Antibiotic resistance represents a growing health crisis that necessitates the immediate discovery of novel treatment strategies. One such strategy is the identification of collateral sensitivities, wherein evolution under a first drug induces susceptibility to a second. Here, we report that sequential drug regimens derived from in vitro evolution experiments may have overstated therapeutic benefit, predicting a collaterally sensitive response where cross-resistance ultimately occurs. We quantify the likelihood of this phenomenon by use of a mathematical model parametrised with combinatorially complete fitness landscapes for Escherichia coli. Through experimental evolution we then verify that a second drug can indeed stochastically exhibit either increased susceptibility or increased resistance when following a first. Genetic divergence is confirmed as the driver of this differential response through targeted and whole genome sequencing. Taken together, these results highlight that the success of evolutionarily-informed therapies is predicated on a rigorous probabilistic understanding of the contingencies that arise during the evolution of drug resistance.

Antibiotic resistance represents a growing health crisis that necessitates the immediate discovery of novel treatment strategies. One such strategy is the identification of collateral sensitivities, wherein evolution under a first drug induces susceptibility to a second. Here, we report that sequential drug regimens derived from in vitro evolution experiments may have overstated therapeutic benefit, predicting a collaterally sensitive response where cross resistance ultimately occurs. We quantify the likelihood of this phenomenon by use of a mathematical model parametrised with combinatorially complete fitness landscapes for Escherichia coli. Through experimental evolution we then verify that a second drug can indeed stochastically exhibit either increased susceptibility or increased resistance when following a first. Genetic divergence is confirmed as the driver of this differential response through targeted and whole genome sequencing. Taken together, these results highlight that the success of evolutionarily-informed therapies is predicated on a rigorous probabilistic understanding of the contingencies that arise during the evolution of drug resistance.

Impeding, as well as reducing, the burden of antimicrobial resistance in Gram-negative pathogens is an urgent public health endeavor. Our current antibiotic armamentarium is dwindling, while major resistance determinants (e.g., extended-spectrum β-lactamases [ESBLs]) continue to evolve and disseminate around the world. One approach to attack this problem is to develop novel therapies that will protect our current agents. AAI101 is a novel penicillanic acid sulfone β-lactamase inhibitor similar in structure to tazobactam, with one important difference. AAI101 possesses a strategically placed methyl group that gives the inhibitor a net neutral charge, enhancing bacterial cell penetration. AAI101 paired with cefepime, also a zwitterion, is in phase III of clinical development for the treatment of serious Gram-negative infections. Here, AAI101 was found to restore the activity of cefepime against class A ESBLs (e.g., CTX-M-15) and demonstrated increased potency compared to that of piperacillin-tazobactam when tested against an established isogenic panel. The enzymological properties of AAI101 further revealed that AAI101 possessed a unique mechanism of β-lactamase inhibition compared to that of tazobactam. Additionally, upon reaction with AAI101, CTX-M-15 was modified to an inactive state. Notably, the in vivo efficacy of cefepime-AAI101 was demonstrated using a mouse septicemia model, indicating the ability of AAI101 to bolster significantly the therapeutic efficacy of cefepime in vivo. The combination of AAI101 with cefepime represents a potential carbapenem-sparing treatment regimen for infections suspected to be caused by Enterobacteriaceae expressing ESBLs.

Multidrug-resistant (MDR) Acinetobacter spp. poses a significant therapeutic challenge in part due to the presence of chromosomally encoded β-lactamases, including class C Acinetobacter-derived cephalosporinases (ADC) and class D oxacillinases (OXA), as well as plasmid-mediated class A β-lactamases. Importantly, OXA-like β-lactamases represent a gap in the spectrum of inhibition by recently approved β-lactamase inhibitors such as avibactam and vaborbactam. ETX2514 is a novel, rationally designed, diazabicyclooctenone inhibitor that effectively targets class A, C, and D β-lactamases. We show that addition of ETX2514 significantly increased the susceptibility of clinical Acinetobacter baumannii isolates to sulbactam. AdeB and AdeJ were identified to be key efflux constituents for ETX2514 in A. baumannii. The combination of sulbactam and ETX2514 was efficacious against A. baumannii carrying blaTEM-1, blaADC-82, blaOXA-23, and blaOXA-66 in a neutropenic murine thigh infection model. We also show that, in vitro, ETX2514 inhibited ADC-7 (k2/Ki 1.0 ± 0.1 × 106 M−1 s−1) and OXA-58 (k2/Ki 2.5 ± 0.3 × 105 M−1 s−1). Cocrystallization of ETX2514 with OXA-24/40 revealed hydrogen bonding interactions between ETX2514 and residues R261, S219, and S128 of OXA-24/40 in addition to a chloride ion occupied in the active site. Further, the C3 methyl group of ETX2514 shifts the position of M223. In conclusion, the sulbactam-ETX2514 combination possesses a broadened inhibitory range to include class D β-lactamases as well as class A and C β-lactamases and is a promising therapeutic candidate for infections caused by MDR Acinetobacter spp. IMPORTANCE The number and diversity of β-lactamases are steadily increasing. The emergence of β-lactamases that hydrolyze carbapenems poses a significant threat to our antibiotic armamentarium. The explosion of OXA enzymes that are carbapenem hydrolyzers is a major challenge (carbapenem-hydrolyzing class D [CHD]). An urgent need exists to discover β-lactamase inhibitors with class D activity. The sulbactam-ETX2514 combination demonstrates the potential to become a treatment regimen of choice for Acinetobacter spp. producing class D β-lactamases.

The presence of β-lactamases (e.g., PDC-3) that have naturally evolved and acquired the ability to break down β-lactam antibiotics (e.g., ceftazidime and ceftolozane) leads to highly resistant and potentially lethal Pseudomonas aeruginosa infections. We show that wild-type PDC-3 β-lactamase forms an acyl enzyme complex with ceftazidime, but it cannot accommodate the structurally similar ceftolozane that has a longer R2 side chain with increased basicity. A single amino acid substitution from a glutamate to a lysine at position 221 in PDC-3 (E221K) causes the tyrosine residue at 223 to adopt a new position poised for efficient hydrolysis of both cephalosporins. The importance of the mechanism of action of the E221K variant, in particular, is underscored by its evolutionary recurrences in multiple bacterial species. Understanding the biochemical and molecular basis for resistance is key to designing effective therapies and developing new β-lactam/β-lactamase inhibitor combinations.