WO2018070874A1 - Means and methods for reducing or avoiding antibiotic resistance and spread of virulence in human pathogens - Google Patents

Abstract

The invention relates to the field of medicine. In particular, it relates to means and methods for reducing or avoiding the acquisition of antibiotic resistance. Provided is an agent capable of reducing the proton motive force (PMF) in bacteria for use in reducing or preventing the acquisition of antibiotic resistance of a commensal bacterium, like Streptococcus pneumoniae, that resides in the nasopharynx of a mammalian subject, wherein the use is not for the purpose of carrying out therapy on the human or animal body.

Description

Title: Means and methods for reducing or avoiding antibiotic resistance and spread of virulence in human pathogens.

The invention relates to the field of medicine. In particular, it relates to means and methods for reducing or avoiding the acquisition of antibiotic resistance or the acquisition of virulence factors in human pathogens, like Streptococcus pneumoniae.

Streptococcus pneumoniae (or pneumococcus) is part of the commensal microbiota of the human upper respiratory tract and may be isolated from the nasopharynx of 5 - 90% of the healthy population, depending on age or underlying diseases.^{1 2}. However, it is a major public health problem because pneumococcus occasionally invades the lungs, bloodstream or brain, causing severe infections that threat the hfe of the patient.^{3 4}. Actually, pneumococcal pneumonia accounts for more deaths than any other infectious disease worldwide.⁵

The introduction of several conjugate vaccines during the last 2 decades effectively reduced resistant infections; Nonetheless, these vaccines target up to 13 of 96 described serotypes, non-vaccine serotype clones have been emerged and have spread worldwide.⁶. Furthermore, several treatment failures of drug-resistant pneumococcal infections have been documented.^{7 9} This fact is accentuated especially among the risk population who undergo multiple courses of antimicrobial therapy per year. For instance, multi-drug resistance in pneumococci isolated from patients with chronic respiratory diseases accounts for a third of the infections.¹⁰

For example, if a patient with a respiratory infection receives antibiotics, the antibiotic can arrive at the nasopharynx at a lower dose than it should, depending on the kind of antibiotic, the diffusion and its life- time. This is a problem because, rather than killing the pneumococci residing harmlessly in the nasopharynx, it induces the competence pathway increasing the probability of the take up of exogenous DNA, including antibiotic resistant genes. This is particularly encountered when antibiotics are administered orally, because these are the antibiotics that patients take at home where there is an increased chance of under-dosing. For instance, patients drink alcohol which can degrade certain antibiotics, patients forget to take the recommended dose at the recommended time interval, or patients do not finish their treatment and stop taking antibiotics

prematurely.

In pneumococci, antimicrobial resistance development results from alteration of the target proteins (i.e. fluoroquinolones),¹¹, or mainly by acquisition of resistant genes by horizontal DNA transfer (HDT) via transformation (i.e. betalactams, macrolides or aminoglycosides).¹².

Transformation, defined as the uptake and assimilation of exogenous DNA, is an important mechanism of genome plasticity throughout evolutionary history and is largely responsible for the rapid spread of antimicrobial resistance and serotype immune evasion in S. pneumoniae.^1:M4. The development of resistance during the course of an infection is very rare, and drug-resistant pneumococcal infections usually occur by the acquisition of new resistant pneumococci from the community.¹⁵ . In fact, it has been shown that HDT occurs mainly during colonization, due to the simultaneous carriage of multiple pneumococcal strains,^{16 17} or by the presence of closely- related Streptococci (such as S. mitis), considered one of the major reservoirs of antimicrobial resistance and virulence genes for S. pneumoniae.¹⁸-²⁰ .

The transformation process requires the induction of a physiological state named competence.^{21 22}. Two key operons are involved in competence activation, comCDE and comAB (see Figure 1). At the heart of the competence regulatory pathway lies the comC-encoded competence- stimulating peptide (CSP), which is matured and exported by the membrane transporter ComAB. When extracellular CSP reaches a certain threshold concentration, the membrane-bound histidine-kinase ComD is triggered to autophosphorylate subsequently activating the response regulator ComE by phosphorylation. Phosphorylated ComE in turn activates the expression of the so-called 'early' competence genes.²³ One of them, comX, codes for a sigma factor (SigX), which is responsible for the activation of over 100 so called 'late' competence genes, including those required for transformation and DNA repair. Strikingly, certain commonly used antimicrobials such as fluoroquinolones, aminoglycosides and co-trimoxazole activate competence development,²⁴-²⁶ and may thereby enhance the acquisition of virulence genes and genes coding for antibiotic resistance of pneumococci passively colonizing the nasopharynx.

Globally, the problem of antibiotic resistance has not been solved yet, mainly due to the high antibiotic consumption of the human and animal populations, and the lack of new antibiotics in the last decades.

The present inventors recognized the urgent need to discover treatments with novel modes of action that can overcome difficult-to-treat bacterial infections. More in particular, they sought a method to protect against the acquisition of antibiotic resistance in commensal bacteria that reside in the respiratory tract.

They surprisingly found that a very low concentration of an agent capable of perturbing the proton motive force (PMF) of a human pathogen is sufficient to inhibit the competence state in human pathogens, and therefore, the acquisition of genes conferring antimicrobial resistance. For example, it was observed that Triclosan [5-chloro-2-(2,4- dichlorophenoxy)phenol], a broad spectrum antimicrobial compound commonly used since the early 1970's for cleaning and hygiene products (including soaps and toothpastes), can completely block transformation of antibiotic-treated, competence activated pneumococci and closely-related Streptococci. Interestingly, this could be achieved at concentrations far below (less than 10% of) the MIC. Similar effects could be demonstrated for related agents capable of reducing the PMF, such as CCCP, triclocarban and pimozide.

Herewith, the invention provides a novel approach to mitigate the acquisition of antibiotic resistance and virulence factors of human

pathogens like S. pneumoniae during the infection processes caused by this pathogen. In case of infection (whether by S. pneumoniae or by any other bacterium), conventional antibiotics may be administrated together with an extremely low dose of the agent, in order to block the induction of

competence and therefore, avoiding the acquisition and spread of antibiotic resistance or other virulence genes such as the capsule in the pneumococci present on and in the human host (mainly colonizing the nasopharynx) .

