Carbapenem antibiotic stress increases blaKPC-2 gene relative copy number and bacterial resistance levels of Klebsiella pneumoniae

Background: The clinical isolation rates of carbapenem-resistant Klebsiella pneumoniae (CR-KP) continue to increase. In China, clinical CR-KP isolates are mainly attributed to the bla(KPC-2) gene carried on plasmids, and the bla(KPC-2) copy number correlates with the expression of KPC enzymes, which can cause elevated carbapenem MICs. Methods: Thirty-seven CR-KP isolates were collected at the Second People's Hospital of Lianyungang City between January 2020 and March 2021, with no duplicate isolates, and were screened for the bla(KPC-2) gene with PCR. Analysis of current CRKP resistance to clinically relevant antimicrobials using the bioMerieux VITEK (R) 2 bacterial identification card. The multilocus sequence types of the strains were confirmed with PCR and DNA sequencing. Recombinant plasmids pET20b-bla(KPC-2) and pET20b-CpsG were constructed, and the copy numbers of the recombinant plasmids per unit volume was calculated based on the molecular weight of the plasmids. After the genomes DNA of clinical isolates of K. pneumoniae carrying the bla(KPC-2) gene were purified, the bla(KPC-2) gene relative copy number in individual K. pneumoniae strains was indicated by the double standard curve method. Detection of MIC values changes of K. pneumoniae under imipenem selection pressure by broth microdilution method. Results: Among the 37 CR-KP strains isolated, only the bla(KPC-2) gene was detected in 30 strains, three strains were positive for the bla(NDM-1) gene, two strains carried both the bla(KPC-2) and bla(NDM-1) genes, and two strains without detectable carbapenem resistance genes. The ST11 clone was predominant among the 37 carbapenem-resistant K. pneumoniae isolates. Drug sensitivity testing showed that except for polymyxins (100% susceptible) and tigecycline (75.7% intermediate), the 37 CR-KP strains were resistant to almost all antimicrobial drugs. The bla(KPC-2) relative copy number in nine ST11 clinical isolates of K. pneumoniae was 7.64 +/- 2.51 when grown on LB plates but 27.67 +/- 13.04 when grown on LB plates containing imipenem. Among these nine isolates, five CRKP strains exhibited elevated MICs to imipenem, while the remaining four strains showed unchanged MIC values to imipenem. Conclusion: Carbapenem-resistant Klebsiella pneumoniae isolates may have multiple pathways to achieve high levels of carbapenem resistance, and moderate carbapenem pressure can increase the copy number of KPC enzyme genes in CRKP strains and enhance the degree of carbapenem resistance in the strains.