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Applied and Environmental Microbiology, November 1998, p. 4194-4201, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Molecular Characterization of Plasmid-Mediated
Oxytetracycline Resistance in Aeromonas
salmonicida
C. A.
Adams,
B.
Austin,*
P. G.
Meaden, and
D.
McIntosh
Department of Biological Sciences,
Heriot-Watt University, Riccarton, Edinburgh, EH14 4AS, Scotland
Received 20 March 1998/Accepted 13 August 1998
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ABSTRACT |
Using broth conjugation, we found that 19 of 29 (66%)
oxytetracycline (OT)-resistant isolates of Aeromonas
salmonicida transferred the OT resistance phenotype to
Escherichia coli. The OT resistance phenotype was encoded
by high-molecular-weight R-plasmids that were capable of transferring
OT resistance to both environmental and clinical isolates of
Aeromonas spp. The molecular basis for antibiotic
resistance in OT-resistant isolates of A. salmonicida was
determined. The OT resistance determinant from one plasmid (pASOT) of
A. salmonicida was cloned and used in Southern blotting and
hybridization experiments as a probe. The determinant was identified on
a 5.4-kb EcoRI fragment on R-plasmids from the 19 OT-resistant isolates of A. salmonicida. Hybridization with
plasmids encoding the five classes (classes A to E) of OT resistance
determinants demonstrated that the OT resistance plasmids of the 19 A. salmonicida isolates carried the class A resistance
determinant. Analysis of data generated from restriction enzyme digests
showed that the OT resistance plasmids were not identical; three
profiles were characterized, two of which showed a high degree of homology.
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INTRODUCTION |
Aeromonas salmonicida is
the causative agent of furunculosis, an economically important disease
of salmonids (5). Control of this disease in aquaculture may
be by prophylaxis (i.e., vaccination) (11) or by
chemotherapy with a wide variety of antimicrobial compounds
(5). Initially when sulfonamides were administered as food
additives, they were successful (12). Subsequently, the
usefulness of oxytetracycline (OT) was reported (29), and this antibiotic is still used extensively for control of furunculosis (5, 28). However, continued and widespread use of
antibiotics has led to the development of resistant strains (3, 8,
15, 23). Moreover, plasmids encoding antibiotic resistance
(R-plasmids) have been isolated from A. salmonicida (2,
15, 26, 27). A second generation of
4-quinolones-fluoroquinolones, notably enrofloxacin and sarofloxacin,
effectively inhibits the pathogen and offers promise for the future
since plasmid-encoded resistance to these compounds has not been
described (7, 14, 19). However, mutational resistance to
this class of compounds can develop in A. salmonicida
(8, 21, 22, 32).
As noted previously (28), it is difficult to make any
definite conclusions about the impact of OT usage in aquaculture
because of the methodological differences described in the
literature. Transferable R-plasmids encoding resistance to tetracycline
in A. salmonicida were described in 1971 (2, 31);
subsequently, studies indicated that the frequency of
OT-resistant strains of A. salmonicida was increasing. In a
survey of 444 A. salmonicida isolates collected from
Scottish salmon farms during 1988 to 1991, 53% of the isolates were
resistant to OT (23). Using a random subsample of these
isolates, researchers determined that 27% contained R-plasmids which
could be transferred to Escherichia coli K-12 by conjugation
(15), although no information concerning the molecular basis
for the resistance was provided. Whereas there is no doubt that the
results of such studies have value, it is necessary to clarify the
situation with regard to the spread of R-plasmids encoding OT
resistance within A. salmonicida populations. Only through
precise molecular characterization of the genes encoding OT resistance
and the plasmids that carry these resistance determinants will it
become clear if aquaculture is facing a real threat from the use of
antibiotics. To address this issue, a collection of OT-resistant
isolates was used in experiments that examined the molecular basis for
OT resistance and the potential environmental impact of the R-plasmids
of these isolates.
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MATERIALS AND METHODS |
Bacterial strains and plasmids.
