Strategy for Mass Production of Lytic Staphylococcus Aureus Bacteriophage pSa-3: Contribution of Multiplicity of Infection

Abstract

BackgroundAntibiotic-resistant bacteria have emerged as a serious problem; bacteriophages have, therefore, been proposed as a therapeutic alternative to antibiotics. Several authorities, such as pharmacopeia, FDA, have confirmed their safety, and some bacteriophages are commercially available worldwide. The demand for bacteriophages is expected to increase exponentially in the future; hence, there is an urgent need to mass-produce bacteriophages economically. Unlike the replication of non-lytic bacteriophages, lytic bacteriophages are replicated by lysing host bacteria, which leads to the termination of phage production; hence, strategies that can prolong the lysis of host bacteria in bacteria-bacteriophage co-cultures are required.ResultsIn the current study, we manipulated the inoculum concentrations of Staphylococcus aureus and phage pSa-3 (multiplicity of infection, MOI), and their energy sources to delay the bactericidal effect while optimizing phage production. We examined an increasing range of bacterial inoculum concentration (2 × 108 to 2 × 109 CFU/mL) to decrease the lag phase, in combination with a decreasing range of phage inoculum (from MOI 0.01 to 0.00000001) to delay the lysis of the host. Bacterial concentration of 2 × 108 CFU/mL and phage MOI of 0.0001 showed the maximum final phage production rate (1.68 × 1010 plaque forming unit (PFU)/mL). With this combination of phage-bacteria inoculum, we selected glycerol, glycine, and calcium as carbon, nitrogen, and divalent ion sources, respectively, for phage production. After optimization using response surface methodology, the final concentration of lytic Staphylococcus phage was 8.63 × 1010 ± 9.71 × 109 PFU/mL (5.13 fold increase).ConclusionsTherefore, Staphylococcus phage pSa-3 production can be maximized by increasing the bacterial inoculum and reducing the seeding phage MOI, which this combinatorial strategy could decrease phage production time. Further, we suggest that response surface methodology has the potential for optimizing mass production of lytic bacteriophages.

Statistical optimization of a podoviral anti-MRSA phage CCASU-L10 generated from an under sampled repository: Chicken rinse

“…Glycerol and peptone were favored by phage CCASU-L10. This agreed partially with a previous study on the staphylococcal Kayvirus psa-3, which stated that glycerol and glycine were the preferred sources (Kim et al, 2020).…”

“…Contrary to expectations, CCASU-L10 leaned towards the inoculum size of 10 7 CFU/ml. This was close to the result announced by Kim et al in the same research, who chose 10 8 CFU/ml to be the optimum level (Kim et al, 2020). Temperature is a crucial culture condition.…”

“…So, to ensure the reproducibility of the production process with a high phage yield, handling the phage-host interaction, and the co-culture could be a promising scaling-up strategy. Hence, optimization of phage CCASU-L10 production on laboratory scale was the last step in this study (Grieco et al, 2012;Gonzaĺez-Meneńdez et al, 2018b;Kim et al, 2020). However, practically deciding on the ideal parameter combination of a certain process is costly and time-consuming.…”