M

Na

EVMa

b

c

d

e

f

a

ARRAA

KSPQMN

I

pmdo

cpl(o(

h0

ARTICLE IN PRESSG ModelICRES-25691; No. of Pages 11

Microbiological Research xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Microbiological Research

j ourna l h om epage: www.elsev ier .com/ locate /micres

ovel insight into antimicrobial resistance and sensitivity phenotypesssociated to qac and norA genotypes in Staphylococcus aureus

mmanuela Marchia, Leonardo Furib, Stefania Arioli c, Ian Morrisseyd,e,aleria Di Lorenzod,e, Diego Morac, Luciana Giovannetti a,arco Rinaldo Oggionib,f, Carlo Viti a,∗

Dipartimento di Scienze delle Produzioni Agroalimentari e dell’Ambiente (DiSPAA), Università di Firenze, Piazzale delle Cascine, 18, Firenze, FI, ItalyLAMMB, Dipartimento di Biotecnologie, Università di Siena, Policlinico Le Scotte (lotto 5, piano 1), Siena, SI, ItalyDipartimento di Scienze per gli Alimenti, la Nutrizione e l’Ambiente, via Mangiagalli, 25, Milano, MI, ItalyQuotient Bioresearch, Newmarket Road, Fordham Cambridgeshire CB7 5WW, UKIHMA Europe Sàrl, Route de la Corniche 9A, Epalinges, SwitzerlandDepartment of Genetics, University of Leicester, Adrian Building, University Road, Leicester, LE1 7RH, UK

r t i c l e i n f o

rticle history:eceived 13 June 2014eceived in revised form 4 July 2014ccepted 6 July 2014vailable online xxx

eywords:taphylococcus aureushenotype microarray

a b s t r a c t

Staphylococcus aureus strains harboring QacA, QacB, QacC, QacG transporters and norA promoterup-regulating mutations were characterized by phenotype microarray (PM), standard methods forsusceptibility testing, and ethidium bromide efflux assays, in order to increase knowledge on phen-otypes associated to efflux pumps and their substrates. PM data and standard susceptibility testinglead to the identification of new potential efflux targets, such as guanidine hydrochloride or 8-hydroxyquinoline for QacA and QacC pumps, respectively. The identification of compounds to which thepresence of efflux pumps induced increased susceptibility opens new perspectives for potential adjunctanti-resistance treatment (i.e. strains bearing QacB transporters showed increased susceptibility to thior-

AC transportersultidrug resistanceorA promoter mutations

idazine, amitriptyline and orphenadrine). Although the tested isolates were characterized by high degreeof heterogeneity, a hallmark of clinical isolates, direct ethidium bromide efflux assays were effective inhighlighting differences in efflux efficiency among strains. These data add to characterization of substratespecificity in the different classes of staphylococcal multidrug efflux systems conferring specific substrateprofiles and efflux features to each of them.

© 2014 Elsevier GmbH. All rights reserved.

ntroduction

Staphylococcus aureus is one of the major opportunistic humanathogens at both nosocomial and community level, especiallyethicillin-resistant S. aureus (MRSA) strains. S. aureus strains pro-

uce several toxins with demonstrated emetic effect, responsiblef staphylococcal food poisoning (SFP).

In S. aureus an important mechanism of antibiotic and bio-ide resistance relies on efflux systems, based on transmembraneroteins able to transport a single class or several structurally unre-

ated compounds, getting energy exploiting either an ion gradient

Please cite this article in press as: Marchi E, et al. Novel insight into aqac and norA genotypes in Staphylococcus aureus. Microbiol Res (2014

H+) or ATP cleavage (Poole 2005). Proteins that transport twor more antimicrobials or biocides are called multidrug-resistantMDR) efflux pumps. Multiresistant strains often bear plasmid

∗ Corresponding author. Tel.: +39 055 3288307; fax: +39 055 3288292.E-mail address: carlo.viti@unifi.it (C. Viti).

ttp://dx.doi.org/10.1016/j.micres.2014.07.001944-5013/© 2014 Elsevier GmbH. All rights reserved.

genes encoding for MDR pumps mediating the efflux of structurallyunrelated disinfectants and biocides. Among biocides, quaternaryammonium compounds (QAC) are widely used in hospital, domes-tic and food industry. Resistance to QACs is often associated withthe presence of plasmid encoded efflux pumps. Four different qacgenes have been found in clinical isolates, including qacA (Rubboet al. 1950; Tennent et al. 1989), qacB (Paulsen et al. 1996), smr(qacC) (Grinius et al. 1992; Paulsen et al. 1995) and qacG, firstlyidentified together with qacH and qacJ in equine and bovine veteri-nary isolates (Bjorland et al. 2003; Heir et al. 1998, 1999), and thenin human isolates (Correa et al. 2008).

Besides the presence of plasmid encoded efflux pumps, mul-tidrug resistance can be associated with increased expression ofthe chromosomal gene encoding for the NorA pump (Ubukata et al.

ntimicrobial resistance and sensitivity phenotypes associated to), http://dx.doi.org/10.1016/j.micres.2014.07.001

1989; Winterberg et al. 2005), mediating efflux of fluoroquinolonesand biocides/disinfectants (Kaatz et al. 1993; Yu et al. 2002).

Substrate specificity of the main S. aureus MDR resistancedeterminants has been widely characterized upon the first

ING ModelM

2 ical R

i1sscitknd

cnerwbPa(etM

M

S

gaBmees5o1aRms

aa2htA(

P

atrappo

cd

ARTICLEICRES-25691; No. of Pages 11

E. Marchi et al. / Microbiolog

dentification of efflux pumps (Leelaporn et al. 1994; Littlejohn et al.992; Mitchell et al. 1998; Mitchell et al. 1999). Afterwards someubstrates were identified and indicated as “top” efflux targets,uch as the QACs benzalkonium chloride, cetylpyridinium chloride,etyltrimethyl ammonium bromide, tetraphenylphosphonium, thentercalating dyes ethidium bromide, acriflavine, proflavine, andhe biguanide chlorhexidine. Nevertheless, the deepening of thenowledge on substrate specificity related to qac or nor determi-ants could be useful in order to open new perspectives for theevelopment of more effective antimicrobial treatments.

The aim of this work was to enlarge the scenario of chemicalompounds toward which the presence of MDR resistance determi-ants influences microbial susceptibility, proposing new potentialfflux targets in S. aureus isolates carrying Qac transporters or up-egulating mutations in the NorA gene promoter. Selected strainsere characterized by phenotype microarray technology (PM) and

y standard methods for susceptibility testing. The advantage ofM approach is that it allows to test at the same time the metabolicctivity of bacterial cells toward a wide panel of toxic compoundsDecorosi et al. 2009; Decorosi et al. 2011; Viti et al. 2007; Vitit al. 2009). Moreover efflux of ethidium bromide, which is one ofhe top substrate to investigate efflux mechanisms associated with

DR phenotypes (Patel et al. 2010), was assayed.

ethods

election of strains

A collection of 1602 S. aureus strains gathered from differenteographical origins, representing hospital and community-cquired infections and hosted at the strain collection of Quotientioresearch (Fordham, UK) were previously characterized at theolecular level and screened to evaluate susceptibility to sev-

ral biocides and for the presence of resistance determinants (Furit al. 2013). Among these strains, we selected a number of repre-entatives for each class of MDR markers: 6 qacA-positive strains,

qacB, 5 qacC, 5 norA promoter mutants. We also included thenly qacG-positive strain found in the collection, QBR102278-191, two strains showing no resistance markers, QBR102278-1619nd QBR102278-1177, and two reference strains, ATCC6538 andN4220. Details of minimum inhibitory concentration (MIC) andinimum bactericidal concentration (MBC) values for selected

trains are described in Table 1.A total of five mutant strains (MO037, MO038, MO042, MO044

nd MO066) were previously selected in vitro by growing RN4220nd ATCC6538 reference strains on biocide–agar media (Furi et al.013). These mutant strains and a RN4220 transconjugant (MO026)arboring the pSK1-qacA+ plasmid (Furi et al. 2013) were selectedo perform the ethidium bromide efflux assay in isogenic strains.ll mutant strains showed mutations in the norA promoter region

Furi et al. 2013).

henotype microarray (PM)

PM technology uses tetrazolium violet reduction as a reporter ofctive cellular metabolism (Bochner et al. 2001, 2008). The reduc-ion of the dye causes the formation of a purple color, which isecorded by a CCD camera every 15 min and provides quantitativend kinetic information about the response of the cells in the PMlates (Bochner et al. 2001). The data obtained are stored in com-uter files and can be analyzed to compare the metabolic kinetics

Please cite this article in press as: Marchi E, et al. Novel insight into aqac and norA genotypes in Staphylococcus aureus. Microbiol Res (2014

f different strains (Koo et al. 2004; Winterberg et al. 2005).The strains were tested by the PM approach for 240 different

ompounds, potentially playing toxic effects on bacterial cells, eachispensed at four increasing concentrations in adjacent wells in

PRESSesearch xxx (2014) xxx–xxx

a panel of ten 96-well plates, identified by codes as PM11–PM20(Table S1). In order to perform PM experiments Biolog Protocol forGram positive bacteria (Biolog Inc.) was used. Briefly the strainswere grown overnight at 37 ◦C on BUG agar (Biolog Inc.) and thencells were picked up with a sterile cotton swab and suspended in15 ml inoculation fluid (IF-0a GN/GP base, Biolog Inc.). Cell densitywas adjusted to 81% transmittance (T) on a Biolog-turbidimeter(Biolog Inc.).

