Growth Characteristics of Bacteriophage

CHAPTER 4

GROWTH CHARACTERISTICS OF BACTERIOPHAGE INFECTING AQUACULTURE BACTERIAL PATHOGENS

4.1 Introduction

Bacteriophage are naturally occurring viruses that predated on bacteria (Clokie et al., 2011). They self-replicate exponentially and leave the commensal flora unaffected makes them useful for industrial application (Tsonos et al., 2014). However, the high number of bacteriophage in environment (Clokie et al., 2011) provide the challenges to the discovery of the most effective phage in treating bacterial pathogens (Lindberg et al., 2014). Even there were many extensive reports on bacteriophages, the clinical outcome of therapy trials are variable (Tsonos et al., 2014). This indicates that there are still many parameters which are unclear that may contributed to efficacy of the phage therapy. Previously, the most common practice to evaluate the therapeutic efficacy of phages was from in vivo studies. However, Lindberg et al. (2014) provide the alternative to evaluate the efficacy the phage treatment. The information could be assessed from the important phage traits such as adsorption, lysis time and burst size (Ackermann et al., 2004). Besides that, there are various physical and chemical factors like temperature, pH and salinity which could determine the occurrence and stability of bacteriophage (Jończyk et al., 2011). These factors reported to cause the inactivation of phage through damage of the phage structure (head, tail or envelope) or DNA structural changes (Ackermann et al., 2004). Therefore, the next section in this study is aimed to characterize the bacteriophage isolates (VALLPKK3, VHLPKM4 and VPLPKK5) based on their adsorption profile, one step growth profile and stability to various range of temperature, pH and bile salt concentration.

4.2 Materials and Methods

4.2.1 Bacteriophage Isolates

The bacteriophage isolates that were used in the third chapter were further characterized in this chapter. The bacteriophage isolates were designated as VALLPKK3, VHLPKM4 and VPLPKK5.

4.2.2 Bacteriophage Adsorption Assay

The bacteriophage adsorption assay was carried out following the method described by Hsieh et al. (2011) with few modification. In adsorption test, the host bacteria was first grown to OD600 1.0 or equivalent to ~10⁸ cfu/ml and diluted to ~10⁵ cfu/ml with TSB media. About nine ml of the host bacteria was mixed with one ml of phage lysate (~10³ pfu/ml) to MOI of 0.001. Then, 100 µl of the bacteria-phage mixture was taken to determine the initial phage titer. The mixture was then incubated at 28°C with no agitation. After 10 min, one ml of the samples was collected and centrifuged at 16,000 xg for 2 min to precipitate the absorbed phages. The same was repeated every 10 min for a period 50 min. The count of unabsorbed free phages in the supernatant was determined. Then, the free phage particles over the initial phage particles was calculated and expressed in percentage. The accuracy of the free phage count was improved by triplicate separate experiments.

4.2.3 Bacteriophage One Step Growth

The one step growth assay was determined following method described Hsieh et al. (2011) with slight modification. First, host bacteria (OD600 1.0) was diluted to ~10⁶ cfu/ml. Then, 100 µl of bacterial suspension was mixed with 100 µl of phage lysate (~10³ pfu/ml) to a 1 ml of final volume with sterile TSB media (MOI 0.001). Second, the phage was allowed to adsorb into bacterial cells for 30 min at 28°C. Then the bacterial cells were precipitated by centrifugation at 16,000 xg for 2 min. Third, the bacterial-phage pellet was suspended in 50 ml sterile TSB. Subsequently, 1 ml of the bacterial-phage suspension was precipitated by centrifugation and plated to determine the initial phage count. Then, two sets of bacterial-phage suspension concurrently collected every 12 min for a period of 84 min (for VALLPKK3 and VPLPKK5) and 132 minutes (VHLPKM4) for the determination of latent period, eclipsed period and burst size. The first set was subjected to above treatment to determine the latent period and burst size while the second set was added with 40 µl chloroform, mixed and incubated at 28°C for 5 min before centrifugation to determine the eclipse period. The free phage count in the supernatant was determined in triplicate. The latent period and burst size was determined according to Middleboe et al. (2010). The eclipse period was determine according to Sillankorva et al. (2008). The accuracy of the free phage count was improved by triplicate separate experiments.

