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

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## Abstract

**:**

_{10}colony-forming unit (CFU)/mL), initial phage concentration (5–8 log

_{10}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 log

_{10}PFU/mL, while that predicted by the model under the optimized conditions (7.07 log

_{10}CFU/mL initial bacterial population and 6.00 log

_{10}PFU/mL initial phage titer) was 9.25 ± 0.30 log

_{10}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.

## 1. Introduction

## 2. Materials and Methods

#### 2.1. Bacterial Strains, Bacteriophage and Media

^{9}colony-forming unit (CFU)/mL) of the host strains were mixed with 100 μL of serial phage dilutions. These mixtures were added to 5 mL of molten TSA overlay (0.7% agar), poured onto TSA plates, incubated at 37 °C for 18–24 h, and the lysis plaques counted [16].

#### 2.2. Biofilm Removal by Phage phiIPLA-RODI Propagated on S. xylosus CTC1642 and S. aureus IPLA1

^{6}CFU/mL into fresh TSB supplemented with 0.25% glucose. Aliquots of 200 μL of each culture were poured into the wells of a polystyrene microtiter plate (TC Microwell 96U w/lid nunclon DSI plates, Thermo Scientific, Madrid, Spain). Biofilms were grown for 24 h at 37 °C. Wells were then washed twice with PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM 138 Na

_{2}HPO

_{4}and 2 mM KH

_{2}PO

_{4}; pH 7.4). To compare the biofilm degradation ability of each phage lysate, 200 μL of phiIPLA-RODI propagated on S. aureus IPLA1 or S. xylosus CTC1642 were added to each well (10

^{8}plaque-forming unit (PFU)/well). SM buffer (20 mM Tris HCl, 10 mM of MgSO

_{4}, 10 mM of Ca(NO

_{3})

_{2}and 0.1 M of NaCl, pH 7.5) was added for control purposes. The microwell plates were incubated for 4 h at 37 °C. The supernatants were removed, and wells washed once with SM buffer (20 mM Tris HCl, 10 mM MgSO

_{4}, 10 mM Ca(NO

_{3})

_{2}and 0.1 M NaCl, pH 7.5) and air-dried for 15 min at room temperature. The biomass adhered to the wells was determined by crystal violet (0.1%, w/v) staining as described previously [25]. All the assays were performed using three biological replicates.

#### 2.3. One-Step Growth Curve

_{3})

_{2}(10 mmol L

^{−1}) and MgSO

_{4}(10 mmol L

^{−1}) using a multiplicity of infection (MOI) of 0.01. Mid-exponential-phase cultures (10 mL) of S. aureus IPLA1 and S. xylosus CTC1642 (OD

_{600}= 0.1) were collected by centrifugation and suspended into 1 mL of fresh TSB. The phage was added and allowed to adsorb for 5 min at 37 °C with shaking. The mixture was then centrifuged, pelleted cells resuspended in 10 mL of TSB, and incubation continued at 37 °C. Samples were first taken at 5 min intervals for 30 min, and subsequently at 10 min intervals. Each sample was immediately diluted and plated for phage titration.

#### 2.4. Bacteriophage Amplification: Conventional Phage Propagation

_{600}= 0.1 (10

^{7}CFU/mL) was reached. Phage was added to the bacterial culture at MOI of 1.0 and incubation proceeded for a further 3.5 h at 37 °C with shaking. Phage preparations were obtained by centrifugation and further filtration to remove bacterial cells and debris.

#### 2.5. Bacteriophage Amplification: Phage Propagation for Optimization Purposes

^{8}CFU/mL) were quickly thawed and used to inoculate at different concentrations (CFU/mL) in 50 mL Falcon tubes filled with 10 mL of TSB broth. The actual viable cell counts were determined immediately after inoculation by plating decimal dilutions of samples onto TSA.

_{10}values) was evaluated after 3.5 h of incubation. The phage titer was determined as described in the previous section.

