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ASM/ESCMID 2019

Apidaecins: Linear Peptide Antibiotics for the Treatment of Serious Gram‐Negative Infections

D. Knappe, A. Gallinat, L. Garoff, D. Hughes, R. Hoffmann, D. Joseph-McCarthy

ABSTRACT | Background: Apidaecins are linear peptide antibiotics (LPAs) with a novel mechanism of action that are aimed at the treatment of serious, drug‐resistant Gram‐negative infections. Apidaecin 1b (GNNRPVYIPQPRPPHPRL‐OH), isolated from Apis melifera (honey bee), is a linear, proline‐rich antimicrobial peptide. Unlike most other classes of antimicrobial peptides (AMPs), apidaecins selectively enter bacterial cellswithout membrane disruption. As a result, they exhibit no mammalian cytotoxicity or hemolytic activity. Apidaecins exert their antibacterial activity through inhibition of protein translation, by binding to the exit tunnel of existing 70S bacterial ribosome which blocks the dissociation of release factors and by preventing the assembly of additional bacterial ribosome.  Methods: MICs were determined under standard CLSI methodology in cation‐adjusted Mueller Hinton Broth. Proteolytic stability was evaluated in plasma, serum, and bronchoalveolar lavage of several mammalian species.  Results: MICs across a set of Gram‐negative strains including MDR, efflux‐defective, and colistin‐resistant strains are quite promising; e.g., MICs in 25% diluted medium for Api137 were ≤0.125 to 8 μg/ml vs. Eco (n=8) and ≤0.125 to 0.5 μg/ml vs. Kpn (n=6). In the limited studies undertaken we have seen correlation between MICs in 25% diluted medium and in vivo efficacy. The frequency of resistance at 4x MIC for Api137 vs. Eco was similar to that for colistin. In addition, the effect of serum on the MICs is minimal, there is only a small MIC shift in an SmbA knockout Eco strain, and there is no evidence of cross‐resistance with strains resistant to ribosome‐targeting antibiotics. Finally, the proteolytic stability was higher overall in non‐rodent matrices.  Conclusions: Taken together with previously reported encouraging in vivo results (in sepsis, thigh infection, and urinary tract infection (UTI) models), this further in vitro characterization of apidaecins supports their continued development as therapeutics, initially for complicated UTIs with a view toward expansion into complicated intra‐abdominal infections and ventilated‐pneumonias.

ASM Microbe 2018

EBX-001:  A Combination of Tobramycin with an Anti-Bacterial Potentiator for the Treatment of Chronic Pseudomonas aeruginosa Infections

M. Koeva, A. D. Gutu, W. Hebert, J.D. Wager, L.M. Yonker, G. A. O’Toole, F. M. Ausubel, S.M. Moskowitz, D. Joseph-McCarthy

ABSTRACT |Background:  Bacteria can enter into a persister state in response to various stresses including antibiotic treatment.  In this metabolically dormant state, bacteria become tolerant or unresponsive to antibiotics, which can lead to chronic, recurrent infections.  A combination of the aminoglycoside tobramycin with fumarate as a potentiator significantly enhances the killing of P. aeruginosa persisters.  Methods:  A planktonic stationary phase time kill potentiation assay was utilized to assess tobramycin-potentiator combinations with respect to in vitro activity by varying the concentration of tobramycin and potentiator.  Ninety-six-well dish biofilm and colony biofilm assays were performed.  An LDH assay was utilized to determine the cytotoxicity of the tobramycin-fumarate combination.  Results:  Enhanced killing of up to 6 orders of magnitude of P. aeruginosa persisters over a range of CF isolates including mucoid and non-mucoid strains was observed for the tobramycin-fumarate combination compared to tobramycin alone.  In addition, significant fumarate-mediated potentiation was seen across a wide range of pH values, as well as in the presence of azithromycin or CF patient sputum.  Fumarate alone had no effect.  Fumarate also reduced the cytotoxicity of tobramycin treated P. aeruginosa to human epithelial airway cells.  Finally, in mucoid and non-mucoid CF isolates, complete eradication of P. aeruginosa biofilm was observed in a naïve colony biofilm assay due to fumarate potentiation (see Figure).  Conclusions:  These data suggest that the combination of tobramycin with fumarate may be an attractive therapeutic for eliminating recurrent P. aeruginosa infections through the eradication of bacterial persisters.  Eliminating bacterial persisters early may be a key to limiting further resistance and prolonging the life time of clinically important anti-infective agents.

