Improving phage titre through examining point of infection


 Bacteriophages are viruses that cause the lysis of bacteria. They have recently been used to combat antimicrobial resistant infections as an alternative therapy to antibiotics. Their production and propagation, however, remains understudied and will be key to obtaining titres required for future clinical studies and research. Previous work suggests that temperature of infection significantly influences the production process and output yield of phage, with a reduction in temperature from 37ºC to 28ºC resulting in significant increases in productivity for multiple host-phage systems. The current study aimed to build upon this previous work by examining different temperature conditions at the point of infection to determine the effect on harvest phage titre improvements. Investigations were conducted at different culture scales ranging from 20mL shake flasks to 3L stirred tank bioreactor cultures to investigate process differences when scaling from laboratory bench-scale to initial industrial scale fermentations. Additionally, the kinetics of phage infection were investigated. In small scale cultures, the greatest phage bursts and harvest titres were generated by maintaining cultures under static 28 o C incubation during infection compared to agitation and temperature reduction from 37 o C. Investigating the dynamics around the point of infection will help to inform scalable processes for manufacture of phage for a variety of applications ranging from direct therapeutic application to self-assembling bio-templates for nanostructure synthesis.


Introduction
It is known that all microbes have a potential to mutate and reduce the effectiveness of antibiotics.
Currently, there are around 50,000 deaths per annum in Europe and the USA, but by 2050, it is estimated that antimicrobial resistant (AMR) bacteria will cause around 10 million deaths per annum

Media and growth conditions
Unless otherwise stated, all reagents were purchased from Sigma-Aldrich, Irvine, UK. Luria broth (LB) media was used for growth of E. coli and T4 phage whilst brain heart infusion (BHI) media was used for growth of S. aureus and phage K. E. coli cultures were agitated at 225rpm whilst S. aureus cultures 4 were agitated at 150rpm. For long term storage, bacteria were stored in a 20% glycerol solution at -80ºC whilst phage were stored at 4ºC but for long term storage, phage were kept in a 50% glycerol solution at -80ºC (Fortier & Moineau, 2009). T4 phage was infected at 0.25OD 600nm at a multiplicity of infection (MOI) 2.5 for 3 hours (225rpm) whilst phage K was infected at 0.25OD 600nm at an MOI 0.1, for 4 hours (150rpm).

Shake flask analysis
A single colony of each bacterium was inoculated in 20ml media overnight in a shake flask in a shaking incubator (Wolflabs midi shaking incubator SQ-4020). After 16 hours, the culture was diluted to an optical density (OD 600nm ) of 0.05 with a Shimadzu biospec mini spectrophotometer. The culture was grown at 37ºC with shaking until it reached an optical density of 0.25OD 600nm and infected with phage.
Three methods of temperature reduction were examined to create differences in temperature reduction profile at the point of infection as follows. Method 1: the culture was placed into a shaking incubator at 37ºC and the temperature of the incubator reduced to 28ºC during the early stages of infection. Method 2: the culture was maintained at room temperature (22ºC) whilst the temperature of the shaking incubator was reduced to 28ºC. Method 3: the culture was placed into a static incubator at 28ºC. Culture temperatures were monitored to record the temperature reduction profile over time. Enumeration, adsorption and burst analyses of early infection were carried out according to the methods described below.

Bioreactor analysis
A 5L Biostat B Plus stirred-tank bioreactor (Sartorius, Göttingen, Germany) was used at 3L working volume and with dO 2 , maintained at 100%, pH maintained at 7.0 and an impellor used for agitation at 150rpm (S. aureus) or 225rpm (E. coli). A single colony of host bacterium was inoculated in 30ml medium (1% working bioreactor volume) and grown for 16 hours in a shake flask under standard conditions. This 1% volume culture was used as the inoculum for the bioreactor and grown to 0.25 OD 600nm at 37ºC. The bioreactor culture was then infected with phage: T4 phage was infected at an 5 MOI 2.5 for 3 hours with 225rpm stirring, whilst phage K was infected at an MOI 0.1 for 4 hours with 225rpm stirring. These conditions were previously determined for achieving high titres at 3L scale (Ali et al, 2019). Immediately upon infection, the temperature of the bioreactor was set to reduce to 28ºC.
Culture temperatures were monitored to record the temperature reduction profile over time.
Adsorption, burst analyses and harvest phage titre enumeration were all carried out according to methods described above.

Phage purification
At the point of harvest, shake flask cultures or the equivalent volume from bioreactor cultures were centrifuged at 4,600g for 10 minutes and filtered using a 0.22μm filter (Millipore, Watford UK). Phage were concentrated using a 20% PEG-8000 overnight at 4 º C. The following morning, the culture was centrifuged at 4,600g for 1 hour and the supernatant aspirated. The pellet was resuspended and stored in 1ml sterile media, LB for T4 phage or BHI for phage K. Harvest phage titre was enumerated by plaque assay described below.

