Cytotoxic Enterotoxin Produced by Aeromonas hydrophila:

INFECTION

AND

Vol. 23, No. 3

IMMUNITY, Mar. 1979, p. 829-837

0019-9567/79/03-0829/09$02.00/0

Cytotoxic Enterotoxin Produced by Aeromonas hydrophila: Relationship of Toxigenic Isolates to Diarrheal Disease NICHOLAS CUMBERBATCH,1 MARC J. GURWITH,2* CLAIRE LANGSTON,3 R. BRADLEY SACK,' AND JAMES L. BRUNTON' Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada'; Departments of Medicine2 and Pathology,3 University of Kansas Medical Center, College of Health Sciences and Hospital, Kansas City, Kansas 66103; Johns Hopkins University International Center for Medical Research, Baltimore, Maryland 21224' Received for publication 26 December 1978

Ninety-six Aeromonas hydrophila isolates were tested for cytotoxin and hemolysin production. Sixty-six (69%) of the isolates were both cytotoxic and hemolytic, whereas the rest produced neither cytotoxin nor hemolysin. No evidence of a separate cytotonic activity could be found in any of the isolates. Cytotoxin activity correlated with enterotoxic activity. Of four cytotoxin-producing strains tested in the isolated rabbit ileal loop, three were definitely positive and one was borderline, whereas two nontoxigenic strains were negative. Cytotoxin activity appeared to be a stable property and could not be associated with any common identified plasmid; only 10 of 21 cytotoxin-producing strains could be shown to have any plasmid by agarose gel electrophoresis. Cytotoxin production correlated with a positive lysine decarboxylase phenotype (98%) or a positive Voges-Proskauer phenotype (94%), compared to 27% lysine decarboxylase-positive and 23% Voges-Proskauer-positive, cytotoxin-negative isolates (P < 0.001 for both). In fecal samples, cytotoxin production correlated with diarrheal disease; of 40 diarrheal isolates, 32 (80%) were toxigenic compared to 9 (41%) of 22 nondiarrheal isolates (P = 0.004). It appears that A. hydrophila can be a cause of diarrhea and that this enteropathogenic potential is mediated by a cytotoxic enterotoxin. Aeromonas hydrophila has been recognized as producing a variety of extracellular products, including enzymes and hemolysins (3, 28). This organism has been considered to be a gastrointestinal pathogen on the basis of reports of isolation from patients with gastroenteritis (1, 5, 15, 17, 25). Nevertheless, there have not been any well-controlled epidemiological studies of the role of A. hydrophila in human disease. In addition, the organism has been isolated from up to 3% of healthy people (26). A possible explanation of the enteropathogenic potential of A. hydrophila has come with the finding that it produces an enterotoxin. This substance, present in the cell-free supernatants of broth cultures, can be identified by production of fluid

secretion in the isolated rabbit leal loop (1, 16). Broth culture supernatants from A. hydrophila cause cell death in several tissue culture systems (13, 16). Similar effects in tissue culture cells have been reported for the gnterotoxins produced by Shigella dysenteriae and Clostridium perfringens (14). The latter enterotoxins have been classified as "cytotoxic" to differentiate them from the "cytotonic" enterotoxins such as those produced by Escherichia coli and Vibrio

cholerae, which cause rounding and increased steroid production, but not cell death, in mouse adrenal tumor cells (14). One group, however, has claimed that A. hydrophila produces both a cytotoxin and a cytotonic enterotoxin which resembles the cholera and E. coli heat-labile enterotoxins by its effect on mouse adrenal tumor cells (16, 28). In their study, heating the broth culture supernatants of A. hydrophila at 56°C for 10 min inactivated the cytotoxic toxin, consequently allowing the cytotonic toxin to be detected by its rounding effect on mouse adrenal cells. These authors claimed also to have separated a cytotoxin, a cytotonic enterotoxin, and a hemolysin from A. hydrophila cell-free filtrates by isoelectric focusing. Both studies investigating the toxigenicity of a large number of A. hydrophila strains isolated from clinical and environmental sources have suggested that all such strains are toxigenic (1, 16). Until recently, no attempt has been made to correlate the biochemical characteristics of A. hydrophila with toxigenicity. Ljungh et al. (16) tested 11 enterotoxigenic strains in more than 90 reactions but could not correlate any biochemical reaction with enterotoxigenicity. Other stud-

