2015b) and that a specific QQ compound will not always be effective against all clinical strains. Also, more effort should be devoted to the study of the implementation of these therapies, since most of them are able to attenuate infections when the QQ compound is administrated shortly before the inoculation (having possible prophylactic effects), but the value of such therapies as a possible cure for an established infection should be better addressed.

Taking the available evidence together, it seems QS interference may be a valuable tool to combat *P. aeruginosa* infections; however, using QS inhibition as the sole therapy may not be an efficient strategy due several potential drawbacks. Hence, before QSI applications are used in the clinic, it is advisable to improve our current understanding of *P. aeruginosa* virulence with an emphasis on clinical strains and also on further defining the roles of QS in the physiology of this remarkable complex pathogen.

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## AUTHOR CONTRIBUTIONS

The author confirms being the sole contributor of this work and approved it for publication.

## FUNDING

This work was supported by grants from SEP/CONACYT-Técnico No. 152794, CONACYT Problemas Nacionales 2015 No. 2015-01-402 and PAPIIT-UNAM No. IA201116.

## ACKNOWLEDGMENTS

I am grateful for critical reading of the manuscript and the valuable suggestions provided by Professor Gloria Soberón-Chávez from the Biomedical Science Institute at UNAM, Mexico and by Professor Thomas K. Wood from Penn State University.

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**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 García-Contreras. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
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CrossMark

# PA3297 Counteracts Antimicrobial Effects of Azithromycin in *Pseudomonas aeruginosa*

Hao Tan¹, Lu Zhang¹, Yuding Weng¹, Ronghao Chen¹, Feng Zhu¹, Yongxin Jin¹, Zhihui Cheng¹, Shouguang Jin¹,²* and Weihui Wu¹*

¹ State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, Department of Microbiology, College of Life Sciences, Nankai University, Tianjin, China, ² Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL, USA

*Pseudomonas aeruginosa* causes acute and chronic infections in human. Its increasing resistance to antibiotics requires alternative treatments that are more effective than available strategies. Among the alternatives is the unconventional usage of conventional antibiotics, of which the macrolide antibiotic azithromycin (AZM) provides a paradigmatic example. AZM therapy is associated with a small but consistent improvement in respiratory function of cystic fibrosis patients suffering from chronic *P. aeruginosa* infection. Besides immunomodulating activities, AZM represses bacterial genes involved in virulence, quorum sensing, biofilm formation, and motility, all of which are due to stalling of ribosome and depletion of cellular tRNA pool. However, how *P. aeruginosa* responds to and counteracts the effects of AZM remain elusive. Here, we found that deficiency of PA3297, a gene encoding a DEAH-box helicase, intensified AZM-mediated bacterial killing, suppression of pyocyanin production and swarming motility, and hypersusceptibility to hydrogen peroxide. We demonstrated that expression of PA3297 is induced by the interaction between AZM and ribosome. Importantly, mutation of PA3297 resulted in elevated levels of unprocessed 23S-5S rRNA in the presence of AZM, which might lead to increased susceptibility to AZM-mediated effects. Our results revealed one of the bacterial responses in counteracting the detrimental effects of AZM.

**Keywords:** RNA helicase, antibiotic resistance, azithromycin, rRNA processing, *Pseudomonas aeruginosa*

**OPEN ACCESS**

**Edited by:**
Etienne Giraud,
Institut National de la Recherche
Agronomique, France

**Reviewed by:**
Francesco Imperi,
Sapienza University of Rome, Italy
Yuji Morita,
Aichi Gakuin University, Japan

*Correspondence:
Weihui Wu
wuweihui@nankai.edu.cn;
Shouguang Jin
sjin@ufl.edu

**Specialty section:**
This article was submitted to *Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology*

Received: 21 December 2015

Accepted: 29 February 2016

Published: 16 March 2016

**Citation:**
Tan H, Zhang L, Weng Y, Chen R, Zhu F, Jin Y, Cheng Z, Jin S and Wu W (2016) PA3297 Counteracts Antimicrobial Effects of Azithromycin in *Pseudomonas aeruginosa*. *Front. Microbiol.* 7:317. doi: 10.3389/fmicb.2016.00317

## INTRODUCTION

*Pseudomonas aeruginosa* is a versatile Gram-negative pathogenic bacterium, that can cause various infections in human (de Bentzmann and Plésiat, 2011; Campa et al., 2012). During infection, *P. aeruginosa* produces multiple virulence factors to facilitate colonization (Sadikot et al., 2005; Kipnis et al., 2006; Hauser, 2009; Liu et al., 2015). Meanwhile, its highly intrinsic antibiotic resistance and biofilm forming ability greatly hinder the eradication of this pathogen (Høiby et al., 2005). In patients suffering from cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD), *P. aeruginosa* caused chronic respiratory infections are responsible for most of the morbidity and mortality (Rabin et al., 2004; Rada and Leto, 2013). Intensive antibiotic treatment has been used to maintain the lung function and extend lifespan of the patients (Doring et al., 2000). However, the increasing antibiotic resistance has been compromising clinical efficacy of traditional antibiotics. Thus, alternatives or unconventional usage of the antibiotics are urgently needed (Breidenstein et al., 2011; Poole, 2011; Imperi et al., 2014).
The macrolide antibiotic azithromycin (AZM) provides a paradigmatic example of an unconventional antibacterial drug for *P. aeruginosa* treatment. Although, *P. aeruginosa* is highly resistance to macrolides owing to its low outer membrane permeability and the resistance-nodulation-cell division (RND) systems, AZM treatment benefits patients suffering from both intermittent and chronic *P. aeruginosa* infections (Saiman et al., 2003; Lister et al., 2009; Blasi et al., 2010; Steel et al., 2012; Aminov, 2013; Morita et al., 2013). AZM has been shown to have immunomodulatory activity, which attenuates the inflammatory response and promotes macrophage phagocytic activity (Legssyer et al., 2006; Steinkamp et al., 2008; Tsai et al., 2009). Furthermore, AZM exhibits bactericidal effect on stationary growth phase *P. aeruginosa* cells (Lovmar et al., 2004, 2009; Imamura et al., 2005; Köhler et al., 2007; Starosta et al., 2010; Gödeke et al., 2013). And sub-inhibitory concentrations of AZM suppress biofilm formation, motility, and production of multiple virulence factors, including proteases, pyocyanin, exotoxin A, phospholipase C (PLC), exopolysaccharides, and other quorum-sensing (QS) regulated genes in *P. aeruginosa* (Molinari et al., 1992, 1993; Tateda et al., 2001; Favre-Bonté et al., 2003; Gillis and Iglewski, 2004). The AZM-mediated killing of stationary-phase bacterial cells and reduced expression of QS-regulated virulence factors require interaction between AZM and ribosome (Köhler et al., 2007). AZM binds in the nascent peptide exit tunnel (NPET), resulting in ribosome stalling and depletion of the intracellular pools of aminoacyl-tRNAs (Lovmar et al., 2004, 2009; Köhler et al., 2007; Starosta et al., 2010; Gödeke et al., 2013). The effects of AZM on *P. aeruginosa* can be counteracted by over expression of ErmBP or a peptidyl-tRNA hydrolase, which blocks the interaction between AZM and ribosome by modifying the 23S rRNA or increases the intracellular aminoacyl-tRNA level, respectively (Köhler et al., 2007; Gödeke et al., 2013). However, how *P. aeruginosa* response to AZM treatment remains unclear. Understanding the mechanisms that *P. aeruginosa* uses to counteract AZM treatment may provide clues to enhance AZM-mediated virulence inhibitory and bacterial killing effects.

A large RNA helicase family named DExD/H box helicases are characterized by a conserved DExD/H box sequence (Cordin et al., 2006; Linder and Jankowsky, 2011), and play crucial roles in rRNA processing, translation initiation, and mRNA decay (Iost et al., 2013; Linder and Fuller-Pace, 2013). In addition, the DExD/H box helicases have been shown to participate in bacterial responses to various stresses, such as cold shock, pH, osmotic, and oxidative stresses (Owttrim, 2013). And several DEAD family RNA helicases, which belong to a specific subfamily of DExD/H box helicases, have been shown to regulate virulence factors in *Escherichia coli*, *Borrelia burgdorferi*, *Staphylococcus aureus*, *Listeria monocytogenes*, and *P. aeruginosa* (Koo et al., 2004; Salman-Dilgimen et al., 2011, 2013; Oun et al., 2013; Bareclev et al., 2014; Intile et al., 2015). The pleiotropic functions of DExD/H box family RNA helicases intrigued us to suspect that they might be involved in the bacterial response to AZM treatment. In this study, we found that deficiency in a DEAH box helicase, PA3297, renders *P. aeruginosa* more susceptible to the killing and virulence suppression by AZM. Our

results suggest that the expression of PA3297 was up regulated in the presence of AZM, which might promote 23S rRNA maturation to counteract the inhibitory effect of AZM on protein elongation.

# MATERIALS AND METHODS

## Strains and Plasmids

The bacterial strains and plasmids used in this study are listed in Table 1 (Simon et al., 1983; Hoang et al., 1998; Choi and Schweizer, 2006; Liberati et al., 2006). The *E. coli* strains DH5α, S17-1 and *P. aeruginosa* strains were routinely cultured in Luria-Bertani (LB) broth at 37°C. Antibiotics were used at the following concentrations: for *E. coli*, ampicillin 100 µg/ml, tetracycline 10 µg/ml, and gentamicin 10 µg/ml; for *P. aeruginosa*, carbenicillin 150 µg/ml, tetracycline 50 µg/ml, and gentamicin 50 µg/ml.