Enumeration of phage
Phages were enumerated using the plaque overlay assay. Briefly, a 20ml overnight culture of host bacteria was centrifuged and re-suspended in 3ml fresh media and added to 5ml 0.6% LB bacteriological agar for T4 phage or 0.7% BHI bacteriological agar for Phage K. The mixture was then poured onto a fresh agar plate and the phage were spotted onto the top agar at appropriate dilutions (Bourdin et al, 2014;Pallavali et al, 2017). All shake flask and bioreactor experiments were carried out in triplicate and enumerated by triplicate plaque assays. . Samples were filtered using a 0.22μm filter and enumerated using the plaque assay technique (triplicate plaque assays per sample).

Statistical tests
All statistical analysis was performed using IBM SPSS 23. Paired t-tests were used to calculate significance with a p value <0.05 considered to be statistically significant.

Small scale evaluation of temperature reduction method
In bench scale phage infection experiments, the simplest method of applying temperature reduction is to allow the shaking incubator to reduce to the desired temperature once culture infection has taken place. However, this will not be an applicable method for a scalable bioprocess for large scale production of phage.
To provide information on the best method for temperature reduction at bench scale (20ml shake flask cultures) with a view towards informing development of scalable culture processes, three methods of culture maintenance during early infection and temperature reduction were investigated.

7
The methods of temperature reduction were Method 1, culture maintained in the shaking incubator during temperature reduction (from 37 -28ºC); Method 2, culture maintained at room temperature (22ºC) during shaking incubator temperature reduction (to 28ºC); and Method 3, culture maintained in a static 28ºC incubator during shaking incubator temperature reduction (to 28ºC). These methods were applied to two different host-phage culture processes; E. coli -T4 phage and S. aureus -Phage Whilst no previous work has examined a similar mechanism, a hypothesis for the improvement in titre may be if the optimal temperature for T4 and phage K infection is 28 o C, maintaining the culture at the most desirable temperature could improve titre by slowing intracellular processes involved in viral propagation, however this is yet to be determined experimentally. Additionally, by preventing a shock to the culture i.e. significant temperature drops which could affect host cell metabolism, efficiency of replication and the ability of phage to propagate. This hypothesis is somewhat supported by the data shown here where reducing the potential for cellular shock using a slower and more controlled temperature reduction profile improved phage titre, but requires further investigation to determine the biological mechanism of action.

Adsorption and burst analysis
An important study in the kinetics of phage infection is the adsorption of the phage to the host cell and the burst size. It is known that phage adsorption is dependent on the culture conditions (Zaburlin et al, 2017) and differences in phage adsorption kinetics may offer some mechanistic explanation for the differences in titre achieved with the various methods of temperature reduction at the point of infection. Figure 3 shows the adsorption of phage to their host cell for each of the infection methods (1)(2)(3). Figure 3A shows that each of the methods had a negligible difference in the adsorption of T4 phage for the first 6 minutes. Thereafter, a statistically significant difference was observed in the rate of adsorption between methods 2 and 3 compared to method 1 where the culture was agitated throughout early infection. There was very little difference between the cultures without agitation (methods 2 and 3) that were held at room temperature or in the static 28ºC incubator respectively. Figure 3B shows a greater degree of variation was observed in the phage K adsorption compared to the T4 phage adsorption. A significantly greater rate of adsorption was observed in incubated cultures (methods 1 and 3) compared to cultures held at room temperature (method 2) from 3 minutes postinfection onwards (p<0.05). Agitation appears play less of a role in influencing phage K adsorption compared to T4 phage adsorption, with no significant difference observed between 7-10 minutes post-infection between methods 1 (agitated) and 3 (static) for phage K whereas only the agitated method influenced T4 phage adsorption.
A further important study in bacteriophage kinetics is the burst size, the increase in phage after the bacterium has been infected with phage (Wang, 2006). Table 1 below shows the average burst size achieved from each phage infection for each of the three temperature reduction methods. Burst size for method 3 was statistically significantly increased compared to methods 1 and 2 (p<0.05) for both phage-host systems. A lower average burst was observed for method 2 compared to method 1 for both phage-host systems although only statistically significant for phage K. This lower burst may be related to reduce control in the temperature reduction profile and possible temperature shock to the culture with method 2 where cultures were removed from 37 o C incubation to room temperature (22 o C). The temperature reduction during early infection is more gradual for method 1, achieving the desired 28 o C culture temperature over 14 minutes compared to 5 minutes for method 2.
Fister et al (2016), previously examined the effect of environmental factors on the adsorption and yield of phage, their data showed how reducing the pH or the addition of salts can negatively influence the adsorption rate of Listeria phage P-100 although it wasn't significant. The data presented in this study shows that culture temperature during early infection and the method of exposure to a change in temperature can influence adsorption rates in multiple host-phage systems.
Although there are differences between the level of influence of temperature on the different hostphage systems, for example different burst sizes for T4 vs. phage K with method 3 temperature reduction and differences in response to agitation for T4 phage. The data shows evidence that a gradual reduction in temperature positively influences adsorption rate and burst size during early infection. Other environmental factors, such as MOI, pH and agitation may also have a significant effect on the phage-host adsorption (Silva & Sauvageau, 2014).   figure 1 showed that under Method 1, where cultures were kept in a shaking incubator during temperature reduction, it took 14 minutes for cultures to stabilise at 28°C. During this experiment, 0.25OD 600nm cultures were inoculated with phage once they were stable at 28°C following Method 1 temperature reduction. The experiment was carried out in triplicate and with duplicate phage plaque assay titre measurements (n=6) and single burst size measurements (n=3). The results showed there was no significant improvement on the T4 or phage K titre, 8.17x10 12 ±4.05x10 12 and 2.67x10 12 ±8.73x10 11 pfu/ml. T4 phage gave a more variable and reduced burst size of 107.7±19.9 whilst phage K gave a reduced burst size of 72.1±6.2 when compared to the bursts achieved at 0.25OD 600nm above (Fig. 4). The results suggest there is no advantage in postponing phage inoculation for culture temperature reduction to stabilise and taken together with the previous phage adsorption results (Fig. 3) that inoculation followed by temperature reduction may be more beneficial to the very early phage infection process.

