Isolation, Enumeration, andCharacterization ofAeromonas - NCBI

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1980, p. 1010-1018 0099-2240/80/05-1010/09$02.00/0

Vol. 39, No. 5

Isolation, Enumeration, and Characterization of Aeromonas from Polluted Waters Encountered in Diving Operations RAMON J.

SEIDLER,'t

D. A.

ALLEN,' H. LOCKMAN,' R. R. COLWELL,' S. W. JOSEPH,2 AND 0. P.

DAILY2

Department of Microbiology, University of Maryland, College Park, Maryland 20742,' and Department of Microbiology, Naval Medical Research Institute, Bethesda, Maryland 200142

Counts of total viable, aerobic, heterotrophic bacteria, indicator organisms, and Aeromonas spp. were made at a diver training site on the Anacostia River in Washington, D.C. The numbers of Aeromonas cells in Anacostia River sediment and water increased during periods of elevated water temperature, to maxima of 4 x 105 cells per g of sediment and 300 cells per ml of water. Correspondingly, Aeromonas counts dropped 2 to 4 logs as the water temperature decreased to 0 to 0.5°C. Cultures taken by sterile swabs from the ears and face masks of divers after a 30-min swim in the Anacostia River yielded bacterial types and numbers similar to those found in the river. The nasal passages of the divers apparently did not become contaminated by swimming, possibly because of the protective effect of the face masks used by the divers. Properties associated with virulence in Aeromonas hydrophila and Aeromonas sobria strains isolated from the river, sediment, and divers were investigated. Nearly 40% of the strains of both species collected during the study produced cytotoxic activity for mouse Y-1 adrenal cells, as well as elastase. Enterotoxin activity, as detected by the Y-1 assay, was observed in 3% (1 of 35) of the strains of A. sobria and in 6% (19 of 330) of the A. hydrophila strains. Fluid accumulation in rabbit ileal loops induced by both species of Aeromonas varied greatly among the 17 strains examined. Fluid accumulation of at least 0.4 ml/cm was correlated with positive cytotoxin- or enterotoxin-like response in the Y-1 tissue culture assay.

It is clear that waste discharges into aquatic environments can have harmful effects on human health. During the past 10 years, a variety of research programs have been established to study the effects of pollution in freshwater, estuarine, and deep ocean environments (15, 19, 20). A hitherto overlooked effect of pollution is the health risk to aquatic sportsmen, as well as military and commercial diving personnel who must work in water contaminated by microorganisms potentially pathogenic for humans. It has been known for many years that Aeromonas species can cause infections in fish and reptiles, as well as in warm-blooded animals. These organisms are ubiquitous in aquatic environments and in warmer climates and warm water conditions can contribute to epizootics in fish (4, 11, 15). The symptomatology of a variety of human Aeromonas infections is becoming increasingly clear (2, 7, 8, 10-14, 21, 26, 29). Once believed to be an opportunistic pathogen of low virulence, Aeromonas is now recognized as a primary pathogen (8, 13, 21). Compromised hosts may experience serious and even fatal t On sabbatical leave from Oregon State University, Cor-

vallis, OR 97331.

infections (8). Clinical strains from various infections elaborate a variety of extracellular virulence factors, including hemolysin, cytotoxic enzymes, and enterotoxin (2, 3, 9, 22). Human gastrointestinal and soft tissue Aeromonas infections are often a result of waterrelated traumas and/or consumption of contaminated water supplies (7, 8, 13, 26). Human infections associated with diving activities in polluted waters have been described (12, 14, 26), and indeed, during the study reported here a diver on a training exercise at the sampling station sustained a leg wound infection caused by two Aeromonas species (17). This study documents the occurrence and seasonal fluctuations of Aeromonas in the Anacostia River in Washington, D.C., where increased numbers of cases of gastroenteritis and wound infections have occurred in personnel undergoing diver training operations throughout the year. Results of the work reported here establish that a significant number of the Aeromonas isolates in the microbial population of a polluted environment demonstrate multiple resistance to antibiotics, are capable of colonizing humans, and produce cytotoxic and enterotoxic substances. Thus, new information concerning the

