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 handful of 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 heatmap 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 E. coli (ExPEC) with antibiotic levels blanketing the minimum inhibitor concentration (MIC) across seven orders of viral titers. Our results suggest that, under certain conditions, phages provide an adjuvating effect by lowering the MIC for drugresistant 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, phage burst size seems to be a significant driver of synergism.Collectively, this data suggests lytic phages can resuscitate an ineffective antibiotic for previously resistant bacteria, while also synergize with antibiotics in a class-dependent manner, processes that may be dampened by lower bacterial growth rates found in host environments. Significance StatementBacteriophage (phage) therapy is a promising approach to combat the rise of multi-drug 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.

Evaluation of Staphylococcal Bacteriophage Sb-1 as an Adjunctive Agent to Antibiotics Against Rifampin-Resistant Staphylococcus aureus Biofilms

“…Previous studies revealed synergistic effects by combining phage with an antibiotic for which the bacteria strain was resistant (Liu et al, 2020). In our study, however, the use of Sb-1 in combination with rifampin against biofilms of RRSA strains, as well as Sb-1/fosfomycin and Sb-1/gentamicin combinations against MRSA3 and MRSA ATCC 43300, respectively, did not reveal an improved anti-biofilm effect.…”

In vitroanalysis of colistin and ciprofloxacin antagonism ofPseudomonas aeruginosaphage PEV2 infection activities

“…In combination therapies, two or more agents possessing different mechanisms of action can enhance each other’s activities 78–80 , broaden activity spectra 78,81–83 , reduce the potential for resistance evolution 84–91 , or improve penetration into biofilms 92 . One common aspect of phage therapy treatments is combining phages with antibiotics 38,92–102 . Here we have assessed the potential of different concentrations of two different classes of antibiotics, colistin and ciprofloxacin, to interfere with infection activities of P. aeruginosa phage PEV2 and other tested phages at bacterial growth-inhibiting antibiotic concentrations (≥MIC).…”

Temperate phage-antibiotic synergy across antibiotic classes reveals new mechanism for preventing lysogeny

“…To test synergy across antibiotics, the standard is a checkerboard assay (Martinez-Irujo et al, 1996), also referred to as synograph (Liu et al, 2020). This previously revealed tPAS reduced the effective ciprofloxacin MIC up to 32-fold, despite the poor effectiveness of phage alone condition (Al-Anany et al, 2021).…”

“…PAS appears to be broadly applicable, spanning phages across the myoviridae (Chan et al, 2016;Comeau et al, 2007;Jansen et al, 2018;Kebriaei et al, 2020;Kirby, 2012;Liu et al, 2021;Malik et al, 2021;Ryan et al, 2012;Simon et al, 2021;Styles et al, 2020;Wang et al, 2020) siphoviridae (Knezevic et al, 2013;Li et al, 2021), and podoviridae families (Manohar et al, 2022;Save et al, 2022;Styles et al, 2020;Torres-Barceló et al, 2014Verma et al, 2009). In addition to phages, PAS has been demonstrated in many hosts: Klebsiella pneumoniae (Bedi et al, 2009;Pacios et al, 2021;, Pseudomonas aeruginosa (Chan et al, 2016;Chaudhry et al, 2017;Coulter et al, 2014;Duplessis et al, 2021;Engeman et al, 2021;Hagens et al, 2006;Oechslin et al, 2017), Actinobacter baumannii (Styles et al, 2020), Staphylococcus aureus (Chhibber et al, 2013;Dickey et al, 2019;Iqbal et al, 2020;Kaur and Chhibber, 2021;Kebriaei et al, 2020;Van Nieuwenhuyse et al, 2021;Yilmaz et al, 2013) and E. coli (Kim et al, 2021;Liu et al, 2020;Moradpour et al, 2020). PAS has also been demonstrated across antibiotics of multiple classes inclu...…”