# Polyamino-isoprenyl derivatives as antibiotic adjuvants and motility inhibitors for 1 Bordetella bronchiseptica porcine pulmonary infection treatment 2 3

Diane Borselli, Jean Michel Brunel, Olivier Gorgé, Jean Michel Bolla

► To cite this version:

Diane Borselli, Jean Michel Brunel, Olivier Gorgé, Jean Michel Bolla. Polyamino-isoprenyl derivatives as antibiotic adjuvants and motility inhibitors for 1 Bordetella bronchiseptica porcine pulmonary infection treatment 2 3. Frontiers in Microbiology, 2019, 10, 10.3389/fmicb.2019.01771. [https://doi.org/10.3389/fmicb.2019.01771](https://doi.org/10.3389/fmicb.2019.01771). This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License.

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1 Polyamino-isoprenyl derivatives as antibiotic adjuvants and motility inhibitors for

2 *Bordetella bronchiseptica* porcine pulmonary infection treatment

3

4 Diane Borselli¹, Jean Michel Brunel¹, Olivier Gorgé¹, Jean Michel Bolla¹*

5 ¹ Aix Marseille Univ, INSERM, SSA, IRBA, MCT, Marseille

6 (*) Corresponding author:

7 JM Bolla, Laboratoire MCT, Faculté de Pharmacie, 27 Bd Jean Moulin, 13385 Marseille

8 cedex 05

9 e-mail: jean-michel.bolla@univ-amu.fr

10 tel : 33 491 324 440; Fax : 33 491 324 606

11

12 Keywords: Antibiotic resistance, Combination therapy, Whole-cell screening, motility,

13 virulence, *Bordetella bronchiseptica*.
14 Abstract

15
16 The spreading of multidrug resistant bacteria and the lack of novel antibiotic molecules are
17 leaving clinicians and veterinarians with very limited options to treat bacterial infections,
18 especially those caused by Gram-negative pathogens. To reduce the selection of antibiotic
19 resistance mechanisms and their transfer to human pathogens, veterinary pharmaceutical
20 companies have dramatically decreased the number of used antibiotics. Among all the
21 investigated alternate solutions, chemosensitizers, which decrease the amount of the used
22 drugs appears to be one of the most promising strategy. In this study, we reported that
23 polyamino-isoprenyl derivatives can potentiate florfenicol activity against veterinary sensitive
24 reference strains as well as clinical isolates. These molecules induce inner membrane
25 depolarization and subsequently inhibit efflux pumps by collapsing the proton motive force
26 (PMF). Considering that *B. bronchiseptica* rotor flagellum is highly PMF dependent and that
27 flagellar motility represents an important factor involved in colonization, we monitored the
28 swimming and swarming motilities of bacteria and showed a strong inhibition in the presence
29 of the lead selected compound. Taken together, our results suggest that this class of molecules
30 are able to increase treatment efficacy and decrease drug consumption.
## Introduction

In Europe, the overall sales of veterinary antimicrobial agents used in food producing animals represented 7680 tons in 2016 (https://bi.ema.europa.eu/analyticsSOAP/saw.dll?PortalPages), and the main use (around 60%) concerns the pig industry (Schwarz et al. 2001; Chauvin et al. 2002). The release of antibiotics in the environment by livestock production is one of the major contributing factors for the emergence of bacterial resistance in pathogens affecting both humans and animals (Jechalke et al. 2014, Wichmann et al. 2014, Bártíková et al. 2016, Albero et al. 2018). Thus, it is crucial to decrease the antibiotic consumption in veterinary field and particularly in livestock without affecting animal health (Wellington et al. 2014, Pruden et al. 2014). Consequently, strategies have been proposed, and one of the most successful consists of a dual drug approach allowing an efficient enhancement of antibacterial activity while minimizing the drug concentration (Brooks and Brooks 2014, Collignon et al. 2016).

The World Health Organization and the European Commission have published guidelines concerning the prudent use, in food production animals, of cephalosporin of 3rd and 4th generation and fluoroquinolones, which are considered as “critically important” antimicrobial agents in human medicine (https://bi.ema.europa.eu/analyticsSOAP/saw.dll?PortalPages, Briyne et al. 2014). According to the international recommendations, the antimicrobials that could be used to treat porcine respiratory tract infections are tetracyclines, phenicols, macrolides, fluoroquinolones or a combination of trimethoprim and sulfonamides (Kadlec et al. 2004, Kadlec et al. 2007). It is noteworthy that respiratory diseases in swine such as pneumonia and atrophic rhinitis are caused by the Gram-negative coccobacillus *Bordetella bronchiseptica* that requires a tetracycline treatment even if resistance can occur (Prüller et al. 2015, Kadlec et al. 2018). Otherwise, florfenicol is a good alternative (Chauvin et al. 2002), because this antibiotic has a good distribution in the body and few side-effects. However, although florfenicol was only used in veterinary medicine, a cross-resistance by efflux with its non-fluorinated analogue, chloramphenicol has been evidenced (Kadlec et al. 2007).

In an ongoing project dedicated to reducing the florfenicol consumption from breeding pig holdings, we screened polyamino-isoprenyl compounds that were previously described (Borselli et al. 2016) for their ability to potentiate florfenicol activity. The objective of this study is to identify a compound with significant activity and to elucidate its mechanism of action on the membrane physiology and to evaluate its impact on the bacterial motility.
## 64 Materials and methods

## 66 Bacterial strains and growth conditions

The *B. bronchiseptica* strain used in this study comes from the Institut Pasteur collection, CIP 55.110. Ten isolates from pig husbandries in France were obtained from Labofarm (Loudéac France) (Table 1). The bacterial identification was carried out by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) Biotyper (Brüker, Billerica, Germany) with comparison with the manufacturer's bacterial database. All the strains were grown either on Columbia agar at 35°C for 24h to 48h or in Columbia broth at 35°C for 24h.

