Phage-Antibiotic Synergy Is Driven by a Unique Combination of Antibacterial Mechanism of Action and Stoichiometry

Abstract

The continued rise in antibiotic resistance is precipitating a medical crisis. Bacteriophage (phage) has been hailed as one possible therapeutic option to augment the efficacy of antibiotics. However, only a few studies have addressed the synergistic relationship between phage and antibiotics. Here, we report a comprehensive analysis of phage-antibiotic interaction that evaluates synergism, additivism, and antagonism for all classes of antibiotics across clinically achievable stoichiometries. We combined an optically based real-time microtiter plate readout with a matrix-like heat map of treatment potencies to measure phage and antibiotic synergy (PAS), a process we term synography. Phage-antibiotic synography was performed against a pandemic drug-resistant clonal group of extraintestinal pathogenic Escherichia coli (ExPEC) with antibiotic levels blanketing the MIC across seven orders of viral titers. Our results suggest that, under certain conditions, phages provide an adjuvating effect by lowering the MIC for drug-resistant strains. Furthermore, synergistic and antagonistic interactions are highly dependent on the mechanism of bacterial inhibition by the class of antibiotic paired to the phage, and when synergism is observed, it suppresses the emergence of resistant cells. Host conditions that simulate the infection environment, including serum and urine, suppress PAS in a bacterial growth-dependent manner. Lastly, two different related phages that differed in their burst sizes produced drastically different synograms. Collectively, these data suggest lytic phages can resuscitate an ineffective antibiotic for previously resistant bacteria while also synergizing with antibiotics in a class-dependent manner, processes that may be dampened by lower bacterial growth rates found in host environments. IMPORTANCE Bacteriophage (phage) therapy is a promising approach to combat the rise of multidrug-resistant bacteria. Currently, the preferred clinical modality is to pair phage with an antibiotic, a practice thought to improve efficacy. However, antagonism between phage and antibiotics has been reported, the choice of phage and antibiotic is not often empirically determined, and the effect of the host factors on the effectiveness is unknown. Here, we interrogate phage-antibiotic interactions across antibiotics with different mechanisms of action. Our results suggest that phage can lower the working MIC for bacterial strains already resistant to the antibiotic, is dependent on the antibiotic class and stoichiometry of the pairing, and is dramatically influenced by the host microenvironment.

Improving the Inhibitory Effect of Phages against Pseudomonas aeruginosa Isolated from a Burn Patient Using a Combination of Phages and Antibiotics

“…Combinations of phages and antibiotics can enhance bacterial pathogen suppression, improve phage and/or antibiotic diffusion through biofilms, and reduce risks of development of resistance [ 25 ]. Phages can induce an evolutionary trade-off in multi-drug-resistant bacteria, leading to increased antibiotic sensitivity in phage-resistant mutants [ 26 , 27 ].…”

“…For example, combining ϕKZ phages with antibiotics that inhibit bacterial RNA polymerases (RNAPs), such as rifampin, should be synergistic, as ϕKZ phages encode their own RNAP and are thus not dependent on the bacterial host transcription for replication [ 28 ]. Synergistic interactions between Caudovirales phages and several antibiotics at different concentrations, including ceftazidime, ciprofloxacin, piperacillin, and meropenem, have been reported in multiple studies [ 24 , 25 , 29 ]. Yet, phage–antibiotic interactions are not always synergistic, and antagonistic and naturalistic interactions have also been described [ 24 , 25 , 30 ].…”

“…Synergistic interactions between Caudovirales phages and several antibiotics at different concentrations, including ceftazidime, ciprofloxacin, piperacillin, and meropenem, have been reported in multiple studies [ 24 , 25 , 29 ]. Yet, phage–antibiotic interactions are not always synergistic, and antagonistic and naturalistic interactions have also been described [ 24 , 25 , 30 ]. For example, bacteriostatic antibiotics such as tetracycline, which inhibit protein synthesis in bacteria, can interfere with phage production [ 25 ].…”

Characterization of a Novel Bacteriophage Henu2 and Evaluation of the Synergistic Antibacterial Activity of Phage-Antibiotics

“…coli , Klebsiella. Pneumoniae , and Burkholderia cepacian by different phage-antibiotic combinations [ 27 , 28 , 29 , 30 , 31 , 32 , 33 ]. Limited reports have confirmed the synergistic effect of phages and antibiotics against S. aureus [ 34 , 35 , 36 , 37 , 38 ].…”

Selection of Disease Targets for Phage Therapy

“…This drives the development of alternative approaches, and phage therapy is considered to be one of the most promising of these (Czaplewski et al 2016). In addition to its direct effects on bacteria, phages also in some cases can restore antibiotic sensitivity to targeted bacteria (Chan et al 2016;Harper 2008;Liu et al 2020). Given the very high specificity of bacteriophage therapeutics, it is necessary to identify a specific target.…”