Accordingly, in one embodiment the invention provides an agent capable of reducing the proton motive force (PMF) in bacteria for use in reducing or preventing the acquisition of antibiotic resistance of a

commensal bacterium that resides in the nasopharynx of a mammalian subject, optionally wherein the use is not for the purpose of carrying out therapy on the human or animal body.

Also provided is an agent capable of reducing the proton motive force (PMF) of a human pathogen for use in a method for reducing or inhibiting in a subject the development of competence in said human pathogen, the method comprising administering the agent to the subject at a concentration below 8% of the minimum inhibitory concentration (MIC) for said human pathogen.

The nasopharynx (the cavity below the mouth/nose) in a mammal contains many different bacteria that reside there without causing any harm. These bacteria are also known as commensals or opportunistic pathogens. Streptococcus pneumoniae is one of these opportunistic

pathogens that is present in the human nasopharynx without causing harm, but which can, under certain conditions, cause disease. For example, when a subject suffering from a respiratory infection is treated with antibiotics, the antibiotic also has a dramatic effect on the commensal bacteria that resides in the nasopharynx. Accordingly, in one embodiment, the invention provides an agent capable of reducing the proton motive force (PMF) in bacteria for use according to any one of the preceding claims, wherein the subject is receiving a treatment with one or more antibiotic compound(s), preferably an antibiotic compound known or suspected to induce antibiotic resistance.

Preferably, said treatment comprises the oral administration of antibiotic(s).

The concept of the present invention thus resides in providing protection against the acquisition of resistance of the commensal bacteria like S. pneumoniae that reside in the nasopharynx during the respiratory infection. It does not aim at inhibiting or killing the bacteria that cause the infection. Thus, provided is the use of an PMF reducing agent to minimize, reduce or fully avoid the unwanted side effect(s) of the antibiotic on the nasopharyngeal microbiota that do not participate in the infection. The proton-motive force (PMF) is generally described as the energy that is generated by the transfer of protons or electrons across an energy- transducing membrane and can be used for chemical, osmotic or mechanical work. The PMF can be generated by a variety of phenomena, including the operation of an electron transport chain, illumination of a purple membrane, and the hydrolysis of ATP by a proton ATPase. The PMF is made up of the sum of two parameters: the electric potential (AW) and the transmembrane proton gradient (ΔρΗ). Interestingly, bacteria exercise exquisite control over AW and ΔρΗ in order to maintain a constant value of PMF. Agents capable of reducing the PMF e.g. by disrupting AW and/or ΔρΗ, in an organism for use in the present invention are known in the art, and include compounds affecting the membrane integrity or the ion exchange across the membrane. In addition, novel inhibitors can be identified by a high-throughput screen to identify molecules that selectively dissipate either component of the PMF, AW or ΔρΗ. See for example Farha et al. ²⁷ Exemplary agents for use in the present invention include

Astemizole, Meclozine dihydrochloride, Terfenadine, Amiodarone

hydrochloride, Fendiline hydrochloride, Lidoflazine, Penbutolol sulfate, Prenylamine lactate, Clofilium tosylate, Propafenone hydrochloride, Verapamil hydrochloride, Methotrimeprazine maleate salt, Quetiapine hemifumarate, Flunarizine dihydrochloride, Fluoxetine hydrochloride, Indatraline hydi chloride, Sertraline, Prochlorperazine dimaleate,

Thiethylperazine dimaleate, Trifluoperazine dihydrochloride,

Triflupromazine hydiOchloride, Metixene hydrochloride, Fluphenazine dihydrochloride, Fluspirilene, Methiothepin maleate, Pimozide, Spiperone, Thioridazine hydrochloride, Zuclopenthixol dihydrochloride, Estradiol Valerate, Norgestimate, Loperamide hydrochloride, Monensin sodium salt, Griseofulvin, Niclosamide, Hexachlorophene, Halofantrine hydrochloride, Proguanil hydrochloride, Pinaverium bromide, Verteporfin, Benzalkonium chloride, Berberine chloride, CCCP, Chlorhexidine, Nigericin, Triclosan and Verapamil.

In one aspect, the agent is an antipsychotic, like pimozide. In a preferred embodiment, the agent is selected from the group consisting of triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol), nigericin, carbonyl cyanide-m-chlorophenylhydrazone (CCCP), triclocarban, pimozide, and functional derivatives thereof. CCCP (affects both AW and ΔρΗ), Nigericin (affects ΔρΗ), Valinomycin (affects AW) and Chlorhexidine isbiocide similar to Triclosan.

In a preferred embodiment, the agent is triclosan or a triclosan derivative. Triclosan derivatives are known in the art and include glycoside derivatives of triclosan. For example, the glycoside derivative of triclosan is a pyranoside derivative. In one embodiment, the triclosan glycoside derivative is selected from triclosan-alpha-D-arabinopyranoside, triclosan- beta-D-arabinopyranoside, triclosan-alpha-D-galactopyranoside, triclosan- beta-D-galactopyranoside, triclosan-alpha-D-glucopyranoside, triclosan- beta-D-glucopyranoside, and triclosan-alpha-D-mannopyranoside. The capacity of a triclosan derivative to affect the PMF in a human pathogen can be readily determined, e.g. by using a fluorescent membrane dye such as DiSCa(5).

It is known in the art that triclosan can have a stimulatory effect on the efficacy of antibiotics. For example, Wignall et al. disclose that sublethal concentrations of triclosan resulted in a significant increase on uropathogen susceptibility to clinically relevant antibiotics, including ciprofloxacin and gentamicin.²⁸

Importantly however, as demonstrated herein below, the inventors surprisingly discovered that administering the agent to the subject at a concentration below 8% of the minimum inhibitory concentration (MIC) for said human pathogen is highly effective to reducing or inhibiting in a subject the competence state / development of competence.

