Browsing by Subject "assay development"

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.