PROJECT SUMMARY
Mycobacterium abscessus complex (MABC) has recently emerged as a significant cause of increasing cases of
both community- and hospital-acquired infections, especially among immunosuppressed populations, including
populations with cystic fibrosis and transplant patients. This situation is worsened by its exceptionally high natural
and acquired antibiotic resistance that complicates treatment, and consequently, complex and ineffective
antibiotic combinations have been tried with success rates below 50%. As a result, there is an urgent need to
improve therapeutic options for these infections. Current treatment recommendations for MABC infection usually
requires a single ß-lactam, either the cephalosporin, cefoxitin, or the carbapenem, imipenem, to be combined
with other drug classes, e.g. clarithromycin and amikacin. Recent studies and our preliminary results
demonstrated that combining two ß-lactams and/or a ß-lactamase inhibitor could be a successful strategy to
treat MABC infections. Our studies showed dual-ß-lactams (ceftazidime-imipenem or ceftazidime-ceftaroline)
had the greatest synergic effects against clinical MABC in vitro and in THP-1 cells, independent of ß-lactamase
inhibition with avibactam. These results provide a compelling scientific basis for our proposal to develop highly
active and targeted dual-ß-lactam combinations against MABC infections. As ß-lactam antibiotics primarily
target peptidoglycan synthesis, we will construct novel conditional peptidoglycan remodeling enzyme repressor
mutants to interrogate the molecular mechanisms underlying dual-ß-lactam synergy, and to probe promising
dual-ß-lactam pairs against MABC infections (Aim 1). As such, we will examine the interactions between various
peptidoglycan remodeling enzymes and a battery of ß-lactams, and build up a gene-compound interaction matrix
of dual-ß-lactam effects. Transcriptional analysis of dual-ß-lactams will be used as a complimentary approach to
reveal additional targets responsive to dual-ß-lactam treatment (Aim 1). The efficacy of putative ß-lactam
combinations will be examined against isolates collected in a well-established MABC clinical collection from over
60 cystic fibrosis clinical centers across the US (Aim 2). Spontaneous mutants conferring dual-ß-lactam
resistance and induced resistance mutants will be subjected to comparative genomic and RNAseq analysis to
identify the resistance mechanisms. MABC peptidoglycan enzyme (e.g. ß-lactamase and transpeptidases)
kinetic and hydrolysis assays will then be used to interrogate the enzymatic mechanism of ß-lactams or a ß-
lactamase inhibitor against MABC infections. Lastly, we will use the state-of-art hollow fiber infection model
(HFIM) and mouse models to test the preferred combinations of ß-lactams and their optimal doses, supported
by pharmacokinetic (PK) and pharmacodynamic (PD) analyses (Aim 3). At the conclusion of this project, we will
have developed novel dual-ß-lactam combination regimens against MABC, and unraveled the molecular
mechanism underlining the synergistic effects. In addition, the effectiveness the dual-ß-lactams against the
MABC isolates across the U.S. will be documented. The genomic output of this study will serve the basis for
future work on MABC drug development, rapid diagnostics and infection control measures. The knowledge
regarding peptidoglycan enzyme and ß-lactam interaction, and the repressor and induced mutant strains will be
a major resource for the MABC research community.