Scale up model evaluation of the temperature kinetics
The small-scale shake flask experiments allow for high throughput approach and fewer resources when compared to larger stirred-tank bioreactor systems. However, small-scale shake flasks are not fully representative of the industrial-scale systems that will be employed for the production of to 28 o C. Figure 5 shows the difference in phage adsorption during early infection between the shake flask and bioreactor culture systems. The shake flask cultures generally gave a greater rate of 13 adsorption for phage K (Fig. 5B), whereas T4 phage showed initially there was a higher level of free phage in the STR cultures but after 7 minutes there was less free phage in the shake flask model (Fig.   5A). The experiments were completed in duplicate and each sample enumerated with duplicate plaque assays. Figure 5 C and D show the temperature reduction in the stirred tank reactor culture.
They show that the culture took around 14 minutes to reduce to the desired 28 o C temperature. The action of the water jacket and agitation will allow the heat transfer throughout the culture and consequently the reduction in temperature. Given that ultimately, manufacturers may look towards continuous culture, future experiments may benefit from replicating this process in a scale up continuous system, i,e feeding cells into a 28 o C reactor.
It was important to note the final titres achieved in the experiments in the 5L bioreactor. The experiment was completed in duplicate with triplicate plaque assay measurements and duplicate burst size measurements. There was a statistically significant increase in titre between method 1 in shake flasks and the 5L scale up model for T4 phage, 5.11 x 10 12 ± 1.5 x 10 12 pfu/ml and 4.33 x 10 14 ± 9.4 x 10 13 pfu/ml p<0.01 using an ANOVA whilst phage K gave a statistically significant increase between method 1 in shake flasks and the 5L scale up model for phage K 1.1x10 12 ±1.02x10 12 and 8.5x10 12 ±8.99x10 12 pfu/ml respectfully, p=0.001 using an ANOVA. This increase in titre between the scale up model and the shake flasks can be accounted for by the change in system used. The stirred tank system offers the advantage of a more controlled environment that the infection can take place in which will consequently an increase in titre is seen. Previous work has shown the improvement in titre between the two culture systems using the same conditions, given the more controlled environment (Ali et al, 2018). Figure 1 showed that the culture took around 14 minutes to reduce from 37 o C to 28 o C and stabilise, similar to the scale up model. The graphs show a statistically significantly higher rate of adsorption in the first 6 minutes for T4 phage in the shake flask compared to the 5L bioreactor, figure 5A and B. It is interesting to note this as although the conditions were kept the same, the agitation between the culture systems will differ despite the same agitation rate being used, 225rpm E. coli, 150rpm, S. aureus. Additionally, phage K showed a statistically significantly higher rate of adsorption in the shake flask after 2 minutes of infection between each time point p<0.05 using a paired t test. The sheer damage to the host cells and difference in mixing patterns seen in the STR due to the impellor may cause differences in the adsorption between systems (Borys et al, 2018). The graphs show that after 10 minutes, around 99% of phage had adsorbed to their host cell. The difference in the mixing between shake flasks and STR, may affect the adsorption of phage to their host organism and it was interesting to note the differences seen between the culture systems. The harsh agitation caused by impellors may account for the reduced higher free phage in the stirred tank culture despite the same agitation rate used across the systems (150rpm -S. The duplicate experiment in the STR gave an average titre of 4.33x10 14 ± 9.4x10 13 pfu/ml and 4x10 12 ± 1.41x10 12 pfu/ml for T4 phage and phage K respectively. The advantage of using a scale up model is two-fold. Firstly, the increase in yield between the shake flask and STR model within their respected bioprocesses and secondly the improvement in maintenance of conditions which improves the overall bioprocess. This can be seen from the titre achieved as in the shake flask model, figure 2, the greatest titres achievable were 7.89x10 12 ± 1.96x10 12 and 1.03x10 13 ± 1.77 x10 12 pfu/ml for T4 phage and phage K respectively. It is interesting to note the burst size achieved in table 1. In the 5L bioreactor, T4 gave a duplicate average burst size of 97.45 ± 7.3 whilst phage K gave an average burst size of 106.1 ± 8.4. The statistically significantly increased burst in the shake flask whilst kept in a static incubator, compared to the 5L for T4 is interesting to note and could be accounted for in the difference in mixing or pH control. Although no significant difference was seen in the phage K burst, a higher titre was achieved in the 5L culture compared to the shake flask culture. The maintenance of the conditions in the STR would allow an overall higher titre to be achieved when the experiment was run to completion. One study has previously shown that reducing the temperature of infection gave a higher titre at 25 o C compared to 37 o C (Shan et al, 2014). Additionally, Vasina & Baneyx (1997) showed that reducing the temperature from 37 o C to 30 o C after E. coli was grown to mid log phase allowed greater protein production to be found. Hypotheses were given stating that reducing the temperature allowed metabolic processes to be extended and therefore the host organisms could replicate for longer periods of time. Monoclonal antibody titres have also been shown to improve when grown at 31 o C compared to 37 o C (Boigard et al, 2018). Previous work has shown significantly improved yields using an STR over shake flask due to pH and oxygen control. Changes in pH will affect the ability of the host organisms to propagate themselves and the phage (Nobrega et al, 2016).
Moreover, there will be differences in the mass transfer within the culture, between the shake flask and 5L STR, which may also cause gradients again limiting the propagation ability which can be mitigated in bioreactors as they have less mass transfer limitations (Klockner & Buchs, 2012). Given the length of the culture and the improved control in the process throughout, compared to the shake flask model, it may help to explain why greater titres and burst sizes were seen in the STR.

