Pan-β-Lactam Resistance Development in Pseudomonas aeruginosa Clinical Strains: Molecular Mechanisms, Penicillin-Binding Protein Profiles, and Binding Affinities

ABSTRACTWe investigated the mechanisms leading toPseudomonas aeruginosapan-β-lactam resistance (PBLR) development during the treatment of nosocomial infections, with a particular focus on the modification of penicillin-binding protein (PBP) profiles and imipenem, ceftazidime, and ceftolozane (former CXA-101) PBP binding affinities. For this purpose, six clonally related pairs of sequential susceptible-PBLR isolates were studied. The presence ofoprD,ampD, anddacBmutations was explored by PCR followed by sequencing and the expression ofampCand efflux pump genes by real-time reverse transcription-PCR. The fluorescent penicillin Bocillin FL was used to determine PBP profiles in membrane preparations from all pairs, and 50% inhibitory concentrations (IC50s) of ceftolozane, ceftazidime, and imipenem were analyzed in 3 of them. Although a certain increase was noted (0 to 5 2-fold dilutions), the MICs of ceftolozane were ≤4 μg/ml in all PBLR isolates. All 6 PBLR isolates lacked OprD and overexpressedampCand one or several efflux pumps, particularlymexBand/ormexY. Additionally, 5 of them showed modified PBP profiles, including a modified pattern (n= 1) or diminished expression (n= 1) of PBP1a and a lack of PBP4 expression (n= 4), which correlated with AmpC overexpression driven bydacBmutation. Analysis of the essential PBP IC50s revealed significant variation of PBP1a/b binding affinities, both within each susceptible-PBLR pair and across the different pairs. Moreover, despite the absence of significant differences in gene expression or sequence, a clear tendency toward increased PBP2 (imipenem) and PBP3 (ceftazidime, ceftolozane, imipenem) IC50s was noted in PBLR isolates. Thus, our results suggest that in addition to AmpC, efflux pumps, and OprD, the modification of PBP patterns appears to play a role in thein vivoemergence of PBLR strains, which still conserve certain susceptibility to the new antipseudomonal cephalosporin ceftolozane.

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Structural Insights into Ceftobiprole Inhibition of Pseudomonas aeruginosa Penicillin-Binding Protein 3

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00106-20 ◽

2020 ◽

Vol 64 (5) ◽

Author(s):

Vijay Kumar ◽

Christie Tang ◽

Christopher R. Bethel ◽

Krisztina M. Papp-Wallace ◽

Jacob Wyatt ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Binding Sites ◽

Binding Protein ◽

Covalent Bond ◽

Penicillin Binding Protein ◽

Content Type ◽

Cephalosporin Antibiotic ◽

Structural Insights ◽

Fine Tune ◽

Advanced Generation

ABSTRACT Ceftobiprole is an advanced-generation broad-spectrum cephalosporin antibiotic with potent and rapid bactericidal activity against Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus, as well as susceptible Gram-negative pathogens, including Pseudomonas sp. pathogens. In the case of Pseudomonas aeruginosa, ceftobiprole acts by inhibiting P. aeruginosa penicillin-binding protein 3 (PBP3). Structural studies were pursued to elucidate the molecular details of this PBP inhibition. The crystal structure of the His-tagged PBP3-ceftobiprole complex revealed a covalent bond between the ligand and the catalytic residue S294. Ceftobiprole binding leads to large active site changes near binding sites for the pyrrolidinone and pyrrolidine rings. The S528 to L536 region adopts a conformation previously not observed in PBP3, including partial unwinding of the α11 helix. These molecular insights can lead to a deeper understanding of β-lactam-PBP interactions that result in major changes in protein structure, as well as suggesting how to fine-tune current inhibitors and to develop novel inhibitors of this PBP.