#### 2.6. Experimental Design

_{10}PFU/mL); initial bacterial concentration (6.0 to 8.0 log

_{10}CFU/mL); temperature (21 to 37 °C) and agitation (20 to 250 rpm). The objective of the second design was focused on the effect of temperature, using a D-optimal design, at fixed agitation (135 rpm). It had the following ranges for the variables: initial phage titer (6.0 to 8.0 log

_{10}PFU/mL); initial bacterial concentration (5.0 to 7.0 log

_{10}CFU/mL) and temperature (34 to 40 °C). Finally, the third design aimed at developing a Response Surface equation, using Central Composite Design, to predict phage production in the region of highest yield. It included only the initial phage titer (5.79 to 7.21 log

_{10}PFU/mL) and initial bacterial concentration (5.59 to 8.41 log

_{10}CFU/mL), while temperature and agitation were fixed at 38 °C and 135 rpm, respectively. The characteristics of these designs (and their respective yields) are summarized in Table 1. The levels of variables for all the designs were given by the program Design-Expert software version 7.0 (State-Ease, Inc., Minneapolis, MN, USA), provided their ranges and type of design. The order of the run performance was always randomly chosen. However, the values for initial bacterial populations, although intended to be those proposed by the designs, were difficult to fix accurately. Therefore, the actual bacterial concentrations reached just after inoculation, as determined by viable cell counts, were used for the statistical analysis.

#### 2.7. Analysis of Results, Model Validation, and Final Response Surface (RS) Equation

_{10}PFU/mL and the initial bacterial populations around the levels of maximal phage yield. A final equation for the model was developed by enlarging the data from the third design with the validation results. This last model was used for obtaining the conditions which maximize the phage yield and amplification.

#### 2.8. Statistical Analysis

_{i}(Y

_{i}) assigns numbers between 0 (undesirable value) and 1 (ideal response) to the possible values of Y

_{i}(phage yield or phage amplification ratio). Usually, the individual desirability values are combined using the geometric mean, which gives the overall desirability (D) which is maximized with respect to the controlled variables.

## 3. Results

#### 3.1. PhiIPLA-RODI Infects Food-Grade S. xylosus Strains and Other Staphylococcal Species

_{10}PFU/mL) was significantly higher (p < 0.05) than the value of suspensions propagated on S. xylosus (8.2 ± 0.2 log

_{10}PFU/mL).

#### 3.2. Identification of Experimental Factors Affecting Phage Yield

_{10}PFU/mL and from 5.00 to 7.00 log

_{10}CFU/mL respectively, to include the experimental regions of high phage yield.

#### 3.3. Response Surface Model for Phage phiIPLA-RODI Yield

_{10}PFU/mL (Table 1), the highest titer found so far. That is, the third design pointed to combinations of the variables which resulted in maximum response (Figure 4), due to the Equation (1) structure.

#### 3.4. Validation of RSM

#### 3.5. Final Equations for the Phage Production and Phage Amplification Ratio

_{10}CFU/mL. The Equation (2) was quite similar to that obtained when using only the data from the third design (Equation (1)) and also reached the maximum at a very close initial bacterial concentration (6.85 vs. 6.82 log

_{10}CFU/mL). The predicted phage yields for the validation data using this final equation were similar to those deduced previously from the third design, but the predictions had a lower dispersion (0.5 vs. 0.3 SE) (Table 5, last two columns).

_{10}PFU/mL, which was not significantly different from that obtained with the reference strain of S. aureus IPLA1 (8.9 ± 0.2 log

_{10}PFU/mL) (p > 0.05).

_{10}) is multiplied during the propagation process). The RS model estimated was also significant, had a non-significant lack of fit (Equation (3)), and was quite similar to that previously deduced for phage yield (Equation (2)). It took the following form:

_{10}CFU/mL (18.63/2 × 1.37, since the first derivative at the maximum, should be null). However, it may also be obtained considering the initial bacteria and phage titer simultaneously. With this objective, the desirability approach was applied, using the following criteria: initial bacterial concentration within the range 6–9 log

_{10}CFU/mL, minimum initial phage titer in the range 6–8 log

_{10}PFU/mL, maximum phage yield and phage amplification ratio. The results indicated that, by using initial bacterial and phage concentrations of about 7.07 log

_{10}CFU/mL and 6.00 log

_{10}PFU/mL, respectively, a remarkable phage yield (9.25 ± 0.9 log

_{10}PFU/mL) and phage amplification ratio (2.72 ± 0.90) could be obtained (Figure 5), with a total desirability of 0.96 (quite close to the ideal value of 1.00). Therefore, the process may reach the highest phage yield and, at the same time, maximum amplification (the initial phage is increased by almost 3 log units).

## 4. Discussion

_{10}CFU/mL. This behavior could be due to the adsorption of a proportion of the new viral progeny to the host cells that were not initially infected, which could partially hamper its detection by phage titration.

