tmRNA kasutamine markermolekulina bakterite tuvastamisel mikrokiibi ja biosensor tehnoloogia kaudu

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Bakterite uurimiseks on traditsiooniliselt kasutatud erinevaid kultuuris kasvatamise meetodeid. Kuigi mainitud meetodid on töökindlad ja täpsed, on nad samas ka küllaltki töö- ja ajamahukad ning ei võimalda kõikide bakterite uurimist. Seetõttu kasutatakse tänapäeval tihti nende uurimiseks erinevaid molekulaarbioloogilisi meetodeid, mis põhinevad spetsiifiliste nukleiinhappe järjestuste tuvastamisel ja kirjeldamisel. Käesolevas töös tutvustatakse erinevaid tehnoloogiaid, mida kasutatakse nukleiinahappe põhises bakteriaalses diagnostikas. Põhirõhk on erinevatel nukleiinhapete paljundamise meetoditel ning hübridisatsiooni-põhistel detektsiooni tehnoloogiatel. Käsitletud on erinevate mikrokiibi ja biosensor tehnoloogiate põhimõtteid ning nende võimalikke kasutusviise bakterite tuvastamisel. Lisaks antakse ülevaade DNA ja RNA järjestustest, mida saab kasutada markerjärjestusena erinevate bakterite tuvastamisel ja üksteisest eristamisel. Pikemalt tutvustatakse tmRNA molekule, mida kasutatakse markerjärjestusena käesoleva doktoritöö raames välja töötatud diagnostiliste meetodite puhul. tmRNA on kõikides bakterites leiduv, keskmiselt 300-400 nukleotiidi pikkune spetsiifiline RNA molekul, mis abistab rakus valgusünteesi mehhanismi, ning mille molekuli järjestuse põhjal on võimalik tuvastada ning eristada erinevaid bakteriliike ja ka kõrgemaid taksonoomilisi üksusi. Töö praktilises osas kirjeldatakse kahte erinevat meetodit, kus tmRNA detektsiooni kaudu tuvastatakse erinevaid baktereid. Nendest esimene põhineb tmRNA molekulide spetsiifilisel paljundamisel NASBA tehnoloogia abil, millele järgneb märgistatud tmRNA molekulide tuvastatamine ja täpne identifitseerimine mikrokiibi tehnoloogia abil. Teise puhul toimub tmRNA-de detektsioon märkevaba reaal-ajas toimiva biosensor süsteemi abil, mis põhineb optilisel mikroring resonaator tehnoloogial. Kuigi mõlema meetodi puhul kasutati testsüsteemina erinevaid hingamisteede haigusi põhjustavaid baktereid ning nende vastavaid liigispetsiifilisi tmRNA molekule, on kirjeldatud tehnoloogiad lihtsasti rakendatavad ka teiste RNA järjestuste ning erinevate bakteri-liikide korral.There is a growing need for faster and more reliable approaches for microorganism detection and identification that could complement or replace conventional rather time- and labor-consuming culture-based technologies. A common tactics nowadays is to analyze the nucleic acid component of analyte solution and determine the bacterial composition according to specific nucleic acid profiles that are detected and identified. Theoretically every bacterial species and strain contain unique characteristic target regions that can be used for their specific identification. In the first part of current thesis a literature overview is given about the different technologies that are used for nucleic acid-based bacterial detection. Main focus is on nucleic acid amplification and hybridization-based detection methods with emphasis on microarray and biosensor technologies, and their practical application in bacterial diagnostics. In second part of the literature overview, a description of different DNA and RNA molecules that have been targeted for bacterial detection and identification is reviewed. Longer explanation is given about the trans-translation mediating RNA molecule called tmRNA that is used as a target marker molecule in the current thesis. The research section describes two different methods that apply tmRNA for bacterial detection and identification. Firstly, a microarray-based technology is described where target tmRNA molecules are amplified using Nucleic Acid Sequence Based Amplification (NASBA) and labeled fluorescently prior the hybridization experiment. The developed method was applied for tmRNA detection from bacterial total RNA samples. In second part of the research tmRNA molecules are specifically targeted using real-time label-free biosensing platform that is based on the optical microring resonator technology. Potential quantitative nature and sensitivity of the biosensor is demonstrated using in vitro synthesized tmRNA molecules

Droplet-based digital antibiotic susceptibility screen reveals single-cell clonal heteroresistance in an isogenic bacterial population

