ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1980, p. 477-483 Vol. 17, No.

3
0066-4804/80/03-0477/07$02.00/0

Isolation of Drug-Resistant Aeromonas hydrophila from

Aquatic Environments
LORE ANNE McNICOL,' K. M. S. AZIZ,2 IMDADUL HUQ,2 JAMES B. KAPER,1 HANK A.
LOCKMAN,lt ELAINE F. REMMERS,' WILLIAM M. SPIRA,3 MARY J. VOLL,' AND RITA R.
COLWELL'*
Department ofMicrobiology, University ofMaryland, College Park, Maryland 207421; International Centre
for Diarrheal Disease Research, Bangladesh, Dacca-2, Bangladesh2; and Division of Geographic Medicine,
School ofMedicine, Johns Hopkins University, Baltimore, Maryland 212243

Antibiotic-resistant strains of Aeromonas hydrophila have been isolated, from

the natural environment in the Chesapeake Bay and areas surrounding Dacca
and the Matlab region of Bangladesh. The Bangladesh strains carried resistance
to chloramphenicol, streptomycin, and tetracycline, and 57% of them had a
multiple streptomycin-tetracycline resistance phenotype correlated with the pres-
ence of a large plasmid. The Chesapeake Bay strains were resistant to polymyxin
B and tetracycline, but showed neither multiple resistance nor R-factor carriage.
Twenty-five percent of the environmental strains were toxigenic in a Y-1 adrenal

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cell assay. Toxigenicity showed no positive correlation with drug resistance or
with plasmid carriage. Environmental areas of heavy human impact appear to be
associated with a higher incidence of antibiotic-resistant strains of aeromonads.

Aeromonas hydrophila is a gram-negative are resistant to chloramphenicol (and other

