JOURNAL OF CLINICAL MICROBIOLOGY, May 1987, p. 916-9210095-1137/87/050916-06$02.00/0Copyright © 1987, American Society for Microbiology

Clinical and Biochemical Significance of Toxin Production byAeromonas hydrophila

MARK KINDSCHUH,' LARRY K. PICKERING,'* THOMAS G. CLEARY,l AND GUILLERMO RUIZ-PALACIOS2Program in Infectious Diseases and Clinical Microbiology and Department of Pediatrics, University of Texas MedicalSchool at Houston, Houston, Texas 77030,1 and Department of Infectious Diseases, Instituto Nacional de la Nutricion,

Mexico City, Mexico2

Received 21 November 1986/Accepted 10 February 1987

Production of cytotoxin and enterotoxin by Aeromonas strains obtained from stools of 50 children in Mexicoand Texas and from blood of 9 children with sepsis was determined. Results were correlated with clinicalfeatures of infected children as well as with biochemical traits of Aeromonas strains. Cytotoxin was producedby 40 of 42 Aeromonas strains (95%) isolated from stools of children with diarrhea, by all 8 isolates from stoolsof well children, and by all 9 isolates from children with sepsis. There was no difference in the quantities(amount of cytotoxin per milligram of protein required to kill 50% of the cells) of cytotoxin produced and inclinical manifestations among the groups. None of the isolates produced a toxin that could be neutralized byantiserum raised against Shiga toxin produced by Shigella dysenteriae 1 60R. Heat-labile-like enterotoxin (LT)was produced by 26 of 42 stool isolates (62%), while only 1 of the 42 isolates (2%) produced enterotoxinlikeactivity in suckling mice; 65% of the cytotoxin-producing strains also produced an LT-like material. All strainsfrom blood produced LT-like material, and 2 of 6 (33%) produced activity in suckling mice. All strainsproduced hemolysin; 37 of 57 (65%) were Voges-Proskauer positive; 27 of 57 (47%) were lysine decarboxylasepositive by API 20E strips, none were positive for lysine decarboxylose production by lysine-iron agar slants at24 h, but 17 of 54 (31%) were positive at 48 h. Thére was no correlation between biochemical reactions andenterotoxin or cytotoxin production. There appears to be no correlation between toxin production byAeromonas spp. and gastroenteritis.

Aeromonas hydrophila is a gram-negative, facultativeanaerobe of the family Vibrionaceae. The organism has beenrecognized as a pathogen associated with several categoriesof human infection which include the following: (i) acutegastroenteritis of both adults and children, ranging fromwatery to bloody diarrhea of either short or prolonged (over2 weeks) duration (1, 2, 6, 11, 15, 16, 18, 19, 22-24, 29, 30,32, 34); (ii) water-contaminated wound infections with rapidonset of cellulitis (33); (iii) septicemia in immunocompro-mised hosts, especially those with leukemia (4, 20, 39); and(iv) other less frequently encountered infections, includingurinary tract infection (28), myositis (13), conjunctivitis (40),osteomyelitis (45), peritonitis (38), meningitis (36),endocarditis (12), and aspiration pneumonia (37). A. hydro-phila is an ubiquitous organism, found in both stagnant andfresh-flowing water, saltwater estuaries, moist soil, anduntreated sewage. Owing to its aquatic predominance, infec-tions caused by A. hydrophila are more frequent in thewarmer months (2).

Various virulence factors have been ascribed to A. hydro-phila to explain the pathogenesis of infection. These includethe production of cytotoxins which are toxic to both HeLaand Y-1 adrenal cells (1, 2, 11, 14, 15, 22, 29, 32, 34),heat-labile-like and heat-stable-like enterotoxins (6, 7, 9, 11,14, 15, 19, 24, 27, 29, 34, 42), alpha and beta hemolysins (43),hemagglutinins (5), and the ability to adhere to and invadeepithelial cells (3, 24-26, 44).The significance ofAeromonas species as intestinal patho-

gens is controversial (1, 2, 6, 11, 15, 16, 19, 30, 34). Todetermine if putative virulence properties of Aeromonasspp. are associated with diarrheal disease, we evaluated the

* Corresponding author.

role of cytotoxin and enterotoxin production by Aeromonasstrains in the pathogenesis of acute gastroenteritis. Our goalswere to determine the frequency of cytotoxin andenterotoxin production by Aeromonas strains obtained fromchildren in different geographic locations; to determinewhether cytotoxin(s) produced was neutralizable by anti-body obtained from a rabbit immunized with purified form-aldehyde-treated Shiga toxin from Shigella dysenteriae 160R; to determine the relationships among enterotoxin pro-duction, the quantity of cytotoxin produced, and the clinicalfeatures ofAeromonas sp.-associated gastroenteritis; and todetermine the relationships between certain biochemicaltraits of Aeromonas spp. and toxin production.

