Efflux Pump Lde Is Associated with Fluoroquinolone Resistance in Listeria monocytogenes

Five Listeria monocytogenes isolates (CLIP 21369, CLIP 73298,CLIP 74811, CLIP 75679, and CLIP 79372) were found to be resistantto fluoroquinolones during the screening for antibiotic resistanceof 488 L. monocytogenes isolates from human cases of listeriosisin France. On the basis of a fourfold or greater decrease inthe ciprofloxacin MIC in the presence of reserpine, fluoroquinoloneresistance was attributed to active efflux of the drugs. Thelde gene (Listeria drug efflux; formerly lmo2741) encodes a12-transmembrane-segment putative efflux pump belonging to themajor facilitator superfamily of secondary transporters thatdisplayed 44% identity with PmrA from Streptococcus pneumoniae.Insertional inactivation of the lde gene in CLIP 21369 indicatedthat the corresponding protein was responsible for fluoroquinoloneresistance and was involved in the level of susceptibility todyes such as ethidium bromide and acridine orange.

INTRODUCTION

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Introduction

Listeria monocytogenes is widely distributed in the environmentand can cause serious human infections, such as bacteremia andcentral nervous system infections, primarily in neonates andimmunocompromised adults, and abortions (22). Food-borne transmissionis recognized as the main route of acquisition of the infectionduring epidemic and sporadic listerioses (3, 18). Listeriosisdiffers from most food-borne diseases by its high fatality rate(20 to 30% of cases), despite the administration of appropriateantibiotics (3, 9). L. monocytogenes is generally susceptibleto a wide range of antibiotics but is not susceptible to cephalosporinsand fosfomycin (8). However, during the last few years, increasingnumbers of strains resistant to one or more antibiotics havebeen reported (1, 2, 8, 11, 19, 20).

Although indications for the use of fluoroquinolones do notinclude listeriosis, they can, due to their increasing use forother pathologies such as respiratory tract infections, selectresistant Listeria. In gram-positive bacteria, resistance usuallyresults from mutational alterations in the so-called quinoloneresistance-determining regions (QRDRs) of the intracellulartargets of fluoroquinolones, the type II DNA topoisomerasesgyrase and topoisomerase IV, or active export of the drugs viaefflux pumps (12). We have detected ciprofloxacin-resistantL. monocytogenes clinical isolates and characterized the effluxpump involved in resistance.

(An initial report of this work was presented at the 41st InterscienceConference on Antimicrobial Agents and Chemotherapy [S. Godreuil,M. Galimand, G. Gerbaud, and P. Courvalin, Abstr. 41st Intersci.Conf. Antimicrob. Agents Chemother., abstr. UL-11, 2001].)

MATERIALS AND METHODS

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Materials and Methods

Strains, plasmids, and growth conditions. Screening of 488 L. monocytogenes isolates responsible for humanlisteriosis in France for resistance to fluoroquinolones wascarried out on brain heart infusion agar (Difco Laboratories,Detroit, Mich.) containing 4 µg of ciprofloxacin per ml.Strains that were found to be resistant and susceptible L. monocytogenesEGD were studied (Table 1). The strains were serotyped withantibodies made in the Laboratoire des Listeria and by the agglutinationtechnique, as described elsewhere (23). Escherichia coli TOP10plasmid PCR2 (Invitrogen, Paisley, United Kingdom), which isa vector used for cloning of PCR products, and plasmid pKSV7,which is thermosensitive for replication (24), were used. TheE. coli strains were grown at 37°C, and the L. monocytogenesstrains were grown at 30, 37, or 43°C. The strains weregrown in brain heart infusion broth and agar supplemented withampicillin at 100 µg/ml and kanamycin at 20 µg/mlfor E. coli and chloramphenicol at 8 µg/ml for L. monocytogenes.

