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In Vitro Phage-antibiotic Synergy Determination Assays

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Phage-antibiotic synergy (PAS) refers to sublethal concentrations of certain antibiotics that enhance the release of progeny phages from bacterial cells. The combination of bacteriophages and antibiotics during treatment is a promising strategy to reduce antibiotic doses and the development of antibiotic resistance. Bacteriophages (phages) have been considered a possible therapeutic option to enhance the efficacy of antibiotics. The use of phages in combination with clinically used antibiotics is one of the most attractive and feasible applications of phages. PAS has been widely used for the detection and validation of various phage antibiotic combinations. However, the methods used to assess these interactions are not defined and, as a result, the methods used vary widely. The detected in vitro interactions are calculated and interpreted as synergistic, additive, irrelevant, or antagonistic, depending on whether the antimicrobial activity of the combined drug is greater than, equal to, or less than that of the drug used alone. The synergistic effects between bacteriophages and antimicrobial agents are determined by broth microdilution limited series method, agar dilution method, or disk diffusion method.

Genetic Mechanism Underlying PAS

The initial infection of phages occurs by binding to receptors such as lipopolysaccharides, teichoic acid, proteins, and flagella on the bacterial surface. Bacterial surface molecules play an important role in the disease phenotype through mechanisms similar to receptor molecules on bacteriophages. Their components are commonly considered virulence factors, antibiotic resistance-related factors, and normal growth factors because they mediate host attachment and injury and antibiotic efflux, respectively. Phages and antibiotics act synergistically on host bacteria to alter the expression of bacterial virulence factors, antibiotic resistance, and growth factor activities. These mechanisms, in turn, can lead to increased antibiotic sensitivity or inhibition of bacterial growth.

Disk Agar Diffusion Assay

Using the disk diffusion method, it is possible to qualitatively examine the interaction, usually antagonistic, between two agents.

The PAS effect of phage ΦMFP on E. coli ΦMFP on Luria-Bertani agar plates.Fig.1 The PAS effect of phage ΦMFP on E. coli ΦMFP on Luria-Bertani agar plates. (Comeau, 2007)

Time-kill Assay

Among the available collaborative assays, a gold standard is a time-killing method. Phage antibiotic synergism obtained by time-killing is defined as a reduction in the number of bacteria when the subinhibitory concentration is combined with two or more logs compared to the number of bacteria treated with only one more potent drug. Again, synergy means a decrease in the number of bacteria relative to the initial number of bacteria. Although time-kill curve analysis is a gold standard, this method is not suitable for rapid and extensive screening of synergies. The reason for the infrequent use of time-killing curves is that the experimental procedure is robust and complex, requires more work, and consistently assays bacterial numbers over short time intervals.

Broth Microdilution Checkerboard Assay

Phage-antibiotic interactions are examined by checkerboard assay in 96-well plates. The broth microdilution checkerboard method is a two-dimensional two-agent broth microdilution test used to test the effects of chemical agents on specific microorganisms.

A microtiter plate with checkerboard to estimate the interaction between antibiotic and phage.Fig.2 A microtiter plate with checkerboard to estimate the interaction between antibiotic and phage. (Nikolic, 2022)

Creative Biolabs has extensive and deep knowledge and experience in the phage field to develop customized detection solutions. If you would like to know more details about our in vitro phage-antibiotic synergy determination assays services, or if you would like to consult the experts at Creative Biolabs, please feel free to contact us.


  1. Comeau, A.M.; et al. Phage-antibiotic synergy (PAS): β-lactam and quinolone antibiotics stimulate virulent phage growth. Plos one. 2007, 2(8): e799.
  2. Nikolic, I.; et al. An Optimized Checkerboard Method for Phage-Antibiotic Synergy Detection. Viruses. 2022. 14(7): 1542.
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