Table
1 shows the OT-resistant strains of
A. salmonicida used, including three isolates which were
obtained from Canada, two isolates which were obtained from the United
States, one isolate which was obtained from Norway, and 24 isolates
which were obtained from Scotland. Table
2 lists environmental isolates and type cultures of Aeromonas spp. OT-sensitive strains of A. salmonicida are shown in Table 3.
E. coli XL-1 Blue MR and HB101 were used in conjugation and
transformation experiments and for maintenance, amplification, and
preparation of plasmid DNA. Table 4 lists the plasmids that contain OT resistance determinant classes A to E.
For routine propagation of
Aeromonas strains, plates of
brain heart infusion agar (BHIA) (Oxoid, Basingstoke, United Kingdom)
were incubated at 15 to 25°C. Strains were subcultured every 3
to 5 days. For long-term storage, most bacterial strains were
stored at
70°C in brain heart infusion broth (Oxoid) supplemented
with 15%
(wt/vol) glycerol.
E. coli was stored at
70°C in
Luria-Bertani
(LB) medium (Oxoid) containing 15% (wt/vol) glycerol.
E. coli transformants were incubated overnight at 37°C on
slants of LB
agar medium containing 1.2% (wt/vol) agar with selection
(15 µg
of tetracycline per ml or 100 µg of ampicillin per ml, as
appropriate)
and were subsequently stored at 4°C. Transformants were
subcultured
every 3 to 4 weeks; stock cultures were also stored at
70°C in
LB medium containing 15% (wt/vol) glycerol (with
selection). The
antibiotic resistance spectra of
E. coli
strains and transformants
were determined by using multidisks (Mast
Diagnostics, Reinfield,
Germany) on
BHIA.
DNA manipulations.
Restriction endonuclease digestion,
transformation of E. coli, agarose gel electrophoresis,
cloning, and ligation of DNA fragments were performed by using standard
methods (25). Preparation of plasmid DNA from E. coli involved growing transformants or transconjugants containing
R-plasmids overnight at 37°C in 5-ml volumes of LB medium with
selection. The cells were pelleted by centrifugation (2,000 × g, 4°C) for 1 to 2 min, and plasmid DNA was prepared by
using Wizard plasmid mini or midi preparation kits (Promega, Southampton, United Kingdom) according to the manufacturer's
instructions. The methods of Kado and Liu (16) and Hiney et
al. (13) were used to prepare A. salmonicida
plasmid and total DNA, respectively. Plasmid preparations which
required further purification were processed by using the Wizard DNA
Clean-Up System (Promega). DNA samples were stored at 20°C until required.
Construction of DIG-labeled probes.
Digoxigenin
(DIG)-labeled probes were prepared by PCR amplification. The templates
used were a 1.2-kb Sau3A fragment (which contained a
sequence encoding OT resistance) from the OT resistance plasmid of
A. salmonicida MT1407 cloned in pUC19 and the class A OT
resistance determinant in pUC18 (purified from E. coli
JM83). For the PCR we used extended versions of the M13 universal
sequencing primer (5'-GTAAAACGACGGCCAGT-3') and reverse
sequencing primer (5'-AACAGCTATGACCATG-3'). Each reaction
mixture (100 µl) contained the following components: 1 ng of template
DNA, 10 µl of 10-fold-concentrated Taq DNA polymerase
buffer (Boehringer Mannheim, Mannheim, Germany), 0.2 mM dATP, 0.2 mM
dCTP, 0.2 mM dGTP, 0.2 mM dTTP, 50 µM DIG-11-dUTP (Boehringer
Mannheim), and each primer at a concentration of 1 µM. The reaction
mixture was mixed, briefly centrifuged, and overlaid with 100 µl of
light mineral oil (Sigma, Poole, United Kingdom). After denaturation of
the reaction mixture at 94°C for 10 min, 1 U of Taq DNA
polymerase (Boehringer Mannheim) was added. The PCR was then carried
out by using the following program: 2 min at 94°C, 2 min at 55°C,
and 3 min at 72°C for 35 cycles. A final extension step consisting of
10 min at 72°C completed the PCR, after which the layer of mineral
oil was carefully removed. The DIG-labeled PCR product was used
directly as a probe in hybridizations without purification.
Southern transfer and hybridization.
