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Multidrug efflux pump overexpression inStaphylococcus aureus after single and multiplein vitro exposures to biocides and dyes Aure´lie A. Huet,1,3 Jose L. Raygada,2 Kabir Mendiratta,1 Susan M. Seo2and Glenn W. Kaatz1,2 1John D. Dingell Department of Veterans Affairs Medical Center, Detroit, MI 48201, USA 2Department of Medicine, Division of Infectious Diseases, Wayne State University School of 3E´cole Supe´rieure de Microbiologie et Se´curite´ Alimentaire de Brest, Universite´ de Bretagne Occidentale, Technopoˆle Brest-Iroise, 29280 Plouzane´, France Biocides and dyes are commonly employed in hospital and laboratory settings. Many of theseagents are substrates for multiple-drug resistance (MDR)-conferring efflux pumps of bothGram-positive and Gram-negative organisms. Several such pumps have been identified inStaphylococcus aureus, and mutants overexpressing the NorA and MepA MDR pumps followingexposure to fluoroquinolones have been identified. The effect of exposure to low concentrations ofbiocides and dyes on the expression of specific pump genes has not been evaluated. Usingquantitative reverse-transcription PCR we found that exposure of clinical isolates to lowconcentrations of a variety of biocides and dyes in a single step, or to gradually increasingconcentrations over several days, resulted in the appearance of mutants overexpressing mepA,mdeA, norA and norC, with mepA overexpression predominating. Overexpression was frequentlyassociated with promoter-region or regulatory protein mutations. Mutants having significantincreases in MICs of common pump substrates but no changes in expression of studied pumpgenes were also observed; in these cases changes in expression of as-yet-unidentified MDRpump genes may have occurred. Strains of S. aureus that exist in relatively protectedenvironments and are repeatedly exposed to sublethal concentrations of biocides can developefflux-related resistance to those agents, and acquisition of such strains poses a threat to patients treated with antimicrobial agents that are also substrates for those pumps, such as ciprofloxacin to contribute to hospital-acquired infections, may berepeatedly exposed to sublethal concentrations of biocides, Biocides are commonly used in the processes of cleaning increasing the possibility of the development of reduced hospital, laboratory and home environments, sterilization susceptibility (Boyce, 2007; Smith et al., 2008). The of medical equipment, and skin decontamination prior to presence of biocide residues on the skin or inanimate surgery (McDonnell & Russell, 1999; Russell, 2003).
objects may also provide such selective pressure.
Pathogenic organisms in the hospital environment maybe sheltered from the effect of these compounds by the One mechanism by which reduced biocide susceptibility concomitant presence of materials that interfere with their can occur is the upregulation of multiple-drug resistance action or their existence in an environmental niche that (MDR) efflux pumps that include these compounds within provides some protection (McBain et al., 2002; McDonnell their substrate profiles. While it is agreed that increases in & Russell, 1999). Such organisms, which have been shown biocide MICs conferred by efflux pumps probably do notconfer true resistance at the concentrations deployed for Abbreviations: AF, acriflavine; BAC, benzalkonium chloride; CET, environmental or cutaneous disinfection, upregulation of cetrimide; CHX, chlorhexidine; CV, crystal violet; DEQ, dequalinium; MDR pumps as a result of biocide exposure may provide a EB, ethidium bromide; MDR, multiple-drug resistance; MFS, major survival advantage as well as resulting in low-level facilitator; MUG, 4-methylumbelliferyl b-D-galactopyranoside; NOR, resistance to antimicrobial agents that also are substrates norfloxacin; PENT, pentamidine; PY, pyronin Y; qRT-PCR, quantitativereverse-transcription-PCR; RD, rhodamine 6G; TPP, tetraphenylphos- for those pumps. Such low-level resistance can have significant consequences (see below).
