R E S EA RCH L E T T E R

Characterization of tetA-like gene encoding for a majorfacilitator superfamily efflux pump in Streptococcus

thermophilus

Stefania Arioli1, Simone Guglielmetti1, Stefano Amalfitano2, Carlo Viti3, Emmanuela Marchi3,Francesca Decorosi3, Luciana Giovannetti3 & Diego Mora1

1Department of Food Environmental and Nutritional Sciences (DeFENS), University of Milan, Milan, Italy; 2Istituto di Ricerca sulle Acque (IRSA-

CNR), Monterotondo, Rome, Italy; and 3Dipartimento di Scienze delle Produzioni Agroalimentari e dell’Ambiente – sezione di Microbiologia,

University of Florence, Florence, Italy

Correspondence: Stefania Arioli,

Department of Food Sciences and Nutrition

(DeFENS), University of Milan, via Mangiagalli

25, 20133 Milan, Italy.

Tel.: 0039 02 503 19133;

fax: 0039 02 503 19138;

e-mail: stefania.arioli@unimi.it

Received 5 March 2014; revised 7 April

2014; accepted 22 April 2014. Final version

published online 12 May 2014.

DOI: 10.1111/1574-6968.12449

Editor: Mark Enright

Keywords

antibiotic resistance; lactic acid bacteria; MFS

efflux pump; Streptococcus thermophilus.

Abstract

Efflux pumps are membrane proteins involved in the active extrusion of a wide

range of structurally dissimilar substrates from cells. A multidrug efflux pump

named TetA belonging to the major facilitator superfamily (MFS) of transport-

ers was identified in the Streptococcus thermophilus DSM 20617T genome. The

tetA-like gene was found in the genomes of a number of S. thermophilus strains

sequenced to date and in Streptococcus macedonicus ACA-DC 198, suggesting a

possible horizontal gene transfer event between these two Streptococcus species,

which are both adapted to the milk environment. Flow cytometry (single-cell)

analysis revealed bistable TetA activity in the S. thermophilus population, and

tetA-like gene over-expression resulted in a reduced susceptibility to ethidium

bromide, tetracycline, and other toxic compounds even when the efflux pump

was over-expressed in a strain naturally lacking tetA-like gene.

Introduction

Both bacterial and eukaryotic cells are usually equipped

with various types of membrane transport systems that

are involved in a number of vital functions. Indeed, such

carriers transport nutrients and ions into the cell, excrete

waste products and toxic compounds from the cell, and

are involved in the maintenance of cellular homeostasis

(Paulsen et al., 1996; Simm et al., 2012). Moreover,

several of these transport/efflux systems have been

demonstrated to play an important role in bacterial and

eukaryotic cells by conferring resistance to antimicrobial

molecules. Export systems that can manage a wide range

of structurally dissimilar substrates are defined as multi-

drug exporters or multidrug resistance (MDR) efflux

pumps (Paulsen et al., 1996; Floyd et al., 2010). Although

genes encoding selective drug efflux pumps are found on

transferable genetic elements or plasmids, those encoding

multidrug efflux pumps are generally located on the bac-

terial chromosome (Putman et al., 2000). Multidrug

transporters are considered to play a significant role in

the resistance of bacteria to antimicrobial compounds (Li

& Nikaido, 2004; Poole, 2007; Simm et al., 2012), and

these systems are energy-dependent membrane proteins

able to extrude a wide array of structurally and

functionally dissimilar compounds. On the basis of

bioenergetic and structural features, multidrug transport-

ers can be divided into two major groups: (1) primary

active ABC transporters, which utilize the free energy of

ATP binding/hydrolysis to efflux toxic substrates; and (2)

secondary active transporters, which derive energy to

mediate drug extrusion from a coupled exchange with H+

and/or Na+ ions (van Veen & Konings, 1997; Putman

et al., 2000; Wang & van Veen, 2012). Approximately

25% of all known membrane transport proteins in

prokaryotes belong to the major facilitator superfamily

(MFS; Saier et al., 1999), the largest and most diverse

superfamily of secondary active transporters (Reddy et al.,

FEMS Microbiol Lett 355 (2014) 61–70 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

MIC

ROBI

OLO

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LET

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2012), comprising 58 families involved in the symport,

antiport, or uniport of various substrate (Floyd et al.,

2010). Streptococcus thermophilus is a lactic acid bacterium

that is widely used for the preparation of several dairy

products, such as fermented milk, yogurt, and cheese

(Hols et al., 2005). Approximately 200 putative mem-

brane transporters have been identified on the basis of

S. thermophilus genome sequencing and according to the

TransportDB database (Ren et al., 2007), with 54% of the

drug efflux pumps belonging to the ABC superfamily and

23% belonging each to the multidrug/oligosaccharide/

polysaccharide superfamily and MFS (Lorca et al., 2007).