## 74 Antibiotics and Chemicals

The antibiotic florfenicol was supplied by the pharmaceutical company Virbac (Carros, France). The antibiotic imipenem was purchased from Sequoia Research Products (Pangbourne, UK). All the chemicals Polymyxin B, Polymyxin B nonapeptide (PMBN), Phenylalanine-arginine beta-naphthylamide (PAβN), Carbonylcyanide m-chlorophenylhydrazone (CCCP), Cetyl trimethylammonium bromide (CTAB), Dimethyl sulfoxide (DMSO), 3,3'-Dipropylthiacarbocyanine iodide (DiSC₃(3)), Hydroxyethyl piperazineethanesulfonic acid (HEPES), Ethylene diamine tetraacetic acid (EDTA) and Benzalkonium chloride were purchased from Sigma Aldrich (St Quentin Fallavier, France). The polyamino-isoprenyl derivatives chemolibrary was described previously (Bolla *et al.* 2012).

## 86 MIC determination

Minimal inhibitory concentrations of antibiotics (MIC) were measured by the microdilution broth method in 96-well microtiter plates using the two-fold dilution method and according to the NCCLS guidelines. The inoculum was adjusted at 5x10⁵ CFU/mL in a final volume of 200 µL. MIC was visually determined after 24 hours incubation at 37°C. Assays were performed in three independent biological experiments.

## 93 High content screening

The polyamino-isoprenyl derivatives chemical library was screened at 10 µM final concentration in the absence and in the presence of sub-inhibitory concentration of florfenicol (MIC/4) to identify only antibiotic adjuvants. We also added control molecules as polymyxin B, PAβN, CCCP, CTAB and benzalkonium chloride at 10 µM. The screening was performed in 96-well microtiter plates in Columbia broth with a final volume of 200 µL and a DMSO final concentration of 2.5%. The bacterial inoculum was adjusted to 5x10⁵ CFU/mL. Microtiter plates were read at 600 nm on an Infinite M200 Pro plate reader (Tecan, Lyon, France) after 24h incubation at 35°C. Assays were performed in three independent biological experiments.

## 104 General procedure for the synthesis of compounds for the hit to lead procedure

The general synthetic pathway is illustrated for the preparation of compound 1. A mixture of farnesal (345 mg, 2.27 mmol), titanium(IV)isopropoxide (645 mg, 2.27 mmol) and spermine (2.27 mmol) in absolute methanol (5 mL) was stirred at room temperature for 12 hours. Sodium borohydride (172 mg, 4.5 mmol) was then added at 0°C and the resulting mixture was stirred for an additional 2 hours. The reaction was then quenched by adding water (1 mL). Stirring was maintained at room temperature for 20 minutes. After filtration over a pad of Celite washing with methanol and ethylacetate, the solvents were removed under vacuum and the crude amine was purified by flash chromatography on silica gel. Using
113 CH2Cl2/MeOH/ NH4OH (7/3/1) as eluent affording the expected coupling product 1 in 64%
114 yield (see also supplementary material).

115

116

117 Checkerboard assay

118 Combinations of polyamino-isoprenyl derivatives with florfenicol were tested by using the
119 previously described microdilution checkerboard method (Kadlec et al. 2018). Briefly, an
120 inoculum (100 µL) of 5x10⁵ CFU/mL of B. bronchiseptica strain was added to 96-well
121 microtiter plates containing a serial two-fold dilution of antimicrobial agents in Columbia
122 broth. After incubation of plates during 24 h at 35°C, we determined the MIC for each
123 combination and calculated the fractional inhibitory concentration indexes (FICIs) allowing
124 us to characterize the level of efficiency of the combination. The FICIs are calculated as
125 follows: FICI = FIC A + FIC B, where FIC A= MIC antibacterial A in combination/ MIC
126 antibacterial A alone and FIC B= MIC antibacterial B in combination/ MIC antibacterial B
127 alone. The antibacterial combination was considered synergistic when the FICI was ≤0.5,
128 indifferent when 0.5 < FICI ≤ 4 and antagonistic when FICI > 4 (Odds 2003).

129

130 Outer membrane permeabilization assay

131 100 milliliters of Columbia broth were inoculated with an overnight culture of a B.
132 bronchiseptica isolate producing β-lactamases (isolate 77-A12). Once the cultures reached an
133 the mid-logarithmic phase, imipenem was added (0.125 mg/L final concentration) for 2 hours,
134 then cells were recovered by centrifugation (3,600 X g, 20 min, 20°C) and washed twice in 20
135 mM potassium phosphate buffer (pH 7.2) 1 mM MgCl2 (PPB) supplemented with 30 µM of
136 CCCP that allows the inactivation of active efflux (Nagano and Nikaido 2009). After the
137 second centrifugation, the cell suspension was adjusted to 0.5 OD 600 nm. One hundred
138 microliters of the bacterial suspension were mixed with 50 µL of compound derivatives
139 dilutions already set up in microplates, to give final concentrations ranging from 1.56 to 200
140 µM. Then, 50 µL of nitrocefin was added to obtain a final concentration of 50 µg/mL. The
141 absorbance at 490 nm was monitored to follow the nitrocefin hydrolysis using a Sunrise
142 microplate reader (Tecan) over 2 hours and 30 minutes. Experiments were performed in
143 triplicate. For each compound, the efficacy of permeation was determined using the slope in
144 the linear range, relatively to the control slope obtained with 100 µM polymyxin-B.
145

146

146 Real Time efflux assay

147 The strain CIP55.110 was inoculated into 20 mL of Columbia broth and grown for 24 h at
148 35°C to reach the stationary phase. Cells were recovered by centrifugation (3,600 X g 20 min,
149 20°C) and washed twice in PPB. The cell suspension was adjusted to 0.5 OD 600 nm and
150 loaded with 1,2' dinaphthylamine (TCI-Europe SA, Zwinjndretch, Belgium) 32 µM final
151 concentration, supplemented with the proton dissipator CCCP 30 µM. After overnight
152 incubation, cells were washed in PPB and the bacterial suspension was adjusted to 0.5 OD
153 600 nm. In a 96-well Greiner black microplate (Greiner, Courtabœuf, France) 50 µL of the
154 desired tested compounds were mixed with 100 µL of the cell suspension yielding final
155 concentration ranging from 12.5 to 200 µM. The dye transport was triggered after addition of
156 5 µL of Columbia broth and the fluorescence was monitored each 22 second during 30 min on
157 an Infinite M200 microplate reader (Tecan) (excitation wavelength 370 nm and emission
158 wavelength 420 nm). Polymyxin B is used as control in our experiments, this antibiotic is able
159 to rapidly disrupt the proton-motive force without interfering with fluorescence
160 measurements. Assays were performed in three independent biological experiments.