## DNA Methods

DNA manipulations were performed according to standard protocols or following manufacturers' instructions (Hoang et al., 1998; Zheng et al., 2004; Choi and Schweizer, 2006). The pEX18Tc::ΔPA3297 (pTH1501) was constructed by cloning the 1002-bp upstream and 964-bp downstream fragments of PA3297 coding region into the KpnI-HindIII sites of plasmid pEX18Tc. The fragments were amplified from the PA14 chromosome with primers PA14-UPA3297-FF, PA14-UPA3297-FR, PA14-DPA3297-FF, and PA14-DPA3297-FR (Table 2), respectively. Deletion of the PA3297 gene was confirmed by PCR with primers PA14-PA3297-FF and PA14-PA3297-FR (Table 2). For the complementation of PA3297, the PA3297 gene was amplified from the PA14 chromosome by PCR with the primers PA14-PA3297-FF and PA14-PA3297-FR (Table 2). The PCR product was ligated into the EcoRI-SacI sites of pUC18t-mini-Tn7T-Gm, resulting in pTH1502. The plasmid was introduced into the ΔPA3297 mutant by electroporation, along with the helper plasmid pTNS3 (Choi and Schweizer, 2006). Insertion of the PA3297 gene into the chromosome was confirmed by PCR with primers $P_{Tn7R}$ and $P_{glmS-down}$ (Table 2; Choi and Schweizer, 2006). The site-directed mutagenesis was performed as previously described (Zheng et al., 2004). The mutation sites were chosen based on the conserved critical residues of other bacterial DExD-box proteins (Koo et al., 2004; Cordin et al., 2006). Briefly, PCR amplification was performed with pTH1502 as template and with primers listed in Table 2, for K101A, D192A, and SAT224-226AAA mutations, respectively. The PCR products were treated by DpnI for 3 h at 37°C and purified before transformation. The correctly mutated clones were identified by DNA sequencing.

## Assay for Pyocyanin Production

The pyocyanin concentration was determined as described previously (Essar et al., 1990). Briefly, 1 ml supernatant from each 24-h-old bacteria culture grown in the absence or presence of AZM was extracted with 0.5 ml of chloroform. Then, 0.4 ml solution from the lower organic phase was re-extracted into
<table><caption>TABLE 1 | Bacterial strains and plasmids used in this study.</caption><thead><tr><th>Strain or plasmid</th><th>Description<sup>a</sup></th><th>Reference or origin</th></tr></thead><tbody><tr><td colspan="3"><strong>E. coli</strong> strains</td></tr><tr><td>DH5α</td><td>F<sup>-</sup>, φ80dlacZ ΔM15, Δ(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(r<sub>K</sub><sup>-</sup>, m<sub>K</sub><sup>+</sup>), phoA, supE44, λ<sup>-</sup>, thi1, gyrA96, relA1</td><td>TransGen</td></tr><tr><td>S17-1</td><td>thi pro hsdR recA Tra<sup>+</sup></td><td>Simon et al., 1983</td></tr><tr><td colspan="3"><strong>P. aeruginosa</strong> strains</td></tr><tr><td>PA14</td><td>Wild type Pseudomonas aeruginosa strain PA14</td><td>Liberati et al., 2006</td></tr><tr><td>PA0426::Tn</td><td>PA14 with a transposon inserted at PA0426</td><td>Liberati et al., 2006</td></tr><tr><td>PA0455::Tn</td><td>PA14 with a transposon inserted at PA0455</td><td>Liberati et al., 2006</td></tr><tr><td>PA2840::Tn</td><td>PA14 with a transposon inserted at PA2840</td><td>Liberati et al., 2006</td></tr><tr><td>PA3002::Tn</td><td>PA14 with a transposon inserted at PA3002</td><td>Liberati et al., 2006</td></tr><tr><td>PA3272::Tn</td><td>PA14 with a transposon inserted at PA3272</td><td>Liberati et al., 2006</td></tr><tr><td>PA3297::Tn</td><td>PA14 with a transposon inserted at PA3297</td><td>Liberati et al., 2006</td></tr><tr><td>PA3308::Tn</td><td>PA14 with a transposon inserted at PA3308</td><td>Liberati et al., 2006</td></tr><tr><td>PA3861::Tn</td><td>PA14 with a transposon inserted at PA3861</td><td>Liberati et al., 2006</td></tr><tr><td>PA3950::Tn</td><td>PA14 with a transposon inserted at PA3950</td><td>Liberati et al., 2006</td></tr><tr><td>ΔPA3297</td><td>PA14 with PA3297 in frame deletion</td><td>This study</td></tr><tr><td>ΔPA3297/att7::PA3297</td><td>PA14 ΔPA3297 with insertion of a single copy of PA3297 driven by its own promoter at attTn7 sites</td><td>This study</td></tr><tr><td>ΔPA3297/att7::PA3297 K101A</td><td>PA14 ΔPA3297 complemented with a single copy of PA3297 acquired a lysine101 mutation to alanine</td><td>This study</td></tr><tr><td>ΔPA3297/att7::PA3297 D192A</td><td>PA14 ΔPA3297 complemented with a single copy of PA3297 acquired an aspartate192 mutation to alanine</td><td>This study</td></tr><tr><td>ΔPA3297/att7::PA3297SAT224AAA</td><td>PA14 ΔPA3297 complemented with a single copy of PA3297 acquired a serine224 and threonine226 mutation to alanines</td><td>This study</td></tr><tr><td colspan="3"><strong>Plasmids</strong></td></tr><tr><td>pEX18Tc</td><td>Broad-host-range gene replacement vector; sacB TET<sup>r</sup></td><td>Hoang et al., 1998</td></tr><tr><td>pUC18t-mini-Tn7T-Gm</td><td>For gene insertion in chromosome; GEN<sup>r</sup></td><td>Choi and Schweizer, 2006</td></tr><tr><td>pTNS3</td><td>Helper plasmid</td><td>Choi and Schweizer, 2006</td></tr><tr><td>pFLP2</td><td>Source of Flp recombinase; sacB, AMP<sup>r</sup>/CAR<sup>r</sup></td><td>Hoang et al., 1998</td></tr><tr><td>pTH1501</td><td>pEX18Tc::ΔPA3297; TET<sup>r</sup></td><td>This study</td></tr><tr><td>pTH1502</td><td>PA3297 gene of PA14 on pUC18T-Mini-Tn7T-Gm with its own promoter; GEN<sup>r</sup></td><td>This study</td></tr></tbody></table>

0.3 ml of 0.2 N HCl to give a pink solution, whose absorbance was measured at 520 nm. Concentrations of pyocyanin (mg/ml) were calculated by multiplying the OD<sub>520</sub> by 32.01 (Kurachi, 1958).

## Antibiotic Susceptibility Assay

Minimum inhibitory concentrations (MICs) of *P. aeruginosa* to antimicrobial agents were determined by serial twofold broth dilution in LB medium, as described previously (Jo et al., 2003). MICs were recorded as the lowest concentration of antibiotic inhibiting visible growth after 24 h of incubation at 37°C.

## Stationary-Phase Bacterial Cell Killing Assay

The killing assay was performed as described previously (Köhler et al., 2007). Briefly, bacteria were inoculated in LB medium and grown for 16 h at 37°C. The culture of each strain was diluted to an OD<sub>600</sub> of 0.05 and cultured at 37°C. After reaching stationary phase (OD<sub>600</sub>, 3.0), indicated concentrations of AZM were added to the 2-ml aliquots of the cultures. Then the bacteria were cultured for 20–22 h at 37°C. The viable bacterial numbers were determined by serial dilution and plating on drug-free LB agar plates.

The survival rate of each strain was calculated as live bacterial number in AZM treated sample divided by the bacterial number of the corresponding untreated sample.

## Biofilm Tolerance to AZM

The biofilm resistance was measured as previously described with minor modifications (Bjerkan et al., 2009; Billings et al., 2013; Liao et al., 2013). Briefly, overnight bacterial cultures were diluted to an OD<sub>600</sub> of 0.025. 150 µl of the bacteria were incubated in each well of a 96-well plate at 37°C without agitation (Brencic et al., 2009). After 24 h, the planktonic bacteria were discarded by aspiration. Then, the biofilms were treated with 150 µl LB medium containing indicated concentrations of AZM for 2 h. The medium in each well was replaced with fresh LB medium, and subjected to sonication at a frequency of 40 kHz, with a power output of 300 W, at 37°C for 5 min. The live bacteria were enumerated by serial dilution and plating.

## Growth Assay

Overnight culture of each strain was diluted into fresh LB (150 µl) to an OD<sub>600</sub> of 0.05 in each well of a 96-well plate without or

<footnote><sup>a</sup>AMP<sup>r</sup>, ampicillin resistant; TET<sup>r</sup>, tetracycline resistant; GEN<sup>r</sup>, gentamicin resistant; CAR<sup>r</sup>, carbenicillin resistant.</footnote>
<table><caption>TABLE 2 | Primers used in this study.</caption><thead><tr><th>Primers<sup>a</sup></th><th>Nucleotide sequence (5'→3')<sup>b</sup></th></tr></thead><tbody><tr><td colspan="2"><strong>Cloning of upstream and downstream fragments for PA3297 deletion</strong></td></tr><tr><td>PA14-UPA3297-FF</td><td>GAAAGCGGTACCGAAGTAAGTCCGCCGCGTTGCC (Kpn I)</td></tr><tr><td>PA14-UPA3297-FR</td><td>CAGCTTTCTAGAGGTGCTGTCGTCGCTCTGGT (Xba I)</td></tr><tr><td>PA14-DPA3297-FF</td><td>TTGCAGTCTAGACGCTGGATGCTGGAGGAGTA (Xba I)</td></tr><tr><td>PA14-DPA3297-FR</td><td>CGCCGGAAGCTTCACCGAGCAGTGGCTGAAGAC (Hind III)</td></tr><tr><td colspan="2"><strong>Cloning of gene PA3297 for complementation</strong></td></tr><tr><td>PA14-PA3297-FF</td><td>TGAAGAGAATTCGCCAGAAGTAAGTCCGCCGCGTTGCC (EcoR I)</td></tr><tr><td>PA14-PA3297-FR</td><td>CACCGGGAGCTCCGACCAGACCGACCTGTTCTTCA CCAT (Sac I)</td></tr><tr><td>P<sub>Tn7R</sub></td><td>CACAGCATAACTGGACTGATTTCT</td></tr><tr><td>P<sub>glmS</sub>-down</td><td>GCACATCGCGACGTCGCTCTC</td></tr><tr><td colspan="2"><strong>Primers used for site-directed mutagenesis of PA3297</strong></td></tr><tr><td>PA3297-K101A-FF</td><td>GCGAGACCGGCTCGGGCGCGACCACCCAG</td></tr><tr><td>PA3297-K101A-FR</td><td>TCGCCGCCGAGCCGGTCTCGCCGGCGATC</td></tr><tr><td>PA3297-D192A-FF</td><td>TACGACACGCTGATCGTCGCCGAAGCCCAC</td></tr><tr><td>PA3297-D192A-FR</td><td>CGCGACGATCAGCGTGTCGTAGCGCTCCAG</td></tr><tr><td>PA3297-SAT224-226AAA-FF</td><td>GCTGATCATCACCGCGCGCGCCATCGACCTGGAG</td></tr><tr><td>PA3297-SAT224-226AAA-FR</td><td>TGCCGCCGCCGGGTGATGATCAGCTTCAGGTCC</td></tr><tr><td colspan="2"><strong>RT-qPCR primers for gene expression measurements</strong></td></tr><tr><td>23S-R-FF</td><td>AAAGATAACCGCTGAAAG</td></tr><tr><td>23S-R-FR</td><td>CTATCAACGTCGTAGTCT</td></tr><tr><td>5S-R-FF</td><td>CGAACTCAGAAGTGAAAC</td></tr><tr><td>5S-R-FR</td><td>CTTGACGATGACCTACTC</td></tr><tr><td>23S-5S-R-FF</td><td>GTACTAATTGCCCGTGAG</td></tr><tr><td>23S-5S-R-FR</td><td>GTTCCAACGCTCTATGAT</td></tr></tbody></table>

with different concentrations of AZM. The plate was incubated at 37°C with constant agitation (Lau et al., 2012; Guénard et al., 2014). The bacterial growth was monitored by measuring the OD<sub>600</sub> every 30 min for 12 h by a Varioskan Flash microplate reader (Thermo Electron Corporation).

