EP4676469A1 - Dequalinium chloride as a mutation-slowing drug to inhibit de novo pathogen evolution including evolution of antibiotic resistance - Google Patents

Abstract

Embodiments of the disclosure encompass methods and compositions related to bacterial infections in mammals. In specific embodiments, an individual that may or may not be receiving one or more antibiotics is provided an effective amount of dequalinium chloride (DEQ) for the purpose of reducing mutagenesis of the bacteria causing the infection. In particular embodiments, this leads to reduction of antibiotic resistance when the individual is receiving the antibiotic(s), or to cross resistance to antibiotics that are not being administered, or this leads to reduction in the rate of evolution of the bacteria that facilitates immune clearance by the immune system in individuals not receiving the antibiotic(s).

Description

DEQUALINIUM CHLORIDE AS A MUTATION-SLOWING DRUG TO INHIBIT DE NOVO PATHOGEN EVOLUTION INCLUDING EVOLUTION OF ANTIBIOTIC RESISTANCE

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63/489215 filed March 9, 2023, and to U.S. Provisional Patent Application Serial No. 63/509395 filed on June 21, 2023, both of which are incorporated by reference herein in their entirety.

[0002] This invention was made with government support under GM122598, GM053158, AG072751, and CA250905 awarded by the National Institutes of Health. The government has certain rights in the invention.

I. Technical Field

[0003] This disclosure relates at least to the fields of bacteriology, immunology, cell biology, molecular biology, and medicine.

II. Background

[0004] Antibiotics have reduced mortality from bacterial infections (1), but, unfortunately, antibiotic resistance now threatens world health with an estimated 1.27 million deaths worldwide from antibiotic-resistant infections in 2019 (2). Evolution of resistance is outpacing introduction of new antibiotics (3, 4). Drugs that could slow the rates of evolution, given with antibiotics, might prolong antibiotic effectiveness (5-7). Evolution- slowing drugs might, additionally, make antibiotics unnecessary if pathogen evolution could be slowed to rates lower than those of somatic evolution of our immune responses. This allows immune clearance while avoiding the destructive effects of antibiotics on human microbiota, now appreciated to underpin many aspects of human health (8). Antibiotic resistance evolves via uptake of resistance genes from other bacteria and/or by mutations in native genes (5), with mutagenesis the main route in priority pathogens designated by the World Health Organization (9). Although potentially transformative (6, 10-15), few mutagenesis-reducing drugs have been reported, and fewer drug targets identified (7, 16-18). Moreover, those reported also reduce viability (16-20), and so favor proliferation of (select) mutants resistant to the evolutionslowing drug and the antibiotic (and see (21) for a similar anti-cancer drug). [0005] The present disclosure provides a solution to the long-felt need of avoiding de novo development of antibiotic resistance.

BRIEF SUMMARY

Evolution of antibiotic resistance is a world health crisis, fueled by new mutations. Drugs to slow mutagenesis could, as co-therapies, prolong the shelf-life of antibiotics, yet evolutionslowing drugs and drug targets have been underexplored and ineffective. The present disclosure concerns a network-based strategy to identify drugs that block hubs of fluoroquinolone antibiotic-induced mutagenesis. Identified herein is an FDA-approved and EMA-approved drug, dequalinium chloride (DEQ), that inhibits activation of the Escherichia coli general stress response, which allows ciprofloxacin- (stress)-induced mutagenic DNA break repair. The step in the pathway that is inhibited is identified herein as activation of the upstream “stringent” starvation stress response, and the disclosure shows that DEQ slows evolution without favoring proliferation of DEQ-resistant mutants. Stress-induced mutagenesis during mouse infections and its inhibition by DEQ are demonstrated herein. The disclosed strategy for drugs to slow evolution in bacteria and generally are useful in the field of medicine.

[0006] Embodiments of the disclosure concern methods and compositions for treatment of individuals having one or more bacterial infections. Embodiments of the disclosure concern methods and compositions for reducing the likelihood of a bacteria in an individual to develop antibiotic resistance to any one or more antibiotics. The antibiotic may be of any kind. The bacteria may be of any kind, including any pathogenic bacteria. The bacteria may cause any type of infection. In specific embodiments, the disclosure concerns use of dequalinium chloride (DEQ) for the purpose of reducing mutagenesis of bacteria, thereby avoiding the likelihood that the bacteria develops antibiotic resistance through one or more mutations. In specific embodiments, the disclosure concerns the use of DEQ for the purpose of reducing mutagenesis, thereby allowing the immune system of the individual more time to naturally clear the infection; in such a case, the individual may or may not be receiving and/or have received one or more antibiotics.

[0007] In certain embodiments, the bacteria may or may not be E. coli. The bacteria in specific embodiments may be Gram-negative bacteria. In specific embodiments, the bacteria are gram-negative enterobacterial pathogens. In certain embodiments, the bacteria have a stringent starvation stress response. In specific embodiments, the bacteria are, or are not, Klebsiella, Acinetobacter, Pseudomonas aeruginosa, Enterobacter, Salmonella, Haemophilus, Proteus, Citrobacter, Yersinia, Shigella, Salmonella, or E. coli. In specific embodiments, the bacteria are Gram-positive. In some embodiments, the bacteria are, or are not, Staphylococcus, Streptococcus, Pneumococcus, Bacillus (including B. sublilis) or Enterococcus. In specific embodiments, the bacteria is, or is not, methicillin-resistant Staphylococcus aureus, cipro- resistant E. coli, vancomycin-resistant Enterococcus, multi-drug-resistant Mycobacterium tuberculosis, carbapenem-resistant Enter obacteriaceae, benzyl penicillin-resistant Staphylococcus aureus, benzyl penicillin-resistant Neisseria gonorrhoeae, and Erythromycin- resistant invasive group A streptococcus.

[0008] In some embodiments, the antibiotics may be of any kind, but in specific cases the antibiotics (regardless of their mechanism of action) induce reactive oxygen species (ROS)- dependent increase in mutagenesis. In some embodiments, the antibiotics induce the stringent response (and/or sigma-S). In some embodiments, the antibiotics induce mutagenesis that is stringent-response (or sigma-S-response or other starvation related stress response or other general stress response) dependent. In particular embodiments, the antibiotics are, or are not, quinolones, penicillins, cephalosporins and other beta-lactams, chloramphenicol, tetracyclines and other inhibitors of protein synthesis, aminoglycosides, macrolides, sulfonamides and other inducers of DNA damage or thymineless death, trimethoprim, bleomycin, phleomycin, ampicillin, gentamicin, norfloxacin, streptomycin, tetracycline, and so forth, and any co- therapeutic enhancers of an antibiotic given with the antibiotic to improve its efficacy.

[0009] In some embodiments there is a method of reducing mutagenesis or the risk of mutagenesis in bacteria in an individual, comprising the step of non-topically administering an effective amount of dequalinium chloride (DEQ) to the individual. The bacteria may or may not be pathogenic, and in specific embodiments the bacteria are Gram-negative or Grampositive. In specific embodiments, the mutagenesis confers resistance to one or more antibiotics. In certain embodiments, the administering also comprises administering to the individual the one or more antibiotics. In some embodiments, the individual will be receiving, has been receiving, and/or is receiving the one or more antibiotics. The individual may or mat not receive the one or more antibiotics when receiving the DEQ. In specific cases, the antibiotic is, or is not, a quinolone or a fluoroquinolone and may or may not be Nalidixic acid, Enoxacin, Norfloxacin, Ciprofloxacin, Ofloxacin, Lomefloxacin, Sparfloxacin, Grepafloxacin, Clinafloxacin, Gatifloxacin, Moxifloxacin, Gemifloxacin, Trovafloxacin, Garenoxacin, or a combination thereof, in some aspects. In specific embodiments, the DEQ is administered orally and/or intravenously. The DEQ may be administered to the individual orally at a dose of about 1-2000 mg/kg or intravenously at a dose of about 0.1-100 mg/kg, as examples. In specific embodiments, the DEQ is administered once a day, more than once a day, once a week, more than once a week, once a month, or more than once a month. The bacteria may or may not cause pneumonia, tuberculosis, blood poisoning, gonorrhea, urinary tract infection, and/or a foodborne disease in the individual. In specific embodiments, the individual previously misused antibiotics, such as by overuse and/or failing to complete a course of antibiotics. In certain embodiments, the individual is, or is not, in a medical facility, such as a hospital, nursing home, skilled nursing care facility, or long-term care facility. The individual may be an infant, greater than 65 years old, or is immunocompromised. In specific embodiments, the bacteria is, or is not, Escherichia coli, Methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), multi-resistant Gram-negative bacteria, Clostridioid.es difficile, Mycobacterium tuberculosis, or a mixture thereof. The individual may be a human or an agricultural animal or a companion animal.

[0010] In particular embodiments, there is a method of reducing the risk or delaying the onset of developing antibiotic resistance of bacteria in an individual, comprising the step of: (a) non-topically administering an effective amount of dequalinium chloride (DEQ) to the individual; and (b) administering one or more antibiotics to the individual. In certain embodiments, (a) and (b) occur at substantially the same time or occur at different times. In specific embodiments, (a) occurs before or after (b). In specific embodiments, DEQ and the one or more antibiotics are in the same formulation, although in some embodiments DEQ and the one or more antibiotics are in different formulations. The bacteria may acquire resistance by mutagenesis or by horizontal gene transfer, for example.

[0011] In particular embodiments, there is a method of facilitating clearance of an infection in an individual, comprising the step of non-topically administering an effective amount of dequalinium chloride (DEQ) to the individual in the absence of also administering one or more antibiotics to the individual. In some embodiments, DEQ in the individual reduces the rate of pathogen evolution by mutagenesis, thereby enhancing immune clearance by the immune system of the individual.

[0012] In certain embodiments, there is a method of protecting native microflora in an individual in need of one or more antibiotics, comprising the step of non-topically administering an effective amount of dequalinium chloride (DEQ) in an individual receiving no antibiotics.