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TABLE 1. Properties of the strains tested

Susceptibility testing. The isolates were tested for their antibiotic susceptibilitiesby disk diffusion on Mueller-Hinton agar (Bio-Rad, Marnes-la-Coquette,France). A minimum of three independent determinations of theMICs of the antimicrobial agents and dyes were determined onMueller-Hinton agar by the E-test (AB Biodisk France, Combourg,France) or by agar dilution with or without reserpine (10 µg/ml)with 10⁴ CFU per spot after 24 h of incubation at 37 or 43°C(25).

DNA preparation and transformation. Purification of plasmid DNA was performed by using the Wizardminipreps DNA kit (Promega, Madison, Wis.). Total DNA of L.monocytogenes was prepared as described previously (20). Restrictionwith endonucleases was according to the recommendations of thesupplier. Extraction of DNA fragments separated by agarose gelelectrophoresis was carried out with a Sephaglas BandPrep kit(Amersham-Pharmacia Biotech, Saint Quentin en Yvelines, France).L. monocytogenes DNA in plugs was digested with AscI or ApaI,and large restriction fragments were separated by electrophoresisin a CHEF-DRIII system (Bio-Rad Laboratories, Richmond, Calif.)by using an electric field of 6 V/cm and an angle of 120°.The initial and final pulse times were 4 and 40 s, respectively.Migration of the DNA fragments was performed in a 1% agarosegel in 0.5x Tris-borate-EDTA at 14°C (6).

Amplification of DNA was performed in a 2400 thermal cycler(Perkin-Elmer Cetus, Norwalk, Conn.), with Pfu DNA polymerase(Stratagene, La Jolla, Calif.), as recommended by the manufacturer.PCR elongation times and temperatures were adjusted accordingto the expected size of the PCR product and the nucleotide sequencesof the primers, respectively.

The QRDRs of the gyrA and gyrB genes for DNA gyrase and theparC and parE genes for DNA topoisomerase IV were amplifiedby PCR with specific primers (Table 2) and chromosomal DNA offluoroquinolone-resistant and -susceptible L. monocytogenesstrains as the template.

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TABLE 2. Primers used for PCR and sequencing

E. coli transformation was performed by the protocol providedby Invitrogen, and electrotransformation of L. monocytogeneswas as described previously (16) and was done with a Gene Pulserapparatus (Bio-Rad).

Insertional mutagenesis of lde gene. A fragment internal to the lde gene of L. monocytogenes CLIP21369 was amplified with the specific oligodeoxynucleotidesLDE-F (5'-GGGGTACCCCAGAATTTGTATGTTGTCTGGG-3'), which harborsa KpnI site (underlined), and LDE-R (5'-CGGGATCCCGTACGAGGAAGATCTCCGTA-3'),which contains a BamHI site (underlined). After digestion ofthe 1,105-bp PCR product with KpnI and BamHI, the fragment wascloned into similarly digested pKSV7 DNA. The recombinant plasmidpAT778 DNA was introduced into CLIP 21369 by electrotransformation,and transformants were selected for chloramphenicol resistanceat 30°C. At 43°C chloramphenicol resistance in transformantscan be maintained only following integrative recombination ofthe plasmid. Total DNA from three chloramphenicol-resistantclones was analyzed by PCR with the M13 reverse and forwardprimers and with two specific primers whose sequences are complementaryto the sequences of the flanking regions of the lde gene inthe L. monocytogenes chromosome (Table 2). The sequences ofthe amplification products were determined.

DNA sequence determination and analysis. DNA sequencing was performed with a CEQ 2000 DNA analysis systemautomatic sequencer (Beckman Coulter, Palo Alto, Calif.). Nucleotidesequence data were analyzed with the GCG sequence analysis softwarepackage (version 10.1; Genetics Computer Group, Madison, Wis.).BLAST program searches were performed by using the NationalCenter for Biotechnology Information website (http://www.ncbi.nlm.nih.gov).Transmembrane sequences (TMSs) were identified by using theTMHMM program at ExPASy (http://www.expasy.ch/). Multiple-sequencealignment was performed with the ClustalW program (version 1.82)at the Institut Pasteur website (http://www.bioweb.pasteur.fr/).

