Molecular mechanisms of colistin-resistant Klebsiella pneumoniae and Escherichia coli isolates / 中华检验医学杂志

ABSTRACT

Objective To elucidate the resistance mechanisms of clinical colistin-resistant Klebsiella pneumoniae and Escherichia coli isolates in China.Methods A total of 964 K.pneumoniae and 1 389 E. coli isolates were retrospectively collected from national surveillance programs from 2011 to 2014 in China. Antimicrobial susceptibility testing was determined by the microdilution method.The PCR amplification followed by sequencing was used to detect the mcr-1 gene and colistin-resistance genes, including mgrB, pmrB and phoQ.Real-time quantitative PCR was performed to examine the relative transcriptional levels of pmrB, pmrC, pmrD, pmrK and pmrE genes in K.pneumoniae, and pmrA, pmrB, pmrC, phoP and phoQ genes in E.coli.Conjugation experiment was used to detect the transferability of the resistance plasmid carrying the mcr-1 gene.Statistical analyses were performed using IBM SPSS Statistics (version 16.0) and a P value <0.05 was considered statistically significant. Results The colistin-resistant rates of K. pneumoniae and E.coli were 0.62% ( 6/964 ) and 1.66% ( 23/1 389 ) , respectively.No amino acids substitutions were identified in mgrB genes among colistin-resistant isolates.Among six colistin-resistant K. pneumoniae isolates, five isolates were identified to have point mutations in pmrB gene, but no point substitution was detected in phoQ gene.One to four point mutations had been found in pmrB and phoQ genes in colistin-resistant E.coli isolates, respectively.The expression level of pmrB, pmrC, pmrD, pmrK and pmrE genes showed no significant difference between colistin-resistant and colistin-susceptible isolates [pmrB, (1.04 ±1.12) vs.(0.94 ±0.67), P=0.945; pmrC, (1.39 ±2.01) vs.(0.16 ±0.27), P=0.101;pmrD, (1.59 ±2.43) vs.(0.88 ±0.34),P=0.445;pmrK, (0.64 ±0.62) vs.(0.04 ±0.10), P=0.051;pmrE, (3 492 833 388.83 ±8 478 977 986.85) vs.(20 771 428.93 ±38 000 732.85), P=0.445].However, the transcriptional level of pmrB genes in colistin-resistant group was 9.5-fold higher than that of the colistin-susceptible group in E.coli isolates.Four in six colistin-resistant K.pneumoniae isolates possessed mcr-1 gene, whereas all of the colistin-resistant E. coli had the mcr-1 gene. The conjugation verified the transferability rate of the plasmid carrying mcr-1 gene was 5.78 ×10-6 , and the MIC value of colistin of the conjugant increased 21-fold than the recipient strain.Conclusions Plasmid-mediated mcr-1 gene was the major reason for colistin resistance in clinical isolates of K.pneumoniae and E.coli. Some other resistance mechanisms such as transcriptional up-regulated pmrB gene also involved in colistin resistance.