Tee PhD thesis presents the study of the response of Mycobacterium tuberculosis, the causative agent of tuberculosis, upon prolonged exposure to lethal concentrations of the first line anti-tuberculosis drug, rifampicin. The study shows that prolonged exposure to lethal concentration of rifampicin causes cell death initially by several log orders of cells, followed by a persistence phase, from where rifampicin resisters emerge carrying mutation in the RRDR locus of the rpoB gene. This phenomenon was found to occur even when the drug concentration is well above MBC levels. Luria-Delbruck experiment, in a modified format, showed that the resisters emerged as fresh mutants, and not due to the growth of pre-existing natural resisters to rifampicin. The per sister cells, which showed high levels of hydroxyl radical generation, were found to have thickened outer layer, unlike the mid-log phase cells, which restricts the permeability of a fluorescent-conjugate of rifampicin, 5-FAM-rifampicin, 10-fold less in per sister cells, as compared to mid-log phase cells. The thickened outer layer has high negative surface charge and is hydrophilic in nature. It is proposed that the hydrophilic-natured thickened outer layer might have restricted the permeability of lethal concentrations of hydrophobic-natured rifampicin. This, in turn, might have ensured the presence of sub-lethal concentration rifampicin inside the per sisters, which in turn might have generated the hydroxyl radical that caused mutagenesis to generate rifampicin resisters. The Chapter 1 is the Introduction to the thesis presented in 4 parts – Part 1.1, 1.2, 1.3 and 1.4, introducing briefly the history of antibiotics, antibiotic resistance, antibiotic persistence and a brief history of tuberculosis respectively. It is concluded with a rationale behind the present study. The Chapter 2 presents the entire Materials and Methods used in the experiments described in the thesis. The Chapter 3 presents the data on the response of M. tuberculosis to rifampicin upon extended exposure. Rifampicin exposure of susceptible M. tuberculosis H37Ra cells showed a decrease in their CFU/ml followed by a persistence phase wherein the CFU/ml remained constant. However, prolonged exposure of rifampicin even at higher concentrations showed regrowth in the culture, which was found to be due to the emergence of rifampicin-resistant bacteria. Screening of rifampicin-resistant mutants showed point mutations in the rifampicin resistance determining region (RRDR) of the rpoB gene in all the mutants. In parallel, using trans formants of M. tuberculosis expressing unstable GFP under the respective native ribosomal RNA promoter, the metabolic status of the per sister cells was determined. When actively growing highly fluorescing cells were exposed to lethal concentration of rifampicin, their metabolism diminished, as illustrated by the decrease in their fluorescence during persistence phase, followed by the emergence of a sub-population of bacteria which were again metabolically active. In order to verify whether the rifampicin resisters are freshly formed mutants or have come from the naturally existing resisters, Luria-Delbruck fluctuation test was performed in a modified manner. The number of rifampicin resisters that emerged from the persistent phase was found to vary amongst different cultures from different days and different times of exposure, showing fluctuation. However, the addition of theorem before persistence phase almost abolished the generation of the rifampicin-resistant bacilli, indicating the role of hydroxyl radical in the emergence of rifampicin resisters. The generation of hydroxyl radical in mycobacteria exposed to rifampicin was confirmed using electron para-magnetic resonance spectrometry (EPR), with the spin trap agent, 5,5- Dimethyl-1-pyrroline N-oxide (DMPO) specific for hydroxyl radical. An increase in the formation DMPO-OH adduct in the persistence phase cells was observed, in comparison to mid-log phase cells. Exposure to theorem significantly diminished the adduct formation. The persistence phase cells also showed significantly high levels of signal specific to the hydroxyl-specific fluorescent dye, hydroxyphenyl fluorescein (HPF), as compared to the mid-log phase cells. In addition we have determined the oxidative stress in the bacilli upon rifampicin exposure using a redox biosensor (Mrx1-roGFP) which also showed high oxidative stress in the persistent phase. These observations confirmed the presence of high levels of oxidative stress and hydroxyl radical in the rifampicin persistent cells, in comparison with mid-log phase cells. Whole genome sequencing of the four independently isolated rifampicin resistant M. tuberculosis showed genome wide mutations having less common mutations with respect to the wild type genome, indicative of the occurrence of random mutagenesis. In addition mutation frequencies were comparable between the samples with respect to the wild type sample. About 69% of the mutations were A-C or T-G, followed by A-T or T-A, which is known to be due to oxidative stress in the cells. Variations in the colony morphology were also observed on the persistent phase cells, indicating the occurrence of mutagenesis in the bacterial genome during rifampicin treatment. The Chapter 4 is on the morphological and ultrastructural studies on rifampicin-exposed M. tuberculosis cells. Transmission electron micrographs of rifampicin per sisters showed significant thickening of the outermost capsular layer (OL), as compared to the mid-log phase cells. This observation was verified by staining the cells with ruthenium red, which specifically stains anionic polysaccharide of the OL. Zeta potential (ZP) measurement of the surface charge of the persistent cells showed high negative ZP, as compared to the mid-log phase cells. The negative ZP value was found to gradually increase during the course of rifampicin treatment and to decrease during the regrowth phase. Hexadecane assay showed larger proportion of per sister cells being retained in the aqueous phase, as compared to the mid-log phase cells. This indicated the higher hydrophilicity of the per sister cells, which was in agreement with the higher surface negative charge of the cells. The permeability of rifampicin per sister cells to 5-FAM-rifampicin (rifampicin conjugated to 5-carboxy fluorescein, which is as hydrophobic as rifampicin) was found to be 10-fold less than that of the mid-log phase cells. However, removal of the thick OL by bead beating (BB) with 4 mm glass beads significantly improved the permeability of per sister cells to 5-FAM-rifampicin. On the contrary, no difference in the 5-FAM-rifampicin uptake was observed in mid-log phase cells, with or without BB. These observations implied that the thick hydrophilic-natured OL with high negative surface charge may be playing a significant role in limiting the permeability of hydrophobic-natured rifampicin entry into the persisted cells. This in turn may ensure the presence of sub-lethal concentration of rifampicin inside the persisted cells that are exposed to lethal concentration of the antibiotic. Exposure of bacteria to sub-lethal concentration of antibiotics has been reported to generate oxidative stress in the bacteria, leading to mutagenesis. A model has been proposed based on these observations in which the persistent mycobacteria are protected from lethal concentrations of the rifampicin by the thick OL which in turn ensures sub-lethal intracellular antibiotic concentration, leading to the generation of hydroxyl radical mediated mutagenesis and thereby emergence of rifampicin resisters. This thesis is concluded with the list of salient findings, publications and references.