All PM plates were incubated at 37 ◦C in an Omnilog Reader(Biolog Inc.) and monitored automatically every 15 min for 72 h forcolor development. To identify phenotypes gained or lost by strains,the kinetic curves obtained were compared using Omnilog-PM soft-ware (release OM PM 109M).

Reproducibility of PM data was investigated by running tworeplicates of 7 strains (ATCC6538, RN4220, 1173 qacA, 2671 qacA,1299 qacC, 1614 qacC, 1027 norA and 1191 qacG) on separate daysusing whole PM11–PM20 panel.

To investigate metabolic activity of strains in the presence ofethidium bromide, which is absent in PM plates, we prepared aseries of ad hoc plates. Using 96-well flat bottom plates, for eachstrain we inoculated 200 �l of inoculation fluid prepared as aboveindicated in 4 adjacent wells in order to have ethidium bromidefinal concentration of 4, 32, 64, 128 �g ml−1. Each strain was inoc-ulated in duplicate and the global experiment was performed intriplicate. Since ethidium bromide is colored per se, in each platewe introduced as “blank” two series of wells inoculated with inoc-ulation fluid with dye and without cells. The color intensity of these“blank” series were averaged and subtracted from kinetic curvesreferring to the strains.

Susceptibility testing

Minimum inhibitory concentration (MIC) was determined usingthe broth microdilution method (CLSI 2009): after over-nightgrowth on Mueller-Hinton broth-II (MHB-II) plates, strains weresuspended in MHB-II until the standardized OD590 of 0.5. Thenthree 10-fold dilutions were performed and each cell suspensionwas inoculated in the presence of toxic compounds at the indicatedconcentrations. The kinetics were followed by measuring OD590 atserial time points. The experiments were performed in triplicate.

Compounds used were methicillin (MET), penicillin (PEN),erythromycin (ERY), vancomycin (VAN), norfloxacin (NOR),ciprofloxacin (CIP), levofloxacin (LVX), tetracycline (TET),trimethoprim–sulfamethoxazole (SXT), ethidium bromide (EB),orphenadrine hydrochloride (ORPH), thioridazine hydrochloride(THIO), amitriptyline hydrochloride (AMI), protamine sulfate (PS)and 8-hydroxyquinoline (8-HQ).

The definition of R/S character for antibiotic susceptibility fol-lowed Clinical Laboratory Standards Institute (CLSI) criteria (CLSI2009).

PCR detection of cadmium transporter

The presence of cadmium resistance determinants was evalu-ated by qPCR amplification of an internal fragment of 272 bp of thecadmium-transporting ATPase, using a couple of primers designedon the basis of the S. aureus plasmid SAP064A DNA sequence(GQ900419): forward primer 5‘-TATGCTGACAGGTGATAATCAAG-3′; reverse primer 5‘-GCTTACTTAAATCATCTCCCAT-3′.

Ethidium bromide efflux assay

ntimicrobial resistance and sensitivity phenotypes associated to), http://dx.doi.org/10.1016/j.micres.2014.07.001

We exploited the ability of S. aureus to efflux ethidium bro-mide, which inside the cell intercalates with double-strandednucleic acids thus determining fluorescence increase when prop-erly excited. Ethidium bromide is a substrate for a variety of efflux

ARTICLE IN PRESSG ModelMICRES-25691; No. of Pages 11

E. Marchi et al. / Microbiological Research xxx (2014) xxx–xxx 3

Table 1Genotypes and phenotypes of S. aureus strains from QBR collection selected for characterization of efflux pumps substrate specificitya.

Isolate Region of host MLSTSTb

norA qacA qacB qacC qacG MIC (�g ml−1) MBC (�g ml−1)

EB CHX BZC EB CHX BZC

QBR102278-1027 Far East nd mut – – – – 16 4 16 16 16 128QBR102278-1881 North America nd mut – – – – 64 4 8 64 4 16QBR102278-1891 North America nd mut – – – – 16 4 8 32 4 16QBR102278-2634 Far East nd mut – – – – 16 4 8 16 4 16QBR102278-2635 Far East nd mut – – – – 128 4 8 128 8 8QBR102278-1024 Far East 20 wt + – – – 64 4 8 64 8 32QBR102278-1173 Europe 8 wt + - - - 256 2 4 256 16 32QBR102278-1205 Europe 30 wt + – – – 256 2 8 256 4 16QBR102278-2106 North America 239 wt + – – – 256 4 8 256 4 8QBR102278-2628 Far East 278 wt + – – – 128 8 8 128 8 8QBR102278-2671 Far East 1498 wt + – – – 64 4 8 64 4 32QBR102278-1064 Far East nd wt – + – – 128 2 4 512 2 16QBR102278-1067 Far East nd mut – + – – 64 4 4 64 4 8QBR102278-1297 Europe nd mut – + – – 256 1 4 512 8 16QBR102278-1828 Europe 5 wt – + – – 256 2 8 256 8 16QBR102278-2507 Far East 8 wt – + – – 512 2 8 512 8 8QBR102278-1209 Europe 9 wt – – + – 32 2 8 128 4 16QBR102278-1299 Europe 26 wt – – + – 16 1 4 16 16 32QBR102278-1390 Europe 9 wt – – + – 32 1 8 64 4 16QBR102278-1614 Europe 8 mut – – + – 64 2 8 64 4 8QBR102278-1889 North America 5 wt – – + – 64 1 8 64 4 16QBR102278-1191 Europe 5 wt – – – + 64 2 4 64 4 4QBR102278-1177 Europe nd wt – – – – 8 2 1 8 4 4QBR102278-1619 Europe nd wt – – – – 4 2 2 8 4 16RN4220 – nd wt – – – – 8 8 2 16 8 2ATCC6538 – nd wt – – – – 4 4 2 16 4 8

t, wib

pobb(PobbmTllietmAas4rowd

S

sIucrF(

a Furi et al. 2013b MLST, Multilocus sequence typing; ST, sequence type; nd, not determined; w

actericidal concentration.

umps so, once inside the cell, it is extruded with the resultf decreasing the overall measurable fluorescence. For ethidiumromide efflux assay, cells of S. aureus strains were grown inrain Heart infusion medium (Oxoid) up to the mid-log phaseOD590 = 3). Cells were washed twice and resuspended in sterileBS (pH 7), in the presence of 20 �g ethidium bromide ml−1. Inrder to disrupt the electrochemical gradient across the mem-rane, which represents the driving force for active ethidiumromide extrusion, 0.1 mM of the protonophore carbonyl cyanide-chlorophenylhydrazone (CCCP) was added (Floyd et al. 2010).

hen cells were incubated for 10 min at 37 ◦C, thus allowing theoading with ethidium bromide. After incubation, cells were col-ected by centrifugation at 18,000 × g for 1 min and resuspendedn PBS. To sustain efflux activity, 1% glucose (w/v) was added asnergy source for the activation of cellular metabolism leading tohe generation of the electrochemical gradient across the plasma

embrane necessary for the efflux pump activity (Floyd et al. 2010).t last, the decreasing fluorescence was measured in the presencend in the absence of 0.1 mM CCCP, by means of a fluorescencepectrophotometer (Victor3, PerkinElmer, Italy) (excitation filter85 nm, emission filter 535 nm) programmed for a reading set of 10epetitions every 3 min at 37 ◦C. Data were expressed as percentagef fluorescence decrease after 30 min of efflux. The reported valuesere calculated as geometric mean ± one SD from three indepen-ent biological assays.

tatistical analysis

To compare PM kinetic curves among all the strains the cho-en parameter was IC50, expressed by PM software in “well units”.C50 is defined as the well or fraction of well at which a partic-lar per-well parameter is at half of its maximal value over the

Please cite this article in press as: Marchi E, et al. Novel insight into aqac and norA genotypes in Staphylococcus aureus. Microbiol Res (2014

oncentration series (Biolog Inc., personal communication). Theeference parameter in our analysis was the area under the curve.or each compound, IC50 values range between a minimum of 0.60no metabolic activity in any of the wells) and a maximum of 4.40

ld type; mut, mutant; MIC, minimum inhibitory concentration; MBC, minimum

(optimal growth in all the wells). When a strain shows highmetabolic activity in all the wells containing the four concentra-tion of a compound without showing any decrease of activity, theIC50 value is established as >4.40, whereas, when metabolic activityis absent in the four wells, IC50 is evaluated as <0.60.

