Richard A Alm1, Michele R Johnstone2, Sushmita D Lahiri2. 1. Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, MA, USA richard.alm@astrazeneca.com. 2. Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, MA, USA.
Abstract
OBJECTIVES: The spread of NDM-1 amongst Enterobacteriaceae has highlighted a significant threat to the clinical management of serious infections. The combination of aztreonam and avibactam, a non-β-lactam β-lactamase inhibitor, may provide a much-needed therapeutic alternative. This combination was potent against most NDM-containing Enterobacteriaceae, although activity was diminished against many Escherichia coli isolates. These E. coli isolates were characterized to elucidate the mechanism of decreased susceptibility to aztreonam/avibactam. METHODS: MIC determinations were performed using broth microdilution, and whole-genome sequencing was performed to enable sequence-based analyses. RESULTS: The decreased susceptibility was not due to avibactam being unable to inhibit the serine β-lactamases found in the E. coli isolates. Rather, it was manifested by a four-amino-acid insertion in PBP3. This same insertion was also found in non-NDM-containing E. coli that had reduced susceptibility to aztreonam/avibactam. Construction of an isogenic mutant confirmed that this insertion resulted in decreased susceptibility to aztreonam and several cephalosporins, but had no impact on carbapenem potency. Structural analysis suggests that this insertion will impact the accessibility of the β-lactam drugs to the transpeptidase pocket of PBP3. CONCLUSIONS: The acquisition of β-lactamases is the predominant mechanism of β-lactam resistance in Enterobacteriaceae. We have demonstrated that small PBP3 changes will affect the susceptibility to a broad range of β-lactams. These changes were identified in multiple MLST lineages of E. coli, and were enriched in NDM-containing isolates. However, they were not present in other key species of Enterobacteriaceae despite significant conservation among the PBP3 proteins.
OBJECTIVES: The spread of NDM-1 amongst Enterobacteriaceae has highlighted a significant threat to the clinical management of serious infections. The combination of aztreonam and avibactam, a non-β-lactam β-lactamase inhibitor, may provide a much-needed therapeutic alternative. This combination was potent against most NDM-containing Enterobacteriaceae, although activity was diminished against many Escherichia coli isolates. These E. coli isolates were characterized to elucidate the mechanism of decreased susceptibility to aztreonam/avibactam. METHODS: MIC determinations were performed using broth microdilution, and whole-genome sequencing was performed to enable sequence-based analyses. RESULTS: The decreased susceptibility was not due to avibactam being unable to inhibit the serine β-lactamases found in the E. coli isolates. Rather, it was manifested by a four-amino-acid insertion in PBP3. This same insertion was also found in non-NDM-containing E. coli that had reduced susceptibility to aztreonam/avibactam. Construction of an isogenic mutant confirmed that this insertion resulted in decreased susceptibility to aztreonam and several cephalosporins, but had no impact on carbapenem potency. Structural analysis suggests that this insertion will impact the accessibility of the β-lactam drugs to the transpeptidase pocket of PBP3. CONCLUSIONS: The acquisition of β-lactamases is the predominant mechanism of β-lactam resistance in Enterobacteriaceae. We have demonstrated that small PBP3 changes will affect the susceptibility to a broad range of β-lactams. These changes were identified in multiple MLST lineages of E. coli, and were enriched in NDM-containing isolates. However, they were not present in other key species of Enterobacteriaceae despite significant conservation among the PBP3 proteins.
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