Final Report Summary - VIRUEVO (The role of protozoa and phage enemies as driving force for bacterial virulence)
The main aim of this project was to study how different protists and phage enemies affect indirectly the evolution of bacterial defensive and virulence traits. In practice, I performed a set of controlled aquatic microcosm experiments (duration for 4 - 8 weeks) where I exposed bacteria to different types of enemies (bacterivorous protists and parasitic phages) in two- and multi-species microbial communities in different environmental conditions. The changes in bacterial defensive traits were measured by using standard laboratory techniques and changes in bacterial virulence determined in Wax moth host system.
I first studied the effects of f2 phage and Tetrahymena thermophila protist on Pseudomonas fluorescens SBW25 bacteria in single-enemy (objective 1) and two-enemy (objective 2) communities in different environmental conditions (objective 3). My results show that selection by both enemies caused evolutionary changes in bacterial life-history traits, while protist and phage enemies also had negative effects on each other. As a result, bacteria evolved the greatest defence against protist enemies in the absence of phage, and greatest resistance against phages in the absence of protists. This can be explained by two important mechanisms: (a) weaker reciprocal selection due to lower densities, and (b) a trade-off between defences against different enemies. Evolution of bacterial defences incurred competitive growth cost, which was in general strongest with bacteria that had evolved in two-enemy community. Thus, P. fluorescens virulence might decrease most in the two-enemy community if lowered growth leads to reduced virulence. Presence of protists also changed the bacteria-phage arms race dynamics to fluctuating coevolutionary dynamics. I later replicated this experiment with T. thermophila protist, PNM-phage and Pseudomonas aeruginosa bacterium community. In a similar manner, evolving defences was costly in terms of reduced growth rate and led to decreased bacterial virulence in wax moth larvae. Together these results support my previous findings: selection by natural enemies leads trade-off with bacterial virulence in variety of opportunistic bacteria.
Next, I concentrated on finding the mechanistic basis of these evolutionary changes at the level of individual clones (objective 4). Phage had no effect on bacterial phenotypic diversification. However, protists selected for highly defensive, small colony types with both bacterial species. With P. fluorescens, increased defence was related to formation of large cell aggregates, which could not be consumed by protist. With P. aeruginosa, most likely mechanism was enhanced growth on culture vessels walls as a mat of bacterial cells (biofilm). It is possible that biofilm growth mode led to lowered growth with both bacteria and could thus explain lowered virulence in Wax moth host. P. aeruginosa bacterium was also toxic for protists in high productivities.
Lastly, I have started to work on defining changes in virulence at the molecular level in collaboration with Steve Paterson (University of Liverpool, objective 5). We are using next generation sequencing technology to see if the phenotypic changes in candidate virulence genes (e.g. biofilm and toxicity) can been detected at the molecular level. I am currently isolating bacterial deoxyribonucleic acid (DNA) for the analyses and the work will be completed during 2012. Besides completing my original objectives, I have studied the evolution of virulence in other smaller projects. Firstly, I studied how protist predation affects social conflict of cooperation and cheating in P. aeruginosa bacterial pathogen. This bacterium cooperates through chemical signalling to express virulence factors more efficiently during infection. Cooperation is however prone to cheating and it is still somewhat unclear why cheaters are not always able to invade cooperative populations. My results show that protist predation can favour cooperation because cheating bacteria pay pleiotropic cost in terms of weaker anti-predatory defence. However, this holds only in low productivity environments, because protists are killed in higher resource concentrations due to exotoxins.
Secondly, I studied how phages could be used to control pathogenic bacteria. In the first project we studied if phage can be used to specifically target bacteria containing conjugative antibiotic plasmids, because these plasmids also encode receptor for the bacteriophage. We found that phages reduced the antibiotic resistance of bacterial populations to less than 5 % within 10 days. In the second project we compared if applying phage species sequentially versus as a mixture affects the clearance and resistance evolution in Pseudomonas aeruginosa. Both methods worked in vitro and in vivo. Mixture yielded best short-term advantages in vivo, while sequential application could be more feasible for long-term treatments because resistance mutations were lost in the absence of the given phage. Together these results show that phage-therapy could be used to complement antibiotic treatments in the future.
Thirdly, I used collection of clinical P. aeruginosa isolates to study how specialisation to within-host environment (lungs of the cystic fibrosis patients) affects bacterial ability to survive in the presence of natural phage and protist enemies when released to external environmental reservoirs. It has been shown previously that long-term adaptation to within-host environment lead to changes in virulence factors, which could be important for bacterial anti-predatory defences. We found that specialisation led to reduced growth and virulence in Wax moth model host. Furthermore, lung-specialists less often killed protist enemies and were less often resistant to natural phages compared to acute clinical P. aeruginosa isolates. As a result, phage-therapy could be especially feasible in treating patients suffering from chronic bacterial infections.
This project demonstrates that protist and phage selection affects bacterial virulence both on short (changes in frequencies or prevalence) and long (evolutionary changes) time scales. In wider socio-economic context, these results offer potential new avenues for the development of alternative or complementary treatments for traditional use of antibiotics. For example, phages could be used in the future to cull out antibiotic resistant bacteria, or to control the density of bacterial pathogens in vitro and in vivo conditions, while the use of protists to treat bacterial infections is still entirely unexamined. Thus, in order to understand and control the evolution of bacterial virulence in the future it is important to learn more about bacterial ecology within- and outside-host environments: fitness trade-offs between virulence and ability to survive under other selective agents are likely to offer novel ways to combat bacterial infections. The main beneficiary of my research is the scientific community. However, the results presented here will also have direct applications for medical microbiologists and researchers.