The minimum inhibitory concentration (MIC) is the lowest concentration of a chemical that prevents visible growth of a bacterium (in other words, at which it has bacteriostatic activity), whereas the minimum bactericidal concentration (MBC) is the concentration that results in microbial death, in other words, the concentration at which it is bactericidal.

The MIC of a compound is typically determined by preparing solutions of the chemical at increasing concentrations, incubating the solutions with the separate batches of cultured bacteria, and measuring the results using agar dilution or broth micro dilution, usually following the guidelines of a reference body such as the CLSI, BSAC or EUCAST

(European Committee on Antimicrobial Susceptibility Testing). In a preferred aspect, the MIC is determined following the CLSI method, using Mueller Hinton medium supplemented with 5% of sheep blood.²⁹ . Based on the CLSI method, the Triclosan MIC of all pneumococcal strains (S. pneumoniae D39, its non-encapsulated variant, and clinical PMEN14 and PMEN18 strains), and other Streptococci (S. sanguis and S. mitis) was determined to be 16 μ^/ml (data not shown). MIC and MBC were identical in all cases, confirming the bactericidal activity of Triclosan.³⁰ .

In one embodiment, the agent for use in a method according to the invention is capable of reducing the proton motive force (PMF) of a bacterium, preferably a bacterium belonging to the commensal microbiota of the human (upper) respiratory tract. For example, said bacterium is of the genus Streptococcus. In a specific aspect, the bacterium is selected from the group consisting of S. pneumoniae, S. sanguis and S. mitis. Also included are S. pseudopneumoniae and other commensal Streptococci belonging to the named Streptococcus from Viridans Group (SVG), which are also present in the nasopharynx and upper respiratory tract. The invention is advantageously used to reduce or inhibit the competence state and/or the development of a multi-drug resistant bacterial strain.

In one embodiment, the agent is used at a concentration to prevent horizontal gene transfer. Horizontal gene transfer (HGT) is the movement of genetic material between unicellular and/or multicellular organisms other than via vertical transmission (i.e. the transmission of DNA from parent to offspring.) HGT is synonymous with lateral gene transfer (LGT) and HDT and the terms are interchangeable. HGT has been shown to be an important factor in the evolution of many organisms and it is the primary reason for the spread of antibiotic resistance in bacteria

In a preferred aspect, the agent is used at a concentration in the range of 1- 8% of the MIC, for example in the range of 1-7%, 2-8%, 2-7% or 2-6% of the MIC for the commensal nasopharyngeal bacterium. In a specific

embodiment, triclosan or a triclosan derivative is used at a concentration of at least 0.25 μg/mL, preferably at least 0.5 μg/mL, more preferably in the range of 1-2 g/mL. In another specific embodiment, CCCP is used at a concentration of at least 0.25 μ£/ηιΙ_-ι, preferably at least 0.5 μ£/ηιΙ,, more preferably in the range of 1-2 g/mL. In yet another specific embodiment, triclocarban is used at a concentration of at least 0.25 μg/mL, preferably at least 0.5 μg/mL, more preferably in the range of 1-3 μ /mL. In a still further embodiment, pimozide is used at a concentration of at least 0.5 μ£/ηιΙ_, preferably at least 1 μg/mL, more preferably in the range of 2-5 μg/mL.

As will be appreciated by a person skilled in the art, the agent capable of reducing the PMF is used in combination with an antibiotic compound that has activity against said human pathogen. This combination is particularly advantageous if the antibiotic compound is known or suspected to induce competence.

In one embodiment, the antibiotic compound belongs to the class of betalactams (including all kind of penicillins, cephalosporins of all generations and monobactams, carbapenems and combinations of betalactams plus inhibitors of betalactamases); quinolones of all

generations, including fluoroquinolones; aminoglycosides; macrolides;

sulfonamides; tetracyclines; chloramphenicol; ansamycines; glycopeptides; lincosamides; oxazolidinones; or lantibiotics.

Hence, also provided herein is a combination formulation in the form of a pharmaceutical composition comprising a combination of (i) an antibiotic compound and (ii) an agent capable of reducing the proton motive force (PMF) in a human pathogen. The agent is preferably present in said composition in an amount to be used at a concentration below 8% of the MIC for said human pathogen. In one embodiment, the pharmaceutical composition comprises triclosan (5- chloro-2-(2,4-dichlorophenoxy)phenol), CCCP, triclocarban, pimozide, or a functional derivative thereof. Any one of these compounds, or any mixture thereof, may be combined in a single formulation with an antibiotic compound selected from the group consisting of betalactams (including all kind of penicillins, cephalosporins of all generations and monobactams, carbapenems and combinations of betalactams plus inhibitors of

betalactamases); quinolones of all generations, including fluoroquinolones; aminoglycosides; macrolides; sulfonamides; tetracychnes; chloramphenicol; ansamycines; glycopep tides; lincosamides; oxazolidinones; or lantibiotics.

A pharmaceutical composition according to the invention finds its use in various preventive or therapeutic applications, in particular for use in a method of treating upper and lower airway infections, and inner ear way infections in a subject.

As will be appreciated by a person skilled in the art, the invention is advantageously used in the treatment of all kind of respiratory infections, not only those caused by pneumococcus. For example, aminoglycosides induce competence in pneumococcus but they are not used to treat infections caused by this bacterium since pneumococcus is highly resistant. Contrary, when they are used for other respiratory pathogens such as Pseudomonas aeruginosa, the antibiotic used to kill the P. aeruginosa can induce competence in the pneumococcus present in the nasopharynx due to its intrinsic resistance to this antibiotic. In the same way, the invention finds its use in non-topical treatments because the antibiotic could reach the nasopharynx with sub -inhibitory concentrations not enough to kill the pneumococcus but induce competence.

LEGEND TO THE FIGURES

Figure 1. Schematic overview of the regulatory network driving competence and transformation in S. pneumoniae.

