WO2022153045A1

WO2022153045A1 - Methods and compositions for antibiotic potentiation

Info

Publication number: WO2022153045A1
Application number: PCT/GB2022/050060
Authority: WO
Original assignee: Quadram Institute Bioscience
Current assignee: Quadram Institute Bioscience
Priority date: 2021-01-12
Filing date: 2022-01-12
Publication date: 2022-07-21
Anticipated expiration: 2023-07-12
Also published as: GB202100378D0

Abstract

A method of potentiating or inducing antibiotic efficacy in one or more compositions or compounds, said method including the step of inducing a substantially cell wall deficient (CWD) state in a bacteria by exposure to at least one antibiotic before and/or in combination with said one or more compositions or compounds.

Description

Methods and Compositions for Antibiotic Potentiation

The present invention relates to the provision of one or more substances for the potentiation or improved efficacy of antibiotics and other compounds or compositions.

Although the present invention refers exclusively potentiation using the β-lactam carbapenem antibiotic meropenem the skilled person will appreciate that other β-lactam antibiotics and in particular other antibiotics from the carbapenem family could be used.

The rising incidence of antimicrobial resistance (AMR) globally is one of the greatest threats to human health (Tacconelli et al. 2018) . The emergence of multi-drug resistant (MDR), extremely drug resistant (XDR), and pan-drug resistant (PDR) bacterial pathogens effectively render most if not all antibiotics ineffective. Unfortunately, there are few new antibiotics under development and no new classes of antibiotics with activity against MDR Gram negative bacteria in the pharmaceutical pipeline (Theuretzbacher et al. 2020) .

The majority of nosocomial infections are caused by the ‘ESKAPE’ group of pathogens — Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanni, Pseudomonas aeruginosa and Enterobacter species (Boucher et al. 2009) . Many of the ‘ESKAPE’ pathogens cause serious respiratory infections and are often associated with ventilator acquired pneumonia (VAP) in the critically ill. However, due to the increasing incidence of AMR in respiratory pathogens, this critical resource is rapidly diminishing. Antibiotics are also a critical defence in the treatment of COVID- 19 patients. In the current COVID-19 pandemic bacterial superinfections may be a major contributor to mortality. A study of 191 hospitalised patients in Wuhan, China showed that 95% of these patients received antibiotics, 50% of non-survivors had secondary bacterial infections compared to only 1 of 137 survivors, and VAP occurred in 32% of patients requiring mechanical ventilation (Zhou et al. 2020).

Of particular concern are the MDR Gram negative bacteria with resistance to third generation cepholosporins and carbapenems. Indeed carbapenem resistant A. baumanii, carbapenem resistant P. aeruginosa and ESBL-producing Enterobacteriaceae (including Klebsiella, Escherichia, Serratia and Proteus spp), which cause severe and often deadly blood-stream infections and pneumonia, have been classified by the WHO as Priority 1 (Critical) for urgent research and development of new antibiotics to treat these MDR Gram negative pathogens (Cheng et al. 2019) .

The mechanisms of antibiotic resistance in bacteria include drug inactivation, modification of the target site, increased drug efflux, reduced cell wall permeability, biofilms, and persister cells. The outer-membrane (OM) of Gram negative bacteria poses a significant permeability barrier to many antibiotics which, to enter the cell, must either diffuse across the OM or enter via porins in the OM. Modification of OM permeability through alteration of lipid or porin composition confers elevated antibiotic resistance (Mulani et al. 2019) .

For example, the Gram negative bacterium P. aeruginosa is able to tolerate high concentrations of β-lactam antibiotics, which despite apparently inhibiting the growth of the organism, exhibit remarkably little bactericidal activity (Monahan et al. 2014) . We have recently determined that when treated with supra-MIC levels of β-lactam antibiotics (carbenicillin, meropenem and imipenem), P. aeruginosa undergoes a rapid and reversible en masse transition to viable, translucent cell wall deficient (CWD) state that confers survival to the β-lactam antibiotics (Monahan et al. 2014) .

It is therefore an aim of the present invention to provide compositions that addresses the abovementioned problems.

It is a further aim of the present invention to provide methods that address the abovementioned problems.