MATERIALS AND METHODSPopulation. Strains of A. hydrophila were obtained from

stool specimens of 50 children, all under 5 years of age. Ofthe 50 children, 42 had diarrhea and 8 did not. The 42children with diarrhea consisted of 32 from Mexico City,Mexico, and 10 from Dallas, Tex. Of the 8 well children, 5were from Mexico City and 3 were from Dallas. The Dallasstrains were kindly provided by John Nelson. We alsoobtained blood culture isolates of A. hydrophila from 9children with sepsis hospitalized at M. D. Anderson Hospitaland Tumor Institute, Houston, Tex.

Clinical data obtained on 50 children from whom entericisolates were identified included age, presence of vomiting,temperature, maximum temperature, number of stools per

day, total number of stools, and duration of illness. Stoolswere tested for the presence of occult blood, fecal leuko-cytes, and enteropathogens, including Salmonella spp., Shi-gella spp., enterotoxigenic Escherichia coli, Campylobacterspp., rotavirus, and protozoal parasites; none were identi-

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TOXIN PRODUCTION BY AEROMONAS HYDROPHILA 917

TABLE 1. Cytotoxin production by Aeromonas strains byclinical condition

Source of initial No. of isolates Amt of cytotoxinisolates producing cytotoxin/no. (CD5c/mg oftested (%) protein)a

Diarrheal stool 40/42 (95) 101.75 + 0.62Nondiarrheal stool 8/8 (100) 1O2.04 +0.32Sepsis 9/9 (100) 101.49+0.22

a Mean + the standard deviation.

fied. Stools were cultured on MacConkey, salmonella-shigella (Difco Laboratories, Detroit, Mich.), tergitol-7(Difco), DNase medium, and Campy-BAP agars (Scott Lab-oratories, Inc., Carson, Calif.). E. coli heat-labileenterotoxin was sought by enzyme-linked immunosorbentassay (ELISA), E. coli heat-stabile enterotoxin was deter-mined by suckling mouse assay, rotavirus was determinedby ELISA, and protozoal parasites were determined bymicroscopy (10).The Aeromonas isolates were evaluated blindly for in vitro

cytotoxin and enterotoxin production. At the completion ofthe toxin studies, the clinical information was retrieved andcorrelations were made.

Bacterial toxin preparation. The strains initially weregrown on charcoal-yeast extract (Difco) plates at 37°C for 24h. Each strain was then inoculated into 50 ml of a Chelex 100(Bio-Rad Laboratories, Richmond, Calif.)-treated iron-depleted syncase broth and incubated for 48 h with shakingat 300 rpm at 37°C. The cells were harvested by centrifuga-tion at 12,000 x g for 10 min and washed twice with 0.85%NaCI. The cell pellet obtained was suspended in phosphate-buffered saline (0.01 M [pH 7.4]) and lysed by sonication.Cell debris was removed by centrifugation at 12,000 x g for30 min, and the supernatant was filter sterilized by using a0.2-,um-pore-size filter (Millipore Corp., Bedford, Mass.).The concentration of protein in the filter-sterilized superna-tant was measured by Bio-Rad protein assay with bovineserum albumin as the standard.HeLa cell cytotoxicity assay. HeLa 229 cells were har-

vested after 7 days in culture by trypsinization. Cells weresuspended in Eagle minimal essential medium with Earlesalts (Hazleton Dutchland Inc., Lenexa, Kans.), 10% fetalcalf serum, 2 mM L-glutamine, and tritiated thymidine (2,uCi; ICN Pharmaceuticals Inc., Irvine, Calif.) and inocu-lated into 96-well plates (approximately 50,000 cells perwell). Ten-fold serial dilutions of filter-sterilized sonic ex-tracts from each Aeromonas strain were assayed in quadru-plicate. After 24 h at 37°C, the wells were washed withphosphate-buffered saline to remove detached cells andunincorporated tritiated thymidine. Cells were lysed with 1N KOH, transferred to liquid-scintillation-counting solution(Liquiscint; National Diagnostics, Somerville, N.J.), andcounted in a beta-scintillation counter. The percent cellsurvival for each dilution was determined by dividing themean counts per minute by the mean counts per minute ofthe medium control, which did not contain toxin. Thecytotoxicity of each isolate was quantified as the amount ofcytotoxin that killed 50% of HeLa cells (CD50) per milligramof bacterial protein. The CD50 was calculated from the linearregression of the logarithm of percent HeLa cell survivalversus the negative logarithm of the cytotoxin dilution.