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VOL. 39, 1980

potential infectious disease risk of Aeromonas species in aquatic environments is provided. MATERIALS AND METHODS Measurement of physical and chemical parameters. Dissolved oxygen and temperature were recorded at stations located in the Anacostia River with a YSI 51B oxygen meter (Yellow Springs Instrument Co., Inc., Yellow Springs, Ohio). Temperature and salinity at the New York Bight stations were recorded by using an Interocean vertical probe aboard the R/V George B. Kelez. Collection of samples. Samples for study were collected over a 10-month period during 1978 and 1979. Samples from the New York Bight, an area of high salinity (27 to 32%o), were collected at three stations; these were near a sewage dump site (longitude, 400 25.4' N; latitude, 730 48.0' W), at a dredge spoils site (longitude, 400 23.4' N; latitude, 730 51.9' W), and at a location northwest of the other two stations near the mouth of New York harbor (longitude, 400 29.4' N; latitude, 730 59.2' W). Samples from the Anacostia River, a freshwater site of low salinity (0 to 2%o), were collected at the U.S. Naval Yard, Washington, D.C. Water samples were collected at three depths at each station. A surface water sample was collected by using a sterile carboy held 2 to 8 cm below the surface. Samples from the top of the water column (ca. 1 m below the surface) and at the bottom of the water column (ca. 1 m above the bottom) were collected with a Niskin sampler (General Oceanics, Inc., Miami, Fla.). Sediment samples were collected by using a Petite Ponar grab sampler (19). Bacterial enumeration. At the Anacostia River station, total viable, aerobic, heterotrophic counts on plate count agar (Difco) were obtained by the spread plate technique, total viable counts of samples collected at the New York Bight were obtained on marine salt yeast extract agar (30). All plates were incubated at 25°C after inoculation, and colonies were counted at 7 days. Sediment samples were diluted with phosphate-buffered saline (7.2 g of NaCl per liter, 1.48 g of Na2HPO4 per liter, 0.43 g of K2HPO4 per liter, pH 7.2) before the treatment described above for water samples. Direct microscopic counts were made by epifluorescent microscopy (16). The total and fecal coliforms in the Anacostia River were enumerated by membrane filtration, using media and methods described in detail elsewhere (1). The total and fecal coliforms in the samples collected in the New York Bight were enumerated by using the three-tube, most-probable number method (1). New York Bight water samples of known volume were concentrated by filtration through nitrocellulose filters (pore size, 0.45 pm; Gelman). Triplicate samples (300, 30, and 3 ml) were filtered, and the filters were placed in tubes of lactose broth. Positive tubes (i.e., those tubes showing acid and gas production after incubation for 48 h at 35°C) were processed through the completed coliform test, inoculated into EC broth at 44.5°C, and incubated for 24 h for the detection of fecal coliforms. Sediment samples (10, 1.0, 0.1, and 0.01 g of each sample) were examined for total and fecal coliforms. The 10-g samples were pipetted into

