Cytotoxin and enterotoxin production as factors delineating enteropathogenicity of Aeromonas caviae.

H Namdari and E J Bottone J. Clin. Microbiol. 1990, 28(8):1796.

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JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1990, p. 1796-1798 0095-1137/90/081796-03$02.00/0 Copyright 1990, American Society for Microbiology

Vol. 28, No. 8

Cytotoxin and Enterotoxin Production as Factors Delineating Enteropathogenicity of Aeromonas caviae

HASSAN NAMDARI AND EDWARD J. BOTTONE* Clinical Microbiology Laboratories, The Mount Sinai Hospital, One Gustave L. Levy Place, New York, New York 10029-6574
Received 10 January 1990/Accepted 17 May 1990

Twenty-one stool and environmental Aeromonas caviae isolates were studied for cytopathogenicity for cultured HEp-2 cells. Cytotoxic activity was demonstrated by the 21 A. caviae strains and by A. caviae ATCC 15468 after challenging HEp-2 cells with filtrates from a 24-h-old broth shake culture composed of double-strength, glucose-free Trypticase soy broth, incubated at 35C. Heat-stable cytotoxicity was observed 5 h after the addition of 1/5 and 1/10 concentrations of filtrates and consisted of cell rounding and cell detachment. Enterotoxin activity was also demonstrated through the suckling mouse assay. These data advance the role of A. caviae as a gastrointestinal pathogen.

Of the genus Aeromonas, A. sobria and A. hydrophila are regarded as gastrointestinal tract pathogens; A. caviae, however, is considered to be nonenteropathogenic (2, 9, 17). Although we (12) and others (1, 8, 16) have reported the recovery of A. caviae from the stools of individuals with diarrhea, its diarrheagenic potential has not been substantiated through experimentation with animals or humans. Whereas several exoproducts (enterotoxin, cytotoxin, and hemolysin) produced by A. hydrophila and A. sobria have been identified to account for their enteropathogenicity (5, 7, 14, 15), to date an exoenteropathic component has not been uniformly detected in A. caviae. In this study, we describe cytotoxin and enterotoxin production among clinical and environmental isolates of A. caviae which further supports its role as an enteropathogen.
MATERIALS AND METHODS Bacterial isolates. A. caviae strains were isolated either from stool specimens of diarrheic individuals at The Mount Sinai Hospital (n = 17) or from a drinking water supply in Israel (n = 4). Additionally, one reference A. caviae culture (ATCC 15468) was included. The isolates were identified to the species level as previously described (11). Cytotoxin and enterotoxin assays. Overnight cultures of A. caviae on 5% sheep blood agar plates (BBL Microbiology Systems, Cockeysville, Md.) were inoculated individually to 5 ml each of glucose-free double-strength tryptic soy broth (TSB-2x), brain heart infusion broth (Difco Laboratories, Detroit, Mich.), and TSB-2x containing 0.4% glucose in 25-ml Erlenmeyer flasks. Individual flasks were incubated at 35C for 8, 16, and 24 h with shaking (200 rpm) on an environmental-water bath shaker (New Brunswick Scientific Co., New Brunswick, N.J.). Cell-free filtrates were prepared by centrifugation (5,000 x g) at 4C for 30 min with subsequent filtration of the supernatants through a 0.45-,umpore-size filter (Millipore Corp.). The filtrates were either refrigerated prior to immediate use or stored at -70C. A. caviae cytotoxin activity in HEp-2 cells. The HEp-2 cell monolayer was prepared by culturing the cells in 12-well tissue culture plates (Costar, Cambridge, Mass.) in minimal essential medium containing 10% fetal calf serum for 48 h. The cell monolayer was maintained in minimal essential
*

were

medium containing 2% fetal calf serum, and culture filtrates added to give final dilutions of 1/5 or 1/10 in each well. Negative controls consisted of TSB-2x or brain heart infusion broth diluted to 1/5 with minimal essential medium. Positive cytotoxin activity was taken as 250% cell rounding and detachment and was determined microscopically after 5 and 24 h of incubation at 37C. Cell death was confirmed by trypan blue (GIBCO Laboratories, Grand Island, N.Y.) dye exclusion. In this technique, viable cells are stained whereas nonviable cells exclude the dye. Suckling mouse assay for enterotoxin activity. Cell filtrates (0.1 ml) containing 0.02% (wt/vol) trypan blue derived from four A. caviae TSB-2x cultures were administered orally to three 3- to 4-day-old Swiss albino mice through a fine polyethylene tube connected to a 1-ml tuberculin syringe. The suckling mice were then individually placed on a layer of filter paper (Whatman, Inc., Clifton, N.J.) in a plastic petri dish and left at room temperature for 3 h. The animals were then sacrificed by cervical dislocation, and the presence of dye in the small intestine and the degree of intestinal distention were recorded. The entire intestinal tract of each infant mouse was then removed and weighed, as was the remaining body. The intestinal weight/body weight ratio was calculated and scored from 0 to 4+ as previously described (4). Additionally, the occurrence of diarrhea in each mouse was assessed by the intensity of blue stain on the filter paper. RESULTS As determined by cytopathogenicity to cultured HEp-2 cells, all clinical and environmental A. caviae isolates as well as strain ATCC 15468 produced cytotoxin, as evidenced by HEp-2 cell monolayer detachment, rounding, and loss of viability (Fig. 1). By using a 1/5 dilution of culture filtrates, cytotoxicity for HEp-2 cells was evident as early as 1 h postexposure and maximized at 5 h postinoculation. Twenty-four hours was required before cytotoxicity was evident with the 1/10 dilution of the A. caviae filtrates. No activity was present in culture filtrates diluted 1/20 or greater or after three consecutive freezing (-20C) and thawing cycles. Cytotoxin was produced by A. caviae in glucose-free TSB2 x but not in TSB-2 x containing 0.4% glucose or brain heart infusion broth supplemented with 0.2% glucose (Table 1). By way of contrast, cytotoxin production by A. hydrophila and A. sobria was present in all of the media. Glucose apparently

