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RESEARCH ARTICLE Polyamino-Isoprenic Derivatives Block Intrinsic Resistance of P. aeruginosa to Doxycycline and Chloramphenicol In Vitro Diane Borselli 1 , Aurélie Lieutaud 1 , Hél
ène Thefenne 1,2
, Eric Garnotel 1,2
, Jean- Marie Pag ès 1
3 , Jean-Michel Bolla 1 *
2 Hôpital d'Instruction des Armées Alphonse-Laveran, 13013 Marseille, France, 3 Centre de Recherche en Cancérologie de Marseille (CRCM), CNRS, UMR7258, Institut Paoli Calmettes, Aix-Marseille Université, UM 105, Inserm, U1068, F-13009, Marseille, France * jean-michel.bolla@univ-amu.fr Abstract Multidrug resistant bacteria have been a worldwide concern for decades. Though new mol- ecules that effectively target Gram-positive bacteria are currently appearing on the market, a gap remains in the treatment of infections caused by Gram-negative bacteria. Therefore, new strategies must be developed against these pathogens. The aim of this study was to select an antibiotic for which a bacterium is naturally resistant and to use an escort molecule to restore susceptibility, similarly to the model of β-lactam/ β-lactamase inhibitors. High-con- tent screening was performed on the reference strain PA01, allowing the selection of four polyamino-isoprenic compounds that acted synergistically with doxycycline. They were assayed against clinical isolates and Multi-Drug-Resistant strains. One of these compounds was able to decrease the MIC of doxycycline on the reference strain, efflux pump overpro- ducers and clinical isolates of P. aeruginosa, to the susceptibility level. Similar results were obtained using chloramphenicol as the antibiotic. Membrane permeation assays and real-time efflux experiments were used to characterize the mechanism of doxycycline potentiation. The results showed that the selected compound strongly decreases the efficiency of glu- cose-triggered efflux associated with a slight destabilization of the outer membrane. Accord- ing to these data, targeting natural resistance may become an interesting way to combat MDR pathogens and could represent an alternative to already devised strategies. Introduction With the current global crisis of antibiotic resistance, any strategy that could improve the ther- apeutic tools used in the fight against bacterial infections must be utilized [ 1 ]. National and International health agencies have successfully established a series of measures to encourage the control of the usage of drugs and the development of new molecules. Although significant progress has been made in the fight against Gram-positive infections in the last decade, a gap PLOS ONE | DOI:10.1371/journal.pone.0154490 May 6, 2016 1 / 16
a11111 OPEN ACCESS Citation: Borselli D, Lieutaud A, Thefenne H, Garnotel E, Pagès J-M, Brunel JM, et al. (2016) Polyamino-Isoprenic Derivatives Block Intrinsic Resistance of P. aeruginosa to Doxycycline and Chloramphenicol In Vitro. PLoS ONE 11(5): e0154490. doi:10.1371/journal.pone.0154490 Editor: Eric Cascales, Centre National de la Recherche Scientifique, Aix-Marseille Université, FRANCE Received: November 3, 2015 Accepted: April 14, 2016 Published: May 6, 2016 Copyright: © 2016 Borselli et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Aix-Marseille University funded the work. Competing Interests: The authors have declared that no competing interests exist. remains in the treatment of Gram-negative bacteria, particularly in the discovery of new active molecules [ 2 ,
]. Effective Gram-positive targeting molecules are globally not very active against Gram-negative bacteria, though the same targets exist in both types of bacteria [ 4 ]. In
the latter, the major problem of antibiotic entry and accumulation of the drug near the target has not yet been solved [ 5 ] This is due, in part, both to the low permeability of Gram-negative bacteria, which are surrounded by the outer membrane, that decreases the entry of compounds and to the constitutive expression of efflux pumps by these bacteria [ 6 ,
]. Moreover, it is well documented that they are able to overproduce these efflux pumps in response to extra-cellular compounds, including drugs [ 8 ]. Thus, screenings based on an in vitro test for a particular tar- get face the recurring problem of the permeability of Gram-negative bacteria [ 9 ]. Therefore, it may be interesting to first identify molecules effective on whole bacteria and then, if the test is positive, search for their mode of action. We used this approach to address the question of the natural resistance of Pseudomonas to doxycycline. The aim of this study was to select an antibiotic for which a bacterium is naturally resistant and to use an escort molecule to restore susceptibility, similarly to the model of β-lactam/ β-lac- tamase inhibitors. This strategy could constitute an opportunity for an old neglected molecule to be rejuvenated by using an adjuvant to improve its action. Pseudomonas aeruginosa represents a serious therapeutic challenge for both community- acquired and nosocomial infections. P. aeruginosa possesses an intrinsic high-level antibiotic resistance due a naturally low permeable outer membrane (1/100 of E. coli outer membrane). It makes the bacterium resistant to many classes of antibiotics [ 8 ]. Together with impermeability, the low susceptibility of P. aeruginosa to β-lactam antibiotics results from the overexpression of the intrinsic AmpC cephalosporinase, and the acquisition of extended-spectrum β-lacta-