161

162 Inner Membrane depolarization assay
An overnight culture of bacterial strain CIP 55.110 was diluted 50-fold into 20 mL Columbia broth. After reaching an OD 600 nm of 0.5, cells were recovered by centrifugation (3,600 X g, 20 min, 20°C) and incubated 5 min at room temperature in 5 mM HEPES-10 mM EDTA (pH 7.0). They were then centrifuged (3,600 X g for 20 min at 20°C) and re-suspended in 5 mM HEPES (pH 7.0) supplemented with Columbia Broth 2.5% and adjusted to an OD 600 nm of 0.29. Fluorescence was monitored every 30 seconds (excitation wavelength 622 nm, emission wavelength 690 nm) after addition of 5 µL of a 160 µM solution of DiSC₃(3) to 100 µL of the cell suspension to reach a final concentration of 7.6 µM. After a period of incubation (16.5 min) to allow the dye incorporation into the polarized membranes, 10 µL of compound dilutions were added. The difference in the relative fluorescence values (RFU) from the control containing only buffer and the control containing untreated bacteria in buffer is taken as the maximum level of depolarization. Assays were performed in three independent experiments.

## Motility assay

Swimming motility was evaluated on tryptone-agar plates (tryptone (1% w/v), 0.5 % NaCl (w/v), yeast extract 0.5 % (w/v)) supplemented with agar 0.3 % (w/v) (Murray *et al.* 2006). Swarming motility was evaluated on tryptone-agar plates supplemented with agar 0.5 % (w/v) (Murray *et al.* 2006). For both assays, sterilized tryptone-agar was dispensed to Petri dishes containing either no compound or compound **1** (Cpd1) at 10 µM and left to dry at room temperature for 2h. An inoculum obtained after dilution of an overnight culture was adjusted to an OD 600 nm of 1.1. The plates were inoculated with a sterile toothpick and incubated in a non-reverse position at 35°C during the indicated times (24h, 48h or 72h) and halo diameters corresponding to bacterial growth were measured and reported. Assays were performed in three independent biological experiments.

## Microscopy live tracking and acquisition

A drop of each bacterial culture in exponential growth was deposited on a microscope slide and Cpd1 was added where indicated and immediately observed. Acquisition was performed in resonant mode with the LEICA SP5 CLS by using a laser HeNe at 633nm and a transmission PMT. The scans were recorded at a 512x512 resolution size and time laps over 200 frames. Their subsequent analysis was realized with the free software ImageJ using the MtrackJ plugging permitting to determine the average speed in µm/sec of each bacterium followed. The results reported correspond to mean values obtained from 15 tracking's measurement for each condition analyzed.

## Time kill study

The effectiveness of Cpd1 and florfenicol combination against the reference strain CIP 55.110 was determined by the time-kill assay. An overnight culture was diluted to 50-fold into 5 mL of Columbia agar and grown until the cell suspension reached the mid-logarithmic phase. The inoculum was then adjusted to 10⁷ CFU/mL in Columbia broth supplemented with either, no addition, sub-inhibitory concentration of florfenicol (MIC/4), sub-inhibitory concentration of Cpd1 (7.5 µM) and sub-inhibitory concentration of both. Each tube containing 1 mL was incubated at 35°C and the bacterial count was determined after several times of incubation: 0, 1, 3, 6, 9, and 12 h by spreading the appropriate dilutions on Columbia agar plates. The plates were incubated overnight at 35°C before colonies counting. The curves obtained are the result of two independent experiments.

## Libraries construction for bioinformatic analysis
212 Libraries were prepared with NebNext kit (New England Biolabs, Ipswich, MA) after Covaris
213 ME220 Covaris Focused-ultrasonicator (Covaris, Woburn, MA) shearing, according to
214 manufacturer protocol, and sequenced on a NextSeq550 (Illumina, San Diego, CA) with High
215 Output 300 cycles V2 cartridge (ref. FC-404-2004). Antibiotic resistance profiles of the four
216 strains were tested against CARD (22) and oneCodex, with confirmation of phenotypic
217 resistance except for florfenicol.
## 218 Results

## 219 High content screening: selection of florfenicol antibiotic chemosensitizer

220 The MICs to florfenicol of 10 clinical strains and the reference one were determined, value ranged from 2 to 32 mg/L showing different level of resistance to this antibiotic (Table 1).

223 A polyamino-isoprenyl chemical library containing 60 molecules has been tested at a 10 µM concentration in combination with florfenicol at sub-inhibitory concentration (MIC/4 = 1 mg/L) on the reference strain of *B. bronchiseptica* CIP 55.110 to identify compounds able to decrease florfenicol MIC below 1mg/L. To avoid compounds with intrinsic activity, a control experiment was performed in the absence of florfenicol. We then identified compounds that could only kill bacteria in the presence of florfenicol. Amongst the tested compounds, only Cpd1 led to an 85% inhibition of the bacterial growth under these experimental conditions.