It is a yet further aim of the present invention to provide a method of improving the efficacy or activity of antibiotics.

In a first aspect of the invention there is provided a method of potentiating or inducing antibiotic efficacy in one or more compositions or compounds, said method including the step of inducing a substantially cell wall deficient (CWD) state in a bacteria by exposure to at least one antibiotic before and/or in combination with said one or more compositions or compounds.

Typically the one or more compositions or compounds are antibiotics and/or antimicrobial compounds.

Typically the bacteria is a Gram negative species.

Preferably the one or more antibiotics that induce the CWD state are β (beta) -lactam antibiotics. Typically the β-lactam antibiotic is from the carbapenem family. Further typically theβ-lactam antibiotic includes meropenem.

In one embodiment the β-lactam antibiotic is delivered in supra- minimum inhibitory concentration (MIC) levels.

In one embodiment the compounds or compositions include antibiotics or antimicrobial compounds. Typically the antibiotics are from the fluoroquinolone, aminoglycoside, tetracycline, polymixin, clofoctol, macrolide, pleuromutilin, quinolone, amphenicol, rifamycin, fosfomycin tromethamine and/or nitroxoline classes or families of compounds.

Typically the compositions or compounds include any one or any combination of the antibiotics listed in Table 1 below.

In one embodiment the compounds or compositions include antibiotics or antimicrobial compounds, typically the antibiotics are from the nitrofuran, penicillin, glycopeptide, fluoroquinolone, aminocyclitol, fusidate sodium, puromycin hydrochloride, lincosamide, cephalosporin, aminocoumarin, trimethoprim, daptomycin, sulfonamide, carbapenem, clofazimine, capreomycin sulfate, mupirocin, oxazolidinone, nitroimidazole, acetohydroxamic acid, dapsone, sodium sulfadiazine, methenamine, sodium 4-aminosalicylate, bacitracin and/or monobactam classes or families of compounds.

Typically the compositions or compounds include any one or any combination of the antibiotics listed in Table 2 below.

Table 2

In one embodiment the compositions or compounds include drugs or approved medications that are not formally recognised as antibiotic or antimicrobial compounds for human or animal use.

Typically the compositions or compounds include any one or any combination from Table 3 below. Table 3

In one embodiment the method is used to treat infection by the ‘ESKAPE’ group of pathogens. The ESKAPE group includes — Enterococcus f aecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanni, Pseudomonas aeruginosa and Enterobacter species

In one embodiment the infection treated is a P. aeruginosa infection. In a second aspect of the invention there is provided β-lactam antibiotic for use in the potentiation of one or more compounds or compositions to treat microbial infections.

Typically the β-lactam antibiotic is provided in a supra- minimum inhibitory concentration (MIC) level. Further typically the β-lactam is a carbapenem.

In a preferred embodiment the β-lactam antibiotic is delivered in a pharmaceutically acceptable carrier.

Further preferable the β-lactam antibiotic is delivered in a concentration to induce the target bacteria to transition to a CWD state.

In one embodiment the CWD state was induced with substantially at least 1 μg/ml meropenem.

In one embodiment 5 μg/ml meropenem (5x MIC) was used to achieve CWD state in the bacteria.

Specific embodiment of the invention are now described.

Materials and Methods

Strains, media and growth conditions.

The meropenem sensitive P. aeruginosa strain PA14 was used to identify antibiotics and novel compounds with bactericidal activity against CWD P. aeruginosa. Broth microdilution was used to determine the MIC of meropenem for this strain and was found to be 1 μg/ml. The concentration of meropenem used to induce P. aeruginosa PA14 to transition to the CWD state was 5 μg/ ml meropenem (5x MIC) .

All bacteria were revived from frozen stocks onto low-salt Lysogeny Broth (LB) Agar (5 g/L yeast extract, 5 g/L sodium chloride, 10 g/L tryptone, 15 g/L bacteriological agar; all Oxoid, Cheshire, UK) and grown at 37 °C overnight. All broth cultures were performed in cation adjusted Mueller Hinton II Broth (CaMHB), prepared as per the manufacturer’s instructions (BD BBL™, Franklin Lakes, NJ) and grown well aerated at 37 °C, shaking at 250 r.p.m overnight.