In this study, we focused our attention on the tetA-like

gene of S. thermophilus DSM 20617T, encoding a putative

MFS efflux pump potentially involved in reduced suscep-

tibility to cytotoxic compounds. To understand the role

and activity of TetA in S. thermophilus DSM 20617T, we

over-expressed the tetA-like gene and evaluated its effects

on the sensitivity of this bacterium to several antimicro-

bial compounds. Moreover, the effect of tetA-like over-

expression on tetracycline (TET) and ethidium bromide

(EB) sensitivity was evaluated in a strain naturally lacking

this efflux pump.

Materials and methods

Bacterial strains, media, and growth conditions

Wild-type S. thermophilus DSM 20617T and MIMSt24

were maintained in M17 broth (Difco Laboratories,

Detroit, MI) supplemented with 20 g L�1 lactose at 37 °C. The recombinant strains MIM27 and MIM28 (tetA

over-expressing recombinant strain) and MIM20 and

MIM21 (harboring the empty vector pMIT5) were culti-

vated in the same medium with chloramphenicol at a

final concentration of 4 lg mL�1. Recombinant strains

MIM20 and MIM27 were derived from strain DSM

20617T, and recombinant strains MIM21 and MIM28

were derived from strain MIMSt24. Escherichia coli cells

harboring plasmid pNZ8048 (de Ruyter et al., 1996),

pMIT5, or pMIT5-tetA were routinely maintained under

aerobic conditions in Luria broth at 37 °C supplemented

with 25 lg mL�1 chloramphenicol.

Vector construction

Plasmid pNZ8048 is a broad-spectrum host shuttle vector

that possesses the nisin-inducible nisA promoter and a

chloramphenicol resistance gene as the selection marker

(de Ruyter et al., 1996). The nisA promoter of pNZ8048

was replaced by a strong phage T5 promoter and lac

operator (Loimaranta et al., 1998; Guglielmetti et al.,

2008), generating pMIT5, as follows: the T5 promoter

was amplified using pCSS945 DNA as the template

(Arioli et al., 2010) with primers T5-BglF (50-TCTAGGAAAGATCTCAACGGAGGACTAGCGTA-30) and T5-

NcoR (50-CCTTGGAACCATGGTCATCATGGATCCCCTCAT-30), which contain the restriction sites of BglII and

NcoR, respectively (underlined). tetA-like gene (EMBL

accession number HG424699) was identified in the

whole-genome sequence of S. thermophilus DSM 20617T.

Pure genomic DNA was extracted and purified using the

UltraCleanTM Microbial DNA Isolation Kit (MO BIO,

CA). tetA was amplified using the primers tetA-BamHI-F

(50-TCTAGGAAGGATCCATTAAATATAATTTTTTCATTATCT-30) and tetA-PstI- (50-CCTTGGAACTGCAGTTAAATATAATTTTTAATAAAGTAATTT-30), which contain

BamHI and PstI restriction sites, respectively (under-

lined). tetA was then ligated to pMIT5 to generate

pMIT5-tetA. The ligation mixture was introduced into

One ShotR MatchTM-T1R Chemically competent E. coli

cells (Invitrogen) by heat shock (30 s at 42 °C). The

transformed cells were plated on Luria-agar containing

chloramphenicol (25 lg mL�1).

Streptococcus thermophilus DSM 20617T or MIMSt24

electrocompetent cells were prepared and transformed

with either pMIT5 or pMIT5-tetA, as previously described

(Arioli et al., 2007). The recombinant strains were

selected by plating on M17 lactose (20 g L�1) agar con-

taining 4 lg mL�1 of chloramphenicol after incubation

of 48 h at 37 °C under anaerobic conditions.

