Download Proteus mirabilis Tigecycline (GAR-936) in with Reduced Levels of

AcrAB Multidrug Efflux Pump Is Associated
with Reduced Levels of Susceptibility to
Tigecycline (GAR-936) in Proteus mirabilis
Melissa A. Visalli, Ellen Murphy, Steven J. Projan and
Patricia A. Bradford
Antimicrob. Agents Chemother. 2003, 47(2):665. DOI:
10.1128/AAC.47.2.665-669.2003.
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 2003, p. 665–669
0066-4804/03/$08.00⫹0 DOI: 10.1128/AAC.47.2.665–669.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 47, No. 2
AcrAB Multidrug Efflux Pump Is Associated with Reduced Levels of
Susceptibility to Tigecycline (GAR-936) in Proteus mirabilis
Melissa A. Visalli,* Ellen Murphy, Steven J. Projan, and Patricia A. Bradford
Wyeth Research, Pearl River, New York
Received 28 June 2002/Returned for modification 7 October 2002/Accepted 10 November 2002
The growing threat of acquired resistance in the Enterobacteriaceae (6, 10, 20) indicates the crucial need for new antibiotics for continued effective treatment of bacterial infection.
Tigecycline, the 9-t-butylglycylamido derivative of minocycline,
is a new antimicrobial agent belonging to a novel class of
tetracyclines, the glycylcyclines (24). It has good activity against
gram-positive pathogens, including penicillin-resistant Streptococcus pneumoniae, vancomycin-resistant enterococci, and methicillin-resistant Staphylococcus aureus (2, 5, 18). Tigecycline
has good activity against most gram-negative pathogens, including Klebsiella pneumoniae and Escherichia coli (4, 18).
Tigecycline also has good activity against organisms with a
resistance determinant from the major facilitator family, including E. coli with tet(A), tet(B), tet(C), tet(D), and tet(M); S.
aureus with tet(K) and tet(M); and Enterococcus faecalis with
tet(M) (18). Proteus mirabilis is a notable exception to the
activity of tigecycline, which routinely shows MICs of 4 ␮g/ml
for the organism in tests.
It is important to identify current and emerging resistance
mechanisms. Identification and mechanistic studies of bacterial resistance mechanisms can help to further reduce health
care costs due to bacterial infection. Therefore, a study was
performed to identify the mechanism responsible for the reduced susceptibility of P. mirabilis to tigecycline (M. A. Visalli,
E. Murphy, S. J. Projan, and P. A. Bradford, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. C1-2019,
2001).
presence of the following selective antibiotics when required: 50 ␮g of kanamycin/ml, 50 ␮g of ampicillin/ml, or 10 ␮g of gentamicin/ml.
Antibiotic and susceptibility testing. Antibiotic and substrate MICs were determined by broth microdilution using twofold dilution in Mueller-Hinton II
broth (BBL, Cockeysville, Md.) according to the procedures established by the
National Committee for Clinical Laboratory Standards (15). The following antibiotics, dyes, and detergents were used in this study: ampicillin, ceftriaxone,
ciprofloxacin, novobiocin, ethidium bromide, chloramphenicol, erythromycin,
acriflavine, and trimethoprim (Sigma Chemical Co., St. Louis, Mo.); imipenem
(Merck, Rahway, N.J.); and tigecycline and minocycline (Wyeth Research, Pearl
River, N.Y.). All substrates and antibiotics tested were prepared fresh on the day
of testing.
Transposon mutagenesis. To make the clinical isolate P. mirabilis G151 electrocompetent, cells were grown overnight in 25 ml of LB broth (without NaCl),
5 g of yeast extract/liter, and 10 g of tryptophan/liter at 37°C with shaking; 12.5
ml of the overnight culture was inoculated into 500 ml of fresh salt-free LB broth.
The culture was grown at 37°C with shaking to an optical density at 600 nm of 0.6.
