Rapid susceptibility profiling of carbapenem-resistant Klebsiella pneumoniae

Abstract

The expanding global distribution of multi-resistant Klebsiella pneumoniae demands faster antimicrobial susceptibility testing (AST) to guide antibiotic treatment. Current ASTs rely on time-consuming differentiation of resistance and susceptibility after initial isolation of bacteria from a clinical specimen. Here we describe a flow cytometry workflow to determine carbapenem susceptibility from bacterial cell characteristics in an international K. pneumoniae isolate collection (n = 48), with a range of carbapenemases. Our flow cytometry-assisted susceptibility test (FAST) method combines rapid qualitative susceptible/non-susceptible classification and quantitative MIC measurement in a single process completed shortly after receipt of a primary isolate (54 and 158 minutes respectively). The qualitative FAST results and FAST-derived MIC (MIC_FAST) correspond closely with broth microdilution MIC (MIC_BMD, Matthew's correlation coefficient 0.887), align with the international AST standard (ISO 200776-1; 2006) and could be used for rapid determination of antimicrobial susceptibility in a wider range of Gram negative and Gram positive bacteria.

Figure 1

Schematic diagram of flow cytometry-assisted susceptibility test method. Demonstration of the workflow used. In brief, an isolate was retrieved from cryopreservation, plated onto blood agar to ensure purity, and inoculated into trypticase soya broth (TSB) overnight to simulate biological fluids. A 1 ml aliquot of this suspension was inoculated into Mueller-Hinton broth (MHB) and incubated at 37 °C to ensure bacteria were actively dividing. One aliquot was subjected to traditional microbroth dilution (MBD) susceptibility testing. Another aliquot was exposed to meropenem for 30 minutes, harvested, stained with SYTO^®9, and then assayed with a flow cytometer. This figure is not covered by the CC-BY licence. [Credit to Life Technologies Corporation, a part of Thermo Fisher Scientific Inc. www.thermofisher.com. ^©2016 Thermo Fisher Scientific Inc. Used under permission.] All rights reserved, used with permission.

Figure 2

Standardised gating applied to raw data. (A) Collected events were gated to include only those with a SYTO®9 (BL1 - 530/30 nm) fluorescence of 10⁴ arbitrary fluorescence units or higher. Doublets were removed via a FSC-A vs FSC-H plot. Background was removed by plotting specific SYTO®9 fluorescence (BL1 – 530/30) against an unused channel (BL3 – 640 LP). (B) In the antibiotic unexposed sample, 10% nearest-neighbour contouring was applied, and a gate (referred to as Unexposed Cell Morphotype) was set to include all clustered events. This gate was then applied to all samples across the antibiotic dilution series.

Figure 3

Susceptibility to meropenem can be identified by AFC by observing a susceptibility-associated signature. (A) Exposure of the K. pneumoniae susceptible type strain (ATCC 700603) increased forward scatter, SYTO®9 fluorescence, reduced overall event numbers, and formed a new contouring focus at the isolate’s MIC (0.25 mg/L). At 32 × MIC, a total of four contouring foci were observed, with an overall shift towards low forward scatter, low fluorescent debris. The progression of these features, when observed in combination, constitutes the susceptibility-associated signature. Colouring on biaxial plots indicates separate contouring foci. Fluorescence micrographs (acquired at 60x magnification) show reduced overall cell numbers and increase aberrant cell morphotypes as meropenem concentration increases. (B) Exposure of highly resistant clinical K. pneumoniae strain K8 to meropenem shows an absence of susceptibility-associated signature across clinically relevant meropenem concentrations by flow cytometer bi-axial plot, and an absence of aberrant cell morphotypes by fluorescence microscopy.

Figure 4

FAST accurately predicted MIC_BMD across our collection of K. pneumoniae isolates. Coloured regions represent the qualitative susceptibility (green)/non-susceptible (red) determination of (A) the initial 10 isolates tested (mean MIC_BMD/FAST and SEM), a strong positive correlation was observed (r = 0.899, p < 0.0001), represents four isolates, all with perfectly concordant MIC_BMD,_FAST coordinates. (B) Full 48 isolate collection. Numerals indicate the number of isolates occupying the same MIC_BMD, MIC_FAST coordinates. Across the full collection of isolates, a strong positive correlation was observed (Matthew’s correlation co-efficient = 0.918).

Figure 5

Differences in MIC_FAST were observed between colony variants of K. pneumoniae isolate K16. When subculturing IMP-4 producing K. pneumoniae isolate K16, a rough and smooth colony variant was observed. The smooth colony variant produced an MIC_FAST of 2 mg/L. The rough colony produced an MIC_FAST of 64 mg/L and, at 2 mg/L, was observed by AFC to contain a population consistent with a non-susceptible phenotype. Both MIC_FAST results were concordant with the MIC_BMD results.

Figure 6

Resistant sub-populations were observed in K. pneumoniae isolate K16 across two days of selective passage and FAST: Day One - IMP-4 producing K. pneumoniae isolate K16 was found, at 2 mg/L meropenem, to contain a minority population of cells with a phenotype consistent with unexposed cells (remaining within the Unexposed Cell Morphotype gate - indicated by arrow). This subpopulation persisted, at a diminished frequency, at 16 mg/L meropenem while the majority of cells display a compromised phenotype (shifted outside the gate). Day 2 – The 2 mg/L culture of K16 from Day 1 was subcultured and subjected to FAST on the following day. The isolate displayed an increased MIC (4 mg/L), delayed progression to the emergence susceptibility-associated signature, with most events consistent with a non-susceptible phenotype at 2 mg/L. Most events at 16 mg/L were consistent with a susceptible phenotype, however a small subpopulation remained inside the Unexposed Cell Morphotype gate.