bacterium classified as a member of the family
Vibrionaceae whose normal habitat is soil and
water (33). Although normally a free-living spe-
cies, A. hydrophila has the unusual potential to
infect both cold- and warn-blooded organisms.
Thus, it causes devastating epidemics in fish,
reptile, and amphibian populations (37, 40) as
well as sporadic water-associated human disease
(37). Although A. hydrophila can be isolated
from a variety of infections, gastroenteritis is the
most frequent clinical syndrome seen in humans
(9), and aeromonads are reported to elaborate
an enterotoxin similar to choleragen (2). In the
past, A. hydrophila was considered an opportun-
istic pathogen, but recent surveys have empha-
sized its emergence as a primary pathogen, par-
ticularly in compromised hosts (9) or in wound
infections (14).
Human clinical isolates of this organism are
consistently reported to be susceptible to chlor-
amphenicol (4, 7, 9, 37, 38), although a few
strains from fish (3) and sewage (29) have been
reported to carry transmissible resistance to this
drug. As part of our studies on the ecology of
pathogenic Vibrionaceae (7, 21; J. Kaper, H.
Lockman, and R. R. Colwell, Abstr. Annu. Meet.
Am. Soc. Microbiol. 1979, N56, p. 188), we have
isolated strains of A. hydrophila from the natu-
ral environment. A number of these organisms
t Present address: Center for Coastal and Environmental
Studies Marine Science Laboratory, Rutgers University, New
Brunswick, NJ 08903.
477
drugs), and many contain plasmid deoxyribo-
nucleic acid (DNA) species. Thus, it appears
that drug resistance is emerging in environmen-
tal isolates of A. hydrophila at a tinme when
significant human infection with this organism
is also increasing.
MATERIALS AND METHODS
The vibrio (V) series organisms were isolated from
the Jones Falls (Baltimore Harbor), Colgate Creek,
Chester River, and Chesapeake Beach stations in the
Upper Chesapeake Bay. Water samples were plated
on thiosulfate-citrate-bile salts agar (Difco, Oxoid) by
the enrichment technique described by Kaper et al.
(21). Two of the 76 organisms isolated (2.6%) were
Aeromonas hydrophila. The cultures were maintained
on tryptic soy agar (Difco) slants under oil.
The Bangladesh enteric (BE) isolates were collected
from river wateri sediment, plankton, and water plants
in Dacca, Bangladesh, and in the Cholera Research
Laboratory rural study area at Matlab (southeast of
Dacca}. Bacteria were isolated on Mueller-Hinton agar
(Difco) plates containing either penicillin (20 ,g/ml),
chloramphenicol (10 ,g/ml), tetracycline (10 jig/ml),
or streptomycin (15 ,tg/ml). One isolate (of each colony
type) from the primary plate was tested for resistance
to each of the four antibiotics. Multiply resistant iso-
lates were examined for Gram stain morphology, and
gram-negative rods were stocked. A total of 295 orga-
nisms resistant to at least two of these antibiotics were
maintained on brain heart infusion (Difco) agar sup-
plemented with one antibiotic, and transported to the
University of Maryland for further identification. Of
the 203 viable cultures, 18 were A. hydrophila (8.9%).
The Bangladesh vibrio (BV) organisms were col-
lected from the same sites as the BE series. Samples
478 McNICOL ET AL.
were plated on thiosulfate-citrate-bile salts agar, pu-
rified, and stored on brain heart infusion agar slants
under oil. Three of the 87 strains (3%) were A. hydro-
phila.
The Chesapeake Bay A. hydrophila (CBAh) strains
were isolated from Bay water by alkaline-peptone
broth enrichment (21), followed by plating on Mac-
Conkey-lactose agar (Difco). The cultures were main-
tained on tryptic soy agar slants under oil.
The A. hydrophila (Ah) group was isolated from
Chesapeake Bay water by a modified membrane filter
technique (35). Thirteen of the 15 bacteria (87%) were
A. hydrophila, and these strains were maintained on
trypic soy agar slants. The A. hydrophila most prob-
able numbers in Bay water at the time of sampling
were as follows: Colgate Creek, 2.4 x 104/100 ml;
Chesapeake Beach, 3 x 103/100 ml; and Chester River,
1.5 x 102/100 ml.
A more detailed description of the strains is given
in Table 1. The BE organisms were collected from
November 1976 to April 1977 at Matlab (BE 10-206)
and Dacca (BE 300). Isolates BE 66, 112, 123, 140, and
ANTIMICROB. AGENTS CHEMOTHER.
113, and 117), Chesapeake Beach (CBAh 21, 26, 41, 42,
and 66), and Chester River (CBAh 77, 80, and 109).
The Ah organisms were collected in 1978 from water
samples at Chester River (Ah 1) and Jones Falls (Ah
2 to 16).
Organisms were identified as A. hydrophila on the
basis of a minimum plexus of characters common to
the major published taxonomic schemes (12, 34, 38):
gram-negative, fermentative, motile rods which are
positive for oxidase, 0/129 resistance, growth in 0%
NaCl, gelatinase, mannitol fermentation, and arginine
dehydrolase and negative for growth in 6% NaCl,
inositol fermentation, and ornithine decarboxylase. A
more extensive numerical taxonomy study of the aero-
monads from environmental sources is in progress
(H.A.L.) and will include the organisms discussed in
this paper. Overall DNA base composition (percent
guanine plus cytosine) was determined by the melting
profile method of Mandel et al. (25).
Antibiotic susceptibility testing was done by the
disk agar diffusion method (Sensi-Discs, BBL Micro-
biology Systems), and resistance was determined from

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155 were in water plant samples; BE 113 was in plank-
ton; BE 183 and 206 were in sediment; and the others
were in water. The BV bacteria were collected in the
spring of 1977 from water at Matlab (BV 11) and river
sediment at Dacca (BV 77 and 78). Both V strains
were isolated in 1977 from Jones Falls water. The
CBAh series was isolated in 1978 from water samples
at Colgate Creek (CBAh 1, 2, 103, 107, 110, 111, 112,
a standard zone size chart (4). Minimal inhibitory
concentrations were determined by the agar plate
dilution method (Sigma chloramphenicol lot 73C1250
and Wyeth ampicillin lot 2740656) with Escherichia
coli ATCC 25922 as the control (4).
Plasmid DNA was prepared by the cleared lysate
method (17, 28) and visualized by agarose gel electro-
phoresis on a Savant horizontal bed apparatus. Gels

