Shang Lu1, Dan Li2, Leilei Wang2, Yingmin Bi2, Minghua Wang3, Fan Yang4. 1. Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, People's Republic of China; Key Laboratory of Clinical Pharmacology of Antibiotics, Ministry of Health, Shanghai, People's Republic of China; Department of Respiratory and Critical Care Medicine, The First people's Hospital of Yancheng, Affiliated Hospital 4 of Nantong University, Yancheng, People's Republic of China. 2. Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, People's Republic of China; Key Laboratory of Clinical Pharmacology of Antibiotics, Ministry of Health, Shanghai, People's Republic of China. 3. Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, People's Republic of China; Key Laboratory of Clinical Pharmacology of Antibiotics, Ministry of Health, Shanghai, People's Republic of China. Electronic address: mhwang007@126.com. 4. Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, People's Republic of China; Key Laboratory of Clinical Pharmacology of Antibiotics, Ministry of Health, Shanghai, People's Republic of China. Electronic address: fanyang9@fudan.edu.cn.
Abstract
OBJECTIVES: Colistin resistance mediated by plasmids for their rapid dissemination in Enterobacteriaceae is alarming. We aimed to characterize the genetic features of mcr-1 gene as well as the role of promoters in gene expression and levels of colistin resistance among clinical isolates of Enterobacteriaceae. METHODS: Clinical isolates of Enterobacteriaceae were collected in thirteen cities in China and screened for mcr-1 gene using polymerase chain reaction (PCR) amplification and sequencing. Antimicrobial susceptibility testing, transformation assay and plasmid sequencing, quantitative real-time PCR were performed for mcr-1-positive isolates. Promoter-probe vector pKK232-8 was utilized to assess the activity of the mcr-1 promoters. RESULTS: This study identified the mcr-1 gene in 15 clinical isolates of Enterobacteriaceae, among which 14 were resistant to colistin, with MICs of 4-8 mg/L, while one mcr-1-bearing isolate EC09 was susceptible to colistin, with an MIC of 0.5 mg/L. Moreover, mcr-1-harbouring plasmids from 10 clinical isolates were transferrable via transformation and belonged to different incompatibility groups (IncI2 and IncX4). Plasmid pEC09 failed to transform and belonged to IncP1. A genetic structure containing the mcr-1-pap2 element was detected in these plasmids. EC09 demonstrated the lowest transcription level of mcr-1 gene, as determined by quantitative real-time PCR, which was in accordance with its susceptibility to colistin. Furthermore, the promoter activity of mcr-1 in pEC09 was the lowest, as determined by promoter-probe vector pKK232-8. CONCLUSION: Promoter variations were associated with expression of the mcr-1 gene and ultimately the levels of colistin resistance.
OBJECTIVES: Colistin resistance mediated by plasmids for their rapid dissemination in Enterobacteriaceae is alarming. We aimed to characterize the genetic features of mcr-1 gene as well as the role of promoters in gene expression and levels of colistin resistance among clinical isolates of Enterobacteriaceae. METHODS: Clinical isolates of Enterobacteriaceae were collected in thirteen cities in China and screened for mcr-1 gene using polymerase chain reaction (PCR) amplification and sequencing. Antimicrobial susceptibility testing, transformation assay and plasmid sequencing, quantitative real-time PCR were performed for mcr-1-positive isolates. Promoter-probe vector pKK232-8 was utilized to assess the activity of the mcr-1 promoters. RESULTS: This study identified the mcr-1 gene in 15 clinical isolates of Enterobacteriaceae, among which 14 were resistant to colistin, with MICs of 4-8 mg/L, while one mcr-1-bearing isolate EC09 was susceptible to colistin, with an MIC of 0.5 mg/L. Moreover, mcr-1-harbouring plasmids from 10 clinical isolates were transferrable via transformation and belonged to different incompatibility groups (IncI2 and IncX4). Plasmid pEC09 failed to transform and belonged to IncP1. A genetic structure containing the mcr-1-pap2 element was detected in these plasmids. EC09 demonstrated the lowest transcription level of mcr-1 gene, as determined by quantitative real-time PCR, which was in accordance with its susceptibility to colistin. Furthermore, the promoter activity of mcr-1 in pEC09 was the lowest, as determined by promoter-probe vector pKK232-8. CONCLUSION: Promoter variations were associated with expression of the mcr-1 gene and ultimately the levels of colistin resistance.
Authors: Carlos Bastidas-Caldes; Jacobus H de Waard; María Soledad Salgado; María José Villacís; Marco Coral-Almeida; Yoshimasa Yamamoto; Manuel Calvopiña Journal: Pathogens Date: 2022-06-08