La perméabilité membranaire joue un rôle important dans la résistance aux antibiotiques chez lesbactéries à Gram négatif.L’objectif de notre travail était de caractériser la fonction tenue par les deux régulateurs globaux dela perméabilité membranaire chez Enterobacer aerogenes: mar et ram. L’objectif initial futd’identifier le répresseur spécifique de RamA qui manquait en tant qu’élément de la cascade derégulation actuellement définie. La sélection de 60 souches nous a permis de confirmer le rôlecentral joué par RamA dans la régulation, ainsi qu’identifier des mutations pouvant être critiques, auniveau de RamR. Ainsi, l’absence variations observées dans le régulon marRAB et l’expressionmodérée des transcrits montrée par qRT-PCR laisse penser, que RamA a un rôle clef dans larégulation de l’expression des porines et des pompes d’efflux chez E. aerogenes.L’autre partie de notre travail reposait sur l’étude de la translocation des antibiotiques au traversdes porines. L’étude des interactions porine-carbapénèmes s’est faite sur la porine sauvage OmpFd'Escherichia coli et deux mutations. Les résultats indiquent également l'importance de l'aspartateen position 113 dans la sélectivité de translocation des carbapénèmes au sein de la porine OmpF.Ce travail montre ainsi que la translocation des pénicillines est aussi sous la dépendance desinteractions qui se créent entre le substrat et le résidu en position 113 de OmpF et limitent alorsleur passage au niveau du canal porine. Nous avons recherché la contribution attribuée à la porineOmp36 d'E. aerogenes dans la translocation de certaines béta-lactamines. Les mesures ont permisde conclure que les deux beta-lactamin / Genetic permeability plays an important role in antibiotic resistance of Gram-negative bacteria.Our work was to characterize and better understand of the genetic regulation of membranepermeability in E. aerogenes. We focused on two global regulators, mar and ram, in about 60clinical isolates. Alterations in the upstream region of ramA and in ramR but no mutations in marAnor marR were observed. Overexpression of ramA or ramR led to an altered antibiotic susceptibilityassociated to decrease of porins expression and over-expression of efflux-pumps. qRT-PCR pointedout the estimated importance of the ram-regulon in the regulation cascade.Another part of this work was to characterize the translocation of compounds through porins andthe role of porins in drug uptake in general. Measurement of the rate of antibiotic action of threecarbapenems in an E. coli strain solely expressing OmpF as porin clearly indicated the importanceof the aspartate at position 113 in antibiotic translocation. A multi-disciplinary three way approachof computer modeling, black-lipid-bilayer assays and measurement of antibiotic action, suggestedthat interactions with residue D113 of E. coli porin OmpF are rate-limiting for transport throughthe porin channel. Combination of biological and biophysical measurements with E. aerogenesporin Omp36 denoted that interactions between the porin channel and the antibiotic facilitate andaccelerate transport.