Corresponding author.
1796

VOL. 28, 1990

A. CAVIAE ENTEROTOXIN AND CYTOTOXIN PRODUCTION

1797

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FIG. 1. Cytotoxin activity of A. caviae in HEp-2 cells as demonstrated by cell rounding and cell death (a), as contrasted with control culture (b) containing broth alone. Phase-contrast magnification, x400.

repressed A. caviae cytotoxin production but not that of A. hydrophila or A. sobria. A. caviae cytotoxin activity was not detected after 8 h of growth in glucose-free TSB-2x but was present at 16 h. A. hydrophila and A. sobria toxin activity was present in the supernatant fluids as early as 8 h postinoculation. The cytotoxic activity of the A. caviae isolates was preserved in heated (100C for 10 min.) culture filtrates. Enterotoxin activity of A. caviae was also demonstrated through the suckling mouse assay, as evidenced by gastric distention and intestinal fluid accumulation. All four strains of A. caviae tested (959, 1585, 15962, and ATCC 15468) as well as a control A. hydrophila strain (223) produced enterotoxin, as determined by the ratio of intestinal weight to body weight. DISCUSSION The role ofA. caviae as a gastrointestinal tract pathogen is widely debated because no clear-cut evidence has been presented to support its diarrheagenic potential. While we
TABLE 1. Cytotoxin production by Aeromonas species in different culture mediaa
Cytotoxin production in:
Species
isolates

(12) and others (6) have documented enteroadherenc of this mesophilic aeromonad, reports documenting enterotoxin production are inconsistent. Burke et al. (3) reported that 2 of 286 strains of A. caviae derived from diverse geographic locales were enterotoxigenic in the suckling mouse assay, and Turnbull et al. (17) found 2 of 18 A. caviae strains to be enterotoxigenic. Barer and colleagues (2), on the other hand, concluded that cytotoxin production was absent in 46 A. caviae strains tested by cultured Vero cell assay. The present study delineates cytotoxin and enterotoxin production to be a constant characteristic of A. caviae.
Culture filtrates of A. caviae grown in glucose-free TSB-2x
were cytotoxic to HEp-2 cells, and as shown, cytotoxicity was heat-stable. Enterotoxigenicity was also observed in the suckling mouse assay, as evidenced by diarrhea and intesti-

No. of

TSB-2x Glucose +0.4% free glucose

nal fluid accumulation. To account, however, for the apparent discrepant results reported previously, we propose that the demonstration of toxin production is time and medium dependent and, more importantly, intrinsically associated with the repressed activity on toxin production by glucose when present in the growth medium. In our hands, A. caviae toxin production was detected in filtrates only after 16 h of growth, in contrast to A. hydrophila and A. sobria, which produced cytotoxin as early as 8 h after culture inoculation. This delay in cytotoxin production could have contributed to negative results reported by authors who tested filtrates from younger cultures

BHIBb
+ +

(17).
A. caviae uniformly produced toxins in glucose-free TSB2x. Initially, we, like other investigators, used filtrates of A. caviae grown in single-strength TSB to demonstrate toxin production and were unsuccessful. The utilization of doublestrength TSB was serendipitous and resulted as a consequence of trying various broth media and formulations to demonstrate toxin production by A. caviae. Interestingly, as noted by others (2, 9, 17), single-strength TSB and brain heart infusion broth were adequate for demonstrating toxin

A. caviaec A. hydrophila A. sobria

21 3 2

+ + +

+ +

a As assessed in HEp-2 cells. +, Produced cytotoxin; -, did not produce cytotoxin. b BHIB, Brain heart infusion broth. C Including ATCC 15468.

