INTRODUCTION

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Since 1976 the genus Aeromonas has been expanded from three phenospecies to 14 nomenspecies. The majority of these newer species were originally discovered when DNA-DNA studies performed on representative strains revealed the presence of genetic heterogeneity. From these studies a number of hybridization groups (HG) were identified, and some were given species names (10). Human infections caused by Aeromonas hydrophila (HG 1), A. caviae (HG 4), and A. veronii biotype sobria (HG 8), formerly phenospecies A. sobria (HG 8), are not uncommon and have been reported in clinical microbiology and infectious disease periodicals worldwide. Susceptibility patterns of these species to various antimicrobial agents are well documented (5, 6, 12, 13, 15, 16, 18, 20), as is an apparent increase in resistance to -lactam antibiotics (11, 14). This increased resistance is attributed to the presence of -lactamases, including those that hydrolyze the carbapenems, in these organisms (3, 7, 17). Other species documented to cause human infections include A. jandaei (HG 9), A. schubertii (HG 12), and A. veronii biotype veronii (HG 10), formerly A. veronii (HG 10) (10). A. trota (HG 13) has been isolated from human clinical sources (feces and appendix) but has not been firmly established as a causative agent of human disease (10). Identification of these organisms is problematic even when widely accepted commercial identification systems, both manual and automated, are used. The identification of isolates of A. schubertii and A. veronii biotype veronii as Vibrio damsela and Vibrio cholerae, respectively, has been reported (2). The antimicrobial susceptibility patterns of these more recently recognized species of aeromonads are not well documented because of the small number of single-isolate cases reported in the scientific literature. No susceptibility studies examining reasonable numbers of these less frequently isolated Aeromonas species have been published to date (10). The purpose of this study was to examine such a collection of reference laboratory-identified isolates of these four species. Determination of antimicrobial susceptibility patterns may also aid in the recognition of these species in the clinical microbiology laboratory.

	MATERIALS AND METHODS

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Fifty-six isolates of four Aeromonas species, which have been documented as causative agents of human infections or isolated from clinical specimens, were subjected to antimicrobial susceptibility testing using a MicroScan WalkAway conventional (overnight incubation) gram-negative panel. The four species tested and the number of each were as follows: A. jandaei, 17; A. schubertii, 12; A. trota, 15; and A. veronii biotype veronii, 12.

The Enteric Unit of the Microbial Diseases Laboratory, California Department of Health Services, Berkeley, Calif., identified all of the isolates by a battery of 65 biochemical tests (1, 9). The Clinical Microbiology Laboratory of the Department of Veterans Affairs Medical Center, Lexington, Kentucky, performed all of the antimicrobial susceptibility tests.

Isolates were shipped in vials containing 2.5 ml of motility medium containing 0.3% agar. Upon receipt, the vials were subcultured to sheep blood agar plates (SBAP) (Becton Dickinson Microbiology Systems, Loveton, Md.) and incubated in an ambient air incubator at 35°C for 18 to 24 h. The SBAP were examined for culture purity, and a Kovács oxidase test was performed with an isolated colony. A second colony isolated from each SBAP was subcultured to a second SBAP and incubated as described above for an additional 18 to 24 h. Colonies from the second subculture were subjected to antimicrobial susceptibility testing using the MicroScan Walkaway 40 system (Dade-Behring, West Sacramento, Calif.). Five well-isolated colonies of each strain were picked by using the MicroScan Prompt Inoculation System-D. The standardized inoculum was added to MicroScan Negative Combo Panel Type 16 microwell trays by using the MicroScan Renok device. Panels were placed in the MicroScan Walkaway 40 for overnight incubation and were read automatically by the instrument.

	RESULTS

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The antibiograms of A. jandaei (HG 9), A. schubertii (HG 12), A. trota (HG 13), and A. veronii biotype veronii (HG 10) are shown in Table 1. The MICs at which 50% of isolates were inhibited (MIC₅₀s) and MIC₉₀s of A. jandaei (HG 9), A. schubertii (HG 12), A. trota (HG 13), and A. veronii biotype veronii (HG 10) are shown in Table 2.

