Mycobacterium abscessus is a rapidly growing non-tuberculous mycobacterium that causes a spectrum of opportunistic infections in humans. M. abscessus can sometimes be considered a respiratory colonizer, however, in the setting of structural lung conditions such as cystic fibrosis, bronchiectasis and COPD, M. abscessus can cause chronic pulmonary disease. In addition, immune competent post-menopausal Caucasian women are also vulnerable to M. abscessus infection. In these settings, Mab infections are often incurable and associated with rapid lung function decline. A growing number of clinical isolates of M. abscessus are resistant to most antibiotics, therefore, new treatment options that are effective against these drug-resistant strains are desperately needed. Like other bacteria, M. abscessus requires its peptidoglycan (bacterial exoskeleton) for viability. But, unlike most bacteria, M. abscessus uses two distinct enzyme classes, namely L,D- and D,D-transpeptidases, for the final step of synthesis of its peptidoglycan. We have generated preliminary data to demonstrate that the two classes of enzymes are bound and inhibited with varying potencies by different sub-classes of β-lactam antibiotics and shown that a combination of two β-lactams, one specific for each enzyme, exhibits synergy in vitro against M. abscessus. The overall objective of this proposal is to leverage these findings to identify combinations of β-lactams that will exhibit synergy in killing M. abscessus in vivo. Data from these exploratory studies will enable us to determine whether dual β-lactam is an effective strategy to treat M. abscessus. If our preliminary observations of in vitro synergy of dual β-lactam is confirmed in vivo (proposed here), we stand a real chance of developing new treatment regimen based on combination of two β-lactams to treat M. abscessus infections.