[0013] In some embodiments, there is a method of treating an individual for a bacterial infection, comprising the step of non-topically administering to the individual an effective amount of dequalinium chloride (DEQ) whether or not the individual receives one or more antibiotics, thereby reducing mutagenesis in the bacteria while also reducing proliferation of DEQ-resistant mutants.

[0014] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the subject matter of the disclosure will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the subject matter of the present disclosure. The subject matter of the present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0016] FIGS. 1A-1G. DEQ inhibits cipro induction of the general stress response and mutagenesis. (FIG. 1A) Diagram of stress-induced mutagenic break repair (MBR) mechanism reviewed (11, 13, 35). Steps (1), (2), and (3) are each necessary but not sufficient for MBR (main text). (FIG. IB) Design of screens for inhibitors of cipro-induced SOS- or o^s -response activity, strategy 493 modified from (71). (1) Primary screen: fluorescence plate-reader for drugs that reduce fluorescence from SOS- or G^S -activity reporters. (2) Secondary screen: more accurate and sensitive flow-cytometry. (FIG. 1C) Data from plate reader screens in MAC cipro (arbitrary fluorescence units or afu). Blue squares, antibiotics, were not tested further because we sought non- antibiotic compounds. Purple triangles, potential stress response inhibitors. Red triangles, positive controls SOS-uninducible lexAInd , or G^S response uninducible ArpoS. (FIG. ID) Identities and approved uses of the primary- screen hits. SOS: Naltrexone (NAL), anti-addiction; niridazole (NIR), antischistosomal (antihelminth, kills parasitic worms). G^S: dequalinium chloride (DEQ) bacteriostatic agent, at high doses unlike those used here (54), used topically in combination with antimicrobial clindamycin for vaginosis (see Example 7); saquinavir (SQV) HIV-protease inhibitor. (FIG. IE) DEQ and NIR reduce cipro-induced G^S- active cell subpopulation. Left, flow-cytometry histograms of fluorescence in cells carrying the yiaG-yfp G^S reporter. Black bar, gate for G^S-activc cells (Methods). Right, means ± SD, 3 independent experiments. (FIG. IF) Fluctuation-test assays for base substitutions (RifR) and any null (AmpR) mutagenesis (35). (FIG. 1G) Cipro-induction of mutagenesis is inhibited by DEQ, not by NIR. Fold induction of mutation rate by cipro: mutations per cell per generation (orange bars (bars on the left of the pairs of bars)) or mutations per chromosome per generation (grey bars (bars on the right of the pairs of bars)) / mutation rate without cipro. Cipro-treated cells contain an average 4.5 chromosomes per cell, compared with 1.5 chromosomes per cell in no drug controls (35). Means ± SEM, 3 experiments. Positive control Edaravone (Edar) is an antioxidant previously shown to inhibit mutagenesis (35). Baseline mutation rates for WT cells (vehicle) not treated with cipro were 4.8 ± 0.6 x 10-9 RifR and 4.5 ± 0.3 x 10-9 AmpR mutations per cell per generation. *p<0.05 (IE), *p<0.001 (1G) one-way ANOVA with Tukey’s post-hoc test; n.s. not significant. Throughout, afu arbitrary fluorescence units.

[0017] FIGS. 2A-2D; DEQ interrupts induction of o^s-activating small RNAs. (FIG. 2A) Summary diagram of observations of (35) that cipro induces DNA double- stranded breaks DSBs and the SOS response in all cells; the SOS response then promotes ROS in a -20% cell subpopulation; the ROS induce transcription of small (s)RNAs DsrA and ArcZ, which allow translation of rpoS mRNA into c^s protein, thereby creating the c^s active “gambler” cell subpopulation that produces nearly all cipro-induced mutants via mutagenic break repair (MBR) (35). Ovals, E. coli cells. (FIG. 2B) Cipro-induced ROS-high cells are not reduced by DEQ. Flow-cytometry of 16 h log-phase cells grown in MAC cipro. DHR, dihydrorhodamine ROS dye. B and C: means ± SEM, 3 experiments. Two-tailed Student’s t-test; n.s. not significant. (FIG. 2C) DEQ reduces cipro induction of dsrA and arcZ promoter activity. |3- galactosidase activity, PdsrAl cZ and P_arczlacZ reporters in log-phase growth in MAC cipro, ± DEQ. *p<0.001, one-way ANOVA with Tukey’s post-hoc test. (FIG. 2D) Summary: DEQ inhibits DsrA/ ArcZ sRNA synthesis and o^s-activc gambler-cell formation and mutagenesis, but does not reduce ROS.

[0018] FIGS. 3A-3D. DEQ inhibits cipro-induced stringent-response and not antibiotic activity. (FIG. 3A) Summary: Edaravone and DEQ inhibit different steps in the newly expanded MBR pathway (35, 39). Antioxidant drug edaravone reduces the ROS-high cell subpopulation and MBR (35), whereas DEQ reduces cipro induction of the stringent response (39) (3B, 3C this figure). (FIG. 3B) DEQ reduces activation of the stringent response, transcriptional reporter P_rmflnCherry (39), shown above. (p)ppGpp is a stringent-response activator that binds RNA polymerase (60-62) and relA spoT mutants lack both E. coli (p)ppGpp synthases, and so are (p)ppGpp-deficient (60-62). Log phase cells in MAC cipro (16 h). Left, representative flow-cytometry histograms. Right, means ± SEM, 3 experiments. *p<0.001, one-way ANOVA with Tukey’s post-hoc test. (FIG. 3C) DEQ inhibits cipro-induced stringent- responsive iraP promoter activity. Log-phase cells in MAC cipro (16 h). |3 galactosidase activity, Pi_raplacZ reporter. Bars as for (FIG. 3B). (FIG. 3D) DEQ does not increase high-dose cipro antibiotic killing activity. Log-phase cells grown with or without 1.5 pg/mL cipro. Means ± SD, 4 independent experiments.

[0019] FIGS. 4A-4C. MBR genes in mutagenesis in mouse infection and inhibition by DEQ and edaravone. (FIG. 4A) Workflow of bacterial mouse thigh infection model of (59). (FIG. 4B) Mutagenesis in mouse infection with cipro requires proteins/functions of cipro- induced MBR: an inducible SOS response (lexAInd- SOS-off mutant); G^S (encoded by rpoS) and the Hfq RNA chaperone (35). Mutants reduced by 6.3 ± 2.3 times relative to the isogenic control strain. Mean ± SEM of the three mutants, three biological replicates each. DEQ and edaravone inhibit mutagenesis to RifR antibiotic cross resistance during mouse infection by 6.4 ± 1.3 times, and 3.7 ± 0.4 times, respectively. Means ± SEM, 3 experiments. Different from control, *p<0.01. One-way ANOVA with Tukey’s post-hoc test. ATCC25922-derived E. coli cells extracted after 48h infection in mouse. (FIG. 4C) STRING interaction diagram (72) of known MBR network genes/proteins highlighting the SOS and G^S responses, including the stringent-response activators in the network of genes from (11), additional required components (44-45) and stringent-response activators RelA, SpoT and DksA (light blue), which acts upstream of G^S activation in cipro-induced MBR (39). Al Mamun et al. (11) estimated that their screen missed about half of the network. Green, shown previously (11, 39) to act upstream of the G^S response sensing stress and transducing signals that activate G^S; light green, known MBR proteins upregulated by G^S. Red, upstream regulators of the SOS-response; and light red, MBR components upregulated by SOS. The c' -activating components of the network (11) are not highlighted here; the GE activator hub is shown in brown. Two negative (upstream) regulators are noted: LexA repressor of the SOS response, and ArcA, loss of which upregulated o^s (11).

[0020] FIGS. 5A-5B. Signal-to-noise ratios in 384-well plate-based assays of SOS and oS activity. Means ± SD of at least 3 experiments with 50 wells each in 384-well plates. (FIG. 5A) Fluorescence detection of cipro-induced SOS-response activity relative to SOS- uninducible ZexAInd" control cells. Per (73), the Z-factor = 0.88 indicates an excellent signal- to-noise ratio with low variation. (FIG. 5B) Fluorescence detection of cipro-induced G^S activity relative to A rpoS control cells. Per (73), the Z factor = 0.7 indicates an excellent signal-to- noise ratio with low variation. Strains: SMR24100 “WT” SOS-reporter; SMR24268 “WT” c^s -reporter; SMR24156 lexAInd"; SMR24312 A rpoS.

[0021] FIG. 6A-6D. The initial SOS but not os inhibitors reduce cell viability in cipro. (FIG. 6A) Growth curves indicate that NAL and NIR inhibit cell proliferation in MAC cipro, whereas DEQ and SQV do not, under conditions of cipro-induced mutagenesis experiments (35) and FIGS. IF, 1G: MAC cipro with or without DEQ used at 30 p M (15.8 pg/ml) in growth over 40 hours. Assays for RifR and AmpR mutagenesis are performed identically to mutation experiments with growth periods of 24h (RifR) and 48h (AmpR), making these conclusions relevant to the conditions of DEQ inhibition of cipro-induced mutagenesis (FIGS. IF, 1G) (35). Genetic ablation of neither stress response in lexAInd' or A rpoS mutants altered growth rates, suggesting that the reductions caused by NAE and NIR resulted from other activities of these drugs. Time, time in MAC cipro (37°C shaking). Strains: SMR24100 “WT” SOS-reporter; SMR24268 “WT” o s-reporter; SMR24312 A rpoS SMR24156 tex llnd . (FIG. 6B) DEQ does not kill cells in stationary phase. Stationary -phase cells were treated with vehicle or DEQ (no cipro). Strain: MG1655. (FIG. 6C) Competition experiments (35) show that DEQ does not retard growth of RifR or AmpR mutant cells, relative to isogenic sensitive cells, in medium with MAC cipro. Initial conditions were 50% sensitive and 50% resistant cells. Means ± range of 2 independent experiments, n.s. not significantly different, one-way ANOVA with Tukey’s post-hoc test. Therefore, we conclude that the DEQ reduction of mutants observed in assays for cipro-induced MBR (FIG. 1G) is caused by DEQ inhibition of mutagenesis, not a disability of RifR and AmpR mutants to proliferate in medium with DEQ. Strains: MG1655, SMR5223, SMR24603, SMR23099. (FIG. 6D) Chemical structure of DEQ.

