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Browsing by Subject "multi-drug tolerance"

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  • Sarekoski, Jenna (2018)
    Most bacteria live as biofilms (99%), which is a population of cells attached to a natural or artificial surface and encased in self-produced exopolysaccharide matrix. The extracellular polymeric substances (EPS) in the matrix can vary greatly between species in chemical and physical properties, but primarily it consists of water, polysaccharides, proteins, nucleic acids and absorbed nutrients from the surrounding area. Biofilm formation appears to be a survival strategy of bacteria and the main purpose of the biofilm matrix is to protect the bacteria. In nature, biofilms have been found in variety of different environments, including humans. Bacterial biofilms demonstrate a decreased susceptibility to antimicrobial agents and several mechanisms have been proposed to be involved in this tolerance. One of the reasons why chronic infections develop is that the immune response fails to remove the biofilm. Most of the bacterial infections currently in developed countries are biofilm related and these infections are often recalcitrant and difficult to eradicate with available treatments. In addition to chronic infections, the treatment of acute infections is shadowed by increasing problems with highly resistant bacteria. The presence of dormant persisters in biofilms accounts for their tolerance to antimicrobials and likely are responsible for latent and chronic infections, such as tuberculosis. Persistence is not primarily an active mechanism of antibiotic tolerance, but a dormant state of the bacteria avoiding the mechanism of action of most antibiotics. Persisters form stochastically only in small numbers, and more relevant physiological explanation is related to the stress responses of the cells. Persisters are distinguish phenotypic variants of the normal population and it is not a heritable feature, as no mutations occur. The dormant, persistent state of the bacteria is largely responsible for the multidrug tolerance of recalcitrant infections. Biofilm cause various diseases in humans, as bacteria are able to attach to practically any surface, such as teeth, heart valves, lungs, middle ear, artificial prosthetics and instruments. Biofilms growing on prosthetic joints can cause also serious infections, which are painful for the patient with high risks for complications, expensive and laborious to replace. Biofilm infections are difficult to treat and a huge burden in the healthcare. Many acute infections can be cured with conventional antibiotic therapies, but this is not case with recalcitrant, chronic infections. B. cenocepacia belongs to the B. cepacia complex (Bcc) which consist of 20 closely related and phenotypically similar species. This species was chosen for this study because of its natural tolerance to antibiotics and ability to form biofilms easily. This species causes fatal lung infections in cystic fibrosis patients, and there is no treatment for it other than inadequate combination antibiotic treatment and lung transplant. In this thesis, a promising method was developed and validated for detecting anti-persister activity against B. cenocepacia. The assay is based on measuring the levels of ATP present in the cultures after treatment and it can be used quantify remaining persisters using B. cenocepacia biofilms. Utilizing the method validated, it was confirmed that mitomycin C is an effective anti-persister compound against highly tolerant B. cenocepacia biofilms even at low concentrations. Doxycycline was found to be ineffective against B. cenocepacia biofilms, although the bacteria are susceptible to it in planktonic form, and ciprofloxacin was proved to be effective at very high concentrations.
  • Miettinen, Ilkka (2016)
    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.