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Journal of Clinical Microbiology, March 2001, p. 1161-1164, Vol. 39, No. 3
Faculty of Agricultural Sciences,
Microbiology Unit, Catholic University of Louvain, B-1348
Louvain-la-Neuve, Belgium,1 and
Akademisch Ziekenhuis, Department of Microbiology, Vrije
Universiteit Brussel, B-1090 Brussels, Belgium2
Received 12 September 2000/Returned for modification 6 November
2000/Accepted 22 December 2000
The ability of the ID 32E system to identify and discriminate 74 Escherichia coli O157 isolates among 106 E. coli non-O157 isolates was evaluated. The results showed atypical
biochemical reactions but accurate identification at the species level
and no unique biochemical profile numbers for E. coli O157,
although these numbers were distinct from those of other serotypes.
Verocytotoxigenic Escherichia
coli (VTEC), a new public health problem worldwide, is represented
by more than 100 serotypes that produce verocytotoxins, with two main
serotypes, O157:H7 and O157:H( Detection of E. coli O157:H7 is based on its recovery from
samples and the presence of its virulence-associated factors
(verocytotoxins) or the detection of its O157 antigens (3, 30,
31). Selective media for isolating O157:H7 strains rely on the
fact that most of these strains display characteristic biochemical
reactions (31): no A new well-based identification system, ID 32E, was recently developed
by bioMérieux Vitek for the identification of
Enterobacteriaceae and other nonfastidious gram-negative
bacteria, after an incubation period of 22 ± 2 h at 37 ± 1°C (17, 23). This system is an upgrading of the API
20E gallery and consists of 32 individual conic test wells (13 enzymatic, 3 biochemical, and 16 sugar utilization tests) in
polystyrene trays (Table 1).
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.3.1161-1164.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Particular Biochemical Profiles for
Enterohemorrhagic Escherichia coli O157:H7 Isolates on the
ID 32E System
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). These last two serotypes, classified
as enterohemorrhagic E. coli (EHEC), are implicated in
hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic
thrombocytopenic purpura. EHEC strains have been described as
important and emergent food-borne pathogens (4, 18, 31).
Other non-O157 VTEC serotypes, not deprived of pathogenicity, have been
increasingly implicated in sporadic enteric diseases and outbreaks
(18, 31).
-glucuronidase activity and no
D-sorbitol fermentation within 24 h at 37°C, except
for some German or U.S. strains (10, 12). However,
these last two biochemical features are not commonly used in
routine clinical and food laboratories because of the simplicity of
well-established commercial identification systems. The use of these
systems on presumptive E. coli strains offers not only the
advantage of confirming strains at the genus and/or species level but
also the possibility to detect the presence of O157 EHEC by identifying
profiles that are unique to these strains. For the Microscan dried
conventional gram-negative identification panel (Dade-MicroScan
International, West Sacramento, Calif.), 90% of the tested E. coli O157:H7 strains shared two common biochemical profiles
(1), whereas in the case of the API 20E (bioMérieux Vitek, Inc., Hazelwood, Mo.), 92% of the tested strains displayed the
same profile (8).
TABLE 1.
E. coli biochemical activity profile
A total of 180 serotyped E. coli strains were tested with
the ID 32E system. These included 55 O157:H7 strains, 19 O157:H() strains confirmed by PCR on the rfbO157 locus
(3), and 106 reference strains and laboratory isolates of
non-O157:H7 or O157H() E. coli harboring (76 strains) or
not harboring (30 strains) verocytotoxin genes, originating from
patients, animals, and food samples. The strains were isolated on
casein soymeal peptone agar (Caso medium; Merck, Darmstadt, Germany).
Conventional identification of strains was done using the
Kligler-Hajna, indole, Voges-Proskauer, Simmon's citrate,
-galactosidase, urease, glutamate decarboxylase, sorbitol fermentation, and -glucuronidase tests (2, 7, 15, 35). A suspension of each strain, adjusted to a 0.5 McFarland standard (theoretical optical density at 550 nm of 0.125) with a
spectrophotometer (Beckman Instruments, Inc., Fullerton, Calif.), was
prepared, and the ID 32E strip was inoculated by pipetting 55 µl of this suspension into each well. After an incubation
of 22 ± 2 h at 37 ± 1°C, James reagent was added to
the indole well, and the variation of color of each well was visually
read according to the manufacturer's instructions. The reactions were
tabulated into an 11-digit numerical profile and entered into the
upgrading APILAB Plus computerized software version 3.3 (bioMérieux Vitek) containing 103 reference bacterial taxa. In
the computer report, identifications are classified according to the
percentage of identification accuracy (%ID), an estimate of how
closely the profile corresponds to the taxon relative to all other taxa
in the database, and the T index (T), an estimate of how closely the
profile corresponds to the most typical set of reactions for the stated
taxon, as follows: excellent (%ID 
99.9, T 0.75), very
good (%ID 99.0, T 0.5), good (%ID 90.0, T 0.25), and acceptable (%ID 80.0, T 0).
Atypical tests, named "supplemental tests," are listed at the end
of the computer report in order to confirm identification by the new
classical tests.
