Department of Medical Biochemistry and Microbiology

Linus Sandegren research group

Available positions in the group:
Degree project: contact linus.sandegren[at]imbim.uu.se for availability.

Dynamics of plasmid-borne antibiotic resistance

We study fundamental aspects of how resistance plasmids are maintained and disseminated between pathogenic bacteria and how they serve as platforms for evolution of antibiotic resistance. The main focus is to understand how factors such as stability, mobility, positive selection and fitness cost influence the evolutionary success of plasmids. The experimental systems used are based on clinically isolated multi-resistance plasmids encoding extended spectrum β-lactamases (ESBLs) in enteric bacteria (Escherichia coli and Klebsiella pneumoniae) that pose an increasing clinical problem by providing bacteria with resistance to the most used antibiotics today, β-lactams such as penicillins and cephalosporins.

Four main themes are of particular interest in these studies:

  1. What impact do low levels of antibiotics have on spread, selection and maintenance of multi-resistance plasmids?
  2. What plasmid factors cause a fitness-cost on the host cell and can the fitness-cost of plasmid carriage be alleviated by the bacterium in the absence of antibiotics?
  3. How common are gene amplifications during treatment, how do they affect the efficacy of antibiotics and does the dynamics of gene amplification on plasmids accelerate evolution of new resistance?
  4. How is resistance development and plasmid dynamics influenced by bacterial growth in biofilms compared to planktonic growth?

From these studies we gain new knowledge of how bacterial cells and plasmids co-evolve and how selection of new resistance can accelerate through gene amplification at different antibiotic concentrations. Such knowledge can be used to design antibiotic treatment regimens that limit selection of resistance and minimize the potential for new resistance to evolve.

ESBL-plasmid evolution during a clinical outbreak of multiresistant Klebsiella pneumoniae

Greta Zaborskyte, Karin Hjort
During 2005-2007 there was a large outbreak of a multi-resistant, ESBL-producing Klebsiella pneumoniae clone at the Uppsala University Hospital. We have been involved in the characterization of the outbreak both with respect to the bacterium and the resistance plasmid. Ongoing projects are dealing with further characterization of how the outbreak clone and the resistance plasmid have changed over time with different selective pressures and how it contributes to evolution of resistance against other antibiotics.

The multi-resistance phenotype of the Klebsiella pneumoniae that caused the outbreak at the Uppsala University Hospital was due to a large multi-resistance plasmid. We have determined the complete sequence of the plasmid using massive parallel sequencing. Analysis of the plasmid shows that it consists of a backbone that is highly similar to a previously sequenced Klebsiella plasmid but has a resistance cassette comprising 45-kbp that instead is highly similar to the resistance cassette from plasmids associated with E. coli belonging to the international outbreak lineage ST131. This combination of a backbone and a resistance cassette from another plasmid have occurred through direct homologous recombination, in part mediated by homology in shared mobile insertion sequences between the two plasmids. We have also detected conjugational transfer of the plasmid from the outbreak Klebsiella to E. coli of the patient’s own intestinal microflora. However, the plasmid is only stable in Klebsiella with an increased loss-rate in E. coli and no further spread of the E. coli transconjugants could be detected during the outbreak. We have now also completed the genome sequence of the outbreak clone and are performing comparative analysis of 110 isolates of the outbreak clone looking into how both the plasmid and the chromosomal sequence has changed during the outbreak and if this has been affected by antibiotic treatment of the individual patients.

Combinatorial effects of plasmid-borne β-lactamases on resistance development to different combination therapies with β-lactamase inhibitors and β-lactam antibiotics

Fredrika Rajer
The main resistance mechanism against β-lactams among Gram-negative bacteria is production of β-lactamases, enzymes that degrade β-lactams. By combining the ineffective β-lactam antibiotics with a β-lactamase inhibitor, which is structurally similar to β-lactams, successful treatment of infections can again be observed. Unfortunately bacteria can circumvent the effect of the combination therapy by overexpressing existing β-lactamases or by altered porin expression, reducing antibiotic entry into the bacterium. We lack full understanding of how bacterial strains carrying resistance plasmids with multiple β-lactamase genes affect the combination therapy. In this study we want to investigate how the well characterized multi-resistance plasmid pUUH239.2, encoding three different β-lactamase genes (blaTEM-1, blaOXA-1 and blaCTX-M-15) and its host overcomes the selective pressure of β-lactam/ β-lactamase inhibitor combinations. The role of gene amplification in the resistance process is studied in particular.

Fitness cost of plasmid-borne antibiotic resistance.

Fredrika Rajer
The evolutionary success of a plasmid is largely determined by its potential to be stably maintained in the host population. Resistance plasmids are widespread among clinically important bacteria due to the beneficial resistance genes encoded on the plasmids. However, plasmids usually confer a fitness cost on the host cell under conditions when the beneficial factors encoded are not needed (i.e. when antibiotics are not present). Why plasmids pose a fitness cost on the bacterium is still unclear. Under such non-selected conditions plasmid-bearing cells will be at a disadvantage and loss of the plasmid will result in more fit segregants that may out-compete the plasmid-containing cells. Stable plasmid maintenance in a bacterial population can therefore only be achieved if the rate of plasmid loss (by segregational loss and/or fitness costs) is balanced by the rate of plasmid gain (by horizontal transfer and/or fitness advantages).

In this project we study the fundamental properties of plasmid fitness costs and how they can be compensated for. The clinical multi-resistance low-copy plasmid, pUUH239.2 confers a fitness cost of 2.7% in competition experiments with plasmid-free cells. This cost is comparable with other clinical ESBL-encoding plasmids tested. Gene-deletion mutants showed the cost to be ameliorated when removing the resistance cassette of the plasmid. The findings indicate the pUUH239.2 backbone to be nearly cost-free, whereas the cost inflicted by the plasmid is largely due to resistance genes. These findings can have an impact on the maintenance of plasmid and resistance genes.

Dynamics of plasmid-borne antibiotic resistance in Klebsiella pneumoniae biofilms

Greta Zaborskyte
Biofilms represent bacterial communities – attached to a surface or not - which are associated with chronic infections. Physiological tolerance to antibiotics leads to survival of the high dosage treatment, and possibly provides a fertile ground for the development of genetically determined resistance. In addition, plasmids – common vehicles for transfer of resistance genes – and biofilms seem to be engaged in a complex relationship. For instance, plasmids can provide structural components for biofilms (e.g. fimbriae, extracellular DNA), while biofilms can in turn affect plasmid biology by increasing conjugation frequency or copy number heterogeneity.

In this project, we are using Klebsiella pneumoniae in vitro biofilms as a model for the study of plasmid biology in surface-attached biofilms. K. pneumoniae is a nosocomial pathogen posing an increasing threat due to the transmission of multiresistance plasmids and it is also able to form biofilms, e.g. on urinary catheters. The overall goal of our study is to find out how antibiotic treatment, especially at low concentrations, affects plasmid-biofilm relationship with the focus on evolution of plasmid-borne antibiotic resistance mechanisms.

The efficiency and rate of resistance enrichment in the biofilm is going to be assessed by in vitro competition experiments between susceptible and resistant bacteria. Experimental evolution might answer whether resistance mechanisms selected in biofilms differ from those prevalent in planktonic cultures. We also plan to set up an in vitro biofilm model system compatible with live in situ imaging to monitor the invasion and spread of resistance plasmids in the biofilms exposed to antibiotics. The knowledge gained in this project might give insights on more efficient treatment of K. pneumoniae biofilm-related infections.