On-chip Carba NP test for accurate and high throughput detection of carbapenemase-producing Enterobacteriaceae

Given the great threat posed by clinical Carbapenemase-producing Enterobacteriaceae (CPE), its rapid and accurate detection is highly required to guide infection treatment and to prevent serious outbreaks. While the Carba NP test (CNPt) is a popular and methodologically simple colorimetric assay for detecting carbapenemase activity, its clinical application is limited by low throughput, poor sensitivity and obscure color interpretation due to the lack of quantitative information on time- and dose-dependent color changes. To address these limitations, we developed a multiplexed microfluidic device (1280 chambers) that is able to automatically generate 16 bacterial concentration gradients and uses green channel intensity to extract quantitative color changes as a function of time. Here we examined the dynamic color changing profiles of 3 reference and 8 clinical bacteria isolates. The results demonstrated linear correlations between color developments and time (within 0-120 min) or bacterial doses (within OD₆₀₀ of 0.8-8.0), enabling accurate and reproducible detection of CPE with over 10⁵ fold improved sensitivity for an NDM-producer (LOD of 2.4 × 10³ cells) and over 10⁴ fold improved sensitivity for an OXA-48-producer (LOD of 7.2 × 10⁴ cells) as compared to the conventional CNPt method (LOD above 1.3 × 10⁹ cells). As the first digital microfluidic platform for CPE, this on-chip CNPt compared favourably with the conventional bench-based method by significantly improved sensitivity, high throughput, low consumption and automatization, favouring its potential clinical applications for rapid screening of CPE with a low cost. Furthermore, the results also revealed dynamic signature associated with bacteria, which may hold promise for a new approach to study divergent bacterial responses to antibiotic treatments.