Models of bacterial persister strains

Abstract

The present invention relates to a recombinant bacterial persister strain, comprising an expression cassette having a nucleic acid sequence of a relA gene or a functional fragment thereof, operably linked to a nucleic acid sequence of an inducible promoter.

Description

MODELS OF BACTERIAL PERSISTER STRAINSFIELD OF INVENTION

[0001] The present invention relates to a recombinant bacterial persister strain, comprising an expression cassette having a nucleic acid sequence of a relA gene or a functional fragment thereof, operably linked to a nucleic acid sequence of an inducible promoter. This bacterial strain has been found to be a model persister strain. This bacterial persister strain is therefore useful in the method of screening for anti-persister drugs, either in-vitro, or in-vivo by using it in an animal model.BACKGROUND OF INVENTION

[0002] According to the World Health Organization (WHO), infectious diseases are responsible for a substantial burden of morbidity and mortality worldwide. This burden can vary depending on factors such as region, population demographics, healthcare infrastructure, and the emergence of new pathogens or antibiotic -resistant strains. In fact, antimicrobial resistance represents a significant global health hazard to human wellbeing.

[0003] With the rise of antibiotic-resistant strains especially due to overuse, there is a pressing need for novel therapeutic approaches for infectious disease. Developing therapies that directly target persisters for elimination or stimulate their return to active growth could reduce the likelihood of disease recurrence and shorten the duration of treatment.

[0004] Bacterial persister cells are defined as a subpopulation exhibiting antibiotic tolerance due to their dormant state. These cells are highly tolerant to stresses such as antibiotics, nutrient limitation, oxidative stress, and heat and have led to a paradigm shift in antimicrobial therapy research. They enter a slow or non-growing state enabling theevasion of the effects of bactericidal antibiotics. Persister cells, which remain dormant in the presence of antibiotics, can reactivate and regrow once the antibiotic treatment ends. This significantly impacts antibiotic effectiveness, as antibiotics targeting growth-related functions fail to eliminate these non-growing cells. This resilience leads to recurrent infections and promotes the emergence and spread of multidrug resistance as persister cells display a higher frequency of mutation.

[0005] Currently, there are limited options for targeting persister cells, as most treatments have been formulated to eradicate active cells, such as conventional antibiotics. Thus, to streamline the development of new anti-persister agents, it appears crucial to establish a platform model that facilitates the screening of such compounds.

[0006] Several genes have been identified as playing a role in the formation of persister cells (Hansen et al., Antimicrobial Agent and Chemotherapy, 2008, p.2718-2726). Yet, despite their potential involvement, the exact mechanism is unclear and this limited understanding could pose challenges in the construction of a persistent bacterial model.

[0007] In this context, it was a remarkable discovery that a bacterial strain expressing RelA (GDP / GTP pyrophosphokinase) in an inducible manner can be used as a dormant persister cell model, enabling us to screen future compounds, such as new antibiotics or new anti-persister agents.

[0008] The present invention relates generally to a bacterial persister model, which expresses the relA gene.SUMMARY

[0009] This invention relates to a recombinant bacterial persister strain comprising an expression cassette, wherein the expression cassette comprises (i) a nucleic acid sequence of a relA gene or a functional variant thereof operably linked to (ii) a nucleic acid sequence of an inducible promoter, and wherein the bacterial strain belongs to the family of Pseudomonadaceae, preferably Pseudomonas aeruginosa.

[0010] In some embodiments, the inducible promoter is selected from the group consisting of: ParaBAD, Ptet, Plac, PlacUV5, Ptac, Ptrc, PT5, PT7, Prha, PprpR, Plux, Pdox, Pmalt, and Prib.

[0011] In some embodiments, the expression cassette is introduced into a vector selected from the group consisting of: a plasmid, an episome, and a bacterial artificial chromosome.

[0012] In some embodiments, the expression cassette is introduced into a vector, preferably the expression cassette and / or the vector is integrative or non-integrative.

[0013] In some embodiments, the bacterial persister strain is persistent to one or more antibiotic families selected from the group consisting of: beta-lactams; such as penicillins, cephalosporins, carbapenems, monobactams; sulfonamides, tetracyclines, oxazolidinones, aminoglycosides, glycopeptides, fluoroquinolones, quinolones, rifamycin, macrolides, lipopeptides, lincosamides, nitrofurans, fosfomycin, trimethoprim, sulfamethoxazole, oxy quinolines, fusidic acid, and mupirocin; preferably beta-lactams; more preferably carbapenem, cephalosporin, and penicillin; even more preferably meropenem, cefepime, and piperacillin.

[0014] This invention also relates to a kit-of-parts for screening a compound of interest for anti-persister activity, comprising:- (a) a first part being a first recombinant bacterial persister strain, and- (b) a second part being a second recombinant bacterial persister strain; wherein said (a) first recombinant bacterial persister strain comprises an expression cassette wherein the expression cassette comprises (ia) a nucleic acid sequence of a relA gene or a functional variant thereof operably linked to (iia) a nucleic acid sequence of an inducible promoter, wherein said (b) second recombinant bacterial persister strain comprises an expression cassette wherein the expression cassette comprises (ib) a second nucleic acid sequence of a relA gene or a functional variant thereof operably linked to (iib) a second nucleic acid sequence of an inducible promoter,and wherein said (a) first recombinant bacterial persister strain belongs to the family of Pseudomonadaceae, preferably Pseudomonas aeruginosa.

[0015] In some embodiments, said (iia) first and / or said (iib) second inducible promoter is selected from the group consisting of: ParaBAD, Ptet, Plac, PlacUV5, Ptac, Ptrc, PT5, PT7, Prha, PprpR, Plux, Pdox, Pmalt, and Prib.

[0016] In some embodiments, the (b) second recombinant bacterial persister strain is a Gram-negative bacterial strain, preferably belonging to the family selected from the group consisting of: Enterobacteriaceae and Pseudomonadaceae.

[0017] In some embodiments, said (a) first recombinant bacterial persister strain, and / or said (b) second recombinant bacterial persister strain, is persistent to one or more antibiotic families selected from the group consisting of: beta-lactams; such as penicillins, cephalosporins, carbapenems, monobactams; sulfonamides, tetracyclines, oxazolidinones, aminoglycosides, glycopeptides, fluoroquinolones, quinolones, rifamycin, macrolides, lipopeptides, lincosamides, nitrofurans, fosfomycin, trimethoprim, sulfamethoxazole, oxy quinolines, fusidic acid and mupirocin.

[0018] This invention further relates to the use of the kit-of-parts as described herein, for screening a compound of interest for anti-persister activity.

[0019] This invention additionally relates to a method for screening a compound of interest for anti-persister activity comprising the following steps: a. culturing the recombinant bacterial persister strain as described herein into a culture medium allowing growth, b. inducing the expression of the re I A gene, c. introducing into the medium a compound of interest to test, and d. quantifying the survival rate of the bacterial persister strain, thereby determining if the compound of interest has anti-persister activity.

[0020] In some embodiments, said method further comprisesthe step (a’) of culturing (b) a second recombinant bacterial persister strain into a second culture medium and allowing the growth and said (b) second recombinant bacterial persister comprises an expressioncassette, wherein the expression cassette comprises (ib) a second nucleic acid sequence of a relA gene or a functional variant thereof operably linked to (iib) a second nucleic acid sequence of an inducible promoter.

[0021] In some embodiments, said (iib) second inducible promoter is selected from the group consisting of: ParaBAD, Ptet, Plac, PlacUV5, Ptac, Ptrc, PT5, PT7, Prha, PprpR, Plux, Pdox, Pmalt, and Prib.

[0022] In some embodiments, said (b) second recombinant bacterial persister strain expressed relA gene endogenously.

[0023] In some embodiments, the recombinant bacterial persister strain as described herein, and / or said (b) second recombinant bacterial persister strain, is persistent to one or more antibiotic families selected from the group consisting of: beta-lactams; such as penicillins, cephalosporins, carbapenems, monobactams; sulfonamides, tetracyclines, oxazolidinones, aminoglycosides, glycopeptides, fluoroquinolones, quinolones, rifamycin, macrolides, lipopeptides, lincosamides, nitrofurans, fosfomycin, trimethoprim, sulfamethoxazole, oxy quinolines, fusidic acid and mupirocin.

[0024] This invention thus relates to a recombinant bacterial persister strain comprising an expression cassette, wherein the expression cassette comprises (i) a nucleic acid sequence of a relA gene or a functional variant thereof operably linked to (ii) a nucleic acid sequence of an inducible promoter.

[0025] In some embodiments, the inducible promoter is selected from the group consisting in: ParaBAD, Ptet, Plac, PlacUV5, Ptac, Ptrc, PT5, PT7, Prha, PprpR, Plux, Pdox, Pmalt, and Prib.

[0026] In some embodiments, the expression cassette is introduced into a vector selected from the group consisting of: a plasmid, an episome, a bacterial artificial chromosome.

[0027] In some embodiments, the expression cassette is introduced into a vector, preferably the expression cassette and / or the vector is integrative or non-integrative.

[0028] In some embodiments, the bacterial persister strain expressed relA gene endogenously.

[0029] In some embodiments, the bacterial strain is a Gram-negative bacterial strain.

[0030] In some embodiments, the bacterial strain belongs to the family selected from the group consisting of: Enterobacteriaceae and Pseudomonadaceae, preferably the species selected in the group consisting of: Escherichia coli and. Pseudomonas aeruginosa.

[0031] In some embodiments, the bacterial persister strain is persistent to one or more antibiotic family selected from the group consisting of: beta-lactams (penicillins, cephalosporins, carbapenems, monobactams), sulfonamides, tetracyclines, oxazolidinones, aminoglycosides, glycopeptides, fluoroquinolones, quinolones, rifamycin, macrolides, lipopeptides, lincosamides, nitrofurans, fosfomycin, trimethoprim, sulfamethoxazole, oxy quinolines, fusidic acid and mupirocin.

[0032] The invention further relates to an insect model, comprising the recombinant bacterial persister strain according to the invention, wherein the insect is preferably Galleria mellonella.

[0033] The invention further relates to a mammalian model, comprising the recombinant bacterial persister strain according to the invention.

[0034] In some embodiments, the mammalian model is a mouse, a rat or a dog.

[0035] The invention further relates to a method for producing a recombinant bacterial persister strain, the method comprising the following steps:(a) transforming at least one bacterium with at least one expression cassette, wherein the at least one expression cassette comprises a (i) nucleic acid sequence of a relA gene and / or a functional fragment thereof operably linked to (ii) a nucleic acid sequence of an inducible promoter, and(b) selecting the bacterium which comprised the expression cassette of the vector.

[0036] The invention further relates to a method for producing an insect model to study bacterial persistence, wherein the at least one recombinant bacterial persister strain according to the invention is administrated to an insect or its larva.

[0037] The invention further relates to a method for producing a mammalian model to study persistent microorganisms, wherein the at least one recombinant bacterial persister strain according to the invention is administrated to a mammalian.

[0038] The invention further relates to a method for screening a compound of interest with an anti-persister activity comprising the following steps: a. culturing the recombinant bacterial persister strain according to the invention or obtained according to the method of the invention into a culture medium allowing the growth, b. inducing the expression of the re I A gene, c. introducing into the medium a compound of interest to test, and d. quantifying the survival rate of the bacterial persister strain, thereby determining if the compound of interest has anti-persister activity.DEFINITIONS

[0039] In the present invention, the following terms have the following meanings:

[0040] “About” refers to mean approximately, roughly, around, or in the region of. The term “about” preceding a figure means more or less 10 % of the value of the figure. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth by 10%.

[0041] “Administration", or a variant thereof (e.g., “administering”), refers to providing a therapeutic agent e.g., a compound of the invention) alone or as part of apharmaceutically acceptable composition, to the patient in whom / which the condition, symptom, or disease is to be treated.

[0042] “Amino acid substitution” is a substitution of one amino acid for another. A conservative amino acid substitution is a substitution of one amino acid for another with similar characteristics. Conservative amino acid substitutions include substitutions within the following groups: valine, alanine and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. The nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (i.e., basic) amino acids include arginine, lysine and histidine. The negatively charged (i.e., acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above- mentioned polar, basic, or acidic groups by another member of the same group can be deemed a conservative substitution. By contrast, a non-conservative substitution is a substitution of one amino acid for another with dissimilar characteristics.

