Conjugation of LasR Quorum-Sensing Inhibitors with Ciprofloxacin Decreases the Antibiotic Tolerance of P . aeruginosa Clinical Strains

Department of Medical Sciences, Section of Microbiology and Medical Genetics, University of Ferrara, Via Luigi Borsari, 46-44121 Ferrara, Italy Department of Chemical and Pharmaceutical Sciences, University of Ferrara, Via Fossato di Mortara, 17, 44121 Ferrara, Italy Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milano, Italy Department of Biomedical and Specialist Surgical Sciences, Section of Medical Biochemistry, Molecular Biology and Genetics, University of Ferrara, Via Luigi Borsari, 46-44121 Ferrara, Italy Department of Life Sciences and Biotechnology, Section of Biology and Evolution, University of Ferrara, Via Luigi Borsari, 46-44121 Ferrara, Italy NC State University, Plants for HumanHealth Institute, Animal Science Deptment, NC Research Campus, Kannapolis, NC, USA LTTA-Electron Microscopy Center, University of Ferrara, Ferrara, Italy


Introduction
Microbial infections can result in complications, such as bacteremia, kidney failure, and toxic shock syndrome.For this, the identification of the systems to counteract bacterial infection is one of the challenges of modern medicine.e absence of novel molecules that control complications due to bacterial infections suggests the defective comprehension of the mechanisms used by bacteria to control host immune response and resist treatment.During host infection, several bacteria organize a bacterial population, and Pseudomonas aeruginosa is one of the most commonly studied.P. aeruginosa is a Gram-negative bacterium that especially infects subjects with weakened immune system causing deadly infections above all at pulmonary level.In fact, cystic fibrosis (CF) or HIV patients exhibit increased susceptibility to P. aeruginosa lung infections.Since P. aeruginosa is a ubiquitous bacterium, exposure to this pathogen in the hospital setting results to be frequent, making it one of the most problematic nosocomial infections.
e antibiotics used to treat P. aeruginosa infection include ciprofloxacin, tobramycin, ceftazidime, gentamicin, and imipenem.P. aeruginosa bacteria present a high degree of resistance to these antibiotics.Interestingly, the response to ciprofloxacin is very effective at the beginning of the treatment, but high-level resistance is rapidly acquired by P. aeruginosa, making the treatment ineffective in the 30% of strains obtained from clinical isolates [1].
P. aeruginosa is characterized by a low permeability of its cell wall that increases its resistance to antibiotics because of a lower drug uptake or higher efflux pumps expression that cause decreased intracellular drug concentrations [5].Alterations in QRDR of the target sites DNA gyrase (gyrA) and topoisomerase IV, encoded by parC and parE subunits, are considered the main reason of bacterial resistance to quinolones [6,7].
e acquisition of resistance to ciprofloxacin is a multistage process in P. aeruginosa: in stage I, the exposure to the drug kills the susceptible cells; in stage II, a small population survives antibiotic exposure without increasing the resistance and maintaining a slow growth; in stage III, a the population is reconstituted by a slow-growing population with an increased drug resistance [8].e appearance of a drug-resistant population limits the possibility to use prolonged therapies with existing or newly developed antibiotic drugs.We suggest the possible use of the inhibitor of quorum-sensing (QS).One of the main defense mechanisms adopted by P. aeruginosa is represented by biofilm formation, which allows the bacteria to avoid both host immune system and antibiotics effects [9,10].QS is a mechanism of gene regulation sensitive to population density that enables host colonization contrasting the immune surveillance through biofilm formation and the expression of virulence factors [10] via the production of self-generated extracellular signal molecules [11][12][13].P. aeruginosa is characterized by two QS systems, Las and Rhl, based on acylhomoserine lactone [14] molecules [15].e las system is controlled by the transcriptional activator LasR and the autoinducer synthase enzyme LasI, which directs the synthesis of N-(3oxododecanoyl) homoserine lactone (3O-C12-HSL).Similarly, the Rhl system is regulated by the transcriptional activator RhlR and the RhlI AI synthase that synthesizes N-butyryl homoserine lactone (C4-HSL).3O-C12-HSL binds LasR and activates the LasR/3O-C12-HSL complex; the multimerization will promote the transcription of RhlR, RhlI, LasI genes, and other virulence genes that are connected to the regulon [16][17][18].In a similar way, RhlR/C4-HSL complex dimerizes and activates the expression of its own regulon and RhlI [19].LasR/3O-C12-HSL regulates the quinolone signal by inducing expression of PqsR, as well as PQS [20].PQS, in turn, increases the transcription of RhlI and the production of C4-HSL [21].Interestingly, PqsR expression is inhibited by RhlR/C4-HSL [21], suggesting that the ratio between 3O-C12-HSL and C4-HSL concentrations controls the Pqs signaling system.
Since QS molecules are important during infection, the interference on QS signaling represents a potential strategy to contrast bacterial virulence, decreasing antibiotics dosage and facilitating the natural bacterial clearance by host immune response.Brackman et al. have shown that QS inhibitors (QSi) increase the susceptibility of bacterial biofilms to different types of antibiotics [22].We report the results of our work on the effect of different QSi compounds, designed on the scaffold of QS inducer 3O-C12-HSL, conjugated with ciprofloxacin.ese results will set up the initial steps developing new strategies that may subvert the ciprofloxacin resistance of P. aeruginosa.