Screen of FDA-Approved Small Chemical Compound library for CWD P. aeruginosa killing activity.

The Sellekchem FDA Approved Small Chemical Compound Library (Sellekchem, Houstan, TX) was supplied at a concentration of 10 mM dissolved in either DMSO or water and stored at -80 °C and thawed prior to use. To induce the CWD state, a log phase culture was prepared from an overnight broth of the strain diluted 1 : 500 in CaMHB supplemented with 0.5 M sucrose to an OD600nm and grown well-aerated at 37 °C shaking at 250 r.p.m. for 2 hr. 175 pl of the log-phase culture was added to the wells of a black-walled flat-bottom p-clear 96 well plate (Greiner Bio-One, Kremunster, Austria) and meropenem trihydrate (Sigma-Aldrich, St. Louis, MO), compound and resazurin disodium salt (Sigma-Aldrich) were added so that the final concentrations were 5 μg/ml, 250 pM and 0.015 mg/ml respectively. A second screen was completed with the meropenem omitted to determine the effect of the compounds in the absence of meropenem. Each plate had duplicate wells with 2.5% DMSO; 5 μg/ml meropenem and 2.5% DMSO; and, 5 μg/ml meropenem, 2.5% DMSO and 64 μg/ml of the antimicrobial peptide nisin (Sigma-Aldrich) was included. Nisin was included as a control for CWD bactericidal activity we have previously shown that this antimicrobial peptide is bactericidal only to CWD P. aeruginosa (Monahan et al. 2014) . The plates were shaken briefly, and then incubated statically at 37 °C for 16 hr before the fluorescence was measured at 530/590 ex/em on an Infinite 200 Pro plate reader (TeCan, Mannedorf, Switzerland) .

Determination of the Minimum Metabolic Activity Concentration (MMAC)

In black-walled flat-bottom p-clear 96 well plate the compounds selected for MMAC titration were serially diluted in quadruplicate in CaMHB with 0.5 M sucrose and the final volume corrected to 100 pl/well. Meropenem (Sigma) was added to the culture and 100 pl added per well to a final concentration of 5 μg/ml meropenem and a compound concentration of 125 pM to 0.49 pM. For compounds that had been identified from the screen as inhibitory in the absence of meropenem, the procedure was repeated without meropenem. All assays included control wells containing 5 μg/ml meropenem with vehicle (2.5% DMSO or water) to enable comparison to the metabolic activity of CWD P. aeruginosa cells. A CWD bactericidal control of 5 μg/ml meropenem and 64 μg/ml nisin was also included. The plates were sealed with semi-permeable membrane and grown without shaking for 20 hr after which resazurin was added to a final concentration of 0.145 μg/well and the plate was incubated for a further 4 hr without shaking. The fluorescence was read at 530/590 ex/em on a fluorescent plate reader. The values were corrected for the blank and then normalised to the appropriate no-meropenem vehicle control. The minimum metabolic inhibitory concentration was the concentration at which a normalised value of <2% was achieved.

Results

Meropenem potentiates activity of other antibiotics against P. aeruginosa

Analysing the effectiveness of antibiotics on CWD bacteria presents many unique challenges. As CWD cells are translucent optical density measurements are not suitable to assess the effects of antibiotics on growth. Supra-MIC levels of meropenem have also been used to induce CWD cells in P. aeruginosa and therefore identifying the MIC of antibiotics on the carbapenem induced CWD cells is impossible. Traditional mechanisms of quantifying the minimal bactericidal concentration (MBC) of compounds remains difficult due to the inherent fragility of CWD cells when plated onto solid media.

To determine if inducing P. aeruginosa to transition to the CWD state potentiates the bactericidal activity of antibiotics, we utilised a resazurin-based assay. Resazurin is a non-toxic, weakly fluorescent compound that is reduced by cellular dehydrogenases to the highly fluorescent compound rezarufin and is used to assess metabolic activity as a measure of cell viability ((Sarker et al. 2007), Methods). 146 unique antibiotics from an FDA- Approved Small Chemical Compound library were screened at 250 mM to identify those that showed inhibition of metabolic activity of P. aeruginosa strain PA14 in the presence of 5 μg/ ml meropenem (5x MIC) . Of these, 46 antibiotics inhibited metabolic activity by at least 50% when normalised to the meropenem-only control (Supplementary Table 1) .