_{10}CFU/mL; and initial phage titer, 6.00 log

_{10}PFU/mL (Figure 5), while maintaining agitation (135 rpm) and temperature (38 °C) at their fixed levels. Using these conditions, it is expected a phage yield of up to 9.25 ± 0.35 log

_{10}CFU/mL along with a phage amplification ratio of 2.72 log units over the initial phage titer. The global desirability, defined as the geometric mean of desirability values of the phage yield and phage amplification ratio, would be in this case high (0.96) and fairly close to the most “desirable” response 1.00. These results support the use of the food-grade strain S. xylosus CTC1642 as an appropriate alternative of the pathogenic strain S. aureus IPLA1 for phiIPLA-RODI propagation at the setting conditions established by the final RS model. The setting parameters could be the starting point for performing the upscaled production of the phage that would be required for its potential use in clinical [28] and food safety [15] applications.

## Supplementary Materials

## Acknowledgments

## Author Contributions

## Conflicts of Interest

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**Figure 1.**One-step growth curves of phiIPLA-RODI on S. aureus IPLA1 (black diamonds) and S. xylosus CTC1642 (grey squares), respectively. Values correspond to the number of plaque-forming unit (PFU) per infected cell. Each data point shows the mean ± standard deviation for three independent experiments.

**Figure 2.**Plot of the first design response. Phage yield (response) as a function of: initial phage titer at fixed temperature (33.76 °C) and agitation (135 rpm) for two levels of bacteria (

**A**); temperature at fixed initial phage titer (6.74 log

_{10}PFU/mL), initial bacterial concentration (7.00 log

_{10}CFU/mL) and agitation (135 rpm) (

**B**); and initial bacterial concentration at fixed initial phage titer (6.43 log

_{10}PFU/mL) and agitation (135 rpm) for two temperature levels (

**C**).

**Figure 3.**Plot of the second design response. Phage yield (response) as a function of temperature, at two initial bacterial concentrations, and fixed initial phage titer (7.32 log

_{10}PFU/mL).

**Figure 4.**Response surface plot of phage yield as a function of the initial bacterial population and phage titer, based on the experiments from the third design, with a maximum at 6.82 log

_{10}CFU/mL initial bacteria.

**Figure 5.**Optimization of both final phage yield and phage multiplication ratio. Final phage yield ( ) and phage multiplication ratio ( ), as a function of initial bacterial populations ( ) and phage titers ( ), using the desirability approach as implemented in Design Expert.

**Table 1.**Experimental designs used for optimizing the phage yield (response) as a function of temperature, initial bacterial concentration, initial phage titer and agitation. Responses are also included.

1st Tentative Design (Central Composite) | 2nd Design (D-Optimal) ^{a} | 3rd Design (Central Composite) ^{b} | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|

Runs | Phage Titer | Bacterial Concentration | Temperature (°C) | Agitation (rpm) | Phage Yield | Phage Titer | Bacterial Concentration | Temperature (°C) | Phage Yield | Phage Titer | Bacterial Concentration | Phage Yield |

1 | 7.39 | 7.56 | 24.2 | 67 | 5.53 | 8.00 | 5.78 | 40.0 | 7.42 | 7.00 | 6.85 | 8.4 |

2 | 6.50 | 7.28 | 29.0 | 135 | 5.67 | 6.00 | 5.95 | 34.0 | 6.61 | 6.50 | 7.72 | 9.0 |

3 | 6.50 | 8.27 | 29.0 | 135 | 4.37 | 7.19 | 7.45 | 37.6 | 8.43 | 6.50 | 7.35 | 9.3 |

4 | 5.61 | 6.96 | 33.8 | 67. | 6.66 | 7.19 | 6.30 | 34.0 | 6.86 | 6.50 | 6.05 | 8.5 |

5 | 5.00 | 7.53 | 29.0 | 135 | 4.86 | 6.00 | 7.44 | 34.0 | 7.57 | 7.21 | 7.61 | 8.9 |

6 | 5.61 | 7.99 | 33.8 | 203 | 4.08 | 7.18 | 6.48 | 37.8 | 7.37 | 5.79 | 7.41 | 9.1 |

7 | 6.50 | 7.51 | 29.0 | 135 | 5.79 | 8.00 | 6.81 | 40.0 | 7.81 | 6.00 | 8.15 | 3.7 |