PM IC50 results were analyzed exploiting Primer 6 software(Plymouth Routines In Multivariate Ecological Research, v. 6.1.13,PRIMER-E Ltd.), performing multi-dimensional scaling (MDS) anal-ysis and cluster analysis (UPGMA) to obtain a comprehensivegraphical view of the distribution of strains on the basis of chemi-cal sensitivity patterns. IC50 values from every sample were usedto generate a resemblance matrix computing Euclidean distancebetween all variables. Euclidean distances were used to obtaintwo dimensional (2D) MDS configuration, in which the closer thesamples are, the more similarly they respond to antimicrobialcompounds; in other words, close strains share similar chemicalsensitivity patterns. The hierarchical clustering dendrogram wasused for the same purpose. Primer 6 software allows to apply clus-ter information on MDS plot, where circles represent the branchesof the dendrogram.

Euclidean distance computation and MDS analysis were per-formed on both strains and compounds, giving the opportunity tohighlight which compounds were associated with discriminatingphenotypic responses.

Data concerning ethidium bromide efflux assay were analyzedas follows: the values of efflux levels for strains harboring thesame or no resistance determinants were averaged and investi-gated for significant differences among five groups applying theKruskal–Wallis test and the Dunn’s multiple comparison post test.

Results

ntimicrobial resistance and sensitivity phenotypes associated to), http://dx.doi.org/10.1016/j.micres.2014.07.001

Phenotype microarray

The S. aureus strains selected from QBR collection (Table 1) wereanalyzed for chemical sensitivity toward 240 compounds present

ARTICLE IN PRESSG ModelMICRES-25691; No. of Pages 11

4 E. Marchi et al. / Microbiological Research xxx (2014) xxx–xxx

Fig. 1. The multi-dimensional scaling (a) and cluster analysis (b) of the phenotype microarray profiles of S. aureus strains for chemical sensitivity patterns to antibiotics.Analysis was performed on the resemblance matrix generated using Euclidean distance. Circles in panel (a) are generated by the software applying clustering on MDS plot.A e marm

ipadttbtocwauetm

nalysis was performed with a cut off at p-val = 0.05 (� = strains with no resistancutant strains).

n PM plates using IC50 parameter (Table S2). To compare PMrofiles we applied MDS analysis on IC50 values, exploring sep-rately data on antibiotics and non antibiotic compounds. Fig. 1(a)escribes the strains distribution as a function of IC50 values inhe presence of antibiotics. Fig. 1(b) shows the dendrogram of clus-er analysis. The circles in Fig. 1(a) are generated by the softwarey applying hierarchical clustering information on MDS distribu-ion. Strains distribution in three clusters highlights the absencef specific association between resistance determinants and anylass of antibiotics. We observed that the 10 strains in cluster IIIere characterized by a higher metabolic activity in the presence of

ntibiotics interfering with protein synthesis (50 S ribosomal sub-

Please cite this article in press as: Marchi E, et al. Novel insight into aqac and norA genotypes in Staphylococcus aureus. Microbiol Res (2014

nit): tylosin, lincomycin, josamycin, spiramycin, oleandomycin,rythromycin and troleandomycin (IC50 > 4.35) (Table S2). Some ofhese strains kept higher metabolic activity in the presence of both

acrolides and lincosamides and were thus associated with the

kers; � = qacA strains; � = qacB strains; � = qacC strains; = qacG strain; = norA

MLS phenotype (Leclercq 2002; Pechere 2001); some other showedhigher metabolic activity only in the presence of 14-memberedmacrolides, and were thus classified by the MSR efflux phenotype(Eady et al. 1993; Pechere 2001).

Fig. 2 describes phenotypic response toward non antibiotic com-pounds, showing the MDS analysis (a) associated with hierarchicalclustering (b) performed on IC50 values. Strains bearing no resis-tance markers, but one (1619 NM), tended to separate from allother strains. The norA mutants formed a specific grouping (clus-ter I), including the strain 1619 NM. Among qac-positive strains,the analysis allowed to identify one cluster formed by strains bear-ing smr (qacC or qacG) determinants (cluster II), except the strain

ntimicrobial resistance and sensitivity phenotypes associated to), http://dx.doi.org/10.1016/j.micres.2014.07.001

1209 qacC, which was included in cluster IV. It was not possibleto highlight a specific grouping for qacA and qacB strains (clus-ters III–IV–V), because of the high heterogeneity of phenotypicresponses to antimicrobial compounds.

ARTICLE IN PRESSG ModelMICRES-25691; No. of Pages 11

E. Marchi et al. / Microbiological Research xxx (2014) xxx–xxx 5

Fig. 2. The multi-dimensional scaling (a) and cluster analysis (b) of the phenotype microarray profiles of S. aureus strains for chemical sensitivity patterns to non-antibioticc uclideo o resi

dhrWseTabrDcdt(

ta

ompounds. Analysis was performed on the resemblance matrix generated using En MDS plot. Analysis was performed with a cut off at p-val = 0.05 (� = strains with n

= norA mutant strains).

In order to reduce the complexity of the large amount of IC50ata for all strains and to identify the non antibiotic compounds thatad the stronger influence on strains distribution, we applied MDSeferring to the non antibiotics compounds as items of the analysis.

e obtained a 2D distribution of compounds (Fig. S1), in which thepatial proximity between two chemicals means that they influ-nce in a similar direction response patterns of analyzed strains.able 2 lists the compounds that played a major role in strains sep-ration (the complete set of IC50 data is given in Table S2). Theyelonged mainly to the classes of QACs (i.e. benzethonium chlo-ide, domiphen bromide, dodecyltrimethylammonium bromide –TAB, cetylpyridinium chloride – CPC, methyltrioctylammoniumhloride – MTAC, chelerythrine), biguanides (chlorhexidine – CHX),yes (ethidium bromide, crystal violet, acriflavine), toward whichhe qac-positive strains were characterized by higher IC50 values

Please cite this article in press as: Marchi E, et al. Novel insight into aqac and norA genotypes in Staphylococcus aureus. Microbiol Res (2014

Table 2).Looking in detail to specific responses, we observed that all

he strains bearing any of the resistance determinants were char-cterized by higher metabolic activity in the presence of the

an distance. Circles in panel (a) are generated by the software applying clusteringstance markers; � = qacA strains; � = qacB strains; � = qacC strains; = qacG strain;

detergent niaproof, the chelator fusaric acid and the guanidinedodine (Table 2). Three qacA-positive strains (1173, 2106, and2671), three qacB (1297, 1828 and 2507) and three norA mutants(1027, 1881 and 2635) showed higher IC50 values in the presenceof 18-crown-6-ether, an organic cyclic compound that binds to avariety of small cations (Gokel et al. 2004).

In comparison to qacAB, qacC-positive strains shared higher sen-sitivity toward crystal violet and tetrazolium violet, to the biocidechelerythrine, and to proflavine and acriflavine. The qacC strainswere also characterized by the average highest metabolic activityin the presence of 8-HQ, orphenadrine and CPC (Table 2). In thepresence of proflavine and acriflavine the qacG strain showed thesame response as qacAB strains (Table 2). The qacG strain was alsocharacterized, together with the qacA strains, by higher metabolicactivity in the presence of the membrane chaotropic agent guani-

ntimicrobial resistance and sensitivity phenotypes associated to), http://dx.doi.org/10.1016/j.micres.2014.07.001

dine hydrochloride (Table 2).All the qac-positive strains showed on average a higher

metabolic activity in the presence of protamine sulfate (PS), acationic peptide identified as biocide because of its activity against

Please cite

this

article in

press

as: M

archi

E, et

al. N

ovel in

sight

into

antim

icrobial resistan

ce an

d sen

sitivity p

hen

otypes

associated to

qac an

d norA

genotyp

es in

Staphylococcus aureus.

Microbiol

Res

(2014), h

ttp://d

x.doi.org/10.1016/j.m

icres.2014.07.001

AR

TIC

LE

IN P

RE

SS

G M

odelM

ICR

ES-25691;

No.

of Pages

11

6

E. M

archi et

al. /

Microbiological

Research

xxx (2014)

xxx–xxx

Table 2IC50 values of S. aureus isolates and control strains in the presence of non-antibiotic compounds associated with differential phenotypes.

