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Antimicrobial Agents and Chemotherapy, May 2001, p. 1550-1552, Vol. 45, No. 5
Antimicrobial Agents Research Group, Division
of Immunity and Infection, University of Birmingham, Birmingham B15
2TT, United Kingdom
Received 7 September 2000/Returned for modification 2 November
2000/Accepted 30 January 2001
The amount of acrB, marA, and soxS mRNA was
determined in 36 fluoroquinolone-resistant E. coli from
humans and animals, 27 of which displayed a multiple-resistance
phenotype. acrB mRNA was elevated in 11 of 36 strains. A
mutation at codon 45 (Arg Multiple antibiotic resistance in
Escherichia coli has been the focus of much recent
attention, and a number of mechanisms that can contribute to this
resistance have been elucidated. One of these is the transport of
diverse substrates out of the cell by the AcrAB efflux transporter
(15). This wide substrate profile leads to a
multiple-antibiotic-resistance (Mar) phenotype; the antibiotics to
which acrAB can confer resistance include tetracycline, ampicillin, chloramphenicol, rifampin, novobiocin, erythromycin, The susceptibilities of all strains to 10 antibiotics, four dyes, four
disinfectants, and six detergents were determined with the agar
doubling dilution method using Iso-Sensitest agar as described
previously (6). At least 27 of the 36 isolates were multiply resistant, i.e., showed resistance to at least three separate
classes of antibiotics (Table 1). Most of
the isolates were resistant to the dyes tested, with some exceptions;
I236, I246, and I276 were more susceptible to acriflavine (MIC of 16 or
32 µg/ml), and I281 and I283 were more susceptible to
ethidium bromide (MICs, 32 to 64 µg/ml). All the isolates
tested were resistant to deoxycholate, cholic acid, taurocholic acid,
3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate
(CHAPS), Triton X-100, cetyltrimethylammonium bromide, dehydroxycholate, sodium dodecyl sulfate, NP-40, and Triton X-114.
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.5.1550-1552.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Absence of Mutations in marRAB or soxRS in
acrB-Overexpressing Fluoroquinolone-Resistant Clinical and
Veterinary Isolates of Escherichia coli
ABSTRACT
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Cys) in acrR was found in 6 of
these 11 strains. Ten of the 36 isolates appeared to overexpress
soxS, and five appeared to overexpress marA. A
number of mutations were found in the marR and
soxR repressor genes, correlating with greater amounts of
marA and soxS mRNA, respectively.
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-lactams, and fluoroquinolones (15, 16, 17). AcrB is a large cytoplasmic membrane protein (10, 11), which
associates with AcrA, a membrane fusion protein (18), and
TolC, a protein thought to be the channel allowing extrusion of the
substrates into the medium (7). The acrA and
acrB genes are transcribed from one operon (11)
with the acrR gene, a repressor of the operon that controls
its activation transcribed divergently from a point downstream of the
acrAB operon (12). The acrAB locus is a member of the mar, sox, and rob regulons
(21). MarA, SoxS, and Rob are related transcriptional
activators of the AraC family (2, 8, 20), and all can
activate acrAB expression, although they are not involved in
regulation of acrAB in response to general stress conditions
(1, 3, 4, 5, 12, 14). Although E. coli
possesses (at least) three transcriptional activators able to activate
an overlapping set of genes in response to different environmental
conditions, little is known about the relationship between the three
genes and their contributions to control of antibiotic resistance. The
importance of mutations in the sox, mar, and rob
loci in antibiotic resistance in clinical isolates is at present
unclear; mar and sox mutations have been
identified in fluoroquinolone-resistant E. coli
(13, 17), but to date only a small number of strains have
been studied (25 by Oethinger et al. and 23 by Maneewannakul and Levy).
We have previously investigated the mechanisms of fluoroquinolone
resistance in 36 E. coli strains from diverse origins
(6); eight were isolated from calves and chickens in
the United Kingdom (I87 to I94); the remainder were human isolates
from hospitals in Argentina and Spain (I236 to I254 and I275 to I283).
All are ciprofloxacin-resistant (MICs, 2 to 128 µg/ml) and have a
substitution of leucine for serine at codon 83 of gyrA; 26 also have additional mutations in gyrA, and 24 possess
single mutations in parC. None of the isolates have any
mutations within gyrB or parE. Twenty-two of the
isolates accumulated significantly less ciprofloxacin than did
wild-type strains. The addition of carbonyl cyanide
m-chlorophenylhydrazone (CCCP) abolished this effect,
suggesting that an efflux pump may be important in the phenotype of
these strains. This study examined the role of genes associated with
efflux-mediated antibiotic resistance, including acrB, tolC,
marA, soxS, and rob, in this set of isolates.
TABLE 1.
Strains with increased expression of efflux-associated
genes, their susceptibilities to antibiotics and dyes, and their
mutations in regulatory genesa
Northern blotting was used to determine the amounts of mRNA
transcripts present in each strain. RNA was prepared and corresponding amounts (determined by spectrophotometry and by agarose gel
electrophoresis followed by analysis with a Gene Genius image
analyzer [SYNGENE, Cambridge, United Kingdom]) were
electrophoresed under denaturing conditions before being
subjected to blotting (19). A constant level of 16S
expression was detected in all the strains (data not shown), indicating
that there was no strain-to-strain variation in mRNA production.
Amounts of acrB mRNA were determined for each strain,
and results were compared to those for wild-type strains and AG102.
Eleven of the 36 strains (31%) consistently produced more
acrB mRNA than the wild-type controls (Fig.
