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
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. 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
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
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
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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-
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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.
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-
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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-
⫹, 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
G151 WT GC 7020 GC 6899 GC 7018 GC 6900 GC 7019 DM1 GC 7021 AG100 GC 7369 AG100A GC 7368
ANTIMICROB. AGENTS CHEMOTHER.
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 64 128 8 128 16 64 512 256 8 8 1 16 2,048 2,048 128 2,048 128 2,048 128 128 512 256 4 512 16 8 ⱕ0.25 8 ⱕ0.25 4 512 256 128 128 1 64 2 2 4 2 2 2 0.25 0.25 ⱕ0.125 ⱕ0.125 ⱕ0.125 0.25 0.06 0.125 ⱕ0.015 0.06 ⱕ0.015 0.06 ⱕ0.015 ⱕ0.5 ⱕ0.015 ⱕ0.015 ⱕ0.015 ⱕ0.015 ⱕ0.06 ⱕ0.06 ⱕ0.06 ⱕ0.06 ⱕ0.06 ⱕ0.06 ⱕ0.06 ⱕ0.06 ⱕ0.06 ⱕ0.06 ⱕ0.06 ⱕ0.06 0.5 2 0.5 4 0.25 2 2 ⬎64 2 64 1 64 0.5 ⬎64 0.25 ⬎64 0.25 ⬎64 2 ⬎64 2 ⬎64 2 ⬎64 32 ⬎64 1 ⬎64 1 ⬎64 0.5 2 1 2 0.125 4 4 16 0.25 16 0.25 16 0.5 0.5 0.5 0.5 0.25 1 ⫹ ⫹⫹ ⫺ ⫹⫹ ⫺ ⫹⫹ ⫹ ⫹ ⫹ ⫹⫹ ⫺ ⫹⫹ P. mirabilis P. mirabilis P. mirabilis P. mirabilis P. mirabilis P. mirabilis E. coli E. coli E. coli E. coli E. coli E. coli
NOV IMP
Strain
c
Organism
acrABa
TGC
MIN
AMP
SAM
CRO
CIP
MIC (g/ml)b
EtBr
CHL
ERY
ACR
TRM
SDS
VISALLI ET AL.
TABLE 2. Substrate profiles of strains expressing or lacking acrAB
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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-
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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. ACKNOWLEDGMENTS We thank David Fruhling for technical assistance with sequencing and Keith Poole for the kind gift of E. coli strains AG100 and AG100A. REFERENCES 1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389–3402. 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 Mar/AcrAB efflux pump of Escherichia coli. J. Bacteriol. 178:5803–5805. 4. Gales, A. C., and R. N. Jones. 2000. Antimicrobial activity and spectrum of the new glycylcycline, GAR-936 tested against 1,203 recent clinical isolates. Diagn. Microbiol. Infect. Dis. 36:19–36. 5. Hoellman, D. B., G. A. Pankuch, M. R. Jacobs, and P. A. Appelbaum. 2000. Antipneumococcal activities of GAR-936 (a new glycylcycline) compared to those of nine other agents against penicillin-susceptible and penicillin-resistant pneumococci. Antimicrob. Agents Chemother. 44:1085–1088. 6. Jacoby, G. A. 1994. Extrachromosomal resistance in gram-negative organisms: the evolution of -lactamase. Trends Microbiol. 2:358–361. 7. Kopytek, S. J., J. C. D. Dyer, G. S. Knapp, and J. C. Hu. 2000. Resistance to methotrexate due to AcrAB-dependent export from Escherichia coli. Antimicrob. Agents Chemother. 44:3210–3212.
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8. Lederberg, J., and E. M. Lederberg. 1952. Replica plating and indirect selection of bacterial mutants. J. Bacteriol. 63:399–406. 9. Lesley, S. A., J. Graziano, C. Y. Cho, M. W. Knuth, and H. E. Klock. 2002. Gene expression response to misfolded protein as a screen for soluble recombinant protein. Protein Eng. 15:153–160. 10. Levy, S. 1994. Balancing the drug-resistance equation. Trends Microbiol. 2:341–342. 11. Linde, H. J., F. Notka, C. Irtenkauf, J. Decker, J. Wild, H. H. Niller, P. Heisig, and N. Lehn. 2002. Increase in MICs of ciprofloxacin in vivo in two closely related clinical isolates of Enterobacter cloacae. J. Antimicrob. Chemother. 49:625–630. 12. Ma, D., D. N. Cook, J. E. Hearst, and H. Nikaido. 1994. Efflux pumps and drug resistance in gram-negative bacteria. Trends Microbiol. 2:489–493. 13. Mazzariol, A., T. Yutaka, T. M. Kanegawa, G. Cornaglia, and H. Nikaido. 2000. High-level flouroquinolone-resistant clinical isolates of Escherichia coli overproduce multidrug efflux protein AcrA. Antimicrob. Agents Chemother. 44:3441–3443. 14. McMurry, L. M., M. Oethinger, and S. Levy. 1998. Overexpression of marA, soxS, or acrAB produces resistance to triclosan in laboratory and clinical strains of Escherichia coli. FEMS Microbiol. Lett. 166:305–309. 15. National Committee for Clinical Laboratory Standards. 2000. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard M7-A5, 5th ed., vol. 17. National Committee for Clinical Laboratory Standards, Wayne, Pa. 16. Nikaido, H. 1996. Multidrug efflux pumps of gram-negative bacteria. J. Bacteriol. 178:5853–5859. 17. Okusu, H., D. Ma, and H. Nikaido. 1996. AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli multiple-antibiotic-resistance (Mar) mutants. J. Bacteriol. 178:306–308. 18. Petersen, P. J., N. V. Jacobus, W. J. Weiss, P. E. Sum, and R. T. Testa. 1999. In vitro and in vivo antibacterial activities of a novel glycylcycline, the 9-t-butylglycylamido derivative of minocycline (GAR-936). Antimicrob. Agents Chemother. 43:738–744. 19. Piddock, L. V., D. G. White, K. Gensberg, L. Pumpbe, and D. J. Griggs. 2000. Evidence for an efflux pump mediating multiple antibiotic resistance in Salmonella enterica serovar Typhimurium. Antimicrob. Agents Chemother. 44:3118–3121. 20. Roberts, M. C. 1994. Epidemiology of tetracycline-resistance determinants. Trends Microbiol. 2:353–357. 21. Sambrook, J. E., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 22. Sanchez, L., W. Pan, M. Vinas, and H. Nikaido. 1997. The acrAB homolog of Haemophilus influenzae codes for a functional multidrug efflux pump. J. Bacteriol. 179:6855–6857. 23. Srikumar, R., T. Kon, N. Gotoh, and K. Poole. 1998. Expression of Pseudomonas aeruginosa multidrug efflux pumps MexA-MexB-OprM and MexCMexD-OprJ in a multidrug-sensitive Escherichia coli strain. Antimicrob. Agents Chemother. 42:65–71. 24. Sum, P. E., and P. J. Petersen. 1999. Synthesis and structure-activity relationship of novel glycylcycline derivatives leading to the discovery of GAR936. Bioorg. Med. Chem. Lett. 9:1459–1462. 25. Van Bambeke, F., E. Balzi, and P. M. Tulkens. 2000. Antibiotic efflux pumps. Biochem. Pharmacol. 60:457–470.