AEROMONAS IN POLLUTED WATERS

1011

10-ml volumes of double-strength lactose broth. Diluted sediment suspensions, which were prepared in phosphate-buffered saline, were processed as described above for water samples. Presumptive Aeromonas species were enumerated by pour plating samples in duplicate on PBG agar (23) for those samples for which counts were expected to exceed 10 cells per ml, and, if necessary, samples were diluted with phosphate-buffered saline. After incubation for 24 h at 37°C, 100 to 200 presumptive Aeromonas colonies appearing on PBG agar were transferred to Aeromonas screening medium (J. B. Kaper, R. J. Seidler, H. Lockman, and R. R. Colwell, submitted for publication). Strains yielding an alkaline surface and acid butt and demonstrating motility were transferred from the screening medium to gelatin agar (32). Oxidase-positive, gelatin-hydrolyzing strains growing in the absence of added sodium chloride were considered to be confirmed Aeromonas spp. Representative strains of confirmed Aeromonas spp. were further tested by using the API 20E identification system (Analytab Products, Inc., Plainview, N.Y.); the results of this testing permitted identification of Aeromonas hydrophila. For those samples yielding Aeromonas counts of fewer than 10 cells per ml, enumeration was accomplished by a most-probable number procedure in which a modified Rimler-Shotts medium was used for initial enrichment (31). The broth medium did not contain L-lysine hydrochloride, L-ornithine hydrochloride, sodium thiosulfate, or agar. Appropriate volumes of water and diluted sediment samples were filtered by using membrane filters (pore size, 0.45 pm; Millipore Corp., Bedford, Mass.), which were placed directly into tubes of modified Rimler-Shotts medium after filtration was completed. The RimlerShotts broth tubes containing the filters were incubated for 24 h at 37°C. Inoculated tubes showing an acid reaction at 24 h were streaked onto MacConkey agar plates and incubated at 37°C for 24 h. Approximately 5 to 10 lactose-negative colonies were picked from each plate; these colonies were transferred to gelatin agar and subsequently to Aeromonas screening medium, as described above. The species A. hydrophila and Aeromonas sobria were identified by using the scheme of Popoff and Veron (25) and tests which have been described previously (17). A. sobria is a new species described after publication of Bergey's Manual of Determinative Bacteriology, 8th ed. (6). Salmonella spp. were isolated by using a threetube, most-probable number, primary, nonselective enrichment involving dulcitol broth (20). Water samples (300, 30, and 3 ml) were concentrated by filtration through 0.45-pum membrane filters. The filters were placed in tubes containing 10 ml of dulcitol broth. Sediment samples (30, 3, and 0.3 g) were processed similarly; the 30-g sample was transferred to 100 ml of broth. All samples were incubated for 24 h at 37°C. Broth tubes showing growth were transferred to selenite cysteine broth (Difco), XLD agar (Difco), and Kligler iron agar (Difco). Salmonella isolates were confirmed with the API 20E system (Analytab Products, Inc.). Vibrio cholerae and Vibrio parahaemolyticus were isolated by using alkaline peptone and ethyl violet broth enrichments, respectively (19). Confirmation of

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APPL. ENVIRON. MICROBIOL.

SEIDLER ET AL.

the isolation of these species was by the API 20E system. Virulence assays. Aeromonas strains were propagated in 10 ml of tryptic soy broth incubated statically at 30°C. Tests for cytotoxin and enterotoxin were performed with culture supernatants added to Y-1 adrenal cells, as previously described (17, 19, 28). A reaction was read as enterotoxic if the morphological appearance after exposure to a supernatant heated at 560C for 10 min resembled that observed with cholera enterotoxin. Cytotoxic activity was revealed by rounding, shrinking, granule formation, and vacuolization of the cells. Rabbit ileal loop tests. Aeromonas strains were grown for 24 h at 37°C on brain heart infusion agar (Difco). Cells were harvested and suspended in 5 ml of brain heart infusion broth, and 1 ml of the suspension, containing 108 to 109 cells, was inoculated into each loop, as previously described (17,19). Rabbits weighing 1 kg were used and sacrificed 8 h after loop inoculation. Statistical analyses. Physical, chemical, and microbiological data were entered in an IBM 370 computer, and multiple linear correlation coefficients were computed by using statistical program packages (19). Antibiotic susceptibility assays. Antibiotic susceptibilities were determined by the disk diffusion technique, as previously described (17). Sampling of divers. Diver training at the sampling station included a timed 1,500-yard (ca. 1,375-tn) surface swim as a measure of physical fitness and swimming ability. Two classes of trainees were sampled for the presence of Aeromonas spp. and other bacteria in their noses, ears, and throats and on their masks before and after a timed surface swim. This permitted determinations of the degree to which exposure to known concentrations of bacteria resulted in transient and/or long-term colonizations. Sterile swabs were used to take samples before and after the timed swim. The swabs were placed in 2 ml of sterile normal saline and transported on ice to our laboratory immediately after collection of the samples. Samples (1 and 0.1 ml) of the saline solutions were placed in petri dishes, and 15 ml of PBG agar was added to each dish. After incubation for 24 h at 37°C, glycogen-fermenting colonies were picked and identified.

RESULTS The Anacostia River sampling site is located in a tidal basin at the U.S. Naval Yard, Washington, D.C. The depth of water at the sampling station in the Anacostia River is ca. 20 feet (6.1 m), and the width of the river in the general area of the study is ca. 1,000 feet (305 m). Throughout the year, the water in the river is turbid (i.e., Secchi disk transparency of 0.34 to 0.17 m), and the temperature of the water fluctuates widely on a seasonal basis, with a range of 0.5 to 28°C. Microbiological enumeration data for water and sediment samples are summarized in Fig. 1 and 2. Aeromonas spp. and total coliforms exhibited similar cell densities and similar fluctuations throughout the sampling period. The number of

104 EPIFLUORESCENT

COUNT (I 103)

103_

-i

fr12 -J

-J

w

V

i lo, a.