## H<sub>2</sub>O<sub>2</sub> Susceptibility Assay

Overnight cultures of the *P. aeruginosa* strains were diluted to an OD<sub>600</sub> of 0.05 and cultured at 37°C. When the OD<sub>600</sub> reached 0.3 (about 1.5–2 h later), AZM (0.5 µg/ml) was added if needed. When the OD<sub>600</sub> reached 2.0 (about 3 h later), bacteria from 500 µl culture were collected and washed twice with phosphate buffered saline (PBS). Then the bacteria were resuspended in PBS with or without 10% H<sub>2</sub>O<sub>2</sub> and incubated for 15 min. The live bacterial numbers were determined by serial dilution and plating.

## Motility Assay

The swarming motility was tested on modified M9 medium plates supplemented with 0.2% glucose, 1 mM MgSO<sub>4</sub>, and 0.05% glutamate as the nitrogen source. 0.5% agar was used

for solidification. Two microliters of exponential growth phase *P. aeruginosa* was deposited on the plates, then incubated for 18 h at 37°C (Köhler et al., 2007).

## Total RNA Isolation and Quantitative Real-Time PCR

Overnight cultures of *P. aeruginosa* strain PA14 and ΔPA3297 were diluted into fresh LB medium to an OD<sub>600</sub> of 0.05. The bacteria were grown at 37°C with agitation (200 rpm). When the OD<sub>600</sub> reached 0.3 (about 1.5–2 h later), AZM was added to reach indicated concentrations. Samples were harvested when the OD<sub>600</sub> reached 2.0 (about 3 h later; Skindersoe et al., 2008). Total RNA was isolated with an RNeasy Minikit (Tiangen Biotech). The cDNA from each RNA sample was synthesized with reverse transcriptase and random primers (Takara). Real-time PCR was performed with SYBR premix Ex Taq (Roche). The conserved hypothetical protein coding gene PA1769 was used as an internal control (Son et al., 2007). The primers used in quantitative real-time PCR were listed in Table 2, with a designation of “RT-qPCR.”

## Statistical Analysis

When indicated, Student's *t*-test (two-tailed) was used to determine whether the deletion of PA3297 resulted in any significant differences compared to the wild-type cells treated with the same concentrations of AZM.

## RESULTS

### Deficiency of PA3297 Intensifies the Effects of AZM on Pyocyanin Production

As pyocanin production is suppressed by AZM (Molinari et al., 1992, 1993; Tateda et al., 2001; Favre-Bonté et al., 2003; Gillis and Iglewski, 2004), which can be easily observed and quantified, we used this phenotype to test whether DExD/H box RNA helicases are involved in bacterial response to AZM treatment. There are 17 DExD/H box RNA helicases in the genome of *P. aeruginosa* strain PA14 (www.pseudomonas.com; Winsor et al., 2011). There are nine DExD/H box helicase mutants in the non-redundant PA14 transposon mutants library, however, the other eight DExD/H box helicase mutants are not available (Table 1; Liberati et al., 2006; Breidenstein et al., 2011). Thus, we examined pyocyanin production of the available mutants in the absence and presence of AZM at an OD<sub>600</sub> of 2.0. A PA3297::Tn mutant displayed a significant decrease in pyocyanin production in the presence of AZM, whereas no difference was observed in the absence of AZM (Figure 1A). To confirm the role of PA3297, we generated an in frame deletion of PA3297 in PA14. The wild type PA14 and the ΔPA3297 mutant were grown in the absence or presence of 2, 5, or 10 µg/ml AZM. When the bacteria reached same density (OD<sub>600</sub> of 2.0), we measured the pyocyanin levels. AZM inhibited the production of pyocyanin in the wild type strain in a dose dependent manner. However, the production of pyocyanin by the mutant was repressed more severely in the presence of AZM.

<footnote><sup>a</sup>The direction of the primer is indicated at the end of the primer designation as follows: FF for forward and FR for reverse. <sup>b</sup>The solid underlines are the sites of listed restriction enzymes; the dotted underlines are the sites for mutagenesis, with all the residues changed into alanine.</footnote>
<figure><img src="image_70.png" /></figure>

**FIGURE 1** | Mutation of PA3297 intensified the AZM mediated inhibition of pyocyanin production. (A) Wild type PA14 and the mutants of DExD/H box helicases were grown to an OD₆₀₀ of 2.0 in the absence or presence of 5 µg/ml AZM at 37°C and the pyocyanin concentrations were measured. (B) Bacteria of wild type PA14, ΔPA3297 mutant and the complementation strain were grown in the absence or presence of 2, 5, or 10 µg/ml AZM. When the OD₆₀₀ reached 2.0, the pyocyanin concentrations were measured. The values are the means of three replicates and the error bars display the standard deviations. “ND” standards for “not detected.” *p* < 0.05 compared to PA14 or the complemented strain by student's t-test.

at all the tested concentrations (Figure 1B). Complementation with a PA3297 gene restored the production of pyocyanin (Figure 1B).

## Increased Killing of the ΔPA3297 Mutant by AZM

Stationary growth phase *P. aeruginosa* cells are susceptible to AZM (Lovmar et al., 2004, 2009; Imamura et al., 2005; Köhler et al., 2007; Starosta et al., 2010; Gödeke et al., 2013). To test whether mutation of PA3297 renders higher susceptibility, we performed the stationary-phase cells killing assay as previously

<figure><img src="image_71.png" /><figcaption>**FIGURE 2** | Increased killing of the ΔPA3297 mutant by AZM.</figcaption></figure>

(A) AZM-mediated killing of wild type PA14, the ΔPA3297 mutant and the mutant complemented with wild type or the mutated PA3297. Stationary-phase bacteria were treated with 2, 5, or 10 µg/ml AZM for 20 h at 37°C. The numbers of live bacteria were determined by serial dilution and plating. (B) AZM-mediated killing of biofilm. 24-h-old biofilms formed by wild type PA14, the ΔPA3297 mutant and the complemented strain were treated with 5 µg/ml, 10 µg/ml AZM or not for 2 h. Bacteria in the biofilm were dissociated from the wells by gentle sonication. The viable bacteria were determined by serial dilution and plating. The averages and associated standard deviations from three replicates are shown. *p* < 0.05 compared to PA14 or the complemented strain by student's t-test.

described (Imamura et al., 2005; Köhler et al., 2007; Gödeke et al., 2013). Stationary-phase cells of PA14, the ΔPA3297 mutant and the complementation strain were subjected to treatment with AZM at the concentrations of 2, 5, and 10 µg/ml. As shown in Figure 2A, the AZM-mediated killing of the ΔPA3297 mutant was significantly increased at all of the AZM concentrations tested. The most significant difference was observed at the concentration of 10 µg/ml, where the survival rate of the mutant was approximately 1% of those of the wild type and complemented strains. In addition, the ΔPA3297 mutant was more susceptible to another macrolid antibiotic, erythromycin, with a fourfold lower MIC compared to the wild type strain PA14 (Table 3).

Next, we tested the susceptibility of the ΔPA3297 mutant to lincosamides, whose bactericidal mechanism is similar to
macrolides (Tenson et al., 2003; Wilson, 2014). Indeed, mutation in PA3297 increased the bacterial susceptibility to lincomycin and clindamycin (Table 3). However, no increase of susceptibility was observed to other antibiotics, including ciprofloxacin, carbenicillin, meropenem, tetracycline, tobramycin, kanamycin, chloramphenicol, or polymyxin B (Supplementary Table S1). These results suggest that PA3297 plays an important role in the resistance against antibiotics targeting the peptide exit tunnel of ribosome.

PA3297 is a putative RNA helicase belonging to the DEAH-box family proteins, which are characterized by the presence of seven to nine conserved motifs (Tanner and Linder, 2001). In the *E. coli* RNA helicases DbpA and HrpA, the conserved residues GETGSGKT in motifI, DEAH in motifII, and SAT in motifIII have been shown to be required for interaction with and hydrolysis of NTP (Koo et al., 2004; Linder and Fuller-Pace, 2013). To determine whether these critical residues within motifs I, II, and III are important for PA3297 in the resistance to macrolides and lincosamides, we altered the residues by site-directed mutagenesis. Specifically, the K101 in motif I or the D167 in motif II was mutated to alanine. The S224 and T226 in motif III were both replaced with alanine. Each mutated PA3297 was transferred into the ΔPA3297 mutant and the susceptibility to antibiotics was tested. None of the mutated PA3297 was able to restore the survival rate of the mutant (Figure 2A). In addition, the mutated PA3297 was unable to restore the resistance of the ΔPA3297 mutant to macrolides, lincomycin, and clindamycin (Table 3). These results suggest that the RNA helicase function of PA3297 is required for its role in the resistance to macrolides and lincosamides.

