Bacteriocins as novel antibiotics against the opportunistic pathogen Pseudomonas aeruginosa

1. Bacteriocins as novel antibiotics against the opportunistic pathogen Pseudomonas aeruginosa Sijmen Schoustra1*, Jonathan Dench1, Shawn Aaron1,2 & Rees Kassen1 1Biology Department, University of Ottawa, Canada; 2Ottawa Health Research Institute, Ottawa, Canada. *Sijmen.Schoustra@uOttawa.ca Bacteriocins Bacteriocins are toxins produced by bacteria directed at killing a limited number of close relatives. They are very abundant in nature, over 95% of bacterial strains tested produce at least one bacteriocin [1]. In this study we explore the possibility of the use of bacteriocins as novel antibiotics using laboratory strains and clinical isolates of the opportunistic pathogen Pseudomonas aeruginosa, whosebacterio-cins are referred to as pyocins [2]. Fig 2.Inhibition of clinical isolates by bacteriocin produced by laboratory strains PA01 and PA14. Fig 3.Inhibition by PA01 bacteriocin as function of relatedness to the victim. Evolution of production and resistance We developed a protocol for experimental evolutionof interaction between multiple strains (Fig 4) to ask if bacteriocin production levels could change by mutation and selection. We let the producing strain evolve over multiple generations, periodically transferring populations to fresh medium. At each transfer, we filtered out the sensitive strain and replaced it with the original strain from a non-evolving stock. Pilot results (Fig 5) of a selection experiment on rich medium show the level of bacteriocin production can change over evolutionary time. We are repeating the selection on minimal medium. We plan to do the same experiment focusing on evolution of the sensitive strain. Fig 4. Protocol for experimental evolution of interaction between multiple strains. Co-evolution By studying the co-evolution of producers and victims we will examine whether a Red-Queen type dynamic between producers and sensitive strains can occur (Fig 6). In the first step of this dynamic, sensitive strains can acquire a resistance, in the second step producers could, by mutation, alter their bacteriocin, being able to target the victims again. When bacteriocins are applied as antibiotics, this dynamic couldbreak antibiotic resistance. Fig.1.Lawn of sensitive bacteria inhibited by colonies of a bacteriocin producing strain (left). Inhibition assays We screened over 150 clinical isolates for their sensitivity to pyocin produced by standard laboratory strains PA01 and PA14. We studied growth inhibition the using a spotting method [3]. Over 85% of our clinical isolates tested were inhibited by at least one of our laboratory strains; on average PA01 inhibits stronger than PA14 (Fig 2). Using banding patterns on PFGE gels, we estimated the level of relatedness of the clinical isolates and PA01. In line with theory on kin selection, inhibition peaks at intermediate relatedness(Fig 3). P P* V V* V** Fig 6.Red Queendynamic of producers and victims Acknowledgements We thank Tracy Giesbrecht and Danna Gifford for help and assistance. The Ontario Provincial Government and the National Science and Research Council of Canada provided funding. References [1]Klaenhammer, T. R. 1988. Biochimie 70:337-349.[2]Michel-Briand, Y., and C. Baysse. 2002. Biochimie 84:499-510.[3]Fyfe, J. A. M., G. Harris, and J. R. W. Govan. 1984. Journal of Clinical Microbiology 20:47-50. April 2008 Fig 5. Killing activity against a common victim of bacteriocin producers with different evolutionary history.