8 | 6.50 | 7.62 | 29.0 | 135 | 6.00 | 8.00 | 5.66 | 36.1 | 8.27 | 7.00 | 8.11 | 8.3 |

9 | 6.50 | 7.38 | 21.0 | 135 | 5.51 | 6.00 | 6.66 | 37.6 | 8.05 | 6.50 | 7.61 | 9.0 |

10 | 6.50 | 6.25 | 29.0 | 135 | 6.13 | 6.00 | 7.82 | 40.0 | 8.12 | 6.50 | 7.34 | 9.0 |

11 | 6.50 | 7.57 | 29.0 | 135 | 5.68 | 6.00 | 7.43 | 40.0 | 8.27 | 6.00 | 6.29 | 8.7 |

12 | 8.00 | 7.11 | 29.0 | 135 | 6.80 | 8.00 | 7.48 | 34.0 | 8.04 | 6.50 | 7.53 | 9.1 |

13 | 5.61 | 7.88 | 24.2 | 203 | 4.23 | 6.00 | 5.77 | 34.0 | 7.58 | 6.50 | 8.40 | 4.1 |

14 | 7.39 | 7.01 | 24.2 | 203 | 7.06 | 6.70 | 5.86 | 40.0 | 7.54 | |||

15 | 5.61 | 6.99 | 24.2 | 67 | 5.25 | |||||||

16 | 6.50 | 7.04 | 29.0 | 20 | 5.02 | |||||||

17 | 6.50 | 7.11 | 29.0 | 135 | 5.26 | |||||||

18 | 6.50 | 6.85 | 37.0 | 135 | 8.13 | |||||||

19 | 7.39 | 7.15 | 33.8 | 203 | 7.34 | |||||||

20 | 7.39 | 7.70 | 33.8 | 67 | 6.83 | |||||||

21 | 6.50 | 7.18 | 29.0 | 250 | 6.43 |

_{10}plaque-forming unit (PFU)/mL and bacterial concentration is expressed as log

_{10}colony-forming unit (CFU)/mL. The bacterial concentrations correspond to the effective levels reached in the experiment. Standard deviations for the design values (as estimated from the analysis of variance (ANOVA) pure error) were 0.34, 0.14, 0.13 log

_{10}PFU/mL, respectively.

^{a,b}Agitation fixed at 135 rpm.

^{b}Temperature fixed at 38 °C.

**Table 2.**ANOVA for Response Surface Reduced Quadratic Model (partial sum of squares type III) of the first design.

Source | Sum of Squares | Degrees of Freedom | Mean Square | F Value | p-Value (Prob > F) |
---|---|---|---|---|---|

Model | 16.99 | 4 | 4.25 | 12.45 | <0.0001 significant |

A-Phage | 4.46 | 1 | 4.46 | 13.09 | 0.0023 |

B-Bacteria | 0.78 | 1 | 0.78 | 2.28 | 0.1508 |

C-Temperature | 3.62 | 1 | 3.62 | 10.62 | 0.0049 |

B^{2} | 1.21 | 1 | 1.21 | 3.56 | 0.0775 |

Residual | 5.46 | 16 | 0.34 | ||

Cor total | 22.44 | 20 |

**Table 3.**ANOVA for Response Surface Reduced Quadratic Model (partial sum of squares type III) of the second design.

Source | Sum of Squares | Degrees of Freedom | Mean Square | F Value | p-Value Prob > F |
---|---|---|---|---|---|

Model | 2.51 | 4 | 0.63 | 4.59 | 0.0271 |

B-Bacteria | 0.13 | 1 | 0.13 | 0.91 | 0.3644 |

C-Temperature | 0.30 | 1 | 0.30 | 2.19 | 0.1734 |

B^{2} | 0.52 | 1 | 0.52 | 3.76 | 0.0844 |

C^{2} | 0.86 | 1 | 0.86 | 6.28 | 0.0336 |

Residual | 1.23 | 9 | 0.14 | ||

Cor total | 3.75 | 13 |

**Table 4.**ANOVA for Response Surface Reduced Quadratic Model (partial sum of squares type III) of the third design.

Source | Sum of Squares | Degrees of Freedom | Mean Square | F Value | p-Value Prob > F |
---|---|---|---|---|---|

Model | 37.26 | 2 | 18.63 | 20.01 | <0.0001 significant |

B-Bacteria | 2.15 | 1 | 2.15 | 2.30 | 0.1455 |

B^{2} | 18.16 | 1 | 18.16 | 19.50 | 0.0003 |

Residual | 1.04 | 18 | 0.058 | ||

Lack of fit | 0.78 | 16 | 0.049 | 0.37 | 0.9042 not significant |

Pure error | 0.26 | 2 | 0.13 | ||

Cor total | 4.03 | 19 |

**Table 5.**Experimental conditions, predicted responses (±SE) according to the RS model developed for the 3rd experimental design, and actual results for the validation experiments.