Chemical Mode ofaction

qacA strains qacB strains qacC strains qacG strain norA mutant strains Control strains

1024 1205 1173 2106 2628 2671 1067 1828 2507 1297 1064 1390 1209 1299 1614 1889 1191 1027 1881 1891 2634 2635 1619 1177 ATCC6538 RN4220

Fusaric acid Chelators,lipophilic

>4.39 >4.31 >4.35 3.86 >4.38 >4.37 >4.37 4.14 >4.32 3.99 4.26 >4.32 4.19 >4.34 >4.39 4.2 >4.39 4.26 3.99 >4.39 4.18 4.27 >4.38 3.76 3.59 3.44

8-HQ 1.9 2.19 >4.4 3.3 1.58 1.55 3.11 2.41 2.57 2.86 1.3 3.01 2.6 3.6 3.34 3.43 3.1 2.99 3.29 1.56 1.58 1.62 2.77 2.58 1.65 1.6

Ethidium bromide Dyes 3.43 3.80 3.80 3.73 3.80 3.47 3.50 3.73 3.80 3.60 3.80 2.63 3.17 3.40 3.40 3.30 2.57 1.80 2.07 2.17 2.37 2.80 1.60 3.30 2.07 1.90Acriflavine >4.4 >4.4 >4.4 >4.4 >4.4 >4.4 >4.4 >4.4 >4.4 >4.4 >4.4 3.7 3.9 3.92 3.76 3.68 >4.4 >4.37 >4.4 4.24 >4.31 >4.36 3.35 2.87 3.64 3.33Tetrazolium violet 1.1 1.2 1.3 0.9 1.2 <0.6 1.3 1.8 1.3 <0.6 1.5 <0.6 <0.6 <0.6 <0.6 <0.6 <0.6 <0.6 <0.6 <0.6 <0.6 <0.6 <0.6 <0.6 <0.6 <0.6Crystal Violet 1.4 1.8 1.8 1.7 1.7 <0.6 <0.6 1.6 1.8 1.5 1.7 <0.6 <0.6 <0.6 <0.6 <0.6 <0.6 <0.6 <0.6 <0.6 <0.6 0.9 <0.6 <0.6 <0.6 <0.6Proflavine >4.4 >4.4 >4.4 >4.36 >4.4 >4.4 >4.4 >4.4 >4.4 >4.36 >4.4 3.79 3.82 3.82 3.87 3.79 >4.4 >4.4 >4.4 3.85 4.15 4.05 3.81 3.85 3.77 3.77

Dichlofluanid Fungicides 2.54 <0.6 <0.6 <0.64 <0.65 1.8 <0.6 <0.6 3.46 <0.61 1.62 2.52 <0.6 <0.6 0.85 <0.66 3.49 1.9 4.29 2.5 3.56 3.45 2.5 <0.66 0.7 <0.6Tolylfluanid <0.61 1.61 <0.63 <0.62 <0.68 <0.6 <0.6 <0.6 1.67 <0.6 <0.65 0.78 <0.64 <0.64 <0.64 <0.62 <0.62 1.6 <0.61 <0.64 1.7 1.6 <0.63 <0.64 <0.68 <0.68

Orphenadrine Anti-cholinergic

2.73 2.83 3.14 3.28 2.47 2.87 2.57 2.64 2.55 2.52 2.61 2.98 3.48 3.34 3.51 3.32 2.73 3.28 3.38 3.14 3.59 3.57 2.92 2.85 2.94 2.57

CHX Membrane,biguanide

>4.4 >4.4 >4.4 >4.4 >4.4 >4.4 >4.4 3.48 3.4 >4.4 >4.4 >4.4 3.26 3.89 >4.4 >4.4 >4.4 3.33 >4.4 >4.4 3.47 3.37 >4.4 >4.4 2.38 2.53

Alexidine >4.4 >4.4 >4.4 3.49 >4.4 >4.4 >4.4 3.56 >4.4 >4.34 4.07 >4.38 4.17 3.66 4.26 >4.4 3.5 >4.39 >4.4 >4.4 >4.4 3.56 >4.37 2.5 >4.4 >4.4

Dodine Membrane,guanidine

2.66 3.5 3.14 2.92 3.5 3.18 2.56 3.49 3.51 2.53 3.54 2.59 3.54 2.74 3.49 2.54 2.55 2.54 2.55 2.54 2.74 3.33 1.98 1.59 2.33 1.56

Protamine sulfate Membraneactiveagents

4.1 >4.4 4.25 4.02 3.56 3.8 4.28 >4.37 3.82 >4.35 >4.32 4.22 4.16 >4.4 3.64 4.12 3.56 2.81 3.4 3.2 3.1 2.83 2.98 1.81 3.19 3.1

Niaproof 1.54 1.58 1.53 1.47 1.68 1.54 1.49 1.53 1.55 1.17 1.51 1.49 1.4 1.54 1.59 1.53 1.57 1.47 1.52 1.53 1.51 1.52 1.52 1.22 0.81 0.88Guanidine hydrochloride 2.26 2.03 2.41 2.57 2 2.4 2.52 1.6 1.52 1.6 1.56 1.64 1.59 1.58 1.64 1.64 2.65 2.56 2.61 2.51 1.58 1.62 1.54 1.54 1.41 1.49Amitriptyline 2.63 3.65 3.43 3.59 2.8 3.47 2.65 2.53 2.94 2.56 2.64 3.38 3.69 3.82 3.6 3.38 3.31 2.72 3.31 3.32 3.95 3.7 2.94 3.42 3.64 3.45

Benzethonium chloride QACs 2.77 2.53 2.65 2.91 2.73 2.98 2.55 2.59 2.52 2.47 3.22 2.5 2.56 2.57 3.35 2.7 2.48 <0.64 2.31 2.22 0.88 <0.61 <0.6 <0.6 <0.6 <0.6DTAB 3.65 3.82 3.7 3.6 3.69 3.54 2.55 3.67 3.83 2.43 3.69 3.69 3.72 3.67 3.71 3.65 3.55 2.49 2.55 2.64 2.4 2.43 1.64 1.57 1.62 1.59CPC 3.41 >4.4 3.56 3.54 >4.4 3.47 3.2 >4.4 >4.4 2.82 >4.4 >4.4 >4.4 >4.4 >4.4 >4.4 >4.39 4.26 2.54 2.51 3.67 2.42 2.4 2.32 3.57 3.9Poly-l-lysine 4.07 3.94 >4.4 2.53 3.78 >4.4 >4.4 >4.4 3.83 4.28 >4.33 >4.32 >4.4 4.1 4.26 3.67 >4.4 4.24 >4.4 >4.4 3.79 3.86 >4.4 3.65 >4.4 >4.4MTAC 4.18 4.23 4.08 4.15 >4.34 4.15 3.67 4.06 3.54 4.1 4.14 4.22 4.24 4.06 4.14 3.93 4.07 3.42 3.45 4 2.56 3.47 4.12 4.05 2.53 2.98Chelerythrine >4.32 >4.36 4.28 4.05 >4.4 >4.39 3.48 4.29 >4.35 4.15 3.79 3.36 4.07 4.04 4.15 2.58 3.52 3.61 3.71 3.41 3.47 3.52 2.95 2.74 3.23 2.56Domiphen bromide 3.48 3.56 3.44 3.47 >4.4 3.47 3.52 3.47 3.5 2.8 3.49 3.38 3.5 3.85 4.2 3.43 3.48 2.47 3.5 2.61 2.6 2.48 1.61 1.29 1.34 1.47

Ornidazole Oxidizingagent

3.56 >4.4 >4.31 >4.4 4.23 3.63 3.48 3.9 3.62 3.67 3.81 3.32 3.58 3.54 4.02 3.98 3.6 3.54 3.59 3.56 3.36 3.58 3.81 3.55 3.69 3.53

18-Crown-6 ether Respirationinhibitors

3.35 3.54 >4.35 >4.37 3.42 4.25 3.53 4.22 4.16 4.07 3.49 3.36 3.45 3.55 3.27 >4.4 3.5 4.26 >4.38 3.54 3.52 >4.38 >4.4 3.55 3.6 3.47

Thioridazine 1.1 1.5 1.3 1.3 <0.6 1.3 <0.6 <0.6 <0.6 <0.6 <0.6 1.3 1.6 1.7 1.6 1.6 1.6 1.6 1.3 1.5 1.5 1.5 1.2 1.5 1.7 <0.6

Sodium selenite Toxic ions 3.75 4.07 2.88 >4.35 2.5 3.78 3.28 3.43 4.04 2.0 3.6 2.8 2.93 4.29 4.2 4.2 4.18 3.99 4.2 >4.35 >4.4 4.28 4.23 2.18 1.66 3.11Cadmium chloride 3.37 >4.36 >4.4 >4.4 >4.4 2.36 3 >4.4 2.89 0.8 1.46 2.39 2.27 2.3 <0.62 3.33 3.67 2.3 3.42 3.2 <0.66 2.15 3.38 <0.62 >4.4 <0.64

ARTICLE IN PRESSG ModelMICRES-25691; No. of Pages 11

E. Marchi et al. / Microbiological Research xxx (2014) xxx–xxx 7

Table 3Efflux phenotypes associated to Qac pumps and overproduction of NorA based on phenotype microarray analysis. Compounds in italic are MDR pumps efflux targets previouslyidentified.

