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The diploma thesis if focused on the study of bioactive hydrogél and nanofiber wound dressings composed of natural biopolymers, which were functionalized by active compounds in the form of analgesic, antibiotics and enzymes. Hydrogél wound dressings were constituted from alginate and chitosan and nanofibers were created from polyhydroxybutyrate. The following 7 active compounds were selected to be added to the wound dressings: ampicillin, streptomycin, ibuprofen, papain, bromelain, collagenase and trypsin. In the theoretical part the structure of the skin and types of wound injuries were described. This part also talks about types of wound dressing and their applications, as well as treatment of skin wounds using enzymes and compounds with analgesic and antimicrobial properties. In addition, this section describes safety assays, in particular cytotoxicity assays on human cells. At the beginning of the experimental part, the process of preparation of hydrogél wound dressing was optimised. Subsequently, the dressings were enriched with active compounds and the rate of gradual releasing of the substances into model environment was monitored. In the case of enzymes, their proteolytic activity was also tested after their incorporation to the wound dressings. Furthermore, the prepared bioactive wound dressings were analyzed for possible cytotoxic effect on human keratinocytes. Finally, the wound dressing with combined content of active substances was created and also characterized for the rate of substance release, proteolytic activity and cytotoxicity. Antimicrobial activity of this wound dressings, against two selected strains of microorganisms: Escherichia coli and Staphylococcus epidermidis, was also evaluated.

Les bactéries doivent s'adapter rapidement aux modifications de leur environnement en ajustant leur modèle d'expression génétique et leurs activités enzymatiques. Dans la plupart des cas, les variations de leur habitat impliquent de petites molécules que les bactéries peuvent détecter et auxquelles elles peuvent réagir. Le ribosome, la machinerie de la cellule qui catalyse la formation de la liaison peptidique, est capable de détecter les métabolites ou les antibiotiques afin de réguler l'expression des gènes, où le peptide naissant au sein du ribosome est capable d’induire l’arrêt de la traduction. Dans ce mécanisme, le peptide en cours de traduction (peptide d'arrêt) bloque le ribosome en interagissant avec les parois du tunnel ribosomal correspondant à la cavité par laquelle le peptide atteint le cytoplasme. L'arrêt peut dépendre uniquement de la séquence du peptide ou bien nécessiter la liaison d’une petite molécule. L’arrêt du ribosome en cours de traduction contrôle à son tour l'expression sur le même ARNm d'un gène situé en aval. Malgré plusieurs études biochimiques et structurales antérieures, le mécanisme exact de détection de ces petits métabolites par le peptide d’arrêt est encore inconnu. Mon travail de doctorat a porté sur : (1) comprendre comment de petites molécules sont détectées par les peptides d'arrêt ribosomaux, et (2) un cas particulier d'arrêt de la traduction dépendant du ligand : la détection des antibiotiques par des peptides d'arrêt courts.Pour répondre au premier problème, j'ai étudié biochimiquement et structurellement un nouveau peptide d'arrêt (appelé SpeFL) qui détecte l’ornithine (un petit métabolite) et qui est codé en amont de l'opéron speF chez Escherichia coli. La structure cryo-EM que j'ai résolue a révélé comment l’ornithine est détectée de manière très spécifique par un complexe ribosomal en cours de traduction. De plus, j'ai montré que le mécanisme d'induction du gène en aval speF implique un arrêt du ribosome au niveau de speFL empêchant ainsi une terminaison prématurée de la transcription Rho-dépendante.Dans la deuxième partie de ma thèse, je me suis concentrée sur la façon dont un antibiotique ciblant les ribosomes, l'érythromycine, est détecté par un peptide d'arrêt court. L'érythromycine est capable de bloquer la traduction de manière séquence-dépendante, où le motif (+)X(+) est le motif principal de blocage. Des données biochimiques publiées antérieurement suggèrent que l'encombrement stérique et électrostatique causé par le premier acide aminé chargé positivement (+) empêche l'addition du second, arrêtant ainsi le ribosome en cours de traduction. La résolution de la structure cryo-EM d'un ribosome arrêté par un peptide MKFR en présence d'érythromycine suggère le contraire, ce qui ouvre la voie à d'autres recherches sur le sujet. / Bacteria need to rapidly adapt to the changing environment by adjusting their gene expression patterns and enzymatic activities. In most cases, the variations in their habitat involve small molecules that bacteria are able to sense and respond to. The ribosome, the machinery of the cell that catalyzes peptide bond formation, is able to detect metabolites or antibiotics to regulate gene expression via nascent-chain mediated translational arrest. In this mechanism, the peptide that is being translated (arrest peptide) stalls the ribosome by interacting with the walls of the ribosomal tunnel, the cavity through which it reaches the cytoplasm. The arrest may depend solely on the sequence of the peptide or need a small molecule to be triggered. Ribosomal stalling in turn, controls the expression of a gene that is located downstream on the same mRNA. Despite previous biochemical and structural studies, the exact mechanism of sensing of small metabolites by the nascent chain is still unknown. My PhD work focused on: (1) understanding how small molecules are sensed by ribosomal arrest peptides, and (2) a special case of ligand-dependent translational arrest: drug sensing by short arrest peptides.To address the first issue, I studied biochemically and structurally a novel L-ornithine sensing arrest peptide (SpeFL) encoded upstream the speF operon in Escherichia coli. The cryo-EM structure that I solved revealed how a small molecule is sensed by a ribosome nascent chain complex in a highly specific manner. Besides, I showed that the mechanism of induction of the downstream gene speF involves ribosomal arrest at speFL preventing premature Rho-dependent transcriptional termination.On the second part of my thesis, I focused on how a ribosome-targeting antibiotic, erythromycin, is sensed by a short arrest peptide. Erythromycin is able to block translation in a sequence dependent manner, with the (+)X(+) motif being the main stalling motif. Previously published biochemical data suggest that steric and static hindrance caused by the first positively charged amino acid prevents the addition of the second one arresting the ribosome. I solved the cryo-EM structure of a ribosome arrested by an MKFR peptide in the presence of erythromycin that shows otherwise and opens up further investigation on the matter.