Since antibiotic resistance is a major threat to global health, recent observations that the traditional test of minimum inhibitory concentration (MIC) is not informative enough to guide effective antibiotic treatment are alarming. Bacterial heteroresistance, in which seemingly susceptible isogenic bacterial populations contain resistant sub-populations, underlies much of this challenge. To close this gap, here we developed a droplet-based digital MIC screen that constitutes a practical analytical platform for quantifying the single-cell distribution of phenotypic responses to antibiotics, as well as for measuring inoculum effect with high accuracy. We found that antibiotic efficacy is determined by the amount of antibiotic used per bacterial colony forming unit (CFU), not by the absolute antibiotic concentration, as shown by the treatment of beta-lactamase-carrying Escherichia coli with cefotaxime. We also noted that cells exhibited a pronounced clustering phenotype when exposed to near-inhibitory amounts of cefotaxime. Overall, our method facilitates research into the interplay between heteroresistance and antibiotic efficacy, as well as research into the origin and stimulation of heterogeneity by exposure to antibiotics. Due to the absolute bacteria quantification in this digital assay, our method provides a platform for developing reference MIC assays that are robust against inoculum-density variations

Detection of tmRNA molecules on microarrays at low temperatures using helper oligonucleotides

<p>Abstract</p> <p>Background</p> <p>The hybridization of synthetic <it>Streptococcus pneumoniae </it>tmRNA on a detection microarray is slow at 34°C resulting in low signal intensities.</p> <p>Results</p> <p>We demonstrate that adding specific DNA helper oligonucleotides (chaperones) to the hybridization buffer increases the signal strength at a given temperature and thus makes the specific detection of <it>Streptococcus pneumoniae </it>tmRNA more sensitive. No loss of specificity was observed at low temperatures compared to hybridization at 46°C. The effect of the chaperones can be explained by disruption of the strong secondary and tertiary structure of the target RNA by the selective hybridization of helper molecules. The amplification of the hybridization signal strength by chaperones is not necessarily local; we observed increased signal intensities in both local and distant regions of the target molecule.</p> <p>Conclusions</p> <p>The sensitivity of the detection of tmRNA at low temperature can be increased by chaperone oligonucleotides. Due to the complexity of RNA secondary and tertiary structures the effect of any individual chaperone is currently not predictable.</p

Physicochemical Properties Predict Retention of Antibiotics in Water-in-Oil Droplets

Water-in-oil droplet microfluidics promises capacity for high-throughput single-cell antimicrobial susceptibility assays and investigation of drug resistance mechanisms. Every droplet must serve as an isolated environment with a controlled antibiotic concentration in such assays. While technologies for generation, incubation, screening, and sorting droplets mature, predictable retention of active molecules inside droplets remains a major outstanding challenge. Here, we analyzed 36 descriptors of the antibiotic molecules against experimental results on the cross-talk of antibiotics in droplets. We show that partition coefficient and fractional polar surface area are the key physicochemical properties that predict antibiotic retention. We verified the prediction by monitoring growth inhibition by antibiotic-loaded neighboring droplets. Our experiments also demonstrate that transfer of antibiotics between droplets is concentration- and distance-dependent. Our findings immediately apply to designing droplet antibiotic assays and give deeper insight into the retention of small molecules in water-in-oil emulsions.National Science Centre, Poland, 2018/30/A/ST4/00036, Maestro 10. Estonian Research Council grants MOBTP109 and PRG620 and from the TTU Development Program 2016−2022 (project no. 2014-2020.4.01.16.0032). the Foundation for Polish Science TEAM NET POIR.04.04.00-00-16ED/18-0

A dual colour FISH method for routine validation of sexed Bos taurus semen

Background: Usage of sexed semen that allows to choose the gender of the calves, is commonly practiced in livestock industry as a profitable breeding alternative, especially in dairy farming. The flow cytometric cell sorting is the only commercially available method for bovine sperm sexing. For validation of the sexing procedure several methods have been developed including sperm fluorescence in situ hybridisation techniques. Latter usually include the use of pre-labelled nucleotides for probe synthesis which is relatively expensive approach compared to combined application of aminoallyl-dUTP and chemical binding of fluorescent dyes. Here a sex determining dual colour bovine sperm fluorescence in situ hybridisation method is presented which is considered more cost-effective technique than the previously reported approaches. Results: The reliability of sex chromosome identifying probes, designed in silico, was proven on bovine metaphase plate chromosomes and through comparison with a commercially available standard method. In the dual colour FISH experiments of unsexed and sexed bovine sperm samples the hybridisation efficiency was at least 98%, whereas the determined sex ratios were not statistically different from the expected. Very few cells carried both of the sex chromosome-specific signals (less than 0.2%). Conclusions: A protocol for a dual colour bovine sperm FISH method is provided which is cost-effective, simple and fast for sex determination of spermatozoa in bull semen samples.Peer reviewe

