Top Lab: Dr. Leen De Gelder

Dynamics of antibiotic-sensitive mutants in a population of resistant bacteria during experimental evolution

Temporarily discontinuing the use of antibiotics has been proposed as a means to eliminate resistant bacteria by allowing sensitive clones to sweep through the population. In this study, we monitored a tetracycline-sensitive subpopulation that emerged during experimental evolution of E. coli K12 carrying the multiresistance plasmid pB10 in the absence of antibiotics. The fraction of tetracycline sensitive mutants increased slowly over 500 generations from 0.1 to 7%, and loss of resistance could be attributed to a recombination event that caused deletion of the tet operon. To help understand the population dynamics of these mutants, three mathematical models were developed that took into consideration recurrent mutations, increased host fitness (selection), or a combination of both mechanisms (full model). The data were best explained by the full model, which estimated a high mutation frequency (λ=3.11×10^-5) and a significant but small selection coefficient (σ=0.007). This study emphasized the combined use of experimental data, mathematical models, and statistical methods to better understand and predict the dynamics of evolving bacterial populations, more specifically the possible consequences of discontinuing the use of antibiotics.

De Gelder et al., 2004, Genetics. DEGELDER GENETICS 04

2. Influence of the bacterial host on plasmid-related properties

   Plasmid transfer in the environment is influenced by the plasmid donor

   Horizontal transfer of broad-host-range (BHR) plasmids in the environment contributes to the adaptation of microbial communities to fluctuating conditions, such as the presence of antibiotics or xenobiotic compounds. Although the host cell is the plasmid’s primary environment, possible effects of the plasmid donor on the range of bacteria to which plasmids spread in microbial communities have not been investigated. In this study, we examined whether the host range of a BHR multiresistance plasmid within an activated sludge microbial community depends on the plasmid carrying donor strain. Three pB10::rfp donors, Pseudomonas putida, Ralstonia eutropha and Sinorhizobium meliloti, were added separately to replicate samples of fresh activated sludge from a wastewater treatment plant. The phylogeny of 306 transconjugants was determined by partial 16S rRNA gene sequence analysis. The transconjugants belonged to 15 genera of the α- and γ-Proteobacteria, of which 8 have not been previously identified as environmental hosts for BHR plasmids. Statistical analysis showed that the phylogenetic diversities of transconjugants obtained using the three donors were significantly different. Most strikingly, the transconjugants obtained after matings in sludge with S. meliloti included eight genera that were not represented among the transconjugants obtained with the other donors. Our results indicate that the spectrum of hosts to which a promiscuous plasmid transfers in a microbial community can be strongly influenced by the donor from which it transfers. This study may stimulate further research on the factors that determine the host range of plasmids in microbial communities.
    

    

   De Gelder et al., 2005, Appl. Envir. Microb.

   
   Detection of pB10::rfp transconjugants on agar plates from the sludge microbial community through red fluorescence.

    

   Plasmid stability of the broad host range plasmid pB10 differs among closely related Proteobacteria.

   Broad-host-range plasmids are known to stably replicate in a wide range of Proteobacteria. Nevertheless, plasmid-free cells can arise through segregational instability and sweep through a population in the absence of selection if the plasmid imposes a burden to its host. Although the host cell is the plasmid’s main environment, possible variation in segregational instability and burden of the same plasmid in different hosts has not been systematically investigated. To examine whether the host can affect plasmid stability, we measured the loss of the IncP-1β plasmid pB10 in 20 different strains belonging to the α-, β- and γ-Proteobacteria, including environmental isolates and lab strains. Three strains (Pseudomonas putida H2, P. koreensis R28 and Stenotrophomonas maltophilia P21) showed a very rapid plasmid loss and four showed slower loss, while for all other strains segregants where either detected sporadically or not at all. Remarkably, all these strains are environmental isolates and the cause of instability was not due to incompatibility with resident plasmids. A mathematical model was developed to capture the segregants’ population dynamics, incorporating segregational instability l and burden s as parameters. For the three strains with high plasmid loss, the Maximum Likelihood estimates of l and s varied from 10^-2 to 10^-9 and from 3.6% to 37% respectively, indicating that the burden was the major cause of the sweep of segregants. The plasmid burden of pB10 in the four strains showing slower loss varied from 0.4% to 4.9%. This study shows that the same plasmid can show high variability in stability in closely related organisms, suggesting that strain specific host-plasmid interactions can influence the persistence of a plasmid.