Chemical Synthesis.
e compounds tested in this work are antagonists of LasR and are designed on 3O-C12-HSL scaffold [23].Previous evaluations of these derivatives showed that S absolute configuration at the 3-position of the homoserine lactone was important for the activity of the compounds.On the contrary, the R derivative was not effective.For this reason, we synthetized all the compounds in the S absolute configuration.
e chemical synthesis was performed as described in Geske et al. [23].e compounds tested in this study derived from libraries of AHL mimics designed to be capable of intercepting the LasR and RhlR QS system which are specific for P. aeruginosa.e LasR antagonists interact with the N-terminal ligand-binding domain of LasR, blocking the binding site for QS molecules [23].

Samples.
Ten sequential P. aeruginosa isolates from four patients with CF were chosen from the strains collection of the CF clinic in Hannover.We selected two strains from patients SG (SG1 and SG58, both LasR wild type (wt)), three strains from patients AA (AA2 and AA12 LasR wt and AA11 LasR mutant), three strains from patient TR (TR2 LasR wt and TR1 and TR66 LasR mutants), and two strains from patient KK (KK1 and KK72, both LasR mutants).ese strains were characterized in previous work [24].Patients were checked after the diagnosis of CF, and meanwhile, the respiratory specimens were sampled.e "early" isolates of P. aeruginosa strains were collected from the first positive cultures, whereas late isolates were collected 7 to 16 years after colonization or prior to death or lung transplantation.CF isolates behaviour was compared with the laboratory strain PAO1 for their phenotypic diversity [25,26].

Phenotypic Analysis for LasR Mutants.
Colony surface iridescence and metallic sheen were evaluated and considered as a phenotypic characteristic of LasR mutants [25,26].
e DNA sequence confirmed the presence of LasR mutations (Table 1).e compounds were dissolved in DMSO added to planktonic cell cultures.Cell growth was evaluated by reading absorbance at 600 nm, while biofilm formation was analysed as previously described [27].Briefly, after 24 hours of treatment, the planktonic cells were gently removed, and the wells were washed three times with PBS 1x.
e plate was let at 60 °C for 1 hour in order to dry the biofilm.e biofilm mass was measured by staining with crystal violet 1% w/v and then resuspended in 200 µl of 33% glacial acetic acid and read at 570 nm.3O-C12-HSL and DMSO were used as positive and negative controls, respectively.Results are reported as mean ± SD and are obtained from three independent experiments.2.5.Syto9 Assay.P.aeruginosa was cultured in 8-chamber slides and treated and left for 24 hours at 37 °C.Planktonic cells were removed, and each chamber was washed with PBS 1x.200 µl of Syto9 working solution prepared by diluting Syto9 stock solution (5 mM in DMSO, Invitrogen) 1 : 1200 was added and kept in dark for 30 minutes at RT. e chambers were then washed two times with PBS 1x, and the biofilm was visualized by fluorescent microscopy (ZOE Fluorescent Cell Imager, Biorad).Pictures of the biofilm were compared to the untreated control.

