An engineered prodrug selectively suppresses β-lactam resistant bacteria in a mixed microbial setting

Abstract The rise of β-lactam resistance necessitates new strategies to combat bacterial infections. We purposefully engineered the β-lactam prodrug AcephPT to exploit β-lactamase activity to selectively suppress resistant bacteria producing extended-spectrum-β-lactamases (ESBLs). Selective targeting of resistant bacteria requires avoiding interaction with penicillin-binding proteins, the conventional targets of β-lactam antibiotics, while maintaining recognition by ESBLs to activate AcephPT only in resistant cells. Computational approaches provide a rationale for structural modifications to the prodrug to achieve this biased activity. We show AcephPT selectively suppresses gram-negative ESBL-producing bacteria in clonal populations and in mixed microbial cultures, with effective selectivity for both lab strains and clinical isolates expressing ESBLs. Time-course NMR experiments confirm hydrolytic activation of AcephPT exclusively by ESBL-producing bacteria. In mixed microbial cultures, AcephPT suppresses proliferation of ESBL-producing strains while sustaining growth of β-lactamase-non-producing bacteria, highlighting its potential to combat β-lactam resistance while promoting antimicrobial stewardship. Competing Interest Statement The authors have declared no competing interest. Abbreviations - Bla - β-lactamase - ESBL - extended-spectrum-β-lactamase - SBL - serine-β-lactamase - MBL - metallo-β-lactamase - PBP - penicillin-binding protein - RMSD - root mean squared deviation - RMSF - root mean squared fluctuation - EC50 - half maximal effective concentration - PT - pyrithione - ARLG - Antibiotic Resistance Leadership Group - DICON - Duke Infection Control Outreach Group - DiRTE - Disinfection, Resistance, and Transmission Epidemiology Lab - NMR - nuclear magnetic resonance - DEPT - distortionless enhancement by polarization transfer - HPLC/MS - high performance liquid chromatography/mass spectrometry - HIC - hydrophobic interaction chromatography - HILIC - hydrophilic interaction chromatography - HRMS - high resolution mass spectrometry - ESI - electrospray ionization - OD600 - optical density at 600 nm - Ex586/Em620 - excitation at 585 nm, emission at 620 nm - CFU - colony forming unit - X-Gal - 5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside - IPTG - isopropyl β-D-1-thiogalactopyranoside - TLC - thin layer chromatography - MD - molecular dynamics - LB - Luria Bertani Broth (Lennox) - MHB - 2 Mueller Hinton Broth 2 - DMSO - dimethyl sulfoxide - DCM - dichloromethane - TFA - trifluoracetic acid - TIPS - triisopropylsilane