Chemicals Mode of action Resistance determinants

QacA QacB QacC QacG norAa

Crystal violet Dyes (monovalent cations) Eb E negc neg negEthidium bromide E E E E varAcriflavine E E neg E EProflavine E E neg E ETetrazolium violet E E neg neg neg

Benzethonium chloride QACs (monovalent cations) E E E E ECPC E vard E E EDTAB E E E E EMTAC E E E E varDomiphen bromide E E E E EChelerythrine E E E E E

CHX Biguanides (divalent cations) E var var E var

Fusaric acid Lipophilic chelators E E E E E5,7-dichloro-8-HQ neg neg neg neg Se

5,7-dichloro-8-hydroxyquinaldine neg neg neg neg S8-HQ E var E E neg

Guanidine hydrochloride Guanidines E neg neg E EDodine E E E E E

Orphenadrine Pharmaceutical drugs neg S E neg EThioridazine neg S neg neg negPromethazine E var E E E

Niaproof Membrane active agents E E E E EProtamine sulfate E E E neg negPoly-l-lysine S neg neg neg varAmitriptyline neg S neg neg neg

Ornidazole Others E neg neg neg negSodium metaborate neg E neg neg negSodium selenite neg neg neg neg E

a Up-regulating mutations in the norA gene promoter.b E, efflux phenotype.c neg, same phenotype as controls.d var, variable phenotypes.

chtmca1e(tpt(

scpmaei1q

ai

e S, increased susceptibility in comparison to control strains.

ell membranes. All the norA promoter mutants specifically showedigh IC50 values in the presence of sodium selenite. In respecto control strains (strains bearing no resistance markers), norA

utants were characterized by higher IC50 values for the bio-ides domiphen bromide, DTAB, chelerythrine and dodine, even ift lower level compared to qac-positive strains. The two strains881 norA and 1891 norA were also more active in the pres-nce of benzethonium chloride, guanidine hydrochloride and CHXTable 2). The norA mutants showed higher metabolic activity inhe presence of the fungicides dichlofluanid and tolyfluanid, com-ared to all other strains. Moreover norA mutants shared withhe qacC strains higher IC50 values in response to orphenadrineTable 2).

Among the compounds that played an important role in strainseparation (Fig. S1 and Table 2) there was the toxic cation cadmiumhloride. The efflux from cells of this cation is often mediated by aump associated to qac determinants on a multiresistance plas-id (Shearer et al. 2011). Therefore qPCR analysis of all strains was

pplied in order to verify the presence of cadmium transporterncoding genes. The strains showing a higher metabolic activ-ty in the presence of cadmium chloride (1173 qacA, 2106 qacA,205 qacA, 2628 qacA, 1828 qacB and 1889 qacC) were positive in

Please cite this article in press as: Marchi E, et al. Novel insight into aqac and norA genotypes in Staphylococcus aureus. Microbiol Res (2014

PCR experiments (data not shown).Global results of efflux phenotypes associated with Qac pumps

nd overproduction of NorA based on PM analysis are summarizedn Table 3.

Susceptibility testing

Based on PM data and referring to standard antimicrobial agentsused for S. aureus strains characterization, all strains were analyzedfor their sensitivity (MIC) toward a series of toxic compounds andantibiotics. Tested antibiotics were MET, PEN, ERY, VAN, NOR, CIP,LVX, TET and SXT. As summarized in Table 4, most of the strainswere classified as MSSA (methicillin sensitive S. aureus), while fourwere MRSA (methicillin resistant S. aureus), comprising one strainbearing none of the investigated resistance markers (1177 NM).Most of the strains showed resistance to PEN, NOR and ERY. Allthe strains were sensitive to VAN and almost all to SXT; 5 strainswere resistant to TET (strains 2106 qacA, 1067 qacB, 1297 qacB,1614 qacC, 1177 NM).

The antibiotic susceptibility characterization did not allow tohighlight any direct association between any class of antibiotics andspecific resistance markers, except for the well known resistanceto NOR conferred by the up-regulated NorA efflux pump (Ubukataet al. 1989; Yoshida et al. 1990).

We also determined the MIC values for all strains in the pres-ence of the lipophilic chelator 8-HQ, the membrane active agentPS, and the three pharmaceutical drugs orphenadrine hydrochlo-

ntimicrobial resistance and sensitivity phenotypes associated to), http://dx.doi.org/10.1016/j.micres.2014.07.001

ride (ORPH), thioridazine hydrochloride (THIO) and amitriptylinehydrochloride (AMI) (Fig. S2). The first observation arising from MICdata was that the strains, also when bearing the same resistancedeterminant, were characterized by heterogeneous responses to

ARTICLE IN PRESSG ModelMICRES-25691; No. of Pages 11

8 E. Marchi et al. / Microbiological Research xxx (2014) xxx–xxx

Table 4Antibiotic susceptibility testing for selected strains described following CLSIa criteria.

Isolate Resistance determinant MET PEN ERY VAN NOR CIP LVX TET SXT

QBR102278-1027 norAb S R S S R S S S SQBR102278-1881 norA S R R S R R R S SQBR102278-1891 norA S R R S R R R S SQBR102278-2634 norA S R R S R S S S SQBR102278-2635 norA S R R S R S S S SQBR102278-1024 qacA S R S S S S S S SQBR102278-1173 qacA S R R S R R R S SQBR102278-1205 qacA S R S S R S S S SQBR102278-2106 qacA S R R S R R I R RQBR102278-2628 qacA S R S S R S S S SQBR102278-2671 qacA S R S S S S S S SQBR102278-1064 qacB R R R S R R S S SQBR102278-1067 qacB norA R R R S R R R R SQBR102278-1297 qacB norA S R R S R R S R SQBR102278-1828 qacB R R S S S S S S SQBR102278-2507 qacB S R S S S S S S SQBR102278-1209 qacC S R R S R S S S SQBR102278-1299 qacC S R S S S S S S SQBR102278-1390 qacC S R R S R I S S SQBR102278-1614 qacC norA S S R S R I S R RQBR102278-1889 qacC S R R S R R R S SQBR102278-1191 qacG S R S S R R S S SQBR102278-1177 control R R R S R R R R SQBR102278-1619 control S R S S S S S S SRN4220 control S S S S S S S S SATCC6538 control S S S S S S S S S

smcmpa1t8anosd

E

mWeatdcwtsiqgabdTsb

a CLSI, Clinical Laboratory Standards Institute.b norA gene promoter mutation.

elected compounds, in particular to PS, except the norA promoterutants, that shared identical MIC values in the presence of all

ompounds, but not of PS. In comparison to control strains, the norAutants resulted concordantly more resistant to 8-HQ. The qacC-

ositive strains showed high homogeneity in the presence of THIOnd also in the presence of AMI and ORPH, except for the strain614, bearing qacC and norA determinants, which was more resis-ant compared to the other qacC strains. In the presence of both-HQ and PS, qacC strains showed variable MIC values, which werenyway higher than those of strains with no resistance determi-ants. The qacAB strains showed high heterogeneity in the presencef all tested compounds. In the presence of AMI, OPRH and THIO,ome qacB strains were characterized by MIC values of one or twoilution lower than control strains.

thidium bromide efflux assay

Ethidium bromide is a useful substrates to investigate effluxechanisms associated with MDR phenotypes (Patel et al. 2010).e thus analyzed all strains performing an ethidium bromide

fflux assay. Fig. 3(a) shows the average ethidium bromide effluxs percentage of fluorescence reduction after 30 min of incuba-ion following cells staining with ethidium bromide. Significantifferences were found between qacB- and qacC-positive strainsompared to control strains, while no statistically significant dataere obtained for strains harboring qacA gene and norA muta-

ions in comparison to all other strains [Fig. 3(a)]. The qacA-positivetrains were characterized by a higher level of heterogeneity (ethid-um bromide efflux between 13% and 77%), while strains harboringacB, qacC genes or norA promoter mutations showed more homo-eneous ethidium bromide efflux phenotypes (Fig. S3). In all thenalyzed strains, ethidium bromide efflux was strongly reducedy the presence of the protonophore CCCP, suggesting that efflux

Please cite this article in press as: Marchi E, et al. Novel insight into aqac and norA genotypes in Staphylococcus aureus. Microbiol Res (2014

epended on the proton gradient across the membrane (Fig. S3).he strains characterized by the absence of resistance markershowed a limited ethidium bromide efflux with values rangingetween 19% and 37% (Fig. S3).

Since this analysis on clinical isolates gave poor discriminatingresults, in order to verify the feasibility of the assay, a time courseanalysis of ethidium bromide efflux was performed on six mutantsderived from the strains ATCC6538 and RN4220. The mutants werecharacterized by an increased resistance to biocides in comparisonwith their respective wild type (Furi et al. 2013). Compared with thewild-type ATCC6538, the ethidium bromide efflux was markedlystronger in the mutants MO037 and MO038 [Fig. 3(b)], selectedin the presence of CHX. Analogous results were obtained for thewild-type RN4220 and mutants MO026, harboring the pSK1-qacA+

vector [Fig. 3(c)], MO042, MO044 and MO066 selected on CHX, ben-zalkonium chloride and ethidium bromide, respectively [Fig. 3(d)].