The cells were chilled on ice for 15 min and then pelleted at 8,000 ⫻ g for 10 min
at 4°C. The supernatant was removed, and the cell pellets were resuspended in
250 ml of ice-cold sterile 10% glycerol. The cells were pelleted and resuspended
three more times in 10% glycerol in decreasing volumes of 100, 50, and, finally,
1 ml. The cells were snap frozen in a dry ice-ethanol bath in 110-␮l aliquots and
stored at ⫺80°C. The electrocompetent cells were used the following day for
transformation by electroporation with the Gene Pulser II system (Bio-Rad,
Hercules, Calif.) using the EZ::TN ⬍R6K␥ori/KAN-2⬎ transposon (Epicentre).
The transposome system was used according to the manufacturer’s protocol. The
optimal electroporation settings with a cuvette gap size of 0.2 cm were 2.5 kV, 25
␮F, 200 ⍀, and ⬃4.7 ms. Transformants were selected on LB agar plates (Difco
Laboratories, Detroit, Mich.) with 2⫻ agar (to inhibit swarming of the P. mirabilis colonies) and 50 ␮g of kanamycin/ml. The transformants were replica plated
as previously described (8), using 50 ␮g of kanamycin/ml and tigecycline at a
concentration of either 2 or 4 ␮g/ml.
Nucleic acid techniques. Standard nucleic acid techniques were performed as
described previously (21). Restriction enzymes (Roche Molecular Biochemicals,
Indianapolis, Ind.) were used according to the manufacturer’s instructions. Ligations were performed using the Fast-Link DNA ligation kit (Epicentre) according to the manufacturer’s instructions. DNA sequencing of transposon insertion sites was performed using the Big Dye version 3 sequencing kit and the
Applied Biosystems (Foster City, Calif.) automated DNA-sequencing system
3700. PCR was performed using the Failsafe PCR system (Epicentre). The PCR
conditions were as follows: one cycle of denaturation at 94°C for 5 min followed
by five cycles at 94°C for 3 min, 55°C for 30 s, and 72°C for 5 min, followed by
30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 5 min. A 5,281-nucleotide
acrRAB gene fragment was amplified from genomic DNA of P. mirabilis G151
using the following primers derived from sequence generated in sequencing the
transposon insertion: forward primer (5⬘-GCGTTTCTGGATGTTGCTCTT-3⬘)
and reverse primer (5⬘-GATTACTTAGTTTGGTGCGGA-3⬘). This gene fragment was then ligated into the pCR 2.1-TOPO TA cloning vector (Invitrogen)
according to the manufacturer’s instructions. The resulting plasmid, pCLL3430,
MATERIALS AND METHODS
Bacterial strains and growth conditions. The bacterial strains, plasmids, and
transposons used in this study are listed in Table 1. A typical clinical isolate of P.
mirablis, G151, was selected from a tigecycline phase II clinical trial. The E. coli
strains used included Top10 (Invitrogen, Carlsbad, Calif.), Transformax EC100D
pir-116 (Epicentre, Madison, Wis.), DM1 (Gibco Life Technologies, Rockville,
Md.), and two laboratory strains, one wild type and the other an acrAB deletion
mutant (17). The strains were grown in Luria-Bertani (LB) broth or agar in the
* Corresponding author. Mailing address: Department of Infectious
Disease, Wyeth Research, Room 3218, Bldg. 200, 401 North Middletown Rd., Pearl River, NY 10965. Phone: (845) 602-5203. Fax: (845)
602-5671. E-mail: [email protected].
665
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Tigecycline has good broad-spectrum activity against many gram-positive and gram-negative pathogens with
the notable exception of the Proteeae. A study was performed to identify the mechanism responsible for the
reduced susceptibility to tigecycline in Proteus mirabilis. Two independent transposon insertion mutants of P.
mirabilis that had 16-fold-increased susceptibility to tigecycline were mapped to the acrB gene homolog of the
Escherichia coli AcrRAB efflux system. Wild-type levels of decreased susceptibility to tigecycline were restored
to the insertion mutants by complementation with a clone containing a PCR-derived fragment from the
parental wild-type acrRAB efflux gene cluster. The AcrAB transport system appears to be associated with the
intrinsic reduced susceptibility to tigecycline in P. mirabilis.