[0043] "Bacterial culture" or simply "culture" denotes the presence of bacteria thriving in an environment conducive to their growth. These cultures can be housed in various containers like flasks, tubes, microwell plates, or similar vessels. Typically, bacteria undergo distinct growth phases. Upon introduction to a nutrient-rich environment, initiating growth, the cells initially adapt to this new setting. This initial phase is termed the lag phase, marked by a slower growth rate as cells acclimate to the environment and gear up for accelerated growth. During the lag phase, there's a heightened production of proteins essential for rapid proliferation. Subsequently, the log phase, or exponential phase, ensues, characterized by the bacteria undergoing rapid, exponential growth. Nutrients are metabolized at their maximum until the exhaustion of a critical nutrient, which becomes limiting. The third phase, termed the stationary phase, results from this nutrient depletion. In certain cases, a bacterial culture in the "stationary phase" indicates that the growth rate and death rate of the bacteria within the culture are approximately equal.

[0044] “Comprise” or a variant thereof (e.g., “comprises”, “comprising”) refers to according to common patent application drafting terminology. “Comprises” is intended to mean “contains”, “encompasses” and “includes”. Hence, “comprise” preceded by an object and followed by a constituent means that the presence of a constituent in the object is required (typically as a component of a composition), but without excluding the presence of any further constituent(s) in the object. Moreover, any occurrence of “comprise” or a variant thereof herein also encompasses narrower expression “substantially consist of’, further narrower expression “consist of’ and any variants thereof (e.g., “consists of’, “consisting of’), unless otherwise stated.

[0045] “Expression cassette" refers to a unit cassette comprising a promoter and a gene of interest, capable of expressing the gene of interest operably linked downstream of the promoter. Inside or outside such expression cassettes can include various factors that can aid in the efficient expression of the gene of interest. The gene expression cassette may typically include, but is not limited to, a transcription termination signal, a ribosome binding site, and a translation termination signal in addition to a promoter operably linked to the gene of interest.

[0046] "Fragment" of a protein, polypeptide, or peptide generally refers to N-terminally and / or C-terminally deleted or truncated forms of said protein, polypeptide or peptide. The term encompasses fragments arising by any mechanism, such as, without limitation, by alternative translation, exo- and / or endo-proteolysis and / or degradation of said peptide, polypeptide or protein, such as, for example, in vivo or in vitro, such as, for example, by physical, chemical and / or enzymatic proteolysis. Without limitation, a fragment of a protein, polypeptide, or peptide may represent at least about 5% (by amino acid number), or at least about 10%, e.g., 20% or more, 30% or more, or 40% or more, such as preferably 50% or more, e.g., 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the amino acid sequence of said protein, polypeptide, or peptide, e.g., a corresponding RelA polypeptide.

[0047] “Functional variant” or “functional fragment” refers to or encompasses variants and / or fragments of proteins, polypeptides or peptides, this denotes variants or fragments which are functionally active or functional, i.e., which at least partly retain thebiological activity or intended functionality of the respective or corresponding proteins, polypeptides, or peptides. By means of an example and not limitation, a functionally active variant or fragment of RelA polypeptide as disclosed herein shall at least partly retain the biological activity of bacterial RelA polypeptide.

[0048] “Homology” or “identity” refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous. Thus, the term "homologous" or "identical", when used in a relationship between the sequences of two or more polypeptides or of two or more nucleic acid molecules, refers to the degree of sequence relatedness between polypeptides or nucleic acid molecules, as determined by the number of matches between strings of two or more amino acid or nucleotide residues. “Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms"). Identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988). Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods of determining identity are described in publicly available computer programs.Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res. \2, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894; Altschul et al., supra). The well-known Smith Waterman algorithm may also be used to determine identity.

[0049] "Mammal" refers to any animal classified as a mammal, including humans, other higher primates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, mouse, rat, etc.

[0050] “Nucleic acid” or “Polynucleotide” refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, these terms include, but are not limited to, single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidates and thus can be an oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphoramidate- phosphodiester oligomer. In addition, a double -stranded polynucleotide can be obtained from the single stranded polynucleotide product of chemical synthesis either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer.

[0051] "Operably linked" refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship withthe second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0052] “Patient” refers to a subject who / which is awaiting the receipt of, or is receiving medical care or was / is / will be the object of a medical procedure, or is monitored for the development of the targeted disease or condition, such as, for example, an infectious disease.

[0053] "Peptide," "Polypeptide," and "Protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0054] “Persisters” or “Persistent bacteria” refer to a subpopulation of bacteria that, without being resistant, is able to survive lethal doses of antibiotic treatment by entering a physiologically dormant state that protects essential cellular processes from antibiotic action. The persister refers to bacteria tolerant to antibiotic treatment. Once the antibiotic is removed, persisters can reactivate, leading to the relapse of an infection. Persistence therefore plays a role in the failure of antibiotic therapy. As used herein, “persister cell” or “persistent cell” refers to a metabolic variant in an isogenic bacterial population that is phenotypically characterized by their slow growth rate coupled with a capacity to survive antibiotic treatment.

[0055] “Persistent infection” refers to any infection in which persister cells are implicated.

[0056] “Pharmaceutically acceptable” means that the ingredients of a composition are compatible with each other and not deleterious to the patient to which / whom it is administered.

[0057] “Pharmaceutically acceptable carrier” refers to an excipient that does not produce an adverse, allergic or other untoward reaction when administered to an animal, preferably a human. It includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by regulatory offices, such as, e.g. , FDA Office or EMA. Examples of pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (for example sodium carboxymethylcellulose), polyethylene glycol, poly acrylates, waxes, polyethylene- polyoxypropylene- block polymers, polyethylene glycol and wool fat.

[0058] Polynucleotide'' refers to a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric "nucleotides." The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e. , the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

[0059] “Promoter” which is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3'-direction) coding sequence. Transcription promoters can include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters”.

[0060] “Inducible promoter” refers to a specific DNA sequence that regulates gene expression in response to external stimuli or inducers. This promoter can be activated or deactivated by certain molecules or environmental factors, allowing precise control over when and to what extent a gene is transcribed and translated within a cell or organism.

[0061] “Selected from” refers to according to common patent application drafting terminology, to introduce a list of elements among which one or more item(s) is (are) selected. Any occurrence of “selected from” in the specification may be replaced by “selected from the group comprising or consisting of’ and reciprocally without changing the meaning thereof.

[0062] “Therapeutic agent”, “active pharmaceutical ingredient” and “active ingredient” refer to a compound for therapeutic use and relating to health. Especially, a therapeutic agent (e.g., a compound of the invention) may be indicated for treating a disease. An active ingredient may also be indicated for improving the therapeutic activity of another therapeutic agent.

[0063] “Transformation” refers to the transfer of one or more polynucleotide(s) into a cell. A cell is “transformed” by or with a polynucleotide when a nucleic acid molecule comprising the polynucleotide is introduced into the cell, and the polynucleotide becomes stably replicated by the cell, either by incorporation of the nucleic acid molecule into the cellular genome, or by episomal replication. As used herein, the term “transformation” encompasses all techniques by which a nucleic acid molecule can be introduced into sucha cell. Examples include, but are not limited to: transfection with viral vectors; transformation with plasmid vectors; electroporation (Fromm et al. , Nature 319:791-3, 1986); lipofection (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7, 1987); microinjection (Mueller et al., Cell 15:579-85, 1978); Agrobacterium- mediated transfer (Fraley et al., Proc. Natl. Acad. Sci. USA 80:4803-7, 1983); direct DNA uptake; conjugation, and microprojectile bombardment (Klein et al., Nature 327:70, 1987).

[0064] "Variant" of a protein, polypeptide or peptide generally refers to proteins, polypeptides or peptides whose amino acid sequence is substantially identical (i.e., largely but not wholly identical) to the sequence of the protein, polypeptide, or peptide, at least about 40% identical, at least about 45% identical, at least about 50% identical, at least about 55% identical, at least about 60% identical, at least about 65% identical, e.g., at least about 70% identical or at least about 75% identical, e.g. preferably at least about 80% identical or at least about 85% identical, e.g., preferably at least about 90% identical, e.g., at least 91% identical, 92% identical, more preferably at least about 93% identical, e.g., at least 94% identical, even more preferably at least about 95% identical, e.g., at least 96% identical, yet more preferably at least about 97% identical, e.g., at least 98% identical, and most preferably at least 99% identical to the sequence of the protein, polypeptide, or peptide, e.g., to the sequence of a corresponding RelA. Preferably, a variant may display such degrees of identity to a recited protein, polypeptide or peptide when the whole sequence of the recited protein, polypeptide or peptide is queried in the sequence alignment (i.e., overall sequence identity). Sequence identity may be determined using suitable algorithms for performing sequence alignments and determination of sequence identity as know per se. Exemplary but non-limiting algorithms include those based on the Basic Focal Alignment Search Tool (BEAST) originally described by Altschul et al. 1990 (J Mol Biol 215: 403-10), such as the "Blast 2 sequences" algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Eett 174: 247- 250), for example using the published default settings or other suitable settings (such as, e.g., for the BEASTN algorithm: cost to open a gap = 5, cost to extend a gap = 2, penalty for a mismatch = - 2, reward for a match = 1 , gap x dropoff = 50, expectation value = 10.0, word size = 28; or for the BEASTP algorithm: matrix = Blosum62 (Henikoff et al., 1992, Proc. Natl. Acad. Sci., 89:10915- 10919), cost to open a gap = 11, cost toextend a gap = 1, expectation value = 10.0, word size = 3). An example procedure to determine the percent identity between a particular amino acid sequence and the amino acid sequence of a query polypeptide (e.g., RelA polypeptide, e.g., as set forth in SEQ ID NO: 2, 4) will entail aligning the two amino acid sequences using the Blast 2 sequences (B12seq) algorithm, available as a web application or as a standalone executable programme (BLAST version 2.2.31+) at the NCBI web site (www.ncbi.nlm.nih.gov), using suitable algorithm parameters. An example of suitable algorithm parameters include: matrix = Blosum62, cost to open a gap = 1 1, cost to extend a gap = 1 , expectation value = 10.0, word size = 3). If the two compared sequences share homology, then the output will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the output will not present aligned sequences. Once aligned, the number of matches will be determined by counting the number of positions where an identical amino acid residue is presented in both sequences. The percent identity is determined by dividing the number of matches by the length of the query polypeptide, followed by multiplying the resulting value by 100.

[0065] “Vector" refers to a DNA preparation comprising the nucleotide sequence of a gene encoding a protein of interest, operably linked thereto, as an artificial DNA molecule carrying genetic material so as to be capable of expressing the gene of interest in a suitable host.

[0066] "Wild-type" as applied to a nucleic acid or polypeptide refers to a nucleic acid or a polypeptide that occurs in, or is produced by, a biological organism as that biological organism exists in nature.

[0067] Unless specifically indicated or implied, the terms “a,” “an,” and “the” signify “at least one,” as used herein.DETAILED DESCRIPTION

[0068] This invention relates to a recombinant bacterial persister strain comprising an expression cassette, wherein the expression cassette comprises (i) a nucleic acid sequenceof a relA gene or a functional variant thereof operably linked to (ii) a nucleic acid sequence of an inducible promoter.

[0069] In some embodiments, the bacterial persister strain is an aerobic strain.

[0070] In some embodiments, the bacterial persister strain is an anaerobic strain.

[0071] In some embodiments, the bacterial persister strain expresses relA gene endogenously. In other words, the bacterial persister strain comprises an endogenous relA gene. Thus, the bacterial persister strain expresses both the endogenous relA gene and the relA gene introduced into the expression cassette described herein above. It allows the overexpression of the relA gene, leading to a persister phenotype. By endogenous expression, it is meant that the relA gene is naturally present within the strain’s genome, is transcribed and translated to produce the RelA protein. This type of gene expression is contrasted with exogenous gene expression, which involves genes introduced from an external source (such as through genetic engineering or viral infection).