Figure 2. At concentrations as low as 3% of the MIC, triclosan blocks competence induction. Panel A: growth curves of Streptococcus

pneumoniae (black line), and in the presence of a range of triclosan concentrations (from

= 3% MIC, to 16μg ml = MIC). Cells were grown in C+Y medium at pH 7.5. Panel B: natural competence induction (as shown by a luciferase production from PssbB-luc) after 100 minutes (black line). With 0.5jig/ml of triclosan or higher, natural competence is blocked along the growth curves. Figure 3. At concentrations as low as 3% of the MIC, triclosan blocks competence induction also in the non-capsulated variant. Panel A: The non-capsulated variant was more susceptible to 10μg/ml of triclosan (white circles). Panel B: the lack of capsule does not affect triclosan abihty blocking competence, and with

of triclosan or higher, natural competence is blocked along the growth curves, as observed for D39 strain.

Figure 4. Growth curves of the pneumococcal clinical strains

PMEN14 (A) and PMEN18 (C), and competence activity (B and D, respectively) in presence of Triclosan. Both strains showed a similar behavior as the D39 variant, with similar susceptibility to Triclosan and similar inhibition pattern of competence.

Figure 5. Growth curves of two Streptococci of Mitis Group, S. mitis (A) and S. sanguis (C), and competence activity (B and D,

respectively) in presence of triclosan. Both strains showed a slight reduced susceptibility to triclosan compared with pneumococci. However, with ^g/ml of triclosan (white squares), competence was also inhibited in these Streptococci species. Figure 6. Growth curves of ADP9 in presence of triclosan, and constitutive expression of luciferase. A strain with constitutive expression of luciferase was created. The addition of Triclosan did not affect the luciferase production, discarding a possible interaction between both products.

Figure 7. Diagram of transformation and horizontal gene transfer (HGT) in planktonic growth (A) and in pneumococci adhered to monolayer of A549 human cells (B). Panel A, the presence of triclosan completely inhibits the uptake of naked DNA and the HGT between two pneumococcal strains growing in a liquid culture (planktonic growth). Panel B, triclosan is also able to block the acquisition of naked DNA and HGT between 2 pneumococci adhered to a human cell hne (A549).

Figure 8. MTT cytotoxicity assay in A549 human cells. The addition of 4 jig/ml of triclosan (TCL) does not affect the viability of A549 cells. In addition, the combination of antibiotics plus triclosan does not reduce the viability compared with the antibiotic alone. The analysis was performed after 8h (black pattern bars) and 24h (white pattern bars). CIP:

Ciprofloxacin, TRIM: trimethoprim and GEN: gentamycin. Figure 9. Competence induction of DLA3 pneumococcal strain (panel A) and the non-capsulated ADP26 variant (panel B) by addition of antibiotics. Growth curves in presence of antibiotics and/or Triclosan (left axis, continuous lines) and competence induction (right axis, in triangles). Concentrations used: 0.45μg/ml 6(p-Hydroxyphenylazo)-uracil (HPUra),

Trimethoprim (TRIM), Ciprofloxacin (CIP), Gentamycin (GEN) and 4μ£/ηι1 Triclosan (TCL). In both cases, competence was not developed in the non-permissive conditions of the control conditions (no luminescence activity along the experiment). In contrast, all antibiotics added alone (without Triclosan) were able to induce competence. However, Triclosan was able to compensate the antibiotic effect by block the competence induction.

Figure 10. Checkerboards of Triclosan with representative

antibiotics belonging to the most common classes. Triclosan shows a small additive effect in presence of betalactams (Cefotaxime),

fluoroquinolones (Ciprofloxacin), macrolides (Azithromycin), lincosamides (Clindamycin) (panels A-D, respectively). However, a slight antagonistic effect between aminoglycosides and 3^g/ml of Triclosan was observed (panels E-F).

Figure 11. Competence inhibition and antagonism to

aminoglycosides. Since in Figure 10 a low level of antagonism was observed with

of triclosan plus aminoglycosides, the competence induction was analyzed for every condition of the checkerboard. Competence was naturally induced in absence of triclosan. Contrary, with lμg/ l of triclosan or higher, no competence induction was observed.

Figure 12. Effect of several compounds on competence inhibition at pH 7.5. Growth curves of Streptococcus pneumoniae (black line), and in the presence of several compounds affecting the proton motive force: 0.5 μg/ml of CCCP (= 1.6% MIC, black dots), triclocarban (O^g/ml = 3% MIC, white dots), pimozide

= 3% MIC, black squares) or chlorhexidine

(0^g/ml = 3% MIC, white squares). CCCP is a compound that disturbs the proton motive force, affecting both ΔρΗ and Δ'Ρ; triclocarban and

chlorhexidine are two biocides with similar effect as triclosan; and pimozide is an anti sychotic drug that also affects the proton motive force. For each of the compounds, a minor growth defect during the late exponential phase is observed. However, competence inhibition occurs with a concentration of O^g/ml or higher for all the compounds tested.

EXPERIMENTAL SECTION Methods Bacterial strains, human cells and growth conditions.

Bacterial strains used in this study are listed in Table 1. Growth conditions of bacterial cells was described previously.²⁵. Briefly, S. pneumoniae was grown in C+Y medium (Martin et al., 1995), at 37°C and stored at -80°C in C+Y with 14.5% glycerol at ODGOO of 0.4. The determination of the minimal inhibitory concentration (MIC) and minimal bactericidal concentration

(MBC), was performed following the Clinical Laboratory Standards Institute (CLSI) methods,²⁹

The cell line A549 (human lung carcinoma cell line) was used for in vitro experiments as follows: Exponential growing cells were plated in 96-well plates at a density of 1.5e⁶ cells/cm² and maintained in GlutaMAX™ media (Gibco®) at 37°C + 5% CO₂.

Table 1. List of strains and plasmids

*Abbreviations of antimicrobials: tet = tetracycline, spec= spectinomycin, gm= gentamicin, chl= chloramphenicol. For antibiotic resistance, xxxR stands for the gene with promoter and terminator. For translational fusion, a dash (-) is used, e.g. PssbB-luc. For transcriptional fusion, an underscore ( _ ) is used, e.g. ssbBjuc. Competence assays.

