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The use of the Reveal™ system to quantitatively determine the efficacy of bacteriophage
ESCMID eLearning. Singh P. 07/09/21; 328841; 2609
Pragya Singh
Pragya Singh
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Abstract
Discussion Forum (0)
Abstract number: 2609

Session Type: ePosters

Session Title: ePosters

Authors(s): H. Siegel, L. Rajeev, P. Singh, P. Rhodes

Authors Affiliations(s): Specific Diagnostics, United States

Background:

Bacteriophage therapy is increasingly appreciated as a valuable  tool in the battle against antibiotic-resistant infections. The narrow strain-specificity of most phages, although of benefit to preserving the host microbiome, also acts as a hurdle, requiring rapid susceptibility testing to screen and select the most appropriate phages for the pathogen concerned. There are currently no commercially available, automated, high-throughput, rapid methods to determine the bacteriophage susceptibility. Specific Diagnostics’ Reveal™ instrument utilizes small molecule sensor technology to detect volatile organic compounds emitted by growing microorganisms to assess antibiotic efficacy, and we hypothesized that the system could be adapted to rapidly assess efficacy of bacteriophage. Here, we demonstrate that Reveal’s capabilities can be leveraged to rapidly determine the bacteriophage susceptibility profile for pathogens.

Methods:

Using well-characterized bacteriophages for known Escherichia coli and Staphylococcus aureus host strains, bacteria and phage were mixed at serial 10-fold ratios in growth medium and aliquoted into 96-well plates.  The plates were sealed with the small molecule sensor sheet and then loaded onto the Reveal system, where the wells were imaged to monitor the sensor responses over time. The gold standard double agar layer plaque assay was used as the reference.  

Results:

The two ATCC-provided E. coli host strains (MG1655 and C600) showed distinct susceptibility to the virulent lambda W60 phage and reduced susceptibility to the lysogenic standard lambda phage (Figure 1). The two ATCC-provided S. aureus host strains showed strong susceptibility to their respective phages and resistance to the alternate phage. A screen of 20 E. coli strains against the lambda W60 phage identified 3 other susceptible strains as hosts for lambda W60 (Figure 2). A similar screen with S. aureus strains identified 14 and 5 strains with varying degrees of susceptibility to phages 27691-B1 and 27702-B1, respectively.  The duration of growth inhibition in the presence of phage quantitatively correlated with the degree of susceptibility reported by the reference assay.  The time to complete the assays (4 to 10 hours) depended upon both strain and phage titer.

Conclusions:

Our results indicate that the Reveal assay may be a rapid and information-rich tool to screen bacterial strains for susceptibility to bacteriophage.

Keyword(s): Bacteriophage susceptibility, Reveal system, Phage therapy

Abstract number: 2609

Session Type: ePosters

Session Title: ePosters

Authors(s): H. Siegel, L. Rajeev, P. Singh, P. Rhodes

Authors Affiliations(s): Specific Diagnostics, United States

Background:

Bacteriophage therapy is increasingly appreciated as a valuable  tool in the battle against antibiotic-resistant infections. The narrow strain-specificity of most phages, although of benefit to preserving the host microbiome, also acts as a hurdle, requiring rapid susceptibility testing to screen and select the most appropriate phages for the pathogen concerned. There are currently no commercially available, automated, high-throughput, rapid methods to determine the bacteriophage susceptibility. Specific Diagnostics’ Reveal™ instrument utilizes small molecule sensor technology to detect volatile organic compounds emitted by growing microorganisms to assess antibiotic efficacy, and we hypothesized that the system could be adapted to rapidly assess efficacy of bacteriophage. Here, we demonstrate that Reveal’s capabilities can be leveraged to rapidly determine the bacteriophage susceptibility profile for pathogens.

Methods:

Using well-characterized bacteriophages for known Escherichia coli and Staphylococcus aureus host strains, bacteria and phage were mixed at serial 10-fold ratios in growth medium and aliquoted into 96-well plates.  The plates were sealed with the small molecule sensor sheet and then loaded onto the Reveal system, where the wells were imaged to monitor the sensor responses over time. The gold standard double agar layer plaque assay was used as the reference.  

Results:

The two ATCC-provided E. coli host strains (MG1655 and C600) showed distinct susceptibility to the virulent lambda W60 phage and reduced susceptibility to the lysogenic standard lambda phage (Figure 1). The two ATCC-provided S. aureus host strains showed strong susceptibility to their respective phages and resistance to the alternate phage. A screen of 20 E. coli strains against the lambda W60 phage identified 3 other susceptible strains as hosts for lambda W60 (Figure 2). A similar screen with S. aureus strains identified 14 and 5 strains with varying degrees of susceptibility to phages 27691-B1 and 27702-B1, respectively.  The duration of growth inhibition in the presence of phage quantitatively correlated with the degree of susceptibility reported by the reference assay.  The time to complete the assays (4 to 10 hours) depended upon both strain and phage titer.

Conclusions:

Our results indicate that the Reveal assay may be a rapid and information-rich tool to screen bacterial strains for susceptibility to bacteriophage.

Keyword(s): Bacteriophage susceptibility, Reveal system, Phage therapy

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