Neutralization studies. Neutralization of cytotoxicity wasattempted by incubating cytotoxin preparations with eithernonimmune rabbit serum or immune serum derived from a

New Zealand White rabbit immunized with formaldehyde-

treated purified Shiga toxin from S. dysenteriae 1 60R aspreviously described (30). For each toxin preparation,cytotoxin (1.5 to 2.0 CD50s) was incubated overnight at 4°Cwith a 1:100 dilution of rabbit immune serum prior to use inthe HeLa cell assay. This concentration was chosen toensure that if Shiga toxin-like material was present, it wouldbe neutralized. Toxin preparations were incubated in parallelwith nonimmune rabbit serum in the same dilution. A toxinpreparation (100 CD50s) of S. dysenteriae 1 60R was used asthe positive control. Assays were performed in quadrupli-cate, and significance of differences was determined by thetwo-tailed unpaired Student t test.

Enterotoxin assays. GM-1 ELISA and Chinese hamsterovary (CHO) cells were used for the detection of heat-labile-enterotoxin-like material (21, 41). Serial dilutions of thesupernatants from 1/10 to 1/320 were made in minimumessential medium modified with Hanks salts (Flow Labora-tories, Inc., McLean, Va.), and the dilutions were incubatedwith 103 cells per well for 18 h at 37°C. A total of 100 cellswere counted, and the percentage of elongated cells wasdetermined; 60% was taken as positive for the presence oftoxin. In each assay, a positive and negative control was run.Ail positive specimens were confirmed by assay in rabbitileal loops. Heat-stable-enterotoxin-like material was as-sayed for by using the suckling mouse model (17).

Biochemical traits. The biochemical traits examined andcorrelated with toxin results were hemolysis, lysine de-carboxylase (LDC) activity, and Voges-Proskauer (VP) re-action. Hemolysis was determined by incubation of eachstrain on sheep blood agar for 24 h at 37°C. The presence ofLDC was determined by incubation of each Aeromonasstrain both in lysine-iron agar slants (Difco) and in API stripsfor 24 h. The VP test was performed by using API substrate(Analytab Products, Plainview, N.Y.).

RESULTSCytotoxin production. Cytotoxin was produced by 40 of 42

strains (95%) of A. hydrophila isolated from stools of chil-dren with diarrhea. Ail 8 isolates from stools of well childrenand all 9 isolates from children with sepsis also producedcytotoxin (Table 1). The mean CD50 per milligram of bacte-rial protein for the 42 diarrheal isolates was 101.75 + 0.62, andthe mean CD50 per milligram of bacterial protein of the 8isolates from well children was 102.04 + 0.32. In addition, themean CD50 per milligram of bacterial protein for childrenwith sepsis was 10149 + 0.39. When grouped together, the 17non-diarrhea-associated isolates (8 well children plus 9 chil-dren with sepsis) had a mean CD50 of 101.75 ± 0.39. None ofthese values was significantly different from the others.For the isolates from the 10 children from Dallas with

Aeromonas-associated diarrhea, there was no significantassociation between the amount of cytotoxin produced invitro and the age of the child, presence or absence of blood,number of bowel movements per day, or duration of diar-rhea. For the 32 Mexican children with Aeromonas-associated acute gastroenteritis, there was no significant

TABLE 2. Heat-labile- and heat-stable-enterotoxin-like activityproduced by Aeromonas strains from various geographic locations

Heat-labile- Heat-stableSource of Isolation location toxin-like toxin-likeisolate activity (%) activity (%)

Stool Mexico City, Mexico 21/34 (62) 1/34 (3)Stool Dallas, Tex. 5/8 (63) 0/8 (0)Blood Houston, Tex. 6/6 (100) 2/6 (33)

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J. CLIN. MICROBIOL.918 KINDSCHUH ET AL.

TABLE 3. Biochemical reactions of Aeromonas strains bycytotoxin production

No. of strains producing (+) and not

Biochemical reaction producing (-) cytotoxin/no. tested (%)+_

Hemolysis 57/57 (100) 2/2LDC

Lysine agar slants + 17/54 (31) 0/248-h incubation

API 20E 27/57 (47) 0/2VP 37/57 (65) 0/2

association between cytotoxicity and the age of the child,presence of vomiting or fever, bloody stools, fecal leuko-cytes, fecal mucus, number of bowel movements per day, or

duration of diarrheal illness.Neutralization. The cytotoxin produced by the 57 strains

was tested for neutralization by antiserum derived from a

rabbit immunized with formaldehyde-treated purified Shigatoxin. Although this antiserum neutralizes 100 CD50 of Shigatoxin (35), none of the 57 Aeromonas strains was neutral-ized.