RESULTS AND DISCUSSION

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Results and Discussion

Properties of the L. monocytogenes strains. Screening of clinical L. monocytogenes isolates for fluoroquinoloneresistance led to the detection of five ciprofloxacin-resistantstrains (Table 1). The disk agar diffusion test showed thatthe five isolates were susceptible to ampicillin, chloramphenicol,erythromycin, gentamicin, kanamycin, minocycline, streptomycin,tetracycline, trimethoprim, and vancomycin (data not shown).The clinical isolates belonged to various serovars (Table 1),and pulsed-field gel electrophoresis of total DNA after digestionwith the AscI or ApaI restriction endonuclease revealed differentprofiles (data not shown). These results indicate that the resistantisolates were not clonally related.

The MICs of fluoroquinolones and dyes for the clinical isolatesand susceptible strain EGDe were determined by the E-test andagar dilution, respectively (Table 1). The clinical isolateswere resistant to ciprofloxacin and norfloxacin, and in thepresence of reserpine, a fourfold or greater decrease in thelevel of resistance to these hydrophilic quinolones was observed.By contrast, the MICs of relatively hydrophilic agents (levofloxacinand moxifloxacin) and hydrophobic drugs (gatifloxacin, sparfloxacin,and trovafloxacin) (Table 1 and data not shown) were not significantlyaltered (onefold or less decrease). These data, together withthe reproducible twofold reduction in the MICs of the dyes tested,suggested that efflux was responsible for the resistance.

Partial sequencing of gyr and par genes. Pairs of primers designed from the genome of L. monocytogenesEGDe (Table 2) were used to amplify and sequence the QRDRs ofgyrA, gyrB, parC, and parE. No differences in the sequencesof susceptible strain EGDe (5, 13) and the five ciprofloxacin-resistantisolates were found (data not shown).

Characterization of lde gene. In gram-positive bacteria, four multidrug efflux transportersthat use the proton motive force present across the cytoplasmicmembrane to extrude a variety of toxic compounds including fluoroquinolonesand dyes have been described: Bmr from Bacillus subtilis (15),NorA from Staphylococcus aureus (26), PmrA from Streptococcuspneumoniae (4), and EmeA from Enterococcus faecalis (10). Theybelong to the major facilitator superfamily (MFS) of secondarymultidrug transporters, share a common structure with 12 TMSs,and are closely related phylogenetically (17, 21).

The family-specific motifs of the MFS proteins were used toidentify structural genes for putative multidrug exporter proteinsin the genome of L. monocytogenes EGDe: (i) a search for thegxxxGPxxGGxl C motif (where x is any amino acid, a capital letterindicates that the amino acid occurs in >70% of the examinedsequences, and a lowercase letter indicates that the amino acidoccurs in <40% of the examined sequences) for multidrug transportersresulted in the identification of 10 genes for multidrug exporterproteins; (ii) Kyte-Doolittle hydropathy plots detected five14-TMS and five 12-TMS exporters; and (iii) Blast comparisonof the 12-TMS putative efflux pumps identified the lde gene(formerly lmo2741 [5]), which encodes a 402-amino-acid proteinthat displayed 44% identity with PmrA from S. pneumoniae (Table3).

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TABLE 3. Identities between the proteins deduced from the lmo gene of L. monocytogenes EGDe and those from 12-TMS MFS-type efflux proteins from gram-positive bacteria

The sequence alignments of the Bmr, NorA, EmeA, PmrA, and Ldeproteins are shown in Fig. 1. TMS prediction with the TMHMMprogram indicated that the 12 TMSs of the Lde efflux pump wereat positions similar to those of other 12-TMS proton-dependentefflux pumps (17), with the N and C termini located in the cytoplasm.The translocase consensus sequence (motif A) located in theloop between TMS 2 and TMS 3 and the drug extrusion consensussequence (motif C) at the end of TMS 5 were conserved in allfive sequences. Other motifs, in particular, D2 and G, thatare exclusive to the 12-TMS family suggested that Lde is a newmember of MFS family 2, which consists of 12-TMS exporters (17,21).