Conclusion
Perhaps the most understudied area within bacteriophage research is its production. Previous research has shown that phage titres can be improved by improving the overall culture conditions. This study examined the conditions at which the T4 and phage K cultures are kept at, at the point of infection. The data presented here shows that maintaining the culture in a static incubator at 28 o C, whilst the shaking incubator cools down to 28 o C, from the pre culture temperature of 37 o C, a significantly improved phage titre can be achieved. Additionally, the burst size is also increased in shake flasks. Whilst minimal differences were seen in the adsorption at each condition, it was interesting to note the differences seen between T4 and phage K. Furthermore, the experiment also studied the point of phage infection. Whilst many infect cultures between 0.2-0.4OD600nm, it was important to show at what infection point the greatest titres and burst sizes were achieved and how the final titres could be statistically significantly improved. This study has shown a further degree of optimisation of two phage bioprocesses and highlights the need to study every part of a bioprocess to improve the final product output. Moreover, an investigation was made into STR phage work, which is severely lacking in the current field. However, future phage therapy will depend on the ability of phage propagation in larger reactors to produce higher yields. Although some of the conditions investigated could not be directly translated into the STR due to differences in the mechanism for cooling, the maintenance of the best conditions in the STR would allow for higher titres to be achieved in experiments run to completion. Further investigation is therefore required into establishing optimal cooling profiles during infection of larger scale cultures and the optimal parameters at infection point for continuous cultures. Future work, must also consider similar studies for other phage as it is well known phage infection kinetics differ between phage-host systems.

Declarations
Ethics approval and consent to participate -Not applicable Availability of data and materials -All data generated or analysed during the study are included in this article The authors declare they have no competing interests   total. Graphs C and D also show the temperature monitoring of the bioreactor cultures (3L working volume, average temperature from duplicate experiments) during reduction to the desirable culture temperature (28°C, solid line).