1). More acrB mRNA signal
was detected in the clinical isolates than in the marR
mutant, AG102. There was a strong association between strains with high
levels of acrB transcripts and ciprofloxacin efflux, with
the isolates that produced most acrB mRNA being those
for which CCCP had the greatest effect on the accumulation of
ciprofloxacin (Table 1). However, there was no direct correlation
between the level of acrB mRNA and MICs of ciprofloxacin
and there was no obvious correlation between acrB mRNA
amounts and the MICs of the other agents. The three strains which were
hypersusceptible to acriflavine and ethidium bromide did not appear to
produce any less acrB mRNA than wild-type strains in
Northern blotting experiments. The contributions of other efflux
systems and non-efflux-based resistance determinants may well mask the
effect of AcrB.
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Most of the strains produced similar amounts of tolC
mRNA compared to the wild-type strains (data not shown). Ten of the
36 strains (28%) showed more soxS mRNA than did the
wild-type strains (Fig. 2). Of these 10 strains, only three appeared to overexpress acrB. Five of
the 36 strains (14%) showed more marA mRNA than did the
wild-type strains, but none of them produced as much as AG102. Only 1 of these 10 strains appeared to also overexpress acrB. All
of the the strains possessed a constitutive level of rob
mRNA comparable with that seen for the wild-type strains (data not
shown). Interestingly all the strains with increased levels of
marA mRNA originated in Argentina and all those with
increased levels of soxS mRNA were from Spain; this may
indicate that different conditions will favor the selection of one type
of mutation over another. None of the veterinary isolates contained
mutations in mar or sox genes; strains with
increased levels of acrB mRNA were detected in isolates
from both animals and humans.
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To identify mutations leading to apparent overexpression of the genes
investigated by Northern blotting, primers were designed to amplify
overlapping products of <350 bp, which covered the genes
themselves or the regulatory genes that control them. The acrR,
soxR (soxS repressor), soxS, marR
(marA repressor), and marA genes were all covered
by overlapping amplimers. Single-stranded conformational polymorphism
and DNA sequencing (6) of all isolates overexpressing
acrB mRNA revealed four silent mutations in
acrRA, six isolates had an amino acid substitution of
cysteine for arginine at codon 45 of acrR, and one strain,
I254, contained an insert of ~1,000 bp in acrR. The
isolates that showed increased marA mRNA all had a
substitution of histidine for tyrosine at codon 137 of marR,
the same as that found by Oethinger et al. (17). One
strain, I283, had a substitution of arginine for cysteine at codon 63 in marA. Analysis of soxRS revealed mutations in
both the soxR and soxS genes among the 10 isolates overproducing soxS mRNA. Five different amino
acid changes were found in soxR (Asp25Glu, Ser31Ala,
Leu65Val, Arg71Ser, and Arg90Gly). An insertion of one base
within codon 106, which changes the reading frame and leads to a stop
codon 2 bp downstream of the insertion, was present in two of the
strains (I248 and I251). Also, two amino acid substitutions were found
within soxS in I237 and I253; one of these was a
threonine-to-serine substitution at codon 38, and the other was a
glycine-to-arginine substitution at codon 74. This diverse pattern of
mutations shows that no single mutation is important in modulating the
function of soxR; it also indicates that the isolates in
this study are genetically diverse, as was seen from the number of
silent mutations and deviations from the sequences deposited in GenBank.
Of the 11 strains that had increased amounts of acrB mRNA 8 appeared to overproduce this mRNA in a manner independent of soxS or marA regulation. Only 3 of the soxS mRNA overexpressing isolates and one marA mRNA overexpressing isolate were also acrB mutants. These data indicate that mutations in the acrR repressor gene are more likely to be involved in acrB derepression than mutations which lead to marA or soxS overexpression. Even when the acrR repressor mutations and mar and sox mutations are combined they do not account for all the strains which overproduced acrB mRNA. These data support previous work by Ma et al. (9) who demonstrated that the acrB operon can be activated in response to various stress conditions in the absence of mar, sox, and rob. The data from the present study indicate that the acrB pump may be involved in ciprofloxacin efflux in both clinical and veterinary isolates of E. coli and that a variety of mutations in different genes associated with efflux can have a role in controlling expression of acrB. No single mechanism is exclusively involved in acrB regulation. Further work is in progress to determine the role, if any, of the mutations in the regulatory genes described above.
The primers used to amplify the DNA used as probes in the blotting were designed from sequences deposited in GenBank (acrB, accession number J00734: nucleotides [nt] 3393 to 4412 [1,019 bp]; marA, accession number M96325: nt 1952 to 2239 [287 bp]; soxS, accession no. X59593: nt 424 to 582 [158 bp]; rob, accession number M97495: nt 423 to 1065 [642 bp]; tolC, accession number X54049: nt 548 to 837 [289 bp]; 16S rRNA, accession number J01859: nt 540 to 1139 [599 bp]).
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ACKNOWLEDGMENTS |
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We are indebted to David White and Stuart Levy for their generous donations of control strains used in this study.
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FOOTNOTES |
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* Corresponding author. Mailing address: Antimicrobial Agents Research Group, Division of Immunity and Infection, University of Birmingham, Birmingham B15 2TT, United Kingdom. Phone: 0121-414-6969. Fax: 0121-414-6966. E-mail: l.j.v.piddock{at}bham.ac.uk.
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Antimicrobial Agents and Chemotherapy, May 2001, p. 1550-1552, Vol. 45, No. 5
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.5.1550-1552.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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