We next assessed if supra-MIC meropenem potentiated the bactericidal activity of these antibiotics. The minimum concentration of the antibiotic that reduced metabolic activity by at least 98% compared to vehicle control was determined by broth microdilution assays with resazurin to assess metabolic activities in the presence and absence of meropenem. Whilst this minimum metabolic activity concentration (MMAC) is likely to be equivalent to the minimum bactericidal concentration (MBC), for clarity we will refer to MMAC as we did not assess viability via the standard agar plate CFU counting method. Broth microdilution assays (2-fold dilution series from 125 mM - 0.49 mM) were performed to determine the MMACs in the absence (MMACo) and presence of 5 μg/ml meropenem (MMACMER) . The potentiation coefficient (MMACO/MMACMER) was calculated for each antibiotic (Table 1) . A potentiation coefficient of 2 or greater indicates that the antibiotic reached our metabolic activity inhibition threshold at a lower concentration in the presence of meropenem than when used without meropenem.

Of the 46 antibiotics assayed for determination of minimal metabolic activity concentrations (MMAC), 13 did not show potentiation with meropenem in this assay (Table 1) . Of these, 7 did not reach 98% inhibition of metabolic activity in the presence or absence of meropenem at the highest concentration tested (125 mM). Sitofloxacin, was extremely potent even at the at the lowest concentration tested (0.49 mM) showing at least 98% inhibition of metabolic activity in both the presence and absence of meropenem. Three antibiotics, for which the MMACMER and the MMACo could be determined, had no change.

Potentiation by supra-MIC meropenem was observed with 35 of the antibiotics (Table 1) . Both the MMACMER and the MMACo could be determined for 13 of the antibiotics with potentiation co-efficient with these ranging from 2-32 fold. The MMACo but not the MMACMER could be determined for 11 antibiotics indicating potentiation by meropenem to below the limit of our assay (0.49 mM) . Interestingly, the MMACMER but not the MMACo could be determined for another 11 antibiotics as these had not reached 98% inhibition of metabolic activity in the absence of meropenem at the highest concentration tested (125 mM) indicating that in the absence of meropenem these antibiotics had little antibiotic activity against P. aeruginosa strain PA14 but that in the presence of supra-MIC these antibiotics were bactericidal. Therefore, from this screen of 146 antibiotics we have identified 35 antibiotics that show potentiation by inducing transition to the CWD state with supra-MIC levels of meropenem. These 35 antibiotics were comprised of 13 fluoroquinolones, 10 aminoglycosides, 5 tetracyclines, 3 macrolides, 1 polymyxin, 1 pleuromutilin (valnemulin) and clofoctol (Table 1) .

Identification of novel bactericidal FDA-approved drugs

Our observations indicate that converting P. aeruginosa to CWD state with supra-MIC meropenem potentiates the activity of many antibiotics, including those to which P. aeruginosa usually has little to no susceptibility. We explored the possibility that there may be other compounds that are not usually recognised as antibacterial, but which may have some bactericidal activity against CWD P. aeruginosa in combination with meropenem. We used the resazurin metabolic activity assay to screen a library of 1443 FDA-Approved Small Chemical Compounds to identify compounds that showed potential bactericidal activity against CWD P. aeruginosa induced by 5 μg/ml meropenem (Supplementary Table 2) . The results were normalised to the 2.5% DMSO vehicle control and compounds which interfered with the fluorescence readout were omitted from further analysis.