[0072] In some embodiments, the bacterial persister strain is not knocked-out of the endogenous relA gene.

[0073] In some embodiments, the bacterial strain is an ESKAPE (also name ESKAPEE) strain, comprising pathogens which are characterized by increased levels of resistance towards multiple classes of first line and last-resort antibiotics.

[0074] In some embodiments, the bacterial persister strain is a Gram-negative strain. Gram-negative bacteria possess an outer membrane, with its characteristic asymmetric bilayer as a hallmark. The outer membrane bilayer consists of an inner monolayer containing phospholipids (primarily phosphatidyl ethanolamine) and an outer monolayer that is mainly composed of a single glycolipid, lipopolysaccharide (LPS).

[0075] In some embodiments, the bacterial persister strain is a Proteobacteria. In one embodiment, the Proteobacteria is selected from the group consisting of: alpha-, beta-, gamma-, delta-, and epsilon- proteobacteria, preferably gamma-proteobacteria.

[0076] In some embodiments, the Gram-negative bacteria are selected from the group consisting of: Enterobacteriaceae, Pseudomonadaceae, Neisseria, Moraxella, Vibrio, Aeromonas, Brucella, Francisella, Bordetella, Legionella, Bartonella, Coxiella, Haemophilus, Pasteurella, Mannheimia, Actinobacillus, Gardnerella, Spirochaetaceae, Leptospiraceae, Campylobacter, Helicobacter, Spirillum, Streptobacillus, Bacteroidaceae, Acinetobacter.

[0077] In some embodiments, the bacteria from Enterobacteriaceae family are selected from the group comprising or consisting of: Escherichia coli, Salmonella enterica, Shigella spp., Klebsiella pneumoniae, Enterobacter spp., Proteus spp., Serratia spp., Citrobacter spp., Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis .

[0078] In some embodiments, the Escherichia coli, also named E. coli, strain includes, but are not limited to, E. coli K-12, E. coli B or E. coli W, such as, for example, MG1655, W3110, BW25113, MC1061, DG1, DG2, ToplO, DH10B, DH5alpha, XL1 Blue, XL10 Gold, HB101, JM109, HMS174, CFT073, BL21, BL21(DE3) and HMS174(DE3).

[0079] In some embodiments, the Escherichia coli strain is selected from the group comprising or consisting of ATCC references: 25922, 35218 and 8739.

[0080] In some embodiments, the Escherichia coli strain has the 16S DNA sequence of SEQ ID NO: 14.

[0081] In some embodiments, the bacteria from Pseudomonadaceae family are selected from the group comprising or consisting of bacteria belonging to the genera Pseudomonas, especially P. aeruginosa, Burkholderia, Stenotrophomonas, Shewanella, Sphingomonas and Comamonas.

[0082] In some embodiments, the bacteria from genera Pseudomonas are selected from the group comprising or consisting of: Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae, Pseudomonas mendocina, Pseudomonas stutzeri, Pseudomonas chlororaphis, Pseudomonas fragi, Pseudomonas pseudoalcaligenes, Pseudomonas citronellolis, preferably Pseudomonas aeruginosa.

[0083] In some embodiments, the Pseudomonas aeruginosa strain has the 16S DNA sequence of SEQ ID NO: 15.

[0084] In some embodiment, the Pseudomonas aeruginosa strain is selected from the group consisting of: PAO1, PAO1-LAC, PA14, PAK, PA103, LESB58, and PA7.

[0085] In some embodiments, the bacteria from Spirochaetaceae family are selected from the group comprising or consisting of: bacteria belonging to the genera Treponema and Borrelia.

[0086] In some embodiments, the bacteria from Bacteroidaceae family are selected from the group comprising or consisting of: bacteria belonging to the genera Bacteroides, Fusobacterium, Prevotella, Porphyromonas.

[0087] In some embodiments, the bacterial strains or a derivative thereof also include those having a 16s rRNA gene sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO: 14 or 15.

[0088] The recombinant bacterial persister strain of the disclosure is persistent to some antibiotics thanks to the presence of the expression cassette which comprises (i) a nucleic acid sequence of relA gene or a functional variant thereof.

[0089] In some embodiments, the expression cassette allows the overexpression of a relA gene.

[0090] In some embodiments, the bacterial persister strain is persistent to at least one antibiotic.

[0091] In some embodiments, the bacterial persister strain is persistent to at least one antibiotic selected from the group comprising or consisting of: beta-lactams (also named P-lactams, i.e. penicillins, cephalosporins, carbapenems, monobactams), sulfonamides, tetracyclines, oxazolidinones, aminoglycosides, glycopeptides, fluoroquinolones, quinolones, rifamycin, macrolides, lipopeptides, lincosamides,, nitrofurans, fosfomycin, trimethoprim, sulfamethoxazole, oxy quinolines, fusidic acid and mupirocin.

[0092] In some embodiments, the penicillin antibiotic is selected from the group comprising or consisting of: ampicillin, amoxicillin, amoxicillin and clavulanic acid, piperacillin, piperacillin / tazobactam, ticarcillin / clavulanic acid, and mecillinam.

[0093] In some embodiments, the cephalosporin antibiotic is selected from the group comprising or consisting of: 1stgeneration (CIG): cefalexin, cefazolin, 2ndgeneration (C2G): cefuroxime, cefoxitin, 3thgeneration (C3G): cefpodoxime, ceftazidime, ceftazidime / avibactam, cefotaxime, ceftriaxone, cefdinir, 4thgeneration (C4G): cefepime, 5thgeneration (C5G): ceftaroline, ceftobiprole, and cefiderocol.

[0094] In some embodiments, the carbapenem antibiotic is selected from the group comprising or consisting of: meropenem, meropenem-vaborbactam, imipenem, imipinem with cilastatin, and ertapenem.

[0095] In some embodiments, the sulfonamide antibiotic is selected from the group comprising or consisting of: sulfamethoxazole and trimethoprim (Bactrim®).

[0096] In some embodiments, the tetracycline antibiotic is selected from the group comprising or consisting of: tetracycline, doxycycline, minocycline and tigecycline.

[0097] In some embodiments, the oxazolidinone antibiotic is linezolid.

[0098] In some embodiments, the aminoglycoside antibiotic is selected from the group comprising or consisting of: tobramycin, amikacin, gentamicin and netilmicin.

[0099] In some embodiments, the glycopeptides antibiotic is selected from the group comprising or consisting of: vancomycin and teicoplanin.

[0100] In some embodiments, the fluoroquinolones antibiotic is selected from the group comprising or consisting of: ciprofloxacin and levofloxacin.

[0101] In some embodiments, the quinolone antibiotic is selected from the group comprising or consisting of: ciprofloxacin, levofloxacin, norfloxacin and ofloxacin.

[0102] In some embodiments, the rifamycin antibiotic is rifampin.

[0103] In some embodiments, the macrolide antibiotic is selected from the group comprising or consisting of: azithromycin, erythromycin, and clarithromycin.

[0104] In some embodiments, the lipopeptide antibiotic is selected from the group comprising or consisting of: colistin and polymyxin B.

[0105] In some embodiments, the lincosamide antibiotic is selected from the group comprising or consisting of: lincomycin and clindamycin.

[0106] In some embodiments, the monobactam antibiotic is the aztreonam.

[0107] In some embodiments, the nitrofurans antibiotic is the nitrofurantoin.

[0108] The group of antibiotics for each family are commonly used in the field and are well-known to the skilled artisan, who will know the group of antibiotics included in each family.

[0109] In some embodiments, the E. coli persister strain is more resistant and / or tolerant to the antibiotic selected from the group comprising or consisting of: beta-lactams (penicillins, cephalosporins, carbapenems, monobactams), sulfonamides, tetracyclines, aminoglycosides, quinolones, rifamycin, macrolides, lipopeptides, nitrofurans, fosfomycin, trimethoprim, sulfamethoxazole and oxy quinolines.

[0110] Table 1: Selection of some antibiotics allowing the characterization of E. coli persister strain

[0111] In some embodiments, the P. aeruginosa persister strain is persistent to the antibiotic selected from the group comprising or consisting of: beta-lactams (penicillins, cephalosporins, carbapenems), aminoglycosides, quinolones, macrolides, lipopeptides, monobactam and fosfomycin.

[0112] Table 2: Selection of some antibiotics allowing the characterization of P. aeruginosa persister strain

[0113] The selection and the concentrations of the antibiotics are commonly used in the field and are well-known to the skilled artisan, who will know in which conditions to cultivate the bacterial persister strain, in order to characterize it.

[0114] In some embodiments, the bacterial persister strain, which is more resistant is characterized by a survival rate after induction of relA gene expression from 5% to 100%, from 6% to 100%, from 7% to 100%, from 8% to 100%, from 9% to 100%, from 10% to 100%, from 20% to 100%, from 30% to 100%, from 40% to 100%, from 50% to 100%, from 60%, to 100%, from 70% to 100%, from 80% to 100% or from 90% to 100%, preferably from 5% to 100%.

[0115] Survival rate is the proportion of bacteria that remain viable after antibiotic treatment. Survival rate is calculated as follows: Survival rate = number of bacterial colonies after treatment / number of bacterial colonies before treatment xlOO expressed as percent (%). Preferably, the survival rate is calculated by dividing the number of CFU / ml after treatment by the number of CFU / ml before adding the antibiotic.

[0116] In some embodiments, survival rate could be evaluated using colorimetric method, dye method, fluorescence method, or luminescence method.

[0117] In some embodiments, the colorimetric method is selected from the group consisting of MTT, XTT, WST-1 and AlamarBlue®, preferably AlamarBlue®.

[0118] In some embodiments, the fluorescence method is selected from the group consisting of calcein-AM, FDA and AlamarBlue®, preferably AlamarBlue®.

[0119] In some embodiments, the treatment is the culture with one or more antibiotics.

[0120] In some embodiments, the treatment is selected from the group of any kind of compound which can be screened for anti-persister activity.

[0121] In some embodiments, the relA gene is induced in early log phase.

[0122] The early log phase is well-known to the skilled artisan, who will know when the early log phase is reached depending on the bacterial strain.

[0123] In some embodiments, the expression of the relA gene (ii) is induced from ODeoo 0.1, to ODeoo 0.5, from ODeoo 0.2, to ODeoo 0.5, from ODeoo 0.3, to ODeoo 0.5, or from ODeoo 0.4, to ODeoo 0.5, ODeoo 0.1, to ODeoo 0.4, ODeoo 0.1, to ODeoo 0.3, ODeoo 0.1, to ODeoo 0.2. In some embodiments, the expression of the relA gene (ii) is preferably induced from ODeoo 0.1, ODeoo 0.2, ODeoo 0.3, ODeoo 0.4, or ODeoo 0.5, more preferably from ODeoo 0.2.

