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Journal of Clinical Microbiology, September 2000, p. 3394-3398, Vol. 38, No. 9
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Multilaboratory Validation of Rapid Spot Tests for
Identification of Escherichia coli
Mary K.
York,1,*
Ellen Jo
Baron,2
Jill E.
Clarridge,3
Richard B.
Thomson,4 and
Melvin
P.
Weinstein5
Department of Laboratory Medicine, University
of California, San Francisco, California 941431;
Department of Pathology, Stanford University Medical School,
Stanford, California 943052; Veterans
Administration Medical Center, Houston, Texas
770303; Department of Pathology and
Laboratory Medicine, Evanston Hospital, Evanston, Illinois
602014; and Departments of Medicine and
Pathology, Robert Wood Johnson Medical School, New Brunswick, New
Jersey 089015
Received 14 February 2000/Returned for modification 18 April
2000/Accepted 4 July 2000
 |
ABSTRACT |
To validate the accuracy of rapid tests for identification of
Escherichia coli, five laboratories sequentially collected
1,064 fresh, clinically significant strains with core criteria of
indole-positive, oxidase-negative, nonspreading organisms on sheep
blood agar plates (BAP), having typical gram-negative rod plate
morphology, defined as good growth on gram-negative rod-selective
media. An algorithm using beta-hemolysis on BAP, lactose reaction on
eosin-methylene blue or MacConkey agar,
L-pyrrolidonyl-
-naphthylamide (PYR), and
4-methylumbelliferyl--D-glucuronide (MUG) was evaluated. Identifications using the algorithm were compared to those obtained using commercial kit system identifications. One thousand strains were
E. coli and 64 were not E. coli by kit
identifications, which were supplemented with conventional biochemical
testing of low probability profiles. Of the 1,064 isolates meeting the
core criteria, 294 were beta-hemolytic and did not require further
testing to be identified as E. coli. None of the 64 non-E. coli strains were hemolytic, although other
indole-positive, lactose-negative species were found to be hemolytic
when further strains were examined in a follow-up study. Of the
remaining strains, 628 were identified as E. coli by a
lactose-positive and PYR-negative reaction. For nonhemolytic,
lactose-negative E. coli, PYR was not helpful, but a
positive MUG reaction identified 65 of 78 isolates as E. coli. The remaining 13 E. coli strains required kit
identifications. This scheme for E. coli identification
misidentified three non-E. coli strains as E. coli, for an error rate of 0.3%. A total of 13 kit
identifications, 657 PYR tests, and 113 MUG tests were needed to
identify 1,000 E. coli strains with the algorithm. The use
of this rapid system saves laboratory resources, provides timely
identifications, and yields rare misidentifications.
| |
INTRODUCTION |
In 1963, Vracko and Sherris
(14) reported the use of a spot indole test for the rapid
identification of members of the family Enterobacteriaceae.
Since then, many studies using rapid testing have been published
(1, 2, 5-9, 13), but the use of rapid methods for the
identification of Escherichia coli has not been validated in
a systematic manner with large numbers of strains from different
geographic areas. E. coli bacteria are among the few species
of lactose (LAC)-positive, oxidase-negative, gram-negative rods that
are indole positive. Due to the infrequent isolation of non-E.
coli strains that are indole positive, the spot indole test has
been used for the rapid, presumptive identification of E. coli. Although the test is not used for LAC-negative isolates, the
error rate in the clinical laboratory associated with using this one
rapid test alone for the identification of LAC-positive E. coli is not known. Rapid hydrolysis of
4-methylumbelliferyl--D-glucuronide (MUG) is also
characteristic of E. coli (5, 7, 8, 9, 11, 13).
However, not all E. coli strains are MUG positive (7,
8, 10, 12), and occasionally other organisms are also MUG
positive (12). E. coli does not hydrolyze
L-pyrrolidonyl--naphthylamide (PYR), while other
LAC-positive Enterobacteriaceae are PYR positive (2,
6). Using spot indole, MUG, and PYR reactions, five laboratories
in different geographic areas participated in a study to differentiate
E. coli from other Enterobacteriaceae by
comparing results from rapid identification methods to those obtained
by standard commercial multicomponent kits. The goal was to find and
validate an algorithm that could be used to accurately identify this
common organism in a clinically useful time frame at low cost.