Enterotoxins. Of the isolates, 48 were evaluated for thepresence of heat-stable- and heat-labile-enterotoxin-like ac-

tivity (Table 2); 26 of the 42 fecal isolates (62%) produced a

heat-labile-like enterotoxin, whereas only one fecal isolateproduced heat-stable-enterotoxin-like material. All five wellcontrols produced a heat-labile-like enterotoxin, as did 21 of37 (57%) of the diarrhea-associated strains. The only stoolisolate to produce a heat-stable-like enterotoxin was from a

child without diarrhea. Of 31 cytotoxic strains, 20 (65%)produced a heat-labile-like enterotoxin, but there was no

significant association between the amount of cytotoxinproduced and the production of heat-labile-like enterotoxin.The two noncytotoxic isolates were negative for bothenterotoxins. All of the isolates from blood produced bothheat-labile-like enterotoxin and cytotoxin; two of the sixblood isolates produced heat-stable-like enterotoxin.

Biochemical tests. Hemolysis of sheep blood agar was

produced by ail 57 of the cytotoxin-producing strains. Inaddition, the two noncytotoxic strains of A. hydrophila alsoexhibited hemolysis (Table 3). The presence of LDC was

examined by using both lysine-iron agar slants and API 20Estrips. After 24 h of incubation at 37°C in lysine-iron agar

slants, all 54 of the cytotoxic and both of the noncytotoxicstrains tested yielded negative results; after 48 h, 17 of 54cytotoxin strains (31%) were positive. The noncytotoxicstrains were negative for LDC as examined by API after 24h of incubation at 37°C. Of 57 cytotoxic strains, 27 (47%)were positive for LDC activity by API examination (Table

3). Of 57 cytotoxic strains, 37 (65%) were positive by the VPreaction after 24 h; both noncytotoxic strains were VPnegative. A correlation of the API results with the VP andLDC reactions revealed that the two noncytotoxic strainswere VP negative and LDC negative. The cytotoxic strainsfell into three major categories: VP positive, LDC positive(46%); VP positive, LDC negative (20%); VP negative, LDCnegative (31%). The mean CD50s per milligram of protein foreach of the above three major groups of cytotoxic strainsshowed no significant variation (VP+/LDC+, 101.88 0.46;VP+/LDC-, 0'54 + 0.59; and VP-/LDC- , i0'7 030)

When examined individually, the CD50s for the LDC-positive (101-89 + 0.46) strains were not significantly differentfrom the CD50s of the VP-positive (101-77 + 0.52) strains. Therewas no significant association between heat-labile- or heat-stabile-enterotoxin-like activity of the Aeromonas strainsand the biochemical tests.

DISCUSSION

The importance of A. hydrophila as an enteropathogen iscontroversial. Although studies have reported the isolationof Aeromonas spp. from stools of patients with diarrhea,recovery from stools does not prove an etiologic role. Instudies in which well controls have been enrolled (Table 4),some investigators have isolated Aeromonas spp. from stoolspecimens from patients with diarrhea more frequently thanfrom stool samples from well controls (2, 6, 23), while inother studies, isolation from these two groups was similar(15, 28, 32).A further method of determining whether Aeromonas spp.

are a cause of diarrhea is to identify in Aeromonas strainsthose virulence characteristics which are recognized as

being important in the pathophysiology of diarrhea and torelate the presence of these characteristics to strains isolatedfrom persons with and without diarrhea. It is known thatinvasion of the intestinal epithelium as well as production ofenterotoxins and cytotoxins in the lumen of the intestine are

important virulence mechanisms for E. coli, Vibrio cholerae,and Shigella species (10, 35). Aeromonas spp. produce a

variety of biologically active extracellular substances, in-cluding hemolysins, cytotoxins, and enterotoxins (Table 5).They also have been shown to be invasive and to possess

hemagglutinins that may be correlated with diarrhea (3, 5,24, 26, 44). There is no general agreement with regard to therole of these factors as virulence mechanisms for Aeromonasspp. In some studies, Aeromonas strains isolated neverproduced enterotoxins (14, 29), while in others, enterotoxin-like activity was noted in 7 to 84% of strains (Table 5).Enterotoxinlike material detected by GM-1 ELISA and byCHO cell and rabbit ileal-loop methods was produced by62% of strains from patients with diarrhea in our study, while