## 231 Efficacy of compounds on animal isolates

232 To extend our study, we evaluated the efficacy of Cpd1 on ten animal isolates. This compound was able to restore the susceptibility of 8/10 isolates to florfenicol. Nevertheless, two of them were still resistant to florfenicol alone and in combination (Figure 1). It is noteworthy that, as already reported in literature (Corbière-Priot and Duménil 1996), a difference in MICs values was observed, depending on the method used for the evaluation; the MIC value being higher by using the microdilution broth method with respect to the one obtained by agar plate method.

## 240 Hit to lead procedure

241 In the perspective of improving the efficiency of Cpd1, several derivatives were synthetized (compounds 2-7, Figure 2) as their corresponding water-soluble tartrate salts to increase their solubility in bacteria growth media and reduce solvent detrimental side-effects. Compounds 1-4 possess a common farnesyl moiety whereas derivatives 5-7 a geranyl one. Moreover, each pair Cpd2 / Cpd6, Cp3 /Cpd5 and Cpd4 /Cpd7 have in common the same polyamine group (Figure 2). The MIC of these compounds were determined for each strain, no significant intrinsic activity was observed for any of these new compounds (Table 1).

## 249 Checkerboard assay

250 To determine both the minimal efficient combinations of the designed compounds and florfenicol that are necessary to inhibit the bacterial growth, we performed a checkerboard assay on animal isolates, the strain CIP 55.110 being used as control. The results are summarized in Table 2. Cpd1 exhibited a FIC index of about 0.5 that reflects the strong synergy between Cpd1 and florfenicol used at MIC/2. We noticed that higher concentrations are necessary to observe a synergy using florfenicol at MIC/4. We also observed the same range of FIC index for the farnesyl compounds (Cpd2-Cpd4). On the other hand, we were unable to determine the FIC indexes for the geranyl ones (Cpd5-Cpd7) due to the high concentrations needed to inhibit the bacterial growth as mentioned by ND in Table 2.

## 260 Presence of an active efflux in *B. bronchiseptica*

261 For all the strains tested, we observed a MIC for florfenicol ranging from 2 to 4 µg/mL except for two strains 42-F10 and SR11-14 exhibiting a MIC over the resistance breakpoint of 8 µg/mL (White *et al.* 2000). We previously reported that efflux mechanisms are produced at a basal level in Gram negative bacteria (Mamelli *et al.* 2007, Borselli *et al.* 2016) and can account for the observed low-level of resistance that can be strongly induced when exposed to toxic compounds (Hay *et al.* 2013, Ghisalberti *et al.* 2005). To evaluate the presence of an
268 active efflux in *B. bronchiseptica*, we measured the ability of the strain CIP55.110 to expel
269 the 1,2'dNA, a fluorescent dye which is a substrate of efflux pumps (Bohnert *et al.* 2011).
270 According to our previous report (Borselli *et al.* 2016), we first observed an accumulation of
271 the dye after addition of CCCP at a 30 µM final concentration whereas no accumulation was
272 observed in its absence. This suggested that de-energization of the cytoplasmic membrane by
273 CCCP blocked dye releasing. Conversely, pre-loaded bacteria incubated with culture media
274 led to a fluorescence decrease suggesting an active efflux as shown in Figure 3 (black lane)
275 where the strain CIP55.110 can expel about 75% of the pre-loaded dye. This result allowed us
276 to compare the ability of the different compounds to inhibit this efflux.
277

278

**Action of the derivatives on dye efflux**

279 Some combinations between compounds 1-7 and florfenicol were found to restore
280 susceptibility of the strains towards this antibiotic. This might suggest that the action of the
281 compounds contributed to a better accumulation of florfenicol into the bacteria, increasing the
282 intracellular concentration over the threshold of susceptibility.
283

283 To understand how the derivatives and especially the farnesyl ones could potentiate
284 the antibiotic activity, we evaluated their ability to block the efflux activity in *B. bronchiseptica* (Figure 3). An example of the results obtained with compounds used at 100
285 µM is presented in Figure 3A. As shown on this graph, the strain CIP55.110 is able to expel
286 the dye (black lane) and this transport is dependent of energy (violet lane) and compounds
287 exhibit different level of efflux-inhibition. To better compare the efficiency of compounds, we
288 performed a dose-response assay ranging from 200µM to 12.5µM, the results are summarized
289 in figure 3B. We observed that inhibition mainly concern compounds 1 to 4 and that no
290 inhibition was found in our conditions, below 25µM. In addition, we observed a significant
291 increase of inhibition, from 25µM to 200µM for compound 1-4. At 100 µM, the dye efflux
292 was inhibited by 50% by using Cpd1 and Cpd2 whereas Cpd3 and Cpd4 led to a 30% and
293 17% efflux inhibition, respectively. Under similar experimental conditions, Cpd5-7 did not
294 present any significant effect.
295

296 The other parameter that could modulate the antibiotic intracellular concentration is
297 the outer-membrane permeability. Thus, we evaluated the permeabilization activity of the
298 polyamino-isoprenyl derivatives by using previously reported nitrocefin hydrolysis method
299 (15). We took advantage of the isolate 77-A12 that produces a β-lactamase (not shown).
300 Interestingly, geranyl compounds (Cpd5-Cpd7) did not exhibit any permeabilization activity
301 while farnesyl ones (Cpd1-Cpd4) increased the permeability of the outer membrane from 30
302 to 50% considering that a maximum rate of permeation was obtained by using polymyxin B
303 (100 µM) (Figure 4A). However, no influence of the amine structure was detected, Cpd4
304 (branched amine) presenting a similar activity with respect to the three other farnesyl
305 compounds bearing a linear amine.
306