This study
Invitrogen
FmcrA ⌬(mrr-hsdRMS-mcrBC) ␾80lacZ ⌬M15
⌬lacX74 deoR recA1 ara⌬139 ⌬(ara-leu)7697
galU ⌬galK ␭ rpsL nupG pir-116
F⫺ mcrA ⌬(mrr-hsdRMS-mcrBC) ␾80lacZ ⌬M15
⌬lacX74 deoR recA1 ara⌬139 ⌬(ara-leu)7697
galU galK rpsL (Strr) endA1 nupG
AcrAB⫺
This study
17
This study
17
This study
Epicentre
Cloning strain
Top 10 with cloned AcrAB
E.
E.
E.
E.
E.
E.
E. coli
E. coli
GC 7021
AG100
GC7369
AG100A
GC 7368
EC100Dpir-116
Top 10
GC 7012
AcrAB deletion parent
AG100 with cloned AcrAB
AcrAB deletion strain
AG100A with cloned AcrAB
Transposon rescue cloning strain
Fdam13::Tn9 (Cmr) dcm mcrB hsdRM⫹ gal-2 ara
lac thr leu (Tonr Tsxr)
DM1 with cloned AcrAB
pCLL3431
pCLL3431
pCLL3431
pCLL3431
coli
coli
coli
coli
coli
coli
pCLL3431
pCLL3431
pCLL3431
EZ::Tn
EZ::Tn
EZ::Tn
EZ::Tn
P. mirabilis
P. mirabilis
P. mirabilis
P. mirabilis
P. mirabilis
P. mirabilis
E. coli
G151
GC 7020
GC 6899
GC 6900
GC 7018
GC 7019
DM1
Clinical isolate
G151 with cloned AcrAB
Insertion mutant
Insertion mutant
Insertion mutant with cloned AcrAB
Insertion mutant with cloned AcrAB
Cloning strain
Genotype
Characteristics
Transposon
Plasmid
Organism
Strain
was then modified by cloning an 800-bp HindIII fragment containing the gentamicin cassette from pUCGm into the HindIII site in the multiple cloning site.
The resulting plasmid, pCLL3431, was used in the complementation assays.
Genomic DNA was prepared from LB broth cultures using the Puregene DNA
isolation kit (Gentra Systems, Minneapolis, Minn.) according to the manufacturer’s instructions.
DNA isolation and transposon mapping. Transposon mapping was performed
by rescue cloning the transposon and flanking chromosomal DNA. This was
achieved by digestion of genomic DNA from insertion mutants using three
individual restriction enzymes, EcoRI, PvuII, and BglII, none of which cut within
the transposon. One microgram of fragmented DNA was self-ligated and then
transformed into pir E. coli. Transformants were selected on LB agar containing
50 ␮g of kanamycin/ml. Plasmid DNA from the transformants was prepared
using a QIAprep Spin Miniprep kit (Qiagen Inc., Chatsworth, Calif.) according
to the manufacturer’s instructions. Only the BglII-cleaved preparation generated
a clone containing the full-length sequence of the disrupted gene.
Southern blot analysis. Transposon insertion was confirmed by the digestion
of the bacterial genomic DNA with restriction enzymes followed by electrophoresis in 1.0% agarose. The DNA fragments were then transferred to a nylon
membrane (Hybond N⫹; Amersham Pharmacia Biotech, Piscataway, N.J.) using
vacuum blotting. Hybridization and chemiluminescent detection were performed
using the ECL Random-Prime Labeling and Detection System (Amersham
Pharmacia Biotech). A 1-kb XbaI-XhoI fragment specific for kanamycin resistance gene sequences was isolated from the EZ:TN construct, labeled by enhanced chemiluminescence, and hybridized to the immobilized fragments.
