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Antimicrob Agents Chemother. May 2005; 49(5): 1865–1871.
PMCID: PMC1087644

A Novel MATE Family Efflux Pump Contributes to the Reduced Susceptibility of Laboratory-Derived Staphylococcus aureus Mutants to Tigecycline

Abstract

Tigecycline, an expanded-broad-spectrum glycylcycline antibiotic is not affected by the classical tetracycline resistance determinants found in Staphylococcus aureus. The in vitro selection of mutants with reduced susceptibility to tigecycline was evaluated for two methicillin-resistant S. aureus strains by serial passage in increasing concentrations of tigecycline. Both strains showed a stepwise elevation in tigecycline MIC over a period of 16 days, resulting in an increase in tigecycline MIC of 16- and 32-fold for N315 and Mu3, respectively. Transcriptional profiling revealed that both mutants exhibited over 100-fold increased expression of a gene cluster, mepRAB (multidrug export protein), encoding a MarR-like transcriptional regulator (mepR), a novel MATE family efflux pump (mepA), and a hypothetical protein of unknown function (mepB). Sequencing of the mepR gene in the mutant strains identified changes that presumably inactivated the MepR protein, which suggested that MepR functions as a repressor of mepA. Overexpression of mepA in a wild-type background caused a decrease in susceptibility to tigecycline and other substrates for MATE-type efflux pumps, although it was not sufficient to confer high-level resistance to tigecycline. Complementation of the mepR defect by overexpressing a wild-type mepR gene reduced mepA transcription and lowered the tigecycline MIC in the mutants. Transcription of tet(M) also increased by over 40-fold in the Mu3 mutant. This was attributed to a deletion in the promoter region of the gene that removed a stem-loop responsible for transcriptional attenuation. However, overexpression of the tet(M) transcript in a tigecycline-susceptible strain was not enough to significantly increase the MIC of tigecycline. These results suggest that the overexpression of mepA but not tet(M) may contribute to decreased susceptibility of tigecycline in S. aureus.

Staphylococcus aureus is an important human pathogen causing infections that range in severity from superficial skin abscesses to more serious invasive diseases (32). Methicillin-resistant S. aureus (MRSA) is a problem in hospitals worldwide (1). The transfer of vancomycin resistance from Enterococcus species into S. aureus (8) and the emerging problem of MRSA infections in the community setting (27) are of particular concern. Tigecycline, a novel glycylcycline antibiotic, exhibits good antimicrobial activity against a broad spectrum of gram-positive and gram-negative pathogens including MRSA and S. aureus strains with intermediate and high levels of resistance to vancomycin (7, 23).

Tigecycline belongs to the glycylcycline class of antibiotics, which are not affected by either of the classical mechanisms of resistance to tetracyclines, specific efflux pumps or ribosomal protection (24). Glycylcyclines have a higher binding affinity for the ribosome than do classical tetracyclines (3, 5), and recent studies have suggested that steric hindrance due to a bulky side group may enable tigecycline to overcome most of the known tetracycline resistance mechanisms (3).

Tigecycline exhibits potent antimicrobial activity against gram-positive and most gram-negative species. However, Pseudomonas aeruginosa and Proteus mirabilis species have demonstrated intrinsic reduced susceptibility to tigecycline, which has been attributed to the resistance nodulation cell division family AcrAB and MexAB-OprM pumps (9, 31). Surveillance studies carried out have not identified any naturally occurring S. aureus isolates with decreased susceptibility to tigecycline to date. However, the ability of S. aureus to develop reduced susceptibility to tigecycline when grown by serial passaging under selective pressure in vitro was demonstrated previously (K. Kuwahara-Arai, H. Hanaki, K. Ohkuma, and K. Hiramatsu, Abstr. 42nd Intersci. Conf. Antimicrob. Agents Chemother., abstr. E-1137, 2002).

