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.

The resistance mechanisms of bacteria against ciprofloxacin and new approaches for enhancing the efficacy of this antibiotic

“…Recently published studies reported additive or synergistic effects of ciprofloxacin and phage combination (134,(218)(219)(220)(221)(222)(223). Gurney et al reported that phages could interact with different structures of P. aeruginosa, such as lipopolysaccharide structure (LPS) and the efflux pumps.…”

Antimicrobial Drug Resistance in Poultry Production: Current Status and Innovative Strategies for Bacterial Control

“…These combinations allow the application of antibiotics in sub-inhibitory doses, leading to a reduction in the harmful effects of antibiotics in animals and humans. In addition, the combination of phages with antibiotics can potentiate antibiotic function, prolonging or restoring their activity against bacteria [105,106]. To the authors' best knowledge, this synergism is not fully characterized regarding the application of phages in poultry production; however, it is a factor that should be considered in the development of protocols for the treatment of bacterial infections using bacteriophages.…”

In Vitro Meropenem/Antibiotic and Meropenem/Bacteriophage Combinations Against Carbapenem-Resistant Gram-Negative Uropathogens

“…The phage lytic efficacy was determined as described by Nikolic et al [22] with few modifications. Briefly, 10-fold dilutions of phage lysates were prepared in 96-well microtiter plates in Mueller-Hinton broth.…”

“…The phage lytic efficacy of bacteriophages was determined by the method described by Nikolic et al [22]. The MOI that prevented bacterial growth was recorded and assigned 'minimal inhibitory MOI' (MIM).…”