A two-component regulatory system interconnects

A Two-Component Regulatory System
Interconnects Resistance to Polymyxins,
Aminoglycosides, Fluoroquinolones, and β
-Lactams in Pseudomonas aeruginosa
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Cédric Muller, Patrick Plésiat and Katy Jeannot
Antimicrob. Agents Chemother. 2011, 55(3):1211. DOI:
10.1128/AAC.01252-10.
Published Ahead of Print 13 December 2010.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 2011, p. 1211–1221
0066-4804/11/$12.00 doi:10.1128/AAC.01252-10
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 55, No. 3
A Two-Component Regulatory System Interconnects Resistance to
Polymyxins, Aminoglycosides, Fluoroquinolones, and
␤-Lactams in Pseudomonas aeruginosa䌤
Cédric Muller, Patrick Plésiat,* and Katy Jeannot
Laboratoire de Bactériologie, Faculté de Médecine, Université de Franche-Comté, 25030 Besançon, France
Constitutive overexpression of the active efflux system MexXY/OprM is a major cause of resistance to
aminoglycosides, fluoroquinolones, and cefepime in clinical strains of Pseudomonas aeruginosa. Upregulation of
this pump often results from mutations occurring in mexZ, the local repressor gene of the mexXY operon. In this
study, analysis of MexXY-overproducing mutants selected in vitro from reference strain PAO1Bes on amikacin
(at a concentration 1.5-fold higher than the MIC) led to identification of a new class of mutants harboring an
intact mexZ gene and exhibiting increased resistance to colistin and imipenem in addition to aminoglycosides,
fluoroquinolones, and cefepime. Reverse transcription-quantitative PCR (RT-qPCR) experiments on a selected
clone named PAOW2 demonstrated that mexXY overexpression was independent of mexZ and the PA5471 gene,
which is required for drug-dependent induction of mexXY. Furthermore, the transcript levels of the oprD gene,
which encodes the carbapenem-selective porin OprD, were found to be reduced drastically in PAOW2. Wholegenome sequencing revealed a single mutation resulting in an M59I substitution in the ParR protein, the
response regulator of the ParRS two-component regulatory system (with ParS being the sensor kinase), which
is required for adaptive resistance of P. aeruginosa to polycationic peptides such as colistin. The multidrug
resistance phenotype was suppressed in PAOW2 by deletion of the parS and parRS genes and conferred to
PAO1Bes by chromosomal insertion of the mutated parRS locus from PAOW2. As shown by transcriptomic
analysis, only a very small number of genes were expressed differentially between PAOW2 and PAO1Bes,
including the lipopolysaccharide (LPS) modification operon arnBCADTEF-ugd, responsible for resistance
to polycationic agents. Exposure of wild-type PAO1Bes to different polycationic peptides, including
colistin, was shown to result in increased mexY and repressed oprD expression via ParRS, independent of
PA5471. In agreement with these results, colistin antagonized activity of the MexXY/OprM substrates in
PAO1Bes but not in a ⌬parRS derivative. Finally, screening of clinical strains exhibiting the PAOW2
resistance phenotype allowed the identification of additional alterations in ParRS. Collectively, our data
indicate that ParRS may promote either induced or constitutive multidrug resistance to four different
classes of antibiotics through the activation of three distinct mechanisms (efflux, porin loss, and LPS
modification).
35). Although produced at very low levels in wild-type bacteria,
the MexXY proteins contribute through their interaction with
OprM to the intrinsic resistance of P. aeruginosa when they are
upregulated as part of the stress response to antibiotics (30).
Expression of the mexXY operon has been found to be induced
strongly when the ribosomal activity is impaired either by mutations (in ribosomal proteins L1 and L25 or in methionyltRNAfmet formyltransferase) or by antibiotics (aminoglycosides, macrolides, tetracyclines, and chloramphenicol) (4, 8, 19,
51). The functional link between the ribosome and MexXY/
OprM was elucidated partially with the demonstration that
a gene (PA5471) coding for a protein of unknown function
was required for drug-dependent induction of mexXY expression (36). Interestingly, PA5471 is cotranscribed with a
gene (PA5470) encoding an alternative peptide releasing
factor (RF) presumed to rescue stalled ribosomes.
MexXY-overproducing mutants with increased resistance
(from 2- to 16-fold) to the pump substrates are quite common
in cystic fibrosis (CF) and non-CF patients (15, 18, 28, 45, 50).
Most of these resistant bacteria exhibit mutations in mexZ, a
gene flanking the mexXY operon and coding for a TetR-like
protein that, when intact, strongly represses mexXY expression
Aminoglycosides are invaluable drugs in the management
of patients with acute or chronic infections caused by
Pseudomonas aeruginosa. Previous studies have shown that
an active efflux mechanism implying a tripartite pump called
MexXY/OprM modulates the activity of aminoglycosides toward this major nosocomial pathogen (1, 35, 51). The RND
(resistance-nodulation-cell division family) transporter MexY
interacts with the outer membrane channel OprM and the
periplasmic adaptor protein MexX to actively extrude aminoglycosides and various unrelated antibiotics (fluoroquinolones,
macrolides, tetracyclines, and zwitterionic ␤-lactams) from the
intracellular compartment to the external medium (31). At the
gene level, the MexX and MexY proteins are encoded by a
single transcriptional unit, mexXY, whereas OprM is encoded
by the third gene of a constitutively expressed operon, mexABoprM, coding for another efflux system (MexAB-OprM) (1, 26,
* Corresponding author. Mailing address: Laboratoire de Bactériologie, Faculté de Médecine, Université de Franche-Comté, 25030
Besançon, France. Phone: (33) 3 81 66 82 86. Fax: (33) 3 81 66 89 14.
E-mail: [email protected].
䌤
Published ahead of print on 13 December 2010.
1211
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Received 10 September 2010/Returned for modification 29 October 2010/Accepted 3 December 2010
1212
MULLER ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Bacterial strains and plasmids used in this study
Phenotype or genotypea
Source or
reference
Strains
P. aeruginosa strains
PAO1Bes
CMZ091
CM090
CM093
CM095
CM096
CM099
CM106
PAOW1
PAOW2
CMZ089
CM092
CM094
CM097
CM098
CM100
CMC
CMCW2
CM107
Wild-type reference strain
PAO1Bes ⌬mexZ
PAO1Bes ⌬PA5470
PAO1Bes ⌬PA5471
PAO1Bes ⌬parS
PAO1Bes ⌬parRS
PAO1Bes ⌬pmrAB
CM096 cis complemented with parRS genes from PAOW2, inserted at the attB site; Tcr
PAO1Bes spontaneous mutant; Amkr
PAO1Bes spontaneous mutant with Met59-to-Ile change in ParR; Amkr
PAOW2 ⌬mexZ
PAOW2 ⌬PA5470
PAOW2 ⌬PA5471
PAOW2 ⌬parS
PAOW2 ⌬parRS
PAOW2 ⌬pmrAB
Wild-type susceptible clinical strain
CMC spontaneous mutant with Leu14-to-Gln change in ParS; Amkr
CM096 cis complemented with parRS genes from CMCW2, inserted at the attB site; Tcr
K. Stover
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
recA thi pro hsdR Tra⫹ Smr
F⫺ supE44 endA1 hsdR17(rK⫺ mK⫺) thi-1 recA1 ⌬ (argF-lacZYA)U169 ␾80dlacZ⌬M15
phoA gyrA96 relA1 deoR ␭⫺
A(ara-leu) araD ⌬lacX74 galE galK phoA20 thi-1 rpsE rpoB argE(Am) recAl
CC118 lysogenized with ␭pir phage
supE44 hsdS20(rB⫺ mB⫺) recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5 mtl-1 leuB6 thi-1
44
Invitrogen
E. coli strains
S17.1
DH5␣
CC118
CC118 ␭pir
HB101
Plasmids
pCR2.1
pKNG101
pRK2013
Mini-CTX1
pKNG⌬mexZ
pKNG⌬5470
pKNG⌬5471
pKNG⌬parS
pKNG⌬parRS
pKNG⌬pmrAB
CTX-W2
CTX-CW2
Cloning vector for PCR products; lacZ⌬ColE1 f1 ori Apr Kmr
Suicide vector in P. aeruginosa; sacB Smr
Helper plasmid; ColE1 ori Tra⫹ Mob⫹ Kmr
Self-proficient integration vector with tet, V-FRT-attPMCS, ori, int, and oriT; Tcr
BamHI/ApaI 1.212-kb fragment composed of sequences flanking 5⬘ and 3⬘ ends of
mexZ, cloned into pKNG101; Smr
BamHI/ApaI 1.194-kb fragment composed of sequences flanking 5⬘ and 3⬘ ends of
PA5470, cloned into pKNG101; Smr
BamHI/ApaI 1.241-kb fragment composed of sequences flanking 5⬘ and 3⬘ ends of
PA5471, cloned into pKNG101; Smr
ApaI/ApaI 1.079-kb fragment composed of sequences flanking 5⬘ and 3⬘ ends of parS,
cloned into pKNG101; Smr
ApaI/ApaI 1.045-kb fragment composed of sequences flanking 5⬘ and 3⬘ ends of parRS,
cloned into pKNG101; Smr
BamHI/ApaI 1.241-kb fragment composed of sequences flanking 5⬘ and 3⬘ ends of
pmrAB, cloned into pKNG101; Smr
parRS from PAOW2 cloned into mini-CTX1 at SpeI/EcoRV sites; Tcr
parRS from CMCW2 cloned into mini-CTX1 at SpeI/EcoRV sites; Tcr
29
12
25
Invitrogen
21
6
14
This study
This study
This study
This study
This study
This study
This study
This study
a
Antibiotic resistance phenotypes; Tcr, tetracycline resistance; Amkr, amikacin resistance; Smr, streptomycin resistance; Apr, ampicillin resistance; Kmr, kanamycin
resistance. MCS, multiple cloning sites.