TABLE 4. Studies evaluating Aeromonas spp. as a cause of gastroenteritis in patients with diarrhea and in controls

No. of persons with Aeromonas spp./total

Reference Isolation location no. tested (%) SignificantWith diarrhea Without diarrhea difference

Cumberbatch et al., 1979 (11) Canada 32/40 (80) 9/22 (41) P < 0.0002Burke et al., 1983 (7) Australia 127/995 (13) 20/975 (2) P < 0.05Pitarangsi et al., 1982 (34) Thailand (Thais) 37/207 (18) 44/367 (12) NSa

Thailand (travelers) 12/39 (31) 3/35 (9) P < 0.025Millership et al., 1983 (30) England 13/123 (11) 28/846 (3) NSAgger et al., 1985 (2) Louisiana 20/1,797 (1) 0/533 (0) P < 0.02Figura et al., 1986 (15) Italy 21/561 (4) 21/561 (4) NS

a NS, Not significant.

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TOXIN PRODUCTION BY AEROMONAS HYDROPHILA 919

TABLE 5. Studies reporting toxin production by Aeromonas isolates

No. of isolates with enterotoxin activity/no. tested (%o)UReference Source of isolate Patient status

LT-like ST-like Cytotoxin

Ljungh et al., 1977 (27) Stool Not stated 11/11 (100) ND NDEnvironmental 0/16 (0) ND ND

Donta and Haddow, 1978 (14) Not stated Not stated 0/23 (0) ND 19/23 (83)Cumberbatch et al., 1979 (11) Stool Diarrhea 4/6 (67) 0/6 (0) 32/40 (80)b

No diarrhea ND ND 9/22 (41)Burke et al., 1984 (5) Stool Diarrhea 115/149 (77)C ND ND

No diarrhea 17/48 (35) ND NDPitarangsi et al., 1982 (34) Stool Diarrhea 16/42 (38)* ND 30/42 (71)*

No diarrhea 8/28 (29) ND 18/28 (64)Burke et al., 1983 (7) Stool and water Not stated 331/686 (48) ND NDJanda et al., 1983 (22) Stool Diarrhea ND ND 12/12 (100)Turnbull et al., 1984 (42) Clinical and environmental Not stated 39/68 (57) ND NDAgger et al., 1985 and 1986 (1, 2) Stool Diarrhea ND ND 10/16 (62)Figura et al., 1986 (15) Stool Diarrhea 7/13 (54)* ND 11/13 (85)*

No diarrhea 4/13 (31) ND 5/13 (38)Chopra et al., 1986 (9) Clinical and environmental Varied 14/23 (61) 19/23 (83) NDMégraud, 1986 (29) Stool Diarrhea 3/44 (7) 0/44 (0) 11/44 (25)Notermans et al., 1986 (32) Stool Not stated ND ND 33/46 (72)

Water 35/62 (56)Kirov et al., 1986 (24) Stool Diarrhea 18/43 (42) ND ND

Water 19/62 (15) ND NDKindschuh et al. (this study) Stool and blood Diarrhea 21/37 (57)* 0/37 (0) 40/42 (95)*

No diarrhea 5/5 (100) 1/5 (20) 8/8 (100)Sepsis 6/6 (100) 2/6 (33) 9/9 (100)

a LT, Heat-labile; ST, heat stable; ND, not done; *, no significant difference from no diarrhea.b Significant difference from no diarrhea, 0.004.c Significant difference from no diarrhea, 0.001.

toxicity for the suckling mouse was not produced by any ofthe diarrheal isolates. In studies in which controls with nodiarrhea were enrolled in addition to patients with diarrhea,no uniformity emerged that could significantly distinguishenterotoxic from nonenterotoxic strains from patients withand without diarrhea (6, 9, 15, 34).

Cytotoxin is produced by the majority of human andenvironmental isolates of Aeromonas spp. (Table 5). Thishigh prevalence of cytotoxin production appears to begreater than that found in random sampling of isolates of thefamily Enterobacteriaceae (14). Studies comparingcytotoxin production from isolates of patients with andwithout diarrhea have not revealed a significant difference(15, 32, 34) except in one study (11). Our observationsextend current knowledge by showing no differences in thequantity of cytotoxin produced by isolates from childrenwith and without diarrhea. In addition, none of the isolatesproduced a toxin which could be neutralized by antibodyraised to Shiga toxin. Once this cytotoxin is purified andantiserum is produced, the degree of homogeneity amongcytotoxic Aeromonas strains can be determined, thiscytotoxin can be compared with cytotoxins produced byother organisms, and secretory antibody response in in-fected people can be ascertained.

