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Applied and Environmental Microbiology, April 2000, p. 1764-1766, Vol. 66, No. 4
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Identification of Motile Aeromonas
Strains with the MicroScan WalkAway System in Conjunction with the
Combo Negative Type 1S Panels
J.
Vivas,1
A. I.
Sáa,1
A.
Tinajas,2
L.
Barbeyto,2 and
L.
A.
Rodríguez1,*
Laboratory of Microbiology, Department of
Functional Biology and Health Sciences, Faculty of Sciences, University
of Vigo, Campus of Ourense,1 and
Laboratory of Microbiology, Cristal Piñor
Hospital,2 Ourense, Spain
Received 7 September 1999/Accepted 7 January 2000
 |
ABSTRACT |
This study was performed to compare the MicroScan WalkAway
automated identification system in conjunction with the new MicroScan Combo Negative type 1S panels with conventional biochemical methods for
identifying 85 environmental, clinical, and reference strains of eight
Aeromonas species.
| |
TEXT |
Over the past two decades, the
number of recognized species in the genus Aeromonas has
expanded from four species (Aeromonas hydrophila, A. sobria, A. cavia, and A. salmonicida) to at
least 16 recognized hybridization groups (6, 8, 16), and
nine of these taxa have been recovered from clinical and environmental samples and therefore could be pathogenic for humans (17, 19, 26).
Motile Aeromonas species are widely distributed in nature
and have been recognized as normal microflora of aquatic and
terrestrial organisms. The most common, A. hydrophila,
causes disease in fish, frogs, and several other animals (10, 13,
17), and Aeromonas spp. have been reported to cause a
wide variety of human infections, including bacteremia,
gastroenteritis, cellulitis, meningitis, soft-tissue infections,
peritonitis, and bronchopulmonary infections (3, 14, 18, 23, 27,
43). Aeromonads may possess several virulence factors, including
cytotoxins, enterotoxins, and the ability to adhere to and invade the
epithelial cells (14, 21, 22, 28, 35, 36), and several
attempts have been made to correlate the biochemical characteristics of
Aeromonas species with toxigenicity (5, 7, 11, 20, 39,
42).
MicroScan (Dade MicroScan Inc., West Sacramento, Calif.) has recently
marketed MicroScan Combo Negative type 1S panels. The panels are
designed to identify, to the species level, aerobic or anaerobic
facultative gram-negative bacilli and to determine susceptibilities to
antimicrobial agents. This system is capable of rapid identification of
commonly encountered human disease organisms and some species of
environmental bacteria, and it has received favorable reports relative
to identification of these bacteria (30, 34, 37). The
purpose of this study was to evaluate the ability of the MicroScan
WalkAway (W/A) system in conjunction with the new Combo Negative type
1S panels to identify motile Aeromonas species.
Eighty-five Aeromonas strains of eight species were selected
for testing. Among them were strains from the species A. hydrophila (n = 53) (29 strains pathogenic for
fish, 16 strains from freshwater, and 5 strains from nonpathogenic
human clinical material), A. veronii biovar
sobria (n = 10) (6 strains pathogenic for
fish and 1 strain from freshwater), A. veronii biovar
veronii (n = 2), A. trota
(n = 1), A. jandaei (n = 3),
A. schubertii (n = 2), and A. caviae (n = 9) (7 strains pathogenic for fish).
The American and Spanish Type Culture Collections strains A. caviae ATCC 15468 and ATCC 13137; A. hydrophila ATCC
13442, ATCC 13136, and CECT 398; A. jandaei ATCC 49568, ATCC
49570, and ATCC 49572; A. schubertii ATCC 43945 and ATCC
43947, A. sobria ATCC 35993, ATCC 43979, and ATCC 9071;
A. trota ATCC 49657; and A. veronii ATCC 35622 and ATCC 35623 were included in the test group.
Also, we used five nonmotile A. media strains (three
isolated from freshwater), including the strains ATCC 33907 and ATCC 35950, which grew well at the incubation temperature used. The strains
were routinely cultured on trypticase soy agar (Cultimed, Barcelona,
Spain) at 37°C for 24 h, stored on trypticase soy agar slants at
4°C under mineral oil, and frozen at 
70°C with 15% glycerol. All
strains were identified in parallel by using the MicroScan W/A system
(34, 37) and by standard reference procedures (1, 9,
10, 15, 34).
The MicroScan W/A system is an automated system, consisting of a reader
incubator module and a data analysis module. The system uses
fluorogenic substrates and a pH indicator to detect bacterial enzymatic
activity. The reagents for the identification were added automatically
by the W/A instruments, and the panels can be removed from the system
for a manual reading after the automatic reading.
MicroScan panels.
Conventional MicroScan panels and Combo
Negative type 1S panels (Dade MicroScan Inc.) were inoculated with the
strains by the turbidity standard technique. The panels were incubated
for a full 24 h at 35°C within the W/A system. All procedures
were performed according to the manufacturer's directions (MicroScan dried gram negative procedural manual, Dade International Inc., West
Sacramento, Calif.).