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Overexpression of AmpC and Efflux Pumps in Pseudomonas aeruginosa Isolates from Bloodstream Infections: Prevalence and Impact on Resistance in a Spanish Multicenter Study

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01645-10 ◽

2011 ◽

Vol 55 (5) ◽

pp. 1906-1911 ◽

Cited By ~ 112

Author(s):

Gabriel Cabot ◽

Alain A. Ocampo-Sosa ◽

Fe Tubau ◽

María D. Macia ◽

Cristina Rodríguez ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Multicenter Study ◽

Efflux Pump ◽

Bloodstream Infections ◽

Efflux Pumps ◽

Complete Collection ◽

Content Type ◽

The Individual ◽

First Time ◽

Inactivating Mutations

ABSTRACTThe prevalence and impact of the overexpression of AmpC and efflux pumps were evaluated with a collection of 190Pseudomonas aeruginosaisolates recovered from bloodstream infections in a 2008 multicenter study (10 hospitals) in Spain. The MICs of a panel of 13 antipseudomonal agents were determined by microdilution, and the expressions ofampC,mexB,mexY,mexD, andmexFwere determined by real-time reverse transcription (RT)-PCR. Up to 39% of the isolates overexpressed at least one of the mechanisms.ampCoverexpression (24.2%) was the most prevalent mechanism, followed bymexY(13.2%),mexB(12.6%),mexF(4.2%), andmexD(2.2%). The overexpression ofmexBplusmexY, documented for 5.3% of the isolates, was the only combination showing a significantly (P= 0.02) higher prevalence than expected from the frequencies of the individual mechanisms (1.6%). Additionally, all imipenem-resistant isolates studied (25 representative isolates) showed inactivating mutations inoprD. Most of the isolates nonsusceptible to piperacillin-tazobactam (96%) and ceftazidime (84%) overexpressedampC, whilemexB(25%) andmexY(29%) overexpressions gained relevance among cefepime-nonsusceptible isolates. Nevertheless, the prevalence ofmexYoverexpression was highest among tobramycin-nonsusceptible isolates (37%), and that ofmexBwas highest among meropenem-nonsusceptible isolates (33%). Regarding ciprofloxacin-resistant isolates, besides the expected increased prevalence of efflux pump overexpression, a highly significant link toampCoverexpression was documented for the first time: up to 52% of ciprofloxacin-nonsusceptible isolates overexpressedampC, sharply contrasting with the 24% documented for the complete collection (P< 0.001). In summary, mutation-driven resistance was frequent inP. aeruginosaisolates from bloodstream infections, whereas metallo-β-lactamases, detected in 2 isolates (1%) producing VIM-2, although with increasing prevalences, were still uncommon.

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Involvement of the AcrAB-TolC Efflux Pump in the Resistance, Fitness, and Virulence of Enterobacter cloacae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05509-11 ◽

2012 ◽

Vol 56 (4) ◽

pp. 2084-2090 ◽

Cited By ~ 68

Author(s):

Astrid Pérez ◽

Margarita Poza ◽

Ana Fernández ◽

Maria del Carmen Fernández ◽

Susana Mallo ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Pathogenic Bacteria ◽

Efflux Pump ◽

Enterobacter Cloacae ◽

Efflux Pumps ◽

Clinical Isolates ◽

Wild Type ◽

Content Type

ABSTRACTMultidrug efflux pumps have emerged as important mechanisms of antimicrobial resistance in bacterial pathogens. In order to cause infection, pathogenic bacteria require mechanisms to avoid the effects of host-produced compounds, and express efflux pumps may accomplish this task. In this study, we evaluated the effect of the inactivation of AcrAB-TolC on antimicrobial resistance, fitness, and virulence inEnterobacter cloacae, an opportunistic pathogen usually involved in nosocomial infections. Two different clinical isolates ofE. cloacaewere used, EcDC64 (multidrug resistance overexpressing the AcrAB-TolC efflux pump) and Jc194 (basal AcrAB-TolC expression). TheacrAandtolCgenes were deleted in strains EcDC64 and Jc194 to produce, respectively, EcΔacrAand EcΔtolCand JcΔacrAand JcΔtolCknockout (KO) derivatives. Antibiotic susceptibility testing was performed with all isolates, and we discovered that these mechanisms are involved in the resistance ofE. cloacaeto several antibiotics. Competition experiments were also performed with wild-type and isogenic KO strains. The competition index (CI), defined as the mutant/wild-type ratio, revealed that theacrAandtolCgenes both affect the fitness ofE. cloacae, as fitness was clearly reduced in theacrAandtolCKO strains. The median CI values obtainedin vitroandin vivowere, respectively, 0.42 and 0.3 for EcDC64/EcΔacrA, 0.24 and 0.38 for EcDC64/EcΔtolC, 0.15 and 0.11 for Jc194/JcΔacrA, and 0.38 and 0.39 for Jc194/JcΔtolC. Use of an intraperitoneal mouse model of systemic infection revealed reduced virulence in bothE. cloacaeclinical strains when either theacrAortolCgene was inactivated. In conclusion, the structural components of the AcrAB-TolC efflux pump appear to play a role in antibiotic resistance as well as environmental adaptation and host virulence in clinical isolates ofE. cloacae.