	DISCUSSION

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Aeromonas species have been the subject of a number of antimicrobial susceptibility studies over the last 30 years. Most of these studies have usually involved the three readily recognized phenospecies, A. hydrophila, A. caviae, and A. veronii biotype sobria (5, 6, 12, 13, 15, 16, 18, 20). In the last decade, increased resistance of these organisms to -lactam antibiotics has been described in Japan (14) and Taiwan (11).

Until now, susceptibility data for these less frequently isolated Aeromonas species have been lacking (10). The results of this study indicated that these species of the genus Aeromonas were more susceptible to narrow-spectrum cephalosporins than the more frequently isolated species. However, resistance also occurred in these less frequently isolated species.

A. jandaei was the most resistant of the four species. Most isolates were resistant to penicillins, including both piperacillin and ticarcillin. Sixty-five percent of the A. jandaei strains were also resistant to imipenem. Previously reported Aeromonas resistance to imipenem varies from 3% (14) to 14% (11) for A. veronii biotype sobria and is 8% for A. hydrophila (11, 14). Recognition of A. jandaei isolates may be enhanced by the increased resistance to penicillins, including piperacillin and ticarcillin, and to imipenem. It is notable that this species which was the most resistant is, of the four species in this study, the one most frequently isolated from blood in clinical infections (10).

A. schubertii isolates had a susceptibility pattern very similar to those that have been reported for A. hydrophila and A. caviae (12).

A. trota was the most susceptible of the four species, with all isolates susceptible to ampicillin. Susceptibility to ampicillin is a characteristic of A. trota (4) and appears to be unique to this species because the more frequently isolated species of Aeromonas are usually resistant to ampicillin (11, 12, 14). Eighty percent of the A. trota strains were also susceptible to cefazolin. With the exception of A. veronii biotype sobria (8), the other species of the genus Aeromonas are resistant to narrow-spectrum cephalosporins (5, 6, 12, 13, 15, 16, 18, 20). Recognition of A. trota isolates may be enhanced by the increased susceptibility to narrow-spectrum cephalosporins and to penicillins, especially ampicillin.

A. veronii biotype veronii isolates displayed 100% susceptibility to piperacillin and 100% resistance to ticarcillin. Sixty-seven percent of the A. veronii biotype veronii strains were also resistant to imipenem. A. veronii biotype veronii was the only species to exhibit any significant aminoglycoside resistance, with 42% of the isolates being resistant to tobramycin. Previously reported values for Aeromonas resistance to tobramycin are 23% for A. veronii biotype sobria and 25% for A. caviae and A. hydrophila (11). Eighty-three percent of the A. veronii biotype veronii isolates were susceptible to cefazolin. Recognition of A. veronii biotype veronii isolates may be enhanced by the susceptibility to piperacillin coupled with resistance to ticarcillin, resistance to imipenem, and susceptibility to narrow-spectrum cephalosporins.

The recommended therapy for infections caused by members of the genus Aeromonas is the use of fluoroquinolones. However, fluoroquinolones should not be used in treating pediatric patients. Alternative therapies include trimethoprim-sulfamethoxazole, aminoglycosides, imipenem, meropenen, parenteral cephalosporins (expanded spectrum and broad spectrum), and tetracyclines (19). The data from the present study indicated the following. (i) A. veronii biotype veronii and A. schubertii had markedly increased resistance to tobramycin. (ii) A. veronii biotype veronii and A. jandaei were generally resistant to imipenem. (iii) A. schubertii and A. trota were less susceptible to cefoxitin, an expanded-spectrum cephalosporin, than to broad spectrum cephalosporins. Perhaps tobramycin, imipenem, and cefoxitin should be removed from the list of alternative therapies.