In this study, the isolation of mutants with reduced susceptibility to tigecycline was evaluated for two MRSA strains by serial passage in increasing concentrations of this antibiotic. The mechanisms of reduced susceptibility were investigated by transcription profile analysis.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

The bacterial strains and plasmids used in this study are listed in Table Table1.1. S. aureus strains were routinely propagated in Trypticase soy broth (BBL, Cockeysville, Md.) or in Mueller-Hinton II broth (MHB II; BBL). Escherichia coli cultures were grown in Luria-Bertani medium (LB; Difco, Detroit, Mich.). Gradient plates were prepared using brain heart infusion agar (Difco). The following antibiotics were incorporated into the medium when appropriate: ampicillin, 50 μg/ml; chloramphenicol, 7.5 or 15 μg/ml; kanamycin, 50 μg/ml.

TABLE 1.
Strains and plasmids used in this study

Antibiotic susceptibility testing.

The MICs of various antibiotics and substrates were determined by broth microdilution or broth macrodilution as indicated, using twofold serial dilutions in MHB II using standard NCCLS procedures (21). Microdilution MIC testing for tigecycline was performed by using the referenced method (using fresh MHB II, <12 h old) (22). The following antibiotics and substrates were used in this study and prepared fresh on the day of testing: minocycline, vancomycin, tobramycin, oxacillin, tetracycline, ciprofloxacin, norfloxacin, ethidium bromide (EtBr), tetraphenylphosphonium bromide (TPP), and erythromycin (Sigma Chemical Co., St. Louis, Mo.); imipenem (United States Pharmacopoeia, Rockville, Md.); levofloxacin (Johnson & Johnson, Spring House, Pa.); and tigecycline, piperacillin-tazobactam, DMG-MINO (9-N,N-dimethylglycylamido-minocycline), and DMG-DMDOT (9-N,N-dimethylglycylamido-6-demethyl-6-deoxytetracycline) (Wyeth Research, Pearl River, N.Y.). DMG-MINO and DMG-DMDOT belong to the glycylcycline family of antibiotics (30). Gradient plates were prepared using brain heart infusion agar and contained gradients of tigecycline, EtBr, TPP, DMG-MINO, DMG-DMDOT, or imipenem as appropriate. Paper strips were soaked in 0.5 McFarland suspensions of overnight cultures grown on agar plates, applied to gradient plates, and removed after 5 min. Plates were incubated for 20 to 22 h at 35°C.

Selection of decreased susceptibility to tigecycline.

Mutants with decreased susceptibility to tigecycline were isolated following serial passage of S. aureus strains Mu3 and N315 in increasing concentrations of this drug. Briefly, tubes containing 2 ml of MHB II with twofold-increasing concentrations of tigecycline were inoculated with 5 × 105 CFU/ml of Mu3 or N315. Following overnight incubation at 35°C without shaking, the MIC was determined by the macrodilution method as the lowest drug concentration to inhibit bacterial growth. The tubes with the highest drug concentration that permitted growth were used to inoculate a series of tubes containing fresh MHB II with twofold-increasing concentrations of tigecycline adjusted to a starting concentration of 5 × 105 CFU/ml. These were incubated overnight at 35°C without shaking as before. Again, the lowest drug concentration that inhibited growth was determined by the macrodilution method to be the MIC, and the culture growing at the highest drug concentration was used to prepare the inoculum for the next passage. This process was repeated for 16 days. The inoculating cultures were saved for each of the 16 passages, and the ribotype of each was determined to confirm the integrity of the selected mutant using the RiboPrinter system (Qualicon, Wilmington, Del.) according to the manufacturer's instructions. Each strain was analyzed using two restriction enzymes, EcoRI and PvuII.

DNA manipulations.

Standard nucleic acid techniques were performed according to the methods of Sambrook et al. (28). The following kits and reagents were used where appropriate and in accordance with the manufacturers' instructions: FailSafe PCR system and Fast-Link DNA ligation kit (Epicentre, Madison, Wis.), QIAquick gel extraction kit (QIAGEN, Inc., Valencia, Calif.), pCR4-TOPO cloning vector (Invitrogen), Spin mini-prep kit (QIAGEN), and Big Dye, version 3.1, sequencing kit (Applied Biosystems, Foster City, Calif.). Oligonucleotide primers (GeneLink, Hawthorne, N.Y.) used in the study are listed in Table Table2.2. For PCRs, DNA was denatured for 1 min at 94°C, primers were annealed for 1 min at a primer-specific temperature, and a 1-min extension at 72°C was allowed per kb of DNA. This was repeated for 30 cycles followed by a final 10-min extension at 72°C. To detect point mutations, gel-purified DNA fragments were cloned into pCR4-TOPO and inserts from three independent PCRs and multiple pCR4-TOPO clones were sequenced in both directions using the M13 forward and reverse primers. For purification of plasmids from S. aureus, lysostaphin was added to buffer P1 at a final concentration of 10 μg per ml of starting culture followed by a 30-min incubation at 37°C.

