Characterization of phage-antibiotic interaction through different in vitro methods: case study of a vibriophage

Climate change and warmer oceans will amplify the impacts on public health of waterborne harmful microorganisms. Phagotherapy offers a promising alternative; but as of today, phages can only be administered to patients when delivered along with antibiotics. Understanding possible interactions between these agents - indifference, synergy or antagonism - is thus a pivotal point. While several methods exist for characterizing such interaction, consensus on a reference method is still lacking. In this work, we screen and compare several in vitro characterization methods, using as a model nt-1, a phage of Vibrio natriegens, and studying its interaction with cefotaxime, a 3G cephalosporine. The different methods highlight different aspects of the interaction, depending whether they focus on phage or bacterial biomass. Overall, we see evidence of antagonism between the studied phage and antibiotic: this antagonism is at its optimum for antibiotic concentration of minimum inhibitory concentration (MIC)/2. Given the non-linear nature of interaction, it appears essential to use multiplexed methods and to cross technics. AUTHOR SUMMARYCurrently, antimicrobial resistance results in close to one million victims per year worldwide. In response to this alarming situation, new antimicrobial drugs and alternative therapies with innovative mechanisms have to be developed, such as phage therapy. It relies on the use of specific bacterial viruses, called bacteriophages (phages), that are therefore natural antibacterial agents. This therapy is strongly investigated for its potential to stop bacteria whenever antibiotics are no longer effective. Phage therapy is a highly personalized approach especially because of the narrow specificity of phages. Understanding how the efficiency of phages could be improved by the use of other antimicrobials, such as antibiotics, is essential in the fight against pathogens. Using a combination of a phage and an antibiotic, instead of only an antibiotic, imposes to think about new in-vitro tests for susceptibility testing. In the particular case of Vibrio bacteria, a common genus of waterborne pathogens, we investigated the efficiency of a phage in presence of cefotaxime, a last resort antibiotic, through different in-vitro methods, in liquid phase as well as on agar media. We observed a decreased efficiency of the phage, in other words an antagonism, especially at the lowest concentrations.