Caracterización de los fagos de Klebsiella pneumoniae con potencial biotecnológico

The extensive use and misuse of antibiotics has led to an increased emergence of multidrug-resistant Klebsiella pneumoniae strains. They are a serious concern worldwide due to their propensity to spread and the scarce effective treatments left. Consequently, phage therapy is garnering renewed interest as an alternative method to defeat antibiotic resistant bacteria. Phages – natural pathogens of bacteria – have several properties: high capacity to replicate and host specificity that turns them into a great advantage over antibiotics. Eight bacteriophages infecting Klebsiella pneumoniae were characterized according to their genetic material and morphology by performing endonuclease digestions and transmission electron microscopy imaging with 1% phosphotungstic acid or 2% uranyl acetate as staining dyes. Then, they were classified in agreement with their morphological characterization. Seven phages (EKP3P1, EKP3P2, EKP3P4, EKP3P5, EKP8P2, EKP8P3 and EKP8P4) were classified into Siphoviridae family showing hexagonal heads with long non- contractile, sometimes flexible tails and closely related restriction patterns. EKP8P1 phage was classified into Podoviridae family showing an icosahedral head with a short non-contractile tail and a different restriction pattern. They all belong to Caudovirales order. Moreover, a prophage was found in EKP8P1 sample, and classified into Siphoviridae family according to its morphology. The genome of EKP3P5 phage, a double stranded DNA of 47,622 bp long, was sequenced and annotated manually. EKP3P5 phage is a temperate phage encoding integrase, holin and endolysin proteins, among others. Therefore, EKP3P5 could not be used in phage therapy due to the risk of transferring virulence and resistance genes to the host bacteria. For all the above reasons, this thesis provides detailed knowledge of the physical structure along with genomic qualities of eight bacteriophages infecting multidrug- resistant Klebsiella pneumoniae strains. This is important for determining the potential of phages as therapeutic agents and the first step to improve phage therapy

Midbiotics : conjugative plasmids for genetic engineering of natural gut flora

ABSTRACTThe possibility to modify gut bacterial flora has become an important goal, and various approaches are used to achieve desirable communities. However, the genetic engineering of existing microbes in the gut, which are already compatible with the rest of the community and host immune system, has not received much attention. Here, we discuss and experimentally evaluate the possibility to use modified and mobilizable CRISPR-Cas9-endocing plasmid as a tool to induce changes in bacterial communities. This plasmid system (briefly midbiotic) is delivered from bacterial vector into target bacteria via conjugation. Compared to, for example, bacteriophage-based applications, the benefits of conjugative plasmids include their independence of any particular receptor(s) on host bacteria and their relative immunity to bacterial defense mechanisms (such as restriction-modification systems) due to the synthesis of the complementary strand with host-specific epigenetic modifications. We show that conjugative plasmid in association with a mobilizable antibiotic resistance gene targeting CRISPR-plasmid efficiently causes ESBL-positive transconjugants to lose their resistance, and multiple gene types can be targeted simultaneously by introducing several CRISPR RNA encoding segments into the transferred plasmids. In the rare cases where the midbiotic plasmids failed to resensitize bacteria to antibiotics, the CRISPR spacer(s) and their adjacent repeats or larger regions were found to be lost. Results also revealed potential caveats in the design of conjugative engineering systems as well as workarounds to minimize these risks.Peer reviewe

Indirect Selection against Antibiotic Resistance via Specialized Plasmid-Dependent Bacteriophages

Antibiotic resistance genes of important Gram-negative bacterial pathogens are residing in mobile genetic elements such as conjugative plasmids. These elements rapidly disperse between cells when antibiotics are present and hence our continuous use of antimicrobials selects for elements that often harbor multiple resistance genes. Plasmid-dependent (or male-specific or, in some cases, pilus-dependent) bacteriophages are bacterial viruses that infect specifically bacteria that carry certain plasmids. The introduction of these specialized phages into a plasmid-abundant bacterial community has many beneficial effects from an anthropocentric viewpoint: the majority of the plasmids are lost while the remaining plasmids acquire mutations that make them untransferable between pathogens. Recently, bacteriophage-based therapies have become a more acceptable choice to treat multi-resistant bacterial infections. Accordingly, there is a possibility to utilize these specialized phages, which are not dependent on any particular pathogenic species or strain but rather on the resistance-providing elements, in order to improve or enlengthen the lifespan of conventional antibiotic approaches. Here, we take a snapshot of the current knowledge of plasmid-dependent bacteriophages