Oral administration of cytotoxic A. hydrophila (109 CFU)fails to cause diarrhea in rhesus monkeys (34). A humanvolunteer study in which volunteers were challenged withdoses of cytotoxic Aeromonas spp. was performed at thismedical center. Cytotoxic strains produced mild to moderatediarrhea in only 2 of 57 human volunteers challenged withdoses ranging from 104 and 1010 CFU (31). Our findingstogether with these animal and human challenge studiesindicate that cytotoxic production does not appear to be a

factor in the pathogenesis of A. hydrophila-relatedgastroenteritis.

Three biochemical traits, i.e., hemolysis, LDC activity,and VP reaction, have been stated to be predictive ofcytotoxin and enterotoxin production by Aeromonas spp.(11, 24). It has been suggested that these easily performedbiochemical tests correlate with enteropathogenicity andwould be suitable for epidemiologic studies of Aeromonasspp. (5, 19). Although a significant correlation between thesebiochemical traits and toxin production has been shown (2,5, 11, 22, 34, 42), other studies failed to demonstrate anycorrelation between any biochemical property and the abilityof strains to produce enterotoxin (15, 27). Our study showedthat all strains were hemolytic and produced cytotoxin andthat no correlation existed between toxin production andLDC or VP reaction. Only 27 of 57 of the cytotoxic strains(47%) were LDC positive. This varied with the test andincubation time used, indicating that differences in methodsmay account for discrepancies among previous reports.Our study further revealed that cytotoxin production did

not correlate with the production of a heat-labile-like orheat-stable-like enterotoxin. Earlier investigators (11, 31)have reported virtually complete correlation of cytotoxicactivity with a positive rabbit ileal-loop assay, indicatingalmost universal production of heat-labile-enterotoxin-likeactivity by cytotoxic strains. The lack of enterotoxin pro-duction by the two noncytotoxic strains in our study sup-ports the results of earlier investigations, as does the lack ofproduction of heat-stable-enterotoxin-like activity by eithercytotoxic or noncytotoxic strains (11). Importantly,Chakraborty et al. (8) have demonstrated that theenterotoxic, cytotoxic, and hemolytic characteristics of A.hydrophila strains are determined by different genes locatedon three different chromosomal segments. Ultimately, thisobservation could explain the existence in different parts ofthe world of strains with different virulence properties.

Conclusions from this study are that the vast majority of

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920 KINDSCHUH ET AL.

A. hydrophila strains produce a cytotoxin and that thiscytotoxin is immunologically distinct from Shiga toxin. Weconclude that there is no correlation between the productionof cytotoxin and gastroenteritis. We found that neitherhemolytic activity nor the presence of LDC- or VP-positivetests are specific as biochemical markers for cytotoxinproduction. We also determined that the vast majority of A.hydrophila strains do not produce heat-stable-enterotoxin-like activity. Finally, although a heat-labile-enterotoxin-likeactivity is produced by the majority of cytotoxic strains,there appears to be no correlation between the amount ofcytotoxin and the production of heat-labile-enterotoxin-likeactivity. The significance of other virulence properties,including adherence and invasion, either alone or in combi-nation with toxin production, remains to be determined.

ACKNOWLEDGMENTS

We appreciate the technical assistance of Deborah Armes, CathyKnisley, and Anne Wright. John Nelson kindly provided severalAeromonas strains.

This work was supported by Public Health Service grant NIH-HD13021 from the National Institutes of Health.

LITERATURE CITED1. Agger, W. A. 1986. Diarrhea associated with Aeromonas hydro-

phila. Pediatr. Infect. Dis. 5(Suppl.):105-108.2. Agger, W. A., J. D. McCormick, and M. J. Gurwith. 1985.

Clinical and microbiological features of Aeromonas hydrophila-associated diarrhea. J. Clin. Microbiol. 21:909-913.

3. Atkinson, H. M., and T. J. Trust. 1980. Hemagglutinationproperties and adherence ability of Aeromonas hydrophila.Infect. Immun. 27:938-946.

4. Bulger, R. J., and J. C. Sherris. 1966. The clinical significance ofAeromonas hydrophila. Arch. Intern. Med. 118:562-564.

5. Burke, V., M. Cooper, J. Robinson, M. Gracey, M. Lesmana, P.Echeverria, and J. M. Janda. 1984. Hemagglutination patternsof Aeromonas spp. in relation to biotype and source. J. Clin.Microbiol. 19:39-43.