Clinically, AZM has been used in the treatment of chronic *P. aeruginosa* infection (Saiman et al., 2003; Blasi et al., 2010), which is characterized by biofilm formation (Singh et al., 2000). The biofilm is notorious for high antibiotic tolerance, which severely hinders eradication of the bacteria (López et al., 2010; Breidenstein et al., 2011). We suspected that mutation of PA3297 might increase the killing efficacy of AZM on the biofilm. Indeed, the survival rates of the ΔPA3297 mutant in biofilm were lower than those of the wild type PA14 at various AZM concentrations, which were restored by complementation with a wild type PA3297 (Figure 2B).

<table><caption>TABLE 3 | Bacterial susceptibilities to macrolides and lincosamides.</caption><thead><tr><th rowspan="2">Strain</th><th colspan="4">MIC (µg/ml)<sup>a</sup></th></tr><tr><th>ERY</th><th>AZM</th><th>LIN</th><th>CLI</th></tr></thead><tbody><tr><td>PA14</td><td>300</td><td>400</td><td>12800</td><td>4800</td></tr><tr><td>ΔPA3297</td><td>75</td><td>100</td><td>3200</td><td>2400</td></tr><tr><td>ΔPA3297/att7::PA3297</td><td>300</td><td>400</td><td>>12800</td><td>9600</td></tr><tr><td>ΔPA3297/att7::PA3297 K101A<sup>b</sup></td><td>75</td><td>100</td><td>6400</td><td>1600</td></tr><tr><td>ΔPA3297/att7::PA3297 D192A<sup>b</sup></td><td>75</td><td>100</td><td>3200</td><td>2400</td></tr><tr><td>ΔPA3297/att7::PA3297 SAT224AAA<sup>b</sup></td><td>75</td><td>100</td><td>6400</td><td>2400</td></tr></tbody></table>

## Mutation of PA3297 Increases the Bacterial Susceptibility to Hydrogen Peroxide in the Presence of AZM

It has been shown that AZM treatment impairs the oxidative stress response in *P. aeruginosa* (Nalca et al., 2006), which intrigued us to test whether mutation of PA3297 leads to further impairment. The $H_2O_2$ susceptibility assay was performed in the presence of 0.5 µg/ml AZM, as the growth rates of wild type PA14 and the ΔPA3297 mutant were similar at this concentration of AZM (Supplementary Figure S1). The wild type PA14, ΔPA3297 mutant and the complemented strain were grown without or with AZM to an OD$_{600}$ of 2.0, and then treated with 10% $H_2O_2$ for 15 min. In the presence of AZM, the ΔPA3297 mutant was more susceptible to $H_2O_2$ than the wild type and complemented strains, whereas no difference was observed in the absence of AZM (Figure 3). These results suggest that PA3297 is involved in the bacterial oxidative stress response in the presence of AZM.

## Mutation of PA3297 Intensifies the Inhibitory Effect of AZM on Swarming Motility

Besides oxidative stress response, AZM suppresses swarming motility (Tateda et al., 2001; Köhler et al., 2007; Gödeke et al., 2013). Same numbers of wild type PA14 and the ΔPA3297 mutant were inoculated on the plates containing various concentrations of AZM. As shown in Figure 4, 10 µg/ml AZM suppressed the swarming motility of wild type PA14 obviously, whereas the lower concentrations of AZM showed no inhibitory effect. However, starting from 2 µg/ml, AZM suppressed the swarming motility of the ΔPA3297 mutant in a dose dependent manner. Complementation with a PA3297 gene restored the swarming motility in the presence of AZM (Figure 4).

<figure><img src="image_72.png" /><figcaption>FIGURE 3 | Hydrogen peroxide ($H_2O_2$) mediated killing of bacteria.</figcaption></figure>

PA14, the ΔPA3297 mutant and complemented strain were grown at 37°C in the absence or presence of 0.5 µg/ml AZM to an OD$_{600}$ of 2.0. Bacteria were collected and washed with PBS. Then the bacteria were incubated in PBS with or without 10% $H_2O_2$ for 15 min. The live bacterial numbers were determined by serial dilution and plating. *p* < 0.05 compared to PA14 or the complemented strain by student's t-test.

<footnote><sup>a</sup>ERY, erythromycin; AZM, azithromycin; LIN, lincomycin; CLI, clindamycin.</footnote>

<footnote><sup>b</sup>Mutants with single or triple amino acid changes.</footnote>
<figure><img src="image_73.png" /><figcaption>**FIGURE 4** | Bacterial swarming motility. PA14 (i), the ΔPA3297 mutant (ii) and the complemented strain (iii) were inoculated on plates containing indicated concentrations of AZM.</figcaption></figure>

## Interaction between AZM and Ribosome Induces the Expression of PA3297

Our results so far suggested that PA3297 is involved in the bacterial response to AZM treatment. To test whether the expression of PA3297 is induced by AZM treatment, wild type PA14 was grown in the absence or presence of AZM at various concentrations and the relative RNA levels of PA3297 were determined by real time PCR. Indeed, the expression of PA3297 was induced by AZM (Figure 5A). However, overexpression of PA3297 in wild type PA14 did not further increase the bacterial

<table><caption>TABLE 4 | Bacterial susceptibilities to macrolides and lincosamides.</caption><thead><tr><th rowspan="2">Strain</th><th colspan="4">MIC (µg/ml)<sup>a</sup></th></tr><tr><th>ERY</th><th>AZM</th><th>LIN</th><th>CLI</th></tr></thead><tbody><tr><td>PA14/pMMB67EH</td><td>600</td><td>200</td><td>12800</td><td>12800</td></tr><tr><td>PA14/pMMB67-PA3297</td><td>600</td><td>200</td><td>12800</td><td>6400</td></tr><tr><td>PA14/pMMB67-ermC</td><td>2400</td><td>800</td><td>&gt;12800</td><td>&gt;12800</td></tr><tr><td>ΔPA3297/pMMB67EH</td><td>75</td><td>50</td><td>6400</td><td>3200</td></tr><tr><td>ΔPA3297/pMMB67-PA3297</td><td>300</td><td>200</td><td>12800</td><td>3200</td></tr><tr><td>ΔPA3297/pMMB67-ermC</td><td>2400</td><td>400</td><td>&gt;12800</td><td>&gt;12800</td></tr></tbody></table>

tolerance to AZM (Table 4). We suspect that since the bacteria at exponential growth phase are highly resistant to macrolides and lincosamides, overexpression of PA3297 might not further increase the resistance significantly in the MIC test. Another possibility is that with the endogenous up regulation of PA3297, additional expression of PA3297 might be redundant.

Köhler et al. (2007) previously demonstrated that ribosome is the only target of AZM in bacteria. To test whether the induction of PA3297 is caused by the interaction between AZM and ribosome, we performed the ribosomal protection assay by overexpressing ErmC, a 23S rRNA methylase that blocks the binding of macrolide antibiotics to the NPET (Köhler et al., 2007; Lawrence et al., 2008). In the presence of 10 µg/ml AZM, the growth speed of the ErmC overexpressing strain was similar with that of the wild type strain containing an empty vector. However, overexpression of ErmC abolished the induction of PA3297 by AZM (Figure 5B). In addition, antibiotics in the other categories, including ciprofloxacin, tobramycin, and carbenicillin did not affect the expression level of PA3297 (Supplementary Figure S2). These results suggest that the expression of PA3297 is regulated in response to AZM-mediated ribosome stalling.

## Ribosome Protection Rescues the ΔPA3297 Mutant from AZM-Mediated Hyperlethality

So far, we have demonstrated that the expression of PA3297 is induced by AZM and that mutation of PA3297 renders *P. aeruginosa* hypersusceptible to AZM. These results suggest that PA3297 might play a role in counteracting the detrimental effects caused by the interaction between AZM and ribosome (Köhler et al., 2007; Gödeke et al., 2013). Thus, ribosome protection should be able to increase the tolerance of the ΔPA3297 mutant to AZM. Indeed, overexpression of ErmC increased both the growth speed and the survival rate of the ΔPA3297 mutant when treated with 10 or 40 µg/ml AZM (Supplementary Figure S3, Figure 6). In addition, overexpression of ErmC increased the MICs of both wild type PA14 and the ΔPA3297 mutant to AZM, erythromycin, lincomycin and clindamycin (Table 4). Mutation of PA3297 did not increase the bacterial susceptibility to a variety of other antibiotics (Supplementary Table S1). These results suggest a specific role of PA3297 in responding to lincosamides and macrolides.

<figure><img src="image_74.png" /></figure>

<figure><img src="image_75.png" /><figcaption>**FIGURE 5** | Expression of PA3297 in the presence of AZM. (A) Wild type PA14 was grown to an OD<sub>600</sub> of 0.3 in LB medium. The bacteria were grown further in the absence or presence of 2, 5, or 10 µg/ml AZM. When the OD<sub>600</sub> reached 2.0, total bacterial RNA was isolated and the mRNA levels of PA3297 were determined with real time PCR. *p* < 0.05 compared to bacteria in the absence of presence of 2 or 5 µg/ml AZM by student's t-test. (B) PA14 harboring pMMB67EH or the ErmC over expressing plasmid (pMMB67EH-ermC) was grown at 37°C in the absence of AZM. When the OD<sub>600</sub> reached 0.3, 1 mM IPTG was added to the medium. Meanwhile, no AZM or AZM at the final concentration of 10 µg/ml was added to the medium. At the OD<sub>600</sub> of 2.0, total RNA was isolated and the mRNA levels of PA3297 were determined with real time PCR. The mRNA levels of lacI from the plasmid were used as internal control. *p* < 0.05 compared to PA14/pMMB67EH by student's t-test.</figcaption></figure>

<footnote><sup>a</sup>ERY, erythromycin; AZM, azithromycin; LIN, lincomycin; CLI, clindamycin.</footnote>
<figure><img src="image_76.png" /><figcaption>**FIGURE 6** | Effects of Ribosome protection on the AZM mediated killing of the ΔPA3297 mutant. Wild type PA14 or the ΔPA3297 mutant harboring pMMB67EH or pMMB67EH-*ermC* was grown to an OD~600~ of 0.5 when 1 mM IPTG was added to the medium. When the OD~600~ reached 2.0, the bacteria were treated without or with 10 or 40 µg/ml AZM for 20 h at 37°C. The numbers of live bacteria were determined by serial dilution and plating. \*p < 0.05 compared to the ΔPA3297 mutant harboring pMMB67EH by student's *t*-test.</figcaption></figure>