DNA samples (1 µg)
were digested with restriction endonucleases and separated on 0.8%
(wt/vol) agarose gels. Gels intended for Southern hybridization were
electrophoresed with 0.01 µg of DIG-labeled 
DNA cut with
HindIII (Boehringer Mannheim) as a molecular weight
marker. The DNA was transferred to a nylon membrane (Stratagene,
Cambridge, United Kingdom) by using a vacuum transfer apparatus
(VacuGene; Pharmacia Biotech, Milton Keynes, United Kingdom) according
to the manufacturer's instructions. Following transfer, the membrane
was removed from the apparatus, subjected to UV cross-linking, and
briefly rinsed in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium
citrate, pH 7.5). The membrane was dried and stored at room temperature.
The 100-cm
2 membrane to which the DNA was bound was placed
in a 100-ml bottle containing 50 ml of prehybridization solution
(Boehringer Mannheim). This bottle was incubated for at least
1 h
at 62°C in a hybridization oven (model Micro 4; Hybaid, Teddington,
United Kingdom). The probe DNA (5 ng per 100-cm
2 membrane)
was denatured just prior to use by adding distilled
water to a final
volume of 100 µl and then boiling the preparation
for 10 min. Probe
DNA was added to the solution in the bottle,
and hybridization was
allowed to take place overnight at 68°C.
After hybridization, the
membrane was washed twice for 5 min each
time at room temperature with
low-stringency wash solution (2×
SSC containing 0.1% [wt/vol]
sodium dodecyl sulfate). This was
followed by two washes (15 min each)
at 62°C with high-stringency
wash solution (0.5× SSC containing
0.1% [wt/vol] sodium dodecyl
sulfate). The DIG-labeled DNA was
detected by an enzyme-linked
immunoassay by using an anti-DIG antibody
conjugated to alkaline
phosphatase as described by the manufacturer
(Boehringer
Mannheim).
Conjugation.
Broth conjugation was performed by mixing equal
volumes (2 ml) of 72-h cultures grown in brain heart infusion broth at
22 or 28°C (Aeromonas species) or in LB broth at 37°C
(E. coli) and then incubating the preparations for 5 h
at 22°C (without shaking). Duplicate plates containing BHIA
supplemented with the appropriate selective and counterselective
antibiotics were inoculated with 100 µl of each conjugation mixture.
The plates were incubated at 18 or 22°C (Aeromonas
recipients) or at 37°C (E. coli recipients) for 2 to 5 days before they were examined for the presence of transconjugants.
MIC of antibiotics.
The MIC of antibiotics were determined
for E. coli XL-1 Blue MR transformants that contained pRAS1
or R-plasmids obtained from the OT-resistant strains of A. salmonicida (Table 5). The antibiotics used for MIC determinations were streptomycin and trimethoprim. Transformants were grown in 10 ml of LB medium containing OT (30 µg ml
1) for 4 to 6 h at 37°C, and a
102 dilution of each culture in LB medium was prepared.
Stock solutions of streptomycin and trimethoprim (50 µg
ml1 in LB medium) were used to prepare dilutions (200 µl in LB medium) containing 25, 12.5, 6.25, 3.13, and 1.56 µg of
each antibiotic per ml. The diluted antibiotic solutions were dispensed
into duplicate wells of a 96-well plate (Nunc; Gibco BRL, Paisley,
United Kingdom) before inoculation with the diluted bacterial
suspension (1 µl). Wells containing LB medium without antibiotics
were also inoculated in duplicate as growth controls. The plates were
incubated overnight at 37°C, and the highest concentration at which
growth occurred was recorded.
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TABLE 5.
Restriction enzyme fragments obtained from plasmid pRAS1
and the R-plasmids from E. coli transformants 718x, MT0320x,
MT0903x, MT0906x, MT1410x, MT1407x, and MT1432x
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RESULTS AND DISCUSSION |
Detection of plasmids in OT-resistant isolates of A. salmonicida and plasmid transfer to E. coli.