[0124] In some embodiments, the survival rate is calculated more than Ih after antibiotic treatment, from about Ih to about lOOh after antibiotic treatment, preferably about 96h. In one embodiment, the survival rate may be measured from about 1 hour to about 100hours, from about 2 hours to about 99 hours, from about 3 hours to about 98 hours, from about 4 hours to about 97 hours, from about 5 hours to about 96 hours, from about 6 hours to about 95 hours, from about 7 hours to about 94 hours, from about 8 hours to about 93 hours, from about 9 hours to about 92 hours, from about 10 hours to about 91 hours, from about 11 hours to about 90 hours, from about 12 hours to about 89 hours, from about 13 hours to about 88 hours, from about 14 hours to about 87 hours, from about 15 hours to about 86 hours, from about 16 hours to about 85 hours, from about 17 hours to about 84 hours, from about 18 hours to about 83 hours, from about 19 hours to about 82 hours, from about 20 hours to about 81 hours, from about 21 hours to about 80 hours, from about 22 hours to about 79 hours, from about 23 hours to about 78 hours, from about 24 hours to about 77 hours, from about 25 hours to about 76 hours, from about 26 hours to about 75 hours, from about 27 hours to about 74 hours, from about 28 hours to about 73 hours, from about 29 hours to about 72 hours, from about 30 hours to about 71 hours, from about 31 hours to about 70 hours, from about 32 hours to about 69 hours, from about 33 hours to about 68 hours, from about 34 hours to about 67 hours, from about 35 hours to about 66 hours, from about 36 hours to about 65 hours, from about 37 hours to about 64 hours, from about 38 hours to about 63 hours, from about 39 hours to about 62 hours, from about 40 hours to about 61 hours, from about 41 hours to about 60 hours, from about 42 hours to about 59 hours, from about 43 hours to about 58 hours, from about 44 hours to about 57 hours, from about 45 hours to about 56 hours, from about 46 hours to about 55 hours, from about 47 hours to about 54 hours, from about 48 hours to about 53 hours, from about 49 hours to about 52 hours, or from about 50 hours to about 51 hours after antibiotic treatment.In some embodiments, the survival rate is evaluated about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, about 72 hours, about 73 hours, about 74 hours, about 75 hours, about 76 hours, about 77 hours, about 78 hours, about 79 hours, about 80 hours, about 81 hours, about 82 hours, about 83 hours, about 84 hours, about 85 hours, about 86 hours, about 87 hours, about 88 hours, about 89 hours, about 90 hours, about 91 hours, about 92 hours, about 93 hours, about 94 hours, about 95 hours, about 96 hours, about 97 hours, about 98 hours, about 99 hours, and about 100 hours after antibiotic treatment, preferably about 96 hours, or about 48 hours of antibiotic treatment. In preferred embodiments, the survival rate is evaluated about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, or about 55 hours after antibiotic treatment. In another embodiment, the survival rate is evaluated about 90 hours, about 91 hours, about 92 hours, about 93 hours, about 94 hours, about 95 hours, about 96 hours, about 97 hours, about 98 hours, about 99 hours, or about 100 hours after antibiotic treatment.

[0125] In some embodiments, the survival rate of the bacteria from Enterobacteriaceae family is evaluated about 90 hours, about 91 hours, about 92 hours, about 93 hours, about 94 hours, about 95 hours, about 96 hours, about 97 hours, about 98 hours, about 99 hours, and about 100 hours after antibiotic treatment, preferably from about 96 hours after the antibiotic treatment.

[0126] In some embodiments, the survival rate of the bacteria from Pseudomonadaceae family is evaluated about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, or about 55 hours after antibiotic treatment, preferably about 48 hours after antibiotic treatment.

[0127] In some embodiments, the method of the characterization of a bacterial persister strain comprises the following steps: a. Cultivating the bacterial persister strain, b. Inducing the expression of the re I A gene,c. Cultivating the culture, d. Optionally, diluting the cells, e. Cultivating the culture with one or more antibiotics, and f. Calculate the survival rate.

[0128] In some embodiments, the step (ii) of inducing the expression of the relA gene corresponds to the expression of the relA gene introduced into the expression cassette as described herein above.

[0129] In some embodiments, the method of the characterization of a bacterial persister strain comprises the following steps: a. Cultivating the bacterial persister strain, b. Inducing the expression of the relA gene, c. Cultivating the culture for one or more hours, preferably 2 hours, more preferably 3 hours, d. Diluting the cells, e. Challenging the cells with ciprofloxacin from about 0.3 pg / ml to about 0.7 pg / ml, more preferably around 0.5 pg / ml, and f. Cultivating the culture for one or more hours, preferably 2 hours, preferably 3 hours, more preferably 4 hours.

[0130] In a preferred embodiment, the bacterial persister strain of the method above is from Enterobacteriaceae family, more preferably from Escherichia coli.

[0131] In some embodiments, the method of the characterization of a bacterial persister strain comprises the following steps: a. Cultivating the bacterial persister strain, b. Inducing the expression of the relA gene, c. Cultivating the culture for one or more hours, preferably 12-18 hours, more preferably 15 hours, d. Diluting the cells, e. Challenging the cells with meropenem from about 1 pg / ml to about 50 pg / ml, more preferably around 25 pg / ml, orf. Challenging the cells with cefepime from about 4 g / ml to about 200 pg / ml, more preferably around 100 pg / ml, or g. Challenging the cells with piperacillin from about 8 pg / ml to about 400 pg / ml, more preferably around 200 pg / ml, and h. Cultivating the culture for one or more hours, preferably 4-6 hours, more preferably 6 hours.

[0132] In one embodiment, the bacterial persister strain of the method above is from Pseudomonadaceae family, more preferably from P. aeruginosa.

[0133] In some embodiments, the method of the characterization of a bacterial persister strain comprises the following steps: a. Cultivating the bacterial persister strain, b. Inducing the expression of the re I A gene, c. Cultivating the culture for one or more hours, preferably 12-18 hours, more preferably 15 hours, d. Diluting the cells, e. Challenging the cells with meropenem, cefepime or piperacillin, from about 5X to 100X the MIC, more preferably 50X the MIC, and f. Cultivating the culture for one or more hours, preferably 4-6 hours, more preferably 6 hours.

[0134] In one embodiment, the bacterial persister strain of the method above is from Pseudomonadaceae family, more preferably from P. aeruginosa.

[0135] In some embodiments, the bacterial persister strain is cultivated in a culture medium.

[0136] In some embodiments, the bacteria are cultivated in a culture medium.

[0137] In some embodiments, the culture medium is selected from the group comprising or consisting of: Lysogeny Broth (LB), LB Broth Miller, LB Agar, M9 Medium, Terrific Broth (TB), MacConkey Agar, Eosin Methylene Blue (EMB) Agar, Tryptic Soy Broth (TSB), Tryptic Soy Agar (TSA), Cystine-Lactose-Electrolyte-Deficient (CLED)Medium, Brain Heart Infusion (BHI) Medium, Raffinose Minimal (RM) Medium, and Mueller Hinton II (MHII) Medium.

[0138] In some embodiments, the culture medium is selected from the group comprising or consisting of: Cetrimide Agar, King's Medium A, King's Medium B, Pseudomonas Isolation Agar, Pseudomonas Agar F, Pseudomonas Agar P, Brain Heart Infusion (BHI) Medium, Tryptic Soy Broth (TSB), Nutrient Agar, LB Agar and LB.

[0139] The culture medium is well known to the skilled artisan, who will know which culture medium has to be used depending on the bacterial strain.

[0140] In some embodiments, the bacterial persister strain comprises an expression cassette comprising (i) a nucleic acid sequence of a re I A gene or a functional fragment thereof operably linked to (ii) a nucleic acid sequence of an inducible promoter.

[0141] In some embodiments, the expression cassette is comprised into an expression vector.

[0142] In some embodiments, the expression vector comprises at least one regulatory element including, without limitations, terminators, translational regulatory sequences (e.g., ribosome binding sites [RBS] and internal ribosome entry sites [IRES]), enhancers, silencers, insulators, boundary elements, matrix attachment sites and locus control regions.

[0143] In some embodiments, the expression cassette comprises a ribosome-binding sequence (RBS). The skilled person would understand and select an appropriate Ribosome Binding Site (RBS) based on known RBS sequences, their strengths, and the desired level of gene expression, optimizing translation efficiency for the specific host organism and expression system used.

[0144] In some embodiments, the expression cassette comprises a transcription terminator. The skilled person would recognize and select a suitable terminator sequence based on known terminators, their efficiency in halting transcription, and compatibility with the host organism and expression system, ensuring proper termination of the transcribed region for optimal gene expression.

[0145] In some embodiments, the expression cassette can be monocistronic or polycistronic, such as bicistronic, tricistronic, tetracistronic, pentacistronic, hexacistronic, heptacistronic, octacistronic, nonacistronic, or decacistronic.

[0146] In the context of the invention, the nucleic acid sequence of the relA gene (i) encodes the RelA polypeptide.

[0147] The RelA polypeptide is a Ribosome-associated protein involved in the stringent response pathway, regulating bacterial adaptation to nutrient limitation by synthesizing (p)ppGpp, a signaling molecule that modulates various cellular processes such as transcription, translation, and metabolism.

[0148] “RelA” or “RelA polypeptide” designates a polypeptide belonging to the family of GDP / GTP pyrophosphokinase, by reference to the amino acid sequence of SEQ ID NO: 2; which corresponds to RelA peptide sequence form E. coli K-12 substr. MG1655 (NCBI reference: NP_417264.1) or the amino acid sequence of SEQ ID NO: 4; which correspond to RelA polypeptide sequence form P. aeruginosa.

[0149] In some embodiments, the RelA polypeptide is a wild-type polypeptide, or a variant thereof.

[0150] In some embodiments, the RelA polypeptide is the wild-type polypeptide from the same family, preferably the same genus, more preferably the same species than the bacterial strain.

[0151] In some embodiments, if the recombinant bacterial persister strain is an Escherichia coli strain, then RelA polypeptide or a functional variant thereof corresponds to RelA peptide sequence from E. coli or a functional variant thereof, such has the RelA polypeptide has the amino acid sequence SEQ ID NO: 2.

[0152] In some embodiments, if the recombinant bacterial persister strain is a Pseudomonas aeruginosa strain, then RelA polypeptide or a functional variant thereof correspond to RelA peptide sequence form P. aeruginosa or a functional variant thereof, such has the RelA polypeptide has the amino acid sequence SEQ ID NO: 4.

[0153] In some embodiments, the wild-type RelA polypeptide has the amino acid sequence SEQ ID NO: 2 (From E. coli K-12 substr. MG1655 with NCBI reference: NP-417264.1).

[0154] In some embodiments, the wild-type relA gene has the nucleic acid sequence SEQ ID NO: 1 (From E. coli K-12 substr. MG1655).

[0155] In some embodiments, the wild-type RelA polypeptide has the amino acid sequence SEQ ID NO: 4 (From P. aeruginosa PA14).

[0156] In some embodiments, the wild-type relA gene has the nucleic acid sequence SEQ ID NO: 3 (From P. aeruginosa PA14).

[0157] The RelA polypeptide or a functional variant thereof refers to proteins, polypeptides or peptides with the amino acid sequence of which is substantially identical (i.e., largely but not wholly identical) to the sequence of the protein, polypeptide, or peptide, e.g., at least 70% identical or at least 75% identical, e.g. preferably at least 80% identical or at least 85% identical, e.g., preferably at least 90% identical, e.g., at least 91% identical, 92% identical, more preferably at least 93% identical, e.g., at least 94% identical, even more preferably at least 95% identical, e.g., at least 96% identical, yet more preferably at least 97% identical, e.g., at least 98% identical, and most preferably at least 99% identical to the sequence of the protein, polypeptide, or peptide, e.g., to the sequence of a corresponding RelA protein. In some embodiments, RelA polypeptide or a functional variant thereof refers to proteins, polypeptides or peptides with the amino acid sequence of which is 100% identical to the sequence of a corresponding RelA polypeptide.

[0158] RelA polypeptide or a functional variant thereof may be a homologue (e.g., orthologue or paralogue) of said protein, polypeptide, or peptide.

[0159] In some embodiments, the RelA polypeptide or a functional variant thereof, i.e. may be conveniently denoted as “modified”, or as “mutated” or “mutant”, or as comprising one or more mutations, i.e., comprising one or more amino acid sequence changes compared to the amino acid sequence of RelA that has not been so-mutated, suchas, particularly, compared to the amino acid sequence of wild-type RelA. Said functional variant may comprise the same number of amino acids as any RelA polypeptide defined above, more preferably as SEQ ID NO: 2 or SEQ ID NO: 4, and thus the mutations and positions described herein are the same for the variant. Alternatively, said variant may comprise a different number of amino acids as SEQ ID NO: 2 or SEQ ID NO: 4. The skilled artisan in the art will know how to place the mutations and positions described herein in the variant.

[0160] In some embodiments, the nucleic acid sequence of the relA gene has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of identity with SEQ ID NO: 1 or SEQ ID NO: 3.

[0161] In some embodiments, the nucleic acid sequence of the relA gene has the nucleic acid sequence SEQ ID NO: 1.

[0162] In some embodiments, the nucleic acid sequence of the relA gene has the nucleic acid sequence SEQ ID NO: 3.

[0163] In some embodiments, the nucleic acid sequence of interest encodes the RelA polypeptide or a functional variant thereof.

[0164] In some embodiments, the RelA polypeptide or a functional variant thereof may be a functional fragment thereof of RelA polypeptide. This fragment can comprise at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the RelA protein sequence.