4.2.4 Bacteriophage Tolerance Test

The stability of the bacteriophage isolates was test in different range of temperature, pH and bile salt concentrations. The temperature test was conducted for one hour, while, the pH and bile salt concentration tests were conducted for 24 hours.

a. Temperature Tolerance Test

The stability of bacteriophage in different temperature was done following method described by Phumkhachorn and Rattanachaikunsopon (2010) with slight modification. The bacteriophage solution was set to approximately 10⁵ pfu ml^-1 in sterile phage buffer. About 900 µl of sterile phage buffer was distributed into sterile empty 1.5 ml microfuge tube. The tube was incubated in the dry bath at desirable temperature (40, 50, 60, 70, 80, 90 and 100°C) at least for 30 minutes. After 30 minutes, about 100 µl of bacteriophage solution (~10⁴ pfu) was added into the preheated tube and mixed immediately. The tube was incubated again at desirable temperature for an hour. After incubation, the tube was placed in ice-warm bath to cool the bacteriophage solution. The titer of the survival phage was calculated by double layer method. The percentage of surviving phage was calculated by dividing the number of survival phage over initial phage count.

b. pH Tolerance Test

The stability of bacteriophage in different pH was done following method described by Hsieh et al. (2011) with slight modification. The pH of phage buffer was adjusted into desirable pH (2, 3, 4, 5, 6, 7, 8 and 9) using pH meter (brand). The phage buffer was sterilized using autoclave machine at 121°C for 15 minutes. The bacteriophage was set to approximately 10⁷ pfu ml^-1 in sterile phage buffer. The bacteriophage suspension was diluted to 10⁵ pfu ml^-1 (1/100) in phage buffer with different pH. The initial phage count was calculated and the bacteriophage solution was incubated at room temperature for 24 hours. After incubation, the bacteriophage solution was diluted using normal phage buffer and plating to calculate the survival phage by double layer method. The percentage of surviving phage was calculated by dividing the number of survival phage over initial phage count.

c. Bile Salt Tolerance Test

The stability of bacteriophage in different bile salt concentration was done following method described by previous. The stock of bile salt (Brand) in phage buffer was prepared by filter sterilized to final concentration of 5 %. Then, the phage buffer was adjusted into desirable bile concentration (5000 ppm, 6000 ppm, 7000 ppm, 8000 ppm and 9000 pm). The phage buffer which used for the dilution of bile concentration was presterilized using autoclave machine at 121°C for 15 minutes. The bacteriophage was set to approximately 10⁷ pfu ml^-1 in sterile phage buffer. The bacteriophage suspension was diluted to 10⁵ pfu ml^-1 (1/100) in phage buffer with different bile concentration. The initial phage count was calculated by serial dilution in normal phage buffer. The treated bacteriophage solution was incubated at room temperature for 24 hours. After incubation, the bacteriophage solution was diluted again using normal phage buffer and plated to calculate the survival phage by double layer method. The percentage of surviving phage was calculated by dividing the number of survival phage over initial phage count.

4.3 Result

4.3.1 Bacteriophage Adsorption Assay

In the adsorption analysis, all isolates have two adsorption phases, rapid and slow adsorptions. The rapid adsorption of VALLPKK3 was occurred within 10 minutes where almost 80% of the phage adsorb to the host (Figure 4.1). This result was similar to the VHLPKM4 (Figure 4.2). Meanwhile, the rapid adsorption of VPLPKK5 showed that around 60 % of the phage adsorbed to the host (Figure 4.3). After 10 minutes, the slow rate was occurred to all isolates. The number of unadsorbed phages was approximately below 20% within 40 minutes in all phages. The increase of phage count in VPLPKK5 was occurred after 40 minutes. The increase in free phages after 50 minutes indicates that the newly formed phages are being release from the infected cells (Figure 4.3).

Figure 4.1: Adsorption of VALLPKK3 to V. alginolyticus ATCC^® 17749^TM

Figure 4.2: Adsorption of VHLPKM4 to V. harveyi VHJR7

Figure 4.3: Adsorption of VPLPKK5 to V. parahaemolyticus VPHG1

4.3.2Bacteriophage One Step Growth

The one step growth was performed to identify different phases of the phage infection process. During the initial stage, the phage-bacteria cell was separated from the free phage during the adsorption since the adsorption result showed the availability of free phage after 30 minutes of incubation. After the infection, the phage growth parameters (latent period, eclipse period and burst size) were determine from the average of three independent curves. The analysis showed that the latency and eclipse periods of VALLPKK3 (Figure 4.4), VHLPKM4 (Figure 4.5) and VPLPKK5 (Figure 4.6) were 48 and 36 minutes, 60 and 36 minutes and, 36 and 24 minutes, respectively. The latent period of VHLPKM4 was longer compared to VALLPKK3 and VPLPKK5. Meanwhile, the eclipse period of VALLPKK3 and VHLPKM4 was similar, while, the eclipse period of VPLPKK5 were shorter than those two isolates. The VALLPKK3, VHLPKM4 and VPLPKK5 showed a burst size of ~174, ~52 and ~180 phage per infected cell, respectively, at the 28°C.