We could therefore conclude that the ethidium bromide effluxassay is suitable to highlight differences in efflux efficiency amongisogenic strains, but it is ineffective to cluster strains on the basisof their qac or norA genotype.

Discussion

Phenotypic characterization of pathogenic bacteria is a funda-mental goal in clinical and public health interventions, not only forbacteria identification but also in order to deepen the knowledgeabout the risks of decreased susceptibility to antibiotics and bio-cides and to identify cross resistance between toxic compounds. It iswell known that antibiotic resistance is selected by the widespreadof use antibiotics, however also biocides might co-select for antibi-otics resistance. A lot of work has been performed during yearsconcerning phenotypic responses to antimicrobial agents in S.aureus strains characterized by the presence of Qac transportersor up-regulating mutations in the promoter of norA gene, as shownin Table S3, which summarizes the known efflux targets of the citedresistance determinants.

Here we exploited the phenotype microarray (PM) high-

ntimicrobial resistance and sensitivity phenotypes associated to), http://dx.doi.org/10.1016/j.micres.2014.07.001

throughput technology in combination with standard methods forsusceptibility testing to enlarge the knowledge on chemical sen-sitivity patterns of 24 S. aureus clinical isolates bearing knownresistance determinants, which were previously characterized for

ARTICLE IN PRESSG ModelMICRES-25691; No. of Pages 11

E. Marchi et al. / Microbiological Research xxx (2014) xxx–xxx 9

Fig. 3. (a) Average efflux of ethidium bromide -stained cells of S. aureus strains, shown as % of fluorescence reduction after 30 min of incubation at 37 ◦C in cells collectedduring the exponential growth phase. Data refer to 6 strains carrying the qacA gene, 5 with qacB, 5 with qacC, 5 with a norA promoter mutation, and 4 control strains (CTRL)characterized by the absence of known resistance markers. Kruskal–Wallis and Dunn’s multiple comparison post tests were performed: ** = p-val < 0.01; * = p-val <0.05. (b)Efflux activity of ethidium bromide-stained cells, in the presence (open shape) or in the absence (filled shape) of 100 �M of CCCP, of S. aureus ATCC6538 (�/�) and relativemutants MO037 (�/©) and MO038 (�/�) selected in the presence of CHX. (c) Efflux activity of ethidium bromide-stained cells of S. aureus RN4220 (�/�) and relativet Effluxm ium cm or eac

tFbdpdbN

fttbrissi2esithsts

ransconjugant MO026 (�/©) harboring the qacA carrying plasmid pSK1 vector. (d)utants MO042 (�/�), MO044 (�/©), and MO066 (�/♦) selected on CHX, benzalkonean of three replicates ± SD calculated from three independent biological assays f

heir susceptibility to widespread used biocides (Ciusa et al. 2012;uri et al. 2013). PM analysis highlighted the absence of associationetween antibiotics and any class of resistance markers in accor-ance with data here obtained by antibiotic susceptibility testserformed following standard CLSI criteria. Most importantly theata showed lack of cross-resistance to fluoroquinolones conferredy any of the MDR pumps, including the strains with up-regulatedorA, which of course conferred resistance to norfloxacin.

Concerning non antibiotic compounds, qac-positive strainsormed separated clusters from qac-negative strains, confirminghe role of a plethora of substrates previously associated to Qacransporters. PM analysis, which allowed to analyze a large num-er of Qac and NorA efflux substrates, confirmed the relevantole of QACs and intercalating dyes in describing the susceptibil-ty profiles, although with a wide range of IC50 values. Variableusceptibility levels were previously observed for compoundsuch as benzalkonium chloride (Zhang et al. 2011), CPC, ethid-um bromide, proflavine, crystal violet, acriflavine (Bay et al.008; Littlejohn et al. 1992; Mayer et al. 2001; Nakaminamit al. 2010; Noguchi et al. 1999). Not all the strains bearing theame resistance determinant shared always the same sensitiv-ty patterns, but it was possible to attribute an efflux phenotypeo Qac or NorA transporters when the majority of isolates kept

Please cite this article in press as: Marchi E, et al. Novel insight into aqac and norA genotypes in Staphylococcus aureus. Microbiol Res (2014

igher metabolic activity in the presence of a specific sub-trate. It was indeed observed that strains bearing Qac resistanceransporters may lack resistance to clearly recognized efflux sub-trates (Noguchi et al. 2005). The wide spectrum of substrates

activity of ethidium bromide-stained cells of S. aureus RN4220 (�/�) and relativehloride, or ethidium bromide, respectively. Data points indicate average geometrich time point.

analyzed with PM technology allowed to identify new targets inthe presence of which Qac and NorA pumps might be associatedto positive phenotypic responses. We hypothesized an efflux phe-notype in all analyzed strains bearing Qac transporters or norApromoter up-regulating mutations toward the QACs benzethoniumchloride, DTAB, domiphen bromide and cheleritrine, the chelatorfusaric acid, the guanidine dodine and the membrane active agentniaproof. The qacC-positive and the norA promoter mutant strainswere more tolerant than the control strains to 8-HQ, while only twoout of six qacA and three out of five qacB strains had higher MIC val-ues. In previous works no efflux phenotype has yet been associatedto 8-HQ, which is recognized as a potent antimicrobial agent actingboth against cell wall structure (Short et al. 2006) and by interferingwith enzymatic activity depending on divalent cations as cofactors(Fraser & Creanor 1975) or inducing metal-poisoning (Zn2+, Fe2+,Cu2+) upon ion chelation and delivery into the cell (Rubbo et al.1950)

The membrane chaotropic agent guanidine hydrochloride,which is one of new potential targets here identified based onPM data, seems to be an efflux substrate for both QacG andQacA transporters, while it is not for QacB and QacC. This resultallowed to hypothesize a new efflux target molecule, together withalready known divalent cations (Mitchell et al. 1999), distinguish-

ntimicrobial resistance and sensitivity phenotypes associated to), http://dx.doi.org/10.1016/j.micres.2014.07.001

ing the substrate specificity between QacA and QacB, which arestructurally very similar, having only seven different amino acids(Paulsen et al. 1996). The qacA and qacB-positive strains were alsodistinguishable by compounds toward which the presence of the

ING ModelM

1 ical R

ttdstQapc(

omao12lpaccseas(sb(oact(cfm

oiqMctfC1twimtacaieioeeeatp

ARTICLEICRES-25691; No. of Pages 11

0 E. Marchi et al. / Microbiolog

ransporter induced an increased sensitivity: QacB was associatedo decreased tolerance to amitriptyline, orphenadrine and thiori-azine (observed by both PM analysis and MIC determination). Thepecific susceptibility of strains expressing the QacB transporter tohese compounds, which are ineffective against strains expressingacA despite the high similarity between the two proteins, could bessociated with the protein sequence specificity, since it has beenreviously observed that a single amino acid substitution is suffi-ient to alter the interaction between inhibitors and target proteinsDey et al. 1999).

The membrane active agent amitriptyline, the anti-cholinergicrphenadrine and the respiration uncoupler thioridazine alterembranes of target cells: (i) amitriptyline is considered

s membrane-stabilizing compound (Corona et al. 1973); (ii)rphenadrine prevents membrane potential decrease (Sureda et al.999) and blocks voltage-gated sodium channels (Desaphy et al.009); (iii) thioridazine seems to influence transcriptional regu-

ation of genes involved in cell wall biogenesis and membraneroteins (Bonde et al. 2011; Dutta et al. 2010). Furthermore bothmitriptyline and thioridazine, which belong to the group of tri-yclic psychopharmacons, have antiplasmid activity. Their effectould be due to an increased membrane permeability or to thetoppage of plasmid replication by binding to plasmid DNA (Molnart al. 1992; Noguchi et al. 2005). It was also shown that thioridazinects as efflux pump inhibitor, preventing or reducing the extru-ion from the cell of common substrates, such as ethidium bromideCouto et al. 2008). Therefore the mechanism of thioridazine actioneems to be multifactorial, involving both perturbation of mem-rane potential and a possible direct interaction with the pumpsKaatz et al. 2003). We cannot exclude that increased sensitivityf some strains to above mentioned compounds (i.e. thioridazine,mitriptyline and orphenadrine) could be due to increased biologi-al costs associated with the production of efflux pumps. It is knownhat antimicrobial resistance might be associated with fitness costsAndersson and Levin 1999), especially when resistance relies onellular target modification, but also because of metabolic expensesor plasmid DNA, mRNA and protein synthesis, competition of plas-

id mRNA for ribosomes, and other effects.To highlight phenotypes specifically associated with each one

f the different Qac transporters has not been obvious. Studiesn literature have reported non-univocal susceptibility profiles forac-positive strains, for example Longtin et al. (2011) referred thatBC values for CHX were higher for strains with smr (qacC) gene

ompared to qacAB-positive strains isolated in Canadian hospi-als, while elsewhere it has been stated that qacC extrudes CHXorm cells with lower efficiency (Noguchi et al. 1999) or even thatHX is not an efflux substrate for qacB and qacC (Littlejohn et al.992; Paulsen et al. 1996). The different substrate response pat-erns highlighted for Qac transporters and the tendency of strainsith no resistance marker to act as solo, could also be due to the

nnate permeability of each strain to specific compounds, which isostly true for clinical isolates, bearing each one its own charac-

eristics, as previously observed for vancomycin (Cui et al. 2000) orcriflavine (Kawai et al. 2009). The ethidium bromide efflux assayonfirmed this aspect, revealing a high degree of heterogeneitymong the isolates here investigated in the ability to extrude ethid-um bromide from cells. However, the results obtained from thexperiments performed on isogenic strains showed consistent datan the comparison between the parental and derivative mutantsr the transconjugant, the latter characterized by more efficientthidium bromide efflux induced by mutations selected by biocidesxposure. We can therefore conclude that the ethidium bromide

