Rough-type and loss of the LPS due to lpx genes deletions are associated with colistin resistance in multidrug-resistant clinical Escherichia coli isolates not harbouring mcr genes.

The emergence of multidrug-resistant Escherichia coli has become a great challenge in treating nosocomial infections. The polymyxin antibiotic colistin is used as a 'last-line' therapy for such strains, but resistance to colistin is increasingly emerging all over the world. In this study, we investigated lipopolysaccharides (LPS) of colistin-resistant isolates and examined mutations in lpx genes in strains not harbouring mcr genes. We examined 351 clinical E. coli isolates with 38 showing reduced susceptibility to colistin. These isolates were collected from different clinical specimens including blood, urine, and wounds, but no stool. After confirmation of the isolates via a BD Phoenix-100 system (Becton Dickinson, USA), we performed antimicrobial susceptibility tests to characterize the resistance pattern of these isolates to different classes of antibiotics, using the disk diffusion test. The Minimum Inhibitory Concentration (MIC) of colistin was determined using E-test strips. The presence of mobile colistin resistance (mcr-1 and mcr-2) genes was tested for all isolates. LPS (including lipid A) were extracted from all isolates and associated lpx genes analyzed by PCR and sequencing. Among the 38 clinical E. coli isolates with reduced susceptibility to colistin, 52% were resistant to colistin. The MICs of colistin ranged from 0.5 μg/ml to ˃256 μg/ml. Within the 20 colistin-resistant strains, six isolates carried the mcr-1 gene, but not mcr-2. Heterologous expression of the mcr-1 gene in susceptible E. coli DH5α increased the MIC of colistin by eight-fold. The remaining 14 isolates, were negative for both mcr genes. Six isolates were further negative for LPS production and five showed rough LPS phenotypes. Here we present evidence that loss of LPS or lipid A-deficiency can lead to colistin-resistance in clinical E. coli isolates not harbouring mcr genes.