Is phage cocktail always the best choice? Antibacterial efficacy of individual and cocktail of phages against antibiotic-resistant Campylobacter jejuni.

Campylobacteriosis is one of the leading foodborne illnesses, arising primarily from consuming undercooked poultry meat due to chickens infected with Campylobacter jejuni. This contamination, from chicken gut to meat can occur during post-harvest processing. As such, there needs to be a mechanism that would maintain the freshness and safety of poultry meat during its shelf life. Bacteriophages (phages) have been well recognised as safe and effective against foodborne pathogens and recently a cocktail of campylobacter phages were granted GRAS status by FDA. We aim to characterize and determine the efficacy of previously isolated campylobacter phages against tetracycline resistant campylobacter jejuni CA-2046 in poultry carcass as well as immobilized in packaging such as absorbent pads. Four phages were selected (CPS1, CPS2, F198, WSS1) and their host range, efficiency of plating, bacteriophage insensitive mutant frequency, thermal stability, and shelf-life stability were determined. All four phages have the same broad host range activity, showing activity against seven out of eighteen strains of C. jejuni and C. coli tested. Transmission electron microscopy was performed to determine morphology of the four phages, all of which were classified as belonging to Group III, Myovirus. They all have icosahederal heads and contractile tails connected with a small terminal globular vesicle at their contracted distal ends. Thermal stability data indicates survival of the four phages at maximum temperature of 60oC for 60 minutes, apart from CPS1 which is active for 30 minutes not 60 minutes. Shelf-life stability was determined at 0, 2, 6 and 9 months. By 9 months, log reduction of 3-5 log PFU/mL was observed for phages CPS2, F198, and WSS1 at 25oC and between 1 and <1 log reduction at -20oC. This was followed by the determination of efficacy of the phages, individually and as a cocktail, against CA-2046 (104 CFU/mL) in broth for 24 hours using optical density and log reduction. The phages were added based on two multiplicities of infections, 10 and 1000. When phages were tested individually, it was observed that at MOI 10, CPS2 achieved highest log reduction of 3.10 log CFU/mL whereas at MOI 1000, CPS1 showed highest log reduction of 4.65 log CFU/mL amongst the four phages. When the four phages were equally combined to form a cocktail, at MOI 10, a log reduction of 1.77 log CFU/mL was observed which corresponded to the log reduction achieved by CPS1 (1.86 log CFU/mL) when it was tested individually at the same MOI. Similarly, at MOI 1000, the cocktail of phages showed a 2.09 log CFU/mL reduction which was almost half that observed when the individual phages (especially CPS2) were tested at the same MOI. The OD600 readings corresponded with the log reductions observed at 24 hours. The two phages that provided the highest log reduction, CPS1 and CPS2, were then selected to create a cocktail and the above experiment repeated. This time, it was observed that with MOI 1000, a 4.11 log CFU/mL reduction was observed which was comparable to the reduction observed with CPS2 when used individually. To the best of our knowledge, this is the first time an antagonistic activity of campylobacter phages in a cocktail has been observed. Bacteriophage insensitive mutant frequency was determined for the two selected phages and observed to be high, 6.49 x 10-3 and 5.80 x 10-3 for CPS1 and CPS2, respectively. However, when these mutant colonies were purified and re-tested for resistance, they appeared to be re-infected with all four phages indicating that while mutants may arise at a higher than usual frequency, they re-vert back to their original virulence and can be re-infected by our phages.