TABLE 2.
Primers and probes used in this study

Transcription profile analysis of RNA and DNA samples.

Chromosomal DNA was prepared and labeled as described by Dunman et al. (10). For RNA preparation, S. aureus cultures grown overnight in 5 ml of Trypticase soy broth were used to inoculate 50 ml of MHB II in a 250-ml Erlenmeyer flask to a starting optical density at 650 nm of 0.05. Cells were grown at 37°C with shaking at 200 rpm for approximately 3.5 h to mid-exponential phase (corresponding to an optical density at 650 nm of 0.6 to 1.0 under the growth conditions described above). An equal volume of an ice-cold acetone-alcohol (1:1) solution was added, and cells were harvested by centrifugation. Cell pellets were resuspended in 500 μl of TE buffer (10 mM Tris, 1 mM EDTA, pH 8) and lysed using the FastPrep system (Qbiogene, Carlsbad, Calif.). RNA was isolated using the RNeasy mini kit (QIAGEN), and on-column DNase treatment was performed using the RNase-free DNase kit (QIAGEN) according to the manufacturers recommendations. Reverse transcription, cDNA fragmentation, and terminal labeling of cDNA fragments with biotin were carried out in accordance with the manufacturer's protocol for antisense prokaryotic arrays (Affymetrix, Inc., Santa Clara, Calif.). Custom-designed S. aureus GeneChips (Affymetrix) are described in detail by Dunman et al. (10). Hybridization and detection of labeled material and analysis of data has been described previously (4, 11).

Construction of plasmids overexpressing tet(M), mepR, or mepA.

tet(M), mepR, and mepA gene fragments were PCR amplified using Mu3 chromosomal DNA template and primers TetMF1, TetMR1, MepRF1, MepRR1, MepAF1, and MepAR1, respectively (as detailed in Table Table2).2). PCR fragments were gel purified, digested with either EcoRI or SacI where appropriate and cloned into pALC2073 cut with either EcoRI or SacI, thus placing these genes under the control of a tetracycline-inducible promoter. Ligation mixtures were transformed into chemically competent Escherichia coli DH5α cells (Invitrogen) as specified by the manufacturer. The orientation and integrity of the inserts were verified by restriction mapping and sequencing. Plasmid constructs containing inserts of the tet(M), mepR, or mepA gene in either the sense or antisense direction are listed in Table Table1.1. The antisense constructs were used as negative controls. Plasmids were electroporated into S. aureus strain RN4220 (12) with selection on chloramphenicol (7.5 μg/ml) and subsequently transduced into the relevant N315 or Mu3 strains using [var phi]80α by standard methods (12). Real-time (RT)-PCR analysis indicated that the mepA, mepR, and tet(M) genes were highly expressed even in the absence of tetracycline inducer, suggesting that the regulation of these constructs was leaky.

RT-PCR.

Oligonucleotide primers and probes used for RT-PCR (Table (Table2)2) were designed with Primer Express software, version 2.0 (Applied Biosystems), and purchased from QIAGEN. The probes were labeled by the manufacturer with the reporter dye 6-carboxyfluorescein at the 5′ end and the quencher dye 6-carboxytetramethylrhodamine at the 3′ end. DNase-treated RNA samples were prepared as described above. RT-PCR was performed using the Taqman one-step RT-PCR master mix reagents kit (Applied Biosystems) with a BioRad iCycler detection system (BioRad, Hercules, Calif.). A typical RT-PCR sample (25 μl) contained 5 μl of a serial dilution of RNA template (range from 0.02 ng/ml to 200 ng/ml) and was run in duplicate. Critical threshold cycle (Ct) numbers were defined by the detection system software. Relative quantification of target gene expression was performed by normalization to an endogenous reference (16S rRNA gene) as recommended by the manufacturer.