We identified 41 FDA-approved drugs that are not recognised as antibiotics (Table 2) that at 250 μ M inhibited P. aeruginosa PA14 metabolic activity to the same extent or greater than our bactericidal control (64 μg/ml nisin) in the presence of 5 μg/ml meropenem but had no inhibition in the absence of meropenem (Supplementary Table 2). We next determined the minimum concentration of these compounds that reduced metabolic activity by at least 98% compared to vehicle control by broth microdilution assays with resazurin to assess metabolic activities in the presence and absence of meropenem. For 34 of these compounds we could determine a MMACMER (minimum concentration for 98% inhibition of metabolic activity relative to untreated control) . The remaining 7 compounds that showed some activity at 250mM in the primary screen did not meet the 98% inhibition cut-off necessary to determine a MMACMER even at the highest concentration assayed (125 mM) in this more sensitive titration.

Discussion

The paucity of antibiotics against Gram-negative pathogens and the lack of any suitable candidates in the therapeutic pipeline has led to the need to investigate already developed compounds for novel uses. Previously we have shown that a combination of supra-MIC concentrations of meropenem has resulted in an en masse conversion of P. aeruginosa to a CWD state which is sensitive to antimicrobial peptides that are ineffective against bacillary P. aeruginosa (Monahan et al. 2014) .

In this study we assessed if inducing P. aeruginosa to transition to the CWD state with supra-MIC levels of meropenem potentiated the bactericidal activity of known antibiotics. We identified 35 antibiotics that were potentiated by supra-MIC levels of meropenem. Interestingly, the bactericidal activity of 11 of these antibiotics occurred only in the presence of meropenem which suggests that inducing P. aeruginosa to transition to the CWD state sensitises it to antibiotics to which it is not normally susceptible. These antibiotics were: flumequine; streptomycin sulphate; azithromycin dihydrate; demeclocycline hydrochloride; paromomycin sulphate; clarithromycin; hygromycin B; minocycline hydrochloride;erythromycin ethylsuccinate, clofoctal; and valnemulin (Table 1).

Macrolides have shown synergistic effects in combination with outer membrane disrupting agents (Buyck et al. 2012) and this is supported by the evidence that the three macrolides were present in our potentiate screen. When used for P. aeruginosa treatment, azithromycin seems to have a greater inhibitory effect than clarithromycin, which in turn has a greater effect than erythromycin (Howe and Spencer 1997) and those findings are in line with our findings which show the macrolides have a MMAC with meropenem of 31.25, 62.5 and 125 respectively (Table 1) which is below the peak serum concentrations from an oral dosing regimen (Foulds et al. 1990; Fraschini et al. 1993; Shanson et al. 1984). Two other macrolides, spiramycin and roxithromycin, had no potentiation when investigated by serial dilution (Table 1). Macrolides are bacteriostatic antibiotics that target protein synthesis and include erythromycin, clarithromycin and azithromycin. They are routinely used in the treatment of P. aeruginosa infections despite P. aeruginosa having intrinsic resistance mechanisms to the macrolide antibiotics (Buyck et al. 2012) . This could be due to macrolide alteration of quorum sensing, virulence factor expression, biofilm formation, serum sensitivity and outer membrane integrity (Gillis and Iglewski 2004; Tateda et al. 1996; Tateda et al. 2001 ; Tateda et al. 1993; Favre-Bonte et al. 2003; Imamura et al. 2005; Molinari et al. 1993)or via inhibition or alteration of host inflammatory processes (Tsai et al. 2004; Cube et al. 2002; Shinkai et al. 2006; Yamasawa et al. 2004; Parnham et al. 2005; Cigana et al. 2006; Fan et al. 2017). Azithromycin, but not clarithromycin, has shown positive effects in trials of chronically infected CF patients ((Saiman et al. 2003; Robinson et al. 2012; Cai et al. 1998) .

Clofoctol is synthetic phenol derivative that is used predominately in France and Italy for the treatment of upper respiratory tract infections by Gram-positive organisms (Danesi and Del Tacca 1985)The mechanism of action against targeted organisms appears to be through a disruption of the permeability of the inner cell membrane leading to collapse of the proton gradient and a decrease in the intracellular pool of ATP required for peptidoglycan synthesis and other enzymatic processes (Yablonsky 1983). The MIC range of clofoctol for susceptible organisms is between 0.3-10 pg/ml (Alessandri et al. 1986; Scaglione et al. 2018). Our data shows that at a clinically relevant serum concentration of meropenem (5x the MIC), clofoctal has an MMAC of 15.63 pM (Table 1) or 5.7 pg/ml which is comparable to other organisms and a concentration therapeutically relevant.