(32). Consequently, inactivation of mexZ and/or MexZ activity
leads to upregulation of MexXY and increased resistance.
However, MexXY overproducers with intact mexZ genes have
repeatedly been reported among clinical strains, suggesting the
presence of an additional regulatory gene(s) for mexXY (18, 28,
45). To our knowledge, none of these strains was demonstrated
to upregulate PA5471. These mutants were named agrW mutants to make a distinction from those exhibiting alterations in
mexZ (called agrZ mutants, for aminoglycoside-resistant mutants dependent on mexZ) (28).
The goal of the present study was to identify new regula-
tors of mexXY expression through a whole-genome sequencing strategy applied to several one-step agrW mutants selected on aminoglycosides.
MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions. The bacterial strains and
plasmids used or constructed for this study are listed in Table 1. Cultures were
grown in Mueller-Hinton broth (MHB) with adjusted concentrations of the
divalent cations Ca2⫹ and Mg2⫹ (Becton Dickinson, Microbiology Systems,
Cockeysville, MD), on Mueller-Hinton agar (MHA) (Bio-Rad, Marnes-la-Coquette, France), or on BM2 glucose minimal medium containing a low (20 ␮M)
Downloaded from http://aac.asm.org/ on February 28, 2014 by INIST-CNRS BiblioVie
Strain or plasmid
VOL. 55, 2011
ParRS-MEDIATED MULTIRESISTANCE IN P. AERUGINOSA
TABLE 2. Oligonucleotides used for gene inactivation, gene
cloning, and RT-qPCR
Oligonucleotide
Sequence (5⬘33⬘)
Oligonucleotides for
gene inactivation
mexZ
PCRimexZC1.......................CGTAGCGCTTGAGCTTGTC
PCRimexZC2.......................GCCCTCACACTGAACGTCCTCACAAGG
PCRimexZC3.......................TTCAGTGTGAGGGCGTCAATCGTCC
PCRimexZC4.......................GGTTCGTAGATCGGCATCTC
PA5471
PCRi5471C1.........................CTCCTGCGCGAGGTCTAC
PCRi5471C2.........................GGCGGTCGCGGACAAAGGCTTGATGTA
PCRi5471C3.........................TGTCCGCGACCGCCAGCTGATCTACG
PCRi5471C4.........................GATCGCCAGGCGTTTGTT
PA5470
PCRi5470C1.........................CTGGAACTGCTCGACATCAA
PCRi5470C2.........................GAAGCTGTCCTCGATTACCTCGACCTC
PCRi5470C3.........................TCGAGGACAGCTTCACTGGCTCTTCG
PCRi5470C4.........................CCGACCTCTTCCACCTGTT
parS
PCRiparSC1.........................AGGAGGACGATCTCGATCAC
PCRiparSC2.........................AGATCTTGGTCGACCACGAAGATCG
PCRiparSC3.........................GTCGACCAAGATCTTCGAACCCTTTTCG
PCRiparSC4.........................GCGTTGATCCTGTGCTGTC
parRS
PCRiParRSC1 .....................GGATTCAGCTTCTGCTCGAC
PCRiParRSC2 .....................AGATCTTCATAGGGTTTCATCGGTCG
PCRiParRSC3 .....................CCCTATGAAGATCTTCGAACCCTTTTCG
PCRiParRSC4 .....................GCGTTGATCCTGTGCTGTC
pmrAB
PCRiPmrABC1....................TGTCGAACTGACCCAGCTAC
PCRiPmrABC2....................CTTCCAGGTCACCCATTCCACGGTATC
PCRiPmrABC3....................GGGTGACCTGGAAGTGCAGGTGTTCCT
PCRiPmrABC4....................GTGCTGAGCTCCTCGATCTT
Oligonucleotides for
complementation
of parRS
CloparRSC1 .............................GAGGGAAAAGCAGAAGTCACC
CloparRSC2 .............................CGAGGTGTCCCATGCTAGG
Oligonucleotides for
RT-qPCR
uvrD1 ........................................GCAGCCTCGCCCTACAGCA
uvrD2 ........................................GGATCTGGAAGTTCTGCTCAGC
mexY1A....................................GCAGCCTCGCCCTACAGCA
mexY1B ....................................GGATCTGGAAGTTCTGCTCAGC
mexZRTA1 ..............................TTACCTCCTCCAGCGGC
mexZRTA2 ..............................GTGAGGCGGGCGTTGTG
PA5470C1.................................CCAAAGAGGAATCCCAGAAA
PA5470C2.................................CAGGCAGACCTCGATCTTGT
PA5471C1.................................TCGAGGTAATCGAGGAGGTG
PA5471C2.................................AGGGTCTGCAAACGGATCTC
oprDRTC1 ...............................CATCTACCGCACAAACGATG
oprDRTC2 ...............................ACAGAGTTGGCGAGGAAAATC
parRSTC1 ................................CGAACTGGAGGAAATGGTCT
parRSTC2 ................................ATGCGGATCTGTTCGACCT
pmrARTC1 ..............................GATACCGTGGAATGGGTGAC
pmrARTC2 ..............................GGCTTGGTCAGGTAGTCGTC
pmrBRTC1...............................ACGAACTCAACCTCCTGCTG
pmrBRTC2...............................ATCTGCTCCATCAAGGTGCT
PA1559RTC1...........................GCAGCAACTGGTGGACTACA
PA1559RTC2...........................CATGCGGAAGACCAGAAGAT
PA1797RTC1...........................GGACCCTTTGCAGATGACTC
PA1797RTC2...........................CGGAGTGTTTCCTGAGAAGC
PA2358RTC1...........................GTACTGTTCGCCGGAACAAC
PA2358RTC2...........................CCTGGAGCAGGAATATCTGG
PA2655RTC1...........................GTGCTGGTGTTCCTGTTGG
PA2655RTC2...........................CGTAGGTCCCCCAGATCG
PA3554RTC1...........................GTGGCTCGAATACCATGTGA
PA3554RTC2...........................TGCCGTATTTCACGCAGTAG
PA4773RTC1...........................CAGTGGATCGAGGAAAGCAT
PA4773RTC2...........................GTACTCCGGCCAGGTATGG
PA4774RTC1...........................CGCCGAACCACTTCTATTTC
PA4774RTC2...........................TCGTGGTACAGCGACTCATC
night culture in 25 ml MHB) to an A600 value of 1 ⫾ 0.05. The bacteria were
collected by centrifugation at 15,000 ⫻ g for 1 min. Total RNA was extracted
from the pellet by using an RNeasy Plus Mini kit (Qiagen SA, Courtaboeuf,
France), treated with DNase (RQ1 RNase-free DNase; Promega, Charbonnières
les Bains, France) for 1 h at 37°C, and purified with an RNeasy Mini Elute
cleanup kit (Qiagen SA). Ten micrograms of RNA was next retrotranscribed into
Downloaded from http://aac.asm.org/ on February 28, 2014 by INIST-CNRS BiblioVie
or high (2 mM) MgSO4 concentration (11). Isolation of one-step mutants with
increased MexXY/OprM efflux activity was performed by plating 100-␮l aliquots
of log-phase P. aeruginosa cultures (A600 equal to 1) on MHA supplemented with
6 ␮g ml⫺1 of amikacin (at a concentration 1.5-fold higher than the MIC for strain
PAO1Bes). Recombinant plasmids were introduced into P. aeruginosa strains by
triparental matings, using the mobilization properties of the broad-host-range
helper plasmid pRK2013 (6). Transconjugants were selected on Pseudomonas
isolation agar (PIA; Becton Dickinson) supplemented with appropriate antibiotic concentrations, as follows: for Escherichia coli, ampicillin at 100 ␮g/ml,
tetracycline at 15 ␮g/ml, and streptomycin at 50 ␮g/ml; and for P. aeruginosa,
ticarcillin at 150 ␮g/ml, tetracycline at 200 ␮g/ml, and streptomycin at 2,000
␮g/ml.