Analysis
of plasmid mini preparations demonstrated that all 29 of the
OT-resistant isolates of A. salmonicida contained
high-molecular-weight plasmids (Fig. 1);
19 (66%) of the plasmids were transferable to E. coli XL-1
Blue MR and HB101 in broth conjugation experiments (Table
6). These plasmid DNA bands were not
present in the OT-sensitive isolates. None of the Canadian or United
States isolates transferred the OT resistance phenotype to E. coli XL-1 Blue MR or HB101, nor did five of the Scottish isolates.
Transconjugants were recovered on plates containing 30 µg of OT per
ml after incubation at 37°C to select against the A. salmonicida donors. The same results were obtained when a filter
mating conjugation method was used instead of the broth conjugation
method (data not shown). Additional conjugation experiments were
carried out by using A. salmonicida OT-resistant strains as
the donors and E. coli XL-1 Blue MR as the recipient to
obtain transfer frequencies. It was determined that the frequencies
ranged from 103 to 106 per donor and
101 to 106 per recipient (Table
7). Sandaa and Enger (27)
reported that the R-plasmid pRAS1, which was isolated from an atypical
isolate of A. salmonicida in Norway, had a frequency of
transfer to E. coli HB101 of 1.4 × 102
per donor. This result is within 1 order of magnitude of the range of
values recorded in the present study. However, it is difficult to
compare the data since in this study we used a broth conjugation method
to determine transfer frequencies, whereas Sandaa and Enger
(27) used a filter mating technique.
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FIG. 1.
Plasmid profiles of OT-resistant and -sensitive isolates
of A. salmonicida. Lanes 1 and 17, markers
(HindIII digest of DNA): lanes 2 and 16, blank;
lanes 4 to 14, plasmid DNA from A. salmonicida OT-resistant
isolates MT1402, MT1431, MT1434, MT1427, MT1401, MT1407, MT1432,
MT0918, MT1425, MT1406, and MT1413, respectively; lanes 3 and 15, plasmid DNA from OT-sensitive isolates 256/91 and AS20, respectively.
The arrow indicates the position of the large plasmids. Not all
plasmids were routinely obtained at sufficiently high yields to be
detected by photography (lanes 6 and 10).
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Several studies have investigated the transfer of R-plasmids from fish
pathogens to other bacteria. Sandaa and Enger (
26,
27)
reported high-frequency transfer of plasmids pRAS1 and RP4
from
A. salmonicida to
E. coli and marine bacteria.
Interestingly,
both plasmids exhibited the highest transfer frequencies
to
E. coli. Similarly, Toranzo et al. (
30)
reported frequencies of
10
4 to 10
7 for
conjugal transfer of plasmids from bacteria isolated from
farmed
rainbow trout to
E. coli. Moreover, Kruse and Sørum
(
17)
described R-plasmid transfer from
A. salmonicida (present in infected
salmon muscle) to
E. coli on a cutting board. In this work, the
highest frequency of
transfer was from
A. salmonicida to
E. coli.
An examination of the plasmid contents of
E. coli
transconjugants showed that they all had acquired a large plasmid. In
some
cases, one, two, or all three of the low-molecular-weight plasmids
common to most
A. salmonicida isolates (
9) were
also detected.
E. coli XL-1 Blue MR was then transformed
(selecting for resistance
to 30 µg of OT per ml) with each of the
large plasmids that had
been prepared from the transconjugants and
purified from low-melting-point
agarose gels. For each of the large
plasmids that could be transferred
to
E. coli from the 19 OT-resistant isolates of
A. salmonicida,
the presence of the
large plasmid alone was demonstrated with
plasmid mini preparations.
This confirmed that the large plasmids
were R-plasmids.
Cloning of the gene encoding OT resistance from an R-plasmid
(pASOT).
A prime objective of this study was to determine the
molecular basis for the observed OT resistance in A. salmonicida. A large R-plasmid that was designated pASOT and was
purified from E. coli transformant MT1407x was used as the
source of the OT resistance determinant in cloning experiments. Cloning
involved the use of Sau3A partial digestion to generate
fragments of pASOT, followed by ligation into the BamHI site
of pUC19 and transformation of E. coli XL-1 Blue MR to OT
resistance. Analysis of plasmid mini preparations from colonies of
OT-resistant transformants revealed that DNA was added to pUC19. A
1.2-kb Sau3A fragment from one transformant (1407xa) was
used to produce the DIG-labeled probe AST for use in hybridization studies.