Biocide- and dye-induced efflux pump overexpression Dyes such as acriflavine (AF), ethidium bromide (EB), associated with the appearance of mepR point mutations rhodamine 6G (RD) and pyronin Y (PY) have significant that inactivated this negative regulator. MDR efflux pump utility in the research laboratory, including nucleic acid overexpression in S. aureus thus is a relatively common staining and the study of transport processes in eukaryotic consequence of biocide and dye exposure.
and microbial cells (Horobin & Kiernan, 2002). Inparticular, EB is a good substrate for many bacterial effluxpumps and susceptibility to it in the presence and absence of an efflux pump inhibitor has been used as a screen for Bacterial strains, plasmids, media and reagents. S. aureus the presence of efflux-related resistance mechanisms SH1000, which is a derivative of S. aureus NCTC 8325-4 in which (DeMarco et al., 2007). Structural features of substrates the rsbU mutation has been repaired, was used as a control strain of bacterial MDR pumps of the major facilitator (MFS), (Horsburgh et al., 2002). Eight clinical strains (three meticillin- small multidrug resistance (SMR), and perhaps the multi- susceptible and five meticillin-resistant) from a collection of unique drug and toxin extrusion (MATE) families include bloodstream isolates of S. aureus used in a study of the frequency of hydrophobic moieties and a positive charge; many dyes baseline MDR efflux pump overexpression (DeMarco et al., 2007),each overexpressing no more than one MDR efflux pump, were and biocides share these characteristics and both groups of selected at random and employed as test strains (Table 1). Strains and compounds are commonly transported by an individual plasmids used for functional analyses of mepR mutants are listed in MDR pump (Hassan et al., 2007; Poole, 2005).
Table 2. Reagents were the highest grade available and along withmedia were obtained from Sigma or BD Biosciences. Unless otherwise Examination of the Staphylococcus aureus genome reveals noted, the incubation temperature for all experiments was 35 uC.
numerous potential MDR efflux-pump-encoding genes (seewww.membranetransport.org). Some of those that have been Antimicrobial susceptibility testing. MICs were determined in studied in detail include QacA and QacB, highly similar MFS duplicate using a microdilution procedure with and without reserpine pumps that are encoded on plasmids, NorA and MdeA, both (20 mg ml21) according to Clinical and Laboratory Standards chromosomally encoded MFS pumps, and MepA, a MATE- Institute (CLSI) guidelines (CLSI, 2006). Compounds selected forevaluation included AF, benzalkonium chloride (BAC), cetrimide family MDR pump that also is chromosomally encoded (CET), chlorhexidine (CHX), crystal violet (CV), dequalinium (Kaatz et al., 2005a; Paulsen et al., 1996b). More recently (DEQ), EB, norfloxacin (NOR), pentamidine (PENT), PY, RD, and described are the NorB, NorC and SdrM MFS pumps, the tetraphenylphosphonium bromide (TPP). One or more of these genes for which are also chromosomal (Truong-Bolduc et al., compounds have been shown to be substrates for many of the known 2005, 2006; Yamada et al., 2006). Of considerable interest is S. aureus MDR efflux pumps (Hassan et al., 2007). A reserpine- the sepA gene, positioned immediately downstream of sdrM, mediated MIC reduction of at least fourfold was considered indicative which encodes a structurally unique transporter that has of efflux of that compound and hereafter will be referred to as asignificant reserpine effect. For production of mutants by the single- some similarity to the SMR family of proteins (Narui et al., exposure approach, MICs were also determined by agar dilution in 2002). Unlike all other S. aureus SMR family pumps order to incorporate accurate drug concentrations in selection plates.
described to date, SepA is encoded on the chromosome.