Antibiotic susceptibility testing. The MICs of selected antibiotics in MHA
were determined by recommended agar dilution procedures (5). Bacterial susceptibility in BM2 glucose minimal medium was determined by the macrodilution broth method at two MgSO4 concentrations (20 ␮M and 2 mM), as described previously (52). Growth was assessed after 24 h of incubation at 37°C.
ParRS-dependent drug induction of the mexY gene was assayed by the disk
diffusion method. MHA plates were inoculated with calibrated suspensions of
strains PAO1Bes and CM096 as recommended by the CLSI. Disks containing
meropenem (MEM; 10 ␮g), cefepime (FEP; 30 ␮g), gentamicin (GEN; 15 ␮g),
and ciprofloxacin (CIP; 10 ␮g) were deposited on the agar surface 3 h after
deposition of the colistin (CST; 50 ␮g) disk (Bio-Rad, Marnes-la-Coquette,
France). The checkerboard technique for investigating antibiotic interactions has
been described elsewhere (5).
Drug killing experiments. Overnight cultures of strain PAO1Bes and its mutant CM096 were diluted 1:50 in fresh MHB containing indolicidin or colistin (at
a concentration equivalent to 0.5⫻ MIC) and grown with constant shaking (250
rpm) at 37°C to an A600 value of 0.5 ⫾ 0.05. The bacteria from 1 ml of culture
were then collected by centrifugation (5,000 ⫻ g) and resuspended in 1 ml of
drug-free MHB. This cell suspension was diluted 1:10 in prewarmed MHB prior
to the addition of 16 ␮g/ml gentamicin (a concentration 8-fold higher than the
MIC) and then was reincubated at 37°C. The survivors of gentamicin action were
counted on MHA plates inoculated with serial 10-fold dilutions of culture aliquots taken at designated time points.
Construction of deletion mutants in strain PAO1 and mutant PAOW2. Singleor multiple-knockout mutants in the mexZ, PA5470, PA5471, parS, parRS, and
pmrAB genes were constructed by using overlapping PCR and recombination
events according to the method reported by Kaniga et al. (21). First, the 5⬘ and
3⬘ regions flanking each of the genes were amplified by PCR (ca. 500 bp in
length) (a list of the primers used is provided in Table 2) under the following
conditions: 5 min at 95°C followed by 30 cycles of 30 s at 95°C, 30 s at 60°C, and
30 s at 72°C, with a final extension step of 5 min at 72°C. The resultant amplicons
were then used as templates for overlapping PCR with external pairs of primers
(Table 2) to generate the mutagenic DNA fragments. The reaction mixtures
contained a 200 ␮M concentration of each deoxynucleoside triphosphate
(dNTP), 6% dimethyl sulfoxide (DMSO), 1⫻ polymerase buffer, a 0.5 ␮M
concentration of each primer, and 0.5 U of BioTaq Red DNA polymerase
(Bioline, London, United Kingdom). The amplified products were cloned into
plasmid pCR2.1 according to the manufacturer’s instructions (Invitrogen,
Carlsbad, CA) and next subcloned into the suicide vector pKNG101 and E.
coli CC118 ␭pir, as BamHI/ApaI or ApaI/ApaI fragments (21). The recombinant plasmids were transferred into P. aeruginosa by conjugation, and the
deletion mutants were selected on PIA plates containing 5% sucrose and
2,000 ␮g ml⫺1 streptomycin. The allelic exchanges were confirmed by PCR.
Nucleotide sequencing experiments confirmed deletion of 627 bp, 522 bp, 957
bp, 1,037 bp, 1,815 bp, and 1,941 bp in mexZ, PA5470, PA5471, parS, parRS,
and pmrAB, respectively.
Chromosomal complementation with full-length parRS. The parRS loci of
mutants PAOW2 and CMCW2 were PCR amplified from genomic DNA by
using the oligonucleotide pair CloparRSC1/CloparRSC2 (Table 2). The resulting
2,314-bp DNA fragments were cloned into pCR2.1 and next subcloned into the
HindIII/SacI restriction sites of plasmid mini-CTX1 (14). The recombinant plasmids were then transferred from E. coli CC118 to P. aeruginosa strain CM096
(PAO1 ⌬parRS) by conjugation, using PIA plates supplemented with tetracycline. Flippase-promoted excision of the chromosomally integrated FRT cassettes (tetracycline resistance and integrase genes) was achieved by conjugational
transfer of plasmid pFLP2 from donor E. coli S17.1 to strain CM096 and subsequent selection of the transconjugants on ticarcillin-MHA plates (13). Plasmid
pFLP2 was subsequently cured by streaking selected recipient clones on MHA
medium supplemented with 5% sucrose.
Transcriptional profiling. Triplicate cultures of mutant strain PAOW2 and its
parent, PAO1Bes, were grown at 37°C with shaking (1:100 dilution of an over-
1213
1214
MULLER ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 3. Drug susceptibilities of various mutants derived from strains PAO1Bes and CMC
MIC (␮g/ml)a
Strain
Wild type
Wild type
PAO1Bes agrW1 mutant
PAO1Bes agrW2 mutant
CMC agrW2 mutant
P〈⌷1 ⌬mexZ
P〈⌷1 ⌬PA5470
P〈⌷1 ⌬PA5471
P〈⌷1 ⌬parS
PAO1 ⌬parRS
P〈⌷1 ⌬pmrAB
P〈⌷W2 ⌬mexZ
P〈⌷W2 ⌬PA5470
P〈⌷W2 ⌬PA5471
P〈⌷W2 ⌬parS
P〈⌷W2 ⌬parRS
P〈⌷W2 ⌬pmrAB
CM096 with parRS from PAOW2
CM096 with parRS from CMCW2
GEN
AMK
TOB
APR
FEP
CIP
IMP
MEM
CST
2
2
16
8
4
8
2
ⱕ0.12
2
2
2
8
8
8
2
2
8
4
4
4
4
32
32
8
16
4
2
4
4
4
32
32
32
4
4
32
8
8
1
1
4
2
2
2
1
0.5
1
1
1
2
2
2
1
1
2
1
1
16
16
64
64
32
32
16
4
16
16
16
64
64
64
16
16
64
32
32
4
4
16
8
8
8
4
4
4
4
4
8
8
8
4
4
8
8
8
0.25
0.25
1
1
0.5
1
0.25
0.12
0.25
0.25
0.25
1
1
1
0.25
0.25
1
0.5
0.5
2
2
2
16
16
2
2
2
2
2
2
16
16
16
2
2
16
16
16
1
1
1
4
4
1
1
1
1
1
1
4
4
4
1
1
4
4
2
1
1
1
2
2
1
1
1
0.5
0.5
0.5
2
2
2
0.5
0.5
1
1
1
a
MICs in bold (or underlined) indicate that the bacteria were at least 2-fold more (or less) resistant than the reference strain PAO1Bes. The data are representative
of four distinct experiments. Abbreviations: GEN, gentamicin; AMK, amikacin; TOB, tobramycin; APR, apramycin; FEP, cefepime; CIP, ciprofloxacin; IMP,
imipenem; MEM, meropenem; and CST, colistin.
cDNA, fragmented, labeled, and hybridized on an Affymetrix P. aeruginosa
GeneChip by DNA Vision (Charleroi, Belgium). Normalization and signal value
extraction were achieved with the Robust Multiarray Average (RMA) software
package (16, 17). Differential gene expression between PAO1Bes and PAOW2
was analyzed with Student’s t test, using a nominal significance level (P) of ⱕ0.05
for each univariate test. Changes in gene expression of ⱖ2-fold were considered
significant.
RT-qPCR. Specific gene expression was measured by real-time reverse
transcription-quantitative PCR (RT-qPCR) as described previously (7).
Briefly, 2 ␮g of total RNA was reverse transcribed with ImpromII reverse
transcriptase as specified by the manufacturer (Promega). The amounts of
specific cDNA were assessed in a Rotor Gene RG6000 real-time PCR instrument (Qiagen) by using a Fast SybrGreen kit (Qiagen) and primers
designed from the sequence of Pseudomonas (Pseudomonas genome database
v2 [http://v2.pseudomonas.com]) (Table 2), with uvrD transcripts as an internal control (19). The transcript levels of a given gene in a given strain were
normalized with those of uvrD (20) and expressed as a ratio (fold change) to
that for wild-type strain PAO1Bes, used as a reference. Gene expression
values were calculated from three independent bacterial cultures, each of
which was tested in duplicate.
SNP identification. Single-nucleotide polymorphisms (SNPs) between the
PAO1 strain used in our laboratory, called PAO1Bes (for Besançon), its derived
mutant PAOW2, and the published PAO1 reference sequence (GenBank accession no. NC-002516) (46) were established with Illumina’s ELAND aligner as
described previously (22). Briefly, the genomic DNAs from PAO1Bes and
PAOW2 were extracted and purified with a QIAamp DNA Mini kit (Qiagen).