Hybridization experiments performed with the cloned OT resistance
determinant from pASOT.
EcoRI-digested plasmid DNA prepared
from E. coli transformants were blotted onto nylon membranes
and incubated with the DIG-labeled probe AST described above. Strong
hybridization with a 5.4-kb EcoRI fragment was observed with
all 19 OT-resistant strains and also with plasmid pRAS1. Moreover,
hybridization was not observed with plasmid preparations produced from
OT-sensitive strains of A. salmonicida or with untransformed
E. coli (Fig. 2a).
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FIG. 2.
(a) Southern blot analysis of plasmid DNA prepared from
E. coli transformants digested with EcoRI and
hybridized with probe AST. Strong hybridization occurred with a 5.4-kb
fragment in lanes 2 to 11 and 13, which contained plasmid DNA from the
following transformants: MT0404x, MT0321x, MT1407x, MT0903x,
MT1410x, MT0900x, MT1431x, MT0164x, Banksx, MT1427x, and 718x,
respectively. Lane 1 contained plasmid DNA from OT-sensitive A. salmonicida isolate 96. Lane 12 contained markers
(HindIII digest of DIG-labeled DNA). (b) Southern
blot analysis of plasmid DNA prepared from E. coli
transformants digested with EcoRI and hybridized with probe
TA. Strong hybridization occurred with a 5.4-kb fragment in lanes 2 to
9, which contained plasmid DNA from the following transformants:
MT1407x, MT1410x, MT0164x, MT1427x, MT0350x, MT1413x, MT0404x, and
718x, respectively. Lane 1 contained markers (HindIII
digest of DIG-labeled DNA), and lane 10 contained plasmid DNA from
the OT-sensitive isolate A. salmonicida 96.
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Determination of the classes of resistance determinants in the
OT-resistant isolates of A. salmonicida.
Plasmid DNA
produced from five strains of E. coli carrying resistance
determinants A to E (Table 4) were used to prepare Southern blots.
These blots were then hybridized with probe AST; hybridization was
observed only with the class A determinant. On the basis of these
results, a second probe was constructed from the class A determinant
carried in pUC18. This probe, probe TA, was used in hybridization
experiments performed with the EcoRI digests described
above. All 19 transferable plasmids (and pRAS1) carried the class A OT
resistance determinant on a 5.4-kb EcoRI fragment previously
detected with probe AST. Hybridization was not observed with DNA
prepared from OT-sensitive isolates or untransformed E. coli
(Fig. 2b).
Andersen and Sandaa (
1) reported that tetracycline
resistance determinant E was the most widespread determinant in
bacterial
isolates obtained from polluted and unpolluted marine
sediments
in Norway and Denmark; it was detected in 63% of the
isolates.
The class A tetracycline resistance determinant was detected
in
only 5% of the samples, and the class D determinant was detected
in
4% of the samples. In Japan, the class D tetracycline resistance
determinant is common in the fish pathogens
Aeromonas
hydrophila,
Edwardsiella tarda, and
Pasteurella
piscicida (=
Photobacterium damselae subsp.
piscicida) (
4). A study of
A. hydrophila in
the United States reported that 50% of OT-resistant
isolates obtained
from aquaculture carried the class E determinant,
whereas 35%
carried the class A determinant (
10).
Interestingly, one isolate
in that study contained both determinants.
It is noteworthy that
all of the transferable plasmids examined in this
study contained
the same resistance determinant on an
EcoRI
fragment of the same
size. The strains of
A. salmonicida
from which these plasmids
were isolated were collected between 1982 to
1993, and, although
they all came from Scottish sites, they were not
otherwise related.
Therefore, it appears that the resistance
determinant identified
in this work established a foothold among
Scottish isolates of
A. salmonicida and persisted for at
least 11
years.
Characterization of the 19 conjugative R-plasmids.
E.
coli transformants exhibited resistance to streptomycin, whereas
transformants carrying pRAS1 displayed resistance to both streptomycin
and trimethoprim. Additional data relating to resistance phenotypes
were obtained by assessing MIC of streptomycin and trimethoprim. There
was some variation in the MIC recorded for the different transformants.
