The role of LpxA/C/D and pmrA/B gene systems in colistin-resistant clinical strains of Acinetobacter baumannii

摘要

Objective: To study LpxA/C/D and pmrA/B gene systems role in colistin resistant clinical strains of Acinetobacter baumannii. Methods: Transmission Electron Microscopy (TEM) was employed to observe changes in the cell wall, inner and outer membranes. GC-Chromatography was applied to quantify the fatty acid content as a result of changes in the LPS in clinical A. baumannii strains. Furthermore, the isolates were subjected to molecular biology approaches employing Real-Time PCR to evaluate the mRNA expression levels of pmrA and pmrB. Colistin-resistant and colistin-dependent A. baumannii isolates were further screened by PCR amplification to determine mutations in lpxA, lpxC and lpxD genes responsible for lipid A biosynthesis. Results: Transmission Electron microscopy of six A. baumannii isolates showed that 2 colistin-resistant (Col-R) and 2 colistin-dependent (Col-D) A. baumannii had decreased integrity of the outer and inner membrane and lost uniformity in the periplasmic space compared with 2 susceptible (Col-S) Acinetobacter baumannii strains. GC-Chromatography indicated that there was a trend of decreased saturated and unsaturated fatty acid biosynthesis, especially long carbon chain (16:0, 17:0 and 18:0 carbon chains) and almost no alcohol substitution on the low carbon chain fatty acid (increased modification on the long chain fatty acid and the loss of most unidentified fatty acid peaks) in Col-D and Col-R strains in comparison with Col-S and ATCC19606 strains. The expression data from RT-PCR of PmrA/B two-component regulatory system suggest that upregulated gene expression in 4 Col-R and 3 Col-D strains may lead to a modification in and/or loss of lipid A. Lipid A biosynthesis genes sequencing results revealed deletion of 11bp nucleotides and change of one nucleotide in lpxA, and a nucleotide point mutation and insertion in lpxC and lpxD of Col-D and Col-R strains resulting in defective lipid A production and outer membrane lipid synthesis. Conclusion: Mechanisms of colistin resistance in clinical strains of A. baumannii show that colistin may not serve as an antibiotic of the last resort for treating MDR A. baumannii infections when other antibiotics are ineffective. The mechanisms of colistin resistance should provide an impetus for future research on the development of newer alternative therapies to treat emerging MDR A. baumannii.