[0022] FIGS. 7A-7D. Dose-responses for NIR, NAL, DEQ, and SQV inhibition of SOS and oS. (FIGS. 7A-7D) Flow cytometry with chromosomal SOS reporter P_suiAmCherry (left half of figure) or oS-rcsponsc reporter yiaG-yfp (right half of figure) at 20-24 h of growth with MAC cipro, per (35), strains SMR24100 or SMR24096. Each half shows a representative histogram and means ± range of at least 2 experiments, right. Response-positive cells are those right of the gate (black bars, Methods). Different from WT no drug, *p<0.05, one-way ANOVA with Tukey’s post-hoc test. Strains: SMR24100 “WT” SOS-reporter; SMR24096 “WT” ct s-reporter; SMR24156 fexAlnd ; SMR24134 A rpoS. (FIG. 7A) DEQ inhibits cipro induction of ct s-active subpopulation cells (right), but not cipro induction of the SOS response (left). (FIG. 7B) NIR reduces cipro-induced cS-activc cells (right) but not SOS-induced cells (left). (FIG. 7C) NAL does not alter cipro-induction of oS- or SOS-response-active cells. (FIG. 7D) SQV does not alter cipro-induction of cS- or SOS-response-active cells.

[0023] FIGS. 8A-8B. DEQ reduces cipro-induced os-p-galactosidase protein level.

Assays of ]3 -galactosidase activity. (FIG. 8A) DEQ inhibits cipro induction of o^s-P- galactosidase protein at 24 h growth in MAC cipro (stationary phase). Means ± SEM, 3 experiments. *p<0.001, two-tailed Student’s t-test. (FIG. 8B) DEQ does not inhibit - galactosidase activity from native lacZ gene at 24 h growth in MAC cipro (stationary phase) with IPTG. This control experiment shows that |3-galactosidase is assayed sensitively in DEQ, and o^s-P- galactosidase protein is itself reduced by DEQ. Means ± SEM, 3 experiments, n.s. not significant, one-way ANOVA with Tukey’s post-hoc test. Strains: (A) SG30013; (B) MG1655.

[0024] FIG. 9. NIR does not inhibit formation of cipro-induced ROS-high cells. Flow cytometry of log-phase (16 h) cells grown in MAC cipro and stained with dihydrorhodamine (DHR) ROS dye. Because NIR reduces numbers of o^s-active cells (FIG. IE), but not ROS, we conclude that NIR interferes with the pathway to o^s-activc gambler cells (FIG. 2A) at one of the steps after formation of ROS. Means ± SEM, 3 experiments. Two-tailed Student’s t-test; n.s. not significant. Strain: MG1655.

[0025] FIG. 10. Raw data for ratios shown in Fig. 2C. -galactosidase activity, P^lacZ and ParczlacZ reporters in log-phase growth in MAC cipro, ± DEQ. Means ±SEM, 3 experiments. Strains: CH2046, PM1450.

DETAILED DESCRIPTION

[0026] In keeping with long-standing patent law convention, the words “a” and “an” when used in the present specification in concert with the word comprising, including the claims, denote “one or more.” Some embodiments of the disclosure may consist of or consist essentially of one or more elements, method steps, and/or methods of the disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments may be combined. [0027] Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that no other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0028] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0029] As used herein, the terms “or” and “and/or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and/or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0030] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0031] The phrases “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as a human, as appropriate. The preparation of a pharmaceutical composition comprising an antibody or additional active ingredient will be known to those of skill in the art in light of the present disclosure. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biological Standards.

[0032] As used herein, “pharmaceutically acceptable carrier” includes any and all aqueous solvents (e.g., water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyloleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.

[0033] The term “subject,” as used herein, generally refers to an individual having a that has or is suspected of having a pathogenic infection, including a bacterial infection. The subject can be any organism or animal subject that is an object of a method or material, including mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens), household pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals. The subject can be a patient, e.g., can have or be suspected of having a bacterial infection. The subject may being undergoing or having undergone treatment. The subject may be healthy individuals but that are desirous of prevention of cancer. The term “individual” may be used interchangeably, in at least some cases. The “subject” or "individual", as used herein, may or may not be housed in a medical facility and may be treated as an outpatient of a medical facility. The individual may be receiving one or more medical compositions via the internet. An individual may comprise any age of a human or non-human animal and therefore includes both adult and juveniles (z.e., children) and infants and includes in utero individuals. It is not intended that the term connote a need for medical treatment, therefore, an individual may voluntarily or involuntarily be part of experimentation whether clinical or in support of basic science studies.

[0034] As used herein “treatment” or “treating,” includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated, e.g., cancer. Treatment can involve optionally either the reduction or amelioration of one or more symptoms of the disease or condition, or the delaying of the progression of the disease or condition. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof. Treating may mean alleviation of at least one symptom of the disease or condition.

I. Embodiments of the Disclosure

[0035] Dequalinium chloride (DEQ) is an off-patent FDA/EMA approved drug used previously as a topical antimicrobial agent in combination with clindamycin (antifungal drug). How it functioned was unknown. The present disclosure concerns its use as an internal agent, taken orally, intraarterially, by injection, intravenously, parenterally, intraperitoneally, intramuscularly, intrastemally, or intraarticularly, for example, to reduce mutagenesis in infecting bacteria, including reducing mutations that lead to antibiotic resistance, and crossresistance to other antibiotics not yet encountered.

[0036] The data show that in culture, DEQ inhibits activation of the Escherichia coli stringent starvation stress response by the quinolone antibiotic ciprofloxacin (cipro). Cipro induction of the stringent response is required for cipro induction of the sigma-S general stress response: a key regulatory hub in a mutagenesis-protein network required for stress-induced mutagenic DNA break repair. In culture this occurs at "subinhibitory" cipro concentration (10% viability), which occurs at the beginning and end of antibiotic therapies, and when doses are missed. In a mouse thigh infection model, in which cipro therapy is used, and is required for survival of the mice, mutagenesis to antibiotic cross-resistance required a functioning sigma-S response in the infecting bacteria, and was reduced by DEQ to a level as low as is observed with a sigma-S protein (rpoS gene) null mutant. Unlike its previous topical use at 2000-4000 mg/kg, it was found that intravenous administration of DEQ into mouse was effective at 2 mg/kg. In previous work, DEQ was tolerated orally in mouse at 2000 mg/kg, indicating that the 2 mg/kg dose is expected to be well tolerated.

[0037] Because de novo evolution of antibiotic resistance is a world health crisis, with many of the pathogens in the recent WHO priority pathogens list obtaining resistance by de novo mutations in native bacterial genes, and because clinically-relevant resistance to fluoroquinolones including cipro occurs by new mutations in three bacterial genes, and is induced by antibiotics, the present disclosure concerns use of DEQ against development of antibiotic resistance and cross resistance at least in two ways. In one embodiment, DEQ is administered with the antibiotic, for example to slow the generation of mutations, including mutations that confer resistance to the antibiotic administered and that confer cross-resistance to antibiotics not yet encountered by the pathogen. In one embodiment, because with or without antibiotics, clearance of infections is achieved by the immune system, which occurs by immune diversification of B and T cells, VDJ-joining, somatic hypermutation and class switch recombination, this disclosure provides a therapeutic strategy of use of DEQ to slow pathogen evolution without coadministration of an antibiotic. In specific embodiments, slowing pathogen evolution with respect to immune adaptation allows immune clearance without antimicrobial drugs. This would avoid deleterious consequences of antibiotics on beneficial native human microflora.

II. Embodiments of Compositions of the Disclosure

[0038] Dequalinium chloride (DEQ) is a is a quaternary ammonium cation having the IUPAC name of l,r-decane-l,10-diylbis(4-amino-2-methylquinolinium) decyl] -2-methyl-4- quinolin-l-iumamine dichloride. In specific embodiments of the disclosure, DEQ is formulated for delivery and use in a route that is other than for topical administration. In specific embodiments, DEQ is formulated for oral or intravenous use. The formulation(s) may comprise different amounts of DEQ dependent on the administration route to be utilized.

[0039] In some embodiments, the DEQ is formulated for oral use, such as at a dose of 1- 2000 mg/kg or at a dose of about 1-2000 mg/kg. The dose may be, or may be about, 1, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, or 200 mg/kg. The dose may be in a range of, or in a range of about, 1-2000, 1-1750, 1-1500, 1-1250, 1-1200, 1-1100, 1-1000, 1-750, 1-500, 1- 250, 1-100, 1-50, 50-2000, 50-1750, 50-1500, 50-1250, 50-1200, 50-1100, 50-1000, 50-750, 50-500, 50-250, 50-100, 100-2000, 100-1750, 100-1500, 100-1250, 100-1100, 100-1000, 100- 750, 100-500, 100-200, 200-2000, 200-1750, 200-1500, 200-1250, 200-1100, 200-1000, 200- 750, 200-500, 500-2000, 500-1750, 500-1500, 500-1250, 500-1100, 500-1000, 500-750, 750- 2000, 750-1750, 750-1500, 750-1250, 750-1100, 750-1000, 1000-2000, 1000-1750, 1000- 1500, 1000-1250, 1000-1100, 1100-2000, 1100-1750, 1100-1500, 1100-1250, 1250-2000, 1250-1750, 1250-1500, 1500-2000, 1500-1750, or 1750-2000 mg/kg, or any range therebetween. [0040] In some embodiments, the DEQ is formulated for intravenous use, such as at a dose of about 0.1-100 mg/kg, including in ranges of about 0.1-75, 0.1-50, 0.1-25, 0.1-10, 0.1-5, 0.1- 2.5, 0.1-2, 1-100, 1-75, 1-50, 1-25, 1-10, 1-5, 1-2, 5-100, 5-75, 5-50, 5-25, 5-10, 10-100, 10- 75, 10-50, 10-25, 25-100, 25-75, 25-50, 50-100, 50-75, or 75-100 mg/kg. The dose may be 0.1, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25. 2.5, 2.75, 5, 5.25, 5.5, 5.75, 10, 10.25, 10.5, 10.75, 25, 30, 40, 50, 60, 70, 75, 80, 90, or 100 mg/kg.