The S. pneumoniae strains were cultured in a Tecan Infinite F200 PRO allowing for real-time monitoring of competence induction in vitro. S.

pneumoniae was grown to OD595 0.4 in C+Y pH 6.8 +/- 0.05 (non-permissive conditions for natural competence induction) at 37°C. This pre-culture was diluted 100-fold in C+Y pH 7.8 +/- 0.05 (permissive conditions for natural competence induction) containing 0.45 mg/ml of luciferine and was

incubated in microliter plates with or without a range of Triclosan

concentrations: 0.5 jig/ml - 64 Growth (OD595) and luciferase activity (RLIJ) were measured every 10 min during 14h, with at least six replicates for each condition. Expression of the luc gene (only if competence is activated) results in the production of luciferase and thereby in the emission of light,²⁴ High Throughput Screen (HTS) of inhibitors of competence in S. pneumoniae.

The Prestwick library was tested in S. pneumoniae (DLA3 strain), in order to identify potential competence inhibitors. This library consists of 1200 compounds, all FDA and EMA approved, including a broad variety of antimicrobials and other human drugs with several therapeutics effects (http://ww-w.prestwickclieinical.com). Two replicates were tested, with an unique concentration of 20 μΜ for all the compounds (data not shown).

In addition, a self-assembled library including 86 relevant clinical

antimicrobials belonging to all important classes and several biocides was designed to increase the potential of getting "high quality" hits, and accelerate lead discovery. Two replicates of a range of 6 serial dilutions were tested for all the compounds.

Checkerboard assays.

In order to find possible interactions (synergies and/or antagonisms) between triclosan and the most common classes of antibiotics, several multi- dose-checkerboards were performed. 8x8 concentrations were done for at least two representative antibiotics of each class: betalactams,

fluoroquinolones, macrolides and aminoglycosides.

Horizont l gene transfer (HGT) between S. pneumoniae strains. We calculated the in vitro HGT efficiency of the chloramphenicol resistant marker in presence of Triclosan. We used the DLA3 (tetracycline resistant) and MK134 (kanamycin resistant) strains, which are naturally competent and are genetically identical, with exception of the locus where PssbB-luc was inserted, and the antimicrobial marker used (table 1). Strains were grown to OD595 0.4 in C+Y pH 6.8 at 37°C (non-permissive conditions for natural competence activation). Then, a mixed 100-fold dilution of both strains were grown in C+Y pH 7.8 (permissive conditions) to OD595 to promote the transfer of genes. Afterwards, serial dilution of cultures were plated without antibiotics (for the recovery of the total viable counts) and with the combination of 250 jig/ml of kanamycin plus 1 jig/ml tetracycline. The experiments were performed adding either 2

of

Triclosan, and comparing with the control without Triclosan. Each experimental condition was independently performed at least three times.

Transformation assays.

The wild-type D39 S. pneumoniae strain was grown to OD595 0.4 in C+Y pH 6.8 at 37°C. This pre-culture was diluted 100-fold in C+Y pH 7.8 (permissive conditions) and 2 g/ml or 4 μg/ml of Triclosan was added to the culture. After 2h30min of incubation at 37°C (time required for natural competence induction, data not shown), 1 g/ml of plasmid pLAl8 (carrying the tetracycline-resistance determinant tetM), was added (table 1 for plasmid information). The cultures were incubated at 37°C for lh30min and then serial dilutions were plated either with or without 1 μg/nll of tetracycline. Transformation efficiency was calculated by dividing the number of transformants by the total number of viable count. Three independent replicates of each condition was performed. In vitro MTT cytotoxicity assays.

Triclosan is a drug approved for the Food and Drug Administration (FDA), which is not toxic at low concentration. Nevertheless, cytotoxic assays with a concentration range of Triclosan in combination of several antimicrobials were performed, using the MTT assay procedure.³¹ A549 cells were incubated in 96-well plates in a density of 1.5e⁶ cells/cm², and grown at 37 °C with 5% CO2. A range of Triclosan concentrations and/or in combination with antimicrobials were added to the wells with fresh colourless culture medium (100 μΐ). Then, plates were incubated for 8h and 24h. At the time indicated, 0.1 ml of MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) was added to the wells following the recommendation of the commercial kit (Vybrant® MTT Cell proliferation assay kit,

ThermoFisher Scientific). After 2h of incubation at 37°C, 50 μΐ of DMSO buffer was added for 10 min, and the optical density was measured as absorbance at 570 nm. Each experiment was repeated at least three times to obtain the mean values.

Transformation and HGT assays in bacteria adhered to human airway cells.

The same experiments previously described were reproduced in S.

pneumoniae adhered to a monolayer of human airway cells. A549 cells were incubated in 96-well plates following the same criteria than in the MTT cytotoxicity assay. After incubation, cells were washed twice with PBS and were fixed in 4 % paraformaldehyde for 10 min. Then they were washed three times with PBS and were inoculated with a S. pneumoniae preculture in C+Y at OD 0.08. After 10 minutes, the 96 well plate was spun down 1 minute at 2000 rpm and the supernatant was removed. Wells were washed with PBS in order to remove planktonic cells, and fresh media

supplemented with Triclosan and/or antibiotics was added in order to reproduce the experiments described above. Statistical analysis.

Statistical analyses were carried out using Graphpad Prism (version 5) and R version 2.15.0 (R Foundation for Statistical Computing). The Fisher exact test was used when appropriate. P<0.05 was considered significant. For growth curves, error bars with 95% confidence interval (CI) were considered.

Results

Identification of a potent inhibitor of pneumococcal competence

In search for a compound that would inhibit pneumococcal competence might mitigate the spread of antimicrobial resistance and could be a potent adjuvant in combination with commonly used antibiotics to treat

pneumococcal infections, we screened a small chemical compound library, as well as the Prestwich library. In total, two independent replicates were performed for all 1286-compounds screened, and growth curves and luminescence activity were compared with 60 control conditions spread out among all the plates. In total, we identified 49 compounds that inhibited competence in sub -inhibitory concentrations (below MIC so). Two main groups of compounds were found based on the therapeutic class and presumed mechanism of action: 28 compounds affecting the membrane and/or ions homeostasis, and 14 antipsychotic drugs. From this initial screen, Triclosan was selected and studied further.