mases, of metallo-carbapenemases, and extended spectrum oxacillinases [ 10 ]. In addition, the inherent resistance of P. aeruginosa is, to a great extent, due to an active efflux of a broad spectrum of molecules. This efflux is mediated by three-component systems that belong to the Resistance Nodulation cell Division family of transporters (RND). MexA- B-OprM and MexXY-OprM are the most studied [ 11 –
] and the most prevalent in clinical isolates [ 14 ]. MexAB-OprM is constitutively expressed and confers the ability to export several classes of drugs [ 15 ]. The expression of MexXY-OprM contributes to both intrinsic and acquired resistance [ 16 ], and this resistance can be induced by antibiotics such as tetracyclines and macrolides [ 16 ]. In an on-going project dedicated to the synthesis of new biologically active molecules, we identified several polyamino-isoprenic derivatives that were able to decrease chloramphenicol- and nalidixic acid-resistance levels of multi-drug-resistant Enterobacterial strains [ 17 ]. In the present study, we describe the use of this chemical library to screen chemosensitizers that could lower the MIC of doxycycline on P. aeruginosa under the threshold of susceptibility. We also present data supporting the concept that polyamino-isoprenic compounds potentiate doxycycline ’s action by circumventing the intrinsic resistance. Materials and Methods Bacterial strains and growth conditions The P. aeruginosa strains used in this study are described in Table 1 . In addition, a series of 20 clinical isolates of P. aeruginosa obtained from the Hôpital d ’Instruction des Armées Laveran, HIA-Laveran (Marseille, France) were also used in this study ( S1 Table
). Strains were stored at -80°C in 15% (v/v) glycerol for cryo-protection. Bacteria were routinely maintained on Muel- ler-Hinton (MH) agar plates and grown in cation-adjusted MH broth (MHBII) at 37°C. Inhibitors of Antibiotic Resistance in Pseudomonas PLOS ONE | DOI:10.1371/journal.pone.0154490 May 6, 2016 2 / 16
Antibiotics and chemicals The antibiotics doxycycline, polymyxin-B polymyxin-B nonapeptide (PMBN) and chloram- phenicol and Phenylalanine-Arginine Beta-Naphthylamide (PAßN), carbonyl cyanide m- chlorophenylhydrazone (CCCP), cetyl trimethylammonium bromide (CTAB), 4-(2-hydro- xyethyl)piperazine-1-ethanesulfonic acid (HEPES) and benzalkonium chloride were purchased from Sigma (St Quentin Fallavier, France). EDTA was from Fisher-Scientific (Quichy-le-châ- teau, France) The antibiotic imipenem was purchased from Pharmacopeia (USA). They were dissolved in water or dimethyl sulfoxide (DMSO) as indicated. The polyamino-isoprenic deriv- atives were described previously [ 18 ]. They were dissolved in DMSO and stored at -20°C until use. Antibiotic susceptibility testing Assays were carried out in 96-well microtiter plates using a two-fold standard broth micro- dilution method, as previously described [ 19 ]. Experiments were performed on the BAC-SC- REEN platform of the UMR-MD1, with a Freedom EVO 150 liquid handling system (Tecan, Lyon, France). The MIC of doxycycline, chloramphenicol and of the 4 compounds for each strain is given in Table 1
. High-content screening The polyamino-isoprenic compounds library [ 18 ] was screened against the P. aeruginosa PA01 reference strain ( Table 1
) in the presence of 4 mg/L doxycycline that corresponds to the con- centration under the threshold of resistance according to the CLSI guidelines, in order to evalu- ate if one of the compound is able to decrease the intrinsic resistance of P. aeruginosa. For further experiments we decided to reduce the concentration of doxycycline at 2mg/L to increase the stringency of the tests. Indeed, this concentration corresponds to the threshold of the susceptibility according to the CLSI. The screening was carried out in 96-well microtiter plates using MHBII, in a final volume of 200 μL. The concentration of library compounds was 10 μM, with a final DMSO concentration of 2.5%. The bacterial inoculum of 5×10 5 CFU/mL
was prepared from an overnight culture. In addition to the polyamino-isoprenic compounds, each plate contained PAßN, CCCP, CTAB, polymyxin-B and benzalkonium chloride as con- trol molecules. Plates were incubated at 37°C under aerobic conditions for 18 hours. Plates were read at 600 nm on an Infinite M200 Pro plate reader (Tecan). Results are summarized in S1 Fig .