[0041] Compositions of the disclosure may also encompass one or more antibiotics, including one or more types of antibiotics. Examples include one or more of quinolones (including fluoroquinolones), penicillins, cephalosporins, beta-lactams, tetracyclines, aminoglycosides, macrolides, sulfonamides, and so forth. Specific examples of antibiotics to which bacteria may become resistant include cipro, methicillin, benzyl penicillin, vancomycin, carbapenem, benzyl penicillin, and Erythromycin. In embodiments wherein the antibiotic is a quinolone, it may be Nalidixic acid, Enoxacin, Norfloxacin, Ciprofloxacin, Ofloxacin, Lomefloxacin, Sparfloxacin, Grepafloxacin, Clinafloxacin, Gatifloxacin, Moxifloxacin, Gemifloxacin, Trovafloxacin, Garenoxacin, or a combination thereof.

[0042] In certain embodiments, the DEQ compositions of the disclosure may be housed in a kit, including DEQ formulations suitable for use in non-topical routes of administration, such as for oral or intravenous use. The dosages of the DEQ formulations may or may not be less than doses typically used for topical administration of DEQ. The DEQ components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one components in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also may typically include a means for containing the DEQ and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained. In some embodiments, the kit may also contain one or more antibiotics, including one or more types of antibiotics, such as those described elsewhere herein. Such antibiotics may or may not be formulated for administration by the same route as the DEQ composition. III. Embodiments of Methods of the Disclosure

[0043] The present disclosure concerns methods of treating an individual for an infection such that the bacteria associated with the infection have a reduced risk of mutagenesis. Embodiments of the disclosure include methods of treating an individual for an infection such that the bacteria associated with the infection do not become resistant to an antibiotic (e.g.. through mutation) being used to treat the infection. Embodiments of the disclosure include methods of treating an individual for an infection such that the bacteria associated with the infection have a reduced risk of becoming resistant to an antibiotic (e.g., through mutation) being used to treat the infection, compared to methods where DEQ is not utilized. Embodiments of the disclosure include methods of treating an individual for infection by enhancing an immune response in the individual, and in specific embodiments there are methods of treating an individual for infection by enhancing an immune response in an individual without using antibiotics. Embodiments of the disclosure include methods of treating an individual for infection by enhancing an immune response relative to pathogen mutation rate in the individual, and in specific embodiments there are methods of treating an individual for infection by enhancing an immune response, relative to pathogen escape from immune response, in an individual without using antibiotics. Embodiments of the disclosure include methods of reducing mutagenesis or the risk of mutagenesis in bacteria in an individual. Embodiments of the disclosure include methods of reducing the risk or delaying the onset of developing antibiotic resistance of bacteria in an individual. Embodiments of the disclosure include methods of facilitating clearance of an infection in an individual, including in the absence of also administering one or more antibiotics to the individual. Embodiments of the disclosure include methods of protecting native microflora in an individual in need of one or more antibiotics. The aforementioned methods comprise at least the step of administering once or more than once to the individual an effective amount of dequalinium chloride (DEQ). The effective amount of DEQ administered to the individual may be dependent on the route of administration. In specific embodiments, the route of administration is not topical, although in alternative embodiments the administration route is topical.

[0044] Embodiments of the disclosure include methods of treating an individual for a bacterial infection, comprising the step of non-topically administering to the individual an effective amount of dequalinium chloride (DEQ) to reduce mutagenesis in the bacteria. Such methods transpire whether or not the individual also receives one or more antibiotics. The methods cause reduction in mutagenesis in the bacteria and in specific embodiments also reduce proliferation of DEQ-resistant mutants. In methods wherein DEQ is administered to the individual but not also one or more antibiotics, the DEQ enhances the immune response of the individual to the infecting bacteria, e.g., because it slows evolution of the bacteria such that the immune system has an increased chance of successfully clearing the infection or an increased chance of clearing the infection at an earlier point in time.

[0045] In methods wherein DEQ is administered to the individual but not also one or more antibiotics, this allows reduced cost of treatment for the individual, an overall reduction in use of antibiotics that lessens impact of antibiotic resistance on public health, and reduces the risk that the antibiotics will deleteriously affect the microbiome of the individual.

[0046] In specific embodiments, an individual in need of treatment for a bacterial infection is provided an effective amount of DEQ, and the individual may be one that has one or more symptoms of a bacterial infection, such as feeling tired or fatigued; having swollen lymph nodes in the neck, armpits, groin or elsewhere; headache; nausea or vomiting; persistent cough; coughing up pus, redness or swelling of the skin; blood in urine, vomit, or stool; severe stomach pain; a cut or burn having pus; or a combination thereof. The individual may or may not also be provided one or more other therapeutic agents, such as one or more antibiotics, steroids, and/or analgesics. In some cases, DEQ is provided before receiving another therapeutic agent(s), while the individual is receiving another therapeutic agent(s), and/or after receiving another therapeutic agent(s).

[0047] Methods of the disclosure may be utilized for any type of bacteria. In specific embodiments, the methods are for bacteria that are Gram-positive or Gram-negative. The bacteria may be Escherichia coli, Methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), multiresistant Gram-negative bacteria, Clostridioid.es difficile, Mycobacterium tuberculosis, carbapenem-resistant Acinetobacter, carbapenem-resistant Enterobacteriaceae (CRE), drug-resistant Neisseria gonorrhoeae, drugresistant Campylobacter, vancomycin-resistant Enterococci (VRE), multidrug-resistant Pseudomonas aeruginosa, drug-resistant nontyphoidal Salmonella, drug-resistant Salmonella serotype Typhi, drug-resistant Shigella, drug-resistant Streptococcus pneumoniae (.S'. pneumoniae), drug-resistant Mycobacterium tuberculosis, erythromycin-resistant group A Streptococcus, clindamycin-resistant group B Streptococcus, or drug-resistant Bordetella pertussis (B. pertussis).

[0048] In some embodiments, the DEQ is administered to the individual once or more than once. When DEQ is administered once to the individual, it may be before an antibiotic, during the duration of time the individual is receiving an antibiotic, and/or after the duration of time the individual is receiving an antibiotic. When DEQ is administered more than once to the individual, it may also be before an antibiotic, during the duration of time the individual is receiving an antibiotic, and/or after the duration of time the individual is receiving an antibiotic. When DEQ is administered more than once to the individual, it may be at substantially the same time as administration of an antibiotic. DEQ may be administered once a day, more than once a day, once a week, more than once a week, once a month, or more than once a month, for example.

[0049] Methods of the disclosure may be utilized for an individual that has pneumonia, tuberculosis, blood poisoning, gonorrhea, urinary tract infection, and/or a foodbome disease. In some embodiments, the individual previously misused antibiotics, such as by taking antibiotics when unnecessary and/or not completing the entire course of antibiotics. The individual may be temporarily or permanently residing in a nursing home, skilled nursing care facility, or long-term care facility. The individual may be in a medical facility, such as a hospital, nursing home, skilled nursing care facility, or long-term care facility. The individual may be of any age, but in specific embodiments the individual is an infant or is greater than 65, 70, 75, 80, 85, 90, 95, or 100 years of age. The individual may or may not be immunocompromised.

[0050] Methods of the disclosure may be utilized for any mammal, including a human, and they may be utilized for an agricultural animal, such as a cow, turkey, chicken, or pig, or a companion animal, such as a dog, cat, or horse.

[0051] In particular embodiments, methods of the disclosure encompass applying an effective amount of DEQ to a surface of any kind, wherein the surface is not a surface of an animal. In specific embodiments, methods of the disclosure encompass applying an effective amount of DEQ to a surface for the purpose of reducing mutagenesis of bacteria thereon. In such cases, DEQ may be formulated in a formulation such as a spray, foam, liquid, gel, or on (and/or in) the surface of a paper, wipe, or cloth sheet, for example. The surface may be a countertop, floor, cabinet surface, furniture of any kind, medical equipment or fixtures, medical device or instrument holder or container (e.g., a tray for scalpels), beds, tables, chairs, stretchers, and so forth. The surface may be metal, glass, granite, plastic, laminate, a composite material, a natural material, a synthetic material, and so forth. DEQ may be used in a medical facility of any kind, a hospital, nursing home, skilled nursing home, long-term care facility, school, plane, ship, train, bus, automobile, hotel, shopping mall, any kind of building of residency, any kind of building of commercial business, a combination thereof, and so forth. [0052] In embodiments wherein an effective amount of DEQ is applied to a non- animal surface, one or more cleaning agents (e.g., disinfectants, antiseptics, sanitizers, sterilizers, decontaminators, and so forth) for surface cleaning may also be utilized. The DEQ and the one or more cleaning agents may or may not be formulated in the same composition. Examples of cleaning agents includes alcohols, chlorine and chlorine compounds, formaldehyde, glutaraldehyde, ortho-phthalaldehyde, hydrogen peroxide, iodophors, peracetic acid, phenolics, quaternary ammonium compounds, or a combination thereof. When DEQ and the cleaning agent(s) are not in the same formulation, they may or may not be applied at the same time. When DEQ and the cleaning agent(s) are not in the same formulation, they may or may not be delivered in the same type of formulation. When they are not applied at the same time, DEQ may be applied to the surface before or after the cleaning agent(s). DEQ may be used in conjunction with ultraviolet radiation. In embodiments, DEQ is applied once or more than once to the surface.

[0053] In some embodiments, an effective amount of DEQ is present in a hand sanitizer, such as a gel hand sanitizer (and may also referred to as hand antiseptic, handrub, or hand rub). The hand sanitizer may be alcohol-based or alcohol-free.