[0165] In some embodiments, the RelA polypeptide or a functional variant thereof may comprise one or more amino acid additions, deletions, or substitutions relative to (i.e., compared with) the corresponding protein or polypeptide, e.g., a corresponding RelA, e.g., RelA polypeptide as set forth in SEQ ID NO: 2 or SEQ ID NO: 4.

[0166] In some embodiments, the substitution is a conservative substitution. A conservative amino acid substitution is a substitution of one amino acid for another with similar characteristics. Conservative amino acid substitutions include substitutions within the following groups: valine, alanine and glycine; leucine, valine, and isoleucine; asparticacid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. The nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (i.e., basic) amino acids include arginine, lysine and histidine. The negatively charged (i.e., acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above- mentioned polar, basic, or acidic groups by another member of the same group can be deemed a conservative substitution. By contrast, a non-conservative substitution is a substitution of one amino acid for another with dissimilar characteristics.

[0167] The nucleic acid sequence of the present disclosure (ii) encodes an inducible promoter.

[0168] In some embodiments, the expression cassette provided herein allows to genetically engineer a cell to increase the expression of RelA polypeptide from (ii) an inducible promoter in the absence of the inducing molecule.

[0169] In some embodiments, the expression cassette provided herein allows to genetically engineer a cell to increase the expression of RelA polypeptide from (ii) an inducible promoter in the presence of the inducing molecule.

[0170] In some embodiments, the relA gene is overexpressed by the strain. In some embodiments, the strain produces more RelA polypeptide than the WT strain.

[0171] In some embodiments, (ii) the inducible promoter is selected in the group comprising or consisting of: ParaBAD (also named pBAD), Ptet, Plac, PlacUV5, Ptac, Ptrc, PT5, PT7, Prha, PprpR, Plux, Pdox, Pmalt, Prib.

[0172] In some embodiments, (ii) the inducible promoter allows the overexpression of the relA gene by the strain.

[0173] In some embodiments, (ii) the inducible promoter is the araBAD promoter (ParaBAD, pBAD). The araBAD promoter is a tightly regulated inducible promoter derived from the araBAD operon of Escherichia coli. It responds to the presence ofarabinose in the surrounding environment. In the absence of arabinose, the araBAD promoter is repressed by the AraC protein, minimizing gene expression.

[0174] In some embodiments, the araBAD promoter is a functional variant of the promoter which is substantially identical (i.e., largely but not wholly identical) to the sequence of the promoter, e.g., at least about 80% identical or at least about 85% identical, e.g., preferably at least about 90% identical, e.g., at least 91% identical, 92% identical, more preferably at least about 93% identical, e.g., at least 94% identical, even more preferably at least about 95% identical, e.g., at least 96% identical, yet more preferably at least about 97% identical, e.g., at least 98% identical, and most preferably at least 99% identical to the sequence of the promoter, e.g., to the sequence of a corresponding ParaBAD.

[0175] In some embodiments, the araBAD promoter has the nucleic acid sequence SEQ ID NO: 5.

[0176] In some embodiments, the lac promoter has the nucleic acid sequence SEQ ID NO: 6.

[0177] In some embodiments, the lac UV5 promoter has the nucleic acid sequence SEQ ID NO: 7.

[0178] In some embodiments, the tac promoter has the nucleic acid sequence SEQ ID NO: 8.

[0179] In some embodiments, the tac promoter is a functional variant of the promoter which is substantially identical (i.e., largely but not wholly identical) to the sequence of the promoter, e.g., at least about 80% identical or at least about 85% identical, e.g., preferably at least about 90% identical, e.g., at least 91% identical, 92% identical, more preferably at least about 93% identical, e.g., at least 94% identical, even more preferably at least about 95% identical, e.g., at least 96% identical, yet more preferably at least about 97% identical, e.g., at least 98% identical, and most preferably at least 99% identical to the sequence of the promoter, e.g., to the sequence of a corresponding tac promoter. In some embodiments, the trc promoter has the nucleic acid sequence SEQ ID NO: 9.

[0180] In some embodiments, the T5 promoter has the nucleic acid sequence SEQ ID NO: 10.

[0181] In some embodiments, the T7 promoter has the nucleic acid sequence SEQ ID NO: 11.

[0182] In some embodiments, the rhaBAD promoter has the nucleic acid sequence SEQ ID NO: 12.

[0183] In some embodiments, the promoter is tet operator and has the nucleic acid sequence SEQ ID NO: 13.

[0184] In some embodiments, the expression cassette is selected in the group comprising or consisting of: a linear or circular DNA or RNA.

[0185] In some embodiments, the expression cassette is comprised in a vector.

[0186] In some embodiments, the vector is selected in the group comprising or consisting of: a plasmid and a bacterial artificial chromosome.

[0187] In some embodiments, the vector is a plasmid.

[0188] In some embodiments, the plasmid is compatible with the replication and / or delivery systems in a bacterium.

[0189] In some embodiments, the plasmid comprises one or more selection markers. A selection marker is used to select a cell transformed with the vector, i.e., to identify whether the polynucleotide is in the cell or not, and markers can be used to confer a selectable phenotype such as drug resistance, auxotrophy, resistance to a cytotoxic agent, or expression of a surface protein.

[0190] In some embodiments, the selection marker is selected from the group comprising or consisting of: antibiotic ssm, nutrient auxotroph, reporter genes, metabolic markers and temperature- sensitive markers.

[0191] In some embodiments, the selection marker is an antibiotic, also named antibioticsm.

[0192] In some embodiments, the antibioticsmis selected in the group comprising or consisting of: ampicillin, carbenicillin, kanamycin, neomycin, streptomycin, spectinomycin, gentamicin, tetracycline, chloramphenicol, erythromycin, bleomycin, hygromycin, apramycin, puromycin, and zeocin.

[0193] In some embodiments, the antibioticsm is a different antibiotic from the one used to characterize the bacterial persister strain.

[0194] In some embodiments, the vector comprises at least one origin of replication (ORI).

[0195] In some embodiments, the plasmid comprises at least one origin of replication selected in the group comprising or consisting of: oriV, oriC, ColE origin, pUC Origin, pBR322 Origin, pl5A Origin, Fl Origin, pMBl Origin, SV40 Origin.

[0196] In some embodiments, the expression cassette and / or the vector is integrative.

[0197] In some embodiments, the expression cassette and / or the vector is non- integrative.

[0198] Integrative or non-integrative vectors are commonly used in the field and are well-known to the skilled artisan, who will know how to design the plasmid construct so that the expression cassette is integrative or not.

[0199] The invention also relates to a method for producing a recombinant bacterial persister strain, the method comprising the following steps:(a) transforming one or more bacterium with one or more expression cassette, wherein the one or more expression cassette comprises a (i) a nucleic acid sequence of a relA gene and / or a functional fragment thereof operably linked to (ii) a nucleic acid sequence of an inducible promoter, and(b) selecting the bacterium which has integrated the expression cassette.

[0200] In some embodiments, the step (b) selecting the bacterium which has integrated the expression cassette comprises a step of culturing the bacteria in a selection medium, such as a medium comprising an antibiotic suitable for the selection (also named here antibioticsm) or a medium comprising an auxotrophic selection.

[0201] The invention also relates to the recombinant bacterial persister strain obtained by the method described herein.

[0202] The transformation (a) comprises all methods of introducing the gene encoding the protein of interest into a cell, and can be performed by selecting suitable standard techniques as known in the art according to the host cell.

[0203] In some embodiments, the bacteria are transformed (a) using techniques selected in the group comprising or consisting of: electroporation, heat shock transformation, also known as chemical transformation and calcium chloride transformation, conjugation, microinjection (i.e., microinjection Polyethylene glycol (PEG)), DEAE-dextran, lipofection (i.e., cationic liposome), lithium acetate-DMSO and Biological Nanoparticles (i.e., Bacteriophage).

[0204] In some embodiments, the selection of the bacterium which has integrated the expression cassette (b) is made with a selection marker. The integration encompassed the introduction of the expression cassette into the bacterial strain in any forms (plasmid, episome, etc.).

[0205] In some embodiments, the integration encompassed the introduction of the expression cassette into the chromosome of the bacterial strain.

[0206] In some embodiments, the selection marker, as mentioned herein, is selected from the group comprising or consisting of: antibiotic ssm, nutrient auxotrophy, reporter genes, metabolic markers and temperature- sensitive markers.

[0207] In some embodiments, the antibioticssmof the step (b) is selected from the group comprising or consisting of: ampicillin, carbenicillin, kanamycin, neomycin, streptomycin, spectinomycin, gentamicin, tetracycline, chloramphenicol, erythromycin, bleomycin, hygromycin, apramycin, puromycin, and zeocin.

[0208] The selection and the concentrations of the antibiotic ssm, and in general, the use of the selection marker, are commonly used in the field and are well-known to the skilled artisan, who will know in which conditions to cultivate the transformed bacterial strain and select the bacteria that has integrated the expression cassette.

[0209] The invention also relates to a model comprising the recombinant bacterial persister strain as described herein.

[0210] In some embodiments, the model is an insect model, such as larva or caterpillar.

[0211] In some embodiments, the model is a Caenorhabditis elegans.

[0212] In some embodiments, the model is a Zebrafish.

[0213] In some embodiments, the model is a mammalian model.

[0214] In some embodiments, the mammalian model is an animal. It may for instance be a rodent or a non-human primate.

[0215] In some embodiments, the mammalian model is a non-human mammalian model.

[0216] In some embodiments, the mammalian model can be a mouse, a rat or a dog.

[0217] In some embodiments, the mammalian model is produced by the administration of the bacterial persister strain to the mammalian model.

[0218] The invention also relates to a method for producing an insect model to study bacterial persistence, wherein the at least one recombinant bacterial persister strain, as described herein, is administrated to an insect.

[0219] The invention also relates to a method for producing a mammalian model to study persistent microorganisms, wherein the at least one recombinant bacterial persister strain, as described herein, is administrated to a mammalian.

[0220] The invention also relates to an insect model comprising the recombinant bacterial persister strain as described herein.

[0221] In some embodiments, the insect is selected in the group comprising or consisting of: Galleria mellonella, Tenebrio molitor, Drosophila melanogaster, Bombyx mori, Apis mellifera, Manduca sexta, Bactrocera dorsalis, Gryllus bimaculatus and Gryllus pennsylvanicus.

[0222] In some embodiments, the insect can be a Galleria mellonella.

[0223] The invention also relates to a method for producing an insect model to study bacterial persistence, wherein the at least one recombinant bacterial persister strain, as described herein, is administrated to an insect.

[0224] In some embodiments, the method for producing an insect model to study bacterial persistence comprises the following steps (in the same order): a) Wash the bacterial persister cells, b) Optionally, adjust the number of cells from about 5 x 107to about 5 x 109preferably 5 x 108CFU / ml, c) Inoculate an aliquot from about 5 x 105to about 5 x 107, more preferably about 5 x 106CFU, from the step b) into an insect, d) Place the inoculated insect from the step c) in petri dishes and incubate from about 30°C to about 45°C, more preferably at 37°C, for at least 30 minutes, more preferably at least 65 minutes, e) Administer one or more antibiotics to the insect incubated in step d), and f) Count the number of the dead and / or live larvae after one or more days.

[0225] In some embodiments, the bacterial persister cells are washed with buffer in step a), preferably PBS.

[0226] In some embodiments, the step a) of washing the bacterial persister cells is repeated several times, once, twice, three times, four, five times or more, preferably three times.

[0227] In some embodiments, the insect model of the step c) is a larva or a caterpillar.

[0228] In some embodiments, inoculation of the step c) is made into hemocoel of the caterpillar via the last proleg. In one embodiment, the last proleg is the left proleg or the right proleg, preferably the left proleg.

[0229] In some embodiments, the antibiotic is administered within the insect (step e), in the same location where the bacterial persister cells were inoculated.

[0230] In some embodiments, the antibiotic is administered within the insect (step e), not in the same location where the bacterial persister cells were inoculated.

[0231] In one embodiment, the bacterial persister cells are inoculated into the hemocoel of each caterpillar via the last left proleg, and the antibiotics are administered into the hemocoel of each caterpillar via the last right proleg.