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Two Regulators, PA3898 and PA2100, Modulate the Pseudomonas aeruginosa Multidrug Resistance MexAB-OprM and EmrAB Efflux Pumps and Biofilm Formation

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01459-18 ◽

2018 ◽

Vol 62 (12) ◽

Cited By ~ 4

Author(s):

Yun Heacock-Kang ◽

Zhenxin Sun ◽

Jan Zarzycki-Siek ◽

Kanchana Poonsuk ◽

Ian A. McMillan ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Efflux Pump ◽

Efflux Pumps ◽

Localized Surface Plasmon ◽

Mouse Lung ◽

Multidrug Efflux ◽

Promoter Regions ◽

Content Type ◽

Multidrug Efflux Pumps

ABSTRACT It is generally believed that the Pseudomonas aeruginosa biofilm matrix itself acts as a molecular sieve or sink that contributes to significant levels of drug resistance, but it is becoming more apparent that multidrug efflux pumps induced during biofilm growth significantly enhance resistance levels. We present here a novel transcriptional regulator, PA3898, which controls biofilm formation and multidrug efflux pumps in P. aeruginosa. A mutant of this regulator significantly reduced the ability of P. aeruginosa to produce biofilm in vitro and affected its in vivo fitness and pathogenesis in Drosophila melanogaster and BALB/c mouse lung infection models. Transcriptome analysis revealed that PA3898 modulates essential virulence genes/pathways, including multidrug efflux pumps and phenazine biosynthesis. Chromatin immunoprecipitation sequencing (ChIP-seq) identified its DNA binding sequences and confirmed that PA3898 directly interacts with promoter regions of four genes/operons, two of which are mexAB-oprM and phz2. Coimmunoprecipitation revealed a regulatory partner of PA3898 as PA2100, and both are required for binding to DNA in electrophoretic mobility shift assays. PA3898 and PA2100 were given the names MdrR1 and MdrR2, respectively, as novel repressors of the mexAB-oprM multidrug efflux operon and activators for another multidrug efflux pump, EmrAB. The interaction between MdrR1 and MdrR2 at the promoter regions of their regulons was further characterized via localized surface plasmon resonance and DNA footprinting. These regulators directly repress the mexAB-oprM operon, independent of its well-established MexR regulator. Mutants of mdrR1 and mdrR2 caused increased resistance to multiple antibiotics in P. aeruginosa, validating the significance of these newly discovered regulators.

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Carbapenem-NonsusceptibleHaemophilus influenzaewith Penicillin-Binding Protein 3 Containing an Amino Acid Insertion

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00671-18 ◽

2018 ◽

Vol 62 (8) ◽

Cited By ~ 7

Author(s):

Kazuki Kitaoka ◽

Kouji Kimura ◽

Hiromitsu Kitanaka ◽

Hirotsugu Banno ◽

Wanchun Jin ◽

...

Keyword(s):