–4. Strains
Relevant phenotype Source and reference Minimal Inhibitory Concentration mg/L ( μM)*
DOX CHL
1 2 3 4 PA01
wt P. Plésiat [ 20 ]
256 > 85 (>250) > 81 (>250) 25 (62.5) 49 (125) PT629
Mex AB overproducer P. Plésiat [ 21 ]
512 > 85 (>250) > 81 (>250) 25 (62.5) 49 (125) PA01 ERY
R Mex CD overproducer P. Plésiat [ 22 ] 16 256
> 85 (>250) > 81 (>250) 25 (62.5) 25 (62.5) PA0-7H Mex EF overproducer P. Plésiat [ 23 ] >32 >1024
85 (250) > 81 (>250) 25 (62.5) 49 (125)
CMZ091 Mex XY overproducer P. Plésiat [ 24 ] 32 256
85 (250) > 81 (>250) 25 (62.5) 49 (125)
FB1 Mex B deleted P. Plésiat [ 25 ] 32 8 85 (250) > 81 (>250) 25 (62.5) 49 (125) DOX, doxycycline; CHL, chloramphenicol. * Results are the mean of 3 independent experiments. doi:10.1371/journal.pone.0154490.t001 Inhibitors of Antibiotic Resistance in Pseudomonas PLOS ONE | DOI:10.1371/journal.pone.0154490 May 6, 2016 3 / 16
Comparison of efficiency between compounds and PAßN Serial dilutions of each compound (from 270 μM to 0.95 μM for PAßN and from 135 μM to 0.06
μM for 3) were prepared in microplates. Doxycycline (2 mg/L) was then added to each well before inoculation of bacteria (5x10 5 cfu/mL). Optical density (600 nm) was measured after 18 hours at 37°C. Only the relevant part of the graphs are shown. Experiments were per- formed in triplicate. Checkerboard assay Serial two-fold dilutions of antimicrobial agents were mixed together in a microtiter plate. The range of concentration of each drug was twice the MIC, down to the eleventh serial two-fold dilu- tions below this amount. Doxycycline was serially diluted along the X-axis, and the polyamino- isoprenic compound or PAßN was diluted along the Y-axis as indicated. Each well was inoculated with a bacterial inoculum of 5 x 10 5 CFU/ml, and the plates were incubated at 37°C for 18 h under aerobic conditions. Each plate also contained a row and a column in which a serial dilution of polyamino-isoprenic compound or doxycycline, respectively, was present alone to determine the MIC. The FIC index was calculated as follows: FIC index = FIC A + FIC B, where FIC A is the MIC of drug A in the combination/MIC of drug A alone and FIC B is the MIC of drug B in the combination/MIC of drug B alone. The combination was considered synergistic when the FIC index is
0.5, and antagonistic when the FIC index is > 4 [ 26 ]. Results are described in S2 Table . Effect of combined antibiotics –polyamino-isoprenic derivatives MH agar plates were prepared, containing 2 mg/L doxycycline and 2 mg/L doxycycline in com- bination with compounds at a concentration of 10 μM. Bacteria, 5 μL of a suspension of each clinical isolate adjusted to 5 x 10 5 colony-forming units per ml were spotted on each plate and growth was monitored after a 18 h period of incubation at 37°C. The isolates that repeatedly grown on the plates containing doxycycline alone and compound alone but that did not grow on both doxycycline and compound, were identified as susceptible of the combination. In a second experiment, 5 μL of logarithmic dilutions of an overnight culture of the 6 strains described in Table 1 were spotted on the MH agar plates prepared as above. An additional assay was also performed with these 6 strains using chloramphenicol as antibiotic instead of doxycycline. Outer membrane permeation assay An overnight culture of PA01 was diluted 100-fold into 10 ml MHII broth containing 0.01 μg/ ml of imipenem to induce a higher level of β-lactamases. After reaching an OD600 nm of 0.5, cells were recovered by centrifugation (4,000 X g for 20 min) and washed twice in 20 mM potassium phosphate buffer (pH 7.2) supplemented with 1 mM MgCl 2 (PPB) at an OD 600 nm of 0.5. 50 μl of each compound was added to 100 μl of the cell suspension, yielding final concentrations ranging from 125 μM to 15.6 μM. Then, 50 μl of nitrocefin was added to obtain a final concentration of 50 μg/ml. Absorbance at 490 nm was monitored by spectrophotometry using an Infinite M200 microplate reader (Tecan) over 60 min. Experiments were performed in triplicate. For each compound, the efficacy of permeation was determined using the slope in the linear range, relatively to the slope obtained with 250 μM polymyxin-B. Real-time efflux assay The experiments were performed as previously described [ 17 ] with slight modifications. For each strain, 20 mL of MHII was inoculated with a single colony from a fresh plate and grown at Inhibitors of Antibiotic Resistance in Pseudomonas PLOS ONE | DOI:10.1371/journal.pone.0154490 May 6, 2016 4 / 16
