Klebsiella pneumoniae is a Gram-negative bacterium commonly giving rise to nosocomial infections especially in immunocompromised individuals. The high occurrence of antibiotic resistant K. pneumoniae strains in combination with the bacterium’s ability to form biofilms that are naturally more tolerant to antibiotic treatment represents a burden for the healthcare system due to potential therapeutic failure. In this study, we were investigating the effect of gentamicin, cefotaxime and ciprofloxacin on biofilm formation and eradication of a multi-resistant ESBL-producing K. pneumoniae strain associated with an outbreak at the Uppsala University Hospital in Uppsala, Sweden, from 2005 to 2007. Multidrug resistance was encoded on the pUUH239.2 plasmid. We also studied how resistant bacteria are selected for in mixed biofilm populations consisting of both resistant and susceptible bacteria under antibiotic treatment. Biofilms were grown in vitro by using an in-the-lab developed peg model. Biofilms were either allowed to form in the presence of the antibiotics or pre- formed and then exposed to antibiotic treatment at different time points. Our results suggest that gentamicin, cefotaxime and ciprofloxacin inhibit biofilm formation at concentrations below the minimum inhibitory concentration (MIC) and that biofilms become more tolerant to antibiotic treatment with increasing maturation. We further observed that increasing antibiotic concentrations select for the presence of the plasmid in less mature biofilms. Overall, these findings highlight the need of early antibiotic treatment during infection and give insight into the dynamics of resistant and susceptible bacteria in mixed biofilm populations in the presence of antibiotics.

Antibiotic resistance is an ongoing global medical crisis and we are in great need of new antibacterial agents to combat rapidly emerging resistant pathogens. Tigecycline is one of few drugs that have been introduced into medicine during the last two decades. It is a broad-spectrum third generation tetracycline that is active against multidrug-resistant bacteria that cause complicated infections. In this thesis I examined the development of tigecycline resistance in Escherichia coli and associated in vitro and in vivo fitness effects. Selections of spontaneous E. coli mutants revealed relatively high accumulation rates of changes in the multidrug efflux system AcrAB-TolC regulation network and in heptose biosynthesis and transport pathways important for lipopolysaccharide (LPS) synthesis. Both groups of mutations led to reduced susceptibility to tigecycline and slower growth compared to the wild-type bacteria. Additional in vitro fitness assays and in vivo competitions showed that LPS mutants were less fit than efflux mutants, providing a possible explanation for why up-regulation of multidrug efflux pumps is the main tigecycline resistance mechanism reported in clinical isolates. Tigecycline was designed to evade the two most common tetracycline resistance mechanisms conferred by Tet proteins, efflux and ribosomal protection. However, tigecycline is a substrate for the tetracycline modifying enzyme Tet(X). Screening of Tet protein mutant libraries showed that it is possible to select Tet mutants with minimal inhibitory concentrations of tigecycline that reach clinically relevant levels. Mutations in Tet proteins that permitted a better protection from tigecycline frequently exhibited reduced activity against earlier generations of tetracyclines, except for the Tet(X) enzyme mutants, which were better at inactivating all tested tetracyclines. This is particularly worrisome because different variants of Tet(X) have recently spread to multidrug-resistant pathogens through horizontal gene transfer. Therefore, Tet(X) mutants with improved activity threaten the medical future of tetracyclines. Multidrug resistance is easily disseminated through horizontally spreading conjugative plasmids. pUUH239.2 is an example of a successful conjugative plasmid that caused the first clonal outbreak of extended spectrum β-lactamase-producing Klebsiella pneumoniae in Scandinavia. This plasmid was formed after rearrangements between two different plasmid backbones and it carries resistance genes to multiple antibiotic classes, heavy metals, and detergents.