Illumina libraries were prepared from 10 ␮g of each DNA preparation by
GATC Biotechn GA (Konstanz, Germany), using a Genome Analyzer I
apparatus. Mapping of the sequence reads (76 bp) of PAOW2 (2,297,644
reads) to the published PAO1 reference led to the identification of potential
sequence variations (SNPs). A SNP was considered reliable if the coverage
was ⱖ5-fold and its percentage was ⱖ75%. The sequence variations predicted
for PAOW2 relative to PAO1 and PAO1Bes (2,289,792 reads) were verified
on both DNA strands in an Applied Biosystems 3130 automatic sequencer
(Applied Biosystems, Courtaboeuf, France) after PCR amplification with
proper primers.
RESULTS AND DISCUSSION
Overexpression of mexY independent of MexZ and PA5471.
A functional link has been established between the efflux system MexXY/OprM and the ribosomal machinery (8, 19). In
order to identify novel physiological functions associated with
this transporter, we selected spontaneously MexXY-overproducing mutants of wild-type strain PAO1Bes (the PAO1 strain
studied in our laboratory) by culture on MHA supplemented
with amikacin at a concentration 1.5-fold higher than the MIC.
Mutants developing on this medium were easily obtained, at
rates ranging from 7.7 ⫻ 10⫺7 to 7.7 ⫻ 10⫺8. Drug susceptibility tests followed by sequencing experiments on 15 randomly
selected colonies led to the identification of MexXY overproducers exhibiting either an intact or mutated mexZ gene (13 agrW and
2 agrZ mutants, respectively). Consistent with MexXY being upregulated in the agrW mutants, all of the colonies tested were
2- to 8-fold more resistant than PAO1Bes to the MexXY/
OprM substrates, including aminoglycosides (gentamicin,
amikacin, tobramycin, and apramycin), cefepime, and ciprofloxacin (28, 45, 50) (Table 3). RT-qPCR data confirmed
overexpression of the mexY gene in these bacteria (data not
shown). More interestingly, the agrW mutants could be divided
into two phenotypic groups, named PAOW1 and PAOW2,
with respect to their susceptibility to carbapenems (imipenem
and meropenem) and colistin (Table 3). While the PAOW1
type (n ⫽ 6) exhibited the same susceptibility as PAO1Bes to
these drugs, the PAOW2 type (n ⫽ 7) was 2- to 8-fold more
resistant. Since MexXY overexpression had thus far never
been associated with a decreased susceptibility to carbapenems
and polymyxins, we focused our attention on a typical PAOW2
mutant (results on PAOW1 will be reported in a separate
paper).
In a first attempt to find out the genetic mechanisms leading
to MexXY upregulation in PAOW2, we sequenced (PA5470,
PA5471, rplA, rplY, and nuoG) and quantified (mexZ, PA5470,
and PA5471) the transcripts of a number of genes known to
influence mexXY operon expression (8, 32, 36, 51). Since all of
these experiments yielded negative results, we looked at whether
some of these genes would be required for mexXY overexpression
Downloaded from http://aac.asm.org/ on February 28, 2014 by INIST-CNRS BiblioVie
PAO1Bes
CMC
PAOW1
PAOW2
CMCW2
CMZ091
CM090
CM093
CM095
CM096
CM099
CMZ089
CM092
CM094
CM097
CM098
CM100
CM106
CM107
Genotype
VOL. 55, 2011
ParRS-MEDIATED MULTIRESISTANCE IN P. AERUGINOSA
in PAOW2. We thus constructed mexZ, PA5470, and PA5471
deletion mutants of PAO1Bes and PAOW2. As expected, inactivation of the repressor gene mexZ in PAO1Bes (mutant
strain CMZ091) resulted in a strong increase in mexY expression
and in more resistance to all of the MexXY/OprM substrates
tested (Fig. 1 and Table 3). In PAOW2, however, deletion of
mexZ (mutant strain CMZ089) upregulated mexY expression only
2-fold, without notable effects on the mutant’s susceptibility (Fig.
1 and Table 3). Concordant with data from the literature (36),
inactivation of PA5471 (mutant strain CM093), but not that of the
adjacent gene PA5470 (mutant strain CM090), significantly reduced mexY expression in wild-type PAO1Bes, leading to supersusceptibility to most of the antibiotics exported by MexXY/
OprM (compare strains CM090, CM093, and PAO1Bes in Fig. 1
and Table 3). In contrast, suppression of PA5471 had no influence on the mexY level and on drug susceptibility in PAOW2
(compare strains CM094 and PAOW2). Altogether, these results
suggested the existence of a new regulatory pathway for MexXY/
OprM that is able to override the control exerted by MexZ and
PA5471.
It is well established that the OprD porin is the specific
uptake pathway for basic amino acids and carbapenems across
the outer membrane of P. aeruginosa (47). Since qualitative
and/or quantitative alterations of this channel are a major
cause of resistance to carbapenems in this organism (24, 27, 38,
42), we sequenced and measured the expression levels of the
oprD gene in PAOW2 and PAO1Bes. PAOW2 turned out to
harbor an intact oprD gene whose expression was 7.1-fold ⫾
0.1-fold less than that in PAO1Bes (Fig. 2). Reminiscent of the
case for nfxC mutants, which overproduce the efflux system
MexEF-OprN while downregulating OprD (23, 39), our results
provided good evidence for coordinated regulation between
two complementary resistance mechanisms aimed at limiting
intracellular drug accumulation (i.e., active efflux and outer
membrane impermeability) in the agrW2 mutants.
Assuming that the elevated rates at which the agrW2 mutants
FIG. 2. Impact of mutations in parR and parS on expression levels
of the mexY and oprD genes. The mRNA levels of the mexY (black
bars) and oprD (white bars) genes were determined by RT-qPCR. The
bars represent means of three independent determinations ⫾ SD.
were recovered on selective medium were the result of single
mutations, we carried out whole-genome sequencing experiments to identify the genetic alteration of PAOW2.
Mutational alteration of ParRS two-component regulatory
system. Because of the genomic polymorphism that may
exist between PAO1 laboratory strains (22), we aligned the
sequence reads from PAO1Bes (2,289,792) and its mutant
PAOW2 (2,297,644) with the PAO1 genome sequenced by
Stover et al. (46). An initial analysis of the sequencing data
pointed to the potential existence of 13 SNPs in PAOW2. Only
one of these was confirmed by PCR and resequencing: a Gto-A change at nucleotide position 177 of the PA1799 gene was
predicted to generate an M59I substitution in the response
regulator (ParR) of a recently described two-component regulatory system, ParRS (for peptide-adaptive resistance regulator and sensor) (9). The mutation was located in the receiver
domain of this regulator, near the conserved phosphorylation
site D57. According to the Pseudomonas genome database
(http://v2.pseudomonas.com), the parS gene (PA1798), which
codes for the sensor kinase ParS, is assumed to be cotranscribed with parR (PA1799). The ParRS system has been
reported to be required for the activation of the lipopolysaccharide (LPS) modification operon arnBCADTEF-ugd in
the presence of subinhibitory concentrations of various bacterial (polymyxin B and colistin) and eukaryotic (indolicidin)
polycationic peptides and, consequently, to be responsible for
adaptive resistance to these peptides (9).
To ascertain the role of mutated ParRS in the multidrug
resistance phenotype of PAOW2, we constructed parS and
parRS deletion mutants of PAOW2 (CM097 and CM098, respectively) and PAO1Bes (CM095 and CM096, respectively).
The MICs of MexXY/OprM substrates (aminoglycosides, ciprofloxacin, and cefepime) and of carbapenems were restored to
wild-type levels for PAOW2 upon inactivation of parS or
parRS, while they remained unchanged for PAO1Bes (Table
3). Notably, these deletions decreased the colistin MIC 2-fold
for PAO1Bes and 4-fold for PAOW2, thus suppressing the
difference in resistance between the two strains. To confirm
these data, we carried out complementation experiments by
inserting a single copy of the mutated parRS operon from
PAOW2 into the chromosome of PAO1Bes ⌬parRS (CM096).
Downloaded from http://aac.asm.org/ on February 28, 2014 by INIST-CNRS BiblioVie
FIG. 1. Mutant strain PAOW2 upregulates the mexXY operon
independently of the mexZ, PA5470, and PA5471 genes. mexY gene
levels in strains PAO1Bes and PAOW2 and in their respective
⌬mexZ (CMZ091 and CMZ089), ⌬PA5470 (CM090 and CM092),
and ⌬PA5471 (CM093 and CM094) mutants were quantified by
RT-qPCR. The bars represent means of three independent determinations ⫾ standard deviations (SD).