Droplet-based digital antibiotic susceptibility screen reveals single-cell clonal heteroresistance in an isogenic bacterial population

Since antibiotic resistance is a major threat to global health, recent observations that the traditional test of minimum inhibitory concentration (MIC) is not informative enough to guide effective antibiotic treatment are alarming. Bacterial heteroresistance, in which seemingly susceptible isogenic bacterial populations contain resistant sub-populations, underlies much of this challenge. To close this gap, here we developed a droplet-based digital MIC screen that constitutes a practical analytical platform for quantifying the single-cell distribution of phenotypic responses to antibiotics, as well as for measuring inoculum effect with high accuracy. We found that antibiotic efficacy is determined by the amount of antibiotic used per bacterial colony forming unit (CFU), not by the absolute antibiotic concentration, as shown by the treatment of beta-lactamase-carrying Escherichia coli with cefotaxime. We also noted that cells exhibited a pronounced clustering phenotype when exposed to near-inhibitory amounts of cefotaxime. Overall, our method facilitates research into the interplay between heteroresistance and antibiotic efficacy, as well as research into the origin and stimulation of heterogeneity by exposure to antibiotics. Due to the absolute bacteria quantification in this digital assay, our method provides a platform for developing reference MIC assays that are robust against inoculum-density variations

Methods for studying microbial acid stress responses:from molecules to populations

The study of how micro-organisms detect and respond to different stresses has a long history of producing fundamental biological insights while being simultaneously of significance in many applied microbiological fields including infection, food and drink manufacture, and industrial and environmental biotechnology. This is well-illustrated by the large body of work on acid stress. Numerous different methods have been used to understand the impacts of low pH on growth and survival of micro-organisms, ranging from studies of single cells to large and heterogeneous populations, from the molecular or biophysical to the computational, and from well-understood model organisms to poorly defined and complex microbial consortia. Much is to be gained from an increased general awareness of these methods, and so the present review looks at examples of the different methods that have been used to study acid resistance, acid tolerance, and acid stress responses, and the insights they can lead to, as well as some of the problems involved in using them. We hope this will be of interest both within and well beyond the acid stress research community

A dual colour FISH method for routine validation of sexed Bos taurus semen

Abstract Background Usage of sexed semen that allows to choose the gender of the calves, is commonly practiced in livestock industry as a profitable breeding alternative, especially in dairy farming. The flow cytometric cell sorting is the only commercially available method for bovine sperm sexing. For validation of the sexing procedure several methods have been developed including sperm fluorescence in situ hybridisation techniques. Latter usually include the use of pre-labelled nucleotides for probe synthesis which is relatively expensive approach compared to combined application of aminoallyl-dUTP and chemical binding of fluorescent dyes. Here a sex determining dual colour bovine sperm fluorescence in situ hybridisation method is presented which is considered more cost-effective technique than the previously reported approaches. Results The reliability of sex chromosome identifying probes, designed in silico, was proven on bovine metaphase plate chromosomes and through comparison with a commercially available standard method. In the dual colour FISH experiments of unsexed and sexed bovine sperm samples the hybridisation efficiency was at least 98%, whereas the determined sex ratios were not statistically different from the expected. Very few cells carried both of the sex chromosome-specific signals (less than 0.2%). Conclusions A protocol for a dual colour bovine sperm FISH method is provided which is cost-effective, simple and fast for sex determination of spermatozoa in bull semen samples

Understanding how micro-organisms respond to acid pH is central to their control and successful exploitation

Microbes from the three domains of life, bacteria, archaea and eukarya, share the need to sense and respond to changes in the external and internal concentrations of protons. When the proton concentration is high, acidic conditions prevail and cells must respond appropriately to ensure that macromolecules and metabolic processes are sufficiently protected to sustain life. While we have learned much in recent decades about the mechanisms that microbes use to cope with acid, including the unique challenges presented by organic acids, there is still much to learn and much to be gained from developing a deeper understanding of the effects and responses to acid in microbes. In this perspective article, we survey the key molecular mechanisms that are known to be important for microbial survival during acid stress. We discuss the research approaches that have been taken to investigate the problem and highlight promising new avenues. We highlight the importance of understanding acid stress in controlling spoilage and pathogenic microbes in the food chain. We discuss the influence of acid on pathogens during the course of infections and highlight the potential of using organic acids in treatments for some types of infection. We explore the influence of acid stress on photosynthetic microbes, and on biotechnological and industrial processes, including those needed to produce organic acids. Finally, we invite colleagues with an interest in microbial responses to low pH to participate in the EU-funded COST Action project called EuroMicropH and contribute to a comprehensive database of literature on this topic that we are making publicly available