Genomic comparison among S. thermophilus and

Streptococcus macedonicus strains was carried out using

rapid annotation using subsystem technology (RAST) Ser-

ver (Aziz et al., 2008) and The SEED VIEWER version 2.0

(Overbeeck et al., 2005).

tetA transcriptional analysis

A transcriptional analysis of tetA-like gene was performed

by RT-qPCR in the S. thermophilus DSM 20617T wild-

type strain and in the recombinant tetA over-expressing

MIM27 strain following a previously described protocol

(Arioli et al., 2010). Total RNA was extracted using the

AurumTM Total RNA Fatty and Fibrous Tissue Kit

(Bio-Rad) from cells collected at an OD600 of 0.5 from

cultures grown in M17 broth with 20 g L�1 lactose in the

presence or absence of 0.125 lg mL�1 of chlorhexidine

(CH) or 0.5 or 0.125 lg mL�1 EB. The concentration

and purity of the purified RNA was analyzed using a

Take3TM Multi-Volume Plate (BioTek). cDNA was syn-

thesized using the iScriptTM cDNA synthesis kit (Bio-Rad)

with random primers. The resultant cDNA was subjected

to qRT-PCR; a control PCR of the same samples without

reverse transcriptase was included to assure the absence

of DNA contamination. The primers used are the

FEMS Microbiol Lett 355 (2014) 61–70ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

62 S. Arioli et al.

following: tetAF 50-tgggattgcagcagcatttgaaag-30 and tetAR

50-ccacctataccactaccgataatc-30 for tetA-like amplification;

ureCF 50-gctgacggtagtaatgcaacaa-30 and ureCR 50-gtgtgtcttaattcctgctgca-30 for ureC gene amplification;

murEF 50-tcaataggtccgatgtggtctgg-30 and murER 50-ccgtatgatggctgaagctgtatc-30 for murE amplification. The

results were normalized using murE and ureC, coding for

UDP-N-acetylmuramoylalanyl-D-glutamate-L-lysine ligase

and urease a-subunit, respectively, as reference genes. The

specificity of the primers was tested with melting curves

during amplification and by 1% agarose gels. For each

condition, we performed the relative quantification of

gene expression in triplicate with the cDNA synthesized

from two independent RNA samples. The PCR reactions

were performed as previously described (Arioli et al.,

2010) using a CFX96 thermocycler (Bio-Rad Laboratories,

Segrate, Italy). The data are expressed as the normalized

expression (DDCT) � standard error of the mean.

EB efflux assay

EB is a common efflux pump substrate that intercalates

with double-stranded nucleic acids in the cytoplasm,

thereby determining a fluorescence increase with the

proper excitation wavelength; as a substrate for a variety

of membrane pumps, EB is extruded across the mem-

brane, thus decreasing the overall measurable fluores-

cence (Lubelski et al., 2007; Czechowska & van der

Meer, 2012). For the EB efflux assay, strains MIM20

and MIM27 were grown until the mid-log phase (OD600

of 0.5). The cells were washed twice and resuspended in

sterile PBS (pH 7) in presence of 0.5 lg mL�1 EB with

and without two commonly employed efflux pump

inhibitors (EPIs): 0.1 mM of the protonophore carbonyl

cyanide m-chlorophenylhydrazone (CCCP) to disrupt

the electrochemical gradient across the membrane, thus

removing the proton gradient (Floyd et al., 2010); or

2 lM of reserpine (Schindler et al., 2013), a known

MDR EPI (Frempong-Manso et al., 2009), both pur-

chased from Sigma-Aldrich (Italy). This approach effec-

tively allowed the loading of the cells with EB after an

incubation time of 10 min at 37 °C; the cells were then

pelleted at 15 000 g for 1 min and resuspended in PBS.

To induce efflux activity, an energy source was applied

to the cells (10 g L�1 lactose, w/v, and urea 1 mM; Ari-

oli et al., 2010), thereby initiating cellular metabolism

and creating the electrochemical gradient across the

membrane that is necessary for efflux pump activity

(Floyd et al., 2010). The EB efflux was assessed at the

single-cell level by flow cytometry (Apogee A50-micro;

Apogee Flow System, UK) and by applying a double-

staining protocol with SYBR Green I (Molecular

Probes�; Invitrogen) and EB (Czechowska & van der

Meer, 2012). Under 488-nm laser beam excitation, the

SYBR signals were registered in the green channel (520/

10 nm), whereas the EB signals were registered in the

red channel (> 610 nm). A compensation factor (4%)

was calculated for a control sample and thus applied to

reduce the contribution of SYBR Green I to the red

channel. The HISTOGRAM software (version 2.6) was used

to plot the density diagrams (color plots and histo-

grams) of the log-transformed fluorescence signals of

each single cell.

Minimum inhibitory concentration and growth

fitness evaluations

The effect of tetA over-expression on the growth kinetic

parameters of MIM20 and MIM27 was determined using a

semi-automated microdilution method performed in a

384-well microtiter plate, as reported by Arioli et al.