Growth curves and competence inhibition in presence of Triclosan

A derivative of the commonly used serotype 2 S. pneumoniae strain D39, strain DLA3, containing the PssbB-luc competence reporter construct stably integrated in the genome,²⁵ was grown in the presence of different concentrations of Triclosan. As shown in Figure 2A, DLA3 was insensitive to Triclosan at a concentration of 2 or lower (a concentration four times below the MIC of S. pneumoniae to Triclosan). However, a slight delay in growth was observed in the presence of 4 Triclosan and the growth delay was augmented at 8 μ£/ηι1. S. pneumoniae was fully susceptible in the presence of 16

or higher of Triclosan, and growth was completely retarded (Figure 2A). At the competence permissive pH condition (pH 7.5), S. pneumoniae was naturally competent in the absence of Triclosan (Figure 2B). However, in at concentrations of 0.5 g/ml or higher of Triclosan, luminescence activity could no longer be observed (Fig. 3B). This confirmed our initial screen demonstrating that at sub-inhibitory concentrations Triclosan (32X below the MIC) inhibits competence development. Often, the nasopharynx is colonized by non-typeable pneumococci, characterised by a lack of capsule.³² To check if the thick exopolysaccharide capsule could interfere with the diffusion of Triclosan into the cell and its activity, we performed the same experiments with a non-encapsulated derivative (strain ADP26). As shown in Figure 3B, competence was inhibited similarly in strain ADP26 although it was slightly more susceptible to Triclosan (Figure 3A).

Since the serotype 2 is currently not commonly found in clinical pandemic isolates, we also performed experiments with two recent clinical multi-drug resistant pneumococcal strains, belonging to two worldwide PMEN lineages: PMEN 14 (sequence type ST236, expressing serotype 19F) and PMEN 18 (ST67, serotype 14).³³ Both strains showed a higher susceptibility to

Triclosan, with lack of growth in the presence of 10 μ£/ηι1 of Triclosan

(Figure 4A-B). Despite this higher susceptibility, Triclosan was also able to potently inhibit competence at concentrations of 1 μ£/ηι1 and higher (Figure 4C-D). Finally, we analysed the effects of Triclosan in two closely-related

Streptococci belonging to the Mitis Group: S. mitis and S. sanguis (Figure 5). In contrast to the pneumococcal strains, the S. mitis strain was less susceptible to Triclosan, being able to grow at concentrations as high as 16 μg/ml. However, the competence inhibition was similar than all the pneumococcal strains (Figure 5). Since the abovementionecl growth experiments were performed using the semi-chemically defined C+Y medium, we also determined the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MIC) of Triclosan following the CLSI methods, with Mueller Hinton medium supplemented with 5% of sheep blood.²⁹. The Triclosan MIC of all pneumococcal strains (S. pneumoniae D39, its non-capsulated variant, and the clinical PMEN14 and PMEN18 strains), and the other Streptococci (S. sanguis and S. mitis) was 16 μ /ml (data not shown). MIC and MBC were identical in all cases, confirming the bactericidal activity of Triclosan. To exclude the possibility that Triclosan does not somehow interfere with our luminescence assay, we tested a strain constitutively expressing luciferase. As shown in Figure 6, luminescence activity was observed at all concentrations of Triclosan. Together, these data suggest that Triclosan specifically inhibits competence expression and not global gene expression. Triclosan inhibits HGT transfer and genetic transformation

While expression of ssbB is a good indicator for transform ability , ^{2 -25} we wanted to test if Triclosan actually inhibits genetic transformabihty. First, we co-cultured two different pneumococcal strains (with a different antibiotic resistant determinant inserted at different chromosomal position) under competence-permissive conditions in liquid culture, allowing competence-dependent HGT to take place via uptake of chromosomal DNA. A drastic block of competence was detected in the presence of Triclosan (Figure 7, Table 2). Table 2. In vitro horizontal gene transfer (HGT) of antimicrobial determinants

Strains DLA3 ( Δ bgaA::(Pssbu-luc, and MK134 (PsxbB-ssbBJuc, kan^R) were used for planktonic assays of HGT. The non-capsular variants of DLA3 (ADP26) and MK134 (ADP46) were used for HGT experiments of adhered bacteria to A549 human airways cells. TCL = Triclosan. In the planktonic assays, the addition of both 2μ¾/ηι1 and 4μ ml of Triclosan blocked the HGT of resistant genes. The total number of cells recovered was similar under all the conditions, showing that the addition of sub -inhibitory concentrations of Triclosan did not affect the viability of the cells.

Second, we tested whether Triclosan would also efficiently block genetic transformation when naked DNA is provided carrying only short homology regions (approximately lkb) directly to the growth medium. For this purpose, we performed standard transformation assays using a plasmid containing the tetraycyline-resistance allele tetM and integrates via double crossover at the non-essential bgaA locus (Figure 7, Table 3). We tested the uptake of plasmid DNA under planktonic growth (strain D39). In both experimental setups, the total viable counts (cfu/ml) were similar with and without the addition of sub inhibitory concentrations of Triclosan (4 μg/ml).

5 Table 3. In vitro transformation assays and effect of Triclosan

* Average of six replicates + standard deviation. D39 was used for the in vitro planktonic experiments, meanwhile the non-capsulated variant ADP25 was used in transformation assays with human cells. Abbreviations: TCL = Triclosan, CIP= Ciprofloxacin, GEN= Gentamycin.

0

However, when 4

of Triclosan was present in the growth medium, no transformants were recovered in all the replicates performed. These results were also reproduced in the presence of ciprofloxacin and kanamycin, two antibiotics that, as showed before, are able to promote competence induction.^{24 25}. In total, our results firmly estabhsh that the sub inhibitory concentrations of Triclosan drastically block competence and subsequent genetic transformation.