I first studied the effects of f2 phage and Tetrahymena thermophila protist on Pseudomonas fluorescens SBW25 bacteria in single-enemy (objective 1) and two-enemy (objective 2) communities in different environmental conditions (objective 3). My results show that selection by both enemies caused evolutionary changes in bacterial life-history traits, while protist and phage enemies also had negative effects on each other. As a result, bacteria evolved the greatest defence against protist enemies in the absence of phage, and greatest resistance against phages in the absence of protists. This can be explained by two important mechanisms: (a) weaker reciprocal selection due to lower densities, and (b) a trade-off between defences against different enemies. Evolution of bacterial defences incurred competitive growth cost, which was in general strongest with bacteria that had evolved in two-enemy community. Thus, P. fluorescens virulence might decrease most in the two-enemy community if lowered growth leads to reduced virulence. Presence of protists also changed the bacteria-phage arms race dynamics to fluctuating coevolutionary dynamics. I later replicated this experiment with T. thermophila protist, PNM-phage and Pseudomonas aeruginosa bacterium community. In a similar manner, evolving defences was costly in terms of reduced growth rate and led to decreased bacterial virulence in wax moth larvae. Together these results support my previous findings: selection by natural enemies leads trade-off with bacterial virulence in variety of opportunistic bacteria.
Next, I concentrated on finding the mechanistic basis of these evolutionary changes at the level of individual clones (objective 4). Phage had no effect on bacterial phenotypic diversification. However, protists selected for highly defensive, small colony types with both bacterial species. With P. fluorescens, increased defence was related to formation of large cell aggregates, which could not be consumed by protist. With P. aeruginosa, most likely mechanism was enhanced growth on culture vessels walls as a mat of bacterial cells (biofilm). It is possible that biofilm growth mode led to lowered growth with both bacteria and could thus explain lowered virulence in Wax moth host. P. aeruginosa bacterium was also toxic for protists in high productivities.
Lastly, I have started to work on defining changes in virulence at the molecular level in collaboration with Steve Paterson (University of Liverpool, objective 5). We are using next generation sequencing technology to see if the phenotypic changes in candidate virulence genes (e.g. biofilm and toxicity) can been detected at the molecular level. I am currently isolating bacterial deoxyribonucleic acid (DNA) for the analyses and the work will be completed during 2012. Besides completing my original objectives, I have studied the evolution of virulence in other smaller projects. Firstly, I studied how protist predation affects social conflict of cooperation and cheating in P. aeruginosa bacterial pathogen. This bacterium cooperates through chemical signalling to express virulence factors more efficiently during infection. Cooperation is however prone to cheating and it is still somewhat unclear why cheaters are not always able to invade cooperative populations. My results show that protist predation can favour cooperation because cheating bacteria pay pleiotropic cost in terms of weaker anti-predatory defence. However, this holds only in low productivity environments, because protists are killed in higher resource concentrations due to exotoxins.
Secondly, I studied how phages could be used to control pathogenic bacteria. In the first project we studied if phage can be used to specifically target bacteria containing conjugative antibiotic plasmids, because these plasmids also encode receptor for the bacteriophage. We found that phages reduced the antibiotic resistance of bacterial populations to less than 5 % within 10 days. In the second project we compared if applying phage species sequentially versus as a mixture affects the clearance and resistance evolution in Pseudomonas aeruginosa. Both methods worked in vitro and in vivo. Mixture yielded best short-term advantages in vivo, while sequential application could be more feasible for long-term treatments because resistance mutations were lost in the absence of the given phage. Together these results show that phage-therapy could be used to complement antibiotic treatments in the future.
Thirdly, I used collection of clinical P. aeruginosa isolates to study how specialisation to within-host environment (lungs of the cystic fibrosis patients) affects bacterial ability to survive in the presence of natural phage and protist enemies when released to external environmental reservoirs. It has been shown previously that long-term adaptation to within-host environment lead to changes in virulence factors, which could be important for bacterial anti-predatory defences. We found that specialisation led to reduced growth and virulence in Wax moth model host. Furthermore, lung-specialists less often killed protist enemies and were less often resistant to natural phages compared to acute clinical P. aeruginosa isolates. As a result, phage-therapy could be especially feasible in treating patients suffering from chronic bacterial infections.
This project demonstrates that protist and phage selection affects bacterial virulence both on short (changes in frequencies or prevalence) and long (evolutionary changes) time scales. In wider socio-economic context, these results offer potential new avenues for the development of alternative or complementary treatments for traditional use of antibiotics. For example, phages could be used in the future to cull out antibiotic resistant bacteria, or to control the density of bacterial pathogens in vitro and in vivo conditions, while the use of protists to treat bacterial infections is still entirely unexamined. Thus, in order to understand and control the evolution of bacterial virulence in the future it is important to learn more about bacterial ecology within- and outside-host environments: fitness trade-offs between virulence and ability to survive under other selective agents are likely to offer novel ways to combat bacterial infections. The main beneficiary of my research is the scientific community. However, the results presented here will also have direct applications for medical microbiologists and researchers.