<p>Normal healthy people are not susceptible to tuberculosis (TB) but immunocompromised and HIV positive patients are at high risk of TB. The treatment regimen (rifampin, isoniazid and amikacin) for TB patients is 6-9 months for normal patients but if <i>Mycobacterium tuberculosis</i> (Mtb) becomes multidrug resistant, it takes 20-30 months to treat. According to the World Health Organization in 2018, there were about half a million new cases among which 78% were multidrug resistant TB. This antibiotic resistance is due in part to its ability to survive in the macrophage in our body by entering a non-replicating persistent state. Mtb also contains efflux pumps that increase antibiotic tolerance by pumping out the drugs. Therefore, if the efflux pump activity can be blocked by using efflux pump inhibitors, then it might increase antibiotic susceptibility of the pathogen. In our study, we used <i>Mycobacterium smegmatis</i> (Msm) as a model organism for Mtb and subjected it to a combination of three stresses (low oxygen, low pH and low nutrients) that mimic the physiological stresses in the human body and report that these conditions produced a non-replicating state in Msm. This is the first report of the use of this combination of stresses to produce a non-replicating state in Msm. Our results show that non-replicating Msm became completely tolerant to isoniazid and displayed increased tolerance to rifampin and clarithromycin by nearly 2-fold when compared to log-phase cells. Moreover, the efflux pump inhibitor verapamil decreased the antibiotic tolerance of the nonreplicating Msm to the antibiotics by 6-10 fold and the efflux pump inhibitor piperine decreased tolerance to the antibiotics by 2-4 fold. Also, in this study we attempted to construct a gene knockout mutant lacking two potential ATP-binding cassette transporters to study their functions as drug exporters. However, we were unable to obtain homologous recombination mutants. Further studies on efflux pump inhibitors could potentially enable greater understanding of antibiotic tolerance mechanisms in non-replicating, drug tolerant Mtb and enable the development of novel therapies that shorten treatment time for tuberculosis.</p>

Methicillinresistent Staphylococcus aureus (MRSA) är en multiresistent bakterie som är ett hälsoproblem över hela världen. Genom mutationer i bakterien skapas en resistens mot antibiotika. Faktorer som bidrar till den ökade spridningen och resistens av MRSA är bland annat dålig handhygien/hygien bland vårdpersonal på sjukhus, samt överanvändning av antibiotika. Personer som är bärare av MRSA riskerar då att inte kunna behandlas med antibiotika vid bakteriella infektioner, vilket kan medföra längre vårdtider eller i värsta fall hot mot hälsan och riskerar då att inte överleva dessa infektioner. Syfte: Att beskriva vårdprogram för MRSA i Sverige, Tyskland och Spanien. Metod: Metoden som använts är litteraturöversikt. Resultat: Vårdprogrammen för att minska spridningen av MRSA består samma riktlinjer i de olika länderna i Europa. Varför spridningen ser olika ut beror i stor utsträckning på vilka förutsättningar som finns i de olika länderna samt hur antibiotikakonsumtionen ser ut. Vissa länder har hög exponering av antibiotika inom djurhållningen, vilket ökar spridningen av MRSA. Konklusion: Trots stora skillnader i spridningen av MRSA ligger inte problemet i vårdprogrammen och dess riktlinjer för MRSA för länder i Europa. Vid jämförelse av länder med hög spridningen av MRSA respektive låg blev slutsatsen att det största problemet ligger i vilka förutsättningar vårdpersonalen har för att genomföra goda hygienrutiner samt brist eller bättre tillgång på kunskap och mer frekventa utbildningar kring MRSA och spridningen av resistenta bakterier.