Compounds used for single- and multiple-exposure mutant produc-tion were chosen from those showing no significant reserpine effect in With the exception of SdrM all the transport proteins just microdilution MIC testing. This approach allowed for simple described are highly conserved among strains for which identification of efflux-related phenotypes by the appearance of a genome sequence data are available (n513; 95–100 % significant reserpine effect for one or more common pump substrates.
omes/lproks.cgi). SdrM is conserved in 10 of 13 sequenced Mutant production. Test organisms were exposed to low to strains (98–100 % homology), but less conservation is moderate concentrations of various biocides and dyes to determineif such exposure resulted in the appearance of mutants overexpressing present in MRSA252 (87 %) and in Mu3 and Mu50, both one or more MDR efflux pumps. For single-exposure mutant of which have a deletion of residues 206–291. Each of the production, organisms were grown overnight in Mueller–Hinton above-described transporters has various biocides and dyes broth (MHB) and then diluted 100-fold into fresh, pre-warmed as substrates and some also efflux hydrophilic fluoroquin- MHB. This culture was incubated with shaking to an OD660 of 0.7, as olone antimicrobial agents such as norfloxacin, ciproflox- previous experiments had shown that this represented the late acin and moxifloxacin. Susceptibility to these clinically exponential growth phase (data not shown). Cells were recovered by relevant agents may be sufficiently reduced by MDR pumps centrifugation and resuspended in 1 ml fresh MHB. Serial dilutionand plating techniques were employed to determine the log such that the emergence of target-based high-level (c.f.u. ml21) of this suspension, and aliquots of the undiluted and resistance is favoured (Markham & Neyfakh, 1996).
serially diluted suspension were plated onto Mueller–Hinton agar(MHA) plates containing two and four times the respective agar In this study we exposed bloodstream isolates of S. aureus dilution MIC of the appropriate compound. Compounds used in this to low to moderate concentrations of several biocides and procedure included DEQ (strains K3043, K3166, K3221 and K3231), dyes and examined the resultant mutants for upregulation CET (strain K3250), BAC (strains SH1000, K3055, K3155, K3221 and of several MDR efflux pumps, mainly those of chro- K3250), EB (strains SH1000, K3043, K3155, K3166, K3225 and mosomal origin. We found that single- and multiple-step K3231) and RD (strains K3055, K3231 and K3250). Plates were exposure led to the appearance of mepA, norA, norC and examined for growth after 48 h incubation.
mdeA-overexpressing mutants, with mepA overexpression Multiple-exposure mutants were produced using the gradient plate predominating. Overexpression of mepA most often was approach (Bryson & Szybalski, 1952). Initial gradients consisted of zero to two times the agar-dilution MIC of BAC (strains K3055, K3221 and K3250), EB (strains SH1000, K3043, K3155, K3166, K3225 and K3231), or RD (all strains). After 24 h incubation organisms from the leading edge of growth were passed again until growthoccurred across the entire plate. This process was repeated using gradually increasing drug gradients to a maximum of 20, 30 and 3 mg ml21 for BAC, EB and RD, respectively, and generally was completed Several colonies from single-exposure plates and the leading edges ofgrowth from the final gradient plates were streaked onto MHA to recover single colonies of potential mutants. One colony from each exposure condition was selected at random and microdilution MICs in the absence and presence of reserpine were determined. These organisms were passed three additional times on MHA followed by repeat MIC testing to ensure maintenance of a significant reserpine effect for at least one test compound compared with the respective parental strain. Mutants meeting this condition were considered qRT-PCR. The expression of mepA, norA-B-C, mdeA, sdrM and sepA in parent and putative MDR pump-overexpressing mutants was determined using quantitative reverse-transcription PCR (qRT-PCR) as described previously (DeMarco et al., 2007). Beacon Designer 7.01 (Premier Biosoft International) was used to design Taqman probes and primers based on the genome sequence of S. aureus 8325, which were purchased commercially (Operon Biotechnologies). qRT-PCR was performed in triplicate using the Superscript III Platinum One- Step kit (Invitrogen) and an ABI 7500 Fast Real-Time PCR system (Applied Biosystems) with parameters of 45 uC for 30 min, 95 uC for2 min, and 40 cycles of 1 min at 95 and positive controls were included and 16S rRNA was used as the endogenous control. The comparative threshold cycle method was used to calculate relative gene expression compared with that of S.
aureus SH1000; expression of each studied gene in this strain was considered to be 1.0 (Livak & Schmittgen, 2001).