TABLE 1. A. hydrophila strains examined in this study

Antibiotic resistance'
Straincbtoi'(0 Entero- Plasmid,d mol wt %G+Ce
G
C 30 (MIOb) E 15 PB 50 S 10 T 30 toxinc (xlO )
BE
10 S (15) S S S S + -
16 S (10) I I S S + -
24 S (10) I S S S + -
35 R (50) S S R S - -
40 S (1) S S S S + -
41 S (5) I S R R + -
45 S (1) S S R R - +,58
49 S (10) I S S S - +,65
66 S (10) R S R R - -
112 S (1) I S R R - +,66and37
113 S (1) I S R R - +,68
123 S (5) I S R R - +,64and46
140 S (10) I S R R - +,65 and 50
155 R (50) S S R R - +,2 58.5 ± 0.5
183 S (10) I I R R - +,65
194 S (5) R I R R - -
206 S (10) I I R R - +, 67 and 1
300 R (50) S S R R - - 57.9 + 0.5
BV
11 S (1) S NDf S S - -
77 S (0.5) I ND I S + -
78 S (0.5) I ND I S + -
V
16 S (ND) I I I S + -
22 S (ND) I I R S + -
VOL. 17, 1980 ANTIBIOTIC-RESISTANT A. HYDROPHILA 479

TABLE 1-continued.
Antibiotic resistance' Entero- Plasmid,d molwt
Strain
C 30 (MIOb) E 15 PB 50 S 10 T 30
Ento-
toiC
Pl1m6,
(x106)
mG+Ce

CBAh
1 S (0.5) S I S R + -
2 S (5) S S S S - -
21 S (5) I S I S - 5.1
26 S (5) I S S S - 5.5
41 S (1) I S S R - -
42 S (5) I S S R - -
66 S (1) S R I S - -
77 S (5) S S S S - -
80 S (10) S S S S - -
103 S (5) S S S R - -
107 S (1) S S S S + -
109 S (1) S S S R + -
110 S (5) I S S S - -
111 S (5) S S S R - -
112 S (5) I S S R - -
113 S (5) I S S S -+,7
117 S (5) S S S S - -

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Ah
1 S (5) S S S S - -
2 S (5) S S S S - - 61.0 0.5
3 S (5) I S S S - -
4 S (5) I S S S - -
5 S (5) I S S S - -
8 S (5) I S S S - -
9 S (5) S S S S - ND
10 S (5) I S S S - -
11 S (5) I S S S - -
12 S (5) I S S S - -
13 S (5) I S S S - -
15 S (5) I S S S + -
16 S (0.5) S S S S - -
aC, Chloramphenicol; E, erythromycin; PB, polymyxin B; S, streptomycin; T, tetracycline. Average of at
least two determinations. In addition to the data shown here, all of the strains were resistant (R) to ampicillin
(10 ug), with MICs greater than 20 ug/ml, and penicillin (10 ug) (the Aeromonas fI-lactamase appears to be
chromosomally mediated [34]), and all were susceptible (S) to gentamicin (10 ,ug), kanamycin (30 ug), and
nalidixic acid (30 ,ug).
b MIC, Minimal inhibitory concentration of chloramphenicol, average of at least three determinations.
C
Average of at least two determinations.
d All strains were extracted at least twice, and each DNA preparation was run on both 1.0 and 0.7% gels.
e%G+C, Percent guanine plus cytosine, average of two determinations.
f ND, Not determined.

were photographed with a Polaroid MP-4 camera on RESULTS

type 55 film with Wratten 9, 2B, and 23A filters, a J344
ultraviolet filter (Ultraviolet Products), and a Blak-
Ray long-wave transilluminator light source (Ultravi-
olet Products). Curing experiments were done accord-
ing to published procedures (1, 44). Molecular weights
were determined relative to standard plasmid species:
E. coli J53 RP4 (34 x 106), J53 Ri (68 x 106), and J53
S-a (25 x 106) (from E. M. Lederberg); and E. coli K-
12 P307 (62 x 106) and K12 H10407 (60 x 106, 40 x
106, and 4 x 106) (from S. Falkow).
Comparison of trait frequencies was done with the
chi-square test, corrected for continuity (43).
Enterotoxin production was tested in Y-1 adrenal
cells by a microculture technique (36). Vibrio cholerae
569B and E. coli H10407 were used as the positive
control; E. coli ATCC 25922 was the negative control.
We have isolated 53 strais of A. hydrophila
in the course of environmental studies on the
ecology of bacteria of the Vibrionaceae family.
As part of the taxonomic study of these orga-
nisms, antibiotic resistance patterns were deter-
mined by the Kirby-Bauer disk method and
mlnimum inhibitory concentrations were deter-
mined fornhiboramphenicotiand ampdcilli
(Tbed 1). In oad n hen ada mshown a int
(Table 1). In addition to the data shown m this
table, all of the organisms were resistant to
ampicillin (10 ,ug) and susceptible to gentamicin
(10,g), kanamycin (30 jg), and nalidixic acid (30
,ug) disks.
480 McNICOL ET AL. ANTIMICROB. AGENTS CHEMOTHER.