(2013), in the absence (positive control) and presence of

six different concentrations of EB, CH, or TET. The effects

of each of the six concentrations of the above-mentioned

toxic compounds were tested in M17 broth in the presence

of two different concentrations of lactose (5 and 20 g L�1).

The strains were inoculated in quadruplicate at a final inoc-

ulum concentration of 105 bacteria mL�1 (with the bacte-

ria from cultures grown overnight to the stationary phase).

The bacterial cell concentration of the overnight culture

was determined microscopically with a Neubauer improved

counting chamber (Marienfeld GmbH, Lauda-K€Onigsho-

fen, Germany). To ensure anaerobic incubation, each well

was covered with 20 lL of sterile mineral oil (Sigma-

Aldrich). The microbial growth was monitored with a spec-

trophotometer (MicroWave RS2; Biotek) programmed for

145 readings (OD600) every 10 min for 24 h at 37 °C. Atthe end of the incubation, the lag time (h) was calculated

using the software GEN5 (Biotek) and reported as the mean

of four independent measurements � standard deviation.

Phenotype microarray assay

Phenotype microarray (PM) technology uses tetrazolium

violet reduction as an indicator of active metabolism; the

reduction of the dye causes the formation of a purple

color, which is recorded by a CCD camera every 15 min

to provide quantitative and kinetic information regarding

the response of the cells in PM plates (Bochner et al.,

2001). Strains MIM20 and MIM27 were tested by the PM

approach for 240 different compounds potentially exert-

ing toxic effects on bacterial cells; each was included at

four concentrations. To perform the PM experiments, the

strains were grown overnight at 37 °C on BUG agar (Bio-

log, Hayward, CA) supplemented with chloramphenicol

4 lg mL�1, and the cells were then collected with a sterile

FEMS Microbiol Lett 355 (2014) 61–70 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

TetA MFS efflux pump in Streptococcus thermophilus 63

cotton swab and suspended in 15-mL inoculation solu-

tion (IF-0; Biolog). The cell density was adjusted to 81%

transmittance using a Biolog turbidimeter. The PM11–PM20 inoculations were performed as described by Deco-

rosi et al. (2011), with the supplementation of chloram-

phenicol 2 lg mL�1. All the PM plates were incubated at

37 °C in an Omnilog Reader (Biolog) and monitored

automatically every 15 min for 72 h for color develop-

ment. Compounds giving bad kinetics over the four wells

(i.e., because the color development depended on the

increasing concentration of the compounds instead of the

real metabolic activity) were excluded from further analy-

sis. To identify the phenotypes gained or lost by MIM27

compared to MIM20, the kinetic curves obtained were

compared for the ‘Darea’ parameter using OMNILOG-PM

software (Biolog Inc) (release OM_PM_109M). Area

under the curve is expressed in arbitrary OmniLog Units

(AOU). We calculated the percentage of increase/decrease

of area of the MIM27 strain compared to MIM20. We

considered as significant all the compounds showing

Darea ≥ 100% in at least one of the four wells. We also

exploited the selection filter of the software, which high-

lights all the wells in which the chosen parameter exceeds

the setup limit. The distance limit for Darea parameter

was the setup at 15 000 AOU. We thus included also the

compounds selected by the software, giving an increase/

decrease of area values of at least 50%.

Results and discussion

Identification, sequence analysis, and tetA

distribution in S. thermophilus

Although MFS-type efflux pumps have been extensively

described, particularly for pathogenic bacteria, this is the

first characterization of this transport system in S. ther-

mophilus, a lactic acid bacterium. A genome analysis of

S. thermophilus DSM 20617T revealed that the 1170-

nucleotide-long tetA-like gene encodes a putative protein

of 389 amino acids. The tetA-like gene was found in the

whole genome of certain strains of S. thermophilus

(CNRZ1066, LMD-9, JIM 8232, and MN-ZLW-002) and

in S. macedonicus ACA-DC 198. In Fig. 1a is reported the

schematic organization of schematic organization of tetA-

like genomic locus as deduced by genomic comparison of

tetA-like gene from S. thermophilus DSM20617, and

S. thermophilus LMD9 and LMG18311, and S. macedoni-

cus ACA-DC198 genomes. Moreover, the nucleotide and

amino acid identities among the two species were found

to be 100% and 99%, respectively. The presence of the

IS1191 (Fig. 1a) could support the hypothesis of a possi-

ble horizontal gene transfer (HGT) event between these

two Streptococcus species adapted to the same milk envi-

ronment (Bolotin et al., 2004; Papadimitriou et al.,

2012).

