A Novel Method to Monitor the Evolution of Antimicrobial Resistance in Acinetobacter baumannii Biofilms

Biofilms are microbial communities embedded in an extracellular matrix that facilitates their attachment to surfaces. This lifestyle provides advantages to pathogenic bacteria, including increased survival in the presence of antibiotics and an enhanced capacity to develop resistance. Once a biofilm is established, infections get difficult to eradicate and frequently become chronic. There is, therefore, an urgent need to develop novel strategies to counteract biofilm-associated antibiotic resistance. Here, we developed a method to monitor the evolution of antimicrobial resistance, aiming to evaluate novel drugs against bacterial resistance to antibiotics. We validated this methodology using an RNase chimera with antibiofilm activity and a reported ability to hinder colistin resistance in planktonic cultures of <i>Acinetobacter baumannii</i> (<i>A. baumannii</i>). We assessed the emergence of resistance in <i>A. baumannii</i> biofilms by repeated cycles of colistin exposure. This method not only preserves biofilm structure throughout treatment but also enables controlled induction of resistance acquisition while monitoring antimicrobial efficacy. Although the RNase enhanced the antibiotic's activity against biofilms by reducing by 50% the effective dose, it did not prevent the emergence of colistin resistance, indicating that the protein may use distinct mechanisms against planktonic and biofilm communities. Nonetheless, our findings highlight the potential of this methodology for evaluating antibiotic-adjuvant candidates to combat antibiotic resistance in biofilms.