Based on earlier experience with the strain set from which test organisms used in this study were selected, qacA/B is very infrequent in Detroit area S. aureus bloodstream isolates (DeMarco et al., 2007).
Thus, using primers designed based on the sequence of qacA (GenBank sequence X56628) that also had complete homology with qacB, routine PCR was employed to detect the presence or absence of EB efflux assay. All parent and each stably resistant single- and multiple-exposure mutant underwent a determination of EB efflux in real time to demonstrate efflux pump function. The procedure wasperformed using a fluorometric approach as described previously (Kaatz et al., 2000). Experiments were performed in duplicate and results were expressed as mean total efflux over a 5 min time-course.
Results for mutant strains were divided by those for the respectiveparent to normalize the data, which for mutants then represented an n-fold change compared with the parent. An increase of twofold or greater was considered significant. The effect of reserpine (20 mg ml21) was also determined and expressed as percentage reduction of RNA slot blotting. Northern analyses were performed to verify qRT- PCR results. RNA from strains exhibiting overexpression of pump genes was immobilized on a nylon membrane using a Bio-Rad Bio- Dot SF apparatus (Bio-Rad) and methods exactly as described previously (Ausubel et al., 2005). A PCR-generated fragment of the appropriate gene served as a probe, and labelling of the probe, hybridization and detection were performed using the BrightStar Psoralen-Biotin labelling, NorthernMax and BrightStar BioDetect kitsaccording to the manufacturer’s directions (Ambion). 16S rRNA was used as the endogenous control and data generated for the Biocide- and dye-induced efflux pump overexpression Table 2. Strains and plasmids used for MepR functional analyses S. aureus vector containing a tetracycline-inducible promoter controlling expression of cloned genes; Cmr *Cmr, confers chloramphenicol resistance.
appropriate parent strain were used as the standard to which mutant were observed for as few as one (CHX for both K3166 and K3231) to as many as five tested substrates (SH1000). Forthose compounds chosen for use in mutant production, Sequencing. Promoter and coding regions of MDR pump genes agar-dilution MICs were within twofold of those deter- having increased expression relative to parent strains were amplifiedby PCR and then sequenced in both directions using an automated mined from the microdilution results (data not shown).
method by the Applied Genomics and Technology Center, Wayne The presence of a significant reserpine effect for at least one State University (Sanger et al., 1977). For strains overexpressing norA- compound in all parent strains indicated that some efflux B-C the sequence of mgrA and its promoter was determined as MgrA pump activity was probably present at baseline.
is known to affect the expression of each of these genes (Luong et al.,2006). In addition, for strains overexpressing mepA the sequence ofmepR, which encodes the repressor of mepA expression and is encoded immediately upstream of mepA, was determined also. DSGene 1.5 (Accelrys) was used for nucleotide sequence analyses. The With the exception of K3225 all parent strains were sequence of the respective parental strain was the standard with which exposed to at least two single-exposure selecting agents, and putative mutants were recovered in all cases except fortwo- and fourfold MIC BAC exposures for strain K3250 MepR functional analyses. Point mutations in mepR resulting in and fourfold MIC RD exposures for all strains. Single- MepR amino acid substitutions were observed in two mepA- exposure mutants appeared at frequencies ranging from 1 overexpressing mutants (see Table 4). The mepR coding region fromeach of these mutants was amplified using PCR and cloned into in 106 to 1 in 109 c.f.u. at twofold MIC exposures and 1 in pALC2073, producing pK580 and pK582 (Table 2). Strains containing 107 to 1 in 1010 c.f.u. at fourfold MIC exposures. Serial pACL2073-based constructs were grown in the absence and presence passage of putative mutants on drug-free media identified of 50 ng tetracycline ml21 to assess the effect of induction