Recently a study of FDA-approved small chemical compound library found that clofoctol inhibited PQS-mediated virulence mechanisms in P. aeruginosa, most likely through inhibiting binding of the cognate molecule to the PqsR protein (D’Angelo 2018) . Interestingly, this study found that a similar mechanism of action occurred with the anti-mycotics clotrimazole and micanozole. Clotrimazole was also identified as a hit in our screen with an MMAC of 31.25 mM (Table 2) . Micanazole was also weakly active in our screen but did not meet the criteria for further testing (Supplementary Table 2). Tioconazole, another antimycotic imadazole derivative was identified in our screen with an MMACMER of 31.25 mM (Table 2). Both anti-mycotics are poorly absorbed from the gut and oral administration of clotrimazole resulted in serum levels less than 0.5 μM (Brugnara et al. 1996) . However, advances in delivery technologies of hydrophobic anti-mycotics to the lung has resulted in concentrations that could prove effective in the future (McConville et al. 2006) .

Valnemulin belongs to the pleuromutilin class of antibiotics which have been used in veterinary applications since 1979 with valnemulin being approved for veterinary use in 1999 (Paukner and Riedl 2017). Pleuromutilins work by inhibiting bacterial protein synthesis through binding to the ribosome and are reported to have activity against Gram positive bacteria and Mycoplasmas but no reported activity against P. aeruginosa (Poulsen et al. 2001). In a study of three patients with primary antibody deficiency who had developed a Mycoplasma sepsis, valnemulin was shown to be effective (Heilmann et al. 2001) . .Mycoplasmas are unusual bacteria in that they have no apparent cell wall, so share some characteristics with CWD variants. Valnemulin has also been found to down-regulate inflammatory responses, suggesting that in vivo benefits may also be due to ameliorating inflammatory damage (Zhang et al. 2009) . Reptamulin was the first pleuromutilin approved for human use but due to unfavourable pharmacochemical properties it is only used topically and, in our assay, had no effect on P. aeruginosa (Supplementary Table 2) . Our observations suggest that pleuromutilin antibiotics may be effective for the treatment of P. aeruginosa infections when used in combination with a CWD inducing antibiotic such as meropenem.

Of the 42 antibiotics selected for further testing which showed potentiation in the presence of meropenem, 13 were aminoglycosides (Table 1) . Aminoglycosides, particularly amikacin, tobramycin, and gentamicin, are one of the most frequently used classes of antibiotic in the treatment of P. aeruginosa and, due to this, resistance is frequently encountered ((Poole 2005). Aminoglycosides act by irreversibly binding to the ribosome of bacteria. The combinatorial effects of β-lactam antibiotics and aminoglycosides have been well established in vitro (Weiss and Lapointe 1995; Nakamura et al. 2000; Tam et al. 2004) so their prevalence in our screen is unsurprising. However, in vitro investigations have failed to translate to better clinical outcomes for patients despite their frequent use together (Paul et al. 2004; Aaron et al. 2005; Zobell et al. 2011) . A study investigating the development of resistance in P. aeruginosa isolates treated with meropenem and tobramycin, either individually or in combination, described a reduction in the frequency of resistance from treatment with the combination(Tam et al. 2005) . Most studies only consider the MIC, not the bactericidal concentration of antibiotics. By definition such MIC concentrations would result in a pool of viable bacteria that could develop resistance. Our study examined the metabolic activity as a measure of viability and as such, the increased bactericidal effects of meropenem with an aminoglycoside could explain the observed decrease in resistance.