Amino Acid ◽

Antimicrobial Susceptibility ◽

Susceptibility Testing ◽

Efflux Pump ◽

Binding Protein ◽

Antimicrobial Susceptibility Testing ◽

Nucleotide Sequence Analysis ◽

Penicillin Binding Protein ◽

Binding Assays ◽

Content Type

ABSTRACTThe prevalence of β-lactamase-negative ampicillin-resistant (BLNAR)Haemophilus influenzaehas become a clinical concern. In BLNAR isolates, amino acid substitutions in penicillin-binding protein 3 (PBP3) are relevant to the β-lactam resistance. Carbapenem-nonsusceptibleH. influenzaeisolates have been rarely reported. Through antimicrobial susceptibility testing, nucleotide sequence analysis offtsI, encoding PBP3, and the utilization of a collection ofH. influenzaeclinical isolates in our laboratory, we obtained a carbapenem-nonsusceptible clinical isolate (NUBL1772) that possesses an altered PBP3 containing V525_N526insM. The aim of this study was to reveal the effect of altered PBP3 containing V525_N526insM on reduced carbapenem susceptibility. After generating recombinant strains with alteredftsI, we performed antimicrobial susceptibility testing and competitive binding assays with fluorescent penicillin (Bocillin FL) and carbapenems. Elevated carbapenem MICs were found for the recombinant strain harboring the entireftsIgene of NUBL1772. The recombinant PBP3 of NUBL1772 also exhibited reduced binding to carbapenems. These results demonstrate that altered PBP3 containing V525_N526insM influences the reduced carbapenem susceptibility. The revertant mutant lacking the V525_N526insM exhibited lower MICs for carbapenems than NUBL1772, suggesting that this insertion affects reduced carbapenem susceptibility. The MICs of β-lactams for NUBL1772 were higher than those for the recombinant possessingftsIof NUBL1772. NUBL1772 harbored AcrR with early termination, resulting in low-level transcription ofacrBand high efflux pump activity. These findings suggest that the disruption of AcrR also contributes to the reduced carbapenem susceptibility found in NUBL1772. Our results provide the first evidence that the altered PBP3 containing V525_N526insM is responsible for the reduced susceptibility to carbapenems inH. influenzae.

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In Vitro and In Vivo Activities of β-Lactams in Combination with the Novel β-Lactam Enhancers Zidebactam and WCK 5153 against Multidrug-Resistant Metallo-β-Lactamase-Producing Klebsiella pneumoniae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00128-19 ◽

2019 ◽

Vol 63 (5) ◽

Cited By ~ 16

Author(s):

Bartolome Moya ◽

Isabel M. Barcelo ◽

Gabriel Cabot ◽

Gabriel Torrens ◽

Snehal Palwe ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Klebsiella Pneumoniae ◽

Molecular Targets ◽

Multidrug Resistant ◽

The Novel ◽

Penicillin Binding Protein ◽

Content Type ◽

Time Kill

ABSTRACT Zidebactam and WCK 5153 are novel bicyclo-acyl hydrazide (BCH) agents that have previously been shown to act as β-lactam enhancer (BLE) antibiotics in Pseudomonas aeruginosa and Acinetobacter baumannii. The objectives of this work were to identify the molecular targets of these BCHs in Klebsiella pneumoniae and to investigate their potential BLE activity for cefepime and aztreonam against metallo-β-lactamase (MBL)-producing strains in vitro and in vivo. Penicillin binding protein (PBP) binding profiles were determined by Bocillin FL assay, and 50% inhibitory concentrations (IC50s) were determined using ImageQuant TL software. MICs and kill kinetics for zidebactam, WCK 5153, and cefepime or aztreonam, alone and in combination, were determined against clinical K. pneumoniae isolates producing MBLs VIM-1 or NDM-1 (plus ESBLs and class C β-lactamases) to assess the in vitro enhancer effect of BCH compounds in conjunction with β-lactams. Additionally, murine systemic and thigh infection studies were conducted to evaluate BLE effects in vivo. Zidebactam and WCK 5153 showed specific, high PBP2 affinity in K. pneumoniae. The MICs of BLEs were >64 μg/ml for all MBL-producing strains. Time-kill studies showed that a combination of these BLEs with either cefepime or aztreonam provided 1 to >3 log10 kill against MBL-producing K. pneumoniae strains. Furthermore, the bactericidal synergy observed for these BLE–β-lactam combinations translated well into in vivo efficacy even in the absence of MBL inhibition by BLEs, a characteristic feature of the β-lactam enhancer mechanism of action. Zidebactam and WCK 5153 are potent PBP2 inhibitors and display in vitro and in vivo BLE effects against multidrug-resistant (MDR) K. pneumoniae clinical isolates producing MBLs.