A blast analysis of the deduced TetA amino acid

sequence revealed high identity with the multidrug trans-

porters of other Gram-positive bacteria, with highly con-

served motifs characteristic of the MFS family (Pao et al.,

1998). A more detailed amino acid sequence analysis

allowed the identification of a conserved structural and

functional domain TetA(P) (ExPxxxxxDxxxRK) (Fig. 1b),

which was previously characterized in the TET-resistant

proteins TetA(P) and Tet(40) of the anaerobes Clostrid-

ium perfringens and Clostridium saccharolyticum (Bannam

& Rood, 1999; Kazimierczak et al., 2008).

Because the tetA-like gene has not been detected in the

genome of all S. thermophilus strains sequenced thus far,

a PCR survey of the tetA-like gene was performed in a

collection of 100 strains isolated from different sources,

revealing the presence of the gene in 34% of the strains

(Table S1). Interestingly, only one of 20 Georgian strains

screened for the tetA-like gene was positive in the PCR

analysis. It is worth noting that all Georgian strains were

isolated from several homemade yogurt samples prepared

without any strain selection or characterization, whereas

industrial strains are usually selected for their robustness,

rapid growth, and acidification of milk.

Single-cell analysis of EB cell efflux

To investigate the role and activity of this putative efflux

pump, we constructed S. thermophilus DSM 20617T rec-

ombinants over-expressing the tetA gene. To this end, we

cloned the gene under the control of the T5 promoter,

thus allowing the constitutive expression of tetA. The

effect of tetA over-expression on the ability of the cell to

pump out EB was evaluated by analyzing the kinetics of

EB efflux at single-cell level by flow cytometry.

The EB efflux assay was performed with an EB concen-

tration that did not in any way affect cell viability, thus

allowing the detection of the extrusion activity of the

efflux pump system (Paix~ao et al., 2009). The tetA over-

expressing mutant MIM27 showed a more efficient EB

efflux compared to that of MIM20. In fact, the results

revealed a higher decrease in fluorescence after 30 min at

37 °C in MIM27 compared to the MIM20 control cells,

suggesting an active extrusion of EB from the cells over-

expressing tetA (Fig. 2b). Following the addition of the

protonophore CCCP (Fig. S1) or reserpine (data not

shown), the fluorescence of both the control and mutant

cells after 30 min was higher compared to the control

without EPIs due to the collapse of the proton gradient

across the membrane. This phenomenon could provide

indirect evidence for an H+-dependent activity of TetA,

as previously indicated for the characterization of LmrS,

FEMS Microbiol Lett 355 (2014) 61–70ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

64 S. Arioli et al.

an MFS multidrug efflux pump from Staphylococcus

aureus (Floyd et al., 2010). Interestingly, flow cytometry

revealed that the MIM27 population is extremely

homogeneous, whereas the MIM20 is composed of two

subpopulations identified on the basis of the ability to

efflux EB. The two populations observed for MIM20 were

also identified in the wild-type DSM 20617T (Fig. S2)

strain and in MIM27 when the cells were treated with

CCCP (Fig. 2), thus suggesting that TetA activity is only

partially blocked by the presence of the inhibitor in the

overall p3opulation over-expressing tetA. However, in the

absence of CCCP, the presence of two MIM20 subpopu-

lations underlined a potential transcriptional bistability of

tetA, a phenomenon observed for other phenotypes in

other bacterial species (Veening et al., 2008).

Substrate spectrum and transcriptional

analysis

Strains MIM20 and MIM27 were analyzed for chemical

sensitivity to 240 compounds, each at four concentrations

(PM11–PM20), using the BIOLOG Phenotype Microarray.

TetA(20617) Streptococcus thermophilus … 53 I L F E F P S G M L S D N W S R K 69… 389aa

TetA(408) Clostridium perfringens … 56 F I F E I P T G I V A D V Y S R K 72… 409aa

TetA(40) Clostridium saccharolyticum … 57 F L F E M P T G V V A D L Y S R R 73… 406aa

TetA(B) Escherichia coli … 55 V I F A P W L G K M S D R F G R R 71… 401aa

TetA(B)Motif G x x x x R x G R R

TetA(P)Motif E x P x x x x x D x x x R K

(a)

(b)

