Over-expression of AdeABC efflux pump stimulated continuously by the mutated AdeRS two component system has been found to result in antimicrobial resistance, even tigecycline (TGC) resistance, in multidrug-resistant Acinetobacter baumannii (MRAB). Although the insertion sequence, ISAba1, contributes to one of the AdeRS mutations, the detail mechanism remains unclear. In the present study we collected 130 TGC-resistant isolates from 317 carbapenem resistant MRAB (MRAB-C) isolates, and 38 of them were characterized with ISAba1 insertion in the adeS gene. The relationship between the expression of AdeABC efflux pump and TGC resistant was verified indirectly by successfully reducing TGC resistance with NMP, an efflux pump inhibitor. Further analysis showed that the remaining gene following the ISAba1 insertion was still transcribed to generate a truncated AdeS protein by the Pout promoter on ISAba1 instead of frame shift or pre-termination. Through introducing a series of recombinant adeRS constructs into a adeRS knockout strain, we demonstrated the truncated AdeS protein was constitutively produced and stimulating the expression of AdeABC efflux pump via interaction with AdeR. Our findings suggest a mechanism of antimicrobial resistance induced by an aberrant cytoplasmic sensor derived from an insertion element.

Thirteen clinical isolates of m ultidrug- r esistant Acinetobacter baumannii r esistant to c arbapenems (MRAB-C) with tigecycline nonsusceptibility were collected from individual patients in this study. None of the 13 isolates shared the same strain characteristics in molecular typing. All of them showed increased adeB transcription, as predicted. However, none of these tigecycline-nonsusceptible MRAB-C isolates were found to possess previously known adeRS mutations. Upregulation of adeB transcription may result from cross stimulation by other mechanisms.

Amino acid substitutions within the AdeRS two-component system are believed to result in overexpression of the AdeABC efflux pump and extensive resistance to antibiotics in clinical Acinetobacter baumannii isolates. However, the exact amino acid substitutions in AdeRS that cause overexpression of the AdeABC efflux pump remain unclear. We elucidated the role of amino acid substitutions in AdeRS by a complementation assay in an adeRS knockout strain of A. baumannii. Five types of adeRS operon from tigecycline-resistant XDR A. baumannii (XDRAB) were cloned and introduced into the adeRS knockout strain to reverse its tigecycline susceptibility. Through shuffling gene segments among those five adeRS operons and performing site-directed mutagenesis, we found that the specific amino acid substitution Gly186Val in AdeS is crucial for reducing tigecycline susceptibility of A. baumannii. Our result demonstrates that a critical amino acid substitution in AdeS alters the AdeABC efflux pump-mediated tigecycline resistance of A. baumannii.