Impact of colistin resistance on adaptive virulence mechanisms of Acinetobacter baumannii

Colistin resistance in Acinetobacter baumannii is an emerging problem that limits antimicrobial therapy options. In this study, we aimed to identify the adaptation of A. baumannii to antimicrobial and environmental stress by interchanging virulence traits. This study included two patient cohorts. The first cohort consists of 2 pairs of colistin-susceptible (K408, K1007) and colistin-resistant (K409, K1006) clonal isolates from 2 individual patients. The second cohort consists of 21 isolates (ColS=11, ColR=10) from 21 patients. Colistin susceptible isolates were exposed to in vitro colistin induction for 50 generations with and without exposure to HgCl2. The selected cell populations of colistin-susceptible, resistant, and in vitro-resistant induced isolates were subjected to DNA and RNA sequencing and phenotypic assays. Comparative virulence traits were examined using SNP analysis, transcriptomic analysis, biofilm formation assay, C. elegans fertility assay, bacterial adherence, cellular internalization, and cytokine expression assays. In the in vitro colistin induction assay, K408 gained colistin resistance on the corresponding day of clinical resistance (K408-G25) and got resensitized to colistin in the consecutive generation (K408-G26) while K1007 did not gain colistin resistance amongst 50 generations. On the other hand, in the in vitro colistin and HgCl2 co-exposure, K408 and K1007 developed resistance on 3rd generation and 5th generation, respectively. A significant upregulation of ompW, ata, and adeFGH genes on K408-G25 was followed by a downregulation upon resensitization to colistin (G26). Despite the upregulation of the ompW gene in transcriptomic analysis, the ompW protein disappeared on K408-G25 and recovered in the resensitized generation (G26). In parallel, disrupted cell membrane integrity and serum resistance activity were recovered in K408-G26. The ΔompW K408 strain exhibited significant downregulation of OMPs (ompA, carO) and adeFGH efflux pump operon (p=0.0026). Bacterial adherence and intercellular internalization assays revealed that colistin-resistant isolates (K408 G25, K408HG3, K409) exhibited lower affinity of adherence and invasion compared to the colistin susceptible cells. In parallel, proinflammatory cytokine expression was significantly higher in colistin susceptible cells (p= 0.0395). In the 2nd cohort, ompW was significantly downregulated with lower protein abundance (p=0.004). Additionally, in the resistant cohort, the pmrCAB and adeFGH operons were significantly upregulated (p<0.001). Eventually, clinically colistin resistant and in vitro colistin resistance induced isolates (K408 G25, K408 HG3, K409, K1006) exhibited lower C. elegans virulence scores compared to the colistin susceptible (K408, K1007) and colistin resensitized (K408 G26) cells. These results were further confirmed by the second cohort with statistically lower virulence scores (p=0.0224). In this study, we showed that, during the early stages of response to colistin stress until the development of chromosomal resistance, Acinetobacter baumannii regulates adhesion, efflux pump, and serum resistance associated virulence traits. Moreover, mercury exposure plays an important role in facilitating colistin resistance. Colistin resistant cells lose their ability to cell invasion and intercellular persistence. Eventually, the ompW protein is an unstable outer membrane protein which may result in suboptimal outcomes for the ompW targeted immunisation strategies in the prevention of colistin resistant A. baumannii infections.