In immunodeficient patients, for example with cystic fibrosis (CF), the opportunistic pathogen Staphylococcus aureus causes chronic infections of respiratory tract that are treated with antibiotics (ATB) in the long term. However, exposure to antibiotics can lead to persistence, thereby result a recurrence of infection. The aim of this work was to examine in selected pairs of S. aureus chronological isolates from the respiratory tract of CF patients how their ability to form persisters is changing in time. I have found that the ability to persist within the clonal pair does not change significantly after two years of survival in the host, and that the ability to persist depends on the adaptative mutations of the isolates. Persister formation may depend on mutations in operon of the alternative sigma B factor (sigB) and the major virulence gene regulator (agr). By dual staining with DioC2(3) and To-pro-3, I was able to determine the changes in membrane potential and membrane permeability during the killing curve with ATBs. The distribution into subpopulations according to these parameters depends primarily on the antibiotic used. I conclude that various antibiotics can induce different mechanisms causing a persistent state. Futhermore, I have constructed plasmids with a labeled promoter to determine...

Background: The method of serum bactericidal assay represents an alternative possibility of optimization of anti-infectious therapy and administration of antibiotics. It mirrors the real activity of one or more administered antibiotics in the complex system of the antibacterial effect of patient's serum. The paper aimed to confirm non-inferiority of bactericidal testing using the broth dilution method according to CLSI M21-A Guidelines (time to results 48, 72 hours) in comparison with modified methods of testing on the basis of turbidimetry (time to result 6, 8, 24 hours) and resazurin color (time to results 8, 24 hours). Methods: Four antibiotics were tested: gentamicin, amikacin, piperacillin/tazobactam and meropenem with 30 Escherichia coli strains isolated from blood cultures of 29 pacients hospitalised in different wards, University Hospital in Hradec Kralove. Human blood sera (n = 76) from ten hematological patients (4th Department of Clinical Medicine, University Hospital, Hradec Kralove) were tested to establish bactericidal titer. Patients' blood was withdrawn prior to and in the course of the first and third day of antibiotic therapy of febrile neutropenia. Testing employed the reference strain Escherichia coli ATCC 25922. Results: A comparison with the standard CSLI showed that the...

Thesis advisor: Kenneth Williams / Due to overuse and misuse of antibiotics, the global threat of antibiotic resistance is a growing crisis. Three critical issues surrounding antibiotic resistance are the lack of rapid testing, treatment failure, and evolution of resistance. However, with new technology facilitating data collection and powerful statistical learning advances, our understanding of the bacterial stress response to antibiotics is rapidly expanding. With a recent influx of omics data, it has become possible to develop powerful computational methods that make the best use of growing systems-level datasets. In this work, I present several such approaches that address the three challenges around resistance. While this body of work was motivated by the antibiotic resistance crisis, the approaches presented here favor generalization, that is, applicability beyond just one context. First, I present ShinyOmics, a web-based application that allow visualization, sharing, exploration and comparison of systems-level data. An overview of transcriptomics data in the bacterial pathogen Streptococcus pneumoniae led to the hypothesis that stress-susceptible strains have more chaotic gene expression patterns than stress-resistant ones. This hypothesis was supported by data from multiple strains, species, antibiotics and non-antibiotic stress factors, leading to the development of a transcriptomic entropy based, general predictor for bacterial fitness. I show the potential utility of this predictor in predicting antibiotic susceptibility phenotype, and drug minimum inhibitory concentrations, which can be applied to bacterial isolates from patients in the near future. Predictors for antibiotic susceptibility are of great value when there is large phenotypic variability across isolates from the same species. Phenotypic variability is accompanied by genomic diversity harbored within a species. I address the genomic diversity by developing BFClust, a software package that for the first time enables pan-genome analysis with confidence scores. Using pan-genome level information, I then develop predictors of essential genes unique to certain strains and predictors for genes that acquire adaptive mutations under prolonged stress exposure. Genes that are essential offer attractive drug targets, and those that are essential only in certain strains would make great targets for very narrow-spectrum antibiotics, potentially leading the way to personalized therapies in infectious disease. Finally, the prediction of adaptive outcome can lead to predictions of future cross-resistance or collateral sensitivities. Overall, this body of work exemplifies how computational methods can complement the increasingly rapid data generation in the lab, and pave the way to the development of more effective antibiotic stewardship practices. / Thesis (PhD) — Boston College, 2020. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

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Staphylococcus aureus is a opportunistic pathogen that can cause severe and chronic infections. The reason of the infections relapse is often the persistence. It is about adapting to stressful conditions by inducing a dormant state, which would allow bacteria to survive exposure to antibiotics and grow again after their elimination. Bacteria that persist in the patient acquire various adaptive mutations, which are transmited creating subpopulations that have a better ability to persist. The aim of this diploma thesis was to compare individual methods of persistent study that could be used in clinical practice in the future, and at the same time to try a closer molecular characterization of the persistent state with using methods for calculating gene expression. I had chronological isolates of Staphylococcus aureus at my disposal, the initial one being the primoisolate, an isolate taken at the diagnostics of cystic fibrosis before the start of antibiotic treatment. Another was taken at a distance of three-quarters of a year and the last with a half-year interval from the previous one. Following whole genome sequencing, genes in which adaptive mutations occurred were identified. The first method determines the degree of persistence by calculating CFU (Colony Forming Units) after antibiotic treatment....