Thus, seven transformants were sensitive to streptomycin; seven
isolates had an MIC of 6.25 µg ml1, four isolates had
an MIC of 12.5 µg ml1, and one isolate had an MIC of 25 µg ml1. Only transformants carrying pRAS1 were
resistant to trimethoprim; the MIC of this antibiotic and the MIC of
streptomycin were both 25 µg ml1.
Midi preparations of plasmid DNA were produced from seven
E. coli transformants containing R-plasmids from
A. salmonicida 718, MT0320, MT0903, MT0906, MT1407,
MT1432, and MT1410, which
were selected on the basis of their
antibiotic resistance profiles.
Single restriction endonuclease
digestion of plasmid DNA was performed
with
EcoRI,
HindIII,
BamHI, and
SalI, and
double digestion was
performed with endonucleases
HindIII plus
PvuI,
BamHI plus
PvuI,
and
BamHI plus
HindIII.
Digestion with
EcoRI generated multiple
fragments for all of
the plasmids. However, it was evident that
there were differences in
the band patterns (Fig.
3). Thus, the
plasmids prepared from transformants MT0903x, MT1407x, and MT1410x
formed a distinct group (profile 1). A band pattern similar to
profile
1 was obtained for plasmids from MT0906x and MT1432x,
but a 2.9-kb
fragment was present in place of the 3.1-kb fragment
present in profile
1; this band pattern was designated profile
2. It was interesting that
the profile 2 plasmids did not encode
streptomycin resistance, whereas
profile 1 plasmids did. It is
possible that streptomycin resistance is
carried on the 3.1-kb
fragment. However, this was not confirmed in the
present work,
and further investigation will be required to determine
the location
of the streptomycin resistance determinant.
EcoRI digestion of
plasmid DNA from MT0320x generated a
unique set of fragments (profile
3), as did
EcoRI digestion
of pRAS1 (718x) (profile 4). However,
these profiles shared with
profiles 1 and 2 the 5.2- and 5.4-kb
doublet; the OT resistance
determinant is located on the 5.4-kb
fragment of this doublet. Data
from additional restriction endonuclease
digestion supported the
findings obtained with
EcoRI, with the
plasmids falling into
the same four groups with each endonuclease
or combination of
endonucleases tested. The sizes of the fragments
generated by these
digestion are shown in Table
5. The remaining
13 transferable
R-plasmids were digested with
EcoRI to determine
if the four
profiles obtained with the seven test isolates were
also produced by
the other R-plasmids. Data from the resulting
digests indicated that
there was some correlation between
EcoRI
fragment profiles
and the expression of a streptomycin resistance
phenotype. Thus,
plasmids from the seven streptomycin-sensitive
isolates produced the
same
EcoRI digestion profile (profile 2).
In contrast, two
profiles were exhibited by the plasmids encoding
streptomycin
resistance, with eight of the plasmids producing
profile 1 and four
plasmids producing profile 3 (Table
8).
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FIG. 3.
Restriction endonuclease fragment patterns generated by
EcoRI digestion of plasmid pRAS1 (from E. coli
transformant 718x) and R-plasmids from E. coli transformants
MT0320x, MT0903x, MT0906x, MT1407x, MT1410x, and MT1432x. Lane 1, markers (HindIII digest of DNA); lane 2, 718x; lane
3, MT0320x; lane 4, MT0903x; lane 5, MT0906x; lane 6, MT1407x; lane 7, MT1410x; lane 8, MT1432x; lane 9, markers (1-kb DNA ladder).
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TABLE 8.
Grouping of OT-resistant isolates of A. salmonicida based on EcoRI digestion profiles of
R-plasmids and antibiotic resistance phenotype
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Assessment of homology among the four plasmid profiles.
