New Mutations Involved in Colistin Resistance in Acinetobacter baumannii

Abstract

Colistin is used as the "last resort" to treat infections caused by multidrug-resistant Acinetobacter baumannii, which is at the top of the World Health Organization's list of the most dangerous bacterial species that threaten human health. Unfortunately, colistin resistance has emerged in A. baumannii To broaden the study of the resistance mechanism of colistin in A. baumannii, we obtained colistin-resistant mutants via two methods: (i) screening and isolation from a mariner-based A. baumannii ATCC 19606 transposon mutant library; (ii) selection from challenge of ATCC 19606 with successively increasing concentrations of colistin. A total of 41 mutants with colistin MIC of 4 μg/ml to 64 μg/ml were obtained by transposon mutant library screening. Five highly resistant mutants with colistin MICs ranging from 256 μg/ml to 512 μg/ml were selected from successive colistin challenges. Genotypic complementation and remodeling of the transposon mutants revealed that the genes inactivated by the transposon insertion were not responsible for resistance. Whole-genome sequence analysis of the colistin-resistant strains revealed that the main causes of the resistance to colistin were mutations in the pmrA-pmrB genes, including pmrA^P102R, pmrB^P233S, and pmrB^T235N and the novel alleles pmrA^I13M and pmrB^Q270P Interestingly, we found that miaA^I221V mutation of A. baumannii strain ATCC 19606 (pmrA^P102R) resulted in 4-fold increases in the colistin MIC, which rose from 32 μg/ml to 128 μg/ml. But miaA^I221V itself had little effect on the colistin susceptibility of ATCC 19606. These data broaden knowledge of the scope of chromosomally encoded mechanisms of resistance to colistin.IMPORTANCEAcinetobacter baumannii is an important Gram-negative opportunistic pathogen commonly infecting critically ill patients. It possesses a remarkable ability to survive in the hospital environment and acquires resistance determinants corresponding to a wide range of antibacterial agents. Given that the current treatment options for multidrug resistant A. baumannii are extremely limited, colistin administration has become the treatment of last resort. However, colistin-resistant A. baumannii strains have recently been reported. The mechanism of resistance to colistin in A. baumannii has rarely been reported. Here, we found two novel mutations in pmrA (I13M) and pmrB (Q270P) that caused colistin resistance. It is also first reported here that the presence of miaA with a I221V mutation enhanced the colistin resistance of pmrA^P102R.

Keywords: Acinetobacter baumannii; colistin; mechanism of resistance.

FIG 1

The transposon plasmid map and genetic map of mariner transposon insertions. (A) pMarinerAb plasmid map. p15A is the replication origin of E. coli. oriAb is the replicon of A. baumannii from pWH1266. Ptrc is used to control the transposase gene “Himar1 C9” expression derived from Haematobia irritans. The transposase recognizes the terminal inverted repeats (TIRs) to perform the excision of the transposon DNA body, which is inserted into a TA target site. TetR is the tetracycline resistance marker for pMarinerAb plasmid-transferred selection. KanR is a transposon of the kanamycin resistance gene used for isolation of transposon mutants. (B) Transposon insertion sites in A. baumannii mutants. Insertions in the plus orientation are marked on the circle exterior. Insertions in the minus orientation are marked on the circle interior. Numbers indicate the precise point of insertion according to genome sequence data for A. baumannii ATCC 19606.

FIG 2

Quantification of relative transcriptional levels of pmrCAB genes in colistin-resistant A. baumannii mutants versus the ATCC 19606 parental strain. The experiments were performed in triplicate. Bars represent means ± standard deviations. *, 0.01≤P ≤ 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant.

FIG 3

Analysis of cytochrome c binding to different A. baumannii strains, including ATCC 19606, ATCC 19606 (pmrA^P102R), ATCC 19606 (miaA^I221V), ATCC 19606 (pmrA^P102R, miaA^I221V), ATCC 19606 (pmrA^I13M), and ATCC 19606 (pmrB^Q270P). The experiments were performed in triplicate. Bars represent means ± standard deviations. *, 0.01≤P ≤ 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant.

FIG 4

Negative-ion mode MALDI-TOF MS mass spectra of lipid A from colistin-susceptible and colistin-resistant A. baumannii strains ATCC 19606, ATCC 19606 (pmrA^P102R, miaA^I221V), ATCC 19606 (pmrA^P102R), ATCC 19606 (pmrA^I13M), and ATCC 19606 (pmrB^Q270P). The insets show partial enlarged views to display the ions at m/z 2,033.