[0232] In some embodiments, one or more antibiotics are administered. The phrase “one or more antibiotic administrations” encompasses the administration of a single antibiotic multiple times or the administration of multiple antibiotics, simultaneous or not.

[0233] In some embodiments, the step f), after one or more days, such as one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, or ten days.

[0234] In some embodiments, in step f), the dead and / or live insects are counted one or more times a day, every day.

[0235] The parameters for cultivation of insects are commonly used in the field and are well-known to the skilled artisan, who will know in which conditions to cultivate the insect, according to the needs of the insect.

[0236] In some embodiments, in step f), the larvae or the caterpillars are kept at 37°C in the dark.

[0237] The invention also relates to a method for producing a mammalian model to study persistent microorganisms, wherein the at least one recombinant bacterial persister strain, as described herein, is administrated to a mammalian.

[0238] The invention also relates to a method for screening a compound of interest with anti-persister activity.

[0239] In some embodiments, said method of screening can comprise the following steps: a. culturing the recombinant bacterial persister strain according to the invention, as described herein, or obtained according to the method of the invention, as described herein, into a medium without any compound with anti-persister activity, b. inducing the expression of the relA gene, c. introducing into the medium a compound of interest, and d. quantifying the survival rate of the bacterial persister strain, thereby determining if the compound of interest has anti-persister activity.

[0240] In some embodiments, said method of screening can comprise the following steps: a. culturing the kit-of-parts as described herein in at least one medium, b. inducing the expression of the relA gene, c. introducing into the medium a compound of interest, and d. quantifying the survival rate of the bacterial persister strain, thereby determining if the compound of interest has anti-persister activity.

[0241] In some embodiments, the expression of the relA gene (ii) is induced in early log phase.

[0242] The early log phase is well-known to the skilled artisan, who will know when the early log phase is depending on the bacterial strain.

[0243] In some embodiments, the expression of the relA gene (ii) is induced from ODeoo 0.1, to ODeoo 0.5, from ODeoo 0.2, to ODeoo 0.5, from ODeoo 0.3, to ODeoo 0.5, or from ODeoo 0.4, to ODeoo 0.5, ODeoo 0.1, to ODeoo 0.4, ODeoo 0.1, to ODeoo 0.3, ODeoo 0.1, to ODeoo 0.2. In some embodiments, the expression of the relA gene (ii) is preferably inducedfrom about ODeoo 0.1, ODeoo 0.2, ODeoo 0.3, ODeoo 0.4, or ODeoo 0.5, more preferably from about ODeoo 0.2.

[0244] In some embodiments, said method of screening further comprises a step (a’) of culturing (b) a second recombinant bacterial persister strain into a second culture medium allowing the growth, said (b) second recombinant bacterial persister comprises an expression cassette, wherein the expression cassette comprises (ib) a second nucleic acid sequence of a relA gene or a functional variant thereof operably linked to (iib) a second nucleic acid sequence of an inducible promoter.

[0245] In some embodiments, the (b) second recombinant bacterial persister strain is an aerobic strain.

[0246] In some embodiments, the (b) second recombinant bacterial persister strain is an anaerobic strain.

[0247] In some embodiments, the (b) second recombinant bacterial persister strain expresses the relA gene endogenously. In other words, the (b) second recombinant bacterial persister strain comprises an endogenous relA gene. Thus, the (b) second recombinant bacterial persister strain can express both the endogenous relA gene and the relA gene introduced into the expression cassette described herein above. It allows the overexpression of the relA gene, leading to a persister phenotype. By endogenous expression, it is meant that the relA gene is naturally present within the strain’s genome, is transcribed and translated to produce the RelA protein. This type of gene expression is contrasted with exogenous gene expression, which involves genes introduced from an external source (such as through genetic engineering or viral infection).

[0248] In some embodiments, the (b) second recombinant bacterial persister strain is not knocked-out of the endogenous relA gene.

[0249] In some embodiments, the (b) second recombinant bacterial strain is an ESKAPE (also name ESKAPEE) strain, comprising pathogens which are characterized by increased levels of resistance towards multiple classes of first line and last-resort antibiotics.

[0250] In some embodiments, the (b) second recombinant bacterial persister strain is a Gram-negative strain. Gram-negative bacteria possess an outer membrane, with its characteristic asymmetric bilayer as a hallmark. The outer membrane bilayer consists of an inner monolayer containing phospholipids (primarily phosphatidyl ethanolamine) and an outer monolayer that is mainly composed of a single glycolipid, lipopolysaccharide (LPS).

[0251] In some embodiments, the (b) second recombinant bacterial persister strain is a Proteobacteria. In one embodiment, the Proteobacteria is selected from the group consisting of: alpha-, beta-, gamma-, delta-, and epsilon- proteobacteria, preferably gamma-proteobacteria.

[0252] In some embodiments, the (b) second recombinant bacterial persister strains or a derivative thereof also include those having a 16s rRNA gene sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO: 14 or 15.

[0253] The (b) second recombinant bacterial persister strain of the disclosure is persistent to some antibiotics thanks to the presence of the expression cassette which comprises (ib) a nucleic acid sequence of a relA gene or a functional variant thereof.

[0254] In some embodiments, the (b) second bacterial persister strain is persistent to at least one antibiotic.

[0255] In some embodiments, said (a) first recombinant bacterial persister strain, and / or said (b) second recombinant bacterial persister strain, is persistent to one or more antibiotic families selected from the group consisting of: beta-lactams (penicillins, cephalosporins, carbapenems, monobactams), sulfonamides, tetracyclines, oxazolidinones, aminoglycosides, glycopeptides, fluoroquinolones, quinolones, rifamycin, macrolides, lipopeptides, lincosamides, nitrofurans, fosfomycin, trimethoprim, sulfamethoxazole, oxy quinolines, fusidic acid and mupirocin.

[0256] In some embodiments, the (b) second bacterial persister strain is more resistant and this is characterized by a survival rate after induction of the relA gene expression from 5% to 100%, from 6% to 100%, from 7% to 100%, from 8% to 100%, from 9% to100%, from 10% to 100%, from 20% to 100%, from 30% to 100%, from 40% to 100%, from 50% to 100%, from 60%, to 100%, from 70% to 100%, from 80% to 100% or from 90% to 100%, preferably from 5% to 100%.

[0257] In some embodiments, the said (a) first recombinant bacterial persister strain, and / or said (b) second recombinant bacterial persister strain are from the same Gramnegative bacteria, more preferably form the same phylum, class, order, family, genus, or species.

[0258] In some embodiments, the said (a) first recombinant bacterial persister strain, and / or said (b) second recombinant bacterial persister strain are from the same Gramnegative bacteria, more preferably form the same phylum, class, order, family, genus, or species; but different strain.

[0259] In some embodiments, the said (a) first recombinant bacterial persister strain, and / or said (b) second recombinant bacterial persister strain are from Gram-negative bacteria, more preferably form the different phylum, class, order, family, genus (genera), species, or different strain.

[0260] In some embodiments, the different strains are selected based on having a common or identical disease origin, preferably an infectious origin.

[0261] In some embodiments, said (a) first recombinant bacterial persister strain, and said (b) second recombinant bacterial persister strain from Pseudomonadaceae family, preferably from P. aeruginosa.

[0262] In some embodiments, said (a) first recombinant bacterial persister strain, and said (b) second recombinant bacterial persister strain are selected from the group consisting of: PAO1, PAO1-LAC, PA14, PAK, PA103, LESB58, and PA7, preferably PAO1, and PA14.

[0263] In some embodiments, the nucleic acid sequence of the relA gene (i) which encodes the RelA polypeptide of the (a) first recombinant bacterial persister strain, and / or of said (b) second recombinant bacterial persister strain are similar, or identical, preferably the “RelA” or “RelA polypeptide” has RelA peptide sequence formEnterobacteriaceae or Pseudomonadaceae, more preferably from Escherichia or Pseudomonas, even more preferably Escherichia, coli or P. aeruginosa.

[0264] In some embodiments, the nucleic acid sequence of the relA gene (i) which encodes the RelA polypeptide of the (a) first recombinant bacterial persister strain, and / or of said (b) second recombinant bacterial persister strain have the amino acid sequence of the endogenous relA gene of said recombinant bacterial strain.

[0265] The invention further relates to a kit-of-parts for screening a compound of interest with an anti-persister activity, comprising:- (a) a first part being a first recombinant bacterial persister strain as described herein, and- (b) a second part being at least one second recombinant bacterial persister strain; wherein said (a) first recombinant bacterial persister strain, comprises an expression cassette, wherein the expression cassette comprises (ia) a nucleic acid sequence of the relA gene or a functional variant thereof operably linked to (iia) a nucleic acid sequence of an inducible promoter, and wherein said (a) first recombinant bacterial persister strain belongs to the family of Pseudomonadaceae, preferably Pseudomonas aeruginosa.

[0266] The invention also relates to a kit-of-parts for screening a compound of interest with an anti-persister activity, comprising:- (a) a first part being a first recombinant bacterial persister strain as described herein, and- (b) a second part being at least one second recombinant bacterial persister strain as described herein; wherein said (a) first recombinant bacterial persister strain comprises an expression cassette, wherein the expression cassette comprises (ia) a nucleic acid sequence of the relA gene or a functional variant thereof operably linked to (iia) a nucleic acid sequence of an inducible promoter, wherein said (b) second recombinant bacterial persister strain comprises an expression cassette, wherein the expression cassette comprises (ib) a second nucleic acid sequence of the relA gene or a functional variant thereof operably linked to (iib) a second nucleic acid sequence of an inducible promoter, and wherein said (a) first recombinant bacterial persister strain belongs to the family of Pseudomonadaceae, preferably Pseudomonas aeruginosa.

[0267] In some embodiments, said kit-of-parts comprises (c) a third part being at least one third recombinant bacterial persister strain as described herein, (d) a fourth part being at least one fourth recombinant bacterial persister strain as described herein, (e) a fifth part being at least one fifth recombinant bacterial persister strain as described herein, (f) a sixth part being at least one sixth recombinant bacterial persister strain as described herein, (g) a seventh part being at least one seventh recombinant bacterial persister strain as described herein, (h) an eighth part being at least one eighth recombinant bacterial persister strain as described herein, (i) a ninth part being at least one ninth recombinant bacterial persister strain as described herein, (j) a tenth part being at least one tenth recombinant bacterial persister strain as described herein.

[0268] In some embodiments, said (b) second recombinant bacterial persister strain is a Gram-negative bacterial strain, preferably belonging to the family selected from the group consisting of: Enterobacteriaceae and Pseudomonadaceae, more preferably to the species of Escherichia coli.

[0269] In some embodiments, said (a) first recombinant bacterial persister strain, and / or said (b) second recombinant bacterial persister strain, is persistent to one or more antibiotic families selected from the group consisting of: beta-lactams (penicillins, cephalosporins, carbapenems, monobactams), sulfonamides, tetracyclines, oxazolidinones, aminoglycosides, glycopeptides, fluoroquinolones, quinolones, rifamycin, macrolides, lipopeptides, lincosamides, nitrofurans, fosfomycin, trimethoprim, sulfamethoxazole, oxy quinolines, fusidic acid and mupirocin.

[0270] The invention further relates to a method for screening a compound of interest for anti-persister activity, using the kit-of-parts as described herein.

[0271] The invention also relates to a use of the kit-of-parts as described herein, for screening a compound of interest with an anti-persister activity.

[0272] In some embodiments, the culture medium allowing the growth of (b) said second recombinant bacterial persister strain as described herein is selected from the list consisting of: selected from the group comprising or consisting of: Lysogeny Broth (LB), LB Broth Miller, LB Agar, M9 Medium, Terrific Broth (TB), MacConkey Agar, EosinMethylene Blue (EMB) Agar, Tryptic Soy Broth (TSB), Tryptic Soy Agar (TSA), Cystine-Lactose-Electrolyte-Deficient (CLED) Medium, Brain Heart Infusion (BHI) Medium, Raffinose Minimal (RM) Medium, and Mueller Hinton II (MHII) Medium.