(A preliminary report of this work was presented previously [M.
K. York, E. J. Baron, J. E. Clarridge, R. B. Thomson,
and M. P. Weinstein, Abstr. 99th Gen. Meet. Am. Soc. Microbiol.,
abstr. C-447, p. 197, 1999].)
| |
MATERIALS AND METHODS |
Strains.
Each of five laboratories in different cities (San
Francisco, Calif.; Stanford, Calif.; Houston, Tex.; Evanston, Ill.; and New Brunswick, N.J.) collected sequential fresh clinical isolates meeting the core criteria of indole-positive, oxidase-negative, nonspreading gram-negative rods, until each had identified at least 200 as E. coli by the commercial kit system in use in that laboratory. The selection process resulted in the collection of 1,064 strains which met the core criteria. Isolates that were considered
clinically significant by the laboratory policies of each site, except
those from stool cultures, were included in the study. Duplicates from
the same patient were avoided; 939 isolates were from urine cultures.
After the completion of the study, laboratory A collected as many
clinical strains as possible that were not identified as E. coli by a commercial kit but were indole positive, oxidase
negative and LAC negative, to further validate the accuracy of the
initial study. These isolates had low representation in the initial
study but could be confused with E. coli.
Laboratory testing.
Selection of strains meeting the core
criteria, observations, and testing were done from the initial plates
(blood agar with 5% sheep blood [blood agar plate (BAP)] and a
gram-negative rod-selective agar) inoculated with the patient specimen,
unless the presence of other flora precluded accurate testing. In the
latter case, testing was performed from a subculture of the isolate
such that observations could be made on both BAP and the selective
medium. Determination of typical gram-negative rod plate morphology and fermentation of LAC was observed as good growth and characteristic color, respectively, on gram-negative rod-selective medium, either eosin-methylene blue (labs A and E) or MacConkey agar (labs B, C, and
D). All other testing, including observation of beta-hemolysis (HEM),
was performed on BAP. Any zone of clearing of the blood around the
colony, but not clearing limited to that under the colony, was
considered positive for HEM. Each of the 1,064 isolates was identified
to species level using commercial kits in each respective laboratory:
Vitek GNR (bioMerieux Vitek, Inc., Hazelwood, Mo.) (labs A and C),
RapID One E (Innovative Diagnostics, Remel, Inc., Lenexa, Kans.) (lab
B), API 20E (bioMerieux Vitek, Inc.) (lab D), and MicroScan
Gram-Negative Breakpoint Combo Overnight panels (type 12;
Dade-MicroScan, West Sacramento, Calif.) (lab E). For indole
production, either 5% p-dimethylaminobenzaldehyde (14) or 1% paradimethylaminocinnamaldehyde (1)
in 10% (vol/vol) concentrated HCl was poured onto filter paper. A
colony >5 mm from the nearest colonies of differing morphology on a
sheep BAP <24 h old was applied to the paper, and red or blue color
development, respectively, was observed. For oxidase production,
tetramethyl-p-phenylenediamine dihydrochloride in water was
poured onto filter paper and allowed to dry. A colony from the BAP was
smeared onto the paper. Development of a blue color in 10 s was
considered a positive reaction. Hydrolysis of PYR was detected by
inoculating a water-moistened substrate disk (Remel, Inc.) with fresh
growth from the BAP. After 2 min, the developer (Remel) was added. A
red color was considered a positive reaction.
For MUG testing, MUG (Sigma, St. Louis, Mo.) was dissolved in 0.05 M
Sorensen's phosphate buffer (pH 7.5) to a final concentration of 31.25 µg/ml. Disks were prepared by adding 1.25 ml of MUG reagent to a vial
of 50 sterile 6-mm-diameter filter paper disks (Becton Dickinson
Microbiology Products, Cockeysville, Md.), to approximately 25 µl per
disk. The disks were thoroughly saturated, then air dried, and stored
at 
20°C until use. This method yielded an approximate final
concentration of 0.8 µg/disk. Disks were moistened with water and
inoculated with fresh growth from the BAP. After 2 h of incubation
at 35°C, the disk was observed under UV (360-nm) light. An intense
blue fluorescence was considered a positive reaction. Weak reactions
were interpreted as negative.