of plasmid- instability of the resistance phenotype for 9 of 17 single- MepR is a repressor of both mepR and mepA expression. This increases in the remaining eight mutants are listed in property was exploited to assess the functional integrity of mutant Table 3. MIC increases ranged from as little as twofold to as MepR proteins using a fluorescent b-galactosidase assay as described high as 32-fold. Interestingly, fourfold or greater MIC previously, employing 4-methylumbelliferyl b-D-galactopyranoside increases were infrequently observed for the selecting agent (MUG) as a substrate (Kaatz et al., 2006). b-Galactosidase activity was (two out of eight mutants). For example, exposure of expressed in MUG units; 1 MUG unit51 pmol MUG cleaved per strains K3043, K3166 and K3221 to DEQ resulted in minute per OD600 unit. Experiments were performed in triplicate,and the expression of chromosomal mepR in SA-K2916-R, SA-K2916- fourfold or greater MIC increases for eight, five and seven R (Q18P), and SA-K2916-R (G97E) was quantified by integrating the substrates, respectively, but these substrates did not include area beneath the expression curves with SigmaPlot 10.0 (Systat The multiple-exposure, or gradient plate, process resultedin the recovery of putative mutants in all cases in which this procedure was applied. As for single-exposuremutants, not all strains maintained their resistance profilesafter serial passage in drug-free media. However, the proportion that did was greater than observed for single- MICs for parent strains with and without reserpine are exposure organisms (14 out of 18 versus 8 out of 17).
given in Table 1. Fourfold or greater decreases in MICs Instability was observed for SH1000 (EB exposure), K3055 Table 3. Susceptibility changes for single- and multiple-exposure mutants (compared with parent strain) *Selection conditions and compound. SE and ME, single- and multiple-exposure. See Table 1 footnote for testcompound abbreviations.
and K3250 (BAC exposure), and K3155 (RD exposure).
increased expression of any pump gene with the exception Fourfold or greater MIC increases for selecting agents were of K3055, K3225 and K3250, which had elevated norA not always observed but were more common than in expression at baseline compared with SH1000 (6.6-, 7.1-, single-exposure mutants (11 out of 14; 79 %).
Increased expression of mepA predominated (8 out of 22 mutants, or 36 %), resulting most commonly after multiple All parental strains and stable mutants were analysed for exposure [BAC (1), EB (3), RD (2)] but also occurring with their expression levels of mepA, norA-B-C, mdeA, sepA and single exposure [DEQ (1) and EB (1)]. Increased norA sdrM compared with that of SH1000. Data for mutants expression was observed in three mutants (14 %) and then were normalized to those of the appropriate parent, followed single exposure to DEQ (1) and multiple exposure and a fourfold or greater increase in expression compared to EB (2). Increased mdeA and norC expression were with the parent was considered significant (Table 4). qRT- observed only after multiple-exposure to RD (one mutant PCR results for genes demonstrating significantly increased each). One multiple-exposure mutant overexpressed two expression were verified by slot-blot analyses (data not pump genes (mepA and norA; K3155 ME-EB). No shown). qacA/B were found to be absent from this strain increased expression of norB, sepA or sdrM was observed.
set using a PCR-based screening approach (data not The magnitude of increased pump gene expression ranged shown). None of the parental strains demonstrated from as little as 7-fold to more than 450-fold. There also Biocide- and dye-induced efflux pump overexpression Table 4. Gene expression, EB efflux, and sequence analyses Premature mepR stop after 10 codons; A302S *Strains with increased norA expression at baseline are indicated (qnorA).
DSelection conditions and compound. SE and ME, single- and multiple-exposure. See Table 1 footnote for test compound abbreviations.
dCompared with parent strain.
§Normalized to parent strain.
||Only differences from parent strain are shown. ND, Not determined.
were five single- and five multiple-exposure mutants in which no change in pump gene expression was identified.
