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The use of the Reveal™ system to detect and characterise heteroresistant bacterial populations in blood samples
ESCMID eLearning. Singh P. 07/09/21; 328761; 2471
Pragya Singh
Pragya Singh
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Abstract
Discussion Forum (0)
Abstract number: 2471

Session Type: ePosters

Session Title: ePosters

Authors(s): F. Tang (1), L. Rajeev (1), P. Rhodes (1), N. Ledeboer (2), R. Humphries (3), P. Singh (1)

Authors Affiliations(s): (1) Specific Diagnostics, United States, (2) Department of Pathology, Medical College of Wisconsin, United States, (3) Dept of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, United States

Background:

Antimicrobial-resistant infections are of growing concern worldwide.  While the current standard for antibiotic susceptibility determination works from isolates implicitly assumed to comprise clonal populations, in some cases heteroresistance, where a small (10-2-10-7) fraction of the population comprises strains of greater resistance, can occur.  A resistant subpopulation may increase risk of treatment failure, as it can be selected for during therapy.  Here we tested the hypothesis that the antibiogram of two distinct populations could be differentiated in Specific Diagnostics’ Reveal rapid AST system by observing the time-lagged emergence of signal from a small resistant subpopulation.  

Methods:

Known resistant and susceptible strains of Escherichia coli or Pseudomonas aeruginosa mixed at frequency ratios of 1:102 to 1:105 were inoculated into 96-well dried antibiotic plates, sealed with the Reveal sensor panel and loaded on the Reveal system.  Reveal uses an array of volatile-sensitive nanoporous indicators to sensitively detect growth in each well, and we reasoned that the resistant subpopulation would be revealed by emergence of signal in a second set of wells corresponding to those only resistant in the second strain after a lag of approximately [doubling time]log2(frequency ratio).  To compare with the information available from conventional methods, the same mixtures were assessed by direct-plating disk diffusion.

Results:

Reveal’s limit of detection was at or below 1:105, consistent with the estimated 50-100,000 CFU present in each well at the initiation of the assay.  The antibiogram of the resistant strain emerged as an “overlay” on the antibiogram of the dominant strain after a delay (Figure 1), with a lag predictable by the ratio of the two populations.  Raw sensor traces confirmed that sensitivity of detection of the subpopulation corresponded to traditional disk diffusion results, a microcolonies were observed within the zones of inhibition at 1:105 as well (Figure 2).

Conclusions:

Reveal may provide a means to detect and characterize heteroresistant mixtures comprising a dominant population with a resistant subpopulation from a positive blood culture bottle sample.  Future studies will aim to use this capability to quantify the degree to which heteroresistance is relevant in clinical practice. 

Keyword(s): Heteroresistance, Reveal rapid AST, Multidrug Resistant Organisms

Abstract number: 2471

Session Type: ePosters

Session Title: ePosters

Authors(s): F. Tang (1), L. Rajeev (1), P. Rhodes (1), N. Ledeboer (2), R. Humphries (3), P. Singh (1)

Authors Affiliations(s): (1) Specific Diagnostics, United States, (2) Department of Pathology, Medical College of Wisconsin, United States, (3) Dept of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, United States

Background:

Antimicrobial-resistant infections are of growing concern worldwide.  While the current standard for antibiotic susceptibility determination works from isolates implicitly assumed to comprise clonal populations, in some cases heteroresistance, where a small (10-2-10-7) fraction of the population comprises strains of greater resistance, can occur.  A resistant subpopulation may increase risk of treatment failure, as it can be selected for during therapy.  Here we tested the hypothesis that the antibiogram of two distinct populations could be differentiated in Specific Diagnostics’ Reveal rapid AST system by observing the time-lagged emergence of signal from a small resistant subpopulation.  

Methods:

Known resistant and susceptible strains of Escherichia coli or Pseudomonas aeruginosa mixed at frequency ratios of 1:102 to 1:105 were inoculated into 96-well dried antibiotic plates, sealed with the Reveal sensor panel and loaded on the Reveal system.  Reveal uses an array of volatile-sensitive nanoporous indicators to sensitively detect growth in each well, and we reasoned that the resistant subpopulation would be revealed by emergence of signal in a second set of wells corresponding to those only resistant in the second strain after a lag of approximately [doubling time]log2(frequency ratio).  To compare with the information available from conventional methods, the same mixtures were assessed by direct-plating disk diffusion.

Results:

Reveal’s limit of detection was at or below 1:105, consistent with the estimated 50-100,000 CFU present in each well at the initiation of the assay.  The antibiogram of the resistant strain emerged as an “overlay” on the antibiogram of the dominant strain after a delay (Figure 1), with a lag predictable by the ratio of the two populations.  Raw sensor traces confirmed that sensitivity of detection of the subpopulation corresponded to traditional disk diffusion results, a microcolonies were observed within the zones of inhibition at 1:105 as well (Figure 2).

Conclusions:

Reveal may provide a means to detect and characterize heteroresistant mixtures comprising a dominant population with a resistant subpopulation from a positive blood culture bottle sample.  Future studies will aim to use this capability to quantify the degree to which heteroresistance is relevant in clinical practice. 

Keyword(s): Heteroresistance, Reveal rapid AST, Multidrug Resistant Organisms

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