All testing was performed in the laboratory of origin of the isolate.
The kit identification was considered the final identification,
unless
it contradicted the expected PYR or MUG result or the probability
of
the identification was below the accepted level for the laboratory
using the kit. Identifications with low probability were confirmed
by
repeating the identification kit test, by using an alternative
kit
method, or by using standard biochemical tests (
3),
depending
on the practice within each laboratory. If the identification
was inconsistent with the rapid results, the strains were sent
to
laboratory A for further testing using one or more of the above
methods.
Evaluation.
Test results for each isolate were recorded on
separate work cards, which listed entries for LAC, HEM, indole,
oxidase, MUG, PYR, the kit identification code number, and the
likelihood of the identification. Space was provided for all repeat
testing, for further testing, and for the final identification, as
reported in the patient report. To ensure patient confidentiality,
patient names were omitted, but each laboratory was asked to provide
verification that the isolate did not represent a duplicate from the
same patient. Obvious clerical errors and missing results were
corrected after communication with the respective laboratory. If
results were discrepant with the expected rapid results and the strain
was available, it was retested in all five laboratories, and if all five laboratories agreed on the result, the retest was counted as the
evaluable result. If the strain could not be retested because it was
not available or all the laboratories did not agree on the result, the
original result was used in the final evaluation.
| |
RESULTS |
Accuracy and reproducibility.
Each laboratory was sent two
separate shipments of indole-positive, oxidase-negative gram-negative
rods to verify that the results achieved were the same regardless of
the media, reagents, and kits used by laboratories in different
geographic areas. There were 10 strains in the first shipment and 12 in
the second, with four E. coli strains duplicated in the
second set. Strains were Morganella morganii (2 strains),
Kluyvera spp., Citrobacter diversus, Proteus vulgaris, Providencia stuartii,
Klebsiella oxytoca (2 strains), and E. coli (10 strains including 1 E. coli O157 strain). Oxidase, PYR, and
indole results were highly reproducible, showing 100% agreement among
all the laboratories. For LAC fermentation, HEM, and MUG, the results
were variable, with 4, 3, and 7 of the 110 results for each test
differing from the majority result, respectively. None of the
discrepant results would have resulted in an erroneous identification
by the rapid methods. All discrepant results were in agreement with
expected variable reactions for the species. The results for LAC
fermentation and HEM were in agreement at all sites for the second
sample testing, after notification that the initial results had varied
by site. The MUG test results continued to be variable by site in the
second testing, despite the fact that all laboratories were using the
same lot of MUG disks.
Consecutive sample testing.
A total of 1,064 isolates of
indole-positive, oxidase-negative gram-negative rods were tested; 1,000 were identified as E. coli. Key reactions are listed in
Table 1. From these results, an algorithm
was developed (Fig. 1), which was then
applied to the strains to see if the E. coli strains could
be rapidly and accurately identified. Confirmatory kit identifications
to verify the identification were 237 by API, 410 by Vitek, 288 by
Dade-MicroScan, and 125 by Innovative Diagnostics Systems. Kit
identifications
for strains that were ultimately identified as
E. colifailed to provide an answer in 10 cases and gave an
incorrect identification in 3 cases. In these latter cases, the
negative PYR reaction indicated that the isolate could not be either
Citrobacter or Enterobacter, as the kit
identification indicated. Conventional tube biochemical testing
verified the isolates as E. coli.
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FIG. 1.
Identification scheme for the separation of E. coli from other indole-positive, oxidase-negative gram-negative
rods. See the text for an explanation of tests. EMB, eosin-methylene
blue; MAC, MacConkey agar; ID, identification. *, LAC-negative,
beta-hemolytic colonies could be M. morganii, E. tarda, or P. vulgaris. In some populations, these may
all be E. coli (see text) and the MUG test is not needed,
especially for isolates from noninvasion sites.
|
|
Application of the algorithm (Table
2)
resulted in the correct identification of 987 of 1,000
E. coli strains without use
of kits and the misidentification of 3 of
the 64 non-
E. coli isolates
as
E. coli (one
strain each of
Kluyvera spp.,
Escherichia
fergusonii,
and
M. morganii).
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TABLE 2.