Time-Kill Studies.
Ciprofloxacin and ET37 time-kill studies were performed at concentrations equal to the theoretical plasma peak (4 μg/mL), then at concentrations equal to 1/2, 2, 4, 8, X MIC 50 of the antibiotic used.Bacteria were cultured for 24 h at 37 °C on Mueller-Hinton agar (MHB, BioMérieux, France) and used to prepare the exponential growth phase at standard inoculum (10 6 CFU/mL) in Mueller-Hinton broth.e inoculum of 10 6 CFU/ml was supplemented with ciprofloxacin or ET37 at the different concentrations and cultured for 24 h at 37 °C.100 ml of culture supernatants were collected at 2, 4, 6, and 24 h and plated on agar for colony counts.
2.8.Pyocyanin and Elastase Assay.P. aeruginosa strains were cultured overnight at 37 °C with shaking.Cultures were back diluted 1 : 1,000 into fresh medium and grown for 18 h.e cells were pelleted by centrifugation, and the culture supernatants were filtered through 0.22 μm filters.e production of pyocyanin was reported as A 380 /A 600 ratio [30].e production of LasB elastase was assessed through the measurement of elastase activity using elastin-Congo red and reported as A 495 /A 600 ratio [31].
e sequences were multiple aligned by ClustalW2 (http://www.ebi.ac.uk/Tools/msa/ clustalw) in order to detect mutations.Nucleotide sequences were translated by Expasy Bioinformatics Resource Portal (http://web.expasy.org/translate/)then compared with P. aeruginosa PAO1 protein sequence using ClustalW2 to find changes in amino acid sequences.

Disk Diffusion Ciprofloxacin Susceptibility Testing.
e antibiotic susceptibility of bacterial isolates was determined using the disk diffusion method standardized according to the EUCAST (http://www.eucast.org/ast_of_bacteria/disk_diffusion_methodology/), with interpretation based on the EUCAST Clinical Breakpoint Tables v. 9.0, valid from 2019 to 01-01, the zone diameter breakpoint >26 for susceptible strains (S), and <26 for resistant strains (R).e bacterial strains were inoculated onto Mueller-Hinton agar.Antibiotic disks were placed on the surface, and this was followed by incubation at 35 ± 1 °C in air for 18 ± 2 h ours.Inhibition zone diameter values were read by a pair of calipers, in millimeters to one decimal place.Ciprofloxacin disks (5 µg) were obtained from Oxoid Ltd (Oxoid AB, Hampshire, UK).

Determination of Minimum Biofilm Inhibitory Concentration (MBIC).
e MBICs of ciprofloxacin and ET37 were determined in PAO1 strain, as previously reported [32].
e experiments were done in 96-well polystyrene microtiter plates with round bottoms.An overnight culture with a turbidity equivalent to that of a 0.5 McFarland standard, obtained with TSB, was aliquoted into the wells of microtiter plates.e plates were incubated for 24 h at 37 °C.
e wells were washed three times with phosphate-buffered saline (PBS) to remove unattached bacteria and dried in an inverted position.Volumes of 100 µL of appropriate twofold dilutions of the ciprofloxacin or ET37 in Mueller-Hinton broth were transferred into the dried wells with established biofilms.e microtiter plates were incubated for 18-20 hours at 37 °C, and minimum biofilm inhibitory concentration (MBIC) was determined, which corresponds to the lowest concentration of antibiotic which inhibits growth of biofilm cells as indicated by absence of visible growth in the wells.A positive control and a negative control were included in all experiments.e experiment was repeated three times.