In this study we have identified 41 FDA-approved drugs that are not recognised as antibiotics that show bactericidal activity in the presence of a supra-MIC concentration of meropenem. Antineoplastic compounds constituted 12 of these compounds that showed bactericidal activity in the presence of supra-MIC meropenem. Clotrimazole and tioconazole, while not used as antineoplastics, have also been shown to have anticancer effects (Benzaquen, 1995) (Liu, 2018). 8 compounds have uses as antimicrobial agents, either as antimycotics (clotrimazole, tioconazole, and ketoconazole), antivirals (efavirenz, lomibuvir, elvitegravir), antiprotozoals (diclurazil) or biocides (triclosan) . 6 compounds exhibit effects on the central nervous system through interaction with the serotonin and dopamine receptors and have been used as antipsychotics (thioridazine, pimavanserin, mesoridazine besylate, and trifluoperazine hydrochloride), antidepressants (sertraline hydrochloride), or a treatment for Alzheimer’s disease (tacrine hydrochloride). 4 compounds were antihypertensives. It is important to note that in this study we have only demonstrated bactericidal activity for these compounds in combination with meropenem in in vitro assays,. However, these observations suggest that these compounds may have immediate clinical utility as antibiotics when used in combination with b-lactam antibiotics or at least may be novel chemical entities that could be further developed as antibacterial agents. Table 1 extendec