Systematic Comparison of Epidemic and Non-Epidemic Carbapenem Resistant Klebsiella pneumoniae Strains

Over the past few decades, extensively drug resistant (XDR) resistant Klebsiella pneumoniae has become a notable burden to healthcare all over the world. Especially carbapenemase-producing strains are problematic due to their capability to withstand even last resort antibiotics. Some sequence types (STs) of K. pneumoniae are significantly more prevalent in hospital settings in comparison to other equally resistant strains. This provokes the question whether or not there are phenotypic characteristics that may render certain K. pneumoniae more suitable for epidemic dispersal between patients, hospitals, and different environments. In this study, we selected seven epidemic and non-epidemic carbapenem resistant K. pneumoniae isolates for extensive systematic characterization for phenotypic and genotypic qualities in order to identify potential factors that precede or emerge from epidemic successfulness. Studied characteristics include growth rates and densities in different conditions (media, temperature, pH, resource levels), tolerance to alcohol and drought, inhibition between strains, ability to compensate pH, as well as various genomic features. Overall, there are clear differences between isolates, yet, only drought tolerance was found to notably associate with non-epidemic K. pneumoniae strains. We further report a preliminary study on the potential to control K. pneumoniae ST11 with an antimicrobial component produced by a non-epidemic K. pneumoniae. This component initially restricts bacterial growth, but stable resistance develops rapidly in vitro

Black Queen Evolution and Trophic Interactions Determine Plasmid Survival after the Disruption of the Conjugation Network

Mobile genetic elements such as conjugative plasmids are responsible for antibiotic resistance phenotypes in many bacterial pathogens. The ability to conjugate, the presence of antibiotics, and ecological interactions all have a notable role in the persistence of plasmids in bacterial populations. Here, we set out to investigate the contribution of these factors when the conjugation network was disturbed by a plasmid-dependent bacteriophage. Phage alone effectively caused the population to lose plasmids, thus rendering them susceptible to antibiotics. Leakiness of the antibiotic resistance mechanism allowing Black Queen evolution (i.e. a “race to the bottom”) was a more significant factor than the antibiotic concentration (lethal vs sublethal) in determining plasmid prevalence. Interestingly, plasmid loss was also prevented by protozoan predation. These results show that outcomes of attempts to resensitize bacterial communities by disrupting the conjugation network are highly dependent on ecological factors and resistance mechanisms.</p

Indirect Selection against Antibiotic Resistance via Specialized Plasmid-Dependent Bacteriophages

Antibiotic resistance genes of important Gram-negative bacterial pathogens are residing in mobile genetic elements such as conjugative plasmids. These elements rapidly disperse between cells when antibiotics are present and hence our continuous use of antimicrobials selects for elements that often harbor multiple resistance genes. Plasmid-dependent (or male-specific or, in some cases, pilus-dependent) bacteriophages are bacterial viruses that infect specifically bacteria that carry certain plasmids. The introduction of these specialized phages into a plasmid-abundant bacterial community has many beneficial effects from an anthropocentric viewpoint: the majority of the plasmids are lost while the remaining plasmids acquire mutations that make them untransferable between pathogens. Recently, bacteriophage-based therapies have become a more acceptable choice to treat multi-resistant bacterial infections. Accordingly, there is a possibility to utilize these specialized phages, which are not dependent on any particular pathogenic species or strain but rather on the resistance-providing elements, in order to improve or enlengthen the lifespan of conventional antibiotic approaches. Here, we take a snapshot of the current knowledge of plasmid-dependent bacteriophages

Taipuisan, montaa elektrodia käyttävän sähkökemiallisen aptasensorin optimointi ja luonnehtiminen malaria biomarkkereiden havaitsemiseen