Determination of Minimum Duration for Killing 99% of the Population (MDK 99 )
. MDK 99 was determined by measuring the time to kill 99% of the population [33].MDK99 was tested in P. aeruginosa clinical strains with concentration 1x the MIC of ciprofloxacin or 1x the MIC ET37 in P. aeruginosa wild-type and LasR mutant clinical strains.

Effect of LasR Antagonists on Biofilm Formation in P.
aeruginosa PAO1 Laboratory Strain.We selected 4 compounds reported to be active antagonists of LasR of P. aeruginosa and designed on 3-O-C12-HLS scaffold (Figure 1, # 1, 2, 3, 4) [23].e synthesis was performed as described in Geske et al. and references cited therein.
We evaluated the effect of the selected 4 LasR antagonists on cell growth and biofilm formation of P. aeruginosa PAO1 laboratory strain.We treated PAO1 immediately after seeding the planktonic bacteria with LasR antagonists at different concentrations (0.1, 1.0, 10.0, 25.0, and 50.0 µM) for 24 hrs.We evaluated cell growth by reading the optical density at 600 nm, and we observed no effect on this parameter (data not shown).Considering biofilm formation, the treatment with number 3 and number 4 compounds reduced the formation of biofilm of more than 50% (Figure 2(a)) (p < 0.0001; Studentʼs t-test).In particular, in the concentration range of 1.0-10.0µM, we observed the highest effect on biofilm inhibition for both number 3 and number 4 compounds.On the contrary, compounds number 1 and 2 failed to significantly inhibit biofilm formation, also increasing the time of exposure to 48-72 hrs (data not shown).On the basis of these results, we selected number 3 and number 4 compounds and the range of concentrations 1.0-10.0µM for further investigations on clinical isolates.