1215
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MULLER ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 4. Microarray analysis of differentially expressed genes (at
least 2-fold) in the PAOW2 mutant compared with PAO1Bes
Gene ID
Name (alias)
Fold
change
arnB (pmrH)
arnC (pmrF)
arnA (pmrI)
arnT (pmrK)
⫺7.5
28.9
14.3
45.7
4.6
25.5
33.6
11.0
11.6
15.3
8.5
PA3557
PA3558
PA3559
arnE (pmrL)
arnF (pmrM)
ugd (pmrE)
3.1
9.2
16.1
PA4773
PA4774
PA4775
oprD
mexY
36.8
12.5
7.1
Fold change in gene expressiona
Description
Gene
PAOW2/PAO1Bes
OprD porin
Hypothetical protein
Hypothetical protein
Hypothetical protein
RND transporter MexY
Hypothetical protein
Hypothetical protein
Hypothetical protein
Probable glycosyl transferase
Hypothetical protein
4-Amino-4-deoxy-L-arabinose
transferase
Hypothetical protein
Hypothetical protein
Probable UDP-glucose
dehydrogenase
Hypothetical protein
Hypothetical protein
Hypothetical protein
The observation that the complemented mutant (CM106) displayed a resistance pattern similar to that of PAOW2 unambiguously established that multidrug resistance may arise in P.
aeruginosa as a result of mutations in parR. Upon complementation, a slight but reproducible difference between the colistin
resistances of CM096 and CM106 could be observed by the
disk method (inhibition zones of 22 and 20 mm, respectively,
for a disk load of 50 ␮g) but failed to produce a significant
(2-fold) difference in the MIC by the microdilution method.
Since the genetic background of strains may potentially influence the phenotypic effects of mutations (22), we selected
new agrW2 mutants on amikacin, using a wild-type clinical
strain (CMC) isolated in our hospital. Sequencing of the parRS
operon from imipenem-resistant clones allowed the identification of a single-step mutant, dubbed CMCW2, harboring
a single T-to-A substitution at position 41 of the parS gene
(L14Q; located in the first transmembrane domain). As for
PAOW2, transfer of mutated parRS from CMCW2 into the
chromosome of CM096 (resulting in mutant CM107) generated a multidrug resistance phenotype due to up- and downregulation of the mexY and oprD genes, respectively (Table 3;
Fig. 2). Taken together, these data indicated that amino acid
changes in ParS could also constitutively coactivate several
resistance mechanisms.
Transcriptomic data. In order to delineate the set of genes
directly or indirectly responding to mutated ParRS, we compared the whole-genome expression profiles of PAOW2 and
PAO1Bes, as determined with Affymetrix microarrays. To our
surprise, only a very small number of open reading frames
(ORFs) (8 in total) were differentially expressed (ⱖ2-fold) in
the two strains, in addition to mexY, arnBCADTEF-ugd, and
oprD (Table 4). Some of the ParRS-regulated genes belong to
operons (PA1559-PA1560, PA4773-PA4775-pmrAB, and
arnBCADTEF-ugd) that are known to be regulated by the
two-component signal transduction system PmrAB (33). With
the exception of the arnBCADTEF-ugd locus, no overlap was
found between the genes responding to mutated ParRS and
PA0958
PA1559
PA1797
PA1799
PA2018
PA2358
PA2655
PA3554
PA4773
PA4774
PA4777
a
(oprD)
(parR)
(mexY)
(arnA)
(pmrB)
⫺7.1 ⫾ 0.1
50.1 ⫾ 14
315.5 ⫾ 83
⫺1.2 ⫾ 0.4
13.1 ⫾ 2.8
27.8 ⫾ 5.6
63.7 ⫾ 14.1
50.6 ⫾ 8.2
90.6 ⫾ 15.7
131.3 ⫾ 25.5
15.9 ⫾ 2.6
CM096/PAO1Bes
1.1 ⫾ 0.3
1.6 ⫾ 0.3
1.1 ⫾ 0.5
⫺1.1 ⫾ 0.1
1.9 ⫾ 0.1
1.2 ⫾ 0.3
2.3 ⫾ 0.4
1.6 ⫾ 0.2
1.5 ⫾ 0.5
1.4 ⫾ 0.2
Values are means of three independent determinations ⫾ SD.
those controlled by PhoP-PhoQ, another regulatory system
which, like PmrAB, is involved in the adaptive response of P.
aeruginosa to Mg2⫹ starvation (33). To validate our transcriptomic data, we measured the mRNA levels of selected genes by
RT-qPCR. As indicated in Table 5, gene expression changes
were concordant, though sometimes more pronounced (up to
15.9 times), for RT-qPCR compared to Affymetrix chips. For
example, the pmrB gene (PA4777), which together with pmrA
(PA4776) is cotranscribed with PA4773 to PA4775, was found to
be upregulated in PAOW2 by RT-qPCR but not with the DNA
chips. Thus, it is possible that some genes of the ParRS regulon
may have been missed by the transcriptomic approach, despite an
excellent (⬎98%) interassay reproducibility.
PmrAB-independent activation of LPS modification. The
signal transduction system PmrAB is able to promote bacterial
resistance to polymyxins and cationic peptides in response
to Mg2⫹ starvation through modification of LPS molecules.
These adaptive changes, which include neutralization of negatively charged phosphate residues of lipid A by addition of
4-aminoarabinose, limit the penetration of polycations across
the bacterial outer membrane (33, 34, 37). Because of this, we
wondered whether PmrAB, whose expression is increased in
PAOW2, would account for the lower susceptibility (2-fold) to
colistin of the mutant than of PAO1Bes. Actually, elimination
of the pmrAB genes had similar effects in PAOW2 (mutant
strain CM100) and PAO1Bes (mutant strain CM099), leading
to a modest 2-fold decrease in the colistin MIC (Table 3). At
the transcriptional level, suppression of the pmrAB locus did
not significantly influence expression of the arnA gene from the
LPS modification operon arnBCADTEF-ugd for both strains
(50.6-fold ⫾ 8.2-fold for PAOW2 versus 40.5-fold ⫾ 7.3-fold
for CM100 and 1-fold for PAO1Bes versus 1.9-fold ⫾ 0.25-fold
for CM099). Contrasting with these results, deletion of the
parRS locus drastically reduced arnA levels in PAOW2 (⫺1.1fold ⫾ 0.15-fold for mutant strain CM098) but not in PAO1Bes
(2.3-fold ⫾ 0.4-fold for mutant strain CM096). Therefore, in
addition to the recent demonstration of the role of ParRS in
adaptive resistance to bacterial and eukaryotic polycationic
peptides (9), we found that the ParRS system may provide P.
aeruginosa with constitutive resistance to these agents, through
upregulation of the arnBCADTEF-ugd operon, when activated
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PA0958
PA1559
PA1560
PA1797
PA2018
PA2358
PA2655
PA3552
PA3553
PA3554
PA3556
TABLE 5. Influence of ParRS on expression of selected genes in
strain PAO1Bes and mutant strain PAOW2
VOL. 55, 2011
ParRS-MEDIATED MULTIRESISTANCE IN P. AERUGINOSA
1217
by mutations affecting either the response regulator ParR or
the sensor kinase ParS.
ParRS mediates multidrug resistance in P. aeruginosa. Since
the oprD and mexY genes are constitutively down- and upregulated in the PAOW2 mutant, respectively, we considered the
possibility that polycationic peptides might induce increased
resistance of wild-type P. aeruginosa strains to carbapenems,
aminoglycosides, cefepime, and fluoroquinolones through their
activation of ParRS. This hypothesis was tested first by measuring the transcriptional levels of the mexY and oprD genes in
bacteria cultured for 4 h in the presence of a concentration
equivalent to a 1:2 ratio of the MIC of colistin (0.5 ␮g/ml),
indolicidin (4 ␮g/ml), polymyxin B (0.5 ␮g/ml), or polymyxin B
nonapeptide (PMBN) (64 ␮g/ml) (Fig. 3A and B). The latter
compound, which lacks the fatty acid tail of polymyxin B, has
lost most of the antibacterial properties of its parent molecule
on P. aeruginosa but retains a strong outer membrane-permeabilizing activity (40, 48, 49). Because the PA5471 gene is
required for drug-induced expression of mexXY in wild-type
bacteria (36), the induction experiments were carried out with
mutants lacking either PA5471 (CM093) or parRS (CM096), in
addition to PAO1Bes. Exposure of PAO1Bes to colistin, the
most therapeutically relevant cationic peptide from this series,
was associated with substantial changes in mexY and oprD
expression compared with that in untreated cells (4.6-fold ⫾
0.6-fold and ⫺2.5-fold ⫾ 0.35-fold, respectively). Suppression
of the PA5471 gene (mutant strain CM093) had no influence
on these transcript levels (4.2-fold ⫾ 0.7-fold and ⫺2.6-fold ⫾
0.2-fold, respectively), indicating a PA5471-independent regulation pathway for mexXY by colistin (Fig. 3A and B). On the
other hand, the colistin-induced responses of mexY and oprD
were completely abolished in the parRS null mutant CM096
(⫺1.1-fold ⫾ 0.15-fold and 1.29-fold ⫾ 0.1-fold changes in
expression, respectively) (Fig. 3A and B and 4). Similar results
were obtained with indolicidin and polymyxin B. Notably,
despite the use of a fairly high concentration (64 ␮g/ml),
PMBN increased mexY activity only marginally (Fig. 3 and 4).