Table 2 summarizes the single antibiotic re- and the E. coli labile toxin (2, 11, 24). Therefore,
sistance frequencies for each isolate series and the toxigenicity of the environmental isolates
compares them to the frequencies reported in was determined (Tables 1 and 3). Overall, 13
the literature. Polymyxin B and erythromycin (25%) of the organisms produced a toxin re-
resistances are decreased, whereas resistances to sponse in Y-1 adrenal cells. A different study by
chloramphenicol, streptomycin, and tetracy- our laboratory of 117 CBAh isolates revealed
cline-widely used and clinically important anti- that 16% produced enterotoxin-like activity and
biotics-are markedly increased in our sample. 57% produced cytotoxin in the Y-1 adrenal cell
The BE strains were responsible for most of this assay (Kaper et al., Abstr. Annu. Meet. Am. Soc.
increased drug resistance (and they were se- Microbiol., 1979, N56, p. 188). The question of
lected for this phenotype). But the occurrence enterotoxigenicity in environmental A. hydro-
of tetracycline resistance was highly significant phila will be discussed more fully (Kaper, Lock-
(P < 0.001) in the CBAh organisms as well. The man, and Colwell, manuscript in preparation),
nonantibiotic selected BV series of Vibrionaceae but caution should be exercised in interpreting
strains collected for comparison with the BE set Y-1 cell reactions, since they have also been
contained only three Aeromonas isolates. These
organisms were not antibiotic resistant. TABLE 3. Multiple drug resistance in
Table 3 summarizes the multiple drug resist- environmental strains of A. hydrophilaa
ance phenotypes in our sample. It is clear that BEsen'es
V and
the BE series has a significant level of multiply Resistance
Resi to o (18 series
BE strains CBAh series
(19 stan

Downloaded from aac.asm.org by on May 17, 2010

resistant organisms (13 of 18), whereas the total) total)
CBAh sample carries only single resistance de-
terminants. Moreover, it is apparent that the One or more anti-
streptomycin-tetracycline pattem is the domi- biotics
nant phenotype, responsible for 12 of the 13 C, E, S, or T 13 (72)
PB, S, or T 9 (47)
multiply resistant strains.
The BE organisms seemed to share a high- Two or more anti- 13 (72) 0
molecular-weight (65 x 106) plasmid species that biotics
was associated with the streptomycin-tetracy- CS 3
chine phenotype (Fig. 1). We were able to find a CT 2
plasmid in only one of the three streptomycin- ES 2
chloramphenicol-resistant bacteria. This strain, ET 2
BE 155, readily lost chloramphenicol resistance ST 12
when treated with the plasmid curing agent so- Three or more 4 (22) 0
dium dodecyl sulfate (44). The Chesapeake aero- antibiotics
monad plasmids were truly cryptic and showed EST 2
no correlation with resistance or toxin pheno- CST 2
types. aData and abbreviations from Table 1.
A. hydrophila is capable of producing an en- b Numbers in parentheses are percentages of total
terotoxin related to the V. cholerae choleragen strains.

