Carbapenemase-Encoding Gene Copy Number Estimator (CCNE): a Tool for Carbapenemase Gene Copy Number Estimation.

Carbapenemase production is one of the leading mechanisms of carbapenem resistance in Gram-negative bacteria. An increase in carbapenemase gene ( bla Carb) copies is an important mechanism of carbapenem resistance. No currently available bioinformatics tools allow for reliable detection and reporting of carbapenemase gene copy numbers. Here, we describe the carbapenemase-encoding gene copy number estimator (CCNE), a ready-to-use bioinformatics tool that was developed to estimate bla Carb copy numbers from whole-genome sequencing data. Its performance on Klebsiella pneumoniae carbapenemase gene ( bla KPC ) copy number estimation was evaluated by simulation and quantitative PCR (qPCR), and the results were compared with available algorithms. CCNE has two components, CCNE-acc and CCNE-fast. CCNE-acc detects bla Carb copy number in a comprehensive and high-accuracy way, while CCNE-fast rapidly screens bla Carb copy numbers. CCNE-acc achieved the best accuracy (100%) and the lowest root mean squared error (RMSE; 0.07) in simulated noise data sets, compared to the assembly-based method (23.4% accuracy, 1.697 RMSE) and the OrthologsBased method (78.9% accuracy, 0.395 RMSE). In the qPCR validation, a high consistency was observed between the bla KPC copy number determined by qPCR and that determined with CCNE. Reverse transcription-qPCR transcriptional analysis of 40 isolates showed that bla KPC expression was positively correlated with the bla KPC copy numbers detected by CCNE ( P  < 0.001). An association study of 357 KPC-producing K. pneumoniae isolates and their antimicrobial susceptibility identified a significant association between the estimated bla KPC copy number and MICs of imipenem ( P  < 0.001) and ceftazidime-avibactam ( P  < 0.001). Overall, CCNE is a useful genomic tool for the analysis of antimicrobial resistance genes copy number; it is available at https://github.com/biojiang/ccne. IMPORTANCE Globally disseminated carbapenem-resistant Enterobacterales is an urgent threat to public health. The most common carbapenem resistance mechanism is the production of carbapenemases. Carbapenemase-producing isolates often exhibit a wide range of carbapenem MICs. Higher carbapenem MICs have been associated with treatment failure. The increase of carbapenemase gene ( bla Carb) copy numbers contributes to increased carbapenem MICs. However, bla Carb gene copy number detection is not routinely conducted during a genomic analysis, in part due to the lack of optimal bioinformatics tools. In this study, we describe a ready-to-use tool we developed and designated the carbapenemase-encoding gene copy number estimator (CCNE) that can be used to estimate the bla Carb copy number directly from whole-genome sequencing data, and we extended the data to support the analysis of all known bla Carb genes and some other antimicrobial resistance genes. Furthermore, CCNE can be used to interrogate the correlations between genotypes and susceptibility phenotypes and to improve our understanding of antimicrobial resistance mechanisms.