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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.

Daniel, J. FERULLO, Jeffrey, 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.