0

FCLCLIFORMS

JUL AUG SEPT

OCT NOV DEC JAN FES MAR APR MAY

JM

MONTH

FIG. 1. Total viable counts, total and fecal coliforms, and number of Aeromonas cells per milliliter of Anacostia River top water.

106

EPIFLUORESCENT

COUNT(:x103) 105 -AeM

\

TOTAL VIABLEE

COUNT lx 102)

\:/

z w w

CD SE w 0. -j -j

w

JUL AUG SEPT OCT NOV

DEC JAN

FEB MAR AR MAY JUN

MONTH

FIG. 2. Total viable counts, total and fecal coliforms, and number of Aeromonas cells per gram of

Anacostia River sediment.

AEROMONAS IN POLLUTED WATERS

VOL. 39, 1980

Aeromonas spp. in the top water samples reached 300 cells per ml in August and declined to 10 cells per ml in February. In May, when the water temperature was 22.50C, the Aeromonas counts began to rise. Fecal coliform counts in the Anacostia River changed very little throughout the study, whereas total viable counts increased fivefold for samples collected in February compared with the samples collected in August. The temperature of the water was 27 to 280C during the summer; this decreased to 10°C in November and 0.50C in February, at which time there was a 0.5-cm-thick cover of ice on the water. Total and fecal coliforms and numbers of Aeromonas cells in the sediment exhibited dramatic changes as the water temperature dropped; maximum numbers were observed in August, when counts of Aeromonas spp. exceeded 105 cells per g of sediment. The numbers of Aeromonas and indicator species declined significantly; there was a 103- to 104-fold decrease from August 1978 to February 1979. In contrast, the total viable counts showed little or no detectable change, remaining at ca. 105 to 106 cells per g of sediment. The ratio of Aeromonas to total coliforms to fecal coliforms changed significantly with decreasing water temperature. During July and August, water samples yielded relative propo;'tions averaging 100:75:5, respectively, wheteas in November the ratio was 100: 100:2 and in February it was 100:400:30. In May the ratio was intermediate between the ratios recorded for November and February (100:120: 16). The strongest correlations among the study parameters were recorded for the bottom water and sediment data. The correlation coefficient between number of Aeromonas cells and temperature was ca. 0.66 and, very likely, was not higher because of the lag in the temperatureAeromonas response observed during the July to November sampling period (Fig. 1 and 2).

TABLE

1013

Aeromonas counts increased from 100 to 300 cells per ml during July and August, even though the temperature of the river water remained at 27 to 280C. The cyclic nature of the Aeromonas counts was most dramatic for the November, February, and May samples, when water temperatures ranging from 0.5 to 22.50C were recorded. The correlation value for bottom water counts of Aeromonas cells and temperature determined for samples collected during August through May was 0.91. Aeromonas counts for bottom water and sediment yielded significant correlations with fecal coliforms (0.90) and total coliforms (0.95 to 0.96); i.e., they were significant at the 1% level (df = n - 2 = 4; r = 0.92). Interestingly, Aeromonas counts for bottom water and sediment exhibited an inverse correlation with concentration of dissolved oxygen (r = -0.63 to -0.68). Large numbers of opportunistic pathogens were recovered from the ears and diving masks of nearly all of the 15 divers after the timed swim (Table 1) when the divers were sampled 60 to 80 min after leaving the water. The noses of all divers except diver 12 appeared to be partially protected from contamination by the face masks. Throat swabs of three divers (divers 13, 14, and 15) yielded significant numbers of organisms, but these divers yielded positive cultures before the swim. More than 70% of the colonies picked from PBG agar and originating from divers or their masks were identified as Enterobacter and Klebsiella spp. The other cultures included Aeromonas spp. (21%) and Escherichia coli (6%). The incidence of Aeromonas was monitored for two separate diving groups, one group diving in August and the other diving in October. Aeromonas spp. were recovered from the ears of more than 90% of the divers of both groups, but were less frequently recovered from the noses and throats of divers participating in the October swim. In October the water was colder, and

appearing on PBG agar after inoculation with samples collected from divers and their diving equipment at the Anacostia Rivera

1. Number of glycogen-fernenting colonies

Avg counts

1-11

12 13 14 15

After 30-min swim

Before swim

Diver Mask

Ear

Nose

Throat

Mask

Ear

Nose

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0.4 0 25 100 20

45 36 30 100 100

136 200 100 1 100

2 100 0 0 0

a Isolates were identified as Enterobacter spp. (55%), Aeromonas spp. (21%), coli (6%).