The possibility that gentamicin might trigger mexXY expression via ParRS was also examined by culturing PAO1Bes,
CM093, and CM096 in the presence of a concentration of the
antibiotic equivalent to 0.5⫻ MIC (1 ␮g/ml, 0.06 ␮g/ml, and 1
␮g/ml, respectively). Confirming previous results from our laboratory (19), sub-MIC gentamicin markedly induced mexY
gene expression (18.5-fold ⫾ 1.7-fold) in wild-type PAO1Bes
compared with untreated cells. However, this adaptive response was independent of parRS, as the mexY levels of CM096
(13.2-fold ⫾ 2-fold) were not really different from those of
PAO1Bes. In agreement with this, using a pPA3552::lux fusion, Fernandez et al. reported no effect of aminoglycosides on
ParRS-dependent activation of the LPS modification operon
arnBCADTEF-ugd (9). Thus, ParRS is unlikely to respond to
the presence of aminoglycosides in the external medium. Similarly, imipenem (2 ␮g/ml), meropenem (0.5 ␮g/ml), and
cefepime (2 ␮g/ml) did not show any ParRS-dependent effects
on mexY and oprD expression (data not presented).
FIG. 4. Interactions of polymyxin B and PMBN with gentamicin.
Antibiograms were created for strain PAO1Bes (A) and ⌬parRS mutant strain CM096 (B) on MHA according to CLSI recommendations.
PB, polymyxin B; PMBN, polymyxin B nonapeptide. As shown in the
upper part of panel A, polymyxin B antagonizes the bacteriostatic
activity of gentamicin (GM) on PAO1Bes.
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FIG. 3. Influence of antibiotic stress on mexY and oprD expression. Changes in expression levels of the mexY (A) and oprD (B) genes were
assessed by RT-qPCR for wild-type strain PAO1Bes (black bars), ⌬PA5471 mutant strain CM093 (white bars), and ⌬parRS mutant strain CM096
(gray bars), which were preincubated for 4 h in the presence of indolicidin, colistin, polymyxin B, PMBN, or gentamicin. An uninduced culture was
performed in parallel as a negative control. Values are means of three independent determinations ⫾ SD.
1218
MULLER ET AL.
Role of ParRS in antagonistic drug interactions. Collectively, the results mentioned above suggested that subinhibitory concentrations of polycationic peptides might promote
antagonistic interactions between antipseudomonal antibiotics
through distinct mechanisms, such as outer membrane impermeability and active drug efflux (Fig. 4). To demonstrate this,
checkerboard assays were performed with wild-type PAO1Bes
and its ⌬parRS mutant CM096 grown in MHB. Consistent
with our gene expression experiments, colistin at concentrations near the MIC strongly antagonized the bacteriostatic
activities of gentamicin, meropenem, cefepime, and ciprofloxacin in PAO1Bes (fractional inhibitory concentration
[FIC] indexes of 2.5, 4.5, 4.5, and 4.5, respectively) but not in
CM096 (FIC indexes of 1, 1, 1.5, and 1, respectively). The FIC
index reflects synergism (value of ⱕ0.5), additivity (value of
0.51 to 0.99), indifference (value of 1 to 2), or antagonism
(value of ⬎2) between the bacteriostatic effects of two drugs.
Such an antagonistic effect of colistin toward aminoglycosides is of considerable clinical importance for cystic fibrosis.
Patients suffering from this inherited genetic disease are indeed treated over long periods with aerosols of colistin and
aminoglycosides, often given sequentially, when they are colonized by P. aeruginosa in the lungs (10). These patients are
also used to receiving repeated courses of intravenous aminoglycosides during infectious exacerbations. To better evaluate
the negative impact of colistin on aminoglycoside efficacy, we
carried out time-kill experiments with gentamicin at 16 ␮g/ml
(a concentration 8-fold higher than the MIC), using PAO1Bes
and CM096 preincubated for 4 h with or without 0.5 ␮g/ml of
colistin (equivalent to a 1:2 ratio of the MIC). Under noninducing conditions, PAO1Bes and its mutant CM096 exhibited
similar killing profiles, with 3 orders of reduction in survivors
after 30 min of gentamicin exposure (Fig. 5). As anticipated
from our checkerboard assays, preincubation with colistin drastically reduced killing of PAO1Bes (⫺1.15-fold ⫾ 0.2 log10-fold),
confirming the development of a refractory state in the bacte-
ria. Of greater importance, such a colistin-induced adaptation
was not observed in mutant strain CM096, which exhibited a
decline in living cells averaging ⫺3.6 ⫾ 0.2 log10 after 30 min
of gentamicin treatment. Similar results were obtained with
amikacin and tobramycin at concentrations 8-fold higher than
the MIC. If confirmed in vivo by clinical trials, these data would
preclude the use of combined therapy with colistin and aminoglycosides in the management of CF lung infection.
Clinical strains with alterations in ParRS. To gain insight
into the clinical relevance of parRS mutants, we screened our
laboratory collection in search of CF and non-CF isolates showing
a typical AgrW2 resistance phenotype. One CF (3020R) and two
non-CF (4922 and 5024) isolates over 6 strains were found to fit
this phenotypic profile. Subsequent determination of the colistin MIC demonstrated a modest but reproducible resistance
to this agent in the three isolates (MIC of 2 ␮g/ml). Compared
to reference strain PAO1Bes, all of the bacteria appeared to
overexpress mexY (10.6-fold ⫾ 1.6-fold, 4.7-fold ⫾ 0.5-fold,
and 7-fold ⫾ 0.7-fold, respectively) and to downregulate oprD
(⫺5.2-fold ⫾ 0.5-fold, ⫺6.4-fold ⫾ 0.8-fold, and ⫺5.7-fold ⫾
0.6-fold, respectively), as assayed by RT-qPCR. Interestingly,
sequence analysis of the parRS loci from these strains
showed nonsynonymous nucleotide substitutions when the
sequences were aligned with the PAO1 reference genome
(http://v2.pseudomonas.com). These changes were predicted
to lead to single amino acid substitutions in the sensor kinase
ParS (V101M in isolate 4922 and L137P in isolate 5024) or the
response regulator ParR (E156K in isolate 3020R). In support
of a role of these alterations in the constitutive activation of
ParRS, we noted that the gene flanking the parRS operon,
PA1797, was overexpressed in the selected strains (108.2fold ⫾ 23.3-fold in 3020R, 33.6-fold ⫾ 7.5-fold in 4922, and
4.6-fold ⫾ 0.3-fold in 5024), as observed in mutant strain
PAOW2.
Conclusions. Among the numerous two-component regulatory systems (64 response regulators, 63 classical histidine kinases, and 16 atypical kinases) possessed by P. aeruginosa (41),
only a few have been reported to provide significant antibiotic
resistance in clinical strains (e.g., PmrB and PhoQ) (3, 43). To
our knowledge, this study is the first showing the role played by
a two-component signal transduction system (ParRS) in bacterial adaptation to no fewer than four different classes of
antibiotics (polymyxins, aminoglycosides, fluoroquinolones,
and ␤-lactams) through three distinct resistance mechanisms
(i.e., LPS modification, increased drug efflux, and a reduced
porin pathway). Such a multiresistance phenotype may be induced by bacterial exposure to polycationic peptides or may be
inherited stably as a result of mutations affecting the sensor
kinase ParS or the response regulator ParR. Adding to the
recent observation that ParRS mediates the adaptive response
of P. aeruginosa to polycations such as polymyxin B, colistin,
and indolicidin at Mg2⫹ concentrations similar to those found
in the human body (1 to 2 mM) (9), this work demonstrates
that when it is activated, ParRS also leads to up- and downregulation of the efflux system MexXY/OprM and the porin
OprD, respectively. Some of our data strongly suggest that the
transcriptional regulator ParR is activated rather than inhibited by ParS once a specific signal is detected in the bacterial
environment. Indeed, deletion of parS or parRS did not affect
antibiotic resistance in wild-type bacteria, except for inducers
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FIG. 5. ParRS is required for colistin-induced resistance to the
killing activity of gentamicin. Survivors of wild-type strain PAO1Bes
(triangles) and its ⌬parRS derivative CM096 (circles) in the presence
of 16 ␮g/ml gentamicin were determined in triplicate after 4 h of
preincubation in drug-free MHB (open symbols) or MHB supplemented with a concentration equivalent to a 1:2 ratio of the MIC of
colistin.