The 10 mutants not overexpressing any studied pump gene Like the qRT-PCR data, EB efflux data for mutants were were omitted from this analysis. For the remaining 12 normalized to the respective parent strain (Table 4).
mutants sequence data are provided in Table 4; only Interestingly, one mutant having a ninefold increase in differences from the respective parent strain are shown. For norC expression had no change in EB efflux (K3225 ME- strains overexpressing mepA a variety of mutations were RD). Similar behaviour with respect to EB efflux was observed in mepR, including four strains having changes evident in 8 of the 10 mutants for which no increased resulting in the creation of premature stop codons very expression of the genes studied here was identified. Where early in the reading frame and two others with point increased EB efflux was observed, reserpine generally was mutations resulting in amino acid substitutions in MepR.
effective in reducing the efflux by at least 50 % (data not The mepRA sequence of the remaining two mepA- overexpressing strains was unchanged from that of the respective parent. A novel point mutation resulting in an A302S MepA substitution was observed for one mutant.
MDR efflux pumps are an important mechanism by which The single mdeA-overexpressing mutant was found to have bacteria can evade the effect(s) of antimicrobial agents. This a point mutation that changed the 235 motif from resistance mechanism has received considerable attention in GTGCTA to TTGCTA as well as a GAT transversion at recent years, and there are ongoing efforts to develop the +2 position of mdeA mRNA (Huang et al., 2004). The inhibitors of MDR pumps. Effective inhibitors would 235 motif change results in a closer match to the expand the antibacterial armamentarium to include pump substrates and reduce the likelihood of the emergence ofhigh-level Three parental strains overexpressed norA and in each case Unfortunately, inhibitors evaluated to date have not been a previously described mutation associated with increased broad-spectrum in their activity. Inhibitors of MFS pumps norA expression, consisting of an insertion of AAT are not active against resistance-nodulation-division (RND) immediately 39 to the 210 promoter motif, was observed family pumps such as AcrB of Escherichia coli or MexB of (DeMarco et al., 2007). Overexpression of norA in two Pseudomonas aeruginosa, organisms of considerable medical mutants (K3155 ME-EB and K3231 ME-EB) was associated importance. Likewise, RND inhibitors do not have activity with a novel potential promoter up-mutation, consisting of against MFS pumps (Kaatz, 2005). Nevertheless, safe and an AAG transition between the 235 and 210 motifs. This effective inhibitors of the major classes of bacterial MDR alteration disturbs an inverted repeat that has been shown pumps, even if separate compounds are required for MFS to be important for the full repressive effect of MgrA and RND inhibition, would be a welcome addition to (Kaatz et al., 2005b). The sequence of mgrA for both of these strains was wild-type. The sequences of norA andmgrA were wild-type in the remaining norA-overexpressing Previous studies in our laboratory have shown that mutant (K3043 SE-DEQ). Likewise, the sequences of mgrA exposure of S. aureus to low concentrations of fluoroquin- and norC were unchanged from those of the parent strain olone antimicrobial agents can result in upregulation of for the norC-overexpressing mutant (K3225 ME-RD).
NorA and MepA, but the effect of biocide exposure ondrug pump expression in this organism has not beenevaluated in detail (Kaatz & Seo, 1995; Kaatz et al., 2000).
Others have shown previously that exposure of E. coli to The induction of wild-type mepR expression in SA-K2916- antiseptic agents such as pine oil and triclosan results in the R resulted in an 83 % reduction in chromosomal mepR upregulation of the AcrB MDR pump, which includes expression, whereas minimal to no effect was observed for several antimicrobial agents in its substrate profile (Levy, strains SA-K2916-R (Q18P) and SA-K2916-R (G97E), 2002; McMurry et al., 1998; Moken et al., 1997). Our data now extend this observation to S. aureus in that we have Fig. 1. Expression of chromosomal mepR in SA-K2916 containing pK434 (SA-K2916-R), pK580 [SA-K2916-R (Q18P)], andpK582 [SA-K2916-R (G97E)]. (a) mepR expression as a function of growth: $ and ., SA-K2916-R; & and X, SA-K2916-R(Q18P); m and {, SA-K2916-R (G97E). In each case data represented by the first and second symbols were obtained in theabsence and presence of 50 ng tetracycline ml”1, respectively. Open symbols illustrate growth of test organisms. Error bars areomitted for the sake of clarity. (b) Cumulative expression of mepR over the entire experiment. Black and white bars illustrateexpression in the absence and presence of tetracycline, respectively. 1 and 2, SA-K2916-R; 3 and 4, SA-K2916-R (Q18P); 5and 6, SA-K2916-R (G97E). Means±SD are plotted.