[0054] In some embodiments, an effective amount of DEQ is present on a medical device. The medical device may be for temporary or permanent use in an individual in need thereof. The medical device may comprise DEQ on all of the device, such as all surfaces of the device, or only part of the device. The DEQ may be applied to the device prior to commercialization or just prior to use in an individual, such as within 1-60 minutes or 1-24 hours, for example. The DEQ may be applied to the device as a spray, foam, liquid, or gel, for example. The DEQ may or may not be applied to the device at the same time as one or more antibiotics. The DEQ may be in the same formulation as one or more antibiotics and applied concomitantly to the device. When DEQ and one or more antibiotics are applied to the device at different times, the DEQ may be applied before or after the one or more antibiotics. The device comprising DEQ may be provided in an off-the-shelf manner and one or more antibiotics added to the device prior to use for the individual. In such cases, the selection of the one or more antibiotics may be tailored to the need of the individual, such as selected to be effective against the particular infection of the individual. Any medical device may comprise DEQ, such as a catheter, stent, syringe, joint prothesis, pacemaker, breast implant, sheath, wire, balloon, tube, drive line, tube, implant, defibrillator, artificial joint, pacemaker, screw, rod, disc, intrauterine device, pin, plate, stent, dental device, eye lens, shunt, valve, neurological or neurosurgical device, gastrointestinal device, genitourinary device, catheter cuff, vascular access device, wound drain, and so forth. DEQ may also be comprised on a bandage, tape, gauze, medical personnel wear, gloves, and so forth , for example.

IV. Examples

[0055] The following examples are included to demonstrate certain embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques to function well in the practice of the subject matter of the disclosure, and thus can be considered to constitute particular modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the subject matter of the invention.

EXAMPLE 1

DRUGGING EVOLUTION OF ANTIBIOTIC RESISTANCE AT A REGULATORY-1 NETWORK HUB

[0056] It was considered that ideal targets for evolution- slowing, or “anti-evolvability,” drugs might include critical non-redundant hubs of functional protein networks that underpin antibiotic-induced mutagenesis (11), reviewed (12, 13, 15). See also (10, 22-24) for mechanisms of antibiotic induction of mutagenesis for which protein networks are not known. Inhibition of hubs could prevent different parallel pathways to mutagenesis simultaneously (11). Herein, this strategy is applied to mutagenesis induced by the widely used fluoroquinolone antibiotic (22) ciprofloxacin (cipro) (10, 23).

[0057] Fluoroquinolones bind and inactivate bacterial type-II topoisomerases midreaction, which kills cells via DNA double-strand breaks (DSBs) (26). Type-II topoisomerases relieve replication- and transcription-generated positive DNA supercoils by cleaving opposite DNA strands, passing a duplex through, then ligating the DSB ends (27). Both clinically and in the laboratory, fluoroquinolone resistance occurs mostly by de novo mutations that alter the topoisomerase, preventing drug binding, or upregulate efflux pumps (28-34). Clinically isolated Escherichia coli that are cipro-resistant (mutants) occur at very high frequencies, ranging from 20% to 50% depending on the geographic area (2).

[0058] Cipro-induced mutagenesis, which can generate resistance (6, 10), including resistance to antibiotics not yet encountered (35). Cipro-induced mutagenesis and occurs by mutagenic repair of DNA breaks, activated by the general stress (o^s) response (35) (outlined in FIG. 1A), reviewed (15). Importantly, the mutagenesis functional protein network is described (11). At 20-million prescriptions per year in the US (25), cipro is the second-most prescribed antibiotic (after b-lactams) (36), and so is relevant clinically, in addition to its utility as a model. For example, a type-II topoisomerase inhibitor, cipro inhibits bacterial topoisomerases similarly to the anticancer drug etoposide mechanism of action on human topoisomerases (26). [0059] The inventors identified a drug, dequalinium chloride (DEQ), that inhibits the G^S response, the central mutagenesis activator (FIG. 1 A), and cipro-induced mutagenesis. A cipro- induced starvation (stringent)-stress response (38), shown recently to activate cipro induction of G^S and mutagenesis (39), is the drug’s point of inhibition. At the low concentrations used herein, 500-1000-times lower than the maximum tolerated dose in rat and mouse, respectively (27), DEQ does not increase cipro-killing activity (See Example 7), which would favor proliferation of resistant mutants, and so is an apparent “stealth” (15) evolution- slowing drug. Furthermore, in mouse thigh infections the occurrence is demonstrated of stress-induced mutagenesis similar to cipro-induced mutagenic break repair (MBR) in culture. Its reduction is shown by this and another drug that inhibits MBR, thus providing a proof-of-concept for drugs to slow evolution of drug resistance and escape from immune responses, found as network-hub inhibitors.

EXAMPLE 2

SCREEN FOR STRESS-RESPONSE INHIBITORS FINDS “ANTI-EVOLVABILITY” DRUG

[0060] The SOS DNA-damage and G^S general stress responses (13, 15, 41-43) are non- redundant hubs in a MBR network of more than 100 proteins (11, 44, 45). Most of these promote mutagenesis by sensing stress and transducing the signals to the stress-response activators that upregulate mutagenesis (11) (FIG. 1 A). Cipro, a type-II topoisomerase inhibitor, induces DNA double-strand breaks (DSBs) and SOS, which upregulates error-prone DNA polymerases and proteins used in DSB repair. However, DSB repair remains non-mutagenic unless another stressor activates G^S (46, 47), e.g., starvation (11) or antibiotic exposure (23, 35, 38). G^S is not needed for DSB repair (46-48) but allows use of, and persistence of errors (49) made by, error-prone DNA polymerases in DSB repair (48) causing mutations (FIG. 1A).

[0061] A small-molecule library was screened of 1120 approved-for-human-use drugs (Prestwick Chemical) for inhibition of the SOS or G^S response. The drugs represent a large spectrum of chemical and pharmacological diversity (50). Cells with either an SOS- or G^S - response fluorescence-reporter gene (11, 51, 52) (FIGS. 1B-1E) were grown in “sub- inhibitory” cipro at the minimum antibiotic concentration (“MAC,” 10% viability, Methods), at which cipro-induced MBR is maximal (35). The primary screen for stress-response inhibition used a fluorescence plate reader (FIGS. 1B-1D, validated FIG. 5), which is high throughput but low resolution. A more accurate, single-cell secondary screen used flow cytometry (FIGS. IB, IE and Example 8), which affords much higher resolution and is low throughput.

[0062] The primary screen identified two potential SOS inhibitors and two potential G^S inhibitors among 1120 compounds. Naltrexone (NAL) and niridazole (NIR) reduced apparent SOS-, and saquinavir (SQV) and dequalinium chloride (DEQ) apparent c^s-rcsponsc fluorescence (FIGS. 1C, ID). NAL and NIR also inhibited growth of cipro- sensitive cells in cipro, an undesirable side effect (which selects resistance, Introduction), whereas SQV and DEQ did not inhibit growth either in MAC cipro (FIG. 6) nor without cipro (FIG. 6B). These experiments (FIG. 6A-6C) mimic the experimental conditions of the mutagenesis experiments described in the following paragraph and used previously in (35), in which MAC cipro is used with or without DEQ, described in the FIG. 6A legend.

[0063] In higher resolution flow cytometry, none inhibited SOS (dose responses, FIG. 7), whereas DEQ inhibited o^s -response activity (FIG. IE, FIG. 7 and Example 8) and G^S protein levels (FIG. 8) robustly. Treatment with DEQ caused cells with a G^S -protein fusion to |3- galactosidase to display reduced |3-galactosidase activity, which was not observed in cells with b galactosidase produced from its native lacZ locus. Surprisingly, NIR also partially reduced GS activation (FIG. IE and FIG. 7) and did not reduce cipro-induction of reactive oxygen species (ROS) (FIG. 9), which induce o^s-activity (35) (discussed, FIG. 9).

[0064] The inventors examined cipro-induced mutagenesis, as per (35), with assays for rifampicin- or ampicillin-cross-resistant mutants (RifR or AmpR) (FIG. IF). These carry specific base substitutions in the rpoB gene, or any null mutation in ampD, respectively. DEQ reduced cipro induction of mutation rates by 7.6-fold (± 2.5), and 2.7-fold (± 0.5, means ± SEM) for RifR and AmpR, respectively (FIG. 1G). NIR reduced neither significantly (FIG. 1G). Induction of mutation rates by cipro is shown as the rate with cipro/rate without cipro (FIG. 1G), and is estimated per cell per generation, and per chromosome per generation, because chromosome copies are increased in cipro-treated cells (35). Mutagenesis, not relative growth rate of RifR or AmpR mutant cells, is reduced by DEQ (FIG. 6C). The data show that DEQ reduces mutagenesis, a validation of the strategy for identification of evolution- slowing drugs. DEQ (structure, FIG. 6D) is used, clinically, at >100-times higher concentration than used here, with the antimicrobial clindamycin as a topical bacteriostatic agent to treat vaginal infection (53, 54). Example 7 reviews the high bacteriostatic and low non-inhibitory DEQ concentrations used here, which do not reduce cell viability (FIG. 8A, 8B). DEQ is well tolerated orally at 1000 mg/kg in rat and 2000 mg/kg in mouse (40), a concentration 1000- times higher than we used here in mouse, described below, and 100-times more than in culture (FIGS. 1-4).

EXAMPLE 3

DEQ INHIBITS SMALL RNAS AND THE STRINGENT RESPONSE

[0065] The step was determined at which DEQ disrupts the pathway, previously elaborated (35, 39), of cipro-induction of the G^S response and mutagenesis. As illustrated in FIG. 2A, cipro induces DSBs and the SOS response in all cells (quantified as foci and fluorescence, respectively) (35). The SOS response then induces ROS in a -20% cell subpopulation (35) (FIG. 2B). The ROS are required for transcription of two small (s)RNAs, DsrA and ArcZ (35), which promote translation of rpoS (o^s) mRNA, activating the GS response. It was found that DEQ prevented cipro-induction of the dsrA and arcZ promoters (FIG. 2C and FIG. 10), and did not reduce ROS-high cells (FIG. 2B). The ROS, G^S response and mutagenesis occur in a -20% “gambler” cell subpopulation (35) (FIG. 2A, 2B). FIG. 2D illustrates the point in the pathway inhibited by DEQ.