   
   Accurate determination of plasmid loss dynamics requires replica picking of large numbers of colonies on selective and non-selective plates.

    

2002:   Master's in Bioengineering, option Cell and Gene Biotechnology, University of Gent, Belgium

Master's thesis: Bioaugmentation strategies for the removal of 3-chloroaniline in wastewater

Research performed at LabMET, Department of Biochemical and Microbial Technology, Faculty of Agricultural and Applied Biological Sciences, University of Gent

Advisers: Dr. Willy Verstraete, Dr. Eva Top

 ·        Peer-reviewed

 De Gelder, L., F. P. J. Vandecasteele, C. J. Brown, L. J. Forney, and E. M. Top. 2005. Plasmid donor affects host range of the promiscuous IncP-1ß plasmid pB10 in a sewage sludge microbial community. Appl. Environ. Microbiol. 71: in press for September

 Joyce, P., Z. Abdo, J. Ponciano, L. De Gelder, L. Forney, and E. M. Top. 2005. Modeling the impact of periodic bottlenecks, unidirectional mutation and observational error in experimental evolution. J. Math. Biol. 50: 645–662.

 De Gelder, L., Z. Abdo, J. M. Ponciano, P. Joyce, L. J. Forney, and E. M. Top. 2004. Combining mathematical models and statistical methods to understand and predict the dynamics of antibiotic sensitive mutants in a population of resistant bacteria during experimental evolution. Genetics 168:1131-1144.

 Boon, N., L. De Gelder, H. Lievens, S. D. Siciliano, E. M. Top, and W. Verstraete. 2002. Bioaugmenting bioreactors for the continuous removal of 3-chloroaniline by a slow release approach. Environ. Sci. Techn. 36:4698-4704.

 ·        Conference Proceedings

 Boon, N., L. De Gelder, H. Lievens, W. Verstraete, S. D. Siciliano, and E. M. Top. 2004. Novel slow release approach to bioaugment more effectively activated sludge systems, p. 93-97. In W. Verstraete (ed.), Proceedings of the European Symposium on Environmental Biotechnology (ESEB 2004), Oostende, Belgium. Balkema Publishers.

·        Abstracts

 De Gelder, L., L. J. Forney, and E. M. Top. 2005. Exploring the host range of the broad-host-range plasmid pB10. Plasmid 53:57-58. (Presented at 2004 Plasmid Biology Meeting, Kanoni, Greece)

 Top, E. M., L. De Gelder, H. Heuer, R. Fox, R. Ganta, L. J. Forney, J. M. Ponciano, Z. Abdo, P. Joyce, R. Lu, and S. Krone. 2005. Ecology and evolution of broad-host-range plasmids: time for new quantitative approaches. Plasmid 53:57. (Presented at 2004 Plasmid Biology Meeting, Kanoni, Greece)

 De Gelder, L., L. J. Forney, and E. M. Top. 2004. Exploring the host range of the broad-host-range plasmid pB10. ISME-10 Abstract Book nr. 1263. (Presented at 2004 ISME-10, Cancun, Mexico)
 

Presentations

Oral presentation at the 2005 Symposium on Bacterial Genetics and Ecology (BAGECO-8), Lyon, France
De Gelder, L., F. P. J. Vandecasteele, C. J. Brown, L. J. Forney, and E. M. Top. Influence of the plasmid donor on transfer of the promiscuous plasmid pB10 in a microbial community.