Establishment of a dual-wavelength spectrophotometric method for analysing and detecting carbapenemase-producing Enterobacteriaceae

Abstract

The spread of carbapenemase-producing Enterobacteriaceae (CPE) is an increasing global public health concern. The development of simple and reliable methods for CPE detection is required in the clinical setting. This study aimed to establish a dual-wavelength measurement method using an ultraviolet-visible spectrophotometer to rapidly quantify imipenem hydrolysis in bacterial cell suspensions. The hydrolytic activities of 148 strains including various CPE strains (Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and Enterobacter aerogenes containing the bla_IMP, bla_KPC, bla_NDM, bla_OXA, and bla_VIM genes) were measured and analysed. A cut-off value was obtained for differentiation between CPE and non-CPE strains, and the method had high sensitivity (100%) and specificity (100%) within 60 min. Our system has potential clinical applications in detecting CPE.

Figure 1

(a) Absorption spectra of imipenem (IPM) between 220 nm and 400 nm at different concentrations. Symbols show the mean of four independent measurements, and bars show standard deviations. (b) Relationship between the imipenem concentration and the absorbance at 297 nm. Curve fitting with linear-regression analysis was performed (r² = 0.9956, β1 = 130.0076 [126.5346, 133.4806]), and the obtained curve is shown as a dotted line. Absorbance measurements were independently performed four times. Each symbol shows a measurement result. (c) Spectral analysis of imipenem hydrolysis before background subtraction. (d) Spectral analysis of imipenem hydrolysis after background subtraction. CPE: imipenem solution incubated with a carbapenemase-producing Enterobacteriaceae (CPE) strain, non-CPE: imipenem solution incubated with a non-CPE strains, Without Bacteria: imipenem solution without bacteria.

Figure 2

(a) A schematic representation of sample preparation for dual-wavelength spectrophotometric measurement. Instead of preparing a background well, the absorbance at 297 nm and 350 nm are measured simultaneously using a single reaction well to calculate the estimated net absorbance of imipenem. IPM = imipenem, Abs. = absorbance. (b) Spectral analysis of bacterial suspensions incubated with (Bac-IPM) or without (Bac-PBS) imipenem. The absorbances at 297 nm of Bac-IPM and Bac-PBS are denoted as A and B, which correspond to the gross absorbance of imipenem and background absorbance, respectively. The absorbance of both suspensions at 350 nm is denoted as C. (c) Relationship between points B and C. The absorbances of 15 representative strains were independently measured six times, at eight different concentrations and after four different incubation times. Curve fitting with linear-regression analysis was performed, and the obtained curve is shown as a dotted line with equation (1) and r² value. The statistical values are summarized in Supplementary Table S2.

Figure 3

(a) The percent hydrolysis of carbapenemase-producing Enterobacteriaceae (CPE; n = 112) and non-CPE (n = 35) strains after a 30-min incubation. The hydrolytic activities were compared among four different kinds of carbapenemase producers, i.e. IMP (n = 13), KPC (n = 11), NDM (n = 52), and OXA-producers (n = 36). A VIM-type carbapenemase producer was not included in the analysis because of the small sample number. The symbols (open circles, black squares, black triangles, and black diamonds) show percent-hydrolysis results for each type of strain tested. The bars show the median with the interquartile range. The Kruskal–Wallis test was performed for global comparisons (p < 0.0001), followed by two-tailed Mann–Whitney testing. *Significantly different. (b) The percent-hydrolysis of CPE (n = 113) and non-CPE (n = 35) strains after the indicated incubation times. The symbols (black circles, black squares, black triangles, black diamonds, open circles, open squares, and open triangles) show the percent hydrolysis results for each type of strain tested. The bars show the median with the interquartile range. The horizontal dashed lines show the cut-off value for each incubation time. The cut-off values are also summarized at the bottom of the figure.

Figure 4

Statistically non-significant relationships between the minimal inhibitory concentrations (MICs) of imipenem and the percent hydrolysis of carbapenemase-producing Enterobacteriaceae (CPE) strains after incubation for 30, 60, 120, or 180 min. CPE strains (n = 113) were divided into seven groups based on the MICs of imipenem, i.e. ≤0.5 (n = 7), 1 (n = 6), 2 (n = 8), 4 (n = 17), 8 (n = 29), 16 (n = 25), and >16 (n = 21). The symbols (black circles, black squares, black triangles, black diamonds, open circles, and open squares) shows percent-hydrolysis results for each type of strain tested. The bars show the median with the interquartile range. Kruskal–Wallis testing was used for comparisons.