Direct determination and differentiation of carbapenemases of A. baumannii from uncultured tracheal samples

The emergence of multiple carbapenemases and the consequent multi drug resistance in bacteria constitute a grave concern in the management of critical care patients and also in community acquired infections. Detection of carbapenamase activity helps to understand the possible mechanism(s) of carbapenem resistance in the microorganism. Identification of carbapenemases is currently being done by various phenotypic methods and molecular methods. However, innovative biochemical and spectrophotometric methods are desirable as they will be easy to perform, affordable and rapid. Recently a novel chromogenic method called CarbaNP test was introduced to screen for carbapenemases in clinical isolates of gram negative pathogens. We adopted this assay: (i) to detect the total carbapenemase activity (ii) to measure the relative rates of hydrolysis of imipenem by class A, B and D carbapenemases with inhibitors (iii) to confirm the genotype by PCR and (iv)for direct differential detection of various carbapenemases in uncultured clinical sample like tracheal aspirate. The study included 75 culture isolates and 153 purulent tracheal aspirates. All isolates were screened by our optimized protocol and also genotyped by PCR. This adopted assay showed good sensitivity and correlation with conventional phenotyping and genotyping. Our protocol offers the fastest way to identify the pathogen by PCR but also its carbapenemase profile directly from uncultured clinical samples in less than 4h. Our protocol is currently being validated on other types of clinical specimens in our laboratory.