[0066] Recently identified in the cipro-induced MBR pathway, the “stringent” starvationstress response is also activated in a roughly 20% cell subpopulation (39). The stringent- activated subpopulation cells are derived from the ROS-containing subpopulation cells, lie downstream of ROS production, and are required for ROS induction of transcription of the sRNAs (39) (illustrated, FIG. 3A). The stringent-response “on” subpopulation cells become (are the same cells as) the G^S response-induced gambler cell subpopulation (39), which, when sorted by FACS produces more than 90% of all cipro-induced mutants (35) (illustrated, FIG. 3A). Activation of the stringent response requires the nucleotide “alarmone” guanosine tetra- (or penta)-phosphate, (p)ppGpp, synthesized by the RelA and SpoT synthases (55) (positive controls, FIGS. 3B, C). It was found that DEQ reduced stringent-response activity assessed with the fluorescence reporter rmflnCherry (39) and flow cytometry (FIG. 3B), and also with the stringent-response reporter P_ira ZacZ, in bulk cultures (56) (FIG. 3C). We conclude that DEQ interrupts the pathway to cipro-induced MBR after ROS (FIG. 2B) by reducing induction of the stringent response (FIG. 3B, C), illustrated in FIG. 3A, which prevents downstream sRNA transcription (FIG. 2C) (39), G^S induction (FIG. IE) and mutagenesis (FIG. 1G). The antioxidant drug edaravone inhibits cipro-induced mutagenesis by reducing ROS (35), a different step from that of DEQ shown here (FIG. 3A).

[0067] DEQ did not alter cell killing by cipro at a high therapeutic dose of 1.5 pg/ml (Fig. 3D), a level comparable with that in patients under cipro therapy (57) (Example 7), and modeled here in mouse, discussed in the following section. It was concluded that DEQ did not alter cipro antibiotic activity (FIG. 3D and FIG. 6A, 6C), nor reduce cell growth rate with or without cipro (FIG. 3D and FIGS. 6A and 6B). This implies that in general, activation of the stringent o^s -response might be points in the cipro-induced MBR pathway that can be inhibited without imposing strong selection for mutants resistant to the evolution-slowing.

EXAMPLE 4

STRESS-INDUCED MUTATION IN MOUSE INFECTIONS AND DRUG INHIBITION

[0068] A consideration of dissection of the molecular mechanisms of antibiotic-induced mutagenesis is that, if found, stealth anti-evolvability drugs might slow evolution of resistance in living animals undergoing infection and antibiotic therapy. This strategy is supported by the demonstration, first, that in an established and well used preclinical mouse thigh-infection model (10, 20, 58, 59) (FIG. 4A), mutagenesis occurs in the infecting bacteria via a mechanism-dependent on proteins of stress-induced MBR (FIG. 4B). In the mouse model, 10⁶ CFU of bacteria are delivered into thigh wounds in neutropenic mice, followed in 2 hours by 1 mg/kg cipro to prevent death of the infected mice, and induce mutagenesis during infection. In our experiments, the candidate anti-evolvability drugs or vehicle were given with the cipro. DEQ was given at a non-toxic 2 mg/kg (1000-times less than mice tolerate orally; Example 7; and 100-times lower than its bacteriostatic dose). Cipro-treated bacteria were recovered after two days of infection and assayed for total CFU and RifR cross-resistant mutants (FIG. 4A); mice not given cipro died earlier (Example 6).

[0069] It was found that mutagenesis occurred in animals, and was reduced to 6.3 ± 2.3 times lower than the control strain (mean ± SEM of three mice per strain, each in three independent experiments, averaged) in isogenic bacteria defective for central stress response regulators of MBR: the SOS response (ZexAInd'); G^S response (ArpoS); and the Hfq RNA chaperone needed for G^S induction by cipro (35) (FIG. 4B). The promotion of mutagenesis by stress response regulators demonstrates stress-induced mutagenesis (mutagenesis promoted by a tress response (13, 15)) in bacteria during infection. Moreover, a mechanism similar to cipro induced MBR is implicated by the dual requirements for SOS and the G^S response (15, 35). The mutant frequencies differ little between those in mouse and those in culture (Example 9), and the genetic dependencies of mutagenesis are the same (Fig. 4B) and (35). This is the first demonstration of G^S response-dependent (stress-induced) mutagenesis in an animal infection model.

[0070] Strikingly, it was found that both DEQ and edaravone reduced frequencies of RifR mutants robustly in mouse, causing reductions similar to those in the corresponding MBR- deficient mutants (FIG. 4B). These data show that small-molecule inhibitors (35) can slow bacterial evolution of resistance during infection, a demonstration of use of evolution-slowing drugs in animal infection, and example of the strategy generally.

EXAMPLE 5

SIGNIFICANCE OF CERTAIN ASPECTS

[0071] The data identify a drug, DEQ (FIG. 6D), that targets the G^S response, a mutagenesis network hub (11), and inhibits cipro-antibiotic-induced mutagenesis (FIG. 1, FIGS. 6 and 7), and define the step at which it acts (FIGS. 2 and 3A-3C) in a defined pathway (35, 39) of cipro- induced mutagenic DNA break repair. DEQ blocks induction of the stringent starvation-stress response (FIG. 3), after its inducer, ROS (39) (FIG. 2B) and before stringent induction (FIGS. 3A-3C) (39) of the two sRNAs (FIG. 2C) that activate G^S in cipro-induced MBR, illustrated in FIG. 3A. The data also provide evidence of stress-induced mutagenesis — mutagenesis upregulated by one or more stress response(s) — occurring in infection of an animal. DEQ reduced mutagenesis during mouse infections (FIG. 4B) and in culture (FIG. 1G). Moreover, when DEQ was used at 1000-times lower concentration in mouse than it is tolerated orally in mouse (40) (Example 10), and >100-times lower than its previous use topically (Example 7), DEQ did not reduce cell viability or growth rate with or without cipro (FIG. 3D and FIGS. 6A- 6C). At this low dose, DEQ is expected, therefore, not to favor proliferation of mutants resistant to DEQ or cipro: a “stealth” (15) evolution slowing drug.

[0072] Antibacterial inhibitors of the stringent response have been identified previously and, unlike DEQ, were analogs of the stringent-response activator (p)ppGpp, which binds RNA polymerase (60-62). Unlike DEQ, those drugs reduced viability, and so favor proliferation of resistant mutants. The (p)ppGpp analogs were developed as anti-bacterials, and their possible effects on mutagenesis were not examined. DEQ is not a (p)ppGpp analog (FIG. 6D), and, unlike the analogs, it inhibits the stringent transcriptional program (FIGS. 3B and 3C, and 2B- 2D) with no measurable reduction of viability when used without cipro (FIG. 6B), or in the presence of a therapeutic high cipro dose (FIGS. 3D) or in MAC cipro (FIG. 6A). MAC is defined as the drug concentration that produces 10% viable CFU (63). MAC is the most mutagenic cipro concentration (35), and will occur at the beginning and end of therapies, and when doses are missed (Example 10). Possible mechanisms of its growth-neutral inhibition of stringent-transcriptional activity (FIG. 3D and FIG. 6A-6C), are considered in Example 11. It is noted that the DEQ dose that reduced of mutagenesis in mouse here (FIGS. 4B) is 2 pg/ml (or mg/kg), which is 1000-times lower than mice tolerate orally (2000 mg/kg (40), Examples 7 and 10), and 100-times lower than the dose we used in culture, throughout, and so might be expected also not to impair viability in animals. Viability cannot be measured directly in mouse. [0073] New antibiotics designed to bind two different target molecules were developed to resist resistance by requiring, presumably, two independent mutations for resistance, one in each of the two target’s gene (64). Cipro also binds two targets, topoisomerases II and IV (37), and one way cells become resistant is by upregulation of efflux pumps (5), which export the drug. The frequencies of mutants resistant to the new drugs are not yet known. Whether or not they are unusually low, combining new or old antibiotics with the evolution-slowing drugs might resist resistance robustly.

[0074] More uniquely, evolution- slowing drugs such as DEQ might make possible treatment of infections without antibiotics. By slowing pathogen evolution, immune-response somatic evolution might outstrip the pathogen to allow clearance of infections without harm to the native human microbiome, e.g., (15), which underpins many aspects of human health (8).

[0075] Whether DEQ (or edaravone) would be useful for slowing bacterial evolution clinically is unknown and requires future clinical studies. However, both support the proposal that non-killing (35) (FIG. 3D and FIG. 6A-6C) “stealth” (15) evolution-slowing drugs can be identified by functional screens for reduced activity of the G^S general stress response. FIG. 4C illustrates the MBR protein network (11, 39, 44, 45) and its SOS- and o^s -response activators, which are critical non-redundant hubs (11). Hubs coordinate multiple inputs that “fan into” a common node (65). Inputs shown to act upstream of G^S activation in MBR (35, 39) are shown as dark green circles in FIG. 4C. At hubs, a common “currency” of few information carriers is used to “fan out” into various outputs (65); those known for G^S and relevant to MBR are shown as light green circles (FIG. 4C). Such “bow tie” informational architectures are robust to perturbations without losing whole-system function, and are flexible to evolution of new inputs and outputs (65); but their inherent weakness is their hubs or “knots”, disruption of which can shut a system down. The hubs in stress-induced MBR, the stress-response activators, are potentially useful targets for evolution-slowing drugs (11) and their identification by functionbased chemical genetic screens. [0076] The data also provide evidence of stress-induced mutagenesis — mutagenesis upregulated by one or more stress response(s) — occurring during infection. Stress-induced MBR, is one of the most well characterized molecular mechanisms of spontaneous or drug- induced mutagenesis based on work in culture, with more than 100 proteins implicated in the MBR protein network (FIG. 4C) (11), reviewed (13, 15). Its apparent occurrence in animal infections identifies a vast trove of potential targets for design of future anti-evolvability drugs for bacteria.