Northern blot analysis. Total P. mirabilis RNA was prepared using the Rneasy
minikit (Qiagen). The RNA was electrophoresed and blotted as described by
Sambrook et al. (21). Hybridization and detection were performed using the
AlkPhos Direct labeling and detection system (Amersham Pharmacia Biotech).
The probe was an 800-nucleotide internal EcoRI fragment generated from the
acrRAB gene fragment of P. mirabilis in pCLL3430.
Bioinformatics. Open reading frames from sequence data were translated
using the EditSeq software program (DNAStar, Inc., Madison, Wis.) and used to
perform a homology search with BLASTP (1).
Nucleotide sequence accession number. The nucleotide and protein sequences
of genes described from P. mirabilis are registered in GenBank under accession
no. AY061647.
RESULTS
Transposon mutagenesis and mapping. A typical clinical
isolate of P. mirabilis, G151, for which the tigecycline MIC is 4
␮g/ml, was selected for identification of the mechanism responsible for decreased susceptibility to tigecycline. Two independent transposon insertion mutants, GC 6899 and GC 6900,
were isolated using the EZ::TN ⬍R6K␥ori/KAN-2⬎ transposon (Epicentre). Southern blot analysis of DNA isolated from
each transposon insertion mutant was performed using a kanamycin resistance gene probe. As shown in Fig. 1, the transposon was inserted into the P. mirabilis chromosome.
The chromosomal DNA flanking the insertion site was rescue cloned, and nucleotide sequencing was performed. A
BLASTP search of the translated open reading frames from
each insertion mutant revealed insertion into a homolog of the
acrB gene of E. coli. GC 6899 had an insertion at bp 1200, and
GC 6900 had an insertion at bp 1925. Sequence analysis of the
BglII rescue clones also revealed E. coli homologs of the acrA
and acrR genes. Figure 2 is a linear view of transposon insertions and gene cluster arrangement.
Susceptibility profiles of insertion mutants. The susceptibilities of the wild-type parental P. mirabilis strain G151 and the
insertion mutants GC 6899 and GC 6900 to a number of
antibiotics were determined (Table 2). Compared to those for
the parent, the MICs of tigecycline for the two insertion mutants showed a 16-fold reduction, and the MICs of minocycline
showed a 32-fold reduction. Transposon insertion did not af-
Downloaded from http://aac.asm.org/ on February 27, 2014 by PENN STATE UNIV
TABLE 1. Bacterial strains used in this study
ANTIMICROB. AGENTS CHEMOTHER.
This study
This study
This study
This study
This study
This study
Invitrogen
VISALLI ET AL.
Reference
666
VOL. 47, 2003
fect susceptibilities to other antibiotics that are not substrates
of the AcrAB efflux system, including ampicillin, ampicillinsulbactam, ceftriaxone, and imipenem. The insertion mutants
were also characterized by their increased susceptibilities to
known AcrAB substrates, which included both antibiotics, such
as ciprofloxacin, trimethoprim, novobiocin, and chloramphenicol, and dyes and detergents, such as ethidium bromide, acriflavin, and sodium dodecyl sulfate. As shown in Table 2, the
MICs of all of the substrates tested for the insertion mutants
showed decreases (range, 2- to 64-fold) compared to those for
the wild-type parent.