As
shown in Table 9, some restriction
endonuclease fragments were common to all four plasmid types. This
indicated that the plasmids (including pRAS1) could be related at the
molecular level. BamHI digestion did not provide any
information concerning the relatedness of the plasmids. However,
digestion with HindIII demonstrated that two fragments
(3.5 and 1.9 kb) were common to plasmid profiles 1 (pASOT) and 2 (pASOT2). SalI digestion indicated that two fragments (12 and 0.6 kb) were also shared by these two plasmid types. A number of
fragments common to all four profiles were identified in
EcoRI digests, as were two fragments at 9.4 and 6.3 kb which were seen only in profiles 1 and 2 (Table 9). Analysis of the fragments
produced from the double digests reinforced the likelihood that the
plasmids had a common ancestry. Thus, four fragments common to pASOT
and pASOT2 were observed following digestion with HindIII plus PvuI, and one of these
fragments (1.5 kb) was present in all four profiles. The results
presented in Table 9 indicate that pRAS1 exhibits significant
homology with pASOT and pASOT2, whereas profile 3 (pASOT3) is as a more
distant relative of the other three profiles.
Estimation of the molecular weights of plasmids pASOT, pASOT2,
pASOT3, and pRAS1.
None of the restriction endonucleases generated
fragments that were all in a size range (0.5 to 10 kb) that allowed
accurate determinations of the molecular weights of plasmids pASOT,
pASOT2, pASOT3, and pRAS1. In all of the digests except the
EcoRI digests there was a restriction endonuclease fragment
which was larger than 23 kb (the largest size for which a marker
fragment was available). However, based on the EcoRI digest
data (Table 5), the plasmids were estimated to have the following
minimum molecular weights (to the nearest whole number): pASOT and
pASOT2, 47 kb; pASOT3, 39 kb; and pRAS1, 44 kb. Sandaa and Enger
(27) determined that pRAS1 had a molecular mass of 25 MDa
(37.5 kb) based on data from EcoRI digestion, although no
figure showing the digest was provided in support of this. Livesley et
al. (18) analyzed plasmid profiles of A. salmonicida isolates by pulsed-field gel electrophoresis. This
method separated a range of plasmids, resulting in resolution of both
high- and low-molecular-weight plasmids (5 to 100 kb) on the same gel.
Thus, pulsed-field gel electrophoresis may provide a means by which the
molecular weights of the R-plasmids identified in this study can be
accurately determined.
Assessment of the potential for intra- and interspecies
transfers of the OT resistance plasmids.
Sandaa and Enger
(26, 27) reported that plasmid pRAS1 could be transferred to
a range of gram-negative and gram-positive bacteria (e.g.,
Lactobacillus sp.). We therefore performed broth conjugation
experiments with plasmids pASOT, pASOT2, and pASOT3 to examine whether
inter- and intraspecies transfers of the OT resistance phenotype could
take place. The results of numerous broth conjugation experiments
performed in this study are summarized in Table 7. We found that 78%
of the OT-sensitive strains of A. salmonicida received the
R-plasmids from two different OT-resistant A. salmonicida
donors (both carrying pASOT). A smaller proportion (55%) of the
OT-sensitive recipients acquired the OT resistance phenotype in crosses
in which an E. coli transconjugant was the donor (Table 7).
In experiments in which OT-sensitive Aeromonas spp.
(predominantly isolated from healthy fish) were used as recipients with
OT-resistant A. salmonicida donors (carrying pASOT), 50% of
the recipients acquired the R-plasmids. The same proportion (50%) was
observed when an E. coli transconjugant carrying pASOT2 was
used as the host, whereas a higher percentage of the recipients (63%)
developed the OT resistance phenotype after crosses in which a
transconjugant carrying pASOT was used as the host (Table 7). Transfer
of the OT resistance phenotype to 44% of the Aeromonas type
cultures was observed with two OT-resistant A. salmonicida donors (both carrying pASOT). E. coli transconjugants
carrying pASOT and E. coli transformants carrying pASOT2
transferred the R-plasmid to 44 and 67% of the type cultures,
respectively (Table 7). The conjugation experiments in which two
strains of E. coli were used indicated that 10 of the
R-plasmids were nontransferable (Table 6). In order to examine the
possibility that this was due to the recipient, conjugation experiments
were performed with the human pathogen Aeromonas trota as an
alternative recipient in crosses with the 29 OT-resistant A. salmonicida isolates. It was observed that the same 19 isolates of
A. salmonicida which conjugated with E. coli also
conjugated with A. trota.
