Bacterial Biofilm Control by Perturbation of Bacterial Signaling Processes

The development of effective strategies to combat biofilm infections by means of either mechanical or chemical approaches could dramatically change today’s treatment procedures for the benefit of thousands of patients. Remarkably, considering the increased focus on biofilms in general, there has still not been invented and/or developed any simple, efficient and reliable methods with which to “chemically” eradicate biofilm infections. This underlines the resilience of infective agents present as biofilms and it further emphasizes the insufficiency of today’s approaches used to combat chronic infections. A potential method for biofilm dismantling is chemical interception of regulatory processes that are specifically involved in the biofilm mode of life. In particular, bacterial cell to cell signaling called “Quorum Sensing” together with intracellular signaling by bis-(3′-5′)-cyclic-dimeric guanosine monophosphate (cyclic-di-GMP) have gained a lot of attention over the last two decades. More recently, regulatory processes governed by two component regulatory systems and small non-coding RNAs have been increasingly investigated. Here, we review novel findings and potentials of using small molecules to target and modulate these regulatory processes in the bacterium Pseudomonas aeruginosa to decrease its pathogenic potential

Kinetic Model for Signal Binding to the Quorum Sensing Regulator LasR

We propose a kinetic model for the activation of the las regulon in the opportunistic pathogen Pseudomonas aeruginosa. The model is based on in vitro data and accounts for the LasR dimerization and consecutive activation by binding of two OdDHL signal molecules. Experimentally, the production of the active LasR quorum-sensing regulator was studied in an Escherichia coli background as a function of signal molecule concentration. The functional activity of the regulator was monitored via a GFP reporter fusion to lasB expressed from the native lasB promoter. The new data shows that the active form of the LasR dimer binds two signal molecules cooperatively and that the timescale for reaching saturation is independent of the signal molecule concentration. This favors a picture where the dimerized regulator is protected against proteases and remains protected as it is activated through binding of two successive signal molecules. In absence of signal molecules, the dimerized regulator can dissociate and degrade through proteolytic turnover of the monomer. This resolves the apparent contradiction between our data and recent reports that the fully protected dimer is able to “degrade” when the induction of LasR ceases

Quorum Sensing and Virulence of Pseudomonas aeruginosa during Lung Infection of Cystic Fibrosis Patients

Pseudomonas aeruginosa is the predominant microorganism in chronic lung infection of cystic fibrosis patients. The chronic lung infection is preceded by intermittent colonization. When the chronic infection becomes established, it is well accepted that the isolated strains differ phenotypically from the intermittent strains. Dominating changes are the switch to mucoidity (alginate overproduction) and loss of epigenetic regulation of virulence such as the Quorum Sensing (QS). To elucidate the dynamics of P. aeruginosa QS systems during long term infection of the CF lung, we have investigated 238 isolates obtained from 152 CF patients at different stages of infection ranging from intermittent to late chronic. Isolates were characterized with regard to QS signal molecules, alginate, rhamnolipid and elastase production and mutant frequency. The genetic basis for change in QS regulation were investigated and identified by sequence analysis of lasR, rhlR, lasI and rhlI. The first QS system to be lost was the one encoded by las system 12 years (median value) after the onset of the lung infection with subsequent loss of the rhl encoded system after 17 years (median value) shown as deficiencies in production of the 3-oxo-C12-HSL and C4-HSL QS signal molecules respectively. The concomitant development of QS malfunction significantly correlated with the reduced production of rhamnolipids and elastase and with the occurrence of mutations in the regulatory genes lasR and rhlR. Accumulation of mutations in both lasR and rhlR correlated with development of hypermutability. Interestingly, a higher number of mucoid isolates were found to produce C4-HSL signal molecules and rhamnolipids compared to the non-mucoid isolates. As seen from the present data, we can conclude that P. aeruginosa and particularly the mucoid strains do not lose the QS regulation or the ability to produce rhamnolipids until the late stage of the chronic infection

Calculated energy performance certificate of a duplex house and energy renovation analysis