6. Burke, V., M. Gracey, J. Robinson, D. Peck, J. Beaman, and C.Bundell. 1983. The microbiology of childhood gastroenteritis:Aeromonas species and other infective agents. J. Infect. Dis.148:68-74.

7. Burke, V., J. Robinson, J. Beaman, M. Gracey, M. Lesmana, R.Rockhill, P. Echeverria, and J. M. Janda. 1983. Correlation ofenterotoxicity with biotype in Aeromonas spp. J. Clin. Micro-biol. 18:1196-1200.

8. Chakraborty, T., M. A. Montenegro, S. C. Sanyal, R. Helmuth,E. Bulling, and K. N. Timmis. 1984. Cloning of enterotoxin genefrom Aeromonas hydrophila provides conclusive evidence ofproduction of a cytotonic enterotoxin. Infect. Immun.46:435-441.

9. Chopra, A. K., C. W. Houston, C. T. Genaux, J. D. Dixon, andA. Kurosky. 1986. Evidence for production of an enterotoxinand cholera toxin cross-reactive factor by Aeromonas hydro-phila. J. Clin. Microbiol. 24:661-664.

10. Cleary, T. G., and L. K. Pickering. 1986. Update of infectiousdiarrhea, p. 117-143. In S. C. Aronoff (ed.), Advances inpediatric infectious diseases. Year Book Medical Publishers,Inc., Chicago.

11. Cumberbatch, N., M. J. Gurwith, C. Langston, R. B. Sack, andJ. L. Brunton. 1979. Cytotoxic enterotoxin produced by Aero-monas hydrophila: relationship of toxigenic isolates to diarrhealdisease. Infect. Immun. 23:829-837.

12. Davis, W. A., II, J. G. Kane, and V. F. Garagusi. 1978. HumanAeromonas infections: a review of the literature and a casereport of endocarditis. Medicine (Baltimore) 57:267-277.

13. Deepe, G. S., and J. D. Coonrod. 1980. Fulminant woundinfection with Aeromonas hydrophila. South. Med. J.73:1546-1547.

14. Donta, S. T., and A. D. Haddow. 1978. Cytotoxic activity ofAeromonas hydrophila. Infect. Immun. 21:989-993.

15. Figura, N., L. Marri, S. Verdiani, C. Ceccherini, and A. Barberi.1986. Prevalence, species differentiation, and toxigenicity ofAeromonas strains in cases of childhood gastroenteritis and incontrols. J. Clin. Microbiol. 23:595-599.

16. George, W. L., M. M. Nakata, J. Thompson, and M. L. White.1985. Aeromonas-related diarrhea in adults. Arch. Intern. Med.145:2207-2211.

17. Giannella, R. A. 1976. Suckling mouse model for detection ofheat-stable Escherichia coli enterotoxin: characteristics of themodel. Infect. Immun. 14:95-99.

18. Goodwin, C. S., W. E. S. Harper, J. K. Steward, M. Gracey, V.Burke, and J. Robinson. 1983. Enterotoxigenic Aeromonashydrophila and diarrhoea in adults. Med. J. Aust. 1:25-26.

19. Gracey, M., V. Burke, and J. Robinson. 1982. Aeromonas-associated gastroenteritis. Lancet i:1304-1306.

20. Harris, R. L., V. Fainstein, L. Elting, R. L. Hopfer, and G. P.Bodey. 1985. Bacteremia caused by Aeromonas species inhospitalized cancer patients. Rev. Infect. Dis. 7:314-320.

21. Honda, T., M. Shimizu, Y. Takeda, and T. Miwatani. 1976.Isolation of a factor causing morphological changes of Chinesehamster ovary cells from the culture filtrate of Vibrioparahaemolyticus. Infect. Immun. 14:1028-1033.

22. Janda, J. M., E. J. Bottone, C. V. Skinner, and D. Calcaterra.1983. Phenotypic markers associated with gastrointestinal Aero-monas hydrophila isolates from symptomatic children. J. Clin.Microbiol. 17:588-591.

23. Kipperman, H., M. Ephros, M. Lambdin, and K. White-Rogers.1984. Aeromonas hydrophila: a treatable cause of diarrhea.Pediatrics 73:253-254.

24. Kirov, S. M., B. Rees, R. C. Wellock, J. M. Goldsmid, and A. D.Van Galen. 1986. Virulence characteristics of Aeromonas spp.in relation to source and biotype. J. Clin. Microbiol. 24:827-834.

25. Lawson, M. A., V. Burke, and B. J. Chang. 1985. Invasion ofHEp-2 cells by fecal isolates of Aeromonas hydrophila. Infect.Immun. 47:680-683.