RESULTS AND DISCUSSION

Isolation of mutants with decreased susceptibility to tigecycline.

Previous attempts to select for spontaneous mutants of S. aureus strain 8325-4 by growth on 4× the MIC of several glycylcyclines were unsuccessful (S. Projan, unpublished results). In the present study, S. aureus strains Mu3 and N315 were grown in increasing concentrations of tigecycline over a period of 16 days to facilitate the selection of mutants with decreased susceptibility to this drug. N315 and Mu3 are closely related MRSA isolates belonging to a clinically prevalent clonotype, and Mu3 exhibits a heterogeneous vancomycin-resistant phenotype (15, 17). Neither strain carries the efflux pump-encoding tet(K) gene, but Mu3 has a copy of tet(M), which encodes a ribosome protection protein. The tigecycline MIC for strain Mu3 increased from 1 to 16 μg/ml over the course of the serial passage experiment as determined by the macrodilution method. This increase in MIC occurred in a stepwise fashion with twofold incremental increases in MIC observed at passage numbers 6, 10, 12, and 15. Similarly, the tigecycline MIC for strain N315 increased from 0.5 to 4 μg/ml in three steps observed at passage numbers 9, 13, and 15. Stepwise mutants for both strains were selected for further study and termed Mu3_mut1 to Mu3_mut4 and N315_mut1 to N315_mut3, respectively. Tigecycline MICs were subsequently redetermined using the reference broth microdilution method and found to be 0.5, 1, 4, 8, and 16 μg/ml for Mu3 and Mu3_mut1 through Mu3_mut4, respectively. Tigecycline MICs for N315 strains were 0.25, 0.5, 2, and 4 μg/ml for the parent and mutant strains N315_mut1 through N315_mut3 (Table (Table3).3). Mu3_mut4 and N315_mut3 were serially passaged daily in drug-free media for a period of 15 days. The decreased susceptibility of these strains to tigecycline was maintained, suggesting that any mutations that may have occurred were stable.

TABLE 3.
Microdilution MIC values for N315 and Mu3 strains

Susceptibility profiles of Mu3 and N315 parent and mutant strain pairs.

The antibiotic susceptibility profile of the Mu3/Mu3_mut4 and N315/N315_mut3 strain pairs was determined to ascertain whether the phenotype of the mutants had changed with respect to agents other than tigecycline (Table (Table3).3). Elevated MICs of two additional glycylcycline compounds, DMG-MINO and DMG-DMDOT, were observed for both mutants, and the MICs of EtBr and TPP increased in the N315 mutant, suggesting that a more general resistance mechanism may be involved. Interestingly, the imipenem MIC for N315_mut3 decreased. In general, MIC changes occurred in a stepwise manner in the intermediate mutants (Table (Table3).3). Several twofold changes in MIC were observed for ciprofloxacin, norfloxacin, minocycline, and tetracycline (data not shown). The MICs of oxacillin, piperacillin-tazobactam, vancomycin, tobramycin, levofloxacin, and erythromycin did not change in the mutant strains (data not shown).

GeneChip analysis of Mu3 and N315 parent and mutant strains.

S. aureus GeneChips were used to perform a global-scale comparison of the DNA and RNA profiles of parent strains Mu3 and N315 and their respective mutants to characterize any changes in the DNA content or gene expression profile that might be responsible for decreased susceptibility to tigecycline in the mutants. No differences were observed between the DNA profiles of Mu3 and Mu3_mut4 or N315 and N315_mut3, indicating that the mutants had not lost any of the genes represented on the GeneChip during the serial passage experiment that may have altered their susceptibility to this antibiotic (data not shown). However, this approach is not sensitive enough to detect any point mutations or small deletions that may have had an impact on gene expression.

To identify any changes in gene expression that might be responsible for the altered susceptibility of the Mu3 and N315 mutants to tigecycline, the transcription profiles of the parent and each of the mutant strains were compared using RNA harvested from the mid-exponential phase of growth. Table Table44 lists the genes whose expression is altered by at least eightfold in the Mu3 or N315 mutants compared to their respective parent strains. An efflux pump-encoding gene, mepA, exhibited dramatically altered expression in all mutants, and the tet(M) gene was overexpressed in Mu3_mut3 and Mu3_mut4. Both seemed likely candidates to play a role in the development of resistance to tigecycline and were investigated further.