Throat 5 2 100 100 30

KlebsielUa spp. (18%), and E.

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APPL. ENVIRON. MICROBIOL.

SEIDLER ET AL.

numbers of Aeromonas were found in adrenal cell assays for cytotoxin and enterotoxin. the water (Table 2). Approximately 39% (123 of 330) of the A. hydroTwo strains of non-01 V. cholerae were iso- phila strains and 37% (13 of 35) of the A. sobria lated from 250-ml samples of top and bottom strains produced cytotoxin, as judged by cell water collected on 9 August 1978. These two rounding, shrinking, and granule formation. strains corresponded to the Heiburg group V When culture supernatants heated for 10 min at phenotype (i.e., they were Voges-Proskauer and 56°C were tested, 6% (20 of 330) of the A. hydrophila strains and 3% (1 of 35) of the A. sobria sucrose negative). Salmonella spp. were also isolated from sediment and water samples col- strains induced cell rounding without shrinking lected in the Anacostia River at the diving school and peripheral granule formation. Such changes study site during July, August, and May. V. were conditionally recorded as resulting from cholerae was not isolated from samples collected enterotoxin activity (21, 22). A total of 17 Aeromonas strains were examin the New York Bight, but Salmonella and Aeromonas spp. were readily isolated from these ined for fluid accumulation response in the rabseawater samples. Aeromonas was present at bit ileal loop test (Table 4); positive control concentrations of approximately 1 to 10 cells per responses were recorded for accumulations of liter of water in the Bight. fluid ranging from 0.6 to 0.8 ml/cm. Sterile culThe biochemical properties of 48 strains of ture fluid, which served as a control for the Aeromonas isolated from Anacostia River water experiments, promoted no detectable fluid acand sediment and from divers after a training cumulation. Five of the nine A. sobria strains swim in the Anacostia River were determined. tested induced accumulations of ca. 0.9 to 1.1 Although the Aeromonas strains were identified ml/cm. Each of the A. sobria culture supernatants containing a detectable enterotoxin or cyas A. hydrophila with the API 20E test system, different responses were exhibited among the totoxin in the Y-1 assay also induced fluid acstrains for the Voges-Proskauer test, lysine de- cumulation in the rabbit ileal loop test. Positive cytotoxin- or enterotoxin-induced Ycarboxylase, citrate utilization, and fermentation of rhamnose, sucrose, amygdalin, and arabinose. 1 adrenal cell responses were noted for the four Biochemical reactions were recorded for 365 strains of A. hydrophila which stimulated the Aeromonas strains. Biotyping distinguished the largest amounts of fluid accumulation in rabbit species A. hydrophila and A. sobria. Of all Aero- loops (Table 4). Interestingly, three strains (2A1, monas strains recovered from the Anacostia F12, and HADSc) induced no morphological River water and sediment and the divers after responses in tissue culture and elicited little or their timed swim, 84 to 85% were A. hydrophila. no fluid accumulation in the rabbit loop test. In comparison, 33 of 34 Aeromonas strains (97%) Also, strain 9 did not produce detectable toxins obtained from the New York Bight were iden- in either system. The latter strain was elastase tified as A. hydrophila. negative and was found to be A. hydrophila During the summer sampling period, 190 biovar X2 (23), since it is Voges-Proskauer negAeromonas strains were collected from Anacos- ative and does not produce gas from glucose. tia River water and sediment, and each strain was tested for resistance to seven antibiotics. DISCUSSION Most of the strains (183 of 190) were resistant to ampicillin, 49 were resistant to erythromycin, 17 From a search of the primary literature, we were resistant to tetracycline, 3 were resistant to concluded that this is the first report to docunalidixic acid, and 1 was resistant to kanamycin. ment the seasonal distribution of Aeromonas None of the isolates was resistant to chloram- spp. in water and sediment at a location where phenicol or gentamicin. a human wound infection caused by Aeromonas Table 3 shows the results of the Y-1 mouse was sustained (17). In addition to enumeration

smaller

TABLE 2. Incidence of Aeromonas spp. recovered from divers and their diving equipment before and after they swam in the Anacostia River No. of No. positive/no. tested Month

Aeromonas cells per

Water temp

Before swim

After swim

(oC)b

Mask mWa Ear Nose Throat August 300 28 0/15 0/15 0/15 3/15 October 50 13 0/10 0/10 0/10 0/10 in Anacostia River water at time of sampling. aCount b Temperature of Anacostia River water at time of sampling.