Figure 4.4: One step growth curve of VALLPKK3 infected with Vibrio alginolyticus ATCC^® 17749^TM at MOI of 0.001. The number of PFU per infected cell in untreated culture () and chloroform-treated culture () are also shown. The burst size, latent period and eclipse are indicated as B, L and E, respectively.

Figure 4.5: One step growth curve of VALLPKK3 infected with Vibrio harveyi VHJR7 at MOI of 0.001. The number of PFU per infected cell in untreated culture () and chloroform-treated culture () are also shown. The burst size, latent period and eclipse are indicated as B, L and E, respectively.

Figure 4.6: One step growth curve of VPLPKK5 infected with V. parahaemolyticus VPHG1 at MOI of 0.001. The number of PFU per infected cell in untreated culture () and chloroform-treated culture () are also shown. The burst size, latent period and eclipse are indicated as B, L and E, respectively.

4.3.3 Bacteriophage Tolerance Test

The activity of all phage isolates was stable at 40°C and declined at 50°C following heating for 60 minutes. The activity was disappeared entirely when heated at more than 60°C for 1 hour (Figure 4.7). When compared among the isolates, the activity of VHLPKM4 were decline dramatically to less than 20 % when incubated at 50°C. The activity of VALLPKK3 and VPLPKK5 were dropped to 80% and 40%, respectively. The activity of bacteriophages VALLPKK3, VHLPKM4 and VPLPKK5 can be measured after incubation at pH 4 to pH 9, but disappear completely at pH 2 and pH 3 (Figure 4.8). When compared among isolates, the VALLPKK3 was sensitive to wide range of pH. Almost all of the VALLPKK3 activity was drop to 20 to 40 % after 24 hours incubation. Meanwhile, the activity of VHLPKM4 was decline to 60 % at pH 4 and 5, relatively stable at pH 6 to pH 8 and decline again to less than 60 % at pH 9. However, the activity of VPLPKK5 relatively stable at wide range of pH (pH 4 to pH 9). Meanwhile, the activity of VALLPKK3, VHLPKM4 and VPLPKK5 can be detected after incubated at bile salt concentration from 5000 ppm to 9000 ppm (Figure 4.9). Among the isolates, VALLPKK3 was more sensitive to the bile compared to VHLPKM4 and VPLPKK5.

Figure 4.7:The temperature stability of VALLPKK3, VHLPKM4 and VPLPKK5. All isolates were incubated at various range of temperature (40°C, 50°C, 60°C, 70°C, 80°C, 90°C and 100°C) for 1 hour. Data are the means from three independent experiments + SD.

Figure 4.8:The temperature stability of VALLPKK3, VHLPKM4 and VPLPKK5. All isolates were incubated at various range of pH (2, 3, 4, 5, 6, 7, 8 and 9) for 24 hours. Data are the means from three independent experiments + SD.

Figure 4.9:The bile salt stability of VALLPKK3, VHLPKM4 and VPLPKK5. All isolates were incubated at various range of bile salt concentration (5000, 6000, 7000, 8000 and 9000 ppm) for 24 hours. Data are the means from three independent experiments + SD.

4.4 Discussion

The phage adsorption of VALLPKK3 and VHLPKM4 was fast (more than 80% after 10 minutes) compared to Vibrio phage PW2 (60% after 10 minutes) (Phumkhachorn and Rattanachaikunsopon, 2010). Meanwhile, the adsorption of VPLPKK5 was comparable to PW2. This might due to both phages were belonged to same family (Sipboviridae). However, the phage adsorption was reported dependent on various condition. According to Binetti et al. (2002), the phage adsorption was shown to be affected by the presence of ion calcium, physiological state of the cell, pH and temperature.