TABLE 4.
Genes with expression changes of >8-fold between Mu3 and N315 parent and mutant strains (P < 0.05)

Overexpression of an efflux pump-encoding gene by N315 and Mu3 mutants.

A previously uncharacterized gene cluster now designated mepRAB (multidrug export protein) (N315 open reading frame numbers SA0322 [mepR], SA0323 [mepA], and SA0324 [mepB]) (17) was dramatically overexpressed (~100-fold) in the Mu3 and N315 mutants (Table (Table4)4) and represents the only genes whose expression was commonly altered by a factor of >8 in both strains. SA0323 was previously identified by Garvis et al. as svrA (staphylococcal virulence regulator); however, it was not identified as a putative efflux pump (13). BLAST analysis revealed that mepR encodes a MarR-like transcriptional regulator, mepA encodes a novel efflux pump belonging to the multidrug and toxin extrusion (MATE) family of efflux pumps (20), and mepB encodes a hypothetical protein of unknown function. MATE pumps are best characterized by the NorM efflux pump or Vibrio parahaemolyticus and Neisseria species (19, 26). These proteins function as Na+-driven efflux pumps. Kyte-Doolittle hydropathy analysis of MepA predicts 12 transmembrane domains, which is typical of this family of pumps (18). The mepRAB genes are closely linked, and it is likely that they are coexpressed and/or coregulated. Transcription of the mepA gene in the N315 and Mu3 mutants was examined by RT-PCR. These results confirm that expression of mepA is increased in each of the stepwise mutants of N315 (198-, 83-, and 155-fold for mut1 to mut3, respectively) and Mu3 (16-, 62-, 82-, and 66-fold for mut1 to mut4, respectively) (data not shown). A fourfold increase in mepA expression between Mu3_mut1 and Mu3_mut2 was detected by RT-PCR analysis that was not observed by transcription profile analysis. This may be related to saturation of the signal detected on the GeneChip.

It was considered that mepR might play a role in regulation of the mepRAB locus. The mepR gene including ~500 bp of the region upstream and the mepRA intergenic region were sequenced for each of the Mu3 and N315 mutants using primers pMepRF1, pMepRR1, MepRF2, and MepRR2. No mutations were identified in the mepR upstream region, but a single T→A transition at base position 123 in the mepR coding sequence was found to occur in N315_mut1 and was maintained in the N315_mut2 and N315_mut3 mutants. This mutation introduces a premature stop codon in the mepR gene, resulting in a predicted truncation of MepR from 139 to 40 amino acid residues. A 4-bp deletion was identified in the mepR gene of Mu3_mut1, Mu3_mut2, Mu3_mut3, and Mu3_mut4. This deletion removes bases 313 to 316 and causes a frameshift that introduces a premature stop codon at position 380, resulting in a MepR truncation from 139 to 125 amino acid residues. These results suggest that MepR may function as a repressor of the mepRAB locus. This is similar to the acrAB pump system in gram-negative bacteria, which is repressed by AcrR and the multiple antibiotic resistance marRAB locus of E. coli which shares a similar organization to mepRAB (14).

Effect of mepA overexpression on the susceptibility profile of parent and mutant strains.

The importance of efflux pumps in the reduced susceptibility of gram-negative organisms such as P. mirabilis and P. aeruginosa to tigecycline has previously been reported (9, 31). The increased expression of mepA detected in strains N315_mut1 and Mu3_mut1 is potentially linked to the two- to fourfold increases in the MIC of tigecycline, EtBr, TPP, DMG-MINO, and DMG-DMDOT observed for these strains (Table (Table3).3). To confirm this, the mepA gene was overexpressed from a multicopy plasmid in the parent strains Mu3 and N315. This caused a fourfold increase in the MIC of tigecycline (Table (Table3).3). A similar result was observed on tigecycline gradient plates (3.2- and 3.5-fold increases for Mu3 and N315, respectively) (data not shown). This suggests that the MepA MATE efflux pump can export tigecycline from the cell. Overexpression of mepA also caused a fourfold increase in N315 resistance to EtBr and TPP and twofold increases in DMG-MINO MICs for both N315 and Mu3, suggesting that this pump can export multiple substrates (Table (Table3).3). These results were confirmed by gradient plate analysis (data not shown). Despite the increased MICs of DMG-DMDOT in the Mu3 and N315 mutants, overexpression of mepA in the parent strains was not sufficient to increase the MIC of this glycylcycline, suggesting that the MepA pump does not alter DMG-DMDOT susceptibility. Overexpression of mepA did not promote any changes in the EtBr, TPP, or imipenem MIC in strain Mu3, which already exhibits high MICs for these compounds (Table (Table3).3). A plasmid overexpressing the mepA gene in the antisense direction was used as a negative control and did not cause any changes in MIC compared to the parent strain (data not shown).