Ster_1914tetA-like

Ster_1913 mccB-like IS1191-trIS1191-tr

Ster_1919

Stu1947Stu1940 Stu1942

S. thermophilus LMD9/DSM20617

S. thermophilus LMG18311

SMA_1914tetA-like mccB-like

IS1191

S. macedonicus ACA-DC 198

Fig. 1. (a) Schematic organization of tetA-like genomic locus as deduced by genomic comparison of tetA-like gene from S. thermophilus

DSM20617, and S. thermophilus LMD9 and LMG18311, and Streptococcus macedonicus ACA-DC 198 genomes (accession numbers CP000419,

CP000023, HE613569). SMA_1914, putative transposase; SMA_1917, tetA-like gene; SMA_1918, mccB, putative bacteriocin biosynthesis gene,

Ster_1913 and its orthologs stu1940, coding for a putative adenylosuccinate synthase; ster_1914, coding for a putative DNA topology

modulation protein; IS1191, coding for IS256 transposase family; IS1191-tr, IS1191 truncated gene; Ster_1919 and its orthologs Stu1947, XRE

family transcript regulator; Stu1942, putative protein with unknown function; Genes identified by black arrows code for putative transposases. S.

thermophilus genes located upstream and the downstream the tetA-like and mccB-like genes are identified by light blue arrows. (b) Alignment of

the regions containing the TetA(P) motif. The prototype TET efflux protein TetA(B) and the previously identified TetA(B) motif are shown. The first

column lists the names of the protein included, followed by the host organism. The name of the first and last residues included is given, as well

as the total length of the protein.

FEMS Microbiol Lett 355 (2014) 61–70 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

TetA MFS efflux pump in Streptococcus thermophilus 65

Strain MIM27 was characterized by increased metabolic

activity compared to MIM20 in the presence of 18

biocidal compounds and eight antibiotic (Table 1).

Some of these compounds belong to classes already

known to be efflux targets of transporters belonging to

the MFS family in pathogenic bacteria, such as QACs

(methyltrioctylammonium chloride) and biguanides (al-

exidine) (Littlejohn et al., 1992; Mitchell et al., 1998).

Furthermore, we found classes of compounds previously

not associated with MFS transporters, such as chelators

(EDTA, 1,10-phenanthroline, 5,7-dichloro-8-hydroxy-

quinaldine) and inhibitors (b-chloro-L-alanine, guanazole,

azathioprine). Interestingly, in comparison with MIM20,

MIM27 showed a higher metabolic activity in the

MIM20CCCP

MIM27CCCP

MIM20 MIM27

MIM20 MIM27

(a)

(b)

Fig. 2. EB efflux as detected by flow cytometry. The density diagrams

(upper panels) show the green (SYBR Green I) and red (EB)

fluorescence levels of different populations of recombinant strains

MIM20 and MIM27, with and without the protonophore carbonyl

cyanide m-chlorophenylhydrazone (CCCP) after EB loading. The

dotted lines discriminate between lower (�) and higher (+) EB cell

content. The frequency histograms (lower panels) show the

decreasing red fluorescence due to enhanced EB efflux over time in

MIM20 (lower left panels) and the low red fluorescence values,

detected already at time 0, of strain MIM27 over-expressing tetA

(lower right panels).

Table 1. Percent increase/decrease in area values in presence of

chemicals as determined by the OMNILOG-PM software (Biolog Inc)

Chemical Mode of action

% Area variation

(MIM27 vs. MIM20)

Nonantibiotic compounds

b-Chloro-L-alanine Alanine analog,

aminotransferase

inhibitor

169

Guanazole Ribonucleotide DP

reductase inhibitor

174

Azathioprine Nucleic acid analog,

purine

74

EDTA Chelator, hydrophilic 68

1,10-Phenanthroline Chelator, lipophilic 78; 144; 111*

5,7-dichloro-8-

hydroxy-quinaldine

Chelator, lipophilic 122

Chloroxylenol Fungicide 139

Protamine sulfate Membrane, ATPase 104

MTAC Membrane, detergent,

cationic

178

Alexidine Membrane, biguanide 57; 78; 248

Diamide Oxidation, glutathione 301

3,4-Dimethoxybenzyl

alcohol

Oxidizing agent 81

Pentachlorophenol Respiration,

ionophore, H+

340; 101

Menadione Respiration, uncoupler 238

Sodium azide Respiration, uncoupler 55; 61

Sodium bromate Toxic anion 69

Sodium nitrite Toxic anion 90

Zinc chloride Toxic cation 94

Antibiotics

Aztreonam Wall, lactam 160

Cefmetazole Wall, cephalosporin 260; 342; 102; 78

Tetracycline Protein synthesis, 30S

ribosomal subunit

154; 79

Penimepicycline Protein synthesis, 30S

ribosomal subunit

113

Furaltadone Nitro-compound,

DNA synthesis

152

Nitrofurantoin Nitro-compound,

DNA synthesis

118

Nitrofurazone Nitro-compound,

oxidizing agent

75; 72; 71

Sulfisoxazole Folate antagonist,

PABA analog

93; 130; 243

MTAC, methyltrioctylammonium chloride.