In terms of frequency of transfer, the highest level of conjugal
transfer was observed when
E. coli XL-1 Blue MR was used
as
the recipient (Table
7). The frequencies of transfer observed
in the
other experiments were similar for the majority of crosses
(that is, in
the range of 10
4 to 10
8 per donor or
recipient).
The results reported above were not entirely unexpected given that
pRAS1 showed a degree of relatedness to the three R-plasmids.
Indeed,
the data gave some support to the strongly debated hypothesis
that
antibiotic-resistant strains of fish pathogens may act as
reservoirs
for resistance genes, which could subsequently be transferred
to
bacteria having public health significance (
17,
20,
28,
31).
Our results suggest that transfer of the R-plasmids between
A. salmonicida isolates could be extended through the presence
of
carrier or mediator bacterial species, such as
E. coli. A
cycle
of resistance transfer could exist in the environment, with
transfer
occurring between
A. salmonicida and a range of
unidentified mediator
bacterial species. The fate of
A. salmonicida in open waters or
in fish farm sediments continues to
be a highly contentious issue
(
5). It is important to
consider that the experimental procedures
used in this study and the
majority of previous studies are not
representative of the conditions
that are encountered in fish
farm environments. The use of cells which
were grown to high densities
in nutritionally rich media and which were
subsequently brought
into contact as pure cultures was a very
artificial means by which
to estimate the environmental implications of
R-plasmid transfer.
Similarly, the use of laboratory strains of
E. coli could be questioned.
In future experiments workers
should aim to employ bacteria isolated
from fish farm environments and
to develop methods that are more
representative of the conditions found
in such locations. In this
context, it has been reported that the gut
contents of fish is
a suitable environment for in vitro
plasmid-mediated transfer
of resistance (
24) and that OT
resistance was transferred successfully
from
A. salmonicida
to
E. coli. However, this result could not
be reproduced in
vivo. The authors speculated that it was unlikely
that human pathogenic
bacteria could acquire antibiotic resistance
by this route. Other
workers have noted that based on a review
of the evidence available at
the time, transfer of R-plasmids
from fish pathogens to potential human
pathogens was a rare event
(
28). No evidence has emerged
since then to contradict this
observation. Therefore, it is probably
inadvisable to attach too
much significance to the data generated by
the in vitro conjugation
experiments in relation to the risk posed by
the release of OT-resistant
A. salmonicida into open waters.
Nevertheless, the observation
that the R-plasmids identified in the
present study were recovered
from samples collected over 11 years from
unrelated sites strongly
suggests that these plasmids are persistent in
fish farm environments
and that they may reside in bacterial species
other than
A. salmonicida.
At present, there is insufficient experimental evidence to allow
definite conclusions to be reached as to the environmental
significance
of OT usage (or the usage of any other antibiotic)
in aquaculture
(
28). However, the present study provided some
data that may
help clarify the situation. In particular, we demonstrated
that the use
of modern molecular techniques can generate highly
specific information
regarding the mechanisms of antibiotic resistance
that are used by fish
pathogens. It is anticipated that the continued
application of such
techniques will help provide some answers
to the many issues relating
to antibiotic usage in aquaculture.
The findings of this study
represent a significant advance in
knowledge concerning the nature of
OT resistance in Scottish isolates
of
A. salmonicida. There
is a need for this research to be extended
to the examination of
isolates from other geographical locations
and, indeed, to other fish
pathogens. In addition, attempts should
be made to perform experiments
to determine the ability of plasmids
to be spread among the bacterial
population present in and beyond
fish farm
environments.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biological Sciences, Heriot-Watt University, Riccarton, Edinburgh, EH14 4AS, United Kingdom. Phone: 44-131-451-3452. Fax: 44-131-451-3009. E-mail: B.Austin{at}hw.ac.uk.
Present address: Departamento de Bioquimica e Biologia Molecular,
Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, 21045-900, Brazil.
| |
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Applied and Environmental Microbiology, November 1998, p. 4194-4201, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