[0273] In some embodiments, the second culture medium allowing the growth of the (b) second recombinant bacterial persister strain as described herein is selected from the list consisting of: selected from the group comprising or consisting of: Lysogeny Broth (LB), LB Broth Miller, LB Agar, M9 Medium, Terrific Broth (TB), MacConkey Agar, Eosin Methylene Blue (EMB) Agar, Tryptic Soy Broth (TSB), Tryptic Soy Agar (TSA), Cystine-Lactose-Electrolyte-Deficient (CLED) Medium, Brain Heart Infusion (BHI) Medium, Raffinose Minimal (RM) Medium, and Mueller Hinton II (MHII) Medium .

[0274] In some embodiments, the (a) recombinant bacterial strain as described herein can be cultured in the one medium, or in a different medium than the (b) second recombinant bacterial strain.

[0275] In some embodiments, the (b) second recombinant bacterial strain as described herein can be cultured in the one medium, or in different medium.

[0276] In some embodiments, the (a) first recombinant bacterial strain as described herein and the (b) second recombinant bacterial strain as described herein may be cultured in the same medium or in different media relative to each other.

[0277] In some embodiments, the (ia) nucleic acid sequence of the relA gene or a functional variant thereof and the (ib) second nucleic acid sequence of the relA gene or a functional variant thereof are identical, similar, or different.

[0278] In some embodiments, the (iia) nucleic acid sequence of an inducible promoter, and the (iib) second nucleic acid sequence of an inducible promoter are identical, similar, or different.BRIEF DESCRIPTION OF THE DRAWINGS

[0279] Figure 1 is a graph showing an example of maps of the pBAD-reZA vector where the relA gene was cloned into the multiple cloning site (MCS) of pBAD30 vector (A) and pMMB209-reZA vector (also named pMMB-relA) where the relA gene was cloned into the multiple cloning site (MCS) of pMMB209 vector (B). pBAD-reZA can be used as a vector for E. coli strain and pMMB209-reZA can be used for P. aeruginosa.

[0280] Figure 2 is a graph showing the effect of protein expression on the growth rate and viability of E. coli TOP 10 cell cultures in RM medium. (A) Growth rate of E. coli cells was monitored over time. 0.5% arabinose was added to the cultures at ODeoo 0.2 and the growth was monitored for another six hours. Arrow indicates the moment when arabinose was added. (B) Viability of E. coli TOP 10 cell cultures expressing the candidate persister genes dnaJ (triangle) and relA (circle). At an ODeoo of 0.2, 0.1% arabinose was added to the cultures and samples were collected over time to determine live cells by colony counts. pBAD: cells carrying the pBAD30 vector; DnaJ: cell carrying pBAD-tZnaJ - DnaJ producing cells; RelA: cell carrying pBAD-reZA - RelA producing cells.

[0281] Figure 3 comprises two graphs, (A) shows the survival rate of E. coli TOP10 cells transformed with either pBAD30 or pBAD-reZA after treatment with ciprofloxacin and after induction with or without arabinose in RM medium. (B) is a graph showing the survival rate of E. coli BW25113 cells transduced with pBAD-reZA after treatment with ciprofloxacin and after induction with or without arabinose. Experiments were carried out in triplicate.

[0282] Figure 4 comprises two graphs, (A) is a graph showing the number of live E. coli BW25113 cells expressing the relA gene with different concentrations of arabinose for induction in RM medium. At an ODeoo of 0.2, arabinose was added to the cultures and samples were collected over time to determine live cells by colony count; and (B) is a graph showing the survival rate of E. coli BW25113 cells producing RelA after induction with different concentrations of arabinose (% ara); cells were challenged with 0.5 pg / mL ciprofloxacin, corresponding to 50 x MIC (Minimal Inhibitory Concentration), and thesurvival rate after four hours of treatment was calculated. Values are averages of three independent experiments carried out in triplicates ± standard deviation.

[0283] Figure 5 is a graph showing a comparison of RelA induced BW25113 persister cells with chemically induced persisters, here carbonyl cyanide m- chlorophenylhydrazone (CCCP). Graph (A) shows the survival rates after the cells were challenged for four hours with 1 pg / mL ciprofloxacin, corresponding to 100 x MIC (Minimal Inhibitory Concentration), in either 1% LB (LB medium diluted 100-fold in 0.85% NaCl) or RM medium with or without CCCP. Graph (B) depicts the cell viability before and after induction of persistence by RelA overexpression and via CCCP treatment. The number of colonies growing after three hours of induction were counted (colony forming units, CFU). Results are depicted as logCFU / ml. pBAD: cells carrying the pBAD30 vector; pBAD-reZA: RelA producing cells; CCCP: cells treated with CCCP (carbonyl cyanide m-chlorophenylhydrazone) at indicated concentrations. Experiments were carried out in triplicate.

[0284] Figure 6 is a graph showing the survival rate of BW25113 E. coli persisters treated with either ciprofloxacin or Pep 19 at 50 x and 5 x multiples of MBC (minimal bactericidal concentrations) respectively. Growing colonies were counted and survival rate was reported as percent relative to number of colonies before treatment. Non- persister E. coli (open bars) and persister E.coli (black bars). Values are averages of three independent experiments.

[0285] Figure 7 (A) is a graph showing the effect of protein expression on the growth rate of P. aeruginosa PA 14 cells cultured in LB medium. The growth rate of P. aeruginosa cells was monitored over 10 hours. 1 mM IPTG was added to the cultures at OD600 0.1, the arrow indicates the moment when IPTG was added. pMMB209 (white squares): cells carrying the empty pMMB209 vector; pMMB209-dnaJ (black triangles): DnaJ producing cells; pMMB209-relA (black circles): RelA producing cells. Figure 7 (B) is a graph showing the effect of protein expression on the growth rate of P. aeruginosa PAO1 cells cultured in LB medium. The growth rate of P. aeruginosa cells was monitored over 10 hours. 1 mM IPTG was added to the cultures at OD600 0.1, the arrow indicates the moment when IPTG was added. pMMB209 (white squares): cells carrying the emptypMMB209 vector; pMMB209-dnak' (black triangles): DnaK producing cells; pMMB209-reZA (black circles): RelA producing cells.

[0286] Figure 8 comprises of two graphs showing the survival rates of P. aeruginosa persisters and non-persisters treated with bactericidal antibiotics at 50X the MIC (minimal inhibitory concentration), (A) survival rates for P. aeruginosa PAO1 with treatment in three different types of media; LB, RM and MHII and (B) survival rates for P. aeruginosa PA 14 using LB medium. Colonies were counted after treatment and survival rate was reported as a percentage relative to the number of colonies before treatment. Both non-persister P. aeruginosa (pMMB209, open bars) and persister P. aeruginosa (pMMB209-relA, black bars) were induced with 1 mM IPTG. Values are averages of three independent experiments.EXAMPLES

[0287] The present invention is further illustrated by the following examples.Example 1: Assessment of initial antibiotic concentrationMaterials and Methods

[0288] Minimal inhibitory concentrations (MIC) & minimal bactericidal concentrations (MBC) were determined by standard protocols known to the skilled artisan. Briefly for MIC determination, bacteria to be tested were streaked on an LB agar plate to obtain single colonies. Three isolated colonies of the same morphological appearance were transferred into a 15 ml tube containing 3 ml of MH2 lx media and incubated at 37°C in a shaker at 160 rpm overnight (O / N). Prepare antibiotic dilutions. From a bacterial suspension with an ODeoo 0.1 corresponding to about 108CFU / mL, dilute bacteria to about 106CFU / mL to obtain 5xl05CFUs / well. Transfer antibiotic solution and bacterial suspension to a 96-well plate with appropriate controls for turbidity determination. An aliquot of each preparation was taken and plated onto MH agar plates for growth control. 96-well plate was incubated for 18-24h at 35°C (± 2 °C). MIC was determined as the lowest concentration of compound leading to no detectable growth.

[0289] For MBC determination: from the wells where there was no visible growth, 50 pl were taken and spotted on a square Petri dish with grids containing MH agar. The following day, the colonies were enumerated.

[0290] MBC is the lowest concentration of an antibacterial agent that either totally prevents growth or results in a >99.9% decrease in the initial inoculum (i.e., a 3-logl0 reduction in colony-forming units, CFU / ml).Results

[0291] Table 4: Minimal inhibitory concentrations (MIC) & minimal bactericidal concentrations (MBC) (pg / mL) determined for E. coli (BW25113).

[0292] Table 4 summarizes the MIC and MBC values that were determined for E. coli transformed with the pBAD plasmid. These values were used as a reference for the concentrations of antibiotics used in the persister assays. The same results are expected for any antibiotic- sensitive strain of E. coli.Example 2: Persister Assay - EscherichiaMaterials and Methods

[0293] To induce persister formation, overnight cultures of E. coli BW25113 / pBAD- relA and BW25113 / pBAD (empty plasmid corresponding to negative control for persistence) are sub-cultured in fresh RM medium supplemented with 100 pg / ml ampicillin with a starting optical density of 0.05 at 600 nm (ODeoo). Same conditions were used for the experiments with E. coli TOP10 strain. The cultures are incubated at 37°C with shaking at 160 rpm until the ODeoo reaches 0.2 and arabinose is added to a final concentration of 0.2%. The cultures are incubated for another 3 h at 37°C with shaking to induce gene expression. Both normal and persister cells are diluted in RM (RM minimal medium (Vazquez-Laslop N., et al. J. Bacteriology, 2006)) to an ODeoo equivalent to IxlO7CFU / ml and the cells are challenged with ciprofloxacin at 0.5 pg / ml, corresponding to 50 x MIC, for 4 h at 37°C with shaking at 160 rpm. Similarly, cells were treated with levofloxacin (50 x MBC), cefepime (100 x MBC), meropenem (100 x MBC), gentamicin (5 x MBC) and polymyxin B (5 x MBC). The cyclic peptide, Pep 19 was tested at 5 x MBC. The treated cells are harvested by centrifugation and washed once with 0.85% NaCl to remove the residual drug. The cells are then resuspended in 0.85% NaCl, serially diluted, and spotted in LB agar plates supplemented with 0.2% glucose for CFU enumeration. The survival rate is calculated by dividing the number of CFU / ml after treatment by the number of CFU / ml before adding the antibiotic.

[0294] Pretreatment with CCCP (carbonyl cyanide m-chlorophenylhydrazone) was used to induce chemical persister formation in E. coli. To enrich a culture of E. coli persisters, cells were incubated with CCCP at a concentration of either 10 or 25 pg / ml for three hours at 37 °C with shaking 160 rpm. Cells were then treated with antibiotics as were re I A overexpressing cells.Results

[0295] Table 5: Survival rate of cells producing RelA. Cells were treated with different antibiotics, at a concentration ranging from 5x to lOOx MBC (Minimal Bactericidal Concentration), and the survival rate after four hours of treatment was calculated. Values are averages of three independent experiments.

[0296] Results show that bacteria overexpressing relA, i.e. after induction with arabinose, survive treatment with ciprofloxacin, levofloxacin, cefepime and meropenem. These results demonstrate that RelA expression renders cells non- susceptible to antibiotic treatment.

[0297] Another feature of persister cells is that they do not actively divide and enter a state of growth arrest or dormancy. The growth rate of RelA expressing bacteria comes to a rest when RelA expression is induced by addition of arabinose during the exponential growth phase (Figure 2A). Overexpression of DnaJ, another candidate for inducing persister cells, did not lead to growth arrest after induction with arabinose (Figure 2A). These data emphasize the specificity of RelA overexpressing for bacteria entering a state of dormancy and persister cell formation. DnaJ overexpression exemplifies that other candidate genes known to be involved in persister formation do not lead to growth arrest, i.e. persister cell formation. To demonstrate that cells in the dormancy state are alive, cells induced to overexpress either RelA or DnaJ were monitored for cell viability over timeafter induction with arabinose (Figure 2B). Both cultures maintained viability over the time of the experiment.

