When does antimicrobial resistance increase bacterial fitness? Effects of dosing, social interactions, and frequency dependence on the benefits of AmpC β-lactamases in broth, biofilms, and a gut infection model

One of the longstanding puzzles of antimicrobial resistance is why the frequency of resistance persists at intermediate levels. Theoretical explanations for the lack of fixation of resistance include cryptic costs of resistance or negative frequency-dependence but are seldom explored experimentally. β-lactamases, which detoxify penicillin-related antibiotics, have well-characterized frequency-dependent dynamics driven by cheating and cooperation. However, bacterial physiology determines whether β-lactamases are cooperative, and we know little about the sociality or fitness of β-lactamase producers in infections. Moreover, media-based experiments constrain how we measure fitness and ignore important parameters such as infectivity and transmission among hosts. Here, we investigated the fitness effects of broad-spectrum AmpC β-lactamases in Enterobacter cloacae in broth, biofilms, and gut infections in a model insect. We quantified frequency- and dose-dependent fitness using cefotaxime, a third-generation cephalosporin. We predicted that infection dynamics would be similar to those observed in biofilms, with social protection extending over a wide dose range. We found evidence for the sociality of β-lactamases in all contexts with negative frequency-dependent selection, ensuring the persistence of wild-type bacteria, although cooperation was less prevalent in biofilms, contrary to predictions. While competitive fitness in gut infections and broth had similar dynamics, incorporating infectivity into measurements of fitness in infections significantly affected conclusions. Resistant bacteria had reduced infectivity, which limited the fitness benefits of resistance to infections challenged with low antibiotic doses and low initial frequencies of resistance. The fitness of resistant bacteria in more physiologically tolerant states (in biofilms, in infections) could be constrained by the presence of wild-type bacteria, high antibiotic doses, and limited availability of β-lactamases. One conclusion is that increased tolerance of β-lactams does not necessarily increase selection pressure for resistance. Overall, both cryptic fitness costs and frequency dependence curtailed the fitness benefits of resistance in this study.