ASM Microbe 2018

A Novel Combination of Tobramycin with a Potentiator for the Treatment of Chronic Pseudomonas aeruginosa Infections

M. Koeva, J.D. Wager, D. Joseph-McCarthy

ABSTRACT |Background:  Bacterial persisters are a quasi-dormant sub-population of cells that are tolerant to antibiotic treatment.  Combination of the aminoglycoside amikacin with bacterial metabolites as potentiators were investigated as therapeutics that reduce recurrent infections such as catheter-associated urinary tract infections (CAUTIs) and persistent lung infections.  Methods:  Pairwise antibiotic and potentiator combinations were screened using the time-kill method (CLSI M26), varying the concentration of amikacin and potentiator.  Cells were enumerated after 4 hr of exposure to the amikacin/potentiator combinations.  Testing involved evaluating combinations vs. a panel of E. coli UTI isolates (13) including extended spectrum beta-lactamase (ESBL)-producing E. coli, K. pneumoniae, E. cloacae, and P. mirabilis UTI isolates, and P. aeruginosa and K. pneumoniae (ESBL and CRE) reference strains. Similarly, the potentiation effect was examined in other species relevant to HAP/VAP and in mycobacteria species.  Results: Enhanced killing of over five orders of magnitude of E. coli persisters was seen for amikacin plus pyruvate (see Figure below) or mannitol as examples.  Essentially no killing of persister cells occurred upon exposure to as high as 128 mg/mL of amikacin alone.  The highest concentration of potentiator tested (40 mM for pyruvate) also did not kill cells when tested alone.  Significant potentiation was also observed in P. aeruginosa, K. pneumoniae, and P. mirablis for specific combinations.  Preliminary data suggest that the killing of mycobacterium persisters may also be potentiated.  Conclusions:  The combination of amikacin with a metabolite potentiator has the capacity to revitalize existing and novel aminoglycosides through eradication of bacterial persisters, with broad spectrum potential.

2018 Dartmouth Cystic Fibrosis Retreat

A Novel Combination of Tobramycin with a Potentiator for the Treatment of Chronic Pseudomonas aeruginosa Infections

M. Koeva, A. D. Gutu, W. Hebert, J.D. Wager, L.M. Yonker, G. A. O’Toole, F. M. Ausubel, S.M. Moskowitz, D. Joseph-McCarthy

ABSTRACT | Bacterial persisters are a quasi-dormant subpopulation of cells that are tolerant to antibiotic treatment.  A combination of the aminoglycoside tobramycin with fumarate as an antibacterial potentiator utilizes an anti-persister strategy aimed at reducing recurrent Pseudomonas aeruginosa infections by enhancing the killing of persisters.  P. aeruginosa cultures in planktonic stationary phase were used to select for persisters.  A range of tobramycin concentrations was tested with a range of metabolite concentrations to determine the potentiation effect of the metabolite under a variety of conditions.  In addition, 96-well dish biofilm and colony biofilm assays were performed, and the cytotoxicity of the tobramycin-fumarate combination was determined utilizing an LDH assay.  Enhanced killing of up to 6 orders of magnitude of P. aeruginosa persisters over a range of CF isolates including mucoid and non-mucoid strains was observed for the tobramycin-fumarate combination compared to tobramycin alone.  Significant fumarate-mediated potentiation was seen in the presence of azithromycin or CF patient sputum.  Fumarate also reduced the cytotoxicity of tobramycin treated P. aeruginosa to human epithelial airway cells.  Finally, in mucoid and non-mucoid CF isolates, complete eradication of P. aeruginosa biofilm was observed in a colony biofilm assay due to fumarate potentiation.  These data suggest that the combination of tobramycin with fumarate may be an attractive therapeutic for eliminating recurrent P. aeruginosa infections through the eradication of bacterial persisters.