306 In Gram negative bacteria, the PMF drives many of the efflux pumps and inner
307 membrane transporters, and our compounds are able to inhibit efflux of the fluorescent dye
308 used (Figure 3). Consequently, it is interesting to determine if the derivatives could alter the
309 proton motive force across the inner membrane of *B. bronchiseptica*. As shown in figure 4B,
310 addition of polyamino-isoprenyl derivatives lead to an increase of the level of fluorescence
311 that reflects the decrease of the proton-motive force. In this case, Cpd1 and Cpd2 cause the
312 most important depolarization at about 60% of the inner membrane. Surprisingly, derivative
313 Cpd6 and Cpd4 were also able to depolarize the inner membrane up to 40%. Nevertheless, we
314 observe a slight effect for Cpd5 and Cp7 which depolarized the inner membrane up to 20%
315 and 10%, respectively. Molecules responsible for the most important depolarization remained
316 the farnesyl ones while the geranyl compounds displayed the lower rate of depolarization. In
317 addition, the presence of a branched amine on the farnesyl moiety greatly influence the
318

activity (compare Cpd1/Cpd4, Cpd2/Cpd4) except for Cpd3, but one must consider that it is
due to the presence of only 3 amino groups instead of Cpd4, as it is encountered for the other
compounds. Thus, conversely to the results obtained from the outer membrane permeation
assays, herein the activity not only depends on the isoprenyl moiety of the compound but also
of the polyamine structure.

319

320

321

322

323

324

Cpd1 and motility inhibition

325

As the derivatives can largely impair the proton-motive force and, particularly Cpd1,
we were wondering if this compound could influence the flagella-driven motility that is
highly dependent from the energy triggered by the PMF. We tested here the ability of Cpd1 to
inhibit the swimming and swarming of two strains of B. bronchiseptica, the CIP 55.110 which
is susceptible to florfenicol and the SR11-14 which is resistant both to florfenicol and to the
combination florfenicol/Cpd1. Thus, we measured the diameter of the motility halo after 24
hours of incubation at 25°C to assess the functioning of the flagella.

326

327

328

329

330

331

332

The motility of each strain was evaluated in the presence and absence of Cpd1 and/or NaCl by
considering that the flagella could use both the proton and the sodium gradient. We observed
that Cpd1 inhibited the swimming motility at about 40% for both strains (Figure 5A) whereas
a slight effect was encountered towards the swarming motility ranging from 20% and 30%
inhibition depending on the considered strain (Figure 5B). Nevertheless, it is noteworthy that
the swarming inhibition by Cpd1 is stronger in the absence of NaCl (up to 40%, (Figure 5B).
To further characterize the motility inhibition by Cpd1, we performed a real time assay by
monitoring the motility speed of bacteria after extemporaneous addition of the compound
under the microscope. As shown in figure 5C, a dramatic decrease of speed motility was
observed for both strains. The observed inhibition of motility for both strains CIP55.110 and
SR11-14 correlated with the phenotype observed previously, strongly suggests that Cpd1
inhibits flagella motilities by disrupting the PMF.

333

334

335

336

337

338

339

340

341

342

343

344

345

Time effect on bacterial growth

346

To further characterize the Cpd1/florfenicol combination, its *in vitro*
pharmacodynamic (PD) parameters were evaluated using time-kill kinetics assays (Fig 6).
Curves were obtained by plotting the number of CFU at every time point for the synergistic
concentrations of the drugs.

347

348

349

350

As expected, the use of florfenicol at a sub-inhibitory concentration (MIC/4) had no
effect on the cell growth such as the Cpd1 used at a concentration of 7.5 µM which
corresponds to its MIC/32. Nevertheless, the combination of both totally inhibits the cell
growth and, after 9 hours of incubation, the reduction of bacterial cell count was around two
Log compared to the bacteria incubated with florfenicol or Cpd1 alone. After 12 hours of
incubation, the cell count was still reduced of four Log demonstrating a significant effect of
the combination compared to the same antimicrobials administered alone (Figure 6). The
combination florfenicol/Cpd1 appeared to be bacteriostatic as florfenicol used alone.

351

352

353

354

355

356

357

358

359

Florfenicol resistance mechanism investigation

360

Two of the strains included in our study (42F10 and SR11-14) exhibited resistance to
florfenicol (MIC = 8 and 32 µg/mL, respectively). Such resistance is not common but has
already been described and the *floR* gene was identified in some isolates as responsible for
this resistance (Kadlec *et al.* 2007, Kadlec *et al.* 2018). To check if *floR* could be associated
with florfenicol resistance in these two strains, we designed primers to amplify the gene
according to the sequence previously described in the study of Kadlec (Kadlec *et al.* 2007).
No amplification was detected despite several attempts suggesting that another mechanism of
resistance was involved in these strains. Taken together, our data suggested that another

361

362

363

364

365

366

367
368 mechanism was involved in florfenicol resistance in these two strains. We thus decided to
369 determine the complete sequenced genomes of those two strains, as well as two other strains,
370 our reference CIP55-110 and 77A12 which was resistant to β-lactams, were realized (SRA
371 accession: PRJNA551886).

372 In order to be able to highlight a new gene of resistance or a *floR* derived one, we mapped our
373 two libraries made from resistant strains against reference strain *B. bronchiseptica* 253, by
374 using BWA MeM software (version 0.7.16a-r1181) with a minimum seed length of 28, all
375 other options kept to standard. Owing the fact that this strain does not have resistance to
376 florfenicol, we selected reads that did not map to this strain. We reconstructed *de novo* the
377 two other strains CIP55-110 and 77H12 with SPAdes version 3.9.1 (Bankevich *et al.* 2012)
378 and we mapped the remaining reads to the obtained contigs with the same goal. We ended
379 with 581 k-reads and 377 k-reads for SR11-14 and 42F10, respectively (Supplemental
380 Material). We reconstructed *de novo* contigs with those remaining reads, keeping in mind the
381 hypothesis that genes responsible for florfenicol resistance would be present in such contigs.
382 We ended with 416 contigs and 331 contigs, respectively, that were mapped against all the
383 florfenicol and/or chloramphenicol resistance associated genes published until now (634
384 sequences). Unfortunately, we found any match.

