(272a) Sequence-Specific Antisense Inhibitors of Antibiotic Resistance and Mechanism of Adaptive Resistance

The recent surge of drug-resistant superbugs and shrinking antibiotic pipeline are serious challenges to global health. In particular, the emergence of β-lactamases has caused extensive resistance against the most frequently prescribed class of β-lactam antibiotics. Furthermore, resistance has also developed to the β-lactam/β-lactamase inhibitor combinations due to extended spectrum β-lactamases such as carbapenemases and New Delhi metallo-β-lactamase 1, providing another avenue for widespread antibiotic resistance. Here, we develop novel synthetic peptide nucleic acid based antisense inhibitors that target the start codon and ribosomal binding site of the TEM-1 β-lactamase transcript and act via translation inhibition mechanism. We show that these antisense inhibitors are capable of re-sensitizing drug-resistant Escherichia coli to β-lactam antibiotics exhibiting 10 fold reduction in the minimum inhibitory concentration (MIC). To study the mechanism of resistance, we adapted E. coli at MIC levels of the β-lactam/antisense inhibitor combination and observed a non-mutational, bet-hedging based adaptive antibiotic resistance response as evidenced by phenotypic heterogeneity as well as heterogeneous expression of key stress response genes. Mutants that developed tolerance to the RNA inhibitor/β-lactam combination exhibited heterogeneous expression of representative stress response genes corresponding to the multiple antibiotic resistance (mar) regulon, general stress response, and SOS response; implying role of gene expression heterogeneity in development of resistance. Our data shows that both the development of new antimicrobials and understanding the cellular response during development of tolerance to could aid in mitigating the impending antibiotic crisis.