## Deficiency in PA3297 Compromises rRNA Processing in the Presence of AZM

Studies in *E. coli* demonstrated that the DExD/H box play crucial roles in rRNA processing (Iost et al., 2013; Linder and Fuller-Pace, 2013). Therefore, we suspected that the up regulated PA3297 might participate in rRNA maturation, which facilitates ribosome biogenesis to compensate for AZM inactivated ribosome. The rRNA coding region in the chromosome of *P. aeruginosa* PA14 is shown in Supplementary Figure S4. To examine the processing of the rRNA transcript, we designed real-time PCR primers to analyze the total 23S and 5S rRNA levels as well as primers across the 23S and 5S rRNA coding region to analyze the level of unprocessed rRNA (Supplementary Figure S4). In wild type PA14, AZM at the concentrations of 5 and 10 µg/ml reduced the 23S and 5S rRNA levels (Figures 7A,B), and 10 µg/ml AZM slightly increased the unprocessed 23S-5S rRNA level (Figure 7C). In the absence of AZM, the total 23S and 5S rRNA levels were higher in the ΔPA3297 mutant than those in the wild type strain (Figures 7A,B), whereas the unprocessed 23S-5S levels were similar between the mutant and wild type strain. Interestingly, treatment with 10 µg/ml AZM resulted in a higher level of unprocessed 23S-5S rRNA in the ΔPA3297 mutant (Figure 7C), although its total 23S and 5S rRNA levels were lower than those in the wild type strain (Figures 7A,B).

Next, we calculated the percentages of unprocessed 23S-5S rRNA in wild type PA14 and the ΔPA3297 mutant with or without AZM treatment. Standard curves were generated to determine the amplification efficiencies of the primer pairs for the detection of total 23S, 5S and unprocessed 23S-5S rRNA levels in real time PCR (Supplementary Figure S5). Considering 5S rRNA is more prone to be lost during RNA purification, we calculated the ratio of unprocessed 23S-5S rRNA by dividing the levels of 23S-5S rRNA by those of 23S rRNA calibrated with the amplification efficiencies (Table 5). In the absence of AZM, the

<figure><img src="image_77.png" /></figure>

<figure><img src="image_78.png" /></figure>

<figure><img src="image_79.png" /><figcaption>**FIGURE 7** | Effects of AZM on the processing of rRNA. PA14, the ΔPA3297 mutant and the complemented strain were grown at 37°C in the absence of AZM. When the OD~600~ reached 0.3, AZM (0, 5, and 10 µg/ml) was added. Total RNA was harvested when the OD~600~ reached 2.0. The levels of total 23S (A), 5S (B), and unprocessed 23S-5S rRNA (C) were determined by real time PCR. The mRNA levels of PA1769 were used as an internal control. \*p < 0.05 compared to PA14 by student's *t*-test.</figcaption></figure>

ratios of unprocessed 23S-5S rRNA were approximately 0.014 and 0.009% in wild type PA14 and the ΔPA3297 mutant, respectively. It seems that, without PA3297, the processing of 23S-5S rRNA is even more efficient. In the presence of 5 and 10 µg/ml AZM, the ratios of unprocessed 23S-5S rRNA rose to approximately 0.05 and 0.09% in wild type PA14, respectively. However, the ratios of unprocessed 23S-5S rRNA in the ΔPA3297 mutant were 0.33 and 1.61%, which were approximately 5- and 18-fold higher than those in PA14 under the same condition (Table 5).

Since, the growth speed of the ΔPA3297 mutant in the presence of 10 µg/ml AZM was similar as that of the wild type PA14 in the presence of 40 µg/ml AZM (Supplementary Figure S3), we compared the rRNAs levels of the two strains
<table><caption>TABLE 5 | The ratio of unprocessed 23S-5S rRNA in total 23S rRNA (%).</caption><thead><tr><th rowspan="2">Strain</th><th colspan="3">AZM (µg/ml)</th></tr><tr><th>0</th><th>5</th><th>10</th></tr></thead><tbody><tr><td>PA14</td><td>0.014</td><td>0.051</td><td>0.089</td></tr><tr><td>ΔPA3297</td><td>0.009</td><td>0.335</td><td>1.608</td></tr></tbody></table>

The unprocessed 23S-5S rRNA ratios were calculated according to the equation: Ratio = (1+e<sup>23S</sup>)<sup>C<sub>t</sub>(23S)</sup>/(1+e<sup>23S-5S</sup>)<sup>C<sub>t</sub>(23S-5S)</sup>; e: Amplification efficiency. C<sub>t</sub>: Cycles when fluorescence intensity reaches detection threshold.

grown under the two conditions. The relative levels of 23S-5S rRNA, total 5S and 23S rRNA in the ΔPA3297 mutant were 150, 55, and 100% of those in the wild type PA14, respectively, indicating a similar rRNA processing status with the two different AZM concentrations. In combination, these results suggest that PA3297 might contribute to rRNA processing in response to AZM. And mutation of PA3297 might impair the biosynthesis of ribosome under AZM treatment, which renders the bacterium more susceptible to AZM.

## DISCUSSION

By binding to 23S rRNA in the 50S subunit of bacterial ribosome, AZM blocks polypeptide elongation and diminishes the intracellular pools of aminoacyl-tRNAs (Tenson et al., 2003; Gödeke et al., 2013; Wilson, 2014). And it has been demonstrated that AZM reduces the expression of *gacA* and the small RNAs *rsmY* and *rsmZ*, as well as quorum sensing genes (Kai et al., 2009; Pérez-Martínez and Haas, 2011). And the stationary phase killing by sub-MIC AZM was demonstrated to be correlated with increased outer membrane permeability (Imamura et al., 2005). Here, we found that mutation of PA3297 intensified the AZM mediated inhibitory effects on pyocyanin production and swarming motility of *P. aeruginosa*. In addition, the PA3297 mutant is more susceptible to oxidative stress in the presence of AZM. During infection, host generated reactive oxygen species (ROS) is an important bacterial killing mechanism. Therefore, inhibition of PA3297 together with the treatment with AZM, might render the bacteria more susceptible to host killing.

The MIC of the ΔPA3297 mutant is a quarter of that of the wild type strain. Consistently, the growth speed of the ΔPA3297 mutant in the presence of 10 µg/ml AZM was similar as that of the wild type strain in the presence of 40 µg/ml AZM (Supplementary Figure S3). However, in the stationary phase cell killing assay, the survival rate of the ΔPA3297 mutant treated with 10 µg/ml AZM was approximately 20% of that of the wild type strain treated with 40 µg/ml AZM (Figure 6). Therefore, PA3297 might play a more important role in the survival of stationary phase cells under AZM treatment.

Macrolides and lincosamides bind to the 50S of ribosome and block the NPET (Tenson et al., 2003). In our experiments, mutation in PA3297 did not alter the bacterial resistance to other antibiotics, including those binding to 30S or other parts of 50S ribosome. In addition, the expression of PA3297 was upregulated

by AZM, which was abolished by ribosome protection. Therefore, PA3297 might specifically play a role in bacterial response to macrolides and lincosamides.

PA3297 is also named HrpA, both in PAO1 and PA14 (Winsor et al., 2016). According to the Profiles from GEO Expression Database at NCBI, the expression level of PA3297 was higher in biofilm than that in planktonic cells (Anderson et al., 2008). Isolates from CF lungs displayed higher expression levels of PA3297 levels than PAO1 (Son et al., 2007; Bielecki et al., 2013). And artificial medium that mimics CF lung sputum could increase the expression level of PA3297 slightly (Fung et al., 2010). In addition, increase of PA3297 expression was also observed in antibiotic-resistant small colony variants (Wei et al., 2011). However, the expression level of PA3297 showed a significant decrease in response to airway epithelia or low oxygen conditions (Alvarez-Ortega and Harwood, 2007; Chugani and Greenberg, 2007). These results indicate that the expression of PA3297 is regulated in response to various environmental stresses.

PA3297 is predicted to locate at the cytoplasmic membrane, with a molecular weight of 149.8 kDa (Winsor et al., 2016). It belongs to the DExD/H box helicase family. Members of this family have been found to play crucial roles in RNA metabolism and gene regulation (Linder and Jankowsky, 2011; Iost et al., 2013; Kaberdin and Bläsi, 2013; Linder and Fuller-Pace, 2013; Putnam and Jankowsky, 2013). According to NCBI protein blast, there are 81 homologous proteins with identities of 78% or more in other microorganisms. The homolog of PA3297 in *Borrelia burgdorferi* was found to be required for mouse infectivity and tick transmission and involved global gene regulation (Salman-Dilgimen et al., 2011; Owttrim, 2013). Another homolog in *E. coli* was found to be involved in fimbrial biogenesis (Koo et al., 2004). The identities they shared with PA3297 are 34 and 49%, respectively. Both of them possess the conserved residues in motif I, motif II, and motif III. Meanwhile, they are also involved in RNA processing. In *Listeria monocytogenes*, it has been reported that defect of a DExD-box RNA helicase, Lmo1722, reduced the maturation of 23S RNA (Bareclev et al., 2014) at low temperatures.

In *E. coli*, the primary transcript rRNA is cleaved by RNase III, yielding precursors of the 16S rRNA (17S rRNA), 23S rRNA, and 5S rRNA (9S rRNA; Shajani et al., 2011). Mutation of RNase III led to slower growth rate, reduction in cell viability and protein synthesis rates in the presence of AZM (Silvers and Champney, 2005). In *E. coli*, under certain stress conditions, DEAD box helicase may substitute for RhlB in the degradosome, such as CsdA under cold shock conditions (Prud'homme-Généreux et al., 2004). We found that mutation of PA3297 increased the percentage of unprocessed 23S-5S rRNA in the presence of AZM, which indicates that the cleaving function of RNase III might be impaired under the stress caused by AZM. Therefore, PA3297 might assist RNase III in rRNA processing in the presence of AZM. In addition, although the growth speed and rRNA processing in the ΔPA3297 mutant treated with 10 µg/ml AZM were similar to those in the wild type strain treated with 40 µg/ml AZM (Supplementary Figure S3), the survival rate of the ΔPA3297 mutant was lower than that of the wild type strain
under those conditions. Thus, PA3297 might play other roles in counteracting the effects of AZM.