26. Levett, P. N., and R. R. Daniel. 1981. Adhesion of vibrios andaeromonads to isolated rabbit brush borders. J. Gen. Microbiol.125:167-172.

27. Ljungh, A., M. Popoff, and T. Wadstrom. 1977. Aeromonashydrophila in acute diarrheal disease: detection of enterotoxinand biotyping of strains. J. Clin. Microbiol. 6:96-100.

28. McCracken, A. W., and R. Barkley. 1972. Isolation of Aeromo-nas species from clinical sources. J. Clin. Pathol. 25:970-975.

29. Mégraud, F. 1986. Incidence and virulence of Aeromonasspecies in feces of children with diarrhea. Eur. J. Clin. Micro-biol. 5:311-316.

30. Millership, S. E., S. R. Curnow, and B. Chattopadhyay. 1983.Faecal carriage rate of Aeromonas hydrophila. J. Clin. Pathol.36:920-923.

31. Morgan, D. R., P. C. Johnson, H. L. DuPont, T. K. Satterwhite,and L. V. Wood. 1985. Lack of correlation between knownvirulence properties ofAeromonas hydrophila and enteropatho-genicity for humans. Infect. Immun. 50:62-65.

32. Notermans, S., A. Havelaar, W. Jansen, S. Kozaki, and P.Guinée. 1986. Production of "Asao toxin" by Aeromonasstrains isolated from feces and drinking water. J. Clin. Micro-biol. 23:1140-1142.

33. Phillips, J. A., H. E. Bernhardt, and S. G. Rosenthal. 1974.Aeromonas hydrophila infections. Pediatrics 53:110-112.

34. Pitarangsi, C., P. Echeverria, R. Whitmire, C. Tirapat, S.Formal, G. J. Dammin, and M. Tingtalapong. 1982.Enteropathogenicity of Aeromonas hydrophila and Plesi-omonas shigelloides: prevalence among individuals with andwithout diarrhea in Thailand. Infect. Immun. 35:666-673.

35. Prado, D., T. G. Cleary, L. K. Pickering, C. D. Ericsson, A. V.Bartlett III, H. L. DuPont, and P. C. Johnson. 1986. Therelationship between production of cytotoxin and clinical fea-tures in shigellosis. J. Infect. Dis. 154:149-155.

36. Qadri, S. M. H., L. P. Gordon, R. D. Wende, and R. P.Williams. 1976. Meningitis due to Aeromonas hydrophila. J.Clin. Microbiol. 3:102-104.

J. CLIN. MICROBIOL.

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TOXIN PRODUCTION BY AEROMONAS HYDROPHILA 921

37. Reines, H. D., and F. V. Cook. 1981. Pneumonia and bacteremiadue to Aeromonas hydrophila. Chest 80:264-267.

38. Siito, R., and S. Schick. 1973. Aeromonas hydrophila peritonitis.Cancer Chemother. Rep. 57:489-491.

39. Sirinavin, S., S. Likitnukul, and S. Lolekha. 1984. Aeromonassepticemia in infants and children. Pediatr. Infect. Dis.3:122-125.

40. Smith, J. A. 1980. Aeromonas hydrophila: analysis of 11 cases.Can. Med. Assoc. J. 122:1270-1272.

41. Svennerholm, A. M., and J. Holmgren. 1978. Identification ofEscherichia coli heat-labile enterotoxin by means of a ganglio-side immunosorbent assay (GM1 ELISA) procedure. Curr.Microbiol. 1:19-23.

42. Turnbull, P. C. B., J. V. Lee, M. D. Miliotis, S. Van de Walle,

H. J. Koornhof, L. Jeffery, and T. N. Bryant. 1984. Enterotoxinproduction in relation to taxonomic grouping and source ofisolation of Aeromonas species. J. Clin. Microbiol. 19:175-180.

43. Wadstrom, T., A. Ljungh, and B. Wretlind. 1976. Enterotoxinhaemolysin and cytotoxic protein in Aeromonas hydrophilafrom human infections. Acta Pathol. Microbiol. Scand. Sect. B84:112-114.

44. Watson, I. M., J. O. Robinson, V. Burke, and M. Gracey. 1985.Invasiveness of Aeromonas spp. in relation to biotype, viru-lence factors, and clinical features. J. Clin. Microbiol. 22:48-51.

45. Weinstock, R. E., S. J. Bass, E. Lauf, and B. A. Sorkin. 1982.Aeromonas hydrophila: a rare and potentially life-threateningpathogen to humans. J. Foot Surg. 21:45-53.

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