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Azole Resistance of Aspergillus fumigatus Biofilms Is Partly Associated with Efflux Pump Activity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01189-10 ◽

2011 ◽

Vol 55 (5) ◽

pp. 2092-2097 ◽

Cited By ~ 82

Author(s):

Ranjith Rajendran ◽

Eilidh Mowat ◽

Elaine McCulloch ◽

David F. Lappin ◽

Brian Jones ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Efflux Pump ◽

Efflux Pumps ◽

Azole Resistance ◽

Dependent Manner ◽

Content Type ◽

Quantitative Expression ◽

Pump Activity ◽

Competitive Substrate

ABSTRACTThis study investigated the phase-dependent expression and activity of efflux pumps inAspergillus fumigatustreated with voriconazole. Fourteen strains were shown to become increasingly resistant in the 12-h (16- to 128-fold) and 24-h (>512-fold) phases compared to 8-h germlings. An Ala-Nap uptake assay demonstrated a significant increase in efflux pump activity in the 12-h and 24-h phases (P< 0.0001). The efflux pump activity of the 8-h germling cells was also significantly induced by voriconazole (P< 0.001) after 24 h of treatment. Inhibition of efflux pump activity with the competitive substrate MC-207,110 reduced the voriconazole MIC values for theA. fumigatusgermling cells by 2- to 8-fold. Quantitative expression analysis ofAfuMDR4mRNA transcripts showed a phase-dependent increase as the mycelial complexity increased, which was coincidental with a strain-dependent increase in azole resistance. Voriconazole also significantly induced this in a time-dependent manner (P< 0.001). Finally, anin vivomouse biofilm model was used to evaluate efflux pump expression, and it was shown thatAfuMDR4was constitutively expressed and significantly induced by treatment with voriconazole after 24 h (P< 0.01). Our results demonstrate that efflux pumps are expressed in complexA. fumigatusbiofilm populations and that this contributes to azole resistance. Moreover, voriconazole treatment induces efflux pump expression. Collectively, these data may provide evidence for azole treatment failures in clinical cases of aspergillosis.

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Activity of Ceftolozane-Tazobactam against Carbapenem-Resistant, Non-Carbapenemase-Producing Pseudomonas aeruginosa and Associated Resistance Mechanisms

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01970-17 ◽

2017 ◽

Vol 62 (1) ◽

Cited By ~ 12

Author(s):

Yu Mi Wi ◽

Kerryl E. Greenwood-Quaintance ◽

Audrey N. Schuetz ◽

Kwan Soo Ko ◽

Kyong Ran Peck ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Efflux Pump ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Resistance Rate ◽

Content Type ◽

Reverse Transcription Pcr ◽

Carbapenem Resistant ◽

Kill Curve ◽

Time Kill

ABSTRACT Although carbapenems are effective for treating serious multidrug-resistant Pseudomonas aeruginosa infections, carbapenem-resistant P. aeruginosa (CRPA) is now being reported worldwide. Ceftolozane-tazobactam (C/T) demonstrates activity against many multidrug-resistant isolates. We evaluated the activity of C/T and compared its activity to that of ceftazidime-avibactam (C/A) using a well-characterized collection of non-carbapenemase-producing CRPA isolates. Forty-two non-carbapenemase-producing CRPA isolates from a previous study (J. Y. Lee and K. S. Ko, Int J Antimicrob Agents 40:168–172, 2012, https://doi.org/10.1016/j.ijantimicag.2012.04.004) were included. All had been previously shown to be negative for bla IMP, bla VIM, bla SPM, bla GIM, bla SIM, and bla KPC by PCR. In the prior study, expression of oprD, ampC, and several efflux pump genes had been defined by quantitative reverse transcription-PCR. Here, antimicrobial susceptibility was determined by broth microdilution according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Time-kill curve assays were performed using three C/T- and C/A-susceptible CRPA isolates. Among 42 non-carbapenemase-producing CRPA isolates, overall susceptibility to C/T was 95.2%, compared to 71.4%, 42.9%, 23.8%, 21.4%, and 2.4% for C/A, ceftazidime, piperacillin-tazobactam, cefepime, and meropenem, respectively. The C/T resistance rate was significantly lower than that of C/A among isolates showing decreased oprD and increased mexB expression (5.1% versus 25.6%, P = 0.025, and 4.3% versus 34.8%, P = 0.022, respectively). In time-kill curve studies, C/T was less bactericidal than C/A against an isolate with decreased oprD and increased ampC expression. C/T was active against 95.2% of non-carbapenemase-producing CRPA clinical isolates. No apparent correlation of C/T MIC values with specific mutation-driven resistance mechanisms was noted.

