Growth Characteristics of Bacteriophage

Growth Characteristics of bacterio bacteriophageCHAPTER 4GROWTH CHARACTERISTICS OF BACTERIOPHAGE INFECTING AQUACULTURE BACTERIAL PATHOGENS4.1IntroductionBacteriophage ar by nature 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 chip 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 put away many parameters which are unclear that may contributed to efficacy of the phage therapy. Previously, the most common practice to evaluate the curative 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 surface assimilation, lysis time and effusion size (Ackermann et al., 2004). Besides that, there are conglomerate physical and chemical factors like temperature, pH and salinity which could determine the occurrence and stability of bacteriophage (Joczyk et al., 2011). These factors report 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 err of temperature, pH and gall common salt concentration.4.2Materials and Methods4.2.1Bacteriophage IsolatesThe bacteriophage isolates that were used in the third chapter were further characte rized in this chapter. The bacteriophage isolates were designated as VALLPKK3, VHLPKM4 and VPLPKK5.4.2.2Bacteriophage Adsorption AssayThe bacteriophage adsorption assay was carried out avocation the system described by Hsieh et al. (2011) with few modification. In adsorption test, the host bacteria was first grown to OD600 1.0 or same to 108 cfu/ml and diluted to 105 cfu/ml with TSB media. About nine ml of the host bacteria was multiform with one ml of phage lysate (103 pfu/ml) to MOI of 0.001. Then, ascorbic acid l of the bacteria-phage mixture was taken to determine the initial phage titer. The mixture was then incubated at 28C with no agitation. aft(prenominal) 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 degree 50 min. The view of unabsorbed free phages in the supernatant was determined. Then, the free phage particles over the initial phage particles was cy pher and expressed in percentage. The accuracy of the free phage search was meliorate by triplicate separate experiments.4.2.3Bacteriophage One Step GrowthThe one step growth assay was determined following system described Hsieh et al. (2011) with slight modification. First, host bacteria (OD600 1.0) was diluted to 106 cfu/ml. Then, 100 l of bacterial suspension was mixed with 100 l of phage lysate (103 pfu/ml) to a 1 ml of final volume with uninventive TSB media (MOI 0.001). Second, the phage was allowed to adsorb into bacterial cells for 30 min at 28C. 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 calculate. Then, devil sets of bacterial-phage suspension concurrently collected every 12 min for a period of 84 min (for VALLPKK3 and VPLPKK5) a nd 132 legal proceeding (VHLPKM4) for the determination of latent period, shadowd period and crumble size. The first set was subjected to above treatment to determine the latent period and break-dance size while the second set was added with 40 l chloroform, mixed and incubated at 28C 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 take fire 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.4.2.4Bacteriophage Tolerance TestThe stability of the bacteriophage isolates was test in assorted 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 TestThe stability of bacteriophage in different temperature was done following system described by Phumkhachorn and Rattanachaikunsopon (2010) with slight modification. The bacteriophage solution was set to roughly 105 pfu ml-1 in sterile phage original. About 900 l 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 100C) at to the lowest degree for 30 transactions. After 30 minutes, about 100 l 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 opined by double layer method. The percentage of living phage was calculated by dividing the number of survival phage over initial phage count.b.pH Tolerance TestThe stability of bacteriophage in differ ent 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 121C 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 TestThe stability of bacteriophage in different bile salt concentration was done following method described by previous. The stock o f 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 121C 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.4.3Res ult4.3.1Bacteriophage Adsorption AssayIn 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 offspring 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 force out from the septic cells (Figure 4.3).Figure 4.1 Adsorption of VALLPKK3 to V. alginolyticus ATCC 17749TMFigure 4.2 Adsorption of VHLPKM4 to V. harveyi VHJR7Figure 4.3 Adsorption of VPLPKK5 to V. parahaemolyticus VPHG14.3.2Bacteriophage One Step GrowthThe 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 sprains. 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 infect cell, r espectively, at the 28C.Figure 4.4 One step growth curve of VALLPKK3 infected with vibrio alginolyticus ATCC 17749TM at MOI of 0.001. The number of PFU per infected cell in untreated farming () 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.4.3.3Bacteriophage Tolerance TestThe activity of all phage isolates was permanen t at 40C and disintegrationd at 50C following heating for 60 minutes. The activity was disappeared entirely when heated at to a greater extent than 60C 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 50C. 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. near 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 4.7The temperature stability of VALLPKK3, VHLPKM4 and VPLPKK5. All isolates were incubated at various range of temperature (40C, 50C, 60C, 70C, 80C, 90C and 100C) for 1 hour. Data are the means from three independent experiments + SD.Figure 4.8The 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 4.9The 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.4.4DiscussionThe phage adsorption of VALLPKK3 and VHLPKM4 was fast (more than 80% a fter 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.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 As51 and A318 (Liu et al., 2014). The V. alginolyticus phage VALLPKK3 show ed 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 H17-7b and H17-8b (Okano et al., 2007). They reported that where the latent period and burst size of H17-7b and 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 sh owed 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 object lens 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 (Joczyk et al., 2011). In this study, the result showed that all phages were stable at 40C. However, the viability was tamed after one hour incubation at 50C. All phage were completely inactivated in temperature over 60C. 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 32C (Albert and Ransangan, 2013). Since the isolates in this study were stable at the temperature up to 40C, 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 fraction 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 m ember of Siphoviridae, which is only active in a press 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 functional 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 cut down 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 brook for long time in gut environment (Ma et al., 2008).4.5ConclusionIn summary, the VALLPKK3, VHLPKM4 and VPLPKK5 were characte rized 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 increase of phage therapy. All phages are inactivated at high temperature but showed stability at temperature 40C. 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.