A method for detecting the presence of bacteria in urine was developed which utilizes the bioluminescent reaction of adenosine triphosphate with luciferin and luciferase derived from the tails of fireflies. The method was derived from work on extraterrestrial life detection. A device was developed which completely automates the assay process

Chappelle, E. W.

Fleig, A. J.

Kelbaugh, B. N.

Picciolo, G. L.

English

NASA Technical Reports Server

188 GODDARD SPACE FLIGHT CENTER
AUTOMATED DETECTION OF BACTERIA IN URINE
Albert J. Fleig
Grace L. Picciolo
Emmett W. Chappelle
Burton N. Kelbaugh
We have developed a method for detecting the presence of bacteria in
urine which may have a substantial impact on present health care
procedures. The incidence of urinary tract infections is second only to that
of infections of the respiratory system. A large hospital laboratory typically
examines up to 4,000 urine specimens a month. The present method of
detection is to dip out a drop of urine and spread it on an agar plate
(Figure 1). The plate is then capped and incubated for from 1 to 4 days.
Then the plate is visually examined (Figure 2) for indication of bacterial
growth, and an estimate of the quantity of bacteria present in the original
specimen is made. At Johns Hopkins Hospital from four to six medical
technicians are occupied full time in performing these assays. It is expensive
in terms of trained personnel, space, and time required for an assay. In
addition, it is a tedious, repetitive, subjective task subject to much human
error.
Our method is derived from work on extraterrestial life detection done
by the former Space Biology Branch. The method utilizes the biolumines-
cent reaction of adenosine triphosphate (ATP) with luciferin and luciferase
derived from the tails of fireflies. All bacteria contain ATP; thus, all
bacterially contaminated urine will contain ATP. However, urine samples
also contain red and white blood cells and skin cells which also contain
ATP. Thus, it is necessary to eliminate all nonbacterial ATP before
performing the bioluminescent assay.
A nonionic detergent is added to the urine sample. This detergent lyses
or ruptures the cell walls of all the nonbacterial cells but does not affect the
bacteria. Then potato apyrase is added, which hydrolizes or destroys all of
the ATP, which has been released. Then perchloric acid is added, which
inhibits the apyrase and lyses any bacterial cells present in the urine. If (and
https://ntrs.nasa.gov/search.jsp?R=19720018150 2020-03-17T03:52:50+00:00Z
TECHNOLOGYACCOMPLISHMENTS, 1970 189
only if) there were bacterial cells, free ATP would be present in the
specimen, and the injection of luciferase and luciferin will produce a flash of
light.
We have developed a device (Figure 3) which completely automates this
process and are conducting preliminary trials of it at Johns Hopkins
Hospital. An entire assay takes 15 minutes, and the device processes samples
at this rate of one per minute. Preliminary test results have been very
encouraging. In a run of several hundred specimens, we have correctly
detected every specimen which was later found positive by the present
laboratory procedure. In addition, we found indication of bacterial in-
fection in roughly 20 percent of specimens considered negative by present
methods and typically found much higher levels of infection than the
present methods do, even when both approaches indicated the presence of
infection.
There are several possible explanations for the difference in results from
the two approaches. A fundamental difference is that our ATP assay detects
bacteria in the specimen as obtained, while culture methods detect only
those bacteria which will grow in the particular culture environment being
used. Thus, bacteria which do not reproduce on agar, at the pH being used,
in contact with oxygen, or for any of a number of other reasons will not
normally be found by present hospital methods but would be with our
ATP-based assay. Urine may contain bacteriostatic agents, either naturally
produced (as might be the case with a low level, long term infection) or as a
result of drug therapy. These bacteriostatic agents may preclude detection
of the bacteria via routine culture methods but would not hinder the ATP
assay. A second basic difference is that we are inferring the presence of
bacteria based upon the existence of ATP in the processed urine. Thus, an
erroneous positive reading would result if there are sources of ATP which
are not deleted by our processing procedure. However, our studies to date
indicate that this is not the case.
There are several important potential implications of the ATP assay for
patient health care. First, it will be possible to reduce the number of urine
specimens which must be cultured by roughly 40 percent by eliminating all
specimens with negative ATP assay results, thus, reducing the personnel and
space (and ultimately the cost) required for the assay. And it will be
possible to return the negative result 1 day sooner which may alter medical
treatment, e.g., administration of antibiotics. The device may also make it
190 GODDARD SPACE FLIGHT CENTER
possible to screen large numbers of patients in classes with above-normal
incidence of bacterial infection, such as teenage girls, pregnant women, and
diabetics. The only requirement for the above is that a thorough clinical
trial should confirm that the ATP assay does not miss any cases of infection
detected by present methods.
The ATP assay also provides information not presently available, and if
further trials establish the correctness of our results, this may be even more
important. In particular, the ATP assay may correctly detect bacteria which
would be missed (or underestimated) by present methods. The ATP assay
provides an accurate, replicable quantitative result in place of a subjective
estimate. Although the impact of this change is impossible to predict, Lord
Kelvin's observation, "...But nearly all the grandest discoveries of science
have been but the rewards of accurate measurement and patient long
continued labor in the minute sifting of numerical results," may apply.
There are still several steps to be completed before this technology is
successfully transferred to the medical community. A thorough clinical trial
is required to establish the accuracy and reliability of the assay and to
resolve the discrepancies in results between the ATP assay and present
standard cultural techniques. Commercial development and marketing of
the device will then complete the process. As a result of discussions with the
National Institute of General Medical Sciences, NIH, we have prepared a
proposal for a clinical trial involving Johns Hopkins Hospital, the NIH
Clinical Center, and GSFC. Several commercial firms have expressed interest
in marketing the device if the clinical trials are successful.
TECHNOLOGY ACCOMPLISHMENTS, 1970 191 
Figure 2 
192 GODDARD SPACE FLIGHT CENTER 
Figure 3 
Figure 4 


Automated detection of bacteria in urine

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