Optimizing Propagation of Staphylococcus aureus Infecting Bacteriophage vB_SauM-phiIPLA-RODI on Staphylococcus xylosus Using Response Surface Methodology

Abstract

The use of bacteriophages for killing pathogenic bacteria is a feasible alternative to antibiotics and disinfectants. To obtain the large quantities of phages required for this application, large-scale production of bacteriophages must be optimized. This study aims to define conditions that maximize the phage yield of the virulent and polyvalent staphylococcal bacteriophage vB_SauM-phiIPLA-RODI in broth culture, using the food-grade species Staphylococcus xylosus as the host strain to reduce the risk of growing massive quantities of pathogenic bacteria and therefore, to ensure the safety of the final phage stock. The effect of four variables, namely initial bacterial concentration (5.66–8.40 log10 colony-forming unit (CFU)/mL), initial phage concentration (5–8 log10 plaque-forming unit (PFU)/mL), temperature (21–40 °C) and agitation (20–250 rpm), on phage yield (response) was studied by using response surface methodology (RSM). Successive experimental designs showed that agitation did not significantly impact phage yield, while temperature did have a significant effect, with 38 °C being the optimum for phage propagation. The results allowed the design of a model to describe phage yield as a function of the initial bacterial and phage concentrations at fixed agitation (135 rpm), and optimum temperature (38 °C). The maximum experimental phage yield obtained was 9.3 log10 PFU/mL, while that predicted by the model under the optimized conditions (7.07 log10 CFU/mL initial bacterial population and 6.00 log10 PFU/mL initial phage titer) was 9.25 ± 0.30 log10 PFU/mL, with the desirability of 0.96. This yield is comparable to that obtained when the phage was propagated on the original host, Staphylococcus aureus. Bacteriophage phiIPLA-RODI showed the same host range and very similar biofilm removal ability regardless of the staphylococcal species used for its propagation. The results presented in this study show the suitability of using a food-grade strain of S. xylosus for the propagation of S. aureus infecting phages and the application of RSM to define the optimal propagation conditions.

Combined Use of the Ab105-2φΔCI Lytic Mutant Phage and Different Antibiotics in Clinical Isolates of Multi-Resistant Acinetobacter baumannii

“…In addition, this effect was observed in the bacterial killing assays, where the growth at 6 h was greater at MOI10 than at MOI1. The mutant lytic phage also presented a latent period of 30 min, and a moderate burst size of 32 ± 2 PFU per cell was obtained with the mutant lytic phage, values in the range of those obtained in several studies for different lytic phages, including phages from A. baumannii [5,36,37,38,39]. The burst size is inversely related to the risk of emergence of phage resistant bacteria [40], which is one of the main objectives of phage therapy research, commonly addressed by the use of phage cocktails [41].…”

A scaled‐down model for the translation of bacteriophage culture to manufacturing scale

“…Fold expansion and population doubling are used within mammalian cell research and allow authors to easily cross compare their data to others. With phage fermentation research increasing cross comparison this will become more important and phage fermentation research may benefit from a similar method (González‐Menéndez et al, ; Kumar & Starly, ; Sanz‐Ruiz et al, )…”

“…Fold expansion and population doubling are used within mammalian cell research and allow authors to easily cross compare their data to others. With phage fermentation research increasing cross comparison this will become more important and phage fermentation research may benefit from a similar method (González-Menéndez et al, 2018;Kumar & Starly, 2015;Sanz-Ruiz et al, 2017) 3.6 | Infection kinetics: adsorption and burst size analysis greatest titer conditions are shown in Figure 4. A statistically significant reduction in the number of free phages was observed at all time points for both T4 and phage K when compared with the baseline process.…”

Combined Use of the Ab105-2ф&Delta;CI Lytic Mutant Phage and Different Antibiotics in Clinical Isolates of Multiresistant <em>Acinetobacter Baumannii</em>

“…At MOI 10, inhibition of growth was greater and occurred earlier than at other MOI, but resistant bacteria emerged earlier than at lower MOI. The mutant lytic phage also presented a latent period of 30 minutes, and a moderate burst size of 32±2 PFU per cell was obtained with the mutant lytic phage including phages from A. baumannii [33][34][35][36]. The burst size is inversely related to the risk of emergence of phage resistant bacteria [37], which is one of the main objectives of phage therapy research, commonly addressed by the use of phage cocktails.…”