[0077] Given that the antibiotic kills by >5-6 logs; then the fate of the remaining bacteria is either clearance by the immune system or generation of new mutations to resistance (66, 67), in specific embodiments those new mutations would be fewer, making clearance more likely before rebound of a resistant (mutant) population. For mutation rates of -10'⁶ per living gambler cell (39), knocked down -10-fold by DEQ, this will matter most when fewer than -10⁷ gamblers (5 xlO⁷ total bacteria) are present, in certain embodiments.

EXAMPLE 6

EXAMPLES OF MATERIALS AND METHODS

[0078] Bacterial strains, media, and growth

[0079] E. coli strains used, and their origins are given in Table 1. Specific strains used in each main figure are listed in the following section. Assays for rifampicin-resistant (RifR) and ampicillin resistant (AmpR) mutants were performed in one of two wild-type (WT) E. coli strains, and their isogenic derivatives. Bacteria were grown in LBH rich medium at 37°C with aeration, and addtives where indicated at the following concentrations: ciprofloxacin (cipro, 1- 8.5 ng/mL, Table 2), ampicillin (lOOpg/ml), chloramphenicol (25pg/ml), kanamycin (50pg/ml), tetracycline (lOpg/ml), rifampicin (l lOpg/ml), and sodium citrate (20mM), lOOpM IPTG. The minimum antibiotic concentration, or MAC, is the concentration at which 10% of treated cells remain viable, compared with cultures with no antibiotic (63), and was determined for each strain experimentally, as previously (35).

[0080] Strains used in FIGS. 1-4

[0081] FIG. 1: (C) SMR24100, SMR24156, SMR24268, SMR24312. (E) SMR24096, SMR24134. (G) MG1655, SMR20479, SMR5223, SMR11641. FIG. 2: (B) MG1655. (C) CH2046, PM1450. FIG. 3: (B) SMR24273, SMR27009. (C) CH1623, CH6485. (D) MG1655. FIG 4: (B) RTC0021, SMR26989, SMR26991, SMR26993.

[0082] Drug screens [0083] The inventors used the Prestwick Chemical Library (PCL) from Prestwick Chemical (Illkirch, France) which contains off-patent approved-for-human use drugs, a gift from the Adam Kuspa lab. The strains SMR24268 with the yiaG-mCherry cS-rcsponsc reporter (11, 35) or SMR24100 with the Aat PsulAmCherry SOS reporter (52), modified from (51), were used for the drug screens. Saturated overnight LBH cultures, started each from a single colony, were diluted l:4xl0⁶ into ml in a 250ml flask in fresh LBH broth and incubated at 37°C with shaking for 3-3.5 h. 17pL of each drug, 1 replicate, 8pg/mL (2x final concentration per Prestwick Chemical recommendations for screening concentration), or vehicle (2% DMSO, 2x final concentration), were pipetted into a 384-well plate, followed by addition of 17 pL of the following mixture: cells (in LBH) carrying either the yiaG-mCherry cS-rcsponsc reporter or the Aat PsulAmCherry SOS reporter, plus cipro (17ng/mL, which is 2x final concentration) or vehicle. To reduce effects from evaporation, the outside rows and columns of each plate were not used. Also, on each plate screened, lexAInd' cells (SOS-response uninducible), or ArpoS cells (no c^s activity) were used as genetic positive controls. After 15 h of growth, the SOS-inhibitor screen was analyzed on a BioTek plate reader (Winooski, VT). The c^s responseinhibitor screen was analyzed after 36 h of growth. For both screens, total mCherry fluorescence and ODeoo were measured. Z-factors were calculated for each assay as described (73). Hits for further analysis met the criteria: (1) not a known antibiotic drug, and (2) inhibiting cipro activation of either the SOS response or G^S response. For secondary flow cytometry validation, new drugs were obtained from Sigma-Aldrich to rule out potential chemical contamination of the drug screening library. The molar concentrations of the hit compounds in the screen were: NIR 1.3pM, DEQ 30pM.

[0084] Assays for ciprofloxacin-induced mutagenesis

[0085] Assays for rifampicin-resistant (RifR) and ampicillin-resistant (AmpR) mutants were performed with cipro at MAC in the wild- type (WT) E. coli strains MG 1655 and SMR5223, and their isogenic derivates, as previously (35). For fluctuation tests performed with the addition of drugs identified in the screen, the final concentrations were 30pM for DEQ and 1.3pM for NIR. Ten to sixty aliquot of log-phase cultures were diluted 1:3 and dispensed into 14-mL tubes with and without the identified drugs and with and without MAC cipro, and then grown at 37°C shaking for the mutagenesis assays for 24 h (RifR) or 48 h (AmpR) then assayed for mutant and total CFU per (35). Mutation rates were estimated with the MSS-MLE algorithm using the FALCOR calculator (74).

[0086] Flow cytometry for SOS-, o^s-, and stringent-response activity

- 1 - [0087] Flow cytometric assays for SOS-, o^s-, and stringent response-regulated promoter activity were as described (35, 39). Quantification of cells that have induced their SOS, stringent, or GS response, and how much they have, was achieved using engineered chromosomal fluorescence reporter genes and flow cytometry, per (51, 52) for SOS, per (11, 35) for oS-response activation, and per (39) for stringent-response activation using AatZ.:PsulAmCherry SOS reporter (51) modified from (52) for SOS, the yiaG-yfp oS-response reporter (11), and the PrmfmCherry stringent-response reporter (39). Strains were grown under fluctuation-test conditions as described for Assays for ciprofloxacin-induced mutagenesis, with or without cipro, at indicated concentration(s), and were harvested in late log phase or stationary phase. For quantification, flow cytometry “gates” were calibrated, for SOS, using the negative-control SOS-off ZexA(Ind-), and SOS-response proficient cells (51) as the dividing place between peaks of the distribution of SOS-proficient cells at which most cells diverge from the spontaneously SOS-induced fluorescent cell subpopulation, usually at between 0.5% and 1% of cells cultured in LBH broth. With this gate, -10'⁴ of SOS-non-inducible ZexAInd- cells cross the gate, scoring as “SOS-positive” (51). For the G^S 652 response, gates were set to the point at which fewer than 0.5% of cells with cipro but without the reporter gene were positive. At this gate, fewer than 10'³ of ArpoS cells, which are o^s -response deficient, cross the gate and would be scored as positive. For all, the percent of the population that scored as positive is reported. For the secondary confirmation of each drug, the inventors used at least 4 doses relative to the molar concentration used in the initial hit screen (see FIGS. 7A-7D). For the stringent response, gates for stringent response-active cells were set to the point at which fewer than 0.5% of cells with cipro but without the reporter gene were positive.

[0088] Single-cell detection of intracellular ROS by flow cytometry

[0089] Cells were grown in the absence or presence of cipro at its MAC (8.5ng/mL) to log phase as for Assays for ciprofloxacin-induced mutagenesis. The ROS measurement protocol was adapted from Pribis et al (35). Cells were grown in the presence of MAC cipro with or without the identified drug as for Assays for ciprofloxacin-induced mutagenesis, then harvested serially from cultures for ROS detection using dihydrorhodamine (DHR, Life Technologies).

[0090] Beta-galactosidase assays

[0091] Cells were grown as for Assays for ciprofloxacin-induced mutagenesis to equivalent ODs and frozen at -20°C until assays were carried out. Determination of the P- galactosidase activity of the PdsrA-lacZ, ParcZ-lacZ, rpoS-lacZ, and PiraP-lacZ fusions was accomplished using the standard assay described by JH Miller, as previously (35, 75, 76).

[0092] Measurement of high-dose cipro antibiotic activity [0093] Cells were grown to log phase OD600 -0.5, then cipro (1.5pg/mL) was added with or without DEQ (30pM) and returned to 37°C incubation shaking. Cells were harvested 0.5, 1, 2, and 3 h later to determine CFU/mL. Cells were washed twice with PBS and then assayed for viable CFU on EB plates.

[0094] Neutropenic murine bacterial thigh infection model

[0095] Animal care and experimental procedures were approved by Baylor College of Medicine Institutional Animal Care and Use Committee in accordance with all guidelines set forth by the US National Institutes of Health. The protocol for neutropenic bacterial thigh infection was modified from (10, 20, 59). Before cyclophosphamide treatment began, mice were administered buprenorphine at Img/kg. Six- to eight- week-old female CD-I mice (Charles River Laboratories; weight: 25-35g) were rendered neutropenic by intraperitoneal (I.P) 686 injection of 150mg/kg cyclophosphamide (sigma) 4 days before infection and lOOmg/kg cyclophosphamide 24 h before infection. LB cultures inoculated from fresh E. coli (ATCC25922) colonies were grown to the log phase (ODeoo approximately 0.3) and diluted in LB broth. Thigh infections were performed by injecting 50pl (approximately 10⁶ CFU) of diluted cultures into the thigh muscle of isoflurane anesthetized mice. Starting 2 h after infection (defined as time zero), mice were administered subcutaneous injections of Img/kg of the antibiotic ciprofloxacin (cipro), or vehicle, and I.P injections of 2mg/kg DEQ or lOmg/kg edaravone, or vehicle, every 24 h for 2 days. Animals given vehicle with no cipro died from untreated infection. Cipro-treated mice were monitored daily for signs of distress. After 48 h, cipro-treated mice were euthanized, and thighs of each were removed and in groups of 3 thighs combined, which were then resuspended in 1ml PBS buffer, from which serial dilutions were plated on LB agar containing kanamycin for total CFU, and undiluted homogenates plated on LB agar containing kanamycin and rifampicin for RifR CFU. Total bacterial colonies were enumerated following 24 h of incubation and RifR colonies were enumerated following 48 h of incubation, and the frequency of RifR mutants calculated. The inventors typically observed 1.1 (± 0.2) x 10⁹ total CFU and 25 + 4 RifR CFU per 3 thighs at 48 h from cipro treated mice given control bacteria. SOS-defective bacteria showed fewer total CFU at 48 h. DEQ-treated mice had 1.5 (+ 0.3) x 10⁹ total CFU and 4 + 0.3 RifR CFU per 3 thighs. Data are means + SEM of 3 independent experiments of three mice each.

