Carbapenemase Inhibition Challenge Method: a novel way for evaluating the presence of carbapenemases – a pilot study

Abstract

Introduction. Resistance to carbapenems represents a public health across the world. Screening for carbapenem resistance can be done starting from the antibiogram, but confirmatory methods are usually performed in reference centers. Methods for rapid identification of carbapenemase activity are required to empower laboratories to detect carbapenemases and raise signals for infectious disease physicians, antibiotic stewardship teams and hospital hygiene specialists. Materials and methods. Eppendorf tubes containing Klebsiella pneumoniae strains with carbapenemase activity (KPC-1, NDM-1, OXA-48) and others with no carbapenemase activity (K. pneumoniae ATCC 700603 and E. coli ATCC 25922), as well as positive and negative controls were incubated at 37 °C with varying concentrations of Meropenem. After incubation, samples were spun down until a pellet formed and the supernatant was used to challenge an exponentially growing E. coli culture. This second culture was followed with nephelometric measurements every half hour. Experiments were carried out in three technical replicates. The statistical analysis was done with R (version 3.3.3) and graphs generated in RStudio (version 1.0.136) with ggplot2 (version 2.1.0). Mann Whitney U Test with the assumption of independent samples was used to analyse the results. Results. The doses of Meropenem were chosen after dose ranging experiments and multiple incubation times (from 30 minutes to two hours) were tested in order to evaluate the robustness of the method. In the cases where the antibiotic was first incubated with strains that had carbapenemase activity, the growth of Escherichia coli ATCC 25 922 was non-hindered, matching that of controls. In the absence of carbapenemase activity and in the case of negative controls, the growth indicator strain was inhibited. Statistically significant differences were identified for 0.3, as well as a 0.5 McFarland index as early as 1.5 hours total work time. Conclusions. The discovery of carbapenem resistance has direct clinical, as well as epidemiological implications. Among the mechanisms implicated in resistance to carbapenems, the presence of a carbapenemase is one of the most worrying from an epidemiological standpoint. This new tool we describe herein can be performed in all clinical microbiology laboratories, with no extra expense (as opposed to CarbaNP and BlueCarba tests).