Initial Bacteria Population | Initial Phage Titer | Phage Yield, Validation Experiments | Predicted Phage Yield, RS 3rd Design | Predicted Phage Yield, RS Enlarged 3rd Design ^{a} |
---|---|---|---|---|

7.51 | 6.50 | 8.8 ± 0.1 | 8.6 ± 0.5 | 8.7 ± 0.3 |

7.42 | 6.50 | 8.8 ± 0.1 | 8.7 ± 0.5 | 8.8 ± 0.3 |

7.63 | 6.50 | 8.8 ± 0.1 | 8.3 ± 0.5 | 8.4 ± 0.3 |

7.56 | 6.50 | 8.7 ± 0.1 | 8.5 ± 0.5 | 8.5 ± 0.3 |

7.72 | 6.50 | 8.8 ± 0.1 | 8.1 ± 0.5 | 8.2 ± 0.3 |

7.62 | 6.50 | 8.8 ± 0.1 | 8.3 ± 0.5 | 8.4 ± 0.3 |

7.35 | 6.50 | 8.6 ± 0.1 | 8.9 ± 0.5 | 8.9 ± 0.3 |

7.28 | 6.50 | 8.5 ± 0.1 | 9.0 ± 0.5 | 9.0 ± 0.3 |

7.39 | 6.50 | 8.8 ± 0.1 | 8.8 ± 0.5 | 8.9 ± 0.3 |

_{10}CFU/mL and 1og

_{10}PFU/mL, respectively.

^{a}Predicted responses (±SE) based on the final Response Surface including the validation data.

**Table 6.**ANOVA for Response Surface Reduced Quadratic Model (partial sum of squares type III) based on the data from the third design and the validation data.

Source | Sum of Squares | Degrees of Freedom | Mean Square | F Value | p-Value Prob > F |
---|---|---|---|---|---|

Model | 37.26 | 2 | 18.63 | 20.01 | <0.0001 significant |

B-Bacteria | 2.15 | 1 | 2.15 | 2.30 | 0.1455 |

B^{2} | 18.16 | 1 | 18.16 | 19.50 | 0.0003 |

Residual | 1.04 | 18 | 0.058 | ||

Lack of fit | 0.78 | 16 | 0.049 | 0.37 | 0.9042 not significant |

Pure error | 0.26 | 2 | 0.13 | ||

Cor total | 4.03 | 19 |

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**MDPI and ACS Style**

González-Menéndez, E.; Arroyo-López, F.N.; Martínez, B.; García, P.; Garrido-Fernández, A.; Rodríguez, A.
Optimizing Propagation of *Staphylococcus aureus* Infecting Bacteriophage vB_SauM-phiIPLA-RODI on *Staphylococcus xylosus* Using Response Surface Methodology. *Viruses* **2018**, *10*, 153.
https://doi.org/10.3390/v10040153

**AMA Style**

González-Menéndez E, Arroyo-López FN, Martínez B, García P, Garrido-Fernández A, Rodríguez A.
Optimizing Propagation of *Staphylococcus aureus* Infecting Bacteriophage vB_SauM-phiIPLA-RODI on *Staphylococcus xylosus* Using Response Surface Methodology. *Viruses*. 2018; 10(4):153.
https://doi.org/10.3390/v10040153

**Chicago/Turabian Style**

González-Menéndez, Eva, Francisco Noé Arroyo-López, Beatriz Martínez, Pilar García, Antonio Garrido-Fernández, and Ana Rodríguez.
2018. "Optimizing Propagation of *Staphylococcus aureus* Infecting Bacteriophage vB_SauM-phiIPLA-RODI on *Staphylococcus xylosus* Using Response Surface Methodology" *Viruses* 10, no. 4: 153.
https://doi.org/10.3390/v10040153