Analysis of colistin resistance in carbapenem-resistant Enterobacterales and XDR Klebsiella pneumoniae

Abstract

Introduction: Increasing occurrence of infections caused by multidrug-resistant Gram-negative bacteria resulted in colistin being the last agent for treatment. Apart from plasmid-mediated mcr genes, mutations involving several genes like mgrB, phoP/phoQ, pmrA, pmrB, pmrC, and crrABC genes, are leading causes of colistin resistance. Four colistin susceptibility testing methods were compared against broth microdilution (BMD) and determined the presence of the mcr1-5 gene.

Methodology: A total of 100 carbapenem-resistant Enterobacterales isolates were tested for colistin susceptibility by commercial broth microdilution (cBMD), E-test, VITEK-2, and rapid polymyxin NP assay (RPNP) and compared with BMD. The presence of the mcr1-5 gene was determined by modified RPNP and PCR. Two non-mcr colistin-resistant XDR isolates were subjected to whole-genome sequencing using Illumina MiSeq sequencing platform.

Results: Among 100 carbapenem-resistant Enterobacterales isolates, 15% were resistant to colistin. Essential agreement, categorical agreement, major error, and very major error for cBMD/E-test/VITEK-2/RPNP were 96%/73%/82%/NA; 99%/86%/88%/91%, 1.2%/9.4%/11.8%/8.2% and 0%/40%/13.3%/13.3%, respectively. Only one Klebsiella pneumoniae isolate harbored the mcr-1 gene, observed by both methods. Whole-genome sequencing of two non-mcr XDR Klebsiella pneumoniae showed multiple mutations in 10 genes responsible for lipopolysaccharide biosynthesis.

Conclusions: The performance of cBMD was excellent, whereas the E-test was unacceptable. VITEK-2 and RPNP performed better but remained unreliable due to high error rates. Multiple mutations in the target proteins involving lipopolysaccharide formation, modification, and regulation were seen, resulting in colistin resistance.

Keywords: Whole-genome sequencing; broth microdilution; carbapenem-resistant Enterobacterales; colistin; mcr-1.

Figure 1.

Scatter diagram of colistin MICs obtained by BMD compared with a) cBMD, b) VITEK-2, c) E-test, and d) CNP for 100 isolates. Essential agreement is highlighted in green (perfect agreement) and yellow (±1 log2 dilution). The solid horizontal and vertical lines represent the clinical breakpoint value established by CLSI/ EUCAST. Blue color signifies susceptible MIC, whereas Red color signifies resistance MIC range. Escherichia coli NCTC 13846 (mcr-1 positive) and Escherichia coli ATCC 25922 (colistin susceptible) are represented by * and †, respectively.

Figure 2.

Correlation plot of colistin MICs obtained by BMD compared with a) cBMD, b) E-test, c) VITEK-2 for 100 isolates. Pearson correlation values show an excellent correlation between BMD and cBMD, whereas it shows a fair correlation for E-test and VITEK-2.

Figure 3.

Modified rapid polymyxin NP test (mRPNP). Wells A1-A6 were filled with colistin-free RPNP solution. Wells B1 to B6 were filled with RPNP solution + colistin sulfate. Wells C1 to C6 were filled with colistin-free NP + EDTA. Wells D1 to D6 were added with RPNP solution + colistin sulfate + EDTA. Wells in column 1 were filled with 0.85% NaCl (negative sterility control), whereas Columns 2 to 7 represent the mRPNP test performed for E. coli ATCC 25922, P. mirabilis ATCC 25933, colistin-resistant mcr-1-5 negative K. pneumoniae, colistin-resistant mcr-1-5 negative E. coli strain, and mcr-1-positive E. coli NCTC 13846, respectively. The plates were incubated at 35 ± 2°C under aerobic conditions for 4 h, and visual changes in the color of the wells were monitored each hour. In wells, B1 to B6, a color change from orange to yellow was considered positive to colistin resistance, whereas the mRPNP test was considered positive to MCR-1 phosphoethanolamine transferase production when the colistin-containing solution supplemented with EDTA (wells D1 to D6) remained orange (i.e. absence of glucose metabolization); this shows that growth of the colistin-resistant E. coli (mcr-1-positive) in the well containing colistin solution (well D6) was inhibited by EDTA. Since mcr-1 translates to PEtN transferase, which is a zinc enzyme, exposure to chelators like EDTA could reduce colistin resistance in mcr-1-producing strains.