[0298] pBAD-reZA bacteria that entered the dormancy state after induction with arabinose are able to survive the treatment with the antibiotic ciprofloxacin (Figure 3 A and B). pBAD bacteria that do not carry the relA gene in the vector, as well as noninduced cells, do not survive treatment with ciprofloxacin at a concentration of 50 x MBC. These results demonstrate that inducible overexpression of relA renders bacteria non- susceptible to antibiotic treatment while putting them into a state of dormancy. Overexpression of relA renders two different strains of E. coli, TOPIO (Figure 3A) and BW25 113 (Figure 3B), non- susceptible to antibiotic treatment. These results indicate that entry into a dormancy state with enhanced survival to antibiotic treatment by overexpressing relA is independent of the E. coli strain used.

[0299] A concentration as low as 0.05% arabinose is enough to enable the survival of pBAD-reZA bacteria in the presence of antibiotics (Figure 4B). Cell viability is not influenced by the arabinose concentration that induces the persister state (Figure 4A).Our induction system is non-toxic to bacteria, in contrast to chemical treatment of bacteria.

[0300] Figure 5A shows the viability of CCCP treated cells compared to RelA overexpressing cells. At concentrations > 50 pg / ml, CCCP starts to be toxic for cells (Figure 5B). Whereas survival of RelA expressing cells is independent of growth medium, CCCP induced bacteria are sensitive to growth medium (1% LB vs RM - Figure 5A), as their survival rate varies with the growth media (Figure 5A). The genetically induced method presented here yields comparable numbers of persisters to that obtained after chemical induction with CCCP. These findings indicate that overexpression of RelA results in a persister bacterial population that is more stable and more consistent than through the CCCP method. Furthermore, CCCP induced persistence is of short duration as CCCP has to be present in the culture medium to maintain survival in RM medium. Thus, this model induced by CCCP will generate less reliable results. Although this model’s suitability to generate persisters in vitro with high efficiency has been demonstrated, this technique cannot be performed in vivo due to the toxicity of themolecule. In contrast, the pBAD-reZA system will enable in vivo testing in infection animal models. The generating of persistence via the overexpression of a gene with an inducer molecule that is non-toxic is a great advantage. Therefore, the results obtained can better translate into in vivo effectiveness and better predict treatment efficacy.

[0301] Persister bacteria, reliably produced by overexpression of RelA represent a valuable tool to test and develop antimicrobial molecules that are able to kill these bacterial populations. Figure 6 shows the results for such a molecule, Pep 19. Pep 19 was engineered from an .S'. aureus toxin, PepAl (Nicolas et al., PLOS Biology (2019), e3000337, 17(7)), rendering this molecule less toxic and more stable. When pBAD-reZA cells in the persister state were incubated with Pep 19, cells were readily eliminated at a concentration of 5 x MBC. Genetically inducible persister bacteria can therefore be used for the development of molecules with a new and innovative treatment modality, active in the context of chronic infections that are not treatable with conventional antibiotics.Example 3: Galleria mellonella infection modelMaterials and Methods

[0302] Persister and non-persister cells obtained as described previously are collected and washed thrice with PBS. Before infection of the larvae, the number of cells is adjusted to 5 x 108CFU / ml in PBS. An aliquot of 10 pl, corresponding to ~5 x 106CFU, is inoculated into the hemocoel of each caterpillar via the last left proleg. After injection, caterpillars are placed in petri dishes and incubated at 37°C for 1 h. The antibiotic is then administered by injection into a different proleg. The larvae are kept at 37°C in the dark for 5 days and the number of live and dead larvae is counted every 24 h.Example 4: Persister Assay - PseudomonasMaterials and Methods

[0303] For persister formation in P. aeruginosa, overnight cultures of P. aeruginosa PAOl / pMMB209-reZA and PAOl / pMMB209 (empty plasmid corresponding to thenegative control for persistence) are sub-cultured in the medium of choice (LB, RM or MHII medium) supplemented with 50 pg / ml gentamicin with a starting optical density of 0.05 at 600 nm (OD600). The same conditions are used for experiments with P. aeruginosa PA14. The cultures are incubated at 37°C with shaking at 200 rpm until the OD600 reaches 0.2 and isopropyl P-D-l -thiogalactopyranoside (IPTG) is added to a final concentration of 1 mM. The cultures are incubated overnight with shaking to induce protein overproduction. Both normal and persister cells are diluted in the chosen medium to an OD600 of 0.1 and a 1 / 100 dilution of this performed before the cells (-5x105 CFU / mL) are challenged with a bactericidal antibiotic at 50X the MIC (meropenem, cefepime or piperacillin). A sample of cells before treatment is washed and resuspended in 0.85% NaCl, serially diluted, and spotted on LB agar plates for CFU enumeration. Antibiotic treatments are conducted for 6 hours at 37 °C with shaking at 200 rpm. The treated cells are harvested by centrifugation and washed three times with 0.85% NaCl to remove the residual inducer and antibiotic. The cells are then resuspended in 0.85% NaCl, serially diluted, and spotted on LB agar plates for CFU enumeration. The survival rate is calculated by dividing the number of CFU / ml after treatment by the number of CFU / ml before treatment.ResultsTable 5: Minimal inhibitory concentrations (MIC) & minimal bactericidal concentrations (MBC) (pg / mL) determined for P. aeruginosa PAO1 and PA14 transformed with the pMMB209 vector.

[0304] Table 5 summarizes the MIC and MBC values that were determined for P. aeruginosa PAO1 and PA14 transformed with the pMMB209 vector. These values were used as a reference for the concentrations of antibiotics used in the persister assays.

[0305] Overexpression of DnaJ, did not lead to growth arrest after induction with IPTG in P. aeruginosa as for E. coli (Figure 7A). Overexpression of DnaK, another candidate for inducing persister cells, did not lead to growth arrest after induction with IPTG (Figure 7B). This data emphasizes the specificity of RelA overexpression for bacteria entering a state of dormancy and persister cell formation. DnaJ and DnaK overexpression exemplifies that candidate genes known to be involved in persister formation do not lead to growth arrest, i.e. persister cell formation.

[0306] Persister bacteria, reliably produced by overexpression of RelA represent a valuable tool to test and develop antimicrobial molecules that are able to kill these bacterial populations. It is important to be able to screen against relevant pathogenic bacteria such as Pseudomonas (Figure 8). When P. aeruginosa cells carrying pMMB209- relA are induced into the persister state and incubated with several antibiotics, the cells survive; in different medium, namely LB, RM, MHII; with different antibiotics, namely meropenem, cefepime, piperacillin; and with different strains, namely PAO1, and PA14. Thus, genetically inducible persister bacteria can therefore be used for the development of molecules with a new and innovative treatment modality, active in specific context, such as wound infection, burn wound infection, or treatment of chronic infections in cystic fibrosis patients.LIST OF THE SEQUENCES

Claims

CLAIMS1. A recombinant bacterial persister strain comprising an expression cassette, wherein the expression cassette comprises (i) a nucleic acid sequence of a re I A gene or a functional variant thereof operably linked to (ii) a nucleic acid sequence of an inducible promoter, and wherein the bacterial strain belongs to the family of Pseudomonadaceae, preferably Pseudomonas aeruginosa.

2. The recombinant bacterial persister strain according to claim 1, wherein the inducible promoter is selected from the group consisting of: ParaBAD, Ptet, Plac, PlacUV5, Ptac, Ptrc, PT5, PT7, Prha, PprpR, Plux, Pdox, Pmalt, and Prib.

3. The recombinant bacterial persister strain according to claim 1 or claim 2, wherein the expression cassette is introduced into a vector selected from the group consisting of: a plasmid, an episome, and a bacterial artificial chromosome.

4. The recombinant bacterial persister strain according to any one of claims 1 to 3, wherein the expression cassette is introduced into a vector, preferably the expression cassette and / or the vector is integrative or non-integrative.

5. The recombinant bacterial persister strain according to any one of claims 1 to 4, wherein the bacterial persister strain is persistent to one or more antibiotic families selected from the group consisting of: beta-lactams; such as penicillins, cephalosporins, carbapenems, monobactams; sulfonamides, tetracyclines, oxazolidinones, aminoglycosides, glycopeptides, fluoroquinolones, quinolones, rifamycin, macrolides, lipopeptides, lincosamides, nitrofurans, fosfomycin, trimethoprim, sulfamethoxazole, oxy quinolines, fusidic acid, and mupirocin; preferably beta-lactams; more preferably carbapenem, cephalosporin, and penicillin; even more preferably meropenem, cefepime, and piperacillin.

6. A kit-of-parts for screening a compound of interest with an anti-persister activity, comprising:- (a) a first part being a first recombinant bacterial persister strain, and- (b) a second part being a second recombinant bacterial persister strain;wherein said (a) first recombinant bacterial persister strain comprises an expression cassette, wherein the expression cassette comprises (ia) a nucleic acid sequence of a relA gene or a functional variant thereof operably linked to (iia) a nucleic acid sequence of an inducible promoter, wherein said (b) second recombinant bacterial persister strain comprises an expression cassette, wherein the expression cassette comprises (ib) a second nucleic acid sequence of a second relA gene or a functional variant thereof operably linked to (iib) a second nucleic acid sequence of a second inducible promoter, and wherein said (a) first recombinant bacterial persister strain belongs to the family of Pseudomonadaceae, preferably Pseudomonas aeruginosa.

7. The kit-of-parts according to claim 6, wherein said (iia) first and / or said (iib) second inducible promoter is selected from the group consisting of: ParaBAD, Ptet, Plac, PlacUV5, Ptac, Ptrc, PT5, PT7, Prha, PprpR, Plux, Pdox, Pmalt, and Prib.

8. The kit-of-parts according to claim 6 or claim 7, wherein said (b) second recombinant bacterial persister strain is a Gram-negative bacterial strain, preferably belonging to the family selected from the group consisting of: Enterobacteriaceae and Pseudomonadaceae.

9. The kit-of-parts according to any one of claims 6 to 8, wherein said (a) first recombinant bacterial persister strain, and / or said (b) second recombinant bacterial persister strain, is persistent to one or more antibiotic families selected from the group consisting of: beta-lactams; such as penicillins, cephalosporins, carbapenems, monobactams; sulfonamides, tetracyclines, oxazolidinones, aminoglycosides, glycopeptides, fluoroquinolones, quinolones, rifamycin, macrolides, lipopeptides, lincosamides, nitrofurans, fosfomycin, trimethoprim, sulfamethoxazole, oxyquinolines, fusidic acid and mupirocin.

10. Use of the kit-of-parts according to any one of claims 6 to 9, for screening a compound of interest with an anti-persister activity.

11. A method for screening a compound of interest with an anti-persister activity comprising the following steps:a. culturing the recombinant bacterial persister strain according to any one of claims 1 to 5 into a culture medium allowing the growth, b. inducing the expression of the relA gene, c. introducing into the medium a compound of interest to test, and d. quantifying the survival rate of the bacterial persister strain, thereby determining if the compound of interest has anti-persister activity.

12. The method according to claim 11, wherein said method further comprises the step of (a’) culturing (b) a second recombinant bacterial persister strain into a second culture medium allowing the growth, wherein said (b) second recombinant bacterial persister comprises an expression cassette, and wherein the expression cassette comprises (ib) a second nucleic acid sequence of a relA gene or a functional variant thereof operably linked to (iib) a second nucleic acid sequence of an inducible promoter.

13. The method according to claim 11 or claim 12, wherein said (iib) second inducible promoter is selected from the group consisting of: ParaBAD, Ptet, Plac, PlacUV5, Ptac, Ptrc, PT5, PT7, Prha, PprpR, Plux, Pdox, Pmalt, and Prib.

14. The method according to any one of claims 11 to 13, wherein said (b) second recombinant bacterial persister strain expressed relA gene endogenously.

15. The method according to any one of claims 11 to 14, wherein the recombinant bacterial persister strain according to any one of claims 1 to 5, and / or said (b) second recombinant bacterial persister strain, is persistent to one or more antibiotic families selected from the group consisting of: beta-lactams; such as penicillins, cephalosporins, carbapenems, monobactams; sulfonamides, tetracyclines, oxazolidinones, aminoglycosides, glycopeptides, fluoroquinolones, quinolones, rifamycin, macrolides, lipopeptides, lincosamides, nitrofurans, fosfomycin, trimethoprim, sulfamethoxazole, oxy quinolines, fusidic acid and mupirocin.

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