Please cite this article in press as: Marchi E, et al. Novel insight into aqac and norA genotypes in Staphylococcus aureus. Microbiol Res (2014

fflux assay is suitable to highlight differences in efflux efficiencymong isogenic strains, but it is ineffective to cluster strains onhe basis of their qac or norA genotype. On the other hand, com-aring MIC/MBC data obtained by standard susceptibility methods

PRESSesearch xxx (2014) xxx–xxx

(Table 1) with averaged IC50 values obtained for ethidium bro-mide (Table 2), the qacA and qacB strains resulted the most tolerantto ethidium bromide, smr strains (qacC and qacG) were character-ized by an intermediate comparable susceptibility level, and thenorA mutants were characterized by only slight higher metabolicactivity in comparison to no marker strains, consistently with thenon-significant efflux ability measured by direct assay.

In conclusion the PM approach produced a great amount of highquality data relating to how stressors affected bacterial activity, andthus made it possible to have a phenotypic profile for each strainusing a single tool. PM profiles were repeatable and gave a globalview of the efflux target extruded by Qac and NorA efflux pumps.PM analysis represented the starting screening upon which wewere able to select some relevant compounds (antibiotics and otherantimicrobial agents) for which we performed further investiga-tions. In fact it is known that it is hard to draw definitive conclusionon clinical strains, as they can have overlapping resistance determi-nants that contribute to a phenotype. PM application permitted toidentified compounds playing potential antimicrobial effect againstclinical isolates bearing specific resistance determinants. The mostinteresting data arising from PM profiles were largely confirmedwith standard susceptibility testing. Besides some hypothesizesconcerning new potential efflux targets, this work led to the iden-tification of compounds toward which the presence of MDR pumpscould be linked to susceptibility compared to the strains with noresistance markers. This result opens new perspectives for thedevelopment of more effective antimicrobial treatments.

The results here obtained should encourage and stimulateefforts to use PM to address specific questions such as the spreadof antibiotic and/or biocide resistances, in order to set up a robustmethodology for investigating problems associated with the devel-opment of cross-resistance between antibiotics and biocides inclinical and environmental isolates.

Acknowledgements

This work is funded by the EU FP7 research grant “BIOHYPO- Confronting the clinical relevance of biocide induced antibioticresistance” (FP7-KBBE-2008-2B). Stefania Arioli was supported bya post-doc grant “Dote Ricerca”: FSE, Regione Lombardia.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.micres.2014.07.001.

References

Andersson DI, Levin BR. The biological cost of antibiotic resistance. Curr Opin Micro-biol 1999;2:489–93.

Bay DC, O’Neil JD, Court DA. The influence of sterols on the conformation of recom-binant mitochondrial porin in detergent. Biochem Cell Biol 2008;86:539–45.

Bjorland J, Steinum T, Sunde M, Waage S, Heir E. Novel plasmid-borne geneqacJ mediates resistance to quaternary ammonium compounds in equineStaphylococcus aureus, Staphylococcus simulans, and Staphylococcus intermedius.Antimicrob Agents Chemother 2003;47:3046–52.

Bochner BR, Gadzinski P, Panomitros E. Phenotype MicroArrays for high-throughputphenotypic testing and assay of gene function. Genome Res 2001;11:1246–55.

Bochner BR, Giovannetti L, Viti C. Important discoveries from analysing bacterialphenotypes. Mol Microbiol 2008;70:274–80.

Bonde M, Hojland DH, Kolmos HJ, Kallipolitis BH, Klitgaard JK. Thioridazine affectstranscription of genes involved in cell wall biosynthesis in methicillin-resistantStaphylococcus aureus. FEMS Microbiol Lett 2011;318:168–76.

Ciusa ML, Furi L, Knight D, Decorosi F, Fondi M, Raggi C, et al. A novel resistancemechanism to triclosan that suggests horizontal gene transfer and demonstrates

ntimicrobial resistance and sensitivity phenotypes associated to), http://dx.doi.org/10.1016/j.micres.2014.07.001

a potential selective pressure for reduced biocide susceptibility in clinical strainsof Staphylococcus aureus. Int J Antimicrob Agents 2012;40:210–20.

CLSI. Clinical and Laboratory Standards Institute. Methods for dilution antimicro-bial susceptibility tests for bacteria that grow aerobically; Approved standard.Wayne, PA, USA: CLSI; 2009.

ING ModelM

ical R

C

C

C

C

D

D

D

D

D

E

F

F

F

G

G

H

H

K

K

K

K

L

L

L

L

ARTICLEICRES-25691; No. of Pages 11

E. Marchi et al. / Microbiolog

orona GL, Santagostino G, Facino Rm, Pirillo D. Cell membrane modifications in rab-bit isolated hepatocytes following a chronic amitryptiline treatment. BiochemPharmacol 1973;22:849–56.

orrea JE, De Paulis A, Predari S, Sordelli DO, Jeric PE. First report of qacG, qacH andqacJ genes in Staphylococcus haemolyticus human clinical isolates. J AntimicrobChemother 2008;62:956–60.

outo I, Costa SS, Viveiros M, Martins M, Amaral L. Efflux-mediated response ofStaphylococcus aureus exposed to ethidium bromide. J Antimicrob Chemother2008;62:504–13.

ui LZ, Murakami H, Kuwahara-Arai K, Hanaki H, Hiramatsu K. Contributionof a thickened cell wall and its glutamine nonamidated component to thevancomycin resistance expressed by Staphylococcus aureus Mu50. AntimicrobAgents Chemother 2000;44:2276–85.

ecorosi F, Lori L, Santopolo L, Tatti E, Giovannetti L, Viti C. Characterization ofa Cr(VI)-sensitive Pseudomonas corrugata 28 mutant impaired in a pyridinenucleotide transhydrogenase gene. Res Microbiol 2011;162:747–55.

ecorosi F, Tatti E, Mini A, Giovannetti L, Viti C. Characterization of twogenes involved in chromate resistance in a Cr(VI)-hyper-resistant bacterium.Extremophiles 2009;13:917–23.

esaphy JF, Dipalma A, De Bellis M, Costanza T, Gaudioso C, Delmas P, et al. Involve-ment of voltage-gated sodium channels blockade in the analgesic effects oforphenadrine. Pain 2009;142:225–35.

ey S, Hafkemeyer P, Pastan I, Gottesman MM. A single amino acid residuecontributes to distinct mechanisms of inhibition of the human multidrugtransporter by stereoisomers of the dopamine receptor antagonist flupentixol.Biochemistry 1999;38:6630–9.

utta NK, Mehra S, Kaushal D. A Mycobacterium tuberculosis sigma factor networkresponds to cell-envelope damage by thepromising anti-mycobacterial thiori-dazine. PLoS One 2010:5.

ady EA, Ross JI, Tipper JL, Walters CE, Cove JH, Noble WC. Distribution of genesencoding erythromycin ribosomal methylases and an erythromycin efflux pumpin epidemiologically distinct groups of staphylococci. J Antimicrob Chemother1993;31:211–7.

loyd JL, Smith KP, Kumar SH, Floyd JT, Varela MF. LmrS is a multidrug efflux pump ofthe major facilitator superfamily from Staphylococcus aureus. Antimicrob AgentsChemother 2010;54:5406–12.

raser RS, Creanor J. The mechanism of inhibition of ribonucleic acid synthesisby 8-hydroxyquinoline and the antibiotic lomofungin. Biochem J 1975;147:401–10.

uri L, Ciusa ML, Knight D, Di Lorenzo V, Tocci N, Cirasola D, et al. Evaluation ofreduced susceptibility to quaternary ammonium compounds and bisbiguanidesin clinical isolates and laboratory-generated mutants of Staphylococcus aureus.Antimicrob Agents Chemother 2013;57:3488–97.