E. coli O157:H7 showed significant divergent biochemical
activities from classical E. coli for ornithine
decarboxylase, arginine dihydrolase, urease, 5-ketogluconate,
-glucuronidase, and sorbitol and, to a lesser extent for rhamnose
(1), adonitol, D-arabitol, trehalose, and
inositol (Table 1). Ten Belgian human and French animal isolates
displayed urease-positive reactions. Identification as E. coli was excellent or very good. Although no single biochemical profile number could account for all E. coli O157:H7 (Table
2) isolates, two-thirds of these isolates
were distributed into only five profiles, with one (i.e., 54465743000)
representing 25% of the tested strains.
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On average, the biochemical activity profiles of O157:H() strains
were more similar to those of the O157:H7 strain than to those of the
non-O157 strain (Table 1). Slight differences between both O157:H
serotypes were noted for rhamnose and ornithine decarboxylase activities. One strain, isolated from a Belgian human, was positive for
the urease test. The quality of identification as E. coli varied from good to very good. Five O157:H() biochemical profiles numbers were also shared by O157:H7 strains (Table 2). Interestingly, O157 E. coli profile numbers were not encountered with other
non-O157 E. coli strains, regardless of their verocytotoxin
production (data not show).
Non-O157 E. coli isolates displayed significant distinct
results compared to the previously reported manufacturer or published data (Table 1), especially for 5-ketogluconate, -glucosidase, and
D-arabitol. The two main biochemical characteristics of
E. coli O157:H7, i.e., -glucuronidase and sorbitol, were
fairly frequent (17.9 and 31.1%, respectively) among non-O157
serotypes. No strikingly different biochemical activity profiles were
found between verocytotoxygenic and non verocytotoxygenic non-O157
E. coli strains (data not shown). As for O157, the quality
of identification of the non-O157 as E. coli was mostly
excellent, especially with the strains not producing verocytotoxins
that displayed less atypical biochemical characters. Sixty-one profiles
for VTEC non-O157 strains (main profile number 54465543400) were observed.
The reproducibility of the ID 32E system was determined by performing repeat analyses, at different days and with different batches for 10 O157 and 10 non-O157 strains. The exact same biotype number was generated for each replicate (100% reproducibility).
In the present study, the biochemical characteristics of O157 E. coli did not influence accuracy of identification and were responsible for the various biochemical profile numbers shown in Table 2. Although phenotypic variants (5, 6, 15, 20, 21, 23) can cause identification problems for some E. coli strains, numerous commercial systems or previous study on ID 32E systems (17, 23) have been shown to identify E. coli with a fairly high degree of accuracy (11, 13, 14, 16, 19-22, 25-29, 32, 33, 36, 37).
One strain serotyped as O74:H52 was found to be indole negative without affecting its identification as E. coli. These atypical characteristics have been previously described in the literature (29) and especially for the pathogenic class of E. coli (10). The O157 E. coli characterized in this work seemed to be more often urease positive than the other E. coli isolates (15 versus 0.9%, respectively). However, no plasmid detection was investigated (24, 34), and it remains to be seen whether this phenotype offers any ecological advantage to these isolates.
Whereas -glucuronidase appears to be a confirmed character to
differentiate between E. coli O157 and non-O157 strains, the sorbitol fermentation is more questionable due to the low number of
positive E. coli non-O157 strains studied thus far.
Interestingly, this observation could explained, at least partially, by
the discrepancy between the classical selective media for O157:H7
E. coli, mainly based on sorbitol fermentation, and
chromogenic agar, based on -glucuronidase and -galactosidase.
Contrary to the data reported for the Microscan (1) or API
20E (8) tests, the results of this study using ID
32E suggested that E. coli O157:H7 or O157:H() strains do
not display a unique profile but instead several particular biochemical
profiles. Nevertheless, a single profile (i.e., 54465743000) accounted
for 25 and 32% of all O157:H7 and O157:H() isolates,
respectively. Moreover, eight profiles (i.e., 54465743000, 54465543000, 54465542000, 54465540000, 54465541000, 54465741000, 54465742000, and 55465743000) represented ~ 80% of all O157:H7 and
O157:H() strains, and these profiles were not found among the
non-O157 E. coli strains.
Finally, it is important to note that the current ID 32E database of reference biochemical profiles has been mostly made with classical E. coli and does not take into account specific biochemical characteristics of the O157 serotype. Incorporation of these particular biochemical profiles or numbers into the ID 32E database could inform customers of presumptive identification of the E. coli serotype O157 and should suggest additional confirmation tests which must be performed.
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FOOTNOTES |
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* Corresponding author. Mailing address: Catholic University of Louvain, Faculty of Agricultural Sciences, Microbiology Unit, Place Croix du Sud, 2, Box 12, B-1348 Louvain-la-Neuve, Belgium. Phone: 32-10-47-34-40. Fax: 32-10-47-34-40. E-mail: Alexandre.Leclercq{at}pasteur-lille.fr.
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Journal of Clinical Microbiology, March 2001, p. 1161-1164, Vol. 39, No. 3
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.3.1161-1164.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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