S
(2)
GENTAMICIN
24 0.25
-0.07
S ND ND ND ND 30 0.125
+0.04
S
(6)
NITROFUR-
18.5
16 -0.05
S ND ND ND ND 25 4 -0.12
S
ANTOIN (50)
COLISTIN 0 >128
+0.88
R ND ND ND ND 0 >128
+0.91
R
(8)
CHLORAM- 24 4 -0.28
S ND ND ND ND 27.5
4 -0.24
S
PHENICOL
(12.5)
__________________________________________________________________________
ND = Not Done
TABLE 2
__________________________________________________________________________
COMPARISON OF RESULTS OBTAINED BY ATP INDEX, AGAR DIFFUSION
(KIRBY-BAUER), AND TUBE DILUTION MIC
MICROORGANISM
Escherichia coli Klebsiella Enterobacter
ATCC 25922 07220 05248
MIC MIC MIC
ANTIBIOTIC
K-B (.mu.g/
ATP K-B (.mu.g/
ATP K-B (.mu.g/
(.mu.g/ml)
(mm.)
ml) INDEX
INTERPR.
(mm.)
ml) INDEX
INTERPR.
(mm.)
ml) INDEX
INTERPR.
__________________________________________________________________________
PENICILLIN G
0 80 +0.99
R 0 >80 +0.99
R 0 >80 +1.00
R
(8)
AMPICILLIN
19 8 -0.33
S 0 >128
+0.93
R 0 >128
+0.93
R
(8)
NAFCILLIN
ND ND ND ND ND ND ND ND ND ND ND ND
(0.6)
CARBENICILLIN
28 32 -0.27
S ND ND ND ND ND ND ND ND
(128)
CEPHALOTHIN
19 32 -0.30
S 21 16 -0.46
S 0 >128
+0.97
R
(16)
TETRACYCLINE
20 4 +0.13
S 19 8 +0.07
S 19 8 +0.04
S
(6)
ERYTHRO- 10.5
64 +0.92
R 11.0
128 +0.98
R 0 >128
+0.98
R
MYCIN (4)
CLINDAMYCIN
0 128 +0.96
R 0 >128
+0.94
R 0 >128
+0.94
S
(2)
GENTAMICIN
22 4 -0.43
S 22 | | |