385 As a last attempt, considering that protein sequence is better preserved than nucleotide
386 sequence, we performed a blastx of all our contigs against all the available florfenicol
387 resistance related protein sequences but even there unsuccessfully. Genes associated with
388 resistance to antibiotics specifically detected in those two strains were found, indicating that
389 our pipeline was able to detect genes present only in those strains and not in the reference and
390 non-resistant ones (sul2 in SR11-14 and TEM-116 in 42F10). Interestingly, genes sharing
391 homology with ABC transporters and OptrA (Tamang *et al.* 2017), that have been described
392 to be associated with florfenicol resistance in *Enterococcus faecium* have been detected co-
393 localized on same contigs, that also exhibit plasmid associated features. Interestingly, OptrA
394 was shown to afford cross-resistance to chloramphenicol and florfenicol in *E. faecium* (Wang
395 *et al.* 2015, Bender *et al.* 2018, Na *et al.* 2019) that can be correlated with the cross-resistance
396 to these two antibiotics in the two strains studied herein (see Supplementary Table 1).
397 **Discussion**

398 The international and European authorities requested to use with parsimony antibiotics
399 which can favor emergence of resistance in human and veterinary medicine. For the first time,
400 an alternate way is studied by using adjuvant molecules that enhance florfenicol antibiotic
401 activity. This strategy allowed us to decrease by four the MIC of the majority of strains tested
402 here with small amounts of polyamino-isoprenyl derivatives, previously known to potentiate
403 antibiotics by acting on membrane functions (Blanchet et al. 2016). Thus, even if florfenicol
404 is only used to treat animal infections it presents cross resistance with chloramphenicol and it
405 appears important to prevent the spread to human pathogens.

406 Today, it is recognized that human health is directly related to animal health and the
407 environment. The dramatic increase in antibiotic resistance and the scarcity of new drugs on
408 the market in recent decades highlight the threat of moving to the past era of antibiotics, with
409 a significant increase in infection mortality. In this context, taking into account the "One
410 Health" problem becomes an urgent need. This must allow collaborative efforts between
411 health actors, but also breeders and those involved in environmental management.

412 This includes the involvement of a research at the interface between chemistry and
413 microbiology, to develop new infection control strategies, by developing molecules that target
414 specific new bacterial processes and / or new molecules that block / inhibit resistance
415 mechanisms already under way. In recent decades, the efflux mechanisms have become
416 increasingly important for human and animal health, we propose to block the mechanisms of
417 resistance by efflux of Gram-negative bacteria.

418 According to our previous work (Borselli et al. 2016), we evaluated enhancing activity
419 of our polyamino-isoprenyl chemical library (used at a 10 µM concentration) in the presence
420 of florfenicol at a sub-inhibitory concentration against B. bronchiseptica, CIP 55.110
421 reference strain. Thus, Cpd1 was identified as an interesting chemosensitizer of B.
422 bronchiseptica to florfenicol and evaluated against ten animal isolates leading to the
423 restoration of susceptibility for 8 of these 10 strains. Two isolates were resistant to the
424 combination, and the mechanism of resistance involved remains unknown.

425 Subsequently, a pharmacomodulation study was performed to try to improve the
426 encountered activity. In this context, six derivatives, three geranyl ones and their
427 corresponding farnesyl homologues were prepared and evaluated in a checkerboard assay.
428 First, we noticed that Cpd1 remained the better tested compound needing the lowest
429 concentration to potentiate florfenicol activity. It is also noteworthy than all farnesyl
430 compounds showed better efficiency than geranyl ones. As polyamines are well known agents
431 to disrupt membranes (Blanchet et al. 2016), we investigated the effect of the compounds on
432 membranes physiology and stability. We investigated the permeability of the outer membrane
433 which play a key role in antibiotic accumulation into Gram-negative bacteria and as expected,
434 all the tested polyamino-isoprenyl compounds increase the outer membrane permeability,
435 farnesyl compounds being better permeabilizers than geranyl ones.

436 The presence of an active efflux in *B. bronchiseptica* was demonstrated by using
437 CCCP allowing an efficient dye loading into the bacterial membranes. These results
438 corroborate the genome sequence analysis from *B. pertussis* (a human and animal pathogen
439 closely related to *B. bronchiseptica*) suggesting the presence of putative RND efflux pumps
440 coding sequences (Andrade *et al.* 2014). To our knowledge, it is also the first time that efflux
441 was determined in *B. bronchiseptica*. Thereby, we evaluated the effect of Cpd1 and its
442 derivatives on the efflux activity of *B. bronchiseptica*. The molecules able to inhibit the dye
443 efflux were the farnesyl ones while the geranyl derivatives demonstrated no ability to block
444 the efflux activity (Cpd1 and Cpd4 leading to 50 % inhibition (used at 100 µM)). Moreover,
445 we observed a dose response effect for each farnesyl compound as encountered in a previous
446 work on *Pseudomonas* (Borselli *et al.* 2016). It also appeared that the farnesyl moiety was
447 essential to obtain high activities. All these data led us to envision that these compounds could
448 act against PMF, which is required for many cell processes, as efflux pump systems but also
449 the flagella motors.

450 The flagella that potentially contribute to virulence, could be considered as a new
451 target for the development of motility inhibitors to circumvent the spread of bacterial
452 infections (Malapaka et al. 2007). Effect of Cpd1 was studied and a significant inhibition on
453 the flagella-dependent motilities was observed. Nevertheless, since B. bronchiseptica can also
454 use the sodium gradient, we were unable to discriminate if Cpd1 targets preferentially the
455 proton or sodium gradient. We concluded that this effect is probably due to a general
456 inhibition. On the other hand, we noticed that the inhibition of motility occurred in an
457 independent way of the resistance level of the strain. Indeed, a resistant strain (SR11-14) and
458 the reference strain CIP55.110 gave the same response to inhibition of motility after treatment
459 with Cpd1. More interestingly, the study of the resistance mechanism of these strains by
460 bioinformatic investigations showed the presence of a gene sharing homologies with the gene
461 coding the ABC-F type ribosomal protection protein OptrA, known to confer florfenicol and
462 chloramphenicol resistance in Enterococcus faecium and to use ATP as a source of energy
463 (Wang et al. 2015, Bender et al. 2018, Na et al. 2019). This is consistent with our results
464 demonstrating that Cpd1 targets the PMF and correlates with the absence of efficacy towards
465 the strains exhibiting this mechanism of resistance. In the strain SR 11-14 one might consider
466 that OptrA and/or efflux pumps are overexpressed in this strain. We can hypothesize that
467 florfenicol resistance could have been acquired through horizontal transfer from
468 *Enterococcus*, and this hypothesis is strengthened by the fact that the contig containing
469 resistance associated genes showed high homology with plasmids found in such bacteria.