The abovementioned experiments were performed in vitro, in planktonic growth. To test whether Triclosan also inhibits competence development in an assay more closely resembling human colonisation, we performed experiments in a human epithehal airway model. For these experiments we used an unencapsulated strain (ADP26) since the lack of capsule enhances adhesion to the human cells.^{3 35} Interestingly, similar results were obtained compared to the mono-culture experiments and antibiotic-induced competence was readily inhibited in pneumococci adhering to human epithelial cells (data not shown). Importantly, competence-dependent genetic transformation was also blocked in this adherence model although slightly higher concentrations of Triclosan (4 μg/ml) were required to fully prevent HGT (Table 2). At this sub-MIC concentration of Triclosan, recombinant bacteria could not be detected in 9 independent replicate experiments (Table 3), highlighting the potency of Triclosan to inhibit competence and subsequent genetic transformation.

Lack of cytotoxicity of Triclosan in h uman airway cells

A real-world adjuvant to commonly used antimicrobials that inhibits competence should not be cytotoxic on its own or in combination with the antimicrobial compound. To evaluate the cytotoxicity, we examined the viability of A459 human lung epithelial cells treated with Triclosan. Figure 8 shows the survival rates of A459 cells after exposure to Triclosan at two time points, 8h and 24h. When cells were exposed to a concentration of Triclosan ten times higher than used in our experiments (40 g ml), the survival rate after 8h and 24h was nearly 90%, with no statistically significant differences with the control growth (P= 1.000). Importantly, the combination of Triclosan plus (ciprofloxacin, gentamycin and trimethoprim) antibiotics did not show additive or synergistic toxicity than the antibiotic alone in both 8h and 24h experiment (Figure 8).

Combination of Triclosan with clinical antibiotics

It is well known that some antimicrobials at sub inhibitory concentrations induce competence,²⁴ thereby increasing the risk of developing or spreading resistance by acquisition and dissemination of genetic

determinants for antimicrobial resistance.¹³ Here, we studied if the addition of Triclosan can bock competence induction in DLA3 pneumococcal strain by the following antimicrobials belonging to three different classes: ciprofloxacin (fluoroquionolone), gentamycin (aminoglycoside) and trimethoprim. As expected, these antimicrobials induced competence under otherwise non-permissive conditions (Figure 9A). Strikingly, when these antimicrobials were supplemented with Triclosan (at concentrations of 4 or higher), competence was no longer induced (Figure 9A).

Similar results were also obtained in combination with the potent competence activator 6 (p-Hydroxyphenylazo) -uracil (HPUra), an analogue that reversibly binds DNA polymerase type III (PolC),³⁶ thereby temporarily stalling DNA leading to overinitiation and subsequent competence activation.²⁵ Together, these results demonstrate that Triclosan, when used in combination with antimicrobials, is an effective agent to block

competence activation. As expected, the same results were observed replicating the experiments with the uncapsulated variant ADP26 adhered to human cells culture (Figure 9B). To demonstrate thatTriclosan is suitably used as adjuvant of the clinical antibiotics to block competence and thereby the acquisition antibiotic resistance, we performed checkerboards experiments with 8x8

concentrations of Triclosan plus several antibiotics in order to test possible antagonisms or synergies that could affect the effectiveness of the antibiotic. An additive effect was observed with Triclosan plus betalactams

(cefotaxime), fluoroquinolones (ciprofloxacin), macrolides (azithromycin), lincosamides (clindamycin) (Figure 10A-D, respectively). However, a slight antagonistic effect between aminoglycosides and 3^g/ml of Triclosan was observed (Figure 10E-F). Nevertheless, despite the slight antagonism observed between triclosan and the aminoglycoside amikacin, competence was inhibited with ljig/ml of triclosan or higher (Figure 11).

Proton motive force is essential for competence development

The slight antagonism effect of Triclosan with aminoglycosides suggest that Triclosan would inhibit competence due to an alteration of the proton motive force (PMF). To confirm this, first we confirmed the results observed in the HTS, testing the inhibition of natural competence by addition of several compounds that disrupt the PMF or one of its components, the electrical component (membrane potential, AW) or chemical component (ΔρΗ). Figure 12 shows that all the compounds tested were also able to block the natural competence development as was observed with Triclosan.

REFERENCES

1. van Deursen, A. M. M., van den Bergh, M. R. & Sanders, E. A. M.

Vaccine 34, 4-6 (2016).

2. Wroe, P. C. et al. Pediatr. Infect. Dis. J. 31, 249-254 (2012).

3. De Lencastre, H. & Tomasz, A. J. Antimicrob. Chemother. 50

Suppl S2, 75-81 (2002).

4. Hausdorff, W. P., Feikin, D. R. & Klugman, K. P. Lancet Infect.

Dis. 5, 83-93 (2005). 5. Prina, E., Ranzani, O. T. & Torres, A. Lancet Loud. Engl. 386, 1097-1108 (2015).

6. Kim, L., McGee, L., Tomczyk, S. & Beall, B. Clin. Microbiol. Rev.

29, 525-552 (2016).

7. Pletz, M. W. R., McGee, L., Burkhardt, O., Lode, H. & Klugman, K. P. Eur. J. Clin. Microbiol. Infect. Dis. Off. Publ. Eur. Soc. Clin. Microbiol. 24, 58-60 (2005).

8. Rzeszutek, M. et al. Int. J. Antimicrob. Agents 24, 95-104 (2004).

9. Zielnik-Jurkiewicz, B. & Bielicka, A. Int. J. Pediatr.

Otorhinolaryngol. 79, 2129-2133 (2015).

10. Domenech, A. et al. J. Antimicrob. Chemother. 69, 932-939 (2014).

11. Stanhope, M. J. et al.. Antimicrob. Agents Chemother. 49, 4315- 4326 (2005).

12. Frost, L. S., Leplae, R., Summers, A. O. & Toussaint, A. Nat. Rev.

Microbiol. 3, 722-732 (2005).

13. Chewapreecha, C. et al, Nat. Genet. 46, 305-309 (2014).

14. Croucher, N. J. et al. Science 331, 430-434 (2011).

15. Sethi, S., Evans, N., Grant, B. J. B. & Murphy, T. F. iV. Engl. J.

Med. 347, 465-471 (2002).