Biocide- and dye-induced efflux pump overexpression shown that exposure of clinical isolates to low concentra- lation. Differences in sensitivity between the qRT-PCR and tions of a variety of biocides and dyes can produce mutants EB efflux assays also may have played a role.
with increased expression of one or more MDR pumps.
Ten mutants demonstrated significant MIC increases but Even though the increases in biocide MICs observed in no change in expression of any of the pumps included in these mutants were moderate, such organisms are likely to our study. It is possible that these MIC changes may be the have a survival advantage in environmental niches where result of a non-pump-related mechanism(s). However, it is more likely that these organisms have increased expression Compounds chosen for mutant production were those of pumps other than those we evaluated as all had a showing no more than a twofold MIC reduction for significant reserpine effect for at least one of the tested parental strains in the presence of the efflux pump substrates. EB efflux of 8 of these 10 strains, including the inhibitor reserpine. This approach resulted in variable RD multiple-exposure mutants of K3043, K3055, K3231 agents being employed for individual test strains. However, and K3250, the RD and DEQ single-exposure mutants of the purpose of our work was not to compare one agent K3055 and K3221, respectively, and EB single- and with another with respect to the frequency of emergence of mutants but rather to determine, in general, if biocides and increased from that of the appropriate parent strain dyes commonly employed in the hospital or laboratory (Table 4). Five of these eight mutants were derived from environments could induce overexpression of MDR efflux parental strains that overexpressed norA; EB efflux in those parental strains was ¢47 %. The baseline activity of NorA The absence of stably raised MICs for the selecting agent in mutants derived from these parents almost certainly employed for some mutants was of considerable interest.
would obscure any contribution to EB efflux from other This observation was more common for single-exposure pumps. It is also conceivable that EB may be a poor than multiple-exposure mutants (6 out of 8 versus 3 out of substrate or not a substrate at all for these as-yet- 14, respectively). The lower incidence of this characteristic unidentified pumps. Baseline increased norA expression in multiple-exposure mutants most likely relates to the and the fact that EB is a relatively poor NorC substrate increased opportunities for accumulation of mutations in probably accounts for the lack of change in EB efflux by the strains repeatedly exposed to a particular compound.
RD multiple-exposure mutant of K3225, which over- Additive or synergistic effects on susceptibility are expected expressed norC (Hassan et al., 2007).
as the number of resistance-conferring mutations increases.
Among the genes included in our screen, overexpression of However, for those mutants demonstrating instability of mepA predominated (8 out of 22 mutants). Mutations in selecting-agent MICs, substrate affinity for the pump(s) in the gene encoding the MepR repressor were responsible in question may play a role. MIC changes for compounds six cases, including four having premature truncations of with lower affinity are likely to be less pronounced than for MepR and two with amino acid substitutions (Q18P and those that are better substrates. Alternatively, it is G97E) resulting in severe impairment in MepR activity conceivable that unidentified mutations may be present (Fig. 1). With respect to the two mutants having no in these mutants affecting susceptibility to selecting agents mutations in the mepRA region, one or more mutations in in a direction opposite to that of the MDR pumps. It also must be recalled that we employed a definition of a stable Overexpression of mepA has been observed in another fourfold or greater MIC increase as significant, but if strain in which the mepRA sequence is wild-type (Kaatz et twofold or greater were employed then the selecting agent al., 2005a). It is apparent that mepA expression can be would be included in all cases (data not shown).