The following tests were compared: fermentation of
D-glucose, sucrose, D-sorbitol, raffinose,
L-rhamnose, L-arabinose,
myo-inositol, D-adonitol, and melibiose; urease
production; hydrogen sulfide production; indole production;
decarboxylation of lysine and ornithine; arginine dihydrolase
production; tryptophan deaminase production; esculin hydrolysis;
Voges-Proskauer; utilization of citrate;
O-nitrophenyl-
,D-galactopyranoside (ONPG);
oxidation-fermentation of glucose and nitrate reduction.
The MicroScan W/A system produced high percentages of similarity for
the majority of tests compared (Table
1).
False reactions
for important tests such as urease production,
utilization of
citrate, esculin hydrolysis, SH
2 production,
and ONPG enabled
some organisms to be classified as
Vibrio
species or unidentifiable
strains (rare biotype) instead of as
Aeromonas. False reactions
obtained with some tests for the
identification of the
A. hydrophila group that produced
variable results, such as the tests for decarboxylation
of lysine and
ornithine and the test for the production of arginine
dihydrolase, were
excluded from the analysis without affecting
the final identification.
The range of percentages for the strains
correctly classified as
members of the
A. hydrophila group (
n = 68)
ranged from 80.09 to 99.99%. Ten strains (11%) were misidentified
by
the MicroScan W/A system and classified as rare biotype. Seven
strains
(8%) were classified as
Vibrio fluvialis (two
A. hydrophila strains, one
A. sobria strain, one
A. trota strain, and three
A. media strains) (Table
2). When the identification level of
the
strain is low, the W/A system proposes several options instead
of
Aeromonas, for example as a
V. fluvialis or other
Vibrio species
(
Vibrio vulnificus,
Vibrio
cholerae, or
Vibrio hollisae).
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Comparison of the MicroScan W/A system and conventional
biochemical tests for the identification of
Aeromonas speciesa
|
|
Most of the
Aeromonas species isolated from either clinical
or environmental sources are being classified with different multitest
identification systems such as the API system (Biomerieux, France),
but
these systems are not always totally adequate for the identification
of
clinical or environmental
Aeromonas isolates (
31,
32,
44).
Recently, automated systems have been developed to identify
gram-negative
bacteria (
24,
29,
33,
34,
45), and these have
often
been unable to accurately identify aeromonads to the species
level
or as part of a
Aeromonas complex (
Aeromonas
hydrophila group);
however, the reports about the evaluation of
these systems do
not include information about a high number of
Aeromonas species.
The failure of commercial systems to satisfactorily identify these
microorganisms has been recognized as a problem, and it
continues to be
a weak area of commercial identification and susceptibility
testing
systems. Often,
Aeromonas species are mistakenly identified
as vibrios, with which they share many phenotypic characteristics
(
2,
16,
32,
38). Our results are consistent with those
previously reported for other identification
systems.
In six tests analyzed, the correlation between the MicroScan system and
conventional tests was less than 90% (urease and indole
production,
lysine decarboxylation esculin hydrolysis, and utilization
of citrate).
We have observed that some of these tests (all except
that for lysine
decarboxylation) can cause a false identification.
This also has
occurred with the ONPG test, though the correlation
for this test has
been increased. Such procedures (standard tube
or plate media) may not
be appropriate reference methods for evaluating
automatic systems,
since variations in incubation temperature
or time produce variable
results (
4,
12).
In summary, the MicroScan W/A system in conjunction with the Combo
Negative type 1S panels can identify 80% of the
A. hydrophila group strains tested, most of which were
A. hydrophila, the most
frequently isolated species in clinical and
veterinary medicine.
As most previous studies were performed with
medically derived
strains, we wanted to gain an insight into the use of
the W/A
system for the identification of environmental bacteria.
Because
this system offers some important advantages over conventional
methods, including reduced labor, increased sample throughput,
and
faster turnaround times for test results, we hope that the
W/A system
can be improved by continued enhancements by the manufacturer
for
applications in veterinary or food
microbiology.
| |
ACKNOWLEDGMENTS |
We thank Nelson P. Moyer for critical reading of the manuscript. We
thank Javier Ducha for providing bacterial strains. The assistance of
the staff of the Clinical Microbiology Laboratory at the Hospital
Cristal Piñor Ourense is greatly appreciated.
This work was supported by a grant (K816 641.02) from the University of Vigo.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Área de
Microbiología, Departamento de Biología Funcional y
Ciencias de la Salud, Universidad de Vigo, Campus de Ourense, As
Lagoas, 32004 Ourense, Spain. Phone: 034 988387006. Fax: 034 988387001. E-mail: lalopez{at}uvigo.es.
| |
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Applied and Environmental Microbiology, April 2000, p. 1764-1766, Vol. 66, No. 4
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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