Mask

Ear

Nose

Throat

13/15 2/10

14/15 9/10

1/15 0/10

4/15 2/10

VOL. 39, 1980

AEROMONAS IN POLLUTED WATERS

1015

termination of A. hydrophila numbers, a conclusion not consistent with the results of the study reported here (i.e., Aeromonas counts were low, but still detectable by our enumeration technique). % Showing positive response No. of In another recent study involving a nationstrains Species wide survey, the number ofAeromonas cells was Cytotoxin Enterotested not correlated with temperature, pH, turbidity, toxin or salinity, but a correlation with conductivity 39 (128)b 6 (19) 330 A. hydrophila was reported (15). In the nationwide survey, 3 (1) 37 (13) 35 A. sobria each of the many stations that were sampled a A cytotoxic response was scored when the tissue possessed a unique set of physical, chemical, and culture cells exhibited shrinking, rounding, and gran- biological parameters, as well as demonstrating ulation. An enterotoxic response was scored when seasonal diversity. Thus, the microbial communheated (560C for 10 min) culture supernatants caused ities at each station could not be identical. cell rounding without granule formation (18, 19, 21, Therefore, it is not surprising that few correla22). b Numbers in parentheses indicate the numbers of tions were detected for Aeromonas and the measured parameters when only a single sample strains exhibiting a positive response. from each station was collected for analysis. Results from the study reported here, in which of Aeromonas spp., microbiological and physi- cultures were collected by swabbing divers becochemical parameters were also monitored. fore and after they swam in the Anacostia River, Total coliform and Aeromonas populations of illustrated dramatically a quantifiable effect of the top and bottom water and sediment ex- exposure to polluted water when the skin microhibited seasonal fluctuations; counts rose during flora of the swimmers was analyzed. A 30-min periods when the water temperature was ele- exposure to river water containing as few as 50 vated and reached a maximum when the tem- to 300 Aeromonas cells per ml, 200 total coliperature of the water exceeded 200C. The counts decreased significantly in February, when the TABLE 4. Rabbit ileal loop fluid accumulation and temperature of the water dropped to 0.50C. FeY-l tissue culture response to A. sobria and A. hydrophila cal coliform counts for top water samples also exhibited some seasonal fluctuation, but the Rabbit ileal changes were smaller (from 400 cells per 100 ml Y-1 reloop fluid ac- sponse' Strain cumulation to 1,000 cells per 100 ml). In contrast, the Aero(nl/cm) monas and total coliform counts ranged from 2,000 cells per 100 ml to 20,000 cells per 100 ml. A. sobria C 1.10 A3 Fecal coliform populations in the sediment flucC 1.10 3A36 tuated nearly 1,000-fold over the study period. C 1.00 I2b It should be pointed out that the minimum C 0.90 G9 temperature for growth of Aeromonas under E 0.35 Ila controlled laboratory conditions is reported to ob 0 D8 ob range from 5 to 150C, depending on the strain 0 G22 (27), whereas that of total and fecal coliforms is 0 0 K6f C 0.90 HA Hosp.c generally lower, ranging from 5 to 100C (6, 24). Thus, it is not surprising that counts were lower A. hydrophila C 0.93 BAll in the natural environment when water temperE 0.74 E10 atures below 200C were recorded. C 0.67 D9 The relationship between water temperature E 0.55 G29 and incidence of Aeromonas and other indica0 0.33 9 tors observed in this study is similar to that 0 0.29 2A2 reported by Fliermans et al. (11) and consistent 0 0.20 F12 E 0 with the results of a recent study reported by HADScc Rippey and Cabelli (S. R. Rippey and V. J. Responses in the Y-1 culture assay: C, cytotoxin; Cabelli, Abstr. Annu. Meet. Am. Soc. Microbiol. E, enterotoxin; 0, no response. bA 1979, p. 179), who found that the concentrations small ml/cm) amount of blood accumu(