TABLE 2. Comparison of the frequency of individual drug resistance phenotypes demonstrated by A. hydro-
phila strains recovered from the natural environment and by clinical and environmental strains reported in
the literature
Orgamams No. of No. of strains resistanta
stram58 C E PB S T
BE 18 3 (17)* 2 (11)* 0* 13 (72)* 12 (67)*
BV, V, CBAh, Ah 35 0 0* 1 (3)* 1 (3) 7 (20)*
Literature
percentageb 222c 0 47 27 5 2
a Numbers in parentheses are percentages. The asterisks mark frequency differences which were significantly
different from the literature values at the 5% level. Abbreviations are as in Table 1.
b
Frequency data were gathered from papers in which adequate taxonomic identification and standard
methods for determining resistance were employed (5, 6, 9, 10, 13, 14-16, 19, 20, 23, 26, 27, 30-32, 39, 41, 45, 48-
50).
c One hundred twenty of these isolates were from environmental sources and 102 (46%) were clinical. The
resistance frequencies of the two groups were identical at the 98% level.
VOL. 17, 1980
reported to be due to a nonenterotoxigenic pro-
tein unrelated to adenyl cyclase stimulation (8,
11).
Choleragen is produced by a chromosomal
gene, whereas the labile toxin gene is generally
carried on a plasmid (47). Thus, it was of interest
to determine whether toxigenicity, as well as
drug resistance, was correlated with plasmid car-
riage in the aeromonads. From Tables 1 and 4 it
can be seen that extrachromosomal DNA ele-
ments were detected in roughly 10% of the Bay
isolates and half the BE strains. Four of the nine
BE organisms with a plasmid carried two dis-
tinct species, whereas the three Bay aeromonads
had single plasmids. Some of the BE plasmids
appear to be R factors, whereas the Chesapeake
plasmids are cryptic. In all series, toxigenicity
ANTIBIOTIC-RESISTANT A. HYDROPHILA 481
shows no evidence of being a plasmid-mediated
trait.
DISCUSSION
The antibiotic resistance patterns of the A.
hydrophila organisms isolated in this study dif-
fer markedly from data in the older literature
(Table 2). In the Bangladesh organisms, we have
observed a 5-fold increase in the frequency of
streptomycin-resistant strains and a 23-fold in-
crease in the frequency of tetracycline resist-
ance. Most of the BE strains carry multiple
antibiotic resistance, with a streptomycin-tetra-
cycline phenotype which is correlated with the
presence of a high-molecular-weight plasmid.
Tetracycline is a widely utsed drug in the Matlab-
Dacca area, whereas streptomycin use has de-
clined since 1976. Thus, tetracycline use pro-
vides the overwhelming selective pressure in this
region.
It is particularly interesting to note that three

Downloaded from aac.asm.org by on May 17, 2010

FIG. 1. Cleared lysate DNA visualized by 0.7%
agarose gel electrophoresis. Well a, BE-45; well b,
BE-35; well c, E. coli H10407; well d, BE-40; and weU
e, BE-24.
of the BE bacteria carried chloramphenicol re-
sistance, an extremely rare trait in Aeromonas
(Table 2). Chloramphenicol resistance was in-
variably accompanied by streptomycin resist-
ance, but the chloramphenicol-resistant strains
did not consistently contain extrachromosomal
DNA.
Two Chesapeake Bay Aeromonas organisms
contained extrachromosomal DNA, but these
plasmids did not appear to be R-factors. The
Chesapeake Bay bacterial isolates carried only
single antibiotic resistance phenotypes. The
most significant resistance was to tetracycline: a
3.5-fold greater frequency than that reported in
the literature.
The sites in Chesapeake Bay from which A.
hydrophila strains were isolated represent two
extremes in water quality. Jones Falls and Col-
gate Creek are located in the relatively polluted
Baltimore Harbor. Bacterial pathogens, includ-
ing Salmonella spp., repeatedly have been iso-
lated from Baltimore Harbor water and sedi-
ment samples. Bacteriological parameters mea-
sured for these stations include fecal coliform
most probable numbers of 240/ml of water, total