37°C to the stationary phase. The cells were then recovered by centrifugation (4,000 X g for 20 min) and washed once in 20 mM PPB. They were then loaded over night with the dye 1,2 ’- dinaphthylamine (TCI-Europe SA, Zwijndrecht, Belgium) at a final concentration of 32 μM, in the presence of 5 μM CCCP. The cell suspension was then recovered by centrifugation (4,000 X g for 20 min) and re-suspended in the same volume of PPB and adjusted to an OD600 nm of 0.5. In a 96-well Greiner black microplate (Greiner, Courtaboeuf, France), a gradient of inhibi- tor (when used) was prepared in PPB, and bacteria were added (100 μL per well). The fluores- cence of the cell suspension, was monitored each 22 sec during 1200 sec on an Infinite M200 microplate reader (Tecan; excitation wavelength 370 nm and emission wavelength 420 nm). In order to trigger the transport, glucose (50 mM final concentration) is added at 200 sec. The maximum efficacy of the efflux is the difference between the value obtained without and with glucose addition after 1200 sec. For each compound, the efficiency of efflux inhibition is calcu- lated as the difference between the maximum efficacy of transport and the residual transport obtained at various concentrations of the compound. Cytotoxicity assessment CHO-K1 cells (ATCC-LGC Standards Sarl (Molsheim, France) and human fibroblasts (Clinis- ciences, Paris, France) were maintained in McCoy ’s 5A and DMEM media, respectively, sup- plemented with 10% bovine calf serum, 2 mM glutamine, and 100 U/mL/10 μg/mL penicillin/ streptomycin. They were incubated at 37°C in a humidified atmosphere containing 5% CO 2 . The cell lines were seeded in 96-well plates and incubated overnight. The cytotoxic effects of compounds were assessed by the colorimetric WST-1 cell proliferation assay as previously described [ 17 ]. Briefly, a range of compound concentrations from 30 μM to 1200 μM was added to triplicate cultures, and cells were incubated at 37°C for 24 h. At the end of the incuba- tion period, cultures were washed three times with phosphate buffer saline (PBS) and incu- bated in fresh culture medium containing 10% WST-1 for an additional 30 min. Cell viability was evaluated by measuring the WST-1 absorbance at 450 nm in a microplate spectrophotom- eter MRX1 II (Dynex technologies, Chantilly, VA, USA). The Inhibitory Concentration 50% (IC 50
50 was defined as the concen- tration of a compound that induced a 50% decrease in viable cells. Doxorubicin was used as a positive control. Statistical analysis Data were analyzed using the student t-test analysis for differences between two groups, and findings were expressed as mean + SD. All assays included 2 replicates and were repeated in at least 2 independent experiments. A p value < 0.05 was considered to be statistically significant. Results
Selection of polyamino-isoprenic compounds A series of 60 compounds, which were described [ 18 ], were tested at a concentration of 10 μM in combination with a sub-inhibitory concentration of doxycycline against the reference strain of P. aeruginosa PA01. In parallel, we evaluated the intrinsic antimicrobial activity of these compounds alone to allow the selection of only doxycycline adjuvants. The results are summa- rized in S1 Fig
. Under these conditions, three compounds, 2 –4 (
Fig 1 ), were able to decrease the MIC of PA01 for doxycycline below the threshold of resistance (4 mg/L), [ 27 ]. Interest- ingly, these three compounds share similar structures with however, very distinct LogD values ( Fig 1 ). Compounds 2 and 4 share the same polyamine and only differ in their isoprenyl Inhibitors of Antibiotic Resistance in Pseudomonas PLOS ONE | DOI:10.1371/journal.pone.0154490 May 6, 2016 5 / 16
moiety. Conversely, 3 and 4 share the same isoprenyl group and differ in their polyamines. In the subsequent experiments, we also considered compound 1, which has the same isoprenyl group as 2 and the same polyamine as 3 but did not show any significant synergy. Cytotoxicity of the selected compounds The cytotoxicity on Chinese Hamster Ovary cells and human normal fibroblasts, was deter- mined using the metabolic WST1 assay for the four compounds ( Table 2 ). Compound 1 appeared to be the least cytotoxic on both cell lines, with values ranging from 20 times for the CHO to 10 times for human fibroblasts compared with the positive control, the doxorubicin. Compounds 2 and 3 showed similar values that were lower than those of 1 but still clearly above those of doxorubicin for the CHO cell line. Compared with 2 and 3, compound 4 exhib- ited the same toxicity on CHO cells and lower toxicity on human fibroblasts. Efficacy comparison of the selected compounds PA01 is a well-studied reference strain of P. aeruginosa, however to extend our study we consid- ered strains recently isolated from hospitalized patients [ 21 –
]. Thus, we decided to test this Yüklə 0,79 Mb. Dostları ilə paylaş:
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