Cloning of the wild-type acrRAB gene complex and complementation studies. Using the information obtained from the
full-length nucleotide sequence generated by sequencing the
insertion clones, a 5,128-bp acrRAB gene fragment from P.
mirabilis G151 was amplified by PCR and ligated into the
pCR2.1-TOPO TA cloning vector. The insert was then sequenced and compared to known homologs. This revealed that
the acrB gene of P. mirabilis had 75% amino acid identity to
the acrB genes found in E. coli, K. pneumoniae, and Entero-
667
bacter aerogenes. Interestingly, the acrB genes of E. coli, K.
pneumoniae, and E. aerogenes have 85 to 88% identity to each
other. The acrA gene of P. mirabilis also had 75% amino acid
identity to the acrA genes found in E. coli, K. pneumoniae, and
E. aerogenes.
The plasmid pCLL3430, containing a 5,128-bp acrRAB gene
fragment from P. mirabilis G151, was altered by inserting a
gentamicin resistance cassette into the multiple cloning site
upstream of the acrRAB gene fragment. The resulting plasmid,
pCLL3431, was then transformed into the wild-type parental P.
mirabilis strain, the two insertion mutants, and four E. coli
strains and selected for on LB agar containing 10 ␮g of gentamicin/ml. The susceptibilities of these strains were determined and are shown in Table 2. The overexpression of the
cloned acrRAB gene fragment in the P. mirabilis wild-type
parent (GC 7020) resulted in a fourfold elevation of the tigecycline MIC. The tigecycline MICs for P. mirabilis insertion
mutants containing pCLL3431 (GC 7018 and GC 7019)
showed a fourfold elevation over that for the wild-type strain.
None of the strains containing pCLL3431 could be evaluated
for a change in the ampicillin or ampicillin-sulbactam MICs
because of the TEM-1 ␤-lactamase expressed as a selection
marker on pCLL3431. The MICs of tigecycline and minocycline for all wild-type E. coli strains containing pCLL3431 and
expressing the P. mirabilis acrAB genes showed no elevation.
However, the tigecycline MIC for the E. coli acrAB deletion
strain containing pCLL3431, GC 7368, showed a fourfold elevation compared to that for the parent strain. Again, all strains
were tested with known substrates of AcrAB (Table 2). Insertion mutants containing pCLL3431 showed a wild-type parental phenotype for all substrates. No wild-type E. coli strains
containing pCLL3431 showed a change in their susceptibilities
to any of the substrates tested compared to their E. coli parent
strains. The MICs of most substrates for the E. coli acrAB
deletion mutant expressing the P. mirabilis AcrAB genes
showed significant elevations compared to those for the parent
(Table 2).
acrRAB expression. To determine if the cloned acrRAB
genes in the E. coli strains for which the tigecycline MIC was
not elevated following transformation with pCLL3431 were
being transcribed, Northern blot analysis of the wild-type parent strain (G151), GC 7020, and both insertion mutants, GC
6899 and GC 6900, along with their complements, GC 7018
and GC 7019, and two E. coli strains with and without
pCLL3431 was performed. Transcripts were detected in all
strains containing pCLL3431, including the E. coli strains (data
not shown). The E. coli acrAB deletion mutant expressing the
P. mirabilis AcrAB genes and its parent strain were not tested,
as they were not available at the time of Northern blot analysis.
The data suggest that expression of the acrRAB gene fragment
from P. mirabilis in the various P. mirabilis strains was correlated with the observed MIC changes. However, the expression
of the P. mirabilis AcrAB efflux system in wild-type E. coli
strains did not affect susceptibilities to the various substrates
tested.
DISCUSSION
FIG. 2. Linear arrangement of P. mirabilis acrAB gene cluster.
Efflux systems have been identified as major contributors to
bacterial resistance. This has furthered studies to identify nu-
Downloaded from http://aac.asm.org/ on February 27, 2014 by PENN STATE UNIV
FIG. 1. Genomic Southern blot showing transposon insertion into
the chromosomes of two independently isolated insertion mutants.
Lane 1, chromosomal DNA from the wild-type parent (G151); lane 2,
positive control plasmid DNA (pCLL2300); lanes 3 and 4, chromosomal DNA from each of the insertion mutants (GC 6899 and
GC6900). The presence of transposon insertion was detected by probing with the kanamycin resistance gene present in the transposable
element.
AcrAB-MEDIATED TIGECYCLINE RESISTANCE
⫹, wild-type acrAB present; ⫹⫹, overexpression of P. mirabilis acrAB; ⫺, not present.