Effect of Number 3 and Number 4 Compounds on
Biofilm Formation in Clinical Strains.First, we evaluate the efficacy of QSi in acting as LasR antagonists in P. aeruginosa clinical strains.We selected two subgroups of P. aeruginosa clinical strains: (i) wild type for the expression of 3O-C12-HSL receptor (LasR); (ii) P. aeruginosa LasR mutant.LasR mutant clinical strains were phenotypically evidenced as producing iridescent and metallic colonies [34,35].We performed our analysis on 10 different isolates from four CF individuals (SG, AA, TR, and KK) wild type or mutant for LasR.In order to evaluate LasR functionality, we treated wild-type (N � 5) and LasR mutant (N � 5) clinical strains immediately after seeding the planktonic bacteria with LasR agonist (1.0, 5.0, 10.0, 25.0, and 50.0 µM) for 24 hours.We analysed cell growth by reading the optical density at 600 nm, and we observed no effect on this parameter (data not shown).Concerning biofilm formation in wild-type strains, we observed biofilm inhibition of 60% with the compound number 3 at the concentrations of 5.0-10.0µM (Figure 2(b)) and compound number 4 reduced biofilm formation of the 30-40% (Figure 2(b)).We observed also a reduction in biofilm formation in LasR mutant clinical strains treated with the same two compounds (number 3 and 4) at the concentrations of 5.0-10.0µM, reporting a decrease of the 30% and 60-70%, when treated with LasR antagonist number 3 and 4, respectively, (Figure 2(c)).To confirm the results obtained by crystal violet staining, we performed the Syto9 assay, based on fluorescence staining.We choose to use Syto9 staining to confirm our data and to overcome the variability of the results that could arise by crystal violet staining of P. aeruginosa biofilm [36].Similarly, Syto9 assay showed a clear decrease of biofilm formation after the addition of the compounds number 3 and 4 in both wild-type and mutant strains (Figure 2(d)), with the highest effect observed at the concentration of 10.0 µM.
Since QS inhibition results in a reduced expression of the virulence factors pyocyanin and elastase and in an alteration of biofilm morphology/phenotype [37], the effect of compound numbers 3 and 4 was tested also on the expression of these genes.We observed that both compound numbers 3 and 4 were able to reduce the production of pyocyanin and elastase in wild-type and LasR mutant strains (Figure 3). between compounds 3 and 4 and cipro oxacin are depicted in Figure 4. e molecules ET37 and ET39 were synthesized as depicted in Figure 5; the decanedioic acid monoethylester 5 was condensed to the amine moiety of the piperazine ring present in the commercially available cipro oxacin antibiotics to obtain compound 6 that was subjected to 2N NaOH saponi cation to achieve acid derivative 7 [38].Finally, compound 7 was condensed to the cyclopentylamine and L-homoserine lactone using WSC/HOBt as activating agents to obtain, respectively, compound ET37 and compound ET39 that were fully characterized by NMR and exact mass spectrometry.ET37 and ET39 were preliminarily tested on PAO1 and P. aeruginosa clinical strains.ET37 maintained its ability to decrease bio lm formation in PAO1 (Figure 6(a)), wild-type (Figure 6(b)) and LasR mutant clinical strains (Figure 6(c)).On the contrary, ET39 lost the e cacy of compound number 4 (Figure 6).Similarly, pyocyanin and elastin were reduced by ET37 treatment in both wild-type (Figure 7(a)) and LasR mutant clinical strains (Figure 7(b)).Starting with PAO1 strain, we evaluated the minimal inhibitory cipro oxacin concentration [28] using the Etest to establish a baseline.e MIC 50 was within the expected ranges, with the PAO1 MIC 50 at 0.5 μg/ml cipro oxacin, corroborating previous research [39]  and at 0.2 μg/ml ET37.
We evaluate the population dynamics in response to cipro oxacin unconjugated and conjugated to compound number 3 (ET37).
e exposure was at concentrations ranging from 0.5 to 8x the MIC in PAO1 strain (Figure 8).
After the treatment with cipro oxacin, a large part of the bacteria was killed by the drug (Figure 8(a)), as the expected response to an antibiotic treatment with an e cacy of 24 h, as illustrated in Figure 5(a).At lower exposure levels, there was a surviving subpopulation of "drug-tolerant" cells, that survived after cipro oxacin treatment (Figure 8  Previous data showed that cipro oxacin reduced virulence factors and bio lm formation decreasing the production of QS signal molecules in P. aeruginosa [40].We tested the minimal bio lm inhibitory concentration (MBIC) of both cipro oxacin and ET37 on PAO1.Results demonstrated that ET37 MBIC was similar to MIC 50 (0.22 μg/ ml) while cipro oxacin MICB was eight times higher (4.0 μg/ml) than MIC 50 (0.5 μg/ml).

ET37 E ect on P. aeruginosa Clinical Strains Susceptibility to Cipro oxacin.
e e cacy of cipro oxacin was then tested also on P. aeruginosa clinical strains.e resulting MIC 50 are reported in Table 2.
When we evaluated the time-kill studies with the ET37 concentration of 4 μg/ml, equal to the theoretical plasma peak [41], we observed 6-log decrease at 24 h for strain SG1, SG58, AA12, and AA11 (Figure 9), which are the most susceptible according to their MICs (Table 2).Interestingly, a bactericidal activity was observed with 3.0-log inoculum reduction at 12 h for strains AA2 and TR66, that were categorized as intermediate and TR2 and KK72, that were considered resistant to cipro oxacin (Table 2) (Figure 9).A bacterial growth reoccurred at 24 h for strains TR1 and KK1 (Figure 9), the most resistant strains according to their MICs (Table 2).

Time-Kill Studies on P. aeruginosa Clinical Strains.
Time-kill studies were performed in parallel, on P. aeruginosa clinical strains, with 1/2, 2, 4, 8, X MIC 50 , for both cipro oxacin and ET37 (Table 2).Following 24 h treatment with cipro oxacin, there was a surviving subpopulation of "drug-tolerant" cells only in low-dose cipro oxacin-treated P. aeruginosa clinical strains (Figure 10(a)), while both wildtype and LasR mutant clinical strains did not change over 48 h treatment with ET37 (Figure 10(b)).
e clinical strains were then tested for their tolerance to cipro oxacin or ET37 treatment.
e measured MDK 99 decreased in the ET37-treated strains in comparison with the cipro oxacin-treated strains (Figure 11).Both P. aeruginosa wild-type and LasR mutant clinical strains showed a MDK 99 of 12 hours after treatment with ET37 in comparison with the MDK 99 of 24 hours observed in the same strains without ET37 treatment (p < 0.1).