ANTIMICROB. AGENTS CHEMOTHER.
VOL. 55, 2011
ParRS-MEDIATED MULTIRESISTANCE IN P. AERUGINOSA
1219
such as colistin. Overexpression of plasmid-borne wild-type
parRS genes did not result in increased resistance either (data
not shown). The nature of the signal sensed by the regulatory
system remains to be identified. However, this signal is unlikely
to be related to disorganization of the outer membrane architecture, since (i) some polycationic peptides (e.g., CP-28 and
LL-37) were unable to activate ParRS-dependent expression of
the arnBCADTEF-ugd operon (9) and (ii) PMBN proved to be
a much weaker inducer of mexY expression than polymyxin B,
despite their having similar outer membrane-permeabilizing
capabilities (Fig. 5). Undoubtedly, identification of the molecular signal perceived by ParS is a prerequisite for the development of innovative molecules able to block the adaptive
resistance of P. aeruginosa to polycations.
The molecular basis of ParRS-dependent regulation of
MexXY/OprM and OprD is another crucial issue that should
be addressed in order to understand and try to prevent the
adaptation of P. aeruginosa to antibiotic stress. A search for
motif elicitation in the promoter regions upstream of the genes
responding to ParRS activation in PAOW2 by use of MEME
software (2) identified a predicted region of 40 nucleotides
(P ⬍ 10⫺10) upstream of mexY, oprD, PA1797, PA2358, and
PA2655. Thus, it is tempting to assume that MexXY/OprM
and OprD act in concert to limit (i.e., by active efflux and by
outer membrane impermeability) the intracellular accumulation of a toxic substrate that directly or indirectly results from
the antibacterial action of polycationic peptides. Since genes
coding for putative homologs of polyamine biosynthetic/
degradative enzymes (e.g., PA4773 and PA4774) are overexpressed in PAOW2 or under polycation exposure, we looked
at whether their inactivation would normalize the expression of
mexY and oprD to baseline levels. The deletion of the PA4773
gene, which is predicted to encode an adenosylmethionine
decarboxylase, failed to produce the expected results (data not
shown). In the same line, plasmid-mediated overexpression or
deletion of the PA2358 and PA2655 genes, whose functions are
virtually unknown, had no influence on mexY or oprD expression in PAO1Bes (not presented). A search for a toxic metabolite recognized by both MexXY/OprM and OprD is currently
under way in our laboratory.
Finally, this work demonstrates the existence of at least two
novel types of agrW mutants in addition to those exhibiting
alterations in the ribosomal machinery (8, 19). A preliminary
study conducted on 94 non-CF, MexXY-overproducing clinical
Downloaded from http://aac.asm.org/ on February 28, 2014 by INIST-CNRS BiblioVie
FIG. 6. Schematic representation of regulation of the mexXY operon in P. aeruginosa. (A) Wild-type strain grown in drug-free medium. During
bacterial growth, a translation attenuation mechanism involving a small peptide, PA5471.1, prevents transcription of PA5471, a downstream gene
of unknown function whose product directly or indirectly modulates the activity of MexZ, the local repressor of the mexXY operon. (B) Growth
of wild-type strain in the presence of MexXY-inducing antibiotics. Following bacterial exposure to ribosome-targeting antimicrobials, MexXY is
produced at high levels conferring intrinsic resistance to the inducer, provided that it is a good substrate for the efflux pump (as is the case for
aminoglycosides but not for chloramphenicol). This MexZ-dependent induction process is linked in part to increased levels of the PA5471 protein
modulating MexZ repressor activity. A second pathway leading to drug-dependent overexpression of mexXY is independent of both the MexZ and
PA5471 proteins. Our results demonstrate that polycationic antibiotics (colistin and polymyxin B) are able to promote expression of the pmrAB,
arnBCDTEF-ugd, and mexXY operons and to coordinately downregulate the oprD gene through the activation of ParRS. This regulation results
in multidrug resistance.
1220
MULLER ET AL.
ACKNOWLEDGMENTS
We are grateful to Fabrice Poncet and Barbara Dehecq of the
Faculty of Medicine, Université de Franche-Comté, Besançon, France,
for their technical assistance.
Funding was obtained from the Ministère de l’Enseignement Supérieur et de la Recherche.
REFERENCES
1. Aires, J. R., T. Köhler, H. Nikaido, and P. Plésiat. 1999. Involvement of an
active efflux system in the natural resistance of Pseudomonas aeruginosa to
aminoglycosides. Antimicrob. Agents Chemother. 43:2624–2628.
2. Bailey, T. L., and C. Elkan. 1994. Fitting a mixture model by expectation
maximization to discover motifs in biopolymers. Proc. Int. Conf. Intell. Syst.
Mol. Biol. 2:28–36.
3. Barrow, K., and D. H. Kwon. 2009. Alterations in two-component regulatory
systems of PhoPQ and PmrAB are associated with polymyxin B resistance in
clinical isolates of Pseudomonas aeruginosa. Antimicrob. Agents Chemother.
53:5150–5154.
4. Caughlan, R. E., et al. 2009. Fmt bypass in Pseudomonas aeruginosa causes
induction of MexXY efflux pump expression. Antimicrob. Agents Chemother. 53:5015–5021.
5. Clinical and Laboratory Standards Institute (CLSI). 2006. Performance
standards for antimicrobial susceptibility testing. CLSI document M100–S16,
16th ed. CLSI, Wayne, PA.
6. Ditta, G., S. Stanfield, D. Corbin, and D. R. Helinski. 1980. Broad-hostrange DNA cloning system for gram-negative bacteria: construction of a
gene bank of Rhizobium meliloti. Proc. Natl. Acad. Sci. U. S. A. 77:7347–
7351.
7. Dumas, J. L., C. van Delden, K. Perron, and T. Köhler. 2006. Analysis of
antibiotic resistance gene expression in Pseudomonas aeruginosa by quantitative real-time-PCR. FEMS Microbiol. Lett. 254:217–225.
8. El’Garch, F., K. Jeannot, D. Hocquet, C. Llanes-Barakat, and P. Plésiat.
2007. Cumulative effects of several nonenzymatic mechanisms on the resistance of Pseudomonas aeruginosa to aminoglycosides. Antimicrob. Agents
Chemother. 51:1016–1021.
9. Fernandez, L., et al. 2010. Adaptive resistance to the “last hope” antibiotics
polymyxin B and colistin in Pseudomonas aeruginosa is mediated by the novel
two-component regulatory system ParR-ParS. Antimicrob. Agents Chemother. 54:3372–3382.
10. Flume, P. A., et al. 2007. Cystic fibrosis pulmonary guidelines: chronic medications for maintenance of lung health. Am. J. Respir. Crit. Care Med.
176:957–969.
11. Gilleland, H. E., J. D. Stinnett, and R. G. Eagon. 1974. Ultrastructural and
chemical alteration of the cell envelope of Pseudomonas aeruginosa, associated with resistance to ethylene diamine tetra acetate resulting from growth
in a Mg2⫹ deficient medium. J. Bacteriol. 117:302–311.
12. Herrero, M., V. de Lorenzo, and K. N. Timmis. 1990. Transposon vectors
containing non-antibiotic resistance selection markers for cloning and stable
chromosomal insertion of foreign genes in gram-negative bacteria. J. Bacteriol. 172:6557–6567.
13. Hoang, T. T., R. R. Karkhoff-Schweizer, A. J. Kutchma, and H. P. Schweizer.
1998. A broad-host-range Flp-FRT recombination system for site-specific
excision of chromosomally-located DNA sequences: application for isolation
of unmarked Pseudomonas aeruginosa mutants. Gene 212:77–86.
14. Hoang, T. T., A. J. Kutchma, A. Becher, and H. P. Schweizer. 2000. Integration-proficient plasmids for Pseudomonas aeruginosa: site-specific integration
and use for engineering of reporter and expression strains. Plasmid 43:59–72.
15. Hocquet, D., et al. 2007. Pseudomonas aeruginosa may accumulate drug
resistance mechanisms without losing its ability to cause bloodstream infections. Antimicrob. Agents Chemother. 51:3531–3536.
16. Irizarry, R. A., et al. 2003. Summaries of Affymetrix GeneChip probe level
data. Nucleic Acids Res. 31:15.
17. Irizarry, R. A., et al. 2003. Exploration, normalization, and summaries of
high density oligonucleotide array probe level data. Biostatistics 4:249–264.
18. Islam, S., S. Jalal, and B. Wretlind. 2004. Expression of the MexXY efflux
pump in amikacin-resistant isolates of Pseudomonas aeruginosa. Clin. Microbiol. Infect. 10:877–883.
19. Jeannot, K., M. L. Sobel, F. El Garch, K. Poole, and P. Plésiat. 2005.
Induction of the MexXY efflux pump in Pseudomonas aeruginosa is dependent on drug-ribosome interaction. J. Bacteriol. 187:5341–5346.