Overall, mutation of PA3297 renders *P. aeruginosa* more susceptible to AZM mediated inhibition on virulence factors and killing effect. Therefore, targeting the regulatory pathway or the function of PA3297 might further increase the beneficial effects of AZM in the treatment of chronic *P. aeruginosa* infections.

## AUTHOR CONTRIBUTIONS

Conceived and designed the experiments: WW, SJ, HT, ZC. Performed the experiments: HT, LZ, YW, RC, FZ, YJ. Analyzed the data: HT, LZ, SJ, WW. Wrote the paper: HT, SJ, WW.

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## FUNDING

This work was supported by National Science Foundation of China (31370168 to WW, and 31370167 to SJ); National Basic Research Program of China (973 Program, 2012CB518700 to SJ), Program of international S&T cooperation (2015DFG32500 to SJ) and Science and Technology Committee of Tianjin (13JCYBJC36700 to WW, 15JCZDJC33000 to SJ).

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Tan, Zhang, Weng, Chen, Zhu, Jin, Cheng, Jin and Wu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
# Efflux Pump Blockers in Gram-Negative Bacteria: The New Generation of Hydantoin Based-Modulators to Improve Antibiotic Activity

Ewa Otrębska-Machaj<fnref n="1" />,<fnref n="2" />, Jacqueline Chevalier<fnref n="2" />, Jadwiga Handzlik<fnref n="1" />, Ewa Szymańska<fnref n="1" />, Jakub Schabikowski<fnref n="1" />, Gérard Boyer<fnref n="2" />, Jean-Michel Bolla<fnref n="2" />, Katarzyna Kieć-Kononowicz<fnref n="1" />, Jean-Marie Pagès<fnref n="2" />* and Sandrine Alibert<fnref n="2" />

## OPEN ACCESS

**Edited by:**
Ivan Rychlík,
Veterinary Research Institute,
Czech Republic

**Reviewed by:**
Helen Zgurskaya,
University of Oklahoma, USA
Alessandra Polissi,
Università degli Studi di
Milano-Bicocca, Italy
Kunihiko Nishino,
The Institute of Scientific and
Industrial Research, Osaka University,
Japan

*Correspondence:
Jean-Marie Pagès
jean-marie.pages@univ-amu.fr

**Specialty section:**
This article was submitted to *Antimicrobials, Resistance and Chemotherapy*, a section of the journal *Frontiers in Microbiology*

**Received:** 07 January 2016
**Accepted:** 15 April 2016
**Published:** 03 May 2016

**Citation:**
Otrębska-Machaj E, Chevalier J, Handzlik J, Szymańska E, Schabikowski J, Boyer G, Bolla J-M, Kieć-Kononowicz K, Pagès J-M and Alibert S (2016) Efflux Pump Blockers in Gram-Negative Bacteria: The New Generation of Hydantoin Based-Modulators to Improve Antibiotic Activity. *Front. Microbiol.* 7:622. doi: 10.3389/fmicb.2016.00622

Multidrug resistant (MDR) bacteria are an increasing health problem with the shortage of new active antibiotic agents. Among effective mechanisms that contribute to the spread of MDR Gram-negative bacteria are drug efflux pumps that expel clinically important antibiotic classes out of the cell. Drug pumps are attractive targets to restore the susceptibility toward the expelled antibiotics by impairing their efflux activity. Arylhydantoin derivatives were investigated for their potentiation of activities of selected antibiotics described as efflux substrates in *Enterobacter aerogenes* expressing or not AcrAB pump. Several compounds increased the bacterial susceptibility toward nalidixic acid, chloramphenicol and sparfloxacin and were further pharmacomodulated to obtain a better activity against the AcrAB producing bacteria.

**Keywords:** antibiotics, AcrAB pump, efflux pump blocker, hydantoin, multidrug resistance

## INTRODUCTION

Multidrug resistance (MDR) described in Gram-negative bacteria is continuously emerging as a prominent worldwide health concern (Chopra et al., 2008; Laxminarayan et al., 2008; Gandhi et al., 2010). One of the most contributing mechanisms is the overexpression of efflux pumps that are involved in bacterial survival, colonization and virulence (Delmar et al., 2014; Blair et al., 2015; Li et al., 2015; Venter et al., 2015; Davin-Regli et al., 2016). Several efflux pump superfamilies, e.g., major-facilitator (MF), multi-drug and toxic efflux (MATE), ATP-binding cassette (ABC), small multidrug resistance (SMR), resistance-nodulation-division (RND) transporters have been classified<fnref n="1" />,<fnref n="2" /> and extensively described in well-documented reviews: they differ by their functional structure and organization, their subcellular location inside the bacterium, their energy source (e.g., membrane potential for RND or ATP for ABC) and the involvement of a coupled antiport during the antibiotic expulsion (e.g., proton for AcrAB pump; Delmar et al., 2014; Blair et al., 2015; Li et al., 2015). The overexpression of Gram-negative efflux pumps, especially those belonging to the

<footnote n="1">¹Department of Technology and Biotechnology of Drugs, Medical College, Jagiellonian University, Krakow, Poland,</footnote>

<footnote n="2">²UMR-MD1, Aix Marseille Université/IRBA, Facultés de Médecine et de Pharmacie, Marseille, France</footnote>

<footnote n="1">¹http://www.membranetransport.org/</footnote>

<footnote n="2">²http://www.tcdb.org/</footnote>
RND family, is now well-described in resistant isolates (Nikaido and Pagès, 2012; Li et al., 2015). This contributes to the acquisition of additional mechanisms of resistance including the mutation in antibiotic targets (e.g., mutation in gyrase/topoisomerase for quinolone) or the production of enzymes that degrade antibiotics (e.g., β-lactamases) and this can be associated or not with the alteration of the outer membrane permeability (Davin-Regli et al., 2008; Pagès et al., 2008). Regarding resistant clinical isolates of Gram-negative bacteria, the archetype of the drug active transporter system is the AcrAB-TolC/MexAB-OprM efflux pumps (Nikaido and Pagès, 2012; Li et al., 2015). The structures of components of efflux systems belonging to RND group, have been solved by X-ray crystallography and models of the pump assembly have been obtained (Yao et al., 2010; Du et al., 2014). The structure and function of the RND efflux pumps must be molecularly deciphered thus allowing the rational design and the synthesis of new compounds to combat MDR. The broad selectivity of efflux pumps makes difficult the identification of precise pharmacophoric groups at the drug surface. However, efflux pumps are attractive target by blocking this efflux mechanism in order to restore the intracellular concentration of antibacterial agents (Bolla et al., 2011; Ruggerone et al., 2013; Dreier and Ruggerone, 2015; Opperman and Nguyen, 2015). Recent computer docking analyses have produced some information about the involvement of certain amino acid residues, but clearly more chemical and biological information are needed to improve models (Schulz et al., 2010; Fischer et al., 2014). This is a 'key' point not only regarding the mode of action and dynamics of the process but also regarding the clinical impact of the design of new antibacterial agents: this last aspect is illustrated by the β-lactamases inhibitors currently used today (Bolla et al., 2011; Chen et al., 2013; Pucci and Bush, 2013).

To enhance the activity of old antibiotics by targeting resistance mechanisms in clinical resistant isolates, they can be combined with adjuvant molecules such as chemosensitizers (e.g., membrane permeabilizer or efflux inhibitor; Jones, 2010; Bolla et al., 2011). In addition, these types of transporter inhibitors may impair the activity of efflux pumps and thus reduce bacterial colonization and virulence (Venter et al., 2015; Davin-Regli et al., 2016). It must be noted that the efficacy of the inhibitors depends on their affinity for transporter binding sites (compared to the antibiotic) and their internal concentration close to the efflux pump. Consequently, due to these parameters associated with penetration rate and affinity for pump sites, some discrepancies can be observed in the level of the internally accumulated antibiotics depending on the bacterial backgrounds (Kaščáková et al., 2012; Cinquin et al., 2015).

Recently, two generations of hydantoin derivatives have been identified as AcrAB-TolC inhibitors with the *Enterobacter aerogenes* CM 64 strain that overproduces AcrAB (Handzlik et al., 2011). Compounds showing chemosensitizing effect on nalidixic acid activity were the starting point for new pharmacomodulations carried out in this study to obtain a new generation of products with an improved activity.

Moreover, tests were extended to other chemically unrelated antibiotics, chloramphenicol and sparfloxacin, for which the antibacterial activity decreased together with the emergence of multidrug-resistant strains. Several chemical derivatives were synthesized to define pharmacophoric groups important for restoring the activity of antibiotics in AcrAB active bacteria.

# MATERIALS AND METHODS

## Bacterial Strains

All generations of hydantoin derivatives were tested against two strains of *E. aerogenes*: the reference strain, ATCC 13048 (basal efflux producer) and its derivative strain, CM 64, a chloramphenicol-selected resistant strain that over-produces the AcrAB-TolC EPs (Ghisalberti et al., 2005). In addition, two isogenic strains EA289 (a clinical MDR strain that overproduced the AcrAB pump) and its EA294 derivative (an acrAB knockout strain) were used (Pradel and Pagès, 2002). These two strains contain additional mechanisms of antibiotic resistance such as β-lactamases, targets mutations, etc. (Malléa et al., 1998; Pradel and Pagès, 2002; Kaščáková et al., 2012).

## Chemicals

Hydantoin derivatives used in the pharmacological assays were obtained by chemical synthesis. The new generations IIIA and B were obtained using 3-4-step synthesis (Handzlik et al., 2014; Matys et al., 2015; **Figure 1** and Supplementary data). Purity and identity of new compounds were confirmed using spectral analysis (H-NMR, IR), elemental analysis and melting point measurements. Phenylalanine-Arginine β-naphthylamide (PAβN, dihydrochloride, Sigma) previously described as efflux pump inhibitor was used as reference (Bolla et al., 2011; Misra et al., 2015).

## Compound Susceptibility Assays

Susceptibilities of ATCC 13048, CM 64, EA294, and EA289 were determined by using the twofold standard microbroth dilution method (microplates and automatic analyses Tecan®; CLSI<fnref n="3" />). Approximately, 10⁵ CFU (colony forming unit) were inoculated in 200 µl of Mueller-Hinton II broth (MH II broth cation adjusted, Becton, Dickinson & Company) containing twofold serial dilutions of the targeted molecule. Experiments were performed in triplicate for each compound and each antibiotic. Results were estimated visually after 18 h incubation at 37°C (Philippe et al., 2015).