TGC, tigecycline; MIN, minocycline; AMP, ampicillin; SAM, ampicillin-sulbactam (2:1); CRO, ceftriaxone; CIP, ciprofloxacin; IMP, imipenem; NOV, novobiocin; EtBr, ethidium bromide; CHL, chloramphenicol;
ERY, erythromycin; ACR, acriflavine; TRM, trimethoprim; SDS, sodium dodecyl sulfate.
c
WT, wild type.
b
a
1,024
512
128
512
128
512
⬎2,048
⬎2,048
⬎2,048
⬎2,048
64
⬎2,048
16
16
ⱕ1.0
16
ⱕ1.0
16
ⱕ1.0
ⱕ1.0
ⱕ1.0
ⱕ1.0
ⱕ1.0
ⱕ1.0
128
64
16
128
16
128
32
32
32
16
2
8
⬎256
⬎256
16
⬎256
8
⬎256
32
128
64
128
4
256
G151 WT
GC 7020
GC 6899
GC 7018
GC 6900
GC 7019
DM1
GC 7021
AG100
GC 7369
AG100A
GC 7368
c
P. mirabilis
P. mirabilis
P. mirabilis
P. mirabilis
P. mirabilis
P. mirabilis
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
⫹
⫹⫹
⫺
⫹⫹
⫺
⫹⫹
⫹
⫹
⫹
⫹⫹
⫺
⫹⫹
4
16
0.25
16
0.25
16
0.5
0.5
0.5
0.5
0.25
1
32
⬎64
1
⬎64
1
⬎64
0.5
2
1
2
0.125
4
0.5
⬎64
0.25
⬎64
0.25
⬎64
2
⬎64
2
⬎64
2
⬎64
0.5
2
0.5
4
0.25
2
2
⬎64
2
64
1
64
ⱕ0.06
ⱕ0.06
ⱕ0.06
ⱕ0.06
ⱕ0.06
ⱕ0.06
ⱕ0.06
ⱕ0.06
ⱕ0.06
ⱕ0.06
ⱕ0.06
ⱕ0.06
0.06
0.125
ⱕ0.015
0.06
ⱕ0.015
0.06
ⱕ0.015
ⱕ0.5
ⱕ0.015
ⱕ0.015
ⱕ0.015
ⱕ0.015
2
2
4
2
2
2
0.25
0.25
ⱕ0.125
ⱕ0.125
ⱕ0.125
0.25
16
8
ⱕ0.25
8
ⱕ0.25
4
512
256
128
128
1
64
2,048
2,048
128
2,048
128
2,048
128
128
512
256
4
512
64
128
8
128
16
64
512
256
8
8
1
16
ACR
CHL
EtBr
NOV
IMP
TGC
MIN
AMP
SAM
CRO
CIP
MIC (␮g/ml)b
acrABa
Organism
Strain
ANTIMICROB. AGENTS CHEMOTHER.
merous efflux systems in a broad range of organisms (25). The
well-characterized AcrAB efflux pump in E. coli confers intrinsic resistance to many structurally diverse lipophilic compounds, including detergents, dyes, and antibiotics (7, 13, 14,
19). In this study, a homolog of the E. coli AcrAB efflux system
was identified in P. mirabilis. This gene cluster appears to be
responsible, at least in part, for the intrinsic reduced susceptibility to tigecycline in P. mirabilis, as shown by transposon
mutagenesis and complementation studies.
The AcrAB efflux system in E. coli is known to transport
hydrophobic substrates, including dyes, detergents, and antibiotics, directly out of the cell without accumulation in the
periplasm (12). This efflux system was previously described as
a tripartite complex consisting of AcrA, a periplasmic lipoprotein; an inner membrane transporter, AcrB; and an outer
membrane channel, TolC (3, 12).
