The most common mechanism of colistin resistance

The most common mechanism of colistin resistance is modification of LPS, a component of cell membrane, which is the first site of action of colistin. To determine whether inner and outer membranes were still present or intact in Col-R and Col-D A. baumannii strains, we visualized the 2 Col-S, 2 Col-R and 2 Col-D strains, by transmission electron microscopy. Some interesting differences between Col-S, Col-R, and Col-D were observed. For example, we found that the outer and inner membranes of 2 Col-S strains had intact, thick and uniform outer and inner membranes. However, 2 Col-R strains and 2 Col-D strains showed disrupted integrity, bifurcated membrane, and lost uniformity, resulting in beaded membranes in most strains, and some revealed loss of irregular periplasmic space. These changes resulted in the loss of defective LPS and defective membrane permeability, poor colistin binding and ultimate colistin resistance. Fatty acids 3-hydroxytetradecanoic equol (3-C14OH), 3-hydroxydodecanoic acid (3-C12OH), 2-hydroxytetradecanoic acid (2-C14OH), and dodecanoic acid (C12), hexadecanoic acid (C16), octadecanoic acid (C18) are crucial chemical compositions of lipid A of A. baumannii. To analyse whether incomplete cell membrane resulted in quantitative change of fatty acid composition in clinical Col-R and Col-D A. baumannii strains, we compared total fatty acid composition of Col-S strains with Col-R, Col-D strains, by Gas Chromatography interfaced with Mass Spectrometry (GC-MS). The results revealed that the composition of critically important long carbon chain saturated fatty acid including C12, C16, C18, altered significantly in the Col-S and Col-D strains. In particular, the C12 fatty acid contents decreased, but that of C16 and C18 increased compared to Col-S respectively. There were apparent distinctions in fatty acid component biosynthesis of alcohol-substituted-side chains and unsaturated fatty acid. Several other component also showed altered quantitative difference in Col-D and Col-R when compared to Col-S. Some colistin-resistant and dependent strains showed almost no alcohol substitution on the low carbon chain fatty acid, indicating decreased fatty acid biosynthesis. In comparing the detailed fatty acid compositions of the three groups of A. baumannii, it is important to recognize that Col-R and Col-D strains can vary greatly with colistin conditions. Expression of the pmrA/B genes was investigated by RT-PCR to determine whether differential expression was associated with colistin resistance in all the resistant and dependent clinical isolates, compared with that in the susceptible ones. Significantly elevated expression of pmrA and pmrB in Col-R and Col-D strains was higher when compared to Col-S. However, the expression patterns in terms of quantitative difference between pmrA and pmrB in each Col-R and Col-D isolates was found to be unaltered. In order to confirm whether colistin-dependent isolates have clearly different expression of pmrA and pmrB in different colistin induction condition or not, 2 Col-D (R3D, R12D) clinical A. baumannii strains were incubated in 10μg/ml, 100μg/ml and 256μg/ml of colistin broth separately. After several culture passages, we observed no significant differences in the expression of pmrA, or pmrB as revealed by RT-PCR. Thus, the upregulation of pmrA and pmrB may be a major mechanism underlying the colistin resistance in clinical Col-R and Col-D A. baumannii strains. The lpxA, lpxC and lpxD genes are involved in the first pivotal three steps in the pathway of lipid A biosynthesis, and the hydrophobic anchor of lipopolysaccharide (LPS). For drug resistant strains, various modifications of the lipid A can occur, which may not be essential for growth, but may strengthen the permeability barrier and influence the virulence of some pathogens. For the purpose of observing the impact of lpxA, lpxC and lpxD gene mutation on lipid A biosynthesis, we employed DNA-sequencing to analyze mutation of lpxA, lpxC and lpxD genes in Col-R and Col-D A. baumannii isolates and compared them with those of Col-S and the reference strain A. baumannii ATCC 19606. A certain number of different mutations were found, ranging from a single point mutation to large deletions of nucleotides within these three genes in 18 Col-R and 3 Col-D isolates. In this study, one of three colistin- dependent strains, R3D had 11bp nucleotides deletion in lpxA from locus 68 resulting in amino acids premature termination of lpxA translation after Valine (V) of locus 23. As a result, lipid A cannot be produced, which may result in loss of LPS. Therefore, not only A. baumannii generated resistance to colistin, but also relied on it for its growth. This evidence has been confirmed by TEM image trial and GC-MS fatty acid quantity. This finding confirmed that the mutation in lpxA and loss of LPS in clinical isolates of A. baumannii have the potential to develop colistin resistance as a result of LPS loss. A Col-D strain (R12D) had 3 nucleotides insertion in lpxC leading to amino acids mistranslation in the second step of lipid A biosynthesis and the failure to produce complete lipid A. Others had single point mutations producing single amino acids change. Among these gene mutations, one mutation (A to G) occurred in a large number of colistin resistant and dependent strains (10 Col-R and 2 Col-D) in lpxD resulting in amino acid change E138K. Analysis of the sequences showed that in almost all of Col-R and Col-D strains, there was a mutation or multiple mutations in one of the genes, lpxA, lpxC, or lpxD.