A 6-year-old girl developed meningitis 1 week after intracranial injury surgery. She was transferred to the surgical intensive care unit (SICU) of Children's Hospital of Nanjing Medical University (Nanjing, China) due to a persistent high fever. Upon arrival at the hospital, the patient was initially treated empirically for 3 days with meropenem (0.88 g IV q8 hours) plus vancomycin (0.22 g IV q6 hours). Three days later, the cerebrospinal fluid (CSF) culture results showed multidrug-resistant (MDR) K. pneumoniae. The antimicrobial susceptibility assay did not provide the sensitive or resistant data for polymyxin and tigecycline due to the limitations of the clinical laboratory. Even though the antimicrobial therapy was changed to meropenem (0.85 g IV q8 hours) plus polymyxin B (24 mg IV q12 hours) and polymyxin B (2 mg IVT qd) for nearly 3 weeks, the severe intracranial infection had not been controlled and the re-cultures of CSF showed the growth of MDR K. pneumoniae. On day 22, the CSF was sent to the First Affiliated Hospital of Nanjing Medical University (Nanjing, China) for further analysis of antimicrobial susceptibility, and the results showed that the MDR K. pneumoniae isolate named 20JS63 was only sensitive to tigecycline (<=1mg/L), but resistant to meropenem (>=128 mg/L), imipenem (>=128 mg/L), even colistin (>=64 mg/L). Thus, the clinicians switched the therapy to meropenem (0.85 g IV q8 hours) plus tigecycline (25 mg IV q12 hours) and added meropenem (5 mg IVT qd) plus tigecycline (4 mg IVT qd) the next day. Accompanied by the insignificant therapeutic effect of dual therapy, the patient manifested a persistent high fever up to 40.3 C and a severe CNS infection. Overall, inflammatory markers (PCT, CRP, WBC count and neutrophilic granulocyte percentage (NEUT%) in his peripheral blood) also peaked during this time (Figure 1). On day 37, polymyxin B was added at a dose of 25 mg IV q12 hours to triple therapy based on dual therapy. Because the local pus in the patient's skull was thick and difficult to drain, the ventricles were irrigated with tigecycline (50 mg qd) and continuous bilateral ventricles drainage to maintain the stability of the brain environment. On day 50, meropenem was replaced with aztreonam (0.65 g IV q8 hours) because long-term use of meropenem leads to changes in cell membrane protein and diminishes the antimicrobial effect. The patient's temperature was maintained under continuous ventricular irrigation and drainage, but she had not achieved a clinical or microbiological cure. On day 69, her parents chose to discontinue treatment and the patient was discharged after the consultation with the parents.
To increase the valuable clinical experience and explore the possibility of anti-infective therapy, we carried out further research on the isolate 20JS63. The entire genomic DNA of the K. pneumoniae 20JS63 was sequenced and deposited in the GenBank database under accession number JAKEDZ000000000. This K. pneumoniae 20JS63 belonged to sequence type 11 (ST11), which harboured antibiotic resistance genes including kpc-2, CTX-M-65, SHV-11, TEM-1B, rmtB, qnrS1, tetA, and dfrA14. Circos (the version number: 0.69) was used to make a circular map of the bacterial genome so that the drug resistance genes can be visualized on the bacterial genome (Figure 2A). In addition, this strain contained virulence genes, including lutA and rmpA. Noteworthy, the isolate possessed a mutated pmrB gene, which encoded the PmrB protein in PmrAB two-component system. The mutant pmrB gene detected in this isolate differed from the wild type featured a single threonine to proline amino acid substitution at position 157 (Figure 2B). DynaMut web server (http://biosig.unimelb.edu.au/dynamut/) was used to analyze the effect of mutation on structure of mutated PmrB protein (Figure 2C). This genetic alteration is known to be responsible for acquired polymyxin B resistance in K. pneumoniae. CAZ-AVI was launched on 21 May 2019 in China after the hospitalization period of this patient (7 March to 14 May 2019). The E-test of CAZ-AVI was performed after the case, and the result showed that this isolate was sensitive to ceftazidime-avibactam (<=0.125 mg/L) in vitro.