Browsing by Author "Miettinen, Ilkka"

Multi-drug tolerance is a phenomenon, in which microorganisms normally susceptible to an antimicrobial agent are able to withstand a treatment via phenotypic alteration. The tolerance is conveyed by a microbial subpopulation that is in a non-replicative and metabolically inactive state also known as persistence. Through this kind of dormancy, the subpopulation may survive an otherwise appropriate course of antimicrobials, since the majority of the drugs target cellular division or metabolism. Upon the reduction of the surrounding antimicrobial concentration the multi-drug tolerant cells - persisters - become resuscitated thus allowing repopulation. As opposed to the more widely acknowledged challenge of antimicrobial resistance, the offspring of the specialist survivor cells are genetically identical to the susceptible majority. Persisters are especially abundant in biofilms, a microbial lifestyle characterized by aggregated microcolonies that are covered in a self-produced slimy matrix known as extracellular polymeric substance (EPS). Partly owning to this protective matrix, biofilms are inherently somewhat tolerant to antimicrobial chemotherapy. Moreover, microbes confined in a biofilm are additionally protected against the components of the host immune system. Conversely, it is assumed that persisters in planktonic, i.e. freely floating state, are easily cleared out by white blood cells. Combined, the immune evasive properties of biofilms and the remarkable multi-drug tolerance of persisters give rise to recalcitrant infections that are immensely difficult to eradicate. The described phenomenon constitutes crucially to the major healthcare challenge of chronic, treatment-resistant infections. Tuberculosis, cystic fibrosis lung disorder, bacterial endocarditis and infections related to indwelling medical devices are only a few examples of such problems. Despite the need for antimicrobials with anti-persister efficacy, no such therapeutics is available and very few are being investigated - one important factor being the lack of relevant drug discovery platforms. Therefore, the aim of this study was to develop an anti-persister assay and to carry out a pilot screening of natural product derived bioactive compounds. Based on the notion that persisters are enriched in bacterial cultures that have reached the stationary phase of growth, a persister model was designed using Staphylococcus aureus ATCC 25923 as the test strain. The bacteria were grown in liquid cultures until they reached the stationary phase and subsequent experimentation was carried out to confirm the tolerant state. After the stationary phase persister model was validated, a small pilot screening of natural products was undertaken in the hope of finding novel anti-persister activity. Mitomycin C, a cytotoxic drug with an existing anti-cancer indication was assigned as the positive control compound because of its previously established anti-persister activity. Since it is common for all of the persister-related diseases that the target microorganisms reside within a protective biofilm, an additional assay based on biofilm regrowth was designed to characterize the hit compounds on a more clinically relevant platform. The persister model culture was shown to be tolerant to conventional antibiotics. The re-induction of metabolic activity by diluting into fresh medium recovered the antimicrobial susceptibility expectedly. A total of 4 compounds were identified as anti-persister hits in the pilot screening campaign. Chromomycin A3, dehydroabietic acid, mithramycin A and oleanolic acid were all able to reduce the viable bacterial count in the stationary phase persister model more than 2 logarithmic units at 100 µM. Mithramycin A was the most potent, reducing the viability over 6 log units. The model compound mitomycin C reduced the viable counts 5.49 (± 0.96) logarithmic units. Out of the 4 hits, dehydroabietic acid was selected for the biofilm relapse assay because of its favourable biocompatibility properties. It reduced regrowth for the treated biofilms by 4 logarithmic.