Penicillin-binding proteins (PBPs) are essential for bacterial cell wall biosynthesis, and several are clinically validated antibacterial targets of β-lactam antibiotics. We identified mutations in the mrdA gene encoding the PBP2 protein in two Escherichia coliblaNDM-1 clinical isolates that reduce susceptibility to carbapenems and to the intrinsic antibacterial activity of a diazabicyclooctane (DBO) PBP2 and β-lactamase inhibitor. These mutations coexisted with previously described mutations in ftsI (encoding PBP3) that reduce susceptibility to monobactams, penicillins, and cephalosporins. Clinical exposure to β-lactams is driving the emergence of multifactorial resistance that may impact the therapeutic usefulness of existing antibacterials and novel compounds that target PBPs.IMPORTANCE Emerging antibacterial resistance is a consequence of the continued use of our current antibacterial therapies, and it is limiting their utility, especially for infections caused by multidrug-resistant isolates. β-Lactams have enjoyed extensive clinical success, but their broad usage is linked to perhaps the most extensive and progressive example of resistance development for any antibacterial scaffold. In Gram-negative pathogens, this largely involves constant evolution of new β-lactamases able to degrade successive generations of this scaffold. In addition, more recently, alterations in the targets of these compounds, penicillin-binding proteins (PBPs), are being described in clinical isolates, which often also have multiple β-lactamases. This study underscores the multifactorial nature of β-lactam resistance by uncovering alterations of PBP2 that reduce susceptibility to carbapenems in E. coli clinical isolates that also have alterations of PBP3 and express the NDM-1 β-lactamase. The changes in PBP2 also reduced susceptibility to the intrinsic antibacterial activity of some diazabicyclooctane (DBO) compounds that can target PBP2. This may have implications for the development and use of the members of this relatively newer scaffold that are inhibitors of PBP2 in addition to their inhibition of serine-β-lactamases.