TABLE 4. Frequency of drug resistance, toxigenicity, and plasmid carriage in strains of A. hydrophila
isolated from the natural environment"
A B C Ratio of re- Ratio of those Ratio of those
Total No. resistant No. toxi- No. with sistant to resistant to
toxigenic to
Series no.
ofto an antibi-
strains
genic piasmid toxigenic ths(A/C)
arry'ng~those carrying a
otic (A/B) plasmnd (B/C,
BE 18 13 (72)b 5 (28) 9 (50) 1/13 (8) 8/13 (62) 0/9 (0)
Ah, BV, CBAh, V 35 9 (26) 6 (17) 3 (9) 2/9 (22) 0/3 (0) 0/3 (0)
a Data from Table 1.
b
Numbers in parentheses are percentages.
 482 McNICOL ET AL.
viable counts of 105/ml of water, and Salmonella
most probable numbers as high as 1.1/g of sed-
iment (22). A. hydrophila counts have been
reported to be as high as 110/ml of water (Kaper
et al., Abstr. Annu. Meet. Am. Soc. Microbiol.
1979, N56, p. 188). In contrast, the Chester River
and Chesapeake Beach stations are relatively
clean areas located in the relatively open water
of Chesapeake Bay. No Salmonella species have
been recovered at either station, and fecal coli-
form counts are usually less than 0.03/ml of
water (21, 22). Total viable counts are usually
102 to 103/ml, aild A. hydrophila counts are
approximately 3/ml.
We do not know the source of drug resistance
in the Chesapeake Bay aeromonads. A recent
study showed that over 10% of the bacteria in
Jones Falls water were resistant to at least one
antibiotic, compared with 1% at Chesapeake
Beach (Allen et al., manuscript in preparation).
The polluted station may have increased poten-
tial for conjugal gene transfer of R factors or
drug resistance transposons from polluting or-
ganisms to native species (17). However, the Bay
isolates carrying a plasmid had no obvious drug
resistance phenotype, and half of the drug-re-
sistant Chesapeake strains in Table 1 were iso-
lated from the clean stations.
Plasmid carriage was higher in the Bangla-
desh organisms (43%) than in the Chesapeake
Aeromonas (9%). In the BE series, plasmid car-
riage was distinctly associated with drug resist-
ance but showed no positive relationship to tox-
igenicity. Curiously, none of the toxigenic strains
identified in this study harbored a plasmid
(Table 4), although three should have been ex-
pected by chance to carry both traits. This is in
agreement with the results of others, who found
no correlation between toxigenicity and plasmid
carriage in A. hydrophila (8), and may be related
to the recent observation of Sinha and Srivas-
tava (42), who found that toxin production in V.
cholerae is suppressed in the presence of P or V
plasmids. However, Cumberbatch et al. (8) ob-
served plasmid carriage in half of their cytotoxic
Aeromonas isolates. Clearly, the role of plasmids
in the toxigenicity of aeromonads needs to be
clarified.
From the studies reported here, it can be
concluded that chloramphenicol-resistant
strains of A. hydrophila can be readily isolated
from the natural environment. This parallels the
recently noted increase of chloramphenicol re-
sistance in other potentially pathogenic bacteria
(46) and may be part of a pandemic spread of
this trait. Resistance was highest in areas of
greatest human impact and could well prove to
be a serious human health problem in these
areas in the future.
ANTIMICROB. AGENTS CHEMOTHER.
ACKNOWLEDGMENTS
This work was funded by Public Health Service grant 1-
R22-AI-14242 from the National Institutes of Health and
supported by the International Centre for Diarrheal Disease
Research, Bangladesh.
LITERATURE CIED
1. Adachi, H., M. Nakano, M. Inuzuka, and M. To-
moeda. 1972. Specific role of sex pill in the effective
eliminatory action of sodium dodecyl sulfate on sex and
drug resistance factors in Escherichia coli. J. Bacteriol.
109:1114-1124.
2. Annapurna, E., and S. C. Sanyal. 1977. Enterotoxicity
of Aeromonas hydrophila. J. Med. Microbiol. 10:317-
323.
3. Aoki, T., S. Egusa, Y. Ogata, and T. Watanabe. 1971.
Detection of resistance factors in fish pathogen Aero-
monas liquefaciens. J.. Gen. Microbiol. 65:343-349.