[0096] Statistics

[0097] For comparisons of two groups, a two-tailed Students t-test was used. For comparisons of 3 or more groups, ANOVA with Tukey post-hoc test was used. Statistics were performed using GraphPad PRISM. EXAMPLE 7

DEQUALINIUM CHLORIDE BACTERIOSTATIC AT HIGHER CONCENTRATIONS

[0098] In this disclosure, we identify dequalinium chloride (DEQ) as an inhibitor of cipro- induced mutagenic DNA break repair (MBR), both in culture (FIG. 1G) and in mouse thigh infections (FIG. 4B), and find that DEQ did not inhibit bacterial viability simultaneously (FIG. 3D and FIG. 6). DEQ did not reduce viability when used with cipro at high (therapeutic) cipro dose (FIG. 3D), at minimal antibiotic concentration cipro (MAC, 10% viability) (FIG. 6A), at which cipro-induced mutagenesis is maximal (35), and when used without cipro (FIG. 3D and FIG. 6B). Although the viability of E. coli in DEQ documented here may appear contradictory to its use as a topical bacteriostatic agent, our use of ~1000-times lower DEQ concentration than is tolerated orally in mouse and ~100-times lower DEQ in culture is most likely to underlie the difference. Whereas DEQ is used clinically as a bacteriostatic agent at 2000-4000 pg/ml (or mg/kg) (40, 54), the inventors used 2 p g/ml (or mg/kg) in mouse and 15.8 pg/ml in culture (FIG. 4B and FIG. 7). Moreover, although used topically, DEQ is tolerated orally in mouse at 2000 mg/kg and in rat at 1000 mg/kg (40), implying that the 2 mg/kg DEQ used to reduce mutagenesis in mouse here (FIG. 4B) would be tolerated well.

EXAMPLE 8

USE OF PLATE-READER AND FLOW-CYTOMETRIC SCREENS

[0099] The flow-cytometric secondary screen for validating drugs that inhibit stress responses and their reporters is more sensitive than the primary screen by fluorescence plate reader because flow cytometry measures single cells rather than the bulk cultures assessed with a plate reader. Whereas the plate-reader detects depressed fluorescence per ODeoounit, with the OD used as a proxy for cell biomass, the flow-cytometric screen detects fluorescence per individual cell. This allows detection of changes to fluorescence that occur in a cell subpopulation, as assayed here, and also avoids potential confounders of OD or fluorescence measurements that are independent of the cells (debris, etc). Plate reader was used first for rapid screening, then flow cytometry, for validating reduced fluorescence per cell.

EXAMPLE 9

MUTANT FREQUENCIES

[0100] The baseline mutant frequencies of the control strain in mouse differed little from those observed with a different strain background in culture. In mouse the inventors found 2.2 ± 0.4 xlO 8 mutants per CFU in the control strain with cipro (FIG. 4B, mean ± SEM), compared with 6.3 ± 0.3 xlO'⁸ mutants per CFU in culture here (FIG. 1G, mean ± SEM), and 7.6 ± 0.9 xlO'⁸ mutants per CFU previously (35) (mean ± SEM, 8 independent experiments). These are small apparent differences considering the different strain backgrounds and different growth environment in mouse versus in shaking liquid culture in LBH medium.

EXAMPLE 10

RELEVANCE OF MAC CIPRO TO MOUSE INFECTION MODEL

[0101] Previously published dose-response curves show MAC cipro (10% viability, 8.5 ng/ml) to be most mutagenic, with induction of mutagenesis dropping over 10-times at 2 ng/ml, and by three times at 14 ng/ml (35). Although MAC differs from the therapeutic cipro dose of 1 mg/kg (1 i. g/ml, 0.001% survival, FIG. 3D), without which the mice die before completion of the infection experiments (Methods), stress-induced mutagenesis was nevertheless observed in mouse (FIG. 4B), as evidenced by the stress-response dependence. This apparent contradiction suggests that most of the mutagenesis in mouse, the SOS- and general (o^s) stress- response-dependent mutagenesis, occurs at a time or place at which cipro concentration is lower. This might be as cipro concentration increases at the beginning of dosing, or between or at the end of the two cipro doses (FIG. 4A), or, alternatively, in a mouse tissue compartment that fails to reach the overall 1 pg/ml during the experiment.

EXAMPLE 11

NON-LETHAL INHIBITION OF THE STRINGENT RESPONSE

[0102] Unlike DEQ, antibacterial inhibitors of the stringent response identified previously were analogs of the nucleotide (p)ppGpp, which activates the stringent-response transcriptional program by binding bacterial RNA polymerase (RNAP) (60-62). Unlike DEQ (FIG. 3D, FIG. 6 and Example 7), those drugs reduce viability, and potential effects on mutagenesis were not tested. How DEQ inhibits the stringent response transcriptional program (FIGS. 3A-3C), and mutagenesis (FIG. 1G), without killing cells might result from any of several mechanisms. DEQ is not a (p)ppGpp analog (FIG. 6D), and so might not compete with (p)ppGpp for binding RNAP, which might be lethal. DEQ might bind RNAP at a different site, and prevent stringent activation, or bind a target molecule other than RNAP (RelA, SpoT, DksA or other). Alternatively, DEQ might be nonlethal because of possible failure to bind a different, essential target of (p)ppGpp that the analogs bind. For example, B. subtilis (p)ppGpp binds an essential DNA-replication protein (77) in addition to RNAP. The (p)ppGpp regulation of important processes other than and in addition to transcription might underlie the reduced viability with (p)ppGpp-analog drugs, and not pertain to DEQ.

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TABLES

[0180] Table 1. Escherichia coli strains and plasmids related to figures, and methods

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[0193] Table 2. MAC ciprofloxacin concentrations related to figures

[0194 ^aMAC (minimum antibiotic concentration) is the lowest concentration of antibiotic at which the final cfu are 10% of those observed without drug after growing for 24 hours. MAC cipro concentration (mode) used most often in fluctuation assays to determine mutation rate. The range reflects inherent variability in experiments to achieve the MAC where final cfu with cipro are 10% of those observed without cipro after growing for 24 hours.

* * *

[0195] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of reducing mutagenesis or the risk of mutagenesis in bacteria in an individual, comprising the step of non-topically administering an effective amount of dequalinium chloride (DEQ) to the individual.

2. The method of claim 1, wherein the bacteria are pathogenic.

3. The method of claim 1 or 2, wherein the bacteria are Gram-negative or Gram-positive.

4. The method of any one of claims 1-3, wherein the mutagenesis confers resistance to one or more antibiotics.

5. The method of claim 4, wherein the administering also comprises administering to the individual the one or more antibiotics.

6. The method of claim 4, wherein the individual will be receiving the one or more antibiotics.

7. The method of claim 4 or 6, wherein the individual does not receive the one or more antibiotics when receiving the DEQ.

8. The method of any one of claims 4-7, wherein the antibiotic is a quinolone or a fluoroquinolone.

9. The method of claim 8, wherein the quinolone is Nalidixic acid, Enoxacin, Norfloxacin, Ciprofloxacin, Ofloxacin, Lomefloxacin, Sparfloxacin, Grepafloxacin, Clinafloxacin, Gatifloxacin, Moxifloxacin, Gemifloxacin, Trovafloxacin, Garenoxacin, or a combination thereof.

10. The method of any one of claims 4-9, wherein the antibiotic is Ciprofloxacin.

11. The method of any one of the preceding claims, wherein DEQ is administered orally or intravenously.

12. The method of any one of the preceding claims, wherein DEQ is administered to the individual orally at a dose of about 1-2000 mg/kg or intravenously at a dose of about 0.1- 100 mg/kg.

13. The method of any one of the preceding claims, wherein DEQ is administered once a day, more than once a day, once a week, more than once a week, once a month, or more than once a month.

14. The method of any one of the preceding claims, wherein the bacteria cause pneumonia, tuberculosis, blood poisoning, gonorrhoea, urinary tract infection, or a foodborne disease.

15. The method of any one of the preceding claims, wherein the individual previously misused antibiotics.

16. The method of any one of the preceding claims, wherein the individual is in a medical facility.

17. The method of claim 16, wherein the medical facility is a hospital, nursing home, skilled nursing care facility, or long-term care facility.

18. The method of any one of the preceding claims, wherein the individual is an infant, greater than 65 years old, or is immunocompromised.

19. The method of any one of the preceding claims, wherein the bacteria is Escherichia coli, Methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), multiresistant Gram-negative bacteria, Clostridioides difficile, Mycobacterium tuberculosis, or a mixture thereof.

20. The method of any one of the preceding claims, wherein the individual is a human or an agricultural animal or a companion animal.

21. A method of reducing the risk or delaying the onset of developing antibiotic resistance of bacteria in an individual, comprising the step of:

(a) non-topically administering an effective amount of dequalinium chloride (DEQ) to the individual;

(b) administering one or more antibiotics to the individual.

22. The method of claim 21, wherein (a) and (b) occur at substantially the same time.

23. The method of claim 21, wherein (a) and (b) occur at different times.

24. The method of claim 23, wherein (a) occurs before (b).

25. The method of claim 23, wherein (a) occurs after (b).

26. The method of claim 21 and 22, wherein DEQ and the one or more antibiotics are in the same formulation.

27. The method of claim 21 and 22, wherein DEQ and the one or more antibiotics are in different formulations.

28. A method of facilitating clearance of an infection in an individual, comprising the step of non-topically administering an effective amount of dequalinium chloride (DEQ) to the individual in the absence of also administering one or more antibiotics to the individual.

29. The method of claim 28, wherein DEQ in the individual reduces the rate of pathogen evolution by mutagenesis, thereby enhancing immune clearance by the immune system of the individual.

30. A method of protecting native microflora in an individual in need of one or more antibiotics, comprising the step of non-topically administering an effective amount of dequalinium chloride (DEQ) in an individual receiving no antibiotics.

31. A method of treating an individual for a bacterial infection, comprising the step of non- topically administering to the individual an effective amount of dequalinium chloride (DEQ) whether or not the individual receives one or more antibiotics, thereby reducing mutagenesis in the bacteria while also reducing proliferation of DEQ-resistant mutants.