Pseudomonas aeruginosa is a Gram-negative, metabolically versatile, opportunistic pathogen that exhibits a multitude of virulence factors, and is extraordinarily resistant to a gamut of clinically significant antibiotics. This ability is in part mediated by two-component systems (TCS) that play a crucial role in regulating virulence mechanisms, metabolism and antibiotic resistance. Our sequence analysis of the P. aeruginosa PAO1 genome revealed the presence of two open reading frames, mifS and mifR, which encodes putative TCS proteins, a histidine sensor kinase MifS and a response regulator MifR, respectively. This two-gene operon was found immediately upstream of the poxAB operon, where poxB encodes a chromosomal ß-lactamase, hinting at the role of MifSR TCS in regulating antibiotic resistance. However, loss of mifSR had no effect on the antibiotic resistance profile when compared to P. aeruginosa parent PAO1 strain. Subsequently, our phenotypic microarray data (BioLOG) and growth profile studies indicated the inability of mifSR mutants to grow in α-ketoglutarate (α-KG), a key tricarboxylic acid (TCA) cycle intermediate, as a sole carbon source. To date, very little is known about the physiology of P. aeruginosa when provided with α-KG as its sole carbon source and the role of MifS and MifR TCS in virulence. Importantly, in the recent years, α-KG has gained notoriety for its newly identified role as a signaling molecule in addition to its conventional role in metabolism. This led us to hypothesize that MifSR TCS is involved in α-KG utilization and virulence in P. aeruginosa. Using mifS, mifR and mifSR clean in-frame deletion strains, our study demonstrates that the MifSR TCS modulates the expression P. aeruginosa kgtP (PA5530) and pcaT (PA0229) genes encoding putative α-KG permeases. In addition, our study shows that the MifSR-regulation of these transporters requires functional sigma factor RpoN (σ54). Loss of mifSR in the presence of α-KG, resulted in differential regulation of P. aeruginosa key virulence determinants including biofilm formation, motility, cell cytoxicity and the production of pyocyanin and pyoverdine. Involvement of multiple regulators and transporters suggests the presence of an intricate circuitry in the transport of α-KG and its importance in P. aeruginosa survival. This is further supported by the α-KG-dependent MifSR regulation of multiple virulence mechanisms. Simultaneous regulation of multiple mechanisms involved in P. aeruginosa pathogenesis suggests a complex mechanism of MifSR action. Understanding the physiological cues and regulation would provide a better stratagem to fight often indomitable P. aeruginosa infections.

No description available.

MSc (Microbiology) / Department of Microbiology / See the attached abstract below

Competition is a very important natural phenomenon, which causes the rivalry of organisms, in cases such as space limitation or lack of nutrients. It occurs mainly in situations where organisms, including microorganisms live in large populations. Multicellular yeast colonies represent an example of such a population. After the population of yeast cells spends nutrients from the environment, the cells in colonies are able to respond to these changes by production of ammonia functioning as a signaling molecule. Subsequently, the cells are able to change their morphology and metabolism and, dependently on their location within the colony, to create a subpopulation of cells with specific characteristics and functions. It is likely that in the case of mixed colonies formed by the two different strains, a competition between the cells of these two strains could exist. Such rivalry can result in changes in the ratio of cells of the two strains within the colony population, so that the cells of one strain outweigh the other. In this diploma thesis, I compared the growth and development of giant colonies and competition between the cells of selected pairs of strains forming mixed colonies. I focused on the parental strain Saccharomyces cerevisiae BY and its variants labeled with fluorescent proteins. For...

Horizontal gene transfer enables acquisition and dissemination of novel traits including antibiotic resistance and virulence among bacteria. Frequently such traits are gained through the acquisition of clusters of functionally related genes, often referred to as genomic islands (GIs). Quantifying horizontal flow of GIs and assessing their contributions to the emergence and evolution of novel metabolic traits in bacterial organisms are central to understanding the evolution of bacteria in general and the evolution of pathogenicity and antibiotic resistance in particular, a focus of this dissertation study. Methods for GI detection have also evolved with advances in sequencing and bioinformatics, however, comprehensive assessment of these methods has been lacking. This motivated us to assess the performance of current methods for identifying islands on broad datasets of well-characterized bacterial genomes and synthetic genomes, and leverage this information to develop a novel approach that circumvents the limitations of the current state-of-the-art in GI detection. The main findings from our assessment studies were 1) the methods have complementary strengths, 2) a gene-clustering method utilizing codon usage bias as the discriminant criterion, namely, JS-CB, is most efficient in localizing genomic islands, specifically the well-studied SCCmec resistance island in methicillin resistant Staphylococcus aureus (MRSA) genomes, and 3) in general, the bottom up, gene by gene analysis methods, are inherently limited in their ability to decipher large structures such as GIs as single entities within bacterial genomes. We adapted a top-down approach based on recursive segmentation and agglomerative clustering and developed a GI prediction tool, GEMINI, which combined compositional features with segment context information to localize GIs in the Liverpool epidemic strain of Pseudomonas aeruginosa. Application of GEMINI to the genome of P. aeruginosa LESB58 demonstrated its ability to delineate experimentally verified GIs in the LESB58 genome. GEMINI identified several novel islands including pathogenicity islands and revealed the mosaic structure of several LESB58 harbored GIs. A new GI identification approach, CAFE, with broad applicability was developed. CAFE incorporates biological information encoded in a genome within the statistical framework of segmentation and clustering to more robustly localize GIs in the genome. CAFE identifies genomic islands lacking markers by virtue of their association with genomic islands with markers originating from the same source. This is made possible by performing marker enrichment and phyletic pattern analyses within the integrated framework of recursive segmentation and clustering. CAFE compared favorably with frequently used methods for genomic island detection on synthetic test datasets and on a test-set of known islands from 15 well-characterized bacterial species. These tools can be readily adapted for cataloging GIs in just sequenced, yet uncharacterized genomes.