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FIG. 1. Alignment of the deduced sequences of 12-TMS MFS proteins from gram-positive bacteria. Bmr, from B. subtilis (GenBank accession number L25604); EmeA, from E. faecalis (10); NorA, from S. aureus (GenBank accession number AB019536); PmrA, from S. pneumoniae (GenBank accession number AJ007367); Lde, from L. monocytogenes EGDe (this study and GenBank accession number AL591984). Identical amino acids are indicated by boldface letters on a black background, and conservative substitutions are indicated by letters on a grey background. The horizontal lines above the alignment indicate the locations of the 12 TMSs (1 to 12). Highly conserved motifs (motifs A, B, C, D2, and G) are displayed below the alignment.

Inactivation of Lde efflux system. To determine if the Lde pump is involved in resistance to fluoroquinolonesand dyes, we inactivated the lde gene by insertion. Two specificprimers, LDE-F and LDE-R, were used to amplify a 1,105-bp fragmentinternal to lde that was ligated into vector pKSV7, which isthermosensitive for replication, to generate pAT778. PlasmidpAT778 DNA was introduced into CLIP 21369 by electrotransformation,and at a high temperature homologous recombination in the chromosomefor disruption of lde was favored. Transformants resistant tochloramphenicol at 43°C were found to be susceptible tofluoroquinolones by disk diffusion. Clones were analyzed byPCR and sequencing; and strain BM4497, which has a single integratedcopy of pAT778 in the lde gene, was studied further (data notshown).

The MICs of fluoroquinolones and dyes for strain BM4497 weredetermined. The strain was three- to fourfold more sensitivethan parental strain CLIP 21369 to ciprofloxacin and norfloxacinand two times more sensitive to dyes, and its susceptibilitywas not affected by the presence of reserpine (Table 1). Theseresults indicate that lde is responsible for fluoroquinoloneresistance and, in part, for acridine orange and ethidium bromideresistance in L. monocytogenes CLIP 21369. The lde gene wasfound by PCR in all six strains of L. monocytogenes examined,irrespective of their susceptibilities to quinolones. It istherefore likely that resistance results from an increase inthe level of expression of the pump.

In summary, we have demonstrated that lde encodes a multidrugefflux pump associated with resistance to hydrophilic fluoroquinolonesin L. monocytogenes. Because of the ubiquitous distributionof L. monocytogenes, this bacterial species is exposed to awide variety of drugs, and thus, active efflux could contributeto the adaptation of Listeria to these environmental challenges(14). Trends in the use of antimicrobials in France between1980-1981 and 1990-1991 revealed that the overall rate of antibioticuse increased by 3.7% a year. During that time, the rate ofuse of all quinolones increased by 5.7% per year, even whenthe rate of use of nonfluorated quinolones decreased by 4.7%(7). Our observations indicate that increasing use of fluoroquinolonescan select resistant mutants in nontarget species.

ACKNOWLEDGMENTS

This work was supported in part by a Bristol-Myers Squibb UnrestrictedBiomedical Research Grant in Infectious Diseases.

We thank P. Glaser, P. Cossart, and the Listeria Consortiumfor access to the L. monocytogenes EGDe sequence prior to publication.

FOOTNOTES

* Corresponding author. Mailing address: Unité des Agents Antibactériens, Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France. Phone: (33) 1 45 68 83 18. Fax: (33) 1 45 68 83 19. E-mail: galimand{at}pasteur.fr.

REFERENCES

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