Supplementary Table 1

Clarithromycin Macrolide No 5.20

Minocycline hydrochloride Tetracycline No 5.20

Dihydro s treptomycin sulfate Aminoglyco side No 5.41

Gentamicin sulfate Aminoglyco side Yes 5.41

Netilmicin sulfate Aminoglyco side Yes 5.62

Flumequine Fluoroquinolone No 6.04

Amikacin disulfate Aminoglyco side Yes 6.47

Sitafloxacin hydrate Fluoroquinolone No 7.04

Levo flox acin Fluoroquinolone Yes 7.07

Azithromycin Macrolide No 7.08

Amikacin hydrate Aminoglyco side Yes 7.69

Azithromycin dihydrate Macrolide No 8.1 1

Norfloxacin Fluoroquinolone No 8.18

Sarafloxacin hydrochloride Quinolone No 8.41

Levo flox acin hydrate Fluoroquinolone Yes 8.46

Enro floxacin Fluoroquinolone No 9.06

Pefloxacin mesylate Quinolone No 9.75

Gatifloxacin Fluoroquinolone No 9.79

Moxifloxacin hydrochloride Fluoroquinolone No 9.85

Polymyxin B sulphate Polymixin Yes 10.04

Marbo floxacin Fluoroquinolone No 10.05

Difloxacin hydrochloride Fluoroquinolone No 10.65

Rifabutin Rifamycin No 10.72

D ano floxacin me sylate Fluoroquinolone No 10.78

Tobramycin Aminoglyco side Yes 10.82

Enoxacin Fluoroquinolone No 11.19

Ofloxacin Fluoroquinolone No 11.39

Neomycin sulfate Aminoglyco side No 11.88

Chlortetracycline hydrochloride Tetracycline No 12.64

Erythromycin Macrolide No 12.82

Sparfloxacin Fluoroquinolone No 13.45

Tylo sin Tartrate Macrolide No 13.53

N adifloxacin Fluoroquinolone No 15.07

Oxytetracyclme dihydrate Tetracycline No 15.82

Thi amp hem col Amphenicol No 15.85

Roxithromycin Macrolide No 16.31

Erythromycin ethyl succinate Macrolide No 17.30

Tetracycline hydrochloride Tetracycline No 18.14

Nitroxoline Ungrouped No 18.19

Doxycycline hydrochloride Tetracycline No 20.07

Valnemulin hydrochloride Pleuromutilin No 20.24

B alo floxacin Fluoroquinolone No 21.95

Clofoctol Ungrouped No 22.88

Florfenicol Amphenicol No 26.40

Supplementary Table 2

Sulis obenzone 94.29 100.19

Pimecrolimus 94.30 102.45

Ethynodiol diacetate 94.31 100.43

Phenindione 94.31 98.61

Eslicarbazepine acetate 94.32 100.99

Perampanel 94.37 97.45

Allopurinol 94.37 101.34

Choline chloride 94.40 102.95

Gallic acid 94.40 102.15

Mubritimb 94.42 100.34

Epinephrine hydrochloride 94.42 88.26

Phenoxybenzamme hydrochloride 94.45 100.42

Voglibose 94.47 101.39

Tranylcypromine hydrochloride 94.50 101.99

Triacetin 94.53 103.35

Arbinoxamine maleate 94.53 101.92

Halothane 94.56 103.09

Orlistat 94.58 102.06

Pemirolast potas sium 94.59 101.26

Emtricitabine 94.62 101.46

Epinephrine bitartrate 94.65 94.40

Povidone iodine 94.65 100.40

Procaine 94.70 98.75

Chloroambucil 94.80 100.45

Alcaftadine 94.80 100.59

Clofibric acid 94.82 101.37

Pemetrexed 94.90 98.61

Roflumilast 94.90 94.11

Pralmorelin 94.91 98.03

Menadione 94.93 95.48

Digoxin 94.98 101.71

Cobimetimb 95.00 99.69

Ibandronate s odium 95.02 48.74

Levobupivacame hydrochloride 95.08 100.80

Eptifibatide acetate 95.13 99.10

Aloxistatin 95.14 102.35

Bemegride 95.16 102.80

Hydroquinone 95.16 100.45

Oxiracetam 95.20 102.17

Alvelestat 95.21 101.10

Rocilinostat 95.24 100.19

Doxylamine succinate 95.26 101.60

Salmon calcitonin acetate 95.30 102.32

Sildenafil 95.31 100.62

Claims

1. A method of potentiating or inducing antibiotic efficacy in one or more compositions or compounds, said method including the step of inducing a substantially cell wall deficient (CWD) state in a bacteria by exposure to at least one antibiotic before and/or in combination with said one or more compositions or compounds.

2. A method according to claim 1 wherein the one or more compositions or compounds are antibiotics and/or antimicrobial compounds.

3. A method according to claim 2 wherein the bacteria is a Gram negative species.

4. A method according to claims 1 -3 wherein the one or more antibiotics that induce the CWD state are β (beta)-lactam antibiotics.

5. A method according to claim 4 wherein the β-lactam antibiotic is from the carbapenem family.

6. A method according to claim 1 wherein the β-lactam antibiotic includes meropenem.

7. A method according to claim 6 wherein the β-lactam antibiotic is delivered in supra-minimum inhibitory concentration (MIC) levels.

8. A method according to claim 2 wherein the compounds or compositions include antibiotics or antimicrobial compounds from any one or any combination of fluoroquinolone, aminoglycoside, tetracycline, polymixin, clofoctol, macrolide, pleuromutilin, quinolone, amphenicol, rifamycin, fosfomycin tromethamine and/or nitroxoline classes or families of compounds.

9. A method according to claim 2 wherein the compounds or compositions include any one or any combination of antibiotics or antimicrobial compounds from the nitrofuran, penicillin, glycopeptide, fluoroquinolone, aminocyclitol, fusidate sodium, puromycin hydrochloride, lincosamide, cephalosporin, aminocoumarin, trimethoprim, daptomycin, sulfonamide, carbapenem, clofazimine, capreomycin sulfate, mupirocin, oxazolidinone, nitroimidazole, acetohydroxamic acid, dapsone, sodium sulfadiazine, methenamine, sodium 4-aminosalicylate, bacitracin and/or monobactam classes or families of compounds.

10. A method according to any preceding claim wherein the method is used to treat infection by Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanni, Pseudomonas aeruginosa and Enterobacter species (the ‘ESKAPE’ group of pathogens).

11. A β-lactam antibiotic for use in the potentiation of one or more compounds or compositions to treat microbial infections.

12. A β-lactam antibiotic according to claim 11 wherein the β - lactam antibiotic is provided in a supra- minimum inhibitory concentration (MIC) level.

13. A β-lactam antibiotic according to claim 12 wherein typically the β-lactam is a carbapenem.

14. A β-lactam antibiotic according to claim 11 wherein β - lactam antibiotic is delivered in a pharmaceutically acceptable carrier.

15. A β-lactam antibiotic according to claim 14 wherein the β - lactam antibiotic is delivered in a concentration to induce the target bacteria to transition to a CWD state.

16. A β-lactam antibiotic according to claim 15 wherein the CWD state was induced with substantially at least 1 μg/ml meropenem.

17. A β-lactam antibiotic according to claim 16 wherein 5 μg/ml meropenem (5x MIC) was used to achieve CWD state in the bacteria.