*In case significant changes were observed in more than one concen-

tration of chemical, all Darea values are given.

FEMS Microbiol Lett 355 (2014) 61–70ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

66 S. Arioli et al.

presence of several antibiotics: TET and penimepicycline

(TETs), furaltadone, nitrofurazone and nitrofurantoine

(nitro-compounds), cefmetazole, aztreonam, and sulfis-

oxazole.

Based on these findings, the transcriptional analysis of

tetA was measured by RT-qPCR in the MIM20 and

MIM27 strains cultivated in the absence and presence of

TET, CH, or EB (the top substrate for studies on MDR

efflux pumps), until the mid-exponential growth phase

(Fig. 3). The results highlighted that the efflux pump was

clearly induced when the cells were exposed to EB and

TET in the wild-type strain, with an increase in fold

expression of 2.1 � 0.4 and 3.6 � 0.5, respectively. No

tetA induction was observed in the presence of CH. As

expected, tetA was over-expressed in MIM27 under all

tested conditions (Fig. 4) due to the activity of the T5

promoter. These results, along with the previous results

of PM, suggest that TetA could be associated with the

resistance to several antimicrobial compounds or, at least,

that it is an efflux pump with a broad range of substrates.

Over-expression of tetA-like gene positively

affects the growth fitness of S. thermophilus

On the basis of the above findings, we determined the

minimum inhibitory concentrations (MICs) of several

antimicrobial compounds, including TET, CH, and EB.

Because efflux pumps could involve either an ion gradient

(commonly H+) or ATP cleavage to provide the required

energy (Poole, 2005), the MIC evaluation was performed

in M17 broth in the presence of 5 and 20 g L�1 of lactose

(that is low and high availabilities of carbon source,

respectively). The obtained results (Table 2) highlighted a

strong correlation between the carbon source concentra-

tion and the sensitivity to the antimicrobial compound,

underlining the role of the carbon source abundance and

H+ or ATP availability in determining the sensitivity

against antimicrobials. Under all tested conditions,

MIM27 showed reduced sensitivity to the cytotoxic com-

pounds compared to MIM20. Specifically, the most sig-

nificant differences between the two strains were in the

presence of TET and EB. The MIC values of TET in

MIM27 were 0.25 and 1 lg mL�1 in the presence of 5

and 20 g L�1 of lactose, respectively; MIM20 displayed

lower MICs, 0.125 and 0.25 lg mL�1, at the lowest and

highest lactose concentrations, respectively. Analogous

results were obtained in the presence of EB (Table 2).

Conversely, the CH MICs were unaffected or only weakly

affected by the over-expression of tetA, and it was there-

fore concluded that CH is not a preferred substrate of

this efflux pump.

To investigate whether the over-expression of tetA

could influence bacterial growth fitness, both MIM20 and

MIM27 were grown in M17 medium, and the growth

Table 2. Effect of tetA over-expression and lactose concentration on S. thermophilus sensitivity to several antimicrobial compounds

Strain

MIC (lg mL�1) for TET MIC (lg mL�1) for CH MIC (lg mL�1) for EB

5 g L�1 lactose 20 g L�1 lactose 5 g L�1 lactose 20 g L�1 lactose 5 g L�1 lactose 20 g L�1 lactose

MIM20 0.125 0.5 0.25 0.5 0.25 1

MIM27 0.25 1 0.5 0.5 0.5 2

MIM21 0.125 0.5 0.125 0.25 0.125 0.5

MIM28 0.5 2 0.125 0.25 1 2

CH, chlorhexidine; EB, ethidium bromide; MIC, minimum inhibitory concentration; TET, tetracycline.

0Control CH EB TET

1

2

3

4

5

6

7

Nor

mal

ized

fold

exp

ress

ionFig. 3. Transcriptional analysis of tetA in wild-

type S. thermophilus DSM 20617T (white bars)

and a recombinant strain over-expressing tetA,

MIM27 (gray bars). Cells cultured in M17

broth (control condition) and in the presence

of CH, EB, or TET were collected at an OD600

of 0.5 and subjected to RNA extraction and

then qRT-PCR analysis. The expression level of

tetA was normalized using murE and ureC as

reference genes. The data were recorded as

the threshold cycle (CT) expressed as the

means � standard deviation, computed using

Bio-Rad CFX MANAGERTM software, and

expressed as the normalized expression

(CT) � standard error of the mean.

