Investigation of the sheltering effect of β-lactam-resistant K.pneumoniae species on twosusceptible E. coli and S. aureus strains

There is increasing evidence that microbial interactions can happen within microbial communities resulting in an increase of the pathogen resistance to antimicrobial therapies. However, the mechanisms of these interactions remain elusive at the present day. Previously, it has been shown that in a polymicrobial community, β-lactam resistant bacteria can protect other non-resistant bacteria from the action of β-lactam drugs without any gene transfer between the resistant and non-resistant bacteria. This phenomenon was named as “sheltering effect” and occurs when the resistant bacteria releases proteins that give protection to the non-resistant bacteria living in the same environment. Klebsiella pneumoniae is one of the world's most dangerous multidrug resistant pathogens. Infections from this bacterium are seriously threatening the public health due to their great ability to quickly become resistant to every antibiotic available today. Even more importantly, K. pneumoniae is found in the blood of patients with polymicrobial infections. This doctoral project focused on the investigation of the sheltering effect in this pathogen. Studies carried out here were designed to elucidate the causes of the variability of this phenomenon observed among different clinical isolates of K. pneumoniae. Furthermore, the role of the Sec-dependent pathway and of the outer membrane vesicles (OMVs) in the sheltering effect was investigated. The present study also examined how the sheltering effect changes in response to different concentrations of β-lactam in the medium. Lastly the presence of sheltering effect in presence of drugs different from the β-lactams was also investigated, The results obtained by these tests also allowed the development of methods for the direct quantification of the sheltering effect based on the features observed on an agar plate. The causes of the variability in the sheltering effect detected on plate were investigated by carrying out Random Amplified Polymorphic DNA (RAPD), Polymerase Chain Reaction (PCR) and Whole Genome Sequencing (WGS) on the genome of the isolates to find a relationship between their genetic features and their potential of sheltering effect. Minimum Inhibitory Concentration (MIC) tests were also performed in the attempt to find a relationship between their resistance profiles and their sheltering effect potential. The results obtained suggest that the sheltering effect is a widespread and variable phenomenon in K. pneumoniae. The protective effect is likely due to the extracellular release of OMV-associated β-lactamases after Sec-mediated translocation of these enzymes from the cytosol to the periplasm. The results also suggest that the sheltering effect diminishes in response to increasing concentrations of β-lactam in the medium. Furthermore, the sheltering effect was not observed in presence of drugs different from the β-lactams. Lastly, no relationship was found between the sheltering effect potential of the isolates and their genetic and resistance features analysed in this project. Therefore more studies will be necessary to elucidate the causes of this variability.