An Optimized Checkerboard Method for Phage-Antibiotic Synergy Detection

Abstract

Phage-antibiotic synergy is a promising therapeutic strategy, but there is no reliable method for synergism estimation. Although the time-kill curve assay is a gold standard, the method is not appropriate for fast and extensive screening of the synergy. The aim is to optimize the checkerboard method to determine phage-chemical agent interactions, to check its applicability by the time-kill curve method, and to examine whether the synergy can be obtained with both simultaneous and successive applications of these agents. In addition, the aim is to determine interactions of the Pseudomonas phage JG024 with ciprofloxacin, gentamicin, or ceftriaxone, as well as the Staphylococcus phage MSA6 and SES43300 with ciprofloxacin, gentamicin, and oxacillin. The results show that the optimized checkerboard method is reliable and that results correspond to those obtained by the time-kill curve. The synergy is detected with the phage JG024 and ciprofloxacin or ceftriaxone against Pseudomonas aeruginosa, and the phage SES43300 with ciprofloxacin against MRSA. The synergy was obtained after simultaneous applications, and in the case of P. aeruginosa, after application of the second agent with delay of one hour, indicating that simultaneous application is the best mode of synergy exploitation for therapy. The checkerboard method can be used for thorough clinical studies on synergy and in the future for personalized therapy when infections are caused by multiple resistant bacteria.

Keywords: checkerboard method; phage-antibiotic synergy; time-kill curve.

Figure 1

A microtiter plate with checkerboard to estimate interaction between antibiotic and phage (the plate represents combination of CIP and phage JG024 against P. aeruginosa). The explanation is in the text.

Figure 2

Effects of different simultaneous combinations of JG024 and CIP (A), GEN (B) or CRO (C) on the growth of PA14 strain obtained with checkerboard (I) and time-kill method (II).

Figure 3

Effects of different simultaneous combinations of SES43300 and CIP (A-I), GEN (B) or OXC (C) on the growth of S. aureus ATCC43300 strain obtained with checkerboard and time-kill method for SES 43300 and CIP (A-II).

Figure 4

Effect of successive treatment with combination of CIP and JG024 with 1 h delay for each agent obtained with checkerboard (I) and time-kill (II); (A) addition of JG024 1 h later; (B) addition of CIP 1 h later.

Figure 5

Effect of successive treatment with combination of CIP and JG024 with 6 h delay for each agent obtained with checkerboard (I) and time-kill (II). (A) addition of JG024 6 h later; (B) addition of CIP 6 h later.

Figure 6

Effect of successive treatment with combination of GEN and JG024 with 1 h delay for each agent obtained with checkerboard (I) and time-kill (II). (A) addition of JG024 1 h later; (B) addition of GEN 1 h later.

Figure 7

Effect of successive treatment with combination of CRO and JG024 with 1 h delay for each agent obtained with checkerboard (I) and time-kill (II). (A) addition of JG024 1 h later; (B) addition of CRO 1 h later.