470 Finally, to better characterize the antibiotic/derivative combination we did time kill
471 assays at the concentrations which have shown synergistic effect. This florfenicol/Cpd1
472 combination reduces the antibiotic efficient concentration four times and so could allow to
473 reduce drastically the amount of the antibiotic used. Because Cpd1 did not modify the
474 bacteriostatic activity of florfenicol, but increased its efficiency, one may consider that Cpd1
475 allows an increase of the intra-cellular concentration of the antibiotic (Vergalli et al. 2018).
476 Experiments are in progress to evaluate this hypothesis and results will be reported in due
477 course.
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585

586 **Conflict of interest Statement**

587 The author(s) declare that the research was conducted in the absence of any commercial or

588 financial relationships that could be construed as a potential conflict of interest.

589

590 **Authors contribution statement**

591 All authors conceived the approach; DB carried out all the experiments related to bacteria; JM

592 Brunel carried out the synthesis, purification and analyses of the compounds; OG performed

593 the DNA sequencing and the sequence analyses; DB, JM Brunel and JM Bolla wrote the main

594 manuscript text; DB and JM Brunel prepared the figures; all authors reviewed the manuscript.

595

596 **Funding**

597 This research was supported by Aix-Marseille Université, Service de Santé des Armées, the

598 SATT-SE and the Virbac company. DB was supported by a French Agency of Research and

599 Technology fellowship (Conventions industrielles de formation par la recherche (Cifre)).

600

601

602 **Acknowledgements**

603 The authors thank Dr Jean-Marie Pagès for helpful discussions in the course of this work and

604 for commenting on the manuscript; Dr Pascal Weber for his numerous advices and his

605 excellent contribution in confocal microscopy.

606
607 **Figures legend**

608

609 Figure 1: The ability of *B. bronchiseptica* reference strain CIP55,110 (5 µL of 5 10⁵ CFU 610 were spotted on the plates) to grow under various experimental conditions was assessed in 611 solid media containing (left to right) no compound, Cpd1 (compound 1) (10 µM), FF 612 (Florfenicol) (2µg/mL), FF (2µg/mL) + Cpd1 (10 µM). 613

614

615 Figure 2: Structure of farnesyl and geranyl polyamine derivatives Cpd1-Cpd7.

616

617 Figure 3: Inhibition of the efflux of the 1,2' dinaphthylamine dye by derivatives 1-7. 3A. 618 Efflux was triggered after 200s by the addition of culture media. The intensity of fluorescence 619 emission for 1,2' dinaphthylamine is given in relative fluorescence units (RFU). Each 620 compound was tested at 100 µM concentration; as controls an experiment without addition of 621 compounds and culture media (purple curve) was also included. 3B. Percentage of efflux 622 inhibition obtained for each compound. Error bars represent standard deviations of 3 623 independent biological experiments. 624

625

626 Figure 4: Effect of polyamine derivatives on membrane functions 4A) Outer membrane 627 permeabilization by the different compounds at 100 µM. Nitrocefin hydrolysis was followed 628 at 490nm. 4B) Inner membrane depolarization measured by the increase fluorescence of 629 DisC₃(5) in the presence of the different compounds at 100 µM. Error bars represent standard 630 deviations of 3 independent biological experiments. 631

632

633 Figure 5: Effect of 10 µM of the hit compound on bacterial motility of a susceptible strain 634 (CIP55.110) and a florfenicol resistant isolate (SR11-14) +/-NaCl. Graph represent the 635 percentage of inhibition of the motility halo in presence of Cpd1. 5A) Picture taken after 24h 636 incubation of the swimming plate. 5B) Swarming motility, halo measured after 48 h 637 incubation. 5C) Picture taken after 24h of incubation of swimming plate with NaCl inoculated 638 with SR11-14, containing no compound (left) and 10 µM of Cpd1 (right). In all cases, error 639 bars represent standard deviations of 3 independent experiments. 640

641

642 Figure 6: Time-kill curves of florfenicol (FF) (0.5 mg/L), Cpd1 (7.5 µM) and of the 643 combination FF (0.5 mg/L)+Cpd1 (7.5 µM) over a 12 hours period against strain CIP55.110. 644 Errors bars represent standard deviations of 2 independent biological experiments.

645
<caption>Table 1: Susceptibilities to florfenicol (FF) and polyamine derivatives (Cpd1-Cpd7) of various *B. bronchiseptica* strains.</caption>