Complementation of the mepR defect.

The presence of mutations in the mepR gene and overexpression of the mepRAB locus in the Mu3 and N315 mutants suggest that MepR may function to repress transcription of these genes. If this is the case, then overexpression of the wild-type mepR gene from pCLL3434 in the mutant strains should complement the defect in the chromosomal mepR copy and cause a reduction in mepA expression. RT-PCR experiments showed that transcription of mepA decreased by 57-fold in Mu3_mut1 and by 87-fold in N315_mut1 when the wild-type mepR gene was overexpressed in these strains (data not shown). This supports the hypothesis that MepR can function as a repressor of mepA expression, but it is not clear whether this is a direct or indirect effect.

The effect of overexpression of a wild-type copy of the mepR gene was of principle interest for N315_mut1, Mu3_mut1, and Mu3_mut2 strains. These are the only strains where mepA expression can potentially be associated with the MIC increases described in the previous section, as mepA expression does not increase further in later-stage mutants, despite continuing increases in tigecycline MICs. Complementation of the mepR defect in N315_mut1 caused a decrease in the MICs of tigecycline (fourfold for N315_mut1 and eightfold for Mu3_mut1) and DMG-MINO (fourfold for N315_mut1 and twofold for Mu3_mut1) and a twofold decrease in the MICs of EtBr and TPP for N315_mut1. This suggests that the elevated MICs observed in these first-step mutants are likely due to the derepression of mepA expression caused by the mepR mutation. These effects were also observed on gradient plates, but the presence of single colonies growing at the high end of the gradient suggests that spontaneous mutants can arise and grow at higher concentrations (data not shown). Interestingly, overexpression of the wild-type mepR gene in N315_mut1 restored the imipenem MIC to the level of the parent strain and caused a two- to fourfold decrease in the MIC of DMG-DMDOT for N315_mut1, Mu3_mut1, and Mu3_mut2 despite the fact that mepA overexpression alone did not impact on the MICs of these compounds in the parent strains. This suggests that mepR can have an effect on antibiotic susceptibility independent of mepA. Complementation of the mepR defect in Mu3_mut2 decreased the MICs of tigecycline and DMG-MINO by only twofold but did not result in parental strain levels, suggesting that other mutations may have accumulated in this strain. Similarly, overexpression of the wild-type mepR gene in the final-step mutants, N315_mut3 and Mu3_mut4, was not sufficient to restore the MICs of tigecycline, DMG-MINO, DMG-DMDOT, EtBr, TPP, or imipenem to the level of the parent strain, which suggests that the altered antibiotic susceptibility profile of the final step mutants cannot be explained solely either by the defect in mepR or the resulting overexpression of the MepA efflux pump and that multiple mutations may be involved. Further experiments such as disruption of mepA are required to define the exact contribution of the mep locus to the susceptibility phenotype of these mutants.

Overexpression of tet(M) by Mu3 mutants.

Expression of the tet(M) gene, which encodes a tetracycline resistance determinant, increased >40-fold in Mu3_mut3 and Mu3_mut4 but did not change in Mu3_mut1 or Mu3_mut2 compared to the parent strain (Table (Table4).4). RT-PCR analysis was used to confirm these results, and increases in tet(M) expression were only observed for Mu3_mut3 and Mu3_mut4 (66- and 64-fold, respectively) (data not shown). The tet(M) gene is regulated by a mechanism of transcription attenuation attributed to the presence of stem-loops in the region 5′ to the start codon (29). Sequencing of the tet(M) promoter region of Mu3_mut3 and Mu3_mut4 using primers pTet MF1 and pTet MR1 revealed an 87-bp deletion (54 to 140 bases upstream of the start codon inclusively) that would remove this stem-loop-forming region and probably explains why more tet(M) transcript was detected in these strains. No changes were detected in the tet(M) promoter regions of Mu3_mut1 and Mu3_mut2.