1798

NAMDARI AND BOTTONE

J. CLIN. MICROBIOL.

production by A. hydrophila and A. sobria but not A. caviae. Perhaps double-strength TSB stabilizes the toxin molecule by increasing phosphate content or supports production of the toxin(s) by the provision of other nutritional factors, thereby allowing its detection. We have long been aware of the deleterious effect (suicide phenomenon) of glucose in the cessation of growth of A. caviae and (variably) A. sobria and A. hydrophila (10). This suicide effect has been attributed to glucose catabolite repression of the tricarboxylic acid cycle enzymes and stimulation of pyruvate dehydrogenase of the suicidal strains (H. Namdari and V. J. Cabelli, J. Bacteriol., in press). Such a regulatory mechanism results in the activation of the fermentation pathway for acetic acid production (with a lowering of pH), which has been shown to be detrimental to A. caviae (13). We postulate that the presence of glucose in broth media used for the cultivation of A. caviae may suppress toxin production via an identical mechanism or as a consequence of slowing cell growth and toxin production below a detectable threshold. These latter parameters are in need of further clarification. In summary, a substantial amount of evidence has mounted incriminating A. caviae as a bona fide enteric pathogen. A. caviae is the most common mesophilic aeromonad recovered from the stools of symptomatic formulafed children (usually less than 1 year of age) in the absence of any other, better-known enteropathogen (12). This species has been shown to adhere to a variety of cultured cell lines, and now we have identified enterotoxin and cytotoxin production as additional virulence factors. Taken together, this constellation of clinical and biologic characteristics evidenced and possessed by A. caviae underscores its enteropathogenic potential.
LITERATURE CITED 1. Altwegg, M. 1985. Aeromonas caviae: an enteric pathogen? Infection 13:228-230. 2. Barer, M. R., S. E. Millership, and S. Tabaqchali. 1986. Relationship of toxin production to species in the genus Aeromonas. J. Med. Microbiol. 22:303-309. 3. Burke, V., J. Robinson, J. Beaman, M. Gracey, M. Lesmana, R. Rockhill, P. Echeverria, and J. M. Janda. 1983. Correlation of enterotoxicity with biotype in Aeromonas spp. J. Clin. Microbiol. 18:1196-1200.

4. Burke, V., J. Robinson, N. J. Berry, and M. Gracey. 1981. Detection of enterotoxins of Aeromonas hydrophila by a suckling-mouse test. J. Med. Microbiol. 14:401-408. 5. Campbell, J. D., and C. W. Houston. 1985. Effect of cultural conditions on the presence of a cholera-toxin cross-reactive factor in culture filtrates of Aeromonas hydrophila. Curr. Microbiol. 12:101-106. 6. Carrello, A., K. A. Silburn, J. R. Budden, and J. B. Chang. 1988. Adhesion of clinical and environmental Aeromonas isolates to HEp-2 cells. J. Med. Microbiol. 26:19-27. 7. Chakraborty, T., M. A. Montenegro, S. C. Sanyal, R. Helmuth, E. Bulling, and K. N. Timmis. 1984. Cloning of enterotoxin gene from Aeromonas hydrophila provides conclusive evidence of production of a cytotonic enterotoxin. Infect. Immun. 46:

435-441.
8. Challapalli, M., B. R. Tess, D. G. Cunningham, A. K. Chopra, and C. W. Houston. 1988. Aeromonas-associated diarrhea in children. Pediatr. Infect. Dis. J. 7:693-698. 9. Millership, S. E., M. R. Barer, and S. Tabaqchali. 1986. Toxin production by Aeromonas spp. from different sources. J. Med. Microbiol. 22:311-314. 10. Namdari, H., and E. J. Bottone. 1988. Correlation of the suicide phenomenon in Aeromonas species with virulence and enteropathogenicity. J. Clin. Microbiol. 26:2615-2619. 11. Namdari, H., and E. J. Bottone. 1989. Suicide phenomenon in mesophilic aeromonads as a basis for species identification. J. Clin. Microbiol. 27:788-789. 12. Namdari, H., and E. J. Bottone. 1990. Microbiologic and clinical evidence supporting the role of Aeromonas caviae as a pediatric enteric pathogen. J. Clin. Microbiol. 28:837-840. 13. Namdari, H., and V. J. Cabelli. 1989. The suicide phenomenon in motile aeromonads. Appl. Environ. Microbiol. 55:543-547. 14. Notermans, S., A. Havelaar, W. Jansen, S. Kozaki, and P. Guine. 1986. Production of "Asao toxin" by Aeromonas strains isolated from feces and drinking water. J. Clin. Microbiol. 23:1140-1142. 15. Pitarangsi, C., P. Echeverria, R. Whitmire, C. Tirapat, S. Formal, G. J. Dammin, and M. Tingtalapong. 1982. Enteropathogenicity of Aeromonas hydrophila and Plesiomonas shigelloides: prevalence among individuals with and without diarrhea in Thailand. Infect. Immun. 35:666-673. 16. San Joaquin, V. H., and D. A. Pickett. 1988. Aeromonas associated gastroenteritis in children. Pediatr. Infect. Dis. 7: 53-57. 17. Turnbull, P. C. B., J. V. Lee, M. D. Miliotis, S. Van De Walle, H. J. Koornhof, L. Jeffery, and T. N. Bryant. 1984. Enterotoxin production in relation to taxonomic grouping and source of isolation of Aeromonas species. J. Clin. Microbiol. 19:175-180.