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ies of the biochemical characteristics of various isolates of A. hydrophila have been reported, but these have been of taxonomic importance only (5, 10, 20). After finding toxigenic A. hydrophila in several severe cases of diarrhea (12, 13) in Canada, we decided to investigate toxin production by A. hydrophila isolated locally and the relationship of toxin production by these isolates to diarrhea. MATERIALS AND METHODS Source of organisms. Al A. hydrophila isolated in the Clinical Microbiology Laboratory in the Health Sciences Centre in Winnipeg, Manitoba, since 1 January 1976 have been saved and stored on Trypticase soy agar slants at 4 and -70'C. Since January 1976, the laboratory has been alerted to look for A. hydrophila isolates; in the stool bacteriology section of the laboratory, all aerobic gram-negative rods, whether lactose positive or negative, were screened by the oxidase reaction. All oxidase-positive organisms were then further investigated. A. hydrophila was defined as oxidase and arginine dehydrolase positive, motile, facultatively anaerobic fermentative organisms (6). A. hydrophila was distinguished from Aeromonas shigelloides by the presence of hemolysis on blood agar, gelatin liquification, and lack of production of ornithine decarboxylase and lack of fermentation of inositol (9). In addition, these organisms were sought in fecal isolates from patients with diarrhea in northern Manitoba and in a prospective family study of diarrhea in Winnipeg and Berens River, Manitoba. In this study, fecal specimens were obtained from all family members at the time of any diarrheal episode as well as every 3 months routinely. Because A. hydrophila was found in the fecal specimens in several Berens River families, samples of drinking water from Berens River were also obtained and cultured, and A. hydrophila was isolated and saved from this water. Two American Type Culture Collection strains of A. hydrophila, A. hydrophila subsp. formicans and A. hydrophila subsp. anaerogenes, and three other A. hydrophila stock cultures were included. All isolates were then reconfirmed as being A. hydrophila and biotyped in a microwell system (8, 24) (API-20E, Analytab Products, Inc.), using a 24-h incubation period at 37°C. Preparation of cell-free supernatants for toxin and hemolysin testing. Four-hour broth subcultures were used to inoculate 8 ml of brain heart infusion (BHI) broth in 50-ml Erlenmeyer flasks incubated at both 25 and 37°C with agitation at 100 rpm for 18 to 24 h. The broth cultures were cleared by centrifugation at 12,000 x g for 45 min at 4°C and filter sterilized through a 0.45-,um filter (Millipore Corp.). These cellfree filtrates were stored at 4°C for use within 3 days. Assay of cytotoxic activity. HeLa cells maintained in minimal essential medium containing 2% fetal calf serum were used for detection of cytotoxicity. The cell-free BHI broth supernatants were added at a final dilution of 1:5 to HeLa cell monolayers in 96well microculture plates (Costar). Negative controls consisted of BHI broth diluted with minimal essential medium. After overnight incubation, the wells were

INFECT. IMMUN. examined microscopically for cell death. Initially, visible cytotoxicity was confirmed by trypan blue dye exclusion, but later it became apparent that the dye exclusion method was not needed for evaluation of cell death. Cytotoxicity was further confirmed and quantitated by measuring the release of 51Cr from prelabeled HeLa cells. Trypsinized HeLa cells, 107, were labeled with 100 XCi of 5"Cr (supplied as sodium chromate, Amherst) at 37°C for 45 min. After four washes with cold minimal essential medium, the cell concentration was adjusted to a concentration of 2 x 105 cells per ml. One hundred microliters of the labeled HeLa cell suspension was dispensed into V-shaped wells of microtiter plates (Linbro). One hundred microliters of

serial dilutions of the A. hydrophila BHI broth culture cell-free filtrates was then added. For 100% and spontaneous 5Cr release, Triton X-100 and cold minimal essential medium, respectively, replaced the test filtrates. Mixtures of labeled cells and filtrates were incubated at 37°C for 2 h. The released 5'Cr, separated from the cell-bound label by centrifugation at 900 x g for 5 min, was counted in 100-pd volumes in an automated gamma counter. Cytotoxicity was estimated from the percentage of released radioactivity. One cytotoxic unit was defined as the reciprocal of the dilution of test material needed to release 50% of radioactivity from the labeled cells. Assay of hemolytic activity. Hemolytic activity was detected in a 1% rabbit erythrocyte suspension in phosphate-buffered saline (pH 7.0). One hundred microliters of diluted test filtrates was incubated with equal volumes of rabbit erythrocytes in V-shaped wells of microtiter plates and incubated at 37°C for 1 h, followed by 1 h at 4°C. Filtrates causing lysis in more than 50% of erythrocytes on visual examination were scored as hemolytic. Hemolytic activity was assayed quantitatively according to the method of Wadstrom (27). Doubling dilutions of test filtrates were incubated at 37 and 4°C for consecutive 1-h periods with equal volumes of the rabbit erythrocyte suspension. The released hemoglobin was measured spectrophotometrically at 530-,m wavelength. One hemolytic unit was defined as the reciprocal of the dilution of test material needed to produce 50% hemolysis. Assay of cytotonic activity. Cytotonic activity of the broth filtrates was assayed in the Y-1 mouse adrenal tumor (MAT) cell system, with greater than 50% rounding considered positive, as previously described (13). Enterotoxin testing. Enterotoxigenicity from six isolates was assayed in the ligated rabbit ileal loop (30). Both live cultures and their corresponding cellfree filtrates were tested for ability to stimulate fluid accumulation (volume/length ratio 2 1.0). Culture filtrates were also tested for enterotoxin by the suckling mouse assay as previously described (13). Plasmid analysis. The presence of plasmid deoxyribonucleic acid in 31 A. hydrophila isolates was assayed using cleared lysates of 30-ml broth cultures, which were prepared and subjected to agarose gel electrophoresis as described by Meyers et al. (19).