Discussion
In this research, for the rst time, we analysed the e ect of selected LasR antagonists, on P. aeruginosa clinical strains, and we identi ed two compounds designed on 3O-C12-HSL sca old, number 3 and number 4, which exhibit an inhibitory e ect on both LasR wild-type and mutant strains.
ese LasR antogonists interact with the N-terminal ligandbinding domain of LasR, blocking the binding site for QS molecules.
Since clinical P. aeruginosa strains are commonly LasR mutants, it is fundamental to obtain QSi with a demonstrated functionality also in LasR mutant clinical strains.Both number 3 and 4 compounds are able to counteract bio lm formation also in LasR mutant strains, possibly due to the shorter aliphatic chain that results in a lower steric hindrance that could facilitate the cross interaction with RhlR [17,18].In fact, it has been reported that LasR antagonist could be e ective also on other QS receptors, as

Journal of Chemistry
RhlR [17,19].We speculate that, in the presence of LasR mutations, LasR antagonists numbers 3 and 4 could interact with other QS receptors that are still functional in LasR mutant strains.It has been described that some virulence factors, such as pyocyanin, are still produced in LasR mutants, con rming that QS system is still functional.is could be explained by the ability of P. aeruginosa to circumvent QS de ciency using rhl and Pqs systems [20].Similarly, 3O-C12-HSL is known to bind not only its receptor LasR, but also other QS receptor, as RhlR and PqsR.
is is in line with the results of Kalaiarasan and coauthors reporting the ability of two anti-QS compounds to decrease bio lm production in two P. aeruginosa clinical isolates, a ecting both lasR and rhlR transcription [42].
e identi cation of new mechanisms to block the appearance of drug resistance in P. aeruginosa is important because it is characterized by a quick adaptation to resist to new drug compounds [1], causing chronic lung infection in individuals with CF [43] or chronic obstructive pulmonary disease (COPD) [44], and the 10% of hospital-acquired infections.
e antibiotics used to treat P. aeruginosa infection include cipro oxacin, tobramycin, ceftazidime, gentamicin, and imipenem.P. aeruginosa bacteria present a high degree of resistance to these antibiotics.Interestingly, the response to cipro oxacin is very e ective at the beginning of the treatment, but high-level resistance is rapidly acquired by P. aeruginosa, making the treatment ine ective in the 30% of strains obtained from clinical isolates [1].e mechanisms that could increase P. aeruginosa antibiotic susceptibility still remain unclear [2][3][4] and few studies tried to develop therapeutic strategies to decrease the insurgence of resistances.10 Journal of Chemistry e onset of resistant mutants seems to be connected with the use of antibiotic concentrations that can select mutants, that ranges between 0.5 and 8 μg ml −1 [45], in line with the concentrations used in our work.As drug concentrations increased, we observed the selection of particular tolerant clones ("drug-tolerant").e use of ET37 reduced the formation of bio lm, the expression of virulence genes (e.g., pyocyanin and elastin), and the onset of tolerant clones.
is was obtained also in those strains that presented a genetic mutation in GyrA and ParC, that are now known to play an integral role in quinolone resistance in P. aeruginosa [6,7].
From the time-kill curves with cipro oxacin and ET37, it seems that the pharmacodynamics of cipro oxacin changes from concentration-/dose-dependent killing, as reported in Figure 8(a), to more time-dependent killing, as we observed similar time-killing curves at di erent ET37 concentrations (1x, 2x, and 4x MICs), as reported in Figure 8(b).is is also evident in the time-kill curves of the clinical isolates presented in Figure 10. is might suggest that the mechanisms involved in cipro oxacin tolerance is quorum-sensing related.In fact, when the clinical strains were tested for their tolerance to cipro oxacin or ET37 treatment, we observed a decrease in MDK 99 after ET37 treatment in comparison with the ciprooxacin (Figure 11).Both P. aeruginosa wild-type and LasR mutant clinical strains showed an MDK 99 of 12 hours after treatment with ET37 in comparison with the MDK 99 of 24 hours observed in the same strains without ET37 treatment.
e reduced incidence of tolerant bacteria after ET37 treatment could be associated with a modi ed gene expression [34].In particular, cipro oxacin treatment induces the production of hydroxyl radicals that might cause oxidation-mediated cell death [46].e onset of drug-tolerant bacteria might be associated with the increase in the phosphorylation of two proteins, PA0265 (succinate-semialdehyde dehydrogenase (SSADH), encoded by GabD) and PA3570 (methylmalonate-semialdehyde dehydrogenase (MMSADH), encoded by MmsA) [8], that seems to induce the production of NADH as a reaction product.NADH is a reducing agent, induced in response to environmental stress [35] to prevent oxidative damage.
erefore, the upregulation of NADH by the phosphorylation of these two proteins might bu er oxidative stress induced by cipro oxacin and facilitate the onset of tolerant bacteria.ET37 compound could facilitate the accumulation of cipro oxacin within the cell, increasing the intracellular superoxide anion (O2 − ) concentration causing more oxidative stress that cannot be bu ered by SSADH/ MMSADH, than in P. aeruginosa strains treated with ciprooxacin alone.e bene t of ET37 in comparison with cipro oxacin is its ability to reduce the formation of bio lm and consequently diminishing the physical barrier to ciprooxacin penetration into bacterial cells.In fact, the MBIC of ET37 was similar to MIC 50 (0.22 μg/ml), while cipro oxacin MBIC was eight times higher (4.0 μg/ml) than MIC 50 (0.5 μg/ ml).Moreover, the inhibition of QS could also a ect the production of rhamnolipid.e production of rhamnolipid in P. aeruginosa is controlled by the transcriptional regulator RhlR of the QS system [47].Even if the role of rhamnolipids in bacterial physiology is not clear, they seem to take part to the assimilation of insoluble substrates [36], to antimicrobial activities [48], to hemolytic activity [49], to the solubilization of the quinolone signal, and to the swarming motility [50].Moreover, rhamnolipids seem to reduce the activation of host innate immunity, facilitating P. aeruginosa survival and colonization on compromised epithelia.e decrease in rhamnolipids production by QSi could facilitate the elimination of the infection by the host's immune system.

