Investigation of Novel pmrB and eptA Mutations in Isogenic Acinetobacter baumannii Isolates Associated with Colistin Resistance and Increased Virulence In Vivo

Abstract

Colistin resistance in Acinetobacter baumannii is of great concern and is a threat to human health. In this study, we investigate the mechanisms of colistin resistance in four isogenic pairs of A. baumannii isolates displaying an increase in colistin MICs. A mutation in pmrB was detected in each colistin-resistant isolate, three of which were novel (A28V, I232T, and ΔL9-G12). Increased expression of pmrC was shown by semi-quantitative reverse transcription-PCR (qRT-PCR) for three colistin-resistant isolates, and the addition of phosphoethanolamine (PEtN) to lipid A by PmrC was revealed by mass spectrometry. Interestingly, PEtN addition was also observed in some colistin-susceptible isolates, indicating that this resistance mechanism might be strain specific and that other factors could contribute to colistin resistance. Furthermore, the introduction of pmrAB carrying the short amino acid deletion ΔL9-G12 into a pmrAB knockout strain resulted in increased pmrC expression and lipid A modification, but colistin MICs remained unchanged, further supporting the strain specificity of this colistin resistance mechanism. Of note, a mutation in the pmrC homologue eptA and a point mutation in ISAba1 upstream of eptA were associated with colistin resistance and increased eptA expression, which is a hitherto undescribed resistance mechanism. Moreover, no cost of fitness was observed for colistin-resistant isolates, while the virulence of these isolates was increased in a Galleria mellonella infection model. Although the mutations in pmrB were associated with colistin resistance, PEtN addition appears not to be the sole factor leading to colistin resistance, indicating that the mechanism of colistin resistance is far more complex than previously suspected and is potentially strain specific.

Abundance of Colistin-Resistant, OXA-23- and ArmA-Producing Acinetobacter baumannii Belonging to International Clone 2 in Greece

“…Similarly, the two ColS isolates from our study had pmrCAB alterations and a modified lipid A, as observed with mass spectrometry. All these observations support the hypothesis that additional and still unknown factors are involved in colistin resistance of clinical A. baumannii isolates (Jeannot et al, 2017;Gerson et al, 2019Gerson et al, , 2020. Determination of the cellenvelope charge could be useful in the elucidation process of the complex mechanism of colistin resistance in A. baumannii (Cafiso et al, 2019).…”

“…Colistin resistance may also result from the overexpression of etpA, a pmrC homolog. This is mediated by insertional inactivation of a gene encoding an H-NS family transcriptional regulator (Lucas et al, 2018) or by integration of insertion sequence elements upstream of the eptA gene itself (Gerson et al, 2019;Potron et al, 2019;Trebosc et al, 2019).…”

Vaccines Against Antimicrobial Resistance

“…This resistance is often associated with changes in the expression of regulators affecting LPS production. For example, the addition of phosphoethanolamine (PE) to lipid A due to overexpression of pmrC results in a substantial reduction of colistin binding as the negative charge of the LPS is hugely lowered (37).…”

Comparison of Fitness Cost and Virulence in Chromosome- and Plasmid-Mediated Colistin-Resistant Escherichia coli

“…In particular, two amino acid alterations in eptA (T148A and K233T) are probably associated with overexpression on the gene, which results in colistin resistance, because the strains with the mutations (E154R, QIA32, QIA33, and EC111) showed obvious high expression of eptA. The association of eptA mutation and colistin resistance has been suggested in several Gram-negative bacteria, including E. coli, K. pneumoniae, and A. baumannii (Lesho et al, 2013;Mathur et al, 2018;Xu et al, 2018;Gerson et al, 2019). The high variation of eptA in E139R may be due to genetic exchange of chromosomal portions including eptA, but its donor could not be identified.…”