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The one step growth is a method to assess the life cycle of the phage (Middleboe et al., (2010). The latent period was the time from adsorption to the release of new progeny from host cell, and the burst size was the number of new virus particles liberated from a single bacterial cell (Bao et al., 2011). When compared to other vibriophage infecting same host species, there were difference in term of the burst size of the phages Ñ„As51 and Ñ„A318 (Liu et al., 2014). The V. alginolyticus phage VALLPKK3 showed high burst size compared to those two (72 and 10 PFU/infected cell). Similar finding with VPLPKK5. This V. parahaemolyticus phage was different to other V. parahaemolyticus phage VP-2 (15 PFU per infected cell) (Silva et al., 2014) where it showed bigger burst size (180 PFU per infected cell). Meanwhile, the VHLPKM4 showed different finding. This study showed smaller burst size and longer latent period compared to previous report on V. harveyi phages Ñ„H17-7b and Ñ„H17-8b (Okano et al., 2007). They reported that where the latent period and burst size of Ñ„H17-7b and Ñ„H17-8b were 35 minutes and 100 particles, and 40 minutes and 170 particles, respectively. Thus, the findings showed that the life cycle of each phage isolates was different from each other. However, the significant of the differences was unclear since the dissimilarity was influenced by the host, medium, temperature and its own growth rate (Carey-Smith et al., 2006). In this study, the bacteriophages VALLPKK3 and VPLPKK5 showed a short period of latent period and large burst size. The shorter latent period and large burst size showed that the bacteriophages replicated more quickly and the new virus particle release more efficiently (Bao et al., 2011). This characteristic showed good candidacy of phage therapy (Silva et al., 2014). Finally, both adsorption and one step growth of phage are important to determine the phage fitness (Wang, 2006) since the phage fitness would determine the efficacy of the phage therapy (Lindberg et al., 2014).

The stability in various stress condition were useful for the application of bacteriophage to inhibit the target bacteria (Lee et al., 2014; Krasowska et al., in press). In this study, the resistance to heat, pH and bile was investigated to determine the efficacy of those phages for biocontrol of V. alginolyticus, V. harveyi and V. parahaemolyticus infections. Phage which can withstand various environmental stress may be useful for the application in aquaculture (Phumkhachorn and Rattanachaikunsopon, 2010).

The temperature is a important factor that affects bacteriophage survivability (Olson et al., 2004). It plays important roles in the bacteriophage attachment, penetration and multiplication (JoÅ„czyk et al., 2011). In this study, the result showed that all phages were stable at 40°C. However, the viability was reduced after one hour incubation at 50°C. All phage were completely inactivated in temperature over 60°C. The phage in this study showed that they are sensitive to high temperature. This findings was different to the findings by Phumkhachorn and Rattanachaikunsopon (2010) where the phage can withstand high temperature. However, in the natural environment, the temperature usually fluctuated at the range of 28 to 32°C (Albert and Ransangan, 2013). Since the isolates in this study were stable at the temperature up to 40°C, the isolates would survive when release to natural environment. Nevertheless, the period of viability of these isolates after release to natural environment was unknown.

In the natural environment, the phage was also facing the other stress factor such as pH. According to Krasowska et al. (in press), the acidity and alkalinity of environment are other important factors influencing phage stability. It was also reported that low pH influences phage aggregation and reduce their adsorption on bacteria cell (Langlet et al., 2007). Therefore, it was important to access the stability of the current phage isolates in different pH. The VALLPKK3 and VHLPKM4 showed resistance to acid (pH 4) and alkaline (pH 9). This showed that the member of Myoviridae family stable at acid and alkaline condition (Krasowska et al., in press). Similar to the other isolates, VPLPKK5 was also showed resistance to acid and alkaline condition. This is similar to the finding by Lasobras et al. (60) where the member of family Siphoviridae were most resistant to adverse conditions. However, this finding was different to phage AR, a member of Siphoviridae, which is only active in a narrow pH range (Krasowska et al., in press). The result of the phage tolerance to pH indicated that they were tolerant to wide range of pH.

In aquaculture, oral administration was the most practical delivery method for immunization (Yasumoto et al., 2006) due to low cost and less stress to fish (Pal et al., 2009). However, the viability of orally administered phage might be rapidly reduced the presence other digestive compounds such as bile (Joerger et al., 2003). In this study, the phage isolates were exposed to various concentration of bile concentrations and result showed that the phage were still survived after incubation. However, there were reduction on the viability of the phage isolates which might showed the adverse effect of bile. With the addition of pH and other enzymes, the phage might not persist for long time in gut environment (Ma et al., 2008).

4.5 Conclusion

In summary, the VALLPKK3, VHLPKM4 and VPLPKK5 were characterized by the growth and tolerance. The life cycle of the current isolates might be different when conducted different time and with different media. Therefore, the optimization was required for optimum phage multiplication which generally required for large scale production. This optimization was also contributed to the development of phage therapy. All phages are inactivated at high temperature but showed stability at temperature 40°C. They are also stable at wide range of pH but not low pH. But, they could tolerate normal fish bile content. However, the study need to be conducted to collect the information of the period of phage survival in fish body. This information would be beneficial for the phage administration of disease treatment.