okel GW, Leevy WM, Weber ME. Crown ethers: sensors for ions and molecularscaffolds for materials and biological models. Chem Rev 2004;104:2723–50.

rinius L, Dreguniene G, Goldberg EB, Liao CH, Projan SJ. A staphylococcal mul-tidrug resistance gene-product is a member of a new-protein family. Plasmid1992;27:119–29.

eir E, Sundheim G, Holck AL. The Staphylococcus qacH gene product: a new mem-ber of the SMR family encoding multidrug resistance. FEMS Microbiol Lett1998;163:49–56.

eir E, Sundheim G, Holck AL. The qacG gene on plasmid pST94 confers resistanceto quaternary ammonium compounds in staphylococci isolated from the foodindustry. J Appl Microbiol 1999;86:378–88.

aatz GW, Moudgal VV, Seo SM, Kristiansen JE. Phenothiazines and thioxanthenesinhibit multidrug efflux pump activity in Staphylococcus aureus. AntimicrobAgents Chemother 2003;47:719–26.

aatz GW, Seo SM, Ruble CA. Efflux-mediated fluoroquinolone resistance in Staphy-lococcus aureus. Antimicrob Agents Chemother 1993;37:1086–94.

awai M, Yamada S, Ishidoshiro A, Oyamada Y, Ito H, Yamagishi J. Cell-wall thickness:possible mechanism of acriflavine resistance in meticillin-resistant Staphylococ-cus aureus. J Med Microbiol 2009;58:331–6.

oo BM, Yoon MJ, Lee CR, Nam TW, Choe YJ, Jaffe H, et al. A novel fermenta-tion/respiration switch protein regulated by enzyme IIA(Glc) in Escherichia coli.J Biol Chem 2004;279:31613–21.

eclercq R. Mechanisms of resistance to macrolides and lincosamides: natureof the resistance elements and their clinical implications. Clin Infect Dis2002;34:482–92.

eelaporn A, Paulsen IT, Tennent JM, Littlejohn TG, Skurray RA. Multidrug-resistanceto antiseptics and disinfectants in coagulase-negative staphylococci. J MedMicrobiol 1994;40:214–20.

Please cite this article in press as: Marchi E, et al. Novel insight into aqac and norA genotypes in Staphylococcus aureus. Microbiol Res (2014

ittlejohn TG, Paulsen IT, Gillespie MT, Tennent JM, Midgley M, Jones IG, et al.Substrate-specificity and energetics of antiseptic and disinfectant resistance inStaphylococcus aureus. FEMS Microbiol Lett 1992;95:259–66.

ongtin J, Seah C, Siebert K, McGeer A, Simor A, Longtin Y, et al. Distributionof antiseptic resistance genes qacA, qacB, and smr in methicillin-resistant

PRESSesearch xxx (2014) xxx–xxx 11

Staphylococcus aureus isolated in Toronto, Canada, from 2005 to 2009. Antimi-crob Agents Chemother 2011;55:2999–3001.

Mayer S, Boos M, Beyer A, Fluit AC, Schmitz FJ. Distribution of the antisep-tic resistance genes qacA, qacB and qacC in 497 methicillin-resistant and-susceptible European isolates of Staphylococcus aureus. J Antimicrob Chemother2001;47:896–7.

Mitchell BA, Brown MH, Skurray RA. QacA multidrug efflux pump from Staphylococ-cus aureus: comparative analysis of resistance to diamidines, biguanidines, andguanylhydrazones. Antimicrob Agents Chemother 1998;42:475–7.

Mitchell BA, Paulsen IT, Brown MH, Skurray RA. Bioenergetics of the staphylococ-cal multidrug export protein QacA – Identification of distinct binding sites formonovalent and divalent cations. J Biol Chem 1999;274:3541–8.

Molnar J, Foldeak S, Nakamura MJ, Rausch H, Domonkos K, Szabo M. Antiplasmidactivity – loss of bacterial-resistance to antibiotics. APMIS 1992;100:24–31.

Nakaminami H, Noguchi N, Sasatsu M. Fluoroquinolone efflux by the plasmid-mediated multidrug efflux pump QacB variant QacBIII in Staphylococcus aureus.Antimicrob Agents Chemother 2010;54:4107–11.

Noguchi N, Hase M, Kitta M, Sasatsu M, Deguchi K, Kono M. Antiseptic suscep-tibility and distribution of antiseptic-resistance genes in methicillin-resistantStaphylococcus aureus. FEMS Microbiol Lett 1999;172:247–53.

Noguchi N, Suwa J, Narui K, Sasatsu M, Ito T, Hiramatsu K, et al. Susceptibilities toantiseptic agents and distribution of antiseptic-resistance genes qacA/B and smrof methicillin-resistant Staphylococcus aureus isolated in Asia during 1998 and1999. J Med Microbiol 2005;54:557–65.

Patel D, Kosmidis C, Seo SM, Kaatz GW. Ethidium bromide MIC screening forenhanced efflux pump gene expression or efflux activity in Staphylococcusaureus. Antimicrob Agents Chemother 2010;54:5070–3.

Paulsen IT, Brown MH, Dunstan SJ, Skurray RA. Molecular characterizationof the staphylococcal multidrug-resistance export protein QacC. J Bacteriol1995;177:2827–33.

Paulsen IT, Brown MH, Littlejohn TG, Mitchell BA, Skurray RA. Multidrug resistanceproteins QacA and QacB from Staphylococcus aureus: membrane topology andidentification of residues involved in substrate specificity. Proc Natl Acad SciUSA 1996;93:3630–5.

Pechere JC. Macrolide resistance mechanisms in Gram-positive cocci. J AntimicrobChemother 2001;18:S25–8.

Poole K. Efflux-mediated antimicrobial resistance. J Antimicrob Chemother2005;56:20–51.

Rubbo CA, Albert A, Gibson MI. The influence of chemical constitution on antibacte-rial activity. V. The antibacterial action of 8-hydroxyquinoline (oxine). Br J ExpPathol 1950;31:425–41.

Shearer JES, Wireman J, Hostetler J, Forberger H, Borman J, Gill J, et al. Major familiesof multiresistant plasmids from geographically and epidemiologically diversestaphylococci. G3 2011;1:581–91.

Short BRD, Vargas MA, Thomas JC, O‘Hanlon S, Enright MC. In vitro activity of anovel compound, the metal ion chelating agent AQ(+), against clinical isolatesof Staphylococcus aureus. J Antimicrob Chemother 2006;57:104–9.

Sureda FX, Gabriel C, Pallas M, Adan J, Martinez JM, Escubedo E, et al. In vitroand in vivo protective effect of orphenadrine on glutamate neurotoxicity. Neu-ropharmacology 1999;38:671–7.

Tennent JM, Lyon BR, Midgley M, Jones IG, Purewal AS, Skurray RA. Physical and bio-chemical characterization of the qacA gene encoding antiseptic and disinfectantresistance in Staphylococcus aureus. J Gen Microbiol 1989;135:1–10.

Ubukata K, Itohyamashita N, Konno M. Cloning and expression of the norA genefor fluoroquinolone resistance in Staphylococcus aureus. Antimicrob AgentsChemother 1989;33:1535–9.

Viti C, Decorosi F, Mini A, Tatti E, Giovannetti L. Involvement of the oscA gene in thesulphur starvation response and in Cr(VI) resistance in Pseudomonas corrugata28. Microbiology 2009;155:95–105.

Viti C, Decorosi F, Tatti E, Giovannetti L. Characterization of chromate-resistantand -reducing bacteria by traditional means and by a high-throughput phe-nomic technique for bioremediation purposes. Biotechnol Prog 2007;23:553–9.

Winterberg KM, Luecke J, Bruegl AS, Reznikoff WS. Phenotypic screening ofEscherichia coli K-12 Tn5 insertion libraries, using whole-genome oligonu-cleotide microarrays. Appl Environ Microbiol 2005;71:451–9.

Yoshida H, Bogaki M, Nakamura S, Ubukata K, Konno M. Nucleotide-sequence andcharacterization of the Staphylococcus aureus norA gene, which confers resis-tance to quinolones. J Bacteriol 1990;172:6942–9.

Yu JL, Grinius L, Hooper DC. NorA functions as a multidrug efflux protein in bothcytoplasmic membrane vesicles and reconstituted proteoliposomes. J Bacteriol

ntimicrobial resistance and sensitivity phenotypes associated to), http://dx.doi.org/10.1016/j.micres.2014.07.001

2002;184:1370–7.Zhang M, O‘Donoghue MM, Ito T, Hiramatsu K, Boost MV. Prevalence of antiseptic-

resistance genes in Staphylococcus aureus and coagulase-negative staphylococcicolonising nurses and the general population in Hong Kong. J Hosp Infect2011;78:113–7.