## Antibiotic Susceptibility Potentiating Assays

To assay the possible chemosensitizing activity of compounds, serial dilutions of antibiotics, nalidixic acid (NAL, Sigma), chloramphenicol (CHL, Sigma), doxycycline (DOX, hyclate, Sigma), erythromycin (ERY, lactobionate, AMDIPHARM) and

<footnote n="3">³http://clsi.org/</footnote>
### Generation IIIA

<table><thead><tr><th>Compound</th><th>R<sup>1</sup></th><th>n</th></tr></thead><tbody><tr><td>29</td><td>CC</td><td>3</td></tr><tr><td>30</td><td>CC</td><td>4</td></tr><tr><td>31</td><td>CC</td><td>5</td></tr><tr><td>32</td><td>CC</td><td>4</td></tr></tbody></table>

<figure><img src="image_80.png" /></figure>

### Generation IIIB

<table><thead><tr><th>Compound</th><th>R<sup>1</sup></th></tr></thead><tbody><tr><td>33</td><td>H</td></tr><tr><td>34</td><td>-CH<sub>3</sub></td></tr><tr><td>35</td><td>-CH<sub>2</sub>COOC<sub>2</sub>H<sub>5</sub></td></tr><tr><td>36</td><td>CH<sub>3</sub></td></tr></tbody></table>

<figure><img src="image_81.png" /><figcaption>FIGURE 1 | Compounds of generation IIIA and generation IIIB of hydantoins.</figcaption></figure>

sparfloxacin (SPX, Sigma), were incubated in the absence or in the presence of compounds. The antibiotics, NAL, CHL, and SPX are substrates of the AcrAB-TolC efflux pump as demonstrated by the increased MIC values obtained in CM 64 strain overexpressing the AcrAB-TolC pump (Table 1).

<table><caption>TABLE 1 | Susceptibility (MIC) of the *Enterobacter aerogenes* reference strain ATCC 13048, the derivative strain CM 64 overexpressing AcrAB-TolC pump, Ea289 overproducing the AcrAB pump and its derivative Ea294 (an acrAB knockout strain) to the different compounds belonging to various generations of hydantoins and to nalidixic acid (NAL), chloramphenicol (CHL), sparfloxacin (SPX), doxycycline (DOX), and erythromycin (ERY).</caption><thead><tr><th>Compound</th><th>MIC [mM]<br>ATCC 13048</th><th>MIC [mM]<br>CM 64</th><th>MIC [mM]<br>Ea294</th><th>MIC [mM]<br>Ea289</th></tr></thead><tbody><tr><td>29-36</td><td>&gt;2</td><td>&gt;2</td><td>&gt;1</td><td>&gt;1</td></tr><tr><td>NAL</td><td>0.034</td><td>0.55 (16)*</td><td>4.4</td><td>&gt;17.6</td></tr><tr><td>CHL</td><td>0.012</td><td>0.79 (66)</td><td>0.2</td><td>3.2</td></tr><tr><td>SPX</td><td>0.00015</td><td>0.0025 (17)</td><td>&gt;2</td><td>&gt;2</td></tr><tr><td>DOX</td><td>0.002</td><td>0.07 (35)</td><td>0.002</td><td>0.07</td></tr><tr><td>ERY</td><td>0.35</td><td>0.7 (2)</td><td>0.087</td><td>0.35</td></tr><tr><td>PAβN</td><td>5</td><td>5 (1)</td><td>0.0625</td><td>3.5</td></tr></tbody></table>

compared to the reference one ATCC 13048. Thus we are able to hypothesize that an efflux blocker may reduce the antibiotic MIC in efflux producer strain. Generations IIIA and IIIB of hydantoin derivatives were tested at a concentration of 0.5 mM according to the intrinsic antibacterial activity of each compound (corresponding to the value of MIC/4). To facilitate the comparison of activity and the performance of a rational SAR analysis, they were additionally tested at the concentration corresponding to that of the best first generation of chemosensitizers (0.0625 mM; Handzlik et al., 2011). PAβN, the reference inhibitor for AcrAB pump, was used at 0.050 mM. Control experiments were carried out without compounds. Experiments were performed in triplicate for each antibiotic, each strain and each condition (without and with compound). The results were assessed after 18 h at 37°C and were presented by using the activity gain parameter A, the ratio of the MIC of the antibiotic (determined in the absence of compound) to its MIC in the presence of the compound.

### Determination of the FIC Index

To determine the fractional inhibitory concentration index (FICi), a two dimensional checkerboard with twofold dilutions of each compound was set up for the study (Berenbaum, 1978;

<footnote>*MIC ratio: efflux overproducer/basal producer strains.</footnote>
<table><caption>TABLE 2 | Effect of the hydantoin derivatives on the susceptibility level of *E. aerogenes* ATCC 13048 and CM 64 strains to nalidixic acid (NAL), chloramphenicol (CHL), and sparfloxacin (SPX).</caption><thead><tr><th rowspan="2">Compound</th><th rowspan="2">Concentration [mM]</th><th colspan="2">A<sub>NAL</sub></th><th colspan="2">A<sub>CHL</sub></th><th colspan="2">A<sub>SPX</sub></th></tr><tr><th>ATCC 13048</th><th>CM 64</th><th>ATCC 13048</th><th>CM 64</th><th>ATCC 13048</th><th>CM 64</th></tr></thead><tbody><tr><td>29</td><td>0.0625</td><td>1</td><td>1</td><td>1</td><td>1</td><td>2</td><td>2</td></tr><tr><td>30</td><td>"</td><td>2</td><td>2</td><td>2</td><td>1</td><td>1</td><td>1</td></tr><tr><td>31</td><td>"</td><td>2</td><td>2</td><td>2</td><td>1</td><td>1</td><td>2</td></tr><tr><td>32</td><td>"</td><td>2</td><td>4</td><td>1</td><td>1</td><td>1</td><td>2</td></tr><tr><td>33</td><td>"</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>34</td><td>"</td><td>1</td><td>0.5</td><td>0.5</td><td>1</td><td>0.5</td><td>1</td></tr><tr><td>35</td><td>"</td><td>1</td><td>1</td><td>0.5</td><td>1</td><td>0.5</td><td>0.5</td></tr><tr><td>36</td><td>"</td><td>1</td><td>1</td><td>1</td><td>1</td><td>0.5</td><td>0.5</td></tr><tr><td>29</td><td>0.5</td><td>8</td><td>4</td><td>2</td><td>4</td><td>4</td><td>2</td></tr><tr><td>30</td><td>"</td><td>8</td><td>4</td><td>4</td><td>4</td><td>8</td><td>4</td></tr><tr><td>31</td><td>"</td><td>16</td><td>4</td><td>4</td><td>4</td><td>4</td><td>4</td></tr><tr><td>32</td><td>"</td><td>16</td><td>32</td><td>4</td><td>32</td><td>8</td><td>8</td></tr><tr><td>33</td><td>"</td><td>2</td><td>2</td><td>1</td><td>2</td><td>2</td><td>1</td></tr><tr><td>34</td><td>"</td><td>2</td><td>1</td><td>1</td><td>1</td><td>2</td><td>2</td></tr><tr><td>35</td><td>"</td><td>2</td><td>1</td><td>2</td><td>1</td><td>2</td><td>1</td></tr><tr><td>36</td><td>"</td><td>2</td><td>1</td><td>2</td><td>1</td><td>2</td><td>1</td></tr><tr><td>PAβN</td><td>0.05</td><td>64</td><td>128</td><td>2</td><td>64</td><td>8</td><td>32</td></tr></tbody></table>

"A" corresponds to the antibacterial activity gain obtained in the presence of the respective compound and evaluated for each antibiotic (A = MIC without compound/MIC with compound).

Allam et al., 2014). For the first clear well in each row of the microplate containing an antimicrobial agent, the FIC was calculated as follows: FIC of compound A (FIC A) = MIC of compound A in combination with B/MIC of compound A alone, and FIC of compound B (FIC B) = MIC of compound B in combination with A/MIC of compound B alone. The FICi was calculated as the sum of the FIC of each compound. The nature of the interaction was classified as follows: synergy FICi ≤ 0.5; additivity 0.5 < FICi ≤ 1; indifference 1 < FICi ≤ 2; and antagonism FICi > 2 (European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID), 2000). For each combination, an isobologram, which graphically illustrates the interaction effect, was constructed: FIC A was showed in the abscissa and FIC B in the ordinate, the profile of the corresponding curve reflects the nature of the interaction.

# RESULTS

## Antibacterial Activity

It is important that putative inhibitors that could be used as "adjuvant molecule" for antibiotics, do not display a high intrinsic antibacterial activity (Davin-Regli et al., 2008; Bolla et al., 2011). The determination of the antibacterial activity for each compound was performed and presented in Table 1. Many of the compounds did not inhibit the growth of bacteria even at the highest tested concentration as the previous generations of hydantoin derivatives (Handzlik et al., 2011). The majority of compounds exhibited a MIC > 2 mM in ATCC13048

and in CM64 strains and MIC ≥ 1 mM in Ea289 and Ea294.

