<i>Enterococcus faecium sagA</i> mutants have cell envelope defects influencing antibiotic resistance and bacteriophage susceptibility

<i>Enterococcus faecium</i> is a gram-positive bacterium that is resident to the intestines of animals including humans. <i>E. faecium</i> is also an opportunistic pathogen that causes multidrug-resistant (MDR) infections. Bacteriophages (phages) have been proposed as therapeutics for the treatment of MDR infections; however, an obstacle for phage therapy is the emergence of phage resistance. Despite this, the development of phage resistance can impact bacterial fitness. Thus, understanding the molecular basis of fitness costs associated with phage resistance can likely be leveraged as an antimicrobial strategy. We discovered that phage-resistant <i>E. faecium</i> harbor mutations in the cell wall hydrolase gene <i>sagA</i>. SagA cleaves crosslinked peptidoglycan (PG) involved in PG remodeling. We show that mutations in <i>sagA</i> compromised <i>E. faecium</i> PG hydrolysis. One <i>sagA</i> mutant, with a defect in cell envelope integrity, increased cellular permeability, and aberrant distribution of penicillin-binding proteins, was also more sensitive to β-lactam antibiotics. These changes correspond to a growth defect where cells have abnormal division septa, membrane blebbing, and aberrant cell shape. The dysregulation of the cell envelope caused by the <i>sagA</i> mutation alters the binding of phages to the <i>E. faecium</i> cell surface, where phage infection of <i>E. faecium</i> requires phages to localize to sites of peptidoglycan remodeling. Our findings show that by altering the function of a single PG hydrolase, <i>E. faecium</i> loses intrinsic β-lactam resistance. This indicates that phage therapy could help revive certain antibiotics when used in combination.IMPORTANCE<i>Enterococcus faecium</i> causes hospital-acquired infections and is frequently resistant to frontline antibiotics, including those that target the cell wall. Bacteriophages represent a promising alternative to combat such infections. However, bacterial adaptation to phage predation often results in resistance. Such resistance is frequently accompanied by fitness trade-offs, most notably altered antibiotic susceptibility. This study provides mechanistic insights into phage resistance-associated antibiotic sensitivity in <i>E. faecium</i>. We show that phage-resistant <i>E. faecium</i> carrying a mutation in the peptidoglycan hydrolase SagA has compromised cell envelope integrity, mislocalized penicillin-binding proteins, and become sensitized to β-lactam antibiotics. These findings highlight the potential of reviving antibiotics when used in combination with phages in the clinical setting.