ASM Microbe 2016

A Novel Combination of Tobramycin with a Potentiator for the Treatment of Chronic Pseudomonas aeruginosa Infections

M. Koeva, A. D. Gutu, F. M. Ausubel, D. Joseph-McCarthy

ABSTRACT | Background:  EBX-001, a combination of tobramycin with a bacterial metabolite as a potentiator, is being developed for the treatment of chronic P. aeruginosa infections in Cystic Fibrosis patients.  The combination utilizes an anti-persisters strategy and is aimed at reducing recurrent infections.  Methods:  P. aeruginosa cultures in the planktonic stationary phase (PSP) were used to select for bacterial persisters, bacteria in a quasi-dormant state.  In these PSP experiments, a range of tobramycin concentrations was tested with a range of metabolite concentrations to determine the potentiation effect of the metabolite under a variety of conditions.  MICs were also determined for a variety of CF clinical isolates to select a diverse set of strains for inclusion in the study. Results:  Enhanced killing of up to 6 orders of magnitude of P. aeruginosa persisters for a range of strains was observed (see example in the figure below).  Conclusions:  A combination of tobramycin with a potentiator remains an attractive therapeutic option for eliminating recurrent P. aeruginosa infections through the eradication of bacterial persisters.

ASM Microbe 2016

Aminoglycoside Potentiation for the Treatment of Catheter-Associated Urinary Tract Infections

M. Koeva, D. Sweeney, D. L. Shinabarger, D. Joseph-McCarthy

ABSTRACT | Background: Bacterial persisters are a quasi-dormant sub-population of cells that are tolerant to antibiotic treatment. EBX-002 is a combination of the aminoglycoside amikacin with a bacterial metabolite as a potentiator. This product is being developed with the goal of reducing recurrent infections in the treatment of catheter-associated urinary tract infections (CAUTIs). Methods: MICs were determined by standard broth microdilution methods (CLSI M7). E. coli cultures in planktonic stationary phase were used to select for bacterial persisters. Experiments were conducted with and without a pre-selection step utilizing the fluoroquinolone ofloxacin. Using the time-kill method (CLSI M26), a panel of 10 E. coli UTI amikacin-sensitive clinical isolates were evaluated, varying the concentration of amikacin and metabolite. Cells were enumerated after 4 hr of exposure to the amikacin/metabolite combinations. Finally, the potentiator effect was similarly examined in other UTI relevant species. Results: The amikacin MIC range for the 10 E. coli isolates was 1-4 μg/mL. Enhanced killing of up to 7 orders of magnitude of E. coli persisters was seen (see example in the figure below). Essentially no killing of persister cells occurred upon exposure to as high as 128 μg/mL of amikacin alone. The highest concentration of metabolite tested (40 mM) also did not kill cells when tested alone. In addition, a potentiation effect was observed, as examples, in P. aeruginosa and K. pneumoniae. Conclusions: The combination of amikacin with a metabolite potentiator has the capacity to revitalize existing and novel aminoglycosides through eradication of bacterial persisters, with broad spectrum potential for the treatment of CAUTIs.

4th World Congress on Targeting Infectious Diseases: Phage Therapy 2016

Gene Insertion in the Podoviridae Phage GRCS Results in Osmotically Sensitive Instability.