## Influence on Antibiotic Susceptibility in ATCC 13048 and CM 64 Strains
### Effect on Nalidixic Acid Susceptibility

Table 2 presents the chemosensitizing effect of compounds on ATCC 13048 and CM 64 susceptibility to nalidixic acid (NAL). The gain on antibiotic activity (A) was calculated for each compound and each antibiotic. Regarding the CM 64 strain, a moderate or a weak effect was observed on the MIC when compounds were used at 0.0625 mM compared to PAβN. Compounds of generations IIIA (29–32) and IIIB (33–36) were used at a concentration of 0.0625 mM and 0.5 mM due to their higher MIC. A higher concentration of compounds of the generation IIIB did not improve the antibiotic activity whereas we observed a significant decrease of the antibiotic MIC, from 4- to 32-fold in CM 64 (see A<sub>NAL</sub> in Table 2), in the case of compounds of generation IIIA (29–32). These compounds decreased the MIC of NAL from 8- to 16-fold comparing to the PAβN effect (64-fold) in the reference ATCC 13048 strain (see A<sub>NAL</sub> in Table 2). In the case of PAβN and derivatives 32 the difference in activity in both tested strains was not significant (only a twofold stronger activity in the strain overexpressing the AcrAB pump than in the reference one) whereas compounds 29–31 showed a better action on antibiotic activity in the reference strain. It must be noted that the compound 32 used at increased concentration exhibits an activity profile similar to PAβN, which has been shown to be an efficient efflux inhibitor at low concentrations (Nikaido and Pagès, 2012; Misra et al., 2015).
<table><caption>TABLE 3 | Effect of the compounds of generation IIIA (29–32) depends on AcrAB context.</caption><thead><tr><th rowspan="2">Compound</th><th colspan="3">Ea294</th><th colspan="3">Ea289</th><th colspan="2">CM 64</th></tr><tr><th>A<sub>DOX</sub></th><th>A<sub>ERY</sub></th><th>A<sub>NAL</sub></th><th>A<sub>DOX</sub></th><th>A<sub>ERY</sub></th><th>A<sub>NAL</sub></th><th>A<sub>DOX</sub></th><th>A<sub>ERY</sub></th></tr></thead><tbody><tr><td>29</td><td>2</td><td>1</td><td>2</td><td>8</td><td>1</td><td>1</td><td>8</td><td>1</td></tr><tr><td>30</td><td>4</td><td>4</td><td>4</td><td>16</td><td>1</td><td>2</td><td>8</td><td>1</td></tr><tr><td>31</td><td>4</td><td>8</td><td>8</td><td>16</td><td>2</td><td>2</td><td>8</td><td>1</td></tr><tr><td>32</td><td>2</td><td>2</td><td>4</td><td>32</td><td>4</td><td>&gt;2</td><td>32</td><td>2</td></tr></tbody></table>

"A" corresponds to the antibacterial activity gain obtained in the presence of the respective compound and evaluated for each antibiotic (A = MIC without compound/MIC with compound). DOX, doxycycline; ERY, erythromycin; NAL, nalidixic acid.

<figure><img src="image_82.png" /><figcaption>FIGURE 2 | Representative isobologram of interactions between compound 32 and nalidixic acid or chloramphenicol. Nalidixic acid, solid line; chloramphenicol, dotted line. The axis numbers correspond to normalized FICs obtained with *Enterobacter aerogenes* CM64 strain.</figcaption></figure>

## Effect on Chloramphenicol Susceptibility

The results are presented in Table 2. In the case of the CM 64 strain, compounds tested at the concentration of 0.0625 mM showed a weak chemosensitizing effect on CHL antibacterial activity compared to PAβN. Compounds of generation IIIA (29–32) examined at a concentration of 0.5 mM decreased the chloramphenicol MIC from 2- to 4-fold in the reference strain and from 4- to 32-fold in the strain overexpressing efflux pump (see A<sub>CHL</sub> in Table 2). We observed a noticeable increase in CHL susceptibility in the AcrAB overproducer CM 64, a 32-fold gain in the susceptibility with the compound 32 compared to compounds 29–31. In this assay, the chemosensitizing effect of compound 32 can be compared to PAβN which was much more active in the strain overexpressing the AcrAB pump than in the parental strain ATCC 13048.

## Effect on Sparfloxacin Susceptibility

Table 2 presents the effects of hydantoins on sparfloxacin (SPX) susceptibility. In CM 64, the chemosensitizing effect of low concentrated hydantoins on SPX was as weak as we observed in

the case of CHL. Compounds of generation IIIA (29–32) tested at the highest concentration 0.5 mM caused a 4–8-fold decrease in the MIC in the ATCC 13048 strain and a 2–8-fold decrease in the MIC in CM 64 one. Comparing the results obtained for the active compounds (29–32) to the results obtained for PAβN (see A<sub>SPX</sub> in Table 2) we observed that the action of hydantoins with SPX was less efficient than PAβN, which was more active in the CM64 strain overexpressing the AcrAB pump than in the reference strain ATCC13048. This may suggest a different conformational site for the two molecules, either for recognition or for binding step, in the AcrB monomer (Delmar et al., 2014; Du et al., 2014; Yamaguchi et al., 2015), inside the pump or a different mode of action on the resistance mechanism.

## Effect of Compounds 29–32 on the Resistance Level in MDR *E. aerogenes* Strains

In order to evaluate the chemosensitizing effect of the compounds 29–32 (the most effective molecules) on the MDR
background, the Ea289 and Ea294 strains were assayed. It
is important to mention that these strains contain various
resistance mechanisms (Malléa et al., 1998; Pradel and Pagès,
2002) such as target mutations (e.g., mutations in QRDR
region of gyrase that increase quinolone resistance) and
expression of modifying enzymes (e.g., β-lactamases that
contribute to β-lactam resistance). **Table 3** presents the
activity gain parameter A tested with doxycycline (DOX),
erythromycin (ERY), and nalidixic acid (NAL) in parental
and AcrAB-derivative strain context. It is interesting to note
that **32** exhibited significant restoring antibiotic activity with
DOX in Ea289 and very weak action in Ea294 that is
devoid of the AcrAB efflux pump components. In contrast,
a weak chemosensitizing activity was observed with ERY,
this effect could be caused by the presence of additional
resistance mechanisms for the macrolide antibiotic class as
previously reported (Chollet et al., 2004). Regarding the
effect on NAL and sparfloxacin susceptibility, the mutations
in the quinolone target (DNA gyrase) previously reported
in Ea289, can explain the weak effect observed toward
these strains (Pradel and Pagès, 2002; Kaščáková et al.,
2012).

Determination of the FIC Index for
Compound 32

To precise the type of interaction (synergistic, additive, or
indifferent) between compound **32** and selected antibiotics, we
carried out analysis based on the FICi as previously described.
Combinations of compound **32** with NAL and CHL respectively
were performed in CM 64 strain. This strain overexpresses
the AcrAB pump and does not contain target mutation that
can impair the effect of compounds on the restoration of
antibiotic activity. The nature of the association was determined
from the FICi average obtained from each combination
and the representation was performed for each combination
(Figure 2). In two cases, the isobologram curve obtained was
concave indicating a synergy association between antibiotic and
compound **32**. The synergistic association corresponds to an
average of calculated FICi of about 0.32 for NAL and 0.44 for
CHL respectively. These curves fitted well with the Table 2
data.

DISCUSSION

The aim of this study was to identify compounds that are able to
modulate the AcrAB pump activity and restore antibiotic activity
on efflux producing strains.

The various molecules combined with NAL and CHL
increased the susceptibility of the CM 64 strain and of the
reference strain ATCC 13048 to antibiotics. On the one
hand, we found compounds that showed stronger activity
in the ATCC 13048 strain than in the CM 64 strain (29–31
tested with NAL, 29, 30 tested with SPX). These findings
could indicate in this case that tested compounds are not
highly selective toward the AcrAB-TolC pump involved
in the efflux of antibiotics. This suggests that they act not

only on the basal AcrAB expressing strain but also that they
may recognize other bacterial targets. In contrast, PAβN
combined with CHL and SPX as well as the 3-aminobutyl-5-
β-naphthylhydantoin **32** tested with CHL exhibited a difference
between the AcrAB overproducer strain and the parental
one. This time these results suggest a significant selectivity of
chemosensitizers for the AcrAB pump expressed in bacterial
strains. Taking into account the influence of concentration
of 3-aminoalkyl-5-naphthylhydantoins (generation IIIA) on
the antibiotic activity, we observed that the higher is the
concentration, the stronger is the chemosensitizing effect,
not only on the CM 64 strain but also on ATCC 13048.
These outcomes could suggest additional mechanisms
of action besides the effect on the AcrAB-TolC efflux
pump.

The comparison of the results in Ea289 and Ea294 (Table 3) indicates the correlation of the restoring activity of antibiotics with the presence of the AcrAB efflux pump. They also illustrate the capability of compound 32 to increase the antibacterial effect of DOX in these strains. The low effect of 32 on the ERY susceptibility when assayed in Ea289 and Ea294 can be due to additional pump, other than AcrB, active in these clinical derivative strains capable to expel this class of drugs (Chollet et al., 2004).

The different structural features of tested compounds
allow showing how the nature and the position of their
diverse molecular fragments modulate on the one hand their
selectivity regarding AcrAB pump and on the other hand their
chemosensitizing activity. The tested compounds exhibiting the
common hydantoin scaffold were modified using two of diverse
substituents at position 5 as well as amine substituents and a
selection of linkers to bind an amine to the hydantoin core.
The location of an amine-alkyl fragment at position N1 or N3
to an aryl fragment at position 5 was modified for comparing
with the activity of the first generations of hydantoin synthesized
(Handzlik et al., 2011). In the case of 32, the hydrophilic primary
amine fragment is focused by terminate location at the end
of longer 3-hydantoin substitution, opposite to the 5-β-nahthyl
one.

The most active compounds (29–32) share the same
pharmacophore profile than PAβN suggesting a similar
physicochemical outline for an identical target. The amphiphilic
nature of the Generation IIIA of optimized derivatives seems to
be crucial to inhibit antibiotic resistance mediated by efflux pump
and open new ways for generating original active compounds
able to inhibit pump activity. With the recent published data
regarding piperazine arylideneimidazolone derivatives as
potential efflux inhibitors in *Escherichia coli* cells (Bohnert et al.,
2016), the compound **32** characterized in this study will be
used for pharmacophoric modulations in order to develop more
potent inhibitors.

AUTHOR CONTRIBUTIONS

JH, KK-K, J-MP, and SA designed research; EO-M, JC, ES,
and SA performed research; JS, GB, and J-MB contributed new
reagents or analytic tools; EO-M, JH, KK-K, J-MP, and SA analyzed data; JH, KK-K, J-MP, and SA wrote the paper.

## FUNDING

This work was supported by French Embassy in Poland, 757/N-POLONIUM/2010/0, by grant ANR-11-BS07-019-01 "IBEF" Agence Nationale de la Recherche (ANR, France) and partly supported by Polish statutory project K/ZDS/005593 and Aix-Marseille University and IRBA.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Otrębska-Machaj, Chevalier, Handzlik, Szymańska, Schabikowski, Boyer, Bolla, Kieć-Kononowicz, Pagès and Alibert. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
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