Demonstration of application of algorithm to isolates in
study (number of tests necessary to arrive at identification)
|
|
Although during the study no laboratory encountered a beta-hemolytic
non-
E. coli strain, laboratory A had previously seen
beta-hemolytic
M. morganii and
P. vulgaris. In
order to determine
the extent of the presence of beta-hemolytic,
indole-positive
non-
E. coli strains, laboratory A collected
strains as follows:
24 of
M. morganii (11 beta-hemolytic),
16 of
P. vulgaris (2 beta-hemolytic
and nonspreading), 17 of
Providencia species (none were beta-hemolytic),
and 8 of
Edwardsiella tarda (all were beta-hemolytic). All strains
were indole positive, oxidase negative, LAC negative, MUG negative,
and
PYR negative and would not have been called
E. coli if the
MUG test were performed. Laboratory B collected five strains of
M. morganii, none of which were beta-hemolytic; however, two
of
the strains were PYR
positive.
| |
DISCUSSION |
Rapid testing was analyzed to determine if indole-positive,
oxidase-negative, LAC-positive isolates could be identified reliably as
E. coli without further testing. The results from five
laboratories indicate that the indole test is sufficient for hemolytic
strains. For nonhemolytic strains, the number of misidentifications
would be low (29 of 656 strains [4.4%]), representing 23 K. oxytoca strains, 5 Citrobacter sp. strains, and 1 Kluyvera sp. strain. The reproducibility studies indicated
that there might be some observer variability in the ability to
accurately observe colonies for hemolysis and LAC fermentation. If
colony morphology could be accurately observed, E. coli
strains are flat, dry, LAC-fermenting colonies on MacConkey agar and
can be differentiated visually from the usual mucoid,
LAC-fermenting colonies of Klebsiella species. With
the use of MacConkey agar, laboratories could use colony characteristics, rather than the PYR test, to differentiate K. oxytoca from E. coli, although there would be 1%
misidentifications (6 of 656 strains) representing the five
Citrobacter sp. strains and the one Kluyvera sp.
strain. Such an algorithm was not tested in this study but could be
validated. However, even a 1% error rate might not be acceptable with
isolates from blood and other normally sterile specimen sources. For
these sources, the 2-min PYR test should be used with all LAC-positive,
nonhemolytic strains. There was a concern that the identification of
E. coli was based on a negative reaction, but in practice,
the PYR test was both sensitive and reproducible.
For LAC-negative strains, 24% of the indole-positive strains were not
E. coli, and one cannot rely on the indole reaction alone.
However, since most of the PYR-negative microorganisms (Morganella, Providencia, and P. vulgaris) that may be misidentified are urea positive, a rapid
2-min urease test along with the PYR test could substitute for the 2-h
MUG test. This option was not tested by the authors, because of the
concern that other PYR-negative microorganisms (e.g., E. tarda and Shigella species) could also be called
E. coli. These strains were not isolated in the study and
are usually found only in stool specimens.
The rapid identification methods used in this study were compared to
four different commercial systems, and it might be argued that the
accepted identification of these strains was not truly accurate.
However, the purpose of this work was to compare the rapid methods to
the generally accepted identification methods routinely used by
clinical laboratories. The use of different confirmatory methods and
different media for LAC fermentation detection was deliberately chosen
to access the greatest number of variables used in clinical
laboratories. Because the identifications using colony morphology and
spot tests were equivalent in accuracy to kit identifications, the use
of the term "presumptive" for these identifications is not indicated.
Differences were seen in the phenotypic characteristics of strains
recovered in different geographic areas. For example, one laboratory
contributed almost no LAC-negative strains to the study. Another
laboratory saw beta-hemolytic M. morganii strains, which were not found in other areas. The differences were not due to the
media used in each laboratory, since each laboratory reported similar
results when a series of test strains were submitted for validation of
methodology. If beta-hemolytic M. morganii strains are known
to be recovered by a laboratory, the MUG test should be used to
avoid misidentifications.
