Work Package 3

"Improving antibiotic activity against tolerant bacteria in biofilm, and strategies for reducing development of antibiotic resistance"



A.  Treatment failure in the absence of resistance. 

Antibiotic therapy can fail without any apparent resistance in the causative microorganisms. Such failure is often due to the induction of general or specific tolerance mechanisms or the formation of biofilms. Little is known about how biofilm formation affects antibiotic action and resistance evolution. We will employ a combination of genetic and molecular biology techniques to identify tolerance mechanisms and factors and regulatory networks important for the microbes response to antimicrobial challenge using the systems biology platform established within DTU. A biofilm model system will be utilized to investigate the dynamics of antibiotic action within microbial biofilms1-3,5-8.
In this context, we will study the role of small regulatory RNAs (sRNA) in bacterial adaptation during chronic infection. As a large collaborative project, the Infectious Microbiology Group has been working together with the Danish Cystic Fibrosis Center (Rigshospitalet, Copenhagen), which has provided a collection of sputum samples collected since 1973 from a large number of CF patients. This unique strain collection represents about 40 years of 'infection history' from a large number of patients, giving the opportunity to analyze the evolution of P. aeruginosa in the CF airway over time. Understanding how P. aeruginosa persists despite the host immune system, the aggressive antibiotic therapy, and changing host pathology may provide with improved treatment strategies.
We will focus on how P. aeruginosa persists in the airways of CF patients despite aggressive and continuous antibiotic therapy. In particular, the molecular mechanisms by which small regulatory RNAs (sRNAs) affect antibiotic susceptibility will be investigated. One of the most important recent advances in biology is the realization that sRNAs, which are encoded in the genomes of all domains of life, can regulate the expression of genes. Little is known about the roles of sRNAs in P. aeruginosa with very few exceptions. The primary objective of the work is to identify and characterize sRNAs in P. aeruginosa laboratory and clinical strains that modulate antibiotic susceptibility. The regulatory functions of identified sRNAs will be investigated and their subsequent utility as antimicrobial targets assessed.
Contributors: Søren Molin, Anders FolkessonKatherine S. LongMaria Gómez-Lozano 

B. Probiotics as a means to prevent antibiotic-associated C. difficile diarrhoea.  

One approach to protect the normal flora from antibiotics will consist in the concomitant administration of probiotics in order to alleviate the ecological impact of antibiotic selection. Particular emphasis will be given to study prevention of C. difficile infections4. We will optimize administration and composition of the probiotic mix on a horse model. The Danish Center for Environment and Toxicology (DHI) will actively collaborate in this study by providing expertise in probiotics and links to networks and industries in this area.
Contributor: Luca Guardabassi 

C. MRSA skin and mucosal decolonization by phage therapy.  

We will study the possible use of a MRSA ST398-specific phage for skin and mucosal decolonization in collaboration with Novolytics. The in vitro bacteriolytic activity of phage isolated from pig slurry will be tested on MRSA ST398 strains available at UC-LIFE. In vivo activity will be studied using a porcine model (PILGRIM). The occurrence of MRSA at different body sites (nasal mucosa, perineum and dorsal skin) will be monitored before and after treatment. Following experiments will be designed to optimize mode of administration and dosage of the phage mix.
Contributor: Luca Guardabassi, Peter Damborg


Reference List

  1. Costerton, W., R. Veeh, M. Shirtliff, M. Pasmore, C. Post, and G. Ehrlich. 2003. The application of biofilm science to the study and control of chronic bacterial infections. J.Clin.Invest 112:1466-1477.
  2. Folkesson, A., J. A. Haagensen, C. Zampaloni, C. Sternberg, and S. Molin. 2008. Biofilm induced tolerance towards antimicrobial peptides. PLoS.One. 3:e1891.
  3. Haagensen, J. A., M. Klausen, R. K. Ernst, S. I. Miller, A. Folkesson, T. Tolker-Nielsen, and S. Molin. 2007. Differentiation and distribution of colistin- and sodium dodecyl sulfate-tolerant cells in Pseudomonas aeruginosa biofilms. J.Bacteriol. 189:28-37.
  4. Hickson, M., A. L. D'Souza, N. Muthu, T. R. Rogers, S. Want, C. Rajkumar, and C. J. Bulpitt. 2007. Use of probiotic Lactobacillus preparation to prevent diarrhoea associated with antibiotics: randomised double blind placebo controlled trial. BMJ 335:80.
  5. Jelsbak, L., H. K. Johansen, A. L. Frost, R. Thogersen, L. E. Thomsen, O. Ciofu, L. Yang, J. A. Haagensen, N. Hoiby, and S. Molin. 2007. Molecular epidemiology and dynamics of Pseudomonas aeruginosa populations in lungs of cystic fibrosis patients. Infect.Immun. 75:2214-2224.
  6. Levin, B. R. and D. E. Rozen. 2006. Non-inherited antibiotic resistance. Nat.Rev.Microbiol. 4:556-562.
  7. Lewis, K. 2001. Riddle of biofilm resistance. Antimicrob.Agents Chemother. 45:999-1007.
  8. Schillaci, D., S. Petruso, S. Cascioferro, M. V. Raimondi, J. A. Haagensen, and S. Molin. 2008. In vitro anti-Gram-positive and antistaphylococcal biofilm activity of newly halogenated pyrroles related to pyrrolomycins. Int.J.Antimicrob.Agents 31:380-382.


Last revised 16 July 2014