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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
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Abstract
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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
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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-beta ,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, SH2 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).

                              
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  		TABLE 1.   Comparison of the MicroScan W/A system and conventional biochemical tests for the identification of Aeromonas speciesa


                              
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  		TABLE 2.   Performance of MicroScan Combo Negative type 1S panels

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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