Using the flow chart in Fig. 1, E. coli can be identified
with 99.7% accuracy, which compares favorably with the identifications with commercial kits. Three species were misidentified as E. coli by rapid testing, compared to three E. coli
strains that were misidentified as not E. coli using the
commercial kits. Identifications by commercial kits had to be repeated
because they failed to yield an identification in 10 cases.
Identifications by commercial kits had to be performed in 13 cases
after the rapid tests did not successfully identify the isolates. Thus,
confirmatory testing was required at a similar frequency for both rapid
and commercial kit methods.
The misidentification of a Kluyvera species as E. coli by the rapid method would have little or no clinical
significance (4). Of concern was the misidentification of a
strain of M. morganii because of a positive MUG reaction.
For convenience, the MUG test was performed by the disk method in each
laboratory, using the same lot of disks. The MUG disk test can be
difficult to read, as evidenced from the differing results in the
validation testing. Not only are tube MUG tests easier to read, but
they can be performed with colonies from selective media, such as
eosin-methylene blue and MacConkey agar (8). The reagent for
the tube test is more concentrated than that used for the disk test
(500 µg/ml) and is used by placing 2 drops of reagent in a Durham
tube, which is incubated at 35°C for 2 h.
Using the algorithm described in Fig. 1, 1,000 E. coli
strains were identified with a cost savings of over $3,000 in reagents, assuming that a kit identification costs $4 each or $4,256 (Table 3). This assumes that the rapid tests
cost approximately $1.00 each; however, in-house-prepared reagents are
much less expensive. If it is assumed that it takes 5 min to perform a
commercial kit identification, the time saved with the rapid algorithm
is 70 h of microbiologist time. The relative cost savings for any
laboratory will depend on the utility of integrating the algorithm into
that laboratory's work flow. For example, if the kit identification system used in the laboratory is combined with the susceptibility test
system, the technologist time saved would not be realized by the rapid
spot testing. The potential for benefit to the patient of immediate
identification based on the rapid tests is unknown.
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TABLE 3.
Cost of identification of 1,064 gram-negative rods by
rapid-testing algorithm compared to commercial kit
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|
It was of interest that most of the isolates were from urine. Many of
the same strains were later found in the blood of these patients, but
only one isolate per patient was studied. This is not surprising,
because the urinary tract is a common focus for subsequent bacteremia.
While the algorithm presented can be used with great accuracy for
LAC-positive isolates from urine or any other source, for LAC-negative
isolates from normally sterile sites, a MUG test should always be
performed (follow the dotted line in the algorithm in Fig. 1). Some
Shigella species and an occasional other species have been
reported to give a positive MUG reaction (12), and the MUG
test is used to screen for E. coli O157 strains, which have
a MUG-negative phenotype (12). Consequently, the algorithm
would be misleading to use in the workup of E. coli in stool specimens.
In summary, these data validate the identification of E. coli based on the use of a small number of rapid tests (Fig. 1)
with an accuracy of greater than 99%, which was equivalent to that for
commercial kit identifications. Confirmatory kit testing was needed for
less than 2% of isolates. Kit identifications performed on over 1,000 strains resulted in no identification for a similar number (2%) of
isolates. The use of rapid testing resulted in a 75% reduction in cost
of reagents and technologist time, with a decrease in time to reporting.
| |
ACKNOWLEDGMENTS |
We thank Barbara Allen, Kim Joho, Durdana Pirwan, Patricia
Rodrigues-Wong, Judy Rothberg, Lynn Schwabe, and the staff of the respective microbiology laboratories for technical assistance with this work.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Laboratory Medicine, L 515, Box 0100, University of California, San
Francisco, CA 94143. Phone: (415) 353-1268. Fax: (415) 353-1829. E-mail: MKYORK{at}WORLDNET.ATT.NET.
| |
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Journal of Clinical Microbiology, September 2000, p. 3394-3398, Vol. 38, No. 9
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Top
Abstract
Introduction