RESULTS Cytotoxicity testing of individual isolates (Table 1). When tested in HeLa cells, 66 (69%)

of the 96 isolates, grown at 370C in BHI broth, produced a toxin causing easily recognizable cell death as manifested by cell shrinkage, loss of adherence, and pyknotic nuclei (Fig. 1). Incubation at 250C was less effective in producing a TABLE 1. Toxin production by 96 A. hydrophila isolates: effects of incubation temperature and heat inactivation Effect in HeLa cells shown by no. of isolates causing: Rounding and/ Cell death or loss of ad- Nomoreoffectg icleft herence 27 3 66

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VOL. 23, 1979

Heated to 560C for 10 min and then rapidly cooled to

cytotoxin. The earliest changes could be seen within 30 min by light microscopy, and in potent toxin preparations the cells were already nonadherent by 2 h. Initially, vital dye exclusion was used to confirm cell death, but this was later not considered to be necessary because the morphological changes associated with cell death were so easily recognizable. These changes were considered cytotoxic or cytolytic and correlated with 5"Cr release. The remaining 30 isolates caused no cytotoxic effect in HeLa cells, although the sterile broth filtrates from 3 to 17 (Table 1) of these 30 cytotoxin-negative isolates caused, in some assays, rounding and loss of adherence of the HeLa cells (Fig. 1). This effect was not reproducibly present in broth filtrates; when present, it was detectable only in low titer (maximum titer was 1:8); and it could be at least partially abolished by inclusion of protease inhibitors (1.0 mM phenylmethyl sulfonyl fluoride and 5.0 mM benzamidine) in the BHI. These broth filtrates of cytotoxic A. hydrophila isolates produced identical morphological

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832

CUMBERBATCH ET AL.

INFECT. IMMUN.

changes in MAT cells (Fig. 2), with the exception of two preparations cytotoxic in HeLa cells but without effect in MAT cells. All 30 preparations not causing cytotoxicity in HeLa cells were also not cytotoxic in MAT cells. The preparations causing "rounding" and loss of adherence in HeLa cells also caused some rounding in MAT cells (Fig. 2). A more striking finding in MAT cells was loss of cells from the cell sheet and loss of adherence of the cell sheet itself, with rolling and folding at its edge. The morphological effect produced by these preparations in MAT and HeLa cells is entirely different from that caused by the adenyl cyclase-stimulating enterotoxins of E. coli and V. cholerae. These latter toxins cause a characteristic rounding of 70 to 80% of the MAT cells without any loss of adherence and are without effect in the HeLa cells (Fig. 1 and 2). By contrast, the A. hydrophila preparations which produce the rounding effect cause rounding in both MAT and HeLa cells. In MAT cells, there is rounding of only approximately 25

to 35% of the cells and focal loss of attachment of the cell sheet (Fig. 2). In HeLa, most cells are rounded and no longer attached (Fig. 1). This rounding effect can be partially inhibited by protease inhibitors and can also be duplicated by leaving trypsin (Baltimore Biological Laboratories) in concentrations from 0.025 to 2.5% on HeLa or MAT cells for 24 h. The effect is not due to production of lesser amounts of cytotoxin by some isolates, since cytotoxin-containing preparations when serially diluted change from positive to negative without ever showing rounding. Because it has been suggested that the cytotoxin of A. hydrophila, by its cytolytic effect, obscures the effect of a separate cytotonic (adenyl cyclase stimulating) enterotoxin, all toxin preparations were also tested in HeLa and MAT cells after heat inactivation of the broth filtrate at 560C for 10 min, as suggested by Wadstrom et al. (28) (Table 1). After this heat inactivation, up to 15 preparations originally cytotoxic caused

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hydrophila isolate (no change from control cells); (B) cytotoxin-positive A. hydrophila isolate (cell death, manifested by pyknotic nuclei, loss of adherence, cell shrinkage); (C) A. hydrophila isolate producing loss of cells from monolayer and a few rounded cells; (D) E. coli heat-labile toxin producing numerous rounded cells without loss of adherence. Fixed in 95% ethanol; stained with hematoxylin and eosin.

either rounding or loss of adherence in HeLa cells, whereas 17 remained still cytotoxic, and the rest were without morphological effect. This suggests that some isolates produced both a cytotoxic toxin and a substance, possibly a proteolytic enzyme, which caused rounding or loss of adherence in low concentration and was masked by the cytotoxin. Similar effects were seen in MAT cells. To further investigate the possible production of a cytotonic enterotoxin similar to E. coli or cholera enterotoxins, sterile broth filtrates from four A. hydrophila isolates were tested for adenyl cyclase stimulation in the lysed pigeon erythrocyte assay (11) (courtesy of D. Robertson, University of Kansas, Lawrence, Kans.), both before and after heat inactivation at 56°C as described above. These broth filtrates, which included one with a cytotoxic effect in HeLa and MAT cells, two which caused rounding in both cell lines, and one which was without any morphological effect in both cell lines, were all negative (