Conclusions
On the basis of our results, we reported, for the rst time, the reduction of bio lm formation and cipro oxacin tolerance in P. aeruginosa clinical strains treated with ET37 compound.is molecule, generated by the conjugation of a QSi and cipro oxacin, could have a wide application in clinical setting.
e possibility to a ect bio lm formation in chronically infected patients, such as CF and COPD patients, and to reduce the onset of cipro oxacin tolerance would improve patient healing and allow to decrease antibiotic drug dosage.

Figure 10 :
Figure10: (a) Time-kill studies with P. aeruginosa clinical strains with concentration ranging from 0.5 to 8x the MIC of cipro oxacin.(b) Time-kill studies with P. aeruginosa wild-type clinical strains with concentration ranging from 0.5 to 8x the MIC of ET37 treatment.(c) Time-kill studies with P. aeruginosa LasR mutant clinical strains with concentration ranging from 0.5 to 8x the MIC of ET37 treatment.Red arrow: "drug-tolerant" cells in cipro oxacin-treated P. aeruginosa clinical strains; blue arrows: "drug-tolerant" cells in ET37-treated P. aeruginosa clinical strains.

Table 1 :
Mutations in LasR in P. aeruginosa isolates.
a LasR status evaluated by phenotypic analysis.bNumberingis based on the sequence of LasR gene and LasR protein from PAO1.