D. J. FERULLO & J.A. RADDING

ABSTRACT | EnBiotix is developing engineered phage expressing biofilm-dispersing enzymes for treating implant-associated infections of Staphylococcus aureus (Sau).  Phage GRCS belongs to a family of related Podoviridae/Picovirinae staphylococcal dsDNA phages utilizing 5’-terminal proteins for replication and packaging [1,2]. GRCS preferably infects clinical isolates of Sau from prosthetic joint infections (PJI) [3].Objective. To insert trans-genes into the GRCS genome, while maintaining stable phage production.Methodology. The 18Kb phage genome, assembled in vitro in a bacterial artificial chromosome (BAC) was transformed into E. coli, amplified and isolated [4]. Transformation of GRCS/BAC into Sau lysed the liquid culture. Plaques from solid agar confirmed as GRCS phage by PCR.  Reporter gene (GFP) inserted into GRCS/BAC yielded phage plaques on Sau for analysis.Results. Plaques isolated from solid agar were natural phage, indicating loss of the GFP-gene.  Liquid cultures resulted in a mixed phage population (natural and engineered). The proportion of engineered phage improved dramatically by increasing osmotic pressure using sucrose, but was lost in plating on solid agar [5].Conclusion. Insertion of trans-genes into GRCS proved unstable. External osmotic pressure can counterbalance this instability.  Stabilizing engineered phage will require selection against natural phage, coupled to evolution of engineered phage by serial passage through decreasing osmolarity.

2016 Dartmouth Cystic Fibrosis Retreat

A novel combination of Tobramycin with a potentiator for the treatment of chronic Pseudomonas aeruginosa infections

M. Koeva, A.D. Gutu, W. Hebert, G.A. O’Toole, F.M. Ausubel, D. Joseph-­McCarthy

Link to Conference Presentation

ABSTRACT | EBX-­‐001,  a  combination  of  tobramycin  with  a  bacterial  metabolite  as  a  potentiator,  is  being developed  for  the  treatment  of  chronic  P.  aeruginosa  infections  in  Cystic  Fibrosis  patients.   The combination  utilizes  an  anti-­‐persisters  strategy  and  is  aimed  at  reducing  recurrent  infections.    P. aeruginosa  cultures  in  the  planktonic  stationary  phase  (PSP)  were  used  to  select  for  bacterial  persisters, bacteria  in  a  quasi-­‐dormant  state.   In  these  PSP  experiments,  a  range  of  tobramycin  concentrations  was tested  with  a  range  of  metabolite  concentrations  to  determine  the  potentiation  effect  of  the  metabolite under  a  variety  of  conditions.   MICs  were  also  determined  for  a  variety  of  CF  clinical  isolates  to  select  a diverse  set  of  strains  for  inclusion  in  the  study.   Enhanced  killing  of  up  to  6  orders  of  magnitude  of  P. aeruginosa  persisters  for  a  range  of  strains  was  observed;  see  example  for  a  mucoid  CF  isolate  in  the figure  below.   A  combination  of  tobramycin  with  a  potentiator  remains  an  attractive  therapeutic  option for  eliminating  recurrent  P.  aeruginosa  infections  through  the  eradication  of  bacterial  persisters.

2014 Bio-IT World Conference

MINE: A Novel Computational Approach for Gene Network Identification

Michael Molla, Diane Joseph-McCarthy, James Costello, Jeffrey A. Radding, Jeffrey D. Wager

ABSTRACT | A perturbation to a biological system results in changes to molecular processes, signaling networks, and the constituent genes. These changes reflect the mode-of-action (MOA) of the perturbation and, if properly characterized, can be used to gain insights into how the perturbation acts.  When gene-expression measurements are coupled with appropriate computational methods, the efficiency and accuracy of MOA determination can be dramatically improved, by reducing the number of targets that need to be probed experimentally for definitive MOA determination.  Our novel MINE approach (Mode-of-action by Iterative Network Expansion), leverages the strengths of two well-established computational methods: MNI (Mode-of-action by Network Identification) [1] and CLR (Context Likelihood of Relatedness) [2].  MNI employs ordinary differential equations to interrogate a data compendium and reverse-engineer a gene network at the resolution of “Metagenes” (groups of genes with similar expression profiles).  CLR utilizes the information theoretic measure of mutual information to determine pairwise gene-to-gene connections.  MINE iteratively uses both methods to allow for enhanced characterization of the biological processes underlying a perturbation.  An expansion/pruning algorithm enables MINE to identify the sub-networks of influence, and derive refined insight from large, compiled compendia of cross-platform gene-expression data.  Details of this approach as well as initial results in microbial and mammalian datasets will be presented.  These in silico methods can elucidate regulatory mechanisms and possible metabolic changes associated with perturbations to any system, which will ultimately lead to improved strategies for targeted therapies in many fields.