FEMS Microbiol Lett 355 (2014) 61–70 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

TetA MFS efflux pump in Streptococcus thermophilus 67

kinetic parameters of MIM20 and the recombinant

mutant MIM27 were compared in the absence and pres-

ence of EB (0.5 lg mL�1), CH (0.25 lg mL�1), or TET

(0.125 lg mL�1), supplemented at subinhibiting concen-

trations. The presence of the antimicrobial compounds

resulted in an increased lag phase duration in MIM20

compared to MIM27. MIM20 showed a delay of 2.2, 7.6,

and 1.59 h in the presence of TET, CH, and EB, respec-

tively. The more extensive lag time duration and the

absence of tetA gene induction (Fig. 3) in the presence of

CH confirmed that TetA has not the same efficacy to

counteract the effect of CH on cell growth as showed for

TET or EB.

Effect of tetA over-expression in a tetA-

negative strain of S. thermophilus

As the tetA-like gene is present in only 34% of S. thermo-

philus strains screened to date, we attempted to over-

express tetA in the S. thermophilus MIMST24 strain,

which is classified as tetA negative. MIMSt24 electrocom-

petent cells were transformed with pMI-T5 or pMIT5-

tetA to obtain strains MIM21 (tetA negative) and MIM28

(over-expressing tetA). We then analyzed the MIC values

of both mutant strains to evaluate the effect of the pres-

ence of the efflux pump TetA. Analogously to the above

results for MIM20 and MIM27, the MICs values of

MIM21 and MIM28 were strongly affected by the lactose

concentration (Table 2). As observed for DSM 20617, the

over-expression of tetA did not appear to affect CH sensi-

tivity in the MIMST24 genetic background.

Conclusion

This study presents the first characterization of an

efflux pump, TetA, in the dairy species S. thermophilus.

The results obtained underline the activity of this efflux

system in the detoxification of the bacteria from several

cytotoxic compounds, including TET and EB. In a

more general view, efflux pumps can accommodate a

variety of substrates, including nutrients, metabolites,

and toxic compounds that bacteria encounter in their

surrounding environment (Martinez et al., 2009; Oliv-

ares et al., 2012). Moreover, the diffusion of this gene

within this species and its potential for transfer to

other species via HGT remains to be clarified to estab-

lish the safety of tetA-like gene harboring strains

according to EFSA recommendations (EFSA, 2012).

Streptococcus thermophilus is perceived as food-adapted

microorganisms that evolved from a commensal ances-

tor through loss-of-function events counterbalanced by

the acquisition of traits essential for the adaptation to

a milk environment. Therefore, the impact of genes

exchange by lateral gene transfer events occurring

among milk-adapted bacterial species should be relevant

on both the physiology and the safety of this dairy

species.

Acknowledgements

This work was supported by European Community FP7

project KBBE-227258 (BIOHYPO), which is a research

project aimed at evaluating the impact of biocide use on

the generation of antibiotic resistance. The funders had

no role in the study design, data collection, and analysis,

decision to publish, or preparation of the manuscript.

Stefania Arioli was supported by a postdoc grant ‘Dote

Ricerca’: FSE, Regione Lombardia. The authors thank

Sacco-Clerici srl for providing the S. thermophilus strains.

The authors have no conflict of interest to declare. SA

thanks Alessandro Mora for his valuable advice.

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TetA MFS efflux pump in Streptococcus thermophilus 69

Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Fig. S1. Ethidium bromide (EB) efflux as detected by

flow cytometry. The density diagrams show the green

(SYBR Green I) and red (EB) fluorescence levels of

MIM20 and MIM27 after 30 min of EB efflux, with and

without the protonophore carbonyl cyanide m-chloro-

phenylhydrazone (CCCP) after EB loading.

Fig. S2. Ethidium bromide (EB) efflux as detected by

flow cytometry. Density diagrams showing the green

(SYBR Green I) and red (EB) fluorescence levels of the

different populations (1 and 2) of wild-type DSM 20617T.

Table S1. LiMIM of S.thermophilus MIMrains of differ-

ent origin screened for the presence of tetA-like gene.

FEMS Microbiol Lett 355 (2014) 61–70ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

70 S. Arioli et al.