Effect of tet(M) overexpression on tigecycline susceptibility.

It has previously been shown that tigecycline can overcome TetM-mediated resistance (24) and that this may be due to tighter binding of this antibiotic class to the ribosome (3, 5). To date, no glycylcycline-resistant tet(M) mutants have been generated in the laboratory (25). Although tet(M) is not sufficient to mediate resistance to tigecycline when expressed from its native promoter, we hypothesized that increased transcription of this gene, as detected in Mu3_mut3 and Mu3_mut4, might produce enough Tet M to dissociate tigecycline from the ribosome. Overexpression of tet(M) by strain Mu3 or N315 only mediated a twofold increase in tigecycline MIC (Table (Table3),3), but no change was detected on gradient plates (data not shown). A plasmid overexpressing tet(M) in the antisense direction was used as a negative control and did not alter the tigecycline MICs in the parent strain (data not shown). As expected, overexpression of tet(M) did not alter the MICs of EtBr, TPP, DMG-MINO, DMG-DMDOT, or imipenem. It was considered that the effects of tet(M) overexpression were only evident when the bacteria were also overproducing the MepA efflux pump. However, overexpression of tet(M) by strains Mu3_mut2 and N315_mut3 (which also overexpress mepA) did not lead to any additional increases in tigecycline MIC (Table (Table3).3). These results suggest that the two mechanisms do not act synergistically despite the evolution of mutants with decreased susceptibility that have developed mutations at both loci in a stepwise fashion. Therefore it appears that the overexpression of Tet M plays no role in decreased susceptibility to tigecycline.

Other gene expression changes in mutant strains.

In addition to mepRAB and tet(M), a number of other genes, of which some are involved in metabolic functions and stress adaptation, had altered expression in the Mu3 and N315 mutant strains (Table (Table44 and data not shown). For example, homologs of the betA and gbsA genes, which showed increased expression in the Mu3 mutants, are important in the adaptation to osmotic stress (6). The Mu3 mutants were found to grow slower than the wild-type strain and did not reach the same optical density in the stationary phase. Mu3_mut4 took 1.75-fold longer to double in A650 units during the exponential phase, and this effect was reproducible during three independent experiments (data not shown). It is likely that the changes in gene expression mentioned above contribute to the decreased tigecycline susceptibility of the mutants and the altered growth characteristics of the Mu3 mutants, but the mechanism is unclear.

Concluding remarks.

Mutation leading to increased transcription of the mepRAB locus occurred during the first stepwise increase in tigecycline MIC for both N315 and Mu3 and suggests that such mutations can occur in a single step. However, despite an ~100-fold increase in transcription of mepA in Mu3_mut1 and N315_mut1, only a twofold increase in tigecycline MIC was observed. Furthermore, overexpression of mepA alone could only promote a fourfold increase in tigecycline MIC. This suggests that tigecycline can function as a substrate for MepA but that this efflux pump alone is unlikely to result in high-level resistance to tigecycline by S. aureus. Experiments presented here also showed that overexpression of tet(M) alone, or in combination with the increased transcription of mepRAB in Mu3_mut2 and N315_mut3, did not contribute to the decreased susceptibility to tigecycline. The reason for selection of the mutation leading to overexpression is unclear. The 16- and 32-fold increases in tigecycline MIC observed here are most likely due to the combination of a number of effects that include MepA and alterations in the expression of other as yet uncharacterized genetic loci. To date, no naturally occurring S. aureus isolates with decreased susceptibility to tigecycline have been identified, and it seems unlikely that clinically significant resistance to tigecycline will emerge in a single step via mutations in either the mepRAB or tet(M) locus.

Acknowledgments

We thank Glenn Kaatz for helpful advice and discussion regarding this study.

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