643

<table><thead><tr><th rowspan="2">Strains</th><th rowspan="2">Source</th><th colspan="2">MIC<br>(µg/mL)</th><th colspan="7">MIC (µM)</th></tr><tr><th>FF</th><th>Cpd1</th><th>Cpd2</th><th>Cpd3</th><th>Cpd4</th><th>Cpd5</th><th>Cpd6</th><th>Cpd7</th></tr></thead><tbody><tr><td>CIP55.110</td><td>Pasteur Institute</td><td>4</td><td>187.5</td><td>50</td><td>50</td><td>50</td><td>&gt;750</td><td>&gt;750</td><td>375</td></tr><tr><td>42-F10</td><td>Labofarm</td><td>8</td><td>187.5</td><td>50</td><td>50</td><td>50</td><td>&gt;750</td><td>&gt;750</td><td>375</td></tr><tr><td>48-J7</td><td>Labofarm</td><td>4</td><td>187.5</td><td>50</td><td>100</td><td>50</td><td>&gt;750</td><td>&gt;750</td><td>375</td></tr><tr><td>77-C4</td><td>Labofarm</td><td>4</td><td>187.5</td><td>50</td><td>100</td><td>100</td><td>&gt;750</td><td>&gt;750</td><td>375</td></tr><tr><td>62-H4</td><td>Labofarm</td><td>4</td><td>187.5</td><td>50</td><td>50</td><td>50</td><td>&gt;750</td><td>&gt;750</td><td>375</td></tr><tr><td>73-B14</td><td>Labofarm</td><td>2</td><td>187.5</td><td>50</td><td>100</td><td>100</td><td>&gt;750</td><td>&gt;750</td><td>375</td></tr><tr><td>67-A11</td><td>Labofarm</td><td>4</td><td>187.5</td><td>50</td><td>100</td><td>50</td><td>&gt;750</td><td>&gt;750</td><td>375</td></tr><tr><td>78-E4</td><td>Labofarm</td><td>4</td><td>187.5</td><td>50</td><td>100</td><td>50</td><td>&gt;750</td><td>&gt;750</td><td>375</td></tr><tr><td>77-A12</td><td>Labofarm</td><td>4</td><td>187.5</td><td>50</td><td>100</td><td>50</td><td>&gt;750</td><td>&gt;750</td><td>375</td></tr><tr><td>SR11-24</td><td>Labofarm</td><td>2</td><td>187.5</td><td>50</td><td>50</td><td>50</td><td>&gt;750</td><td>&gt;750</td><td>375</td></tr><tr><td>SR11-14</td><td>Labofarm</td><td>32</td><td>187.5</td><td>50</td><td>50</td><td>50</td><td>&gt;750</td><td>&gt;750</td><td>375</td></tr></tbody></table>

644
Table 2: FIC index obtained for each compound tested leading to a decrease of florfenicol concentration of 50% and 75% respectively, under conditions A and B. Condition A (MIC/2). Condition B (MIC/4). ND (Not determined).

648

649

<table><thead><tr><th></th><th colspan="2">Cpd1</th><th colspan="2">Cpd2</th><th colspan="2">Cpd3</th><th colspan="2">Cpd4</th><th colspan="2">Cpd5</th><th colspan="2">Cpd6</th><th colspan="2">Cpd7</th></tr><tr><th></th><th>A</th><th>B</th><th>A</th><th>B</th><th>A</th><th>B</th><th>A</th><th>B</th><th>A</th><th>B</th><th>A</th><th>B</th><th>A</th><th>B</th></tr></thead><tbody><tr><td>CIP 55.110</td><td>0.50</td><td>0.29</td><td>0.56</td><td>0.75</td><td>0.56</td><td>0.50</td><td>0.63</td><td>0.38</td><td>0.53</td><td>ND</td><td>0.50</td><td>ND</td><td>0.53</td><td>ND</td></tr><tr><td>42-F10</td><td>0.51</td><td>ND</td><td>0.56</td><td>1.25</td><td>0.53</td><td>0.75</td><td>0.63</td><td>0.75</td><td>0.63</td><td>ND</td><td>0.57</td><td>ND</td><td>0.63</td><td>ND</td></tr><tr><td>48-J7</td><td>0.54</td><td>0.33</td><td>0.75</td><td>1.25</td><td>0.63</td><td>0.75</td><td>1.00</td><td>1.25</td><td>0.77</td><td>ND</td><td>0.63</td><td>ND</td><td>0.63</td><td>ND</td></tr><tr><td>77-C4</td><td>0.52</td><td>ND</td><td>0.56</td><td>0.75</td><td>0.53</td><td>1.25</td><td>0.63</td><td>1.25</td><td>0.57</td><td>ND</td><td>0.52</td><td>ND</td><td>0.57</td><td>ND</td></tr><tr><td>62-H4</td><td>0.54</td><td>ND</td><td>1.00</td><td>0.50</td><td>0.75</td><td>1.25</td><td>0.75</td><td>1.25</td><td>0.77</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>73-B14</td><td>0.51</td><td>ND</td><td>0.63</td><td>0.75</td><td>0.53</td><td>0.75</td><td>0.63</td><td>1.25</td><td>ND</td><td>ND</td><td>0.63</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>67-A11</td><td>0.50</td><td>0.26</td><td>0.53</td><td>0.50</td><td>0.52</td><td>0.38</td><td>0.53</td><td>0.50</td><td>0.63</td><td>ND</td><td>0.50</td><td>0.28</td><td>0.57</td><td>ND</td></tr><tr><td>78-E4</td><td>0.51</td><td>ND</td><td>0.75</td><td>0.25</td><td>0.52</td><td>0.75</td><td>0.53</td><td>0.50</td><td>0.51</td><td>0.38</td><td>0.50</td><td>0.38</td><td>0.57</td><td>ND</td></tr><tr><td>77-A12</td><td>0.50</td><td>0.26</td><td>0.53</td><td>0.50</td><td>0.52</td><td>0.50</td><td>0.53</td><td>0.50</td><td>0.52</td><td>0.32</td><td>0.52</td><td>0.32</td><td>0.57</td><td>ND</td></tr><tr><td>SR11-24</td><td>0.52</td><td>ND</td><td>0.75</td><td>0.75</td><td>0.53</td><td>1.25</td><td>0.56</td><td>0.75</td><td>0.63</td><td>ND</td><td>0.57</td><td>ND</td><td>0.57</td><td>ND</td></tr><tr><td>SR11-14</td><td>0.51</td><td>ND</td><td>0.63</td><td>0.75</td><td>0.56</td><td>0.75</td><td>1.00</td><td>0.75</td><td>0.63</td><td>ND</td><td>0.57</td><td>ND</td><td>0.57</td><td>ND</td></tr></tbody></table>

650