Instability of the resistance phenotype to serial passage in In addition to augmented mepA expression, an A302S the absence of drug was intriguing but not necessarily MepA substitution was identified in K3043 ME-EB. It surprising. Pump overexpression in the absence of seems unlikely that this change contributed to the 12-fold substrate could confer a selective disadvantage by way of increase in EB efflux observed for this mutant. Single wasted resources or perturbed growth kinetics. Reversion amino acid differences, both within transmembrane helices to a wild-type phenotype in the absence of selective and in extramembrane loops, are known to alter substrate pressure would reverse these potential disadvantages.
specificity, as has been shown for QacA/B and the The magnitude of pump gene overexpression did not tetracycline efflux transporter TetA (Paulsen et al., 1996a; necessarily correlate with increases in EB efflux observed.
Sapunaric & Levy, 2005). In addition, there are conserved This discrepancy is exemplified by the BAC and RD charged residues in the extramembrane loop between multiple-exposure mutants of K3221, where mepA expres- transmembrane helices 2 and 3 in many MFS efflux sion was 12- and 450-fold increased, respectively, with proteins, changes in which can adversely affect protein similar increases in EB efflux (Table 4). Transcript quantity function (Yamaguchi et al., 1992). However, to the best of may not correlate with translated protein. There may be a our knowledge, mutations resulting in increased efficiency maximal amount of MDR pump tolerated and in this of transport for a particular substrate have not been situation excess transcripts are degraded prior to trans- identified. Introduction of the A302S mutation into MepA and a comparison of the kinetics of EB efflux with that of DeMarco, C. E., Cushing, L. A., Frempong-Manso, E., Seo, S. M., wild-type MepA would be required to establish or refute Jaravaza, T. A. & Kaatz, G. W. (2007). Efflux-related resistance to Staphylococcus aureus. Antimicrob Agents Chemother 51, 3235–3239.
Two of the three mutants overexpressing norA were found to Hassan, K. A., Skurray, R. A. & Brown, M. H. (2007). Active export have the same mutation in the promoter region, consisting proteins mediating drug resistance in staphylococci. J Mol Microbiol of an AAG transition between the 235 and 210 motifs.
This change disrupts an inverted repeat, but whether or not Horobin, R. W. & Kiernan, J. A. (2002). Conn’s Biological Stains, 10th it affects norA expression requires further study. Likewise, edn. Oxford, UK: BIOS Scientific Publishers.
the mdeA-overexpressing mutant had a mutation resulting Horsburgh, M. J., Aish, J. L., White, I. J., Shaw, L., Lithgow, J. K. & in the conversion of the 235 motif into a sequence that Foster, S. J. (2002). sB modulates virulence determinant expression more closely matches the preferred consensus sequence and and stress resistance: characterization of a functional rsbU strainderived from Staphylococcus aureus 8325-4. J Bacteriol 184, 5457– may improve promoter strength (Szoke et al., 1987). It also had a GAT transversion at the +2 position that could affect Huang, J., O’Toole, P. W., Shen, W., Amrine-Madsen, H., Jiang, X., mRNA stability, but further work would be required to Lobo, N., Palmer, L. M., Voelker, L., Fan, F. & other authors (2004).
assess this possibility formally. Increased norA and norC Novel chromosomally encoded multidrug efflux transporter MdeA in expression not associated with sequence changes in known Staphylococcus aureus. Antimicrob Agents Chemother 48, 909–917.
regulatory elements, such as the promoter regions or known Kaatz, G. W. (2005). Bacterial efflux pump inhibition. Curr Opin regulatory loci such as mgrA, is likely to result from mutational alterations in as-yet-uncharacterized loci that Kaatz, G. W. & Seo, S. M. (1995). Inducible NorA-mediated affect the expression of these genes.
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