This thesis aims to optimize and characterize a multi-target testing method for malaria detection. Malaria is a mosquito-borne infectious disease caused by Plasmodium parasites. It is a leading cause of death and disease in many developing countries. Electrochemical aptamer-based biosensors (aptasensors) offer lower cost, more mobile and easier to manufacture and operate detection tests compared to other testing methods such as microscopy and polymerase chain reaction. Aptamers are artificially prepared short single-stranded oligonucleotides which are used as bioreceptors for aptasensors. Chronocoulometry, cyclic voltammetry and differential pulse voltammetry (DPV) were used for fabrication optimization and parameter analysis and DPV was used for characterization and detection. Diffusion based spontaneous self-assembled monolayer (SAM) formation was used as bio-functionalization method. By using four different aptamers synthetized with multiple thiol groups, 2008s, pL1, LDHp11 and 2106s, a multi-target aptasensor was fabricated by SAM deposi-tion forming an aptamer/PEG mixed monolayer offering high sensitivity, selectivi-ty, and specificity. The resulting multi-target aptasensors have sensitivities > 80% at 5 parasites/μL, clearly surpassing the WHO’s clinical minimum of > 75% at 200 parasites/μL. Furthermore, the multi-target aptasensors have also demonstrated specificities of 100% for all the aptamers, therefore sufficing the WHO’s clinical minimum of 90% for malaria detection. Long-term stability of the aptasensors was enhanced with commonly used stabilizer pullulan. The experiments were pullulan was introduced to the aptasensors showed great promise of enhanced stability. Fabrication of the flexMEA multi-target electrochemical aptasensor for malaria biomarker detection was further optimized with aptamers synthetized with multi-ple thiol groups and the resulting aptasensor was characterized based on its sensitivity, selectivity, specificity, and stability. Combination test that could detect and distinguish between P. falciparum and P. vivax is in high demand since distinguishing between the parasites is a key factor for correct treatment and preventing the parasites from developing further resistance towards antimalaria medication, a phenomenon recently observed in certain malaria-endemic areas. With further research, this detection method that detects not only HRP-2 but also PfLDH and PvLDH biomarkers for P. falciparum and P. vivax detection has potential to offer a low-cost, high affinity and sensitive approach for highly specific malaria detection.Tämän diplomityön tavoite on malarialoisten aiheuttaman malariainfektion havaitsemiseen käytetyn testausmenetelmän valmistuksen parantaminen ja luonnehtiminen. Tämä testausmenetelmä hyödyntää useita malarialoisten proteiineja tartuntalähteen tunnistamiseksi. Malaria on yksi johtavista kuolin- ja sairastumissyistä kehitysmaissa. Sähkökemialliset aptasensorit tarjoavat edullisemman, liikkuvamman ja helpommin valmistettavan ja käytettävän testausmenetelmän kuin mikroskopia ja polymeraasiketjureaktio. Valmistuksen parantamiseen ja analysointiin käytettiin biosensoreille tyypillisiä sähkökemiallisia luonnehtimis- ja testausmenetelmiä. Diffuusioon perustavaa itsestään kokoontuvan monomolekulaarisen kerroksen muodostumista käytettiin biotoiminnallisuuden saavuttamiseksi. Neljä aptameeria, 2008s, pL1, LDHp11 ja 2106s, toimivat työssä reseptorimolekyyleinä. Nämä aptameerit syntetisoitiin käyttäen useita tioliryhmiä aptameeri/PEG sekalaisen kerroksen muodostamiseksi, mikä tarjosi korkean signaalin, havaintokyvyn, selektiivisyyden ja havaitsemistarkkuuden. Tuotetun aptasensorin havaintokyky oli yli 80% kun parasiittejä oli 5/μL ylittäen selkeästi WHO:n kliinisen minimin, yli 75% kun parasiittejä on 200/ μL. Sen lisäksi, aptasensorin havaitsemistarkkuus oli 100% kaikkien aptameerien kohdalla täyttäen WHO:n kliinisen vaatimuksen, 90%. Pitkäaikaisen vakauden parantaminen pullulaanilla näytti myös lupaavia tuloksia. Taipuisan useita elektroneja sisältävän kokoonpanon käyttöä useita malaria biomarkkereita havaitsevana sähkökemiallisena aptasensorina paranneltiin käyttämällä useita tioliryhmiä ja lukuisia piirteitä havaitsevia menetelmiä tutkittiin tämän diplomityön aikana. Yhdistelmätesteille, jotka pystyvät erottamaan P. falciparumin ja P. vivaxin välillä, on tällä hetkellä tarvetta ja kysyntää. Parasiittien erottaminen on tärkeää oikean hoidon tarjoamisen kannalta. Parasiittien erottelu ja oikea diagnoosi estää parasiittejä kehittämästä vastustuskykyä malarialääkkeitä vastaan, sillä eri parasiittien aiheuttama infektio hoidetaan oikealla tavalla. Tutkimalla lisää tätä menetelmää, joka käyttää HRP-2:n lisäksi PfLDH:ta ja PvLDH:ta biomarkkereina P. falciparumin ja P. vivaxin havaitsemiseen, on mahdollista tarjota edullinen, korkea affiniteettinen ja tarkka menettelytapa malarian havaitsemiselle