Members of several other genera, including E. coli, K. pneumoniae, and E. aerogenes, have close homologs of the AcrAB
efflux system (11, 16, 22) yet do not show decreased susceptibility to tigecycline. The reason for the functional differences
has yet to be found.
The transfer of resistance determinants among bacterial
genera is a major concern for the spread of resistance. In this
study, the possibility that the transfer and subsequent expression of the P. mirabilis AcrAB efflux system in different genera
would result in decreased susceptibilty to tigecycline or other
known substrates was investigated. When the AcrAB efflux
system of P. mirabilis was expressed in various E. coli strains,
wild-type strains did not show a change in their susceptibilities
to any of the antibiotics or substrates tested. Although we did
not express TolC from P. mirabilis in the E. coli clones expressing the P. mirabilis acrAB genes to determine the effect of the
third component of this efflux system on tigecycline susceptibility, it is unlikely that the genes encoding TolC would be
transferred to E. coli in a natural setting because they are
located some distance from the AcrAB operon on the P. mirabilis chromosome. This suggests that the mobilization of the
AcrAB pump from Proteus onto a plasmid does not pose an
immediate threat of acquired resistance to tigecycline in E.
coli.
It appears that even though the P. mirabilis AcrAB system
was expressed in wild-type E. coli, it had no effect on susceptibilities to these substrates. Similar results have been previously reported in which the MexAB-OprM system from
Pseudomonas aeruginosa was expressed in E. coli but showed
no susceptibility changes. However, when the same P. aeruginosa efflux system was expressed in an E. coli strain containing
an acrAB deletion, there were decreases in susceptibilities to a
variety of substrates tested (23). We show a similar phenomenon in the E. coli acrAB deletion strain expressing the P.
mirabilis AcrAB efflux system.
There are several possible explanations for the apparent lack
of effect of P. mirabilis AcrAB expression in wild-type E. coli
strains. First, there could be a conformational preference for
the E. coli proteins over the P. mirabilis proteins. Regulation by
the E. coli system might be preventing the expression of the P.
mirabilis AcrAB efflux system. We looked at expression of the
P. mirabilis AcrAB efflux system in E. coli via RNA levels.
Since we did not look at actual protein levels, it is possible that
the P. mirabilis transcripts are not being translated (9). Fur-
Downloaded from http://aac.asm.org/ on February 27, 2014 by PENN STATE UNIV
ERY
TRM
SDS
VISALLI ET AL.
TABLE 2. Substrate profiles of strains expressing or lacking acrAB
668
VOL. 47, 2003
AcrAB-MEDIATED TIGECYCLINE RESISTANCE
ACKNOWLEDGMENTS
We thank David Fruhling for technical assistance with sequencing
and Keith Poole for the kind gift of E. coli strains AG100 and
AG100A.
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and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation
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2. Boucher, H. W., C. B. Wennersten, and G. M. Eliopoulos. 2000. In vitro
activities of the glycylcycline GAR-936 against gram-positive bacteria. Antimicrob. Agents Chemother. 44:2225–2229.
3. Fralick, J. A. 1996. Evidence that TolC is required for functioning of the
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thermore, it is possible that there are other, yet-undefined
factors involved in this efflux system.
The reason for the differences between the MIC s for wildtype and acrAB deletion E. coli strains expressing the P. mirabilis AcrAB efflux system has not yet been explained and deserves further exploration. However, these experiments
suggest that the simple transfer of acrRAB genes from P. mirabilis to other genera may not play a significant role in acquired
resistance to glycylcyclines.
Although tigecycline is unaffected by classical tetracycline
resistance determinants in E. coli, the identification of the
AcrAB efflux pump in P. mirabilis, which is related to the
reduced susceptibility of the organism to tigecycline, suggests
that an efflux pump which can act upon tigecycline exists. The
reason that the AcrAB pump acts on tigecycline in P. mirabilis
but not in other genera has yet to be determined. Fortunately,
there does not appear to be an immediate threat of the spread
of resistance to tigecycline.
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