4. Blair, J. E., E. M. Lennette, and J. P. Truant (ed.).
1974. Manual of clinical microbiology, 2nd ed. American
Society for Microbiology, Washington, D.C.
5. Caselitz, F. H., D. Krebs, and W. Maass. 1961. Unter-
suchungen zur Differenzierung von Mikroorganismen
Downloaded from aac.asm.org by on May 17, 2010
der Genera Aeromonas und Vibrio Miiller (Antibiotika,
Sulfonaniide, Vibriostatikum 0/129). Z. Tropenmed.
Parasitol. 12:325-329.
6. Caselitz, F. H., and W. Maass. 1962. Pathogenic strains
of Aeromonas and their sensitivity to antibiotics and
sulfonamides. Ger. Med. Monthly 7:195-197.
7. Colwell, R. R., J. Kaper, and S. W. Joseph. 1977.
Vibrio cholerae, Vibrio parahaemolyticus, and other
vibrios: occurrence and distribution in Chesapeake Bay.
Science 198:394-396.
8. Cumberbatch, N., M. J. Gurwith, C. Langston, R. B.
Sack, and J. I. Brunton. 1979. Cytotoxic enterotoxin
produced by Aeromonas hydrophila: relationship of
toxigenic isolates to diarrheal disease. Infect. Immun.
23:829-837.
9. Davis, W. A., J. G. Kane, and V. F. Garagusi. 1978.
Human aeromonas infections: a review of the literature
and a case report of endocarditis. Medicine 57:267-277.
10. Dean, H. M., and R. M. Post. 1967. Fatal infections with
Aeromonas hydrophila in a patient with acute my-
elogenous leukemia. Ann. Intern. Med. 66:1177-1179.
11. Donta, S. T., and A. D. Haddon. 1978. Cytotoxic activity
of Aeromonas hydrophila. Infect. Immun. 21:989-993.
12. Ewing, W. H., R. Hugh, and J. C. Johnson. 1961.
Studies on the Aeromonas group, p. 1-37. Center for
Disease Control, Atlanta.
13. Feaster, F. T., R. M. Nisbet, and J. C. Barber. 1978.
Aeromonas hydrophila corneal ulcer. Am. J. Ophthal-
mol. 85:114-117.
14. Fraire, A. E. 1978. Aeromonas hydrophila infection. J.
Am. Med. Assoc. 239:192.
15. Fulghujm, D. D., W. R. Linton, and D. Taplin. 1978.
Fatal Aeromonas hydrophila infection of the skin.
South. Med. J. 71:739-741.
16. Gilardi, G. L. 1967. Morphological and biochemical char-
acteristics of Aeromonas punctata (hydrophila, lique-
faciens) isolated from human sources. Appl. Microbiol.
15:417-421.
17. Guerry, P., and R. R. Colwell. 1976. Isolation of cryptic
plasmid deoxyribonucleic acid from Kanagawa strains
of Vibrio parahaemolyticus. Infect. Immun. 16:328-
334.
18. Guerry P., D. J. LeBlanc, and S. Falkow. 1973. General
method for the isolation of plasmid deoxyribonucleic
acid. J. Bacteriol. 116:1064-1066.
19. Hanson, P. G., J. Standridge, F. Jarrett, and D. G.
Maki. 1977. Freshwater wound infection due to Aero-
monas hydrophila. J. Am. Med. Assoc. 238:1053-1054.
VOL. 17, 1980 ANTIBIOTIC-RESISTANT A. HYDROPHILA
20. Hussain Qadri, S. M., L P. Gordon, R. D. Wende, and
R. P. Wifliams. 1976. Meningitis due to Aeromonas
hydrophila. J. Clin. Microbiol. 3:102-104.
21. Kaper, J., H. Lockman, R. R. Colwell, and S. W.
Joseph. 1979. Ecology, serology, and enterotoxin pro-
duction of Vibrio cholerae in Chesapeake Bay. Appl.
Environ. Microbiol. 37:91-103.
22. Kaper, J. B., G. S. Sayler, M. M. Baldini, and R. R.
Colwell. 1977. Ambient-temperature primary non-se-
lective enrichment for isolation of Salnonella spp. from
an estuarine environment. Appl. Environ. Microbiol.
33:829-835.
23. Ketover, B. P., L S. Young, and D. Armstrong. 1973.
Septicemia due to Aeromonas hydrophila: clinical and
immunologic aspects. J. Infect. Dis. 127:284-290.
24. Ijungh, A., B. Wretlind, and T. Wadstrom. 1978.
Evidence for enterotoxin and two cytolytic toxins in
human isolates of Aeromonas hydrophila, p. 947-960.
In P. Rosenberg (ed.), Toxins: animal, plant, and micro-
biol. Pergamon Press, Elmsford, N.Y.
25. MandeL M., L Igambi, J. Bergendahl, M. L Dodson,
and E. Scheitgen. 1970. Correlation of melting tem-
perature and cesium chloride buoyant density of bac-
terial deoxyribonucleic acid. J. Bacteriol. 101:333-
338.
26. McGrath, V. A., S. B. Overman, and T. L Overman.
1977. Media-dependent oxidase reaction in a strain of
Aeromonas hydrophila. J. Clin. Microbiol. 6:112-113.
27. Meyer, F. P. 1964. Field treatment of Aeromonas lique-
faciens infections in golden shiners. Prog. Fish. Cult., p.