V diplomski nalogi je predstavljena izdelava računske energetske izkaznice s poudarkom na priporočilih za energetsko sanacijo dvostanovanjske stavbe, locirane v Medvodah. Stavba je bila zgrajena leta 1985. V tem obdobju se je začela uporabljati toplotna izolacija, saj so se že pojavljala vprašanja o energetski učinkovitosti stavb. Energetska izkaznica stavb je potrebna pri prodaji in najemu stavbe, če je obdobje najema daljše od enega leta. Obvezna je tudi pri novogradnji kot del projekta izvedenih del. V uvodnem delu sem predstavil veljavno zakonodajo, ki ureja področje učinkovite rabe energije v stavbah. Potem sem s pomočjo programa TEDI in TOST izdelal računsko energetsko izkaznico za obravnavani objekt v obstoječem stanju, nato pa sem se osredotočil na učinkovitost posameznega ukrepa energetske sanacije. Pri vsakem ukrepu sem komentiral dobljene rezultate in se odločil o smiselnosti njegove izvedbe v odvisnosti od tehnične zahtevnosti in posledičnega prihranka energije. Pri obravnavani stavbi se največ energije, potrebne za ogrevanje z menjavo transparentnega dela in mehanskim prezračevanjem, prihrani z vračanjem toplote odpadnega zraka, in sicer blizu 50 %. Z izborom vseh ukrepov, za katere menim, da so smiselni, pa se prihrani preko 80 % potrebne letne toplote za ogrevanje stavbe. Tudi koeficient specifičnih transmisijskih izgub se zmanjša za 60 %. Poveča pa se potreba po hlajenju, kar je tipična stranska škoda zelo dobro toplotno izoliranih stavb. V zaključnem delu so predstavljene ugotovitve, nastale tekom diplomske naloge.In the undergraduate thesis, the production of the calculated energy performance certificate for the specific building is undertaken. This is done with the emphasis on recommendations for energy renovation of the duplex residential building, which is located in Medvode, Slovenia and was built in 1985. When the building was constructed, energy efficiency was already gaining importance. Consequently many buildings constructed in this period were moderately thermally insulated. An energy performance certificate is legally required when a building is offered for purchase and when it is leased for a period longer than one year. Furthermore the certificate is mandatory for newly built properties, as a basic requirement for the project plan. The thesis initially examines the current legislation, which regulates the area of energy efficiency in buildings. Next the energy performance for the considered building will be produced. This will be done with TEDI and TOST computer software. After that, individual technical measures will be analysed. The viability of these measures will be assessed in consideration of technical complexity and estimated energy savings potential. For the considered building, the greatest energy savings can be realised with exterior joinery renovation and installation of heat recovery ventilation. The implementation of booth measures accounts for about 50% of energy savings. With the comprehensive renovation, more than 80% of the annual heat required for the building would be preserved. In this case the specific heat transfer coefficient would decrease by about 60 %. On the contrary the requirements for cooling would increase, which is the common downside of a well-insulated building

Oxidative stress response plays a role in antibiotic tolerance of <i>Streptococcus mutans</i> biofilms

Knowledge about biofilm-associated antibiotic tolerance mechanisms is warranted in order to develop effective treatments against biofilm infections. We performed a screen of a Streptococcus mutans transposon mutant library for mutants with reduced biofilm-associated antimicrobial tolerance, and found that the spxA1 gene plays a role in tolerance towards gentamicin and other antibiotics such as vancomycin and linezolid. SpxA1 is a regulator of genes involved in the oxidative stress response in S. mutans. The oxidative stress response genes gor and ahpC were found to be up-regulated upon antibiotic treatment of S. mutans wild-type biofilms, but not spxA1 mutant biofilms. The gor gene product catalyses the formation of glutathione which functions as an important antioxidant during oxidative stress, and accordingly biofilm-associated antibiotic tolerance of the spxA1 mutant could be restored by exogenous addition of glutathione. Our results indicate that the oxidative stress response plays a role in biofilm-associated antibiotic tolerance of S. mutans, and add to the on-going debate on the role of reactive oxygen species in antibiotic mediated killing of bacteria.Published versio