16. Donkor, E. S. et al. niBio 2, e00040- 11 (2011).

17. Marks, L. R., Reddinger, R. M. & Hakansson, A. PmBio 3, e00200- 12 (2012).

18. Bryskier, A. Clin. Microbiol. Infect. Off. Publ. Eur. Soc. Clin.

Microbiol. Infect. Dis. 8, 65-69 (2002).

19. Janoir, C, Podgiajen, I., Kitzis, M. D., Poyart, C. & Gutmann, L. J.

Infect. Dis. 180, 555-558 (1999).

20. Reichmann, P. et al. J. Infect. Dis. 176, 1001-1012 (1997).

21. Claverys, J.-P., Martin, B. & Polard, P. The genetic transformation machinery: composition, localization, and mechanism. FEMS Microbiol. Rev. 33, 643-656 (2009).

22. Johnston, C, Martin, B., Fichant, G., Polard, P. & Claverys, J.-P.

Nat. Rev. Microbiol. 12, 181-196 (2014).

23. Martin, B. et al. Mol. Microbiol. 87, 394-411 (2013).

24. Prudhomme, M., Attaiech, L., Sanchez, G., Martin, B. & Claverys, J.-P. Science 313, 89-92 (2006).

25. Slager, J., Kjos, M., Attaiech, L. & Veening, J.-W. Cell 157, 395- 406 (2014).

26. Stevens, K. E., Chang, D., Zwack, E. E. & Sebert, M. E. mBio 2, (2011).

27. Farha, M. A., Verschoor, C. P., Bowdish, D. & Brown, E. D. Chem, Biol. 20, 1168-1178 (2013).

28. WignaU, G. R., Goneau, L. W., Chew, B. H., Denstedt, J. D. &

Cadieux, P. A. J. Endourol. 22, 2349-2356 (2008). 29. Clinical Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Eighteenth Informational Supplement M100-S 18. (2010).

30. Heath, R. J. & Rock, C. O. Nature 406, 145-146 (2000).

31. Mosmann, T. J. Immunol. Methods 65, 55-63 (1983).

32. Sa-Leao, R. et al. Microbiology 152, 367-376 (2006).

33. McGee, L. et al. J. Clin. Microbiol. 39, 2565-2571 (2001).

34. Hammerschmidt, S. et al. Infect. Immun. 73, 4653-4667 (2005).

35. Kjos, M. et al. J. Bacteriol. 197, 807-818 (2015).

36. Brown, N. C. Proc. Natl. Acad. Sci. U. S. A. 67, 1454-1461 (1970).

37. Taber, H. W., Mueller, J. P., Miller, P. F. & Arrow, A. S. Microbiol.

Rev. 51, 439-457 (1987).

38. Martin-Galiano, AJ, et al. Molecular Microbiology 41, 1327- 1338 (2001)

Claims

Claims

1. An agent capable of reducing the proton motive force (PMF) in bacteria for use in reducing or preventing the acquisition of antibiotic resistance of a commensal bacterium that resides in the nasopharynx of a mammalian subject, wherein the use is not for the purpose of carrying out therapy on the human or animal body.

2. Agent for use according to claim 1, wherein said agent is selected from the group consisting of triclosan (5-chloro-2-(2,4- dichlorophenoxy)phenol), CCCP, triclocarban, pimozide, and functional derivatives thereof.

3. Agent for use according to claim 2, wherein said agent is triclosan or a triclosan derivative.

4. Agent for use according to any one of the preceding claims, wherein the subject is receiving a treatment with one or more antibiotic

compound(s), preferably an antibiotic compound known or suspected to induce antibiotic resistance.

5. Agent for use according to claim 4, wherein said treatment comprises the oral administration of antibiotic(s).

6. Agent for use according to any one of the preceding claims, wherein the subject is suffering from a respiratory infection.

7. Agent for use according to any one of the preceding claims, wherein said commensal nasopharyngeal bacterium is of the genus Streptococcus.

8. Agent for use according to claim 7, wherein said bacterium is selected from the group consisting of S. pneumoniae, S. sanguis and S.

mitis.

9. Agent for use according to any one of the preceding claims, wherein said agent is used at a concentration to prevent horizontal gene transfer.

10. Agent for use according to any one of the preceding claims, wherein said agent is used at a concentration in the range of 1-8% of the MIC for the commensal nasopharyngeal bacterium, preferably 2-6% of said MIC.

11. Agent for use according to any one of the preceding claims, wherein triclosan is used at a concentration of at least 0.25 g mL, preferably at least 0.5 μg/mL, more preferably in the range of 1-2 g mL

12. Agent for use according to any one of the preceding claims, wherein said agent is used in combination with an antibiotic compound, preferably an antibiotic compound known or suspected to induce antibiotic resistance.

13. Agent for use according to claim 12, wherein said antibiotic compound is a betalactam, monobactam, fluoroquinolone, aminoglycoside, macrolide, cephalosporin, sulfonamide, tetracycline chloramphenicol, ansamycin, glycopeptide, lincosamide, or lantibiotic.

14. A pharmaceutical composition comprising a combination of (i) an antibiotic compound and (ii) an agent capable of reducing the proton motive force (PMF) in a human pathogen and wherein said agent is present in said composition in an amount to be used at a concentration below 8% of the MIC for said human pathogen.

15. Pharmaceutical composition of claim 14, comprising triclosan (5- chloro-2-(2,4-dichlorophenoxy)phenol), CCCP, triclocarban, pimozide, or a functional derivative thereof.

16. Pharmaceutical composition of claim 14 or 15, comprising an antibiotic compound selected from the group consisting of a betalactam, monobactam, fluoroquinolone, aminoglycoside, macrolide, cephalosporin, sulfonamide, tetracycline chloramphenicol, ansamycin, glycopeptide, lincosamide, and lantibiotic.

17. Pharmaceutical composition according to claim 14-16, for method of treating a bacterial infection in a subject.