33-35.
28. Meyers, J. A., D. Sanchez, L P. Elwell, and S. Fal-
kow. 1976. Simple agarose gel electrophoretic method
for identification and characterization of plasmid deoxy-
ribonucleic acid. J. Bacteriol. 127:1529-1537.
29. Mizon, F. M., G. R. Gerbaud, H. Leclerc, and Y. A.
Chabbert. 1978. Pr6sence de plasmides de resistance
appartenant au groupe d'incompatibilite C chez Aero-
monas hydrophila isole d'eaux residuaires. Ann. Micro-
biol. 129B:19-26.
30. Neilson, A. H. 1978. The occurrence of Aeromonads in
activated sludge: isolation of Aeromonas sobria and its
possible confusion with Escherichia coli. J. Appl. Bac-
teriol. 44:259-264.
31. Park, R. W. A. 1962. A study of certain heterotrophic
polarly flagellate water bacteria: Aeromonas, Pseudo-
monas, and Comomonas. J. Gen. Microbiol. 27:121-
133.
32. Pauckova, V., and A. Fukalova. 1967. Occurrence (sic)
of Aeromonas hydrophila and Aeromonas shigelloides
in feces. Zentralbl. Bakteriol. Parasitenkd. Infektionskr.
Hyg. Abt. 1 Orig. 206:212-216.
33. Popoff, M., and M. Veron. 1976. A taxonomic study of
the Aeromonas hydrophila-Aeromonas punctata
group. J. Gen. Microbiol. 94:11-22.
34. Porazikova, T., M. Michalus, and V. Kremery. 1978.
R plasmids in vibrionaceae-Beta-lactamases in Vibrio
cholerae (NAG-Heiberg II) and A. hydrophila. Zen-
483
tralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt.
1 Orig. Reihe A 242:481-486.
35. Rippey, S. R., and V. J. Cabelli. 1979. Membrane filter
procedure for enumeration of Aeromonas hydrophila
in fresh waters. Appl. Environ. Microbiol. 38:108-113.
36. Sack, D. A., and R. B. Sack. 1975. Test for enterotoxi-
genic Escherichia coli using Y1 adrenal cells in mini-
culture. Infect. Immun. 11:334-336.
37. Schubert, R. H. W. 1967. Die Pathogenitat der Aero-
monaden fur Mensch und Tier. Arch. Hyg. 160:709-
716.
38. Schubert, R. H. W. 1974. Genus Aeromonas Kluyver and
van Niel, p. 345-348. In R. E. Buchanan and N. E.
Gibbons (ed.), Bergey's manual of determinative bac-
teriology, 8th ed. Williams and Wilkins Co., Baltimore.
39. Shackelford, P. G., S. A. Ratzan, and W. T. Shearer.
1973. Ecthyma gangrenosum produced by Aeromonas
hydrophila. J. Pediatr. 83:199-201.
40. Shotts, E. B., J. L. Gaines, L. Martin, and A. K.
Prestwood, 1975. Aeromonas-induced deaths among
fish and reptiles in an eutrophic inland lake. J. Am. Vet.
Med. Assoc. 161:603-607.
41. Simidu, V., and E. Kaneko. 1973. A numerical taxonomy
of Vibrio and Aeromonas from normal and diseased
marine fish. Bull. Jpn. Soc. Sci. Fish. 39:689-703.
42. Sinha, B. V., and B. S. Srivastava. 1978. Plasmid-
induced loss of virulence in Vibrio cholerae. Nature
Downloaded from aac.asm.org by on May 17, 2010
(London) 276:708-709.
43. Snedecor, G. W., and W. G. Cochran. 1967. Statistical
methods, 6th ed., p. 215-218. Iowa State University
Press, Ames.
44. Sonstein, S. A., and J. N. Baldwin. 1972. Nature of the
elimination of the penicillinase plasmid from Staphy-
lococcus aureus by surface-active agents. J. Bacteriol.
111:152-155.
45. Tapper, M. L., L. R. McCarthy, J. B. Mayo, and D.
Armstrong. 1975. Recurrent Aeromonas sepsis in a
patient with leukemia. Am. J. Clin. Pathol. 64:525-530.
46. Van, A. D., G. Tiiaby, F. F. Acar, W. V. Shaw, and D.
M. Bouanchaud. 1978. Chloramphenicol resistance in
Streptococcus pneumonia: enzymatic acetylation and
possible plasmid linkage. Antimicrob. Agents Chemo-
ther. 13:577-583.
47. Vasil, AL L, R. K. Holmes, and R. A. Finkelstein.
1975. Conjugal transfer of a chromosomal gene deter-
mining production of enterotoxin in Vibrio cholerae.
Science 187:849-850.
48. von Graevenitz, A., and A. H. Mensh. 1968. The genus
Aeromonas in human bacteriology: report of 30 cases
and review of the literature. N. Engl. J. Med. 278:245-
249.
49. Washington, J. A. 1972. Aeromonas hydrophila in clin-
ical bacteriologic specimens. Ann. Inten. Med. 76:611-
614.
50. Zajc-Satler, J. 1971. Morphological and biochemical
studies of 27 strains belonging to the genus Aeromonas
isolated from clinical sources. J. Med. Microbiol. 5:263-
265.