Tracking emergence and outbreak of Klebsiella pneumoniae co-producing NDM-1 and KPC-2 after sulfamethoxazole-trimethoprim treatment: Insights from genetic analysis

The co-production of KPC and NDM carbapenemases in carbapenem-resistant Klebsiella pneumoniae (CRKP) complicates clinical treatment and increases mortality rates. The emergence of KPC-NDM CRKP is believed to result from acquiring an NDM plasmid by KPC-CRKP, especially under the selective pressure of ceftazidime-avibactam (CZA). In this study, a CRKP-producing KPC-2 (JNP990) was isolated from a patient at a tertiary hospital in Shandong Province. Following sulfamethoxazole-trimethoprim (SXT) treatment, the isolate evolved into a strain that co-produces KPC and NDM (JNP989), accompanied by resistance to SXT (MIC>2/38 μg/mL) and CZA (dd≤14 mm). Whole-genome sequencing (WGS) and S1 pulsed-field gel electrophoresis (PFGE) revealed that JNP989 acquired an IncC plasmid (NDM plasmid) spanning 197kb carrying sul1 and blaNDM-1 genes. The NDM plasmid could be successfully transferred into E. coli J53 at a conjugation frequency of (8.70±2.47) × 10−4. The IncFⅡ/IncR plasmid carrying the blaKPC-2 gene in JNP990 could only be transferred in the presence of the NDM plasmid at a conjugation frequency of (1.93±0.41) × 10−5. Five CRKP strains with the same resistance pattern as JNP989 were isolated from other patients in the same hospital, belonging to the same clone as JNP989, with a sequence type of ST11. Two strains lost resistance to CZA due to the loss of blaNDM-1-carrying fragment mediated by insertion sequence 26. Plasmid stability testing indicated that the IncC plasmid was more stable than the blaNDM-1 genes in the hosts. Our study describes the evolution of KPC-NDM-CRKP and its spread in hospitalized patients following antibiotic treatment, highlighting the severity of the current resistance spread.