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Triclosan depletes the membrane potential in Pseudomonas aeruginosa biofilms inhibiting aminoglycoside induced adaptive resistance

10.1101/2020.04.09.034033 ◽

2020 ◽

Author(s):

Michael M. Maiden ◽

Christopher M. Waters

Keyword(s):

Pseudomonas Aeruginosa ◽

Membrane Potential ◽

Efflux Pump ◽

Antibacterial Properties ◽

Efflux Pumps ◽

Chronic Infections ◽

Onset Of Action ◽

Adaptive Resistance

AbstractBiofilm-based infections are difficult to treat due to their inherent resistance to antibiotic treatment. Discovering new approaches to enhance antibiotic efficacy in biofilms would be highly significant in treating many chronic infections. Exposure to aminoglycosides induces adaptive resistance in Pseudomonas aeruginosa biofilms. Adaptive resistance is primarily the result of active antibiotic export by RND-type efflux pumps, which use the proton motive force as an energy source. We show that the protonophore uncoupler triclosan depletes the membrane potential of biofilm growing P. aeruginosa, leading to decreased activity of RND-type efflux pumps. This disruption results in increased intracellular accumulation of tobramycin and enhanced antimicrobial activity in vitro. In addition, we show that triclosan enhances tobramycin effectiveness in vivo using a mouse wound model. Combining triclosan with tobramycin is a new anti-biofilm strategy that targets bacterial energetics, increasing the susceptibility of P. aeruginosa biofilms to aminoglycosides.Author summaryAdaptive resistance is a phenotypic response that allows P. aeruginosa to transiently survive aminoglycosides such as tobramycin. To date, few compounds have been identified that target adaptive resistance. Here, we show the protonophore uncoupler triclosan disrupts the membrane potential of P. aeruginosa. The depletion of the membrane potential reduces efflux pump activity, which is essential for adaptive resistance, leading to increased tobramycin accumulation and a shorter onset of action. Our results demonstrate that in addition to its canonical mechanism inhibiting membrane biosynthesis, triclosan can exert antibacterial properties by functioning as a protonophore that targets P. aeruginosa energetics.

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Biolog Phenotype Microarray Is a Tool for the Identification of Multidrug Resistance Efflux Pump Inducers

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01263-18 ◽

2018 ◽

Vol 62 (11) ◽

Cited By ~ 8

Author(s):

P. Blanco ◽

F. Corona ◽

J. L. Martínez

Keyword(s):

Multidrug Resistance ◽

Fluorescent Protein ◽

Efflux Pump ◽

Yellow Fluorescent Protein ◽

Efflux Pumps ◽

Phenotype Microarray ◽

Content Type ◽

Environmental Signals ◽

Reverse Transcription Pcr ◽

Biolog Phenotype Microarray

ABSTRACTMultidrug resistance efflux pumps frequently present low levels of basal expression. However, antibiotic-resistant mutants that overexpress these resistance determinants are selected during infection. In addition, increased expression of efflux pumps can be induced by environmental signals/cues, which can lead to situations of transient antibiotic resistance. In this study, we have applied a novel high-throughput methodology in order to identify inducers able to trigger the expression of theStenotrophomonas maltophiliaSmeVWX and SmeYZ efflux pumps. To that end, bioreporters in which the expression of the yellow fluorescent protein (YFP) is linked to the activity of eithersmeVWXorsmeYZpromoters were developed and used for the screening of potential inducers of the expression of these efflux pumps using Biolog phenotype microarrays. YFP production was also measured by flow cytometry, and the levels of expression ofsmeVandsmeYin the presence of a set of selected compounds were also determined by real-time reverse transcription-PCR (RT-PCR). The expression ofsmeVWXwas induced by iodoacetate, clioquinol, and selenite, while boric acid, erythromycin, chloramphenicol, and lincomycin triggeredsmeYZexpression. The susceptibility to antibiotics that are known substrates of the efflux pumps decreased in the presence of the inducers. However, the analyzed multidrug efflux systems did not contribute toS. maltophiliaresistance to the studied inducers. To sum up, the use of fluorescent bioreporters in combination with Biolog plates is a valuable tool for identifying inducers of the expression of bacterial multidrug resistance efflux pumps, and likely of other bacterial systems whose expression is regulated in response to signals/cues.

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