20. Jo, J. T., F. S. Brinkman, and R. E. Hancock. 2003. Aminoglycoside efflux in
Pseudomonas aeruginosa: involvement of novel outer membrane proteins.
Antimicrob. Agents Chemother. 47:1101–1111.
21. Kaniga, K., I. Delor, and G. R. Cornelis. 1991. A wide-host-range suicide
vector for improving reverse genetics in gram-negative bacteria: inactivation
of the blaA gene of Yersinia enterocolitica. Gene 109:137–141.
22. Klockgether, J., et al. 2010. Genome diversity of Pseudomonas aeruginosa
PAO1 laboratory strains. J. Bacteriol. 192:1113–1121.
23. Köhler, T., S. F. Epp, L. K. Curty, and J.-C. Pechère. 1999. Characterization
of MexT, the regulator of the MexE-MexF-OprN multidrug efflux system of
Pseudomonas aeruginosa. J. Bacteriol. 181:6300–6305.
24. Köhler, T., M. Michéa-Hamzehpour, S. F. Epp, and J. C. Pechère. 1999.
Carbapenem activities against Pseudomonas aeruginosa: respective contributions of OprD and efflux systems. Antimicrob. Agents Chemother. 43:424–
427.
25. Lacks, S., and B. Greenberg. 1977. Complementary specificity of restriction
endonucleases of Diplococcus pneumoniae with respect to DNA methylation.
J. Mol. Biol. 114:153–168.
26. Li, X. Z., H. Nikaido, and K. Poole. 1995. Role of MexA-MexB-OprM in
antibiotic efflux in Pseudomonas aeruginosa. Antimicrob. Agents Chemother.
39:1948–1953.
27. Livermore, D. M. 2002. Multiple mechanisms of antimicrobial resistance in
Pseudomonas aeruginosa: our worst nightmare? Clin. Infect. Dis. 34:634–640.
28. Llanes, C., et al. 2004. Clinical strains of Pseudomonas aeruginosa overproducing MexAB-OprM and MexXY efflux pumps simultaneously. Antimicrob. Agents Chemother. 48:1797–1802.
29. Manoil, C., and J. Beckwith. 1985. TnphoA: a transposon probe for protein
export signals. Proc. Natl. Acad. Sci. U. S. A. 82:8129–8133.
30. Masuda, N., et al. 2000. Contribution of the MexX-MexY-OprM efflux
system to intrinsic resistance in Pseudomonas aeruginosa. Antimicrob.
Agents Chemother. 44:2242–2246.
31. Masuda, N., et al. 2000. Substrate specificities of MexAB-OprM, MexCDOprJ, and MexXY-OprM efflux pumps in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 44:3322–3327.
32. Matsuo, Y., S. Eda, N. Gotoh, E. Yoshihara, and T. Nakae. 2004. MexZmediated regulation of mexXY multidrug efflux pump expression in Pseudomonas aeruginosa by binding on the mexZ-mexX intergenic DNA. FEMS
Microbiol. Lett. 238:23–28.
33. McPhee, J. B., et al. 2006. Contribution of the PhoP-PhoQ and PmrA-PmrB
two-component regulatory systems to Mg2⫹-induced gene regulation in
Pseudomonas aeruginosa. J. Bacteriol. 188:3995–4006.
34. McPhee, J. B., S. Lewenza, and R. E. Hancock. 2003. Cationic antimicrobial
peptides activate a two-component regulatory system, PmrA-PmrB, that
regulates resistance to polymyxin B and cationic antimicrobial peptides in
Pseudomonas aeruginosa. Mol. Microbiol. 50:205–217.
35. Mine, T., Y. Morita, A. Kataoka, T. Mizushima, and T. Tsuchiya. 1999.
Expression in Escherichia coli of a new multidrug efflux pump MexXY from
Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 43:415–417.
36. Morita, Y., M. L. Sobel, and K. Poole. 2006. Antibiotic inducibility of the
MexXY multidrug efflux system of Pseudomonas aeruginosa: involvement of
the antibiotic-inducible PA5471 gene product. J. Bacteriol. 188:1847–1855.
37. Moskowitz, S. M., R. K. Ernst, and S. I. Miller. 2004. PmrAB, a twocomponent regulatory system of Pseudomonas aeruginosa that modulates
resistance to cationic antimicrobial peptides and addition of aminoarabinose
to lipid A. J. Bacteriol. 186:575–579.
38. Mushtaq, S., Y. Ge, and D. M. Livermore. 2004. Doripenem versus Pseudomonas aeruginosa in vitro: activity against characterized isolates, mutants,
and transconjugants and resistance selection potential. Antimicrob. Agents
Chemother. 48:3086–3092.
39. Ochs, M. M., M. P. McCusker, M. Bains, and R. E. Hancock. 1999. Negative
regulation of the Pseudomonas aeruginosa outer membrane porin OprD
selective for imipenem and basic amino acids. Antimicrob. Agents Chemother. 43:1085–1090.
40. Plésiat, P., J. R. Aires, C. Godard, and T. Köhler. 1997. Use of steroids to
monitor alterations in the outer membrane of Pseudomonas aeruginosa. J.
Bacteriol. 179:7004–7010.
41. Rodrigue, A., Y. Quentin, A. Lazdunski, V. Mejean, and M. Foglino. 2000.
Two-component systems in Pseudomonas aeruginosa: why so many? Trends
Microbiol. 8:498–504.
42. Satake, S., H. Yoneyama, and T. Nakae. 1991. Role of OmpD2 and chromosomal beta-lactamase in carbapenem resistance in clinical isolates of
Pseudomonas aeruginosa. J. Antimicrob. Chemother. 28:199–207.
43. Schurek, K. N., et al. 2009. Involvement of PmrAB and PhoPQ in polymyxin B adaptation and inducible resistance in non-cystic fibrosis clinical
isolates of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 53:
4345–4351.
Downloaded from http://aac.asm.org/ on February 28, 2014 by INIST-CNRS BiblioVie
strains showed that 21 (22%) were agrW mutants and 73 (78%)
were of the agrZ type. The characterization of these mutants is
in progress. The involvement of some of the agrW isolates in
severe infections (e.g., bacteremia) suggests that they are fully
virulent. From a physiological perspective, the active efflux
system MexXY/OprM now appears to be a key element of
bacterial adaptation to antibiotics targeting the ribosome (aminoglycosides, tetracyclines, macrolides, and chloramphenicol)
or the cellular envelope (colistin and polymyxin B). Figure 6
summarizes our current knowledge of the complex regulation
of MexXY.
ANTIMICROB. AGENTS CHEMOTHER.
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ParRS-MEDIATED MULTIRESISTANCE IN P. AERUGINOSA
44. Simon, R., M. O’Connell, M. Labes, and A. Puhler. 1986. Plasmid vectors
for the genetic analysis and manipulation of Rhizobia and other gramnegative bacteria. Methods Enzymol. 118:640–659.
45. Sobel, M. L., G. A. McKay, and K. Poole. 2003. Contribution of the MexXY
multidrug transporter to aminoglycoside resistance in Pseudomonas aeruginosa clinical isolates. Antimicrob. Agents Chemother. 47:3202–3207.
46. Stover, C. K., et al. 2000. Complete genome sequence of Pseudomonas
aeruginosa PAO1, an opportunistic pathogen. Nature 406:959–964.
47. Trias, J., and H. Nikaido. 1990. Outer membrane protein D2 catalyzes facilitated diffusion of carbapenems and penems through the outer membrane of
Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 34:52–57.
48. Vaara, M., P. Viljanen, T. Vaara, and P. H. Makela. 1984. An outer membrane-disorganizing peptide PMBN sensitizes E. coli strains to serum bactericidal action. J. Immunol. 132:2582–2589.
1221
49. Viljanen, P., and M. Vaara. 1984. Susceptibility of gram-negative bacteria
to polymyxin B nonapeptide. Antimicrob. Agents Chemother. 25:701–
705.
50. Vogne, C., J. R. Aires, C. Bailly, D. Hocquet, and P. Plésiat. 2004. Role of the
multidrug efflux system MexXY in the emergence of moderate resistance to
aminoglycosides among Pseudomonas aeruginosa isolates from patients with
cystic fibrosis. Antimicrob. Agents Chemother. 48:1676–1680.
51. Westbrock-Wadman, S., et al. 1999. Characterization of a Pseudomonas
aeruginosa efflux pump contributing to aminoglycoside impermeability. Antimicrob. Agents Chemother. 43:2975–2983.
52. Zhang, L., M. G. Scott, H. Yan, L. D. Mayer, and R. E. Hancock. 2000.
Interaction of polyphemusin I and structural analogs with bacterial membranes, lipopolysaccharide, and lipid monolayers. Biochemistry 39:14504–
14514.
Downloaded from http://aac.asm.org/ on February 28, 2014 by INIST-CNRS BiblioVie