The Gram-positive bacterium Staphylococcus aureus, a frequent colonizer of humans, can also cause infections such as bacteremia, osteomyelitis or sepsis. Along with its pathogenicity, S. aureus also has the ability to be resistant to antibiotics such as β-lactams, making it one of the leading causes of global mortality. Classically, β-lactam resistance is attributed to mecA (or mecC), as seen in methicillin resistant S. aureus (MRSA). Prior studies identified PBP4 (Penicillin Binding Protein-4), a protein involved in cell wall synthesis, as a non-classical mediator of resistance. Mutations associated with the regulatory region of the gene (Ppbp4) led to increased expression of PBP4 that subsequently caused increased cell wall cross-linking, while missense mutations in the pbp4 gene led to structural alterations, both of which resulted in β-lactam resistance. Using techniques in microbiology and molecular genetics, this study substantiated the potential of PBP4 as a novel mediator of β-lactam resistance by first demonstrating the clinical relevance of pbp4-associated mutations and their corresponding phenotypic alterations. The Ppbp4 region is shared with a divergently transcribed gene, abcA, which encodes a multidrug exporter. This study confirmed that Ppbp4 mutations caused an upregulation of pbp4 and downregulation of abcA, confirming that the resistant phenotype was attributed to PBP4 overexpression. In prior experiments, pbp4-associated mutations in resistant strains were largely accompanied by loss-of-function mutations of gdpP, a phosphodiesterase. This study demonstrated that alterations of PBP4 and GdpP led to synergistic, broad-spectrum resistance to β-lactams at levels comparable to that seen by MRSA. Lastly, the effect of PBP4 on bacterial virulence was inspected using C. elegans, the results of which indicated that β-lactam resistance mediated via PBP4-overexpression likely came at the cost of virulence. This study emphasizes the relevance of pbp4-associated mutations as a clinically significant cause of β-lactam resistance, not only by itself but also in synergy with altered GdpP. The experimental findings put forth the suggestion of considering PBP4 as an important target not only for development of therapeutics, but also during diagnosis of S. aureus infections.