[ES] Las bacterias resistentes a múltiples antibióticos, como el Enterococo resistente a vancomicina (ERV), son un problema creciente en los pacientes hospitalizados, por lo que se necesita estrategias alternativas para combatir estos patógenos. Las infecciones causadas por ERV suelen comenzar con la colonización del tracto intestinal, un paso crucial que se afectado por la presencia de la microbiota. Sin embargo, los antibióticos alteran la microbiota y esto promueve la colonización de ERV. Una vez que el patógeno ha colonizado el intestino, alcanza niveles muy altos pudiendo diseminar a otros órganos y pacientes. A pesar de su importancia, se sabe muy poco sobre los genes que codifica para colonizar el intestino y sobre el mecanismo por el cual la microbiota suprime su colonización intestinal, siendo los dos objetivos principales. En primer lugar hemos utilizado una metodología previamente descrita (Zhang et al., 2017, BMC Genomics), basada en la generación de una librería de mutantes por transposición junto a secuenciación masiva, con el fin de identificar los genes codificados por ERV necesarios para la colonización del intestino en ratones. Además, hemos realizado análisis metatranscriptómicos para identificar aquellos genes más expresados. El análisis ha identificado genes cuya interrupción reduce significativamente la colonización intestinal en el intestino grueso. Los genes que más afectaron a la colonización codifican proteínas relacionadas con la absorción o el transporte de diversos nutrientes como los carbohidratos (subunidad EIIAB del transportador PTS de manosa, el regulador transcripcional de la familia LacI, ácido N-acetilmurámico 6-fosfato eterasa) o iones (proteína transportadora dependiente de ATP (ABC) y proteínas del grupo [Fe-S]). El papel de estos genes en la colonización se ha confirmado mediante experimentos de mutagénesis directa y de competición con la cepa salvaje. Además, estos genes afectan a la colonización intestinal con diferentes antibióticos (clindamicina y vancomicina). Para identificar el mecanismo molecular por el cual cada gen afecta a la colonización, hemos realizado experimentos in vitro y ex vivo además del análisis transcriptómico. Los experimentos in vitro confirman que las proteínas del grupo [Fe-S] están involucradas en el transporte iones de hierro, principalmente Fe3+. Por otra parte, los genes de la subunidad EIIAB del transportador de manosa y del ácido N-acetilmurámico 6-fosfato eterasa son necesarios para la utilización de la manosa y el ácido N-acetilmurámico, respectivamente, azúcares que suelen estar presentes en el intestino. También confirmamos que el regulador transcripcional de la familia LacI es un represor que afecta a proteínas transportadoras ABC, probablemente implicadas en la absorción de carbohidratos. Además, algunos de estos genes están codificados principalmente por cepas clínicas de E. faecium y en menor medida por cepas comensales. En segundo lugar, estudiamos los mecanismos de protección de un consorcio de cinco bacterias comensales, que anteriormente se había demostrado que disminuían la colonización intestinal por ERV en ratones. Mediante transcriptómica, metabolómica y los ensayos in vivo observamos que el consorcio bacteriano inhibe el crecimiento de ERV mediante la reducción de nutrientes, concretamente fructosa. Por último, el análisis ARN-Seq in vivo de cada aislado en combinación con los ensayos ex vivo e in vivo demostraron que una sola bacteria (Olsenella sp.) proporciona protección. En conjunto, los resultados obtenidos han identificado la función de genes específicos requeridos por ERV para colonizar el intestino. Además, hemos identificado un mecanismo mediante el cual la microbiota confiere protección. Estos resultados podrían conducir a nuevos enfoques terapéuticos para prevenir las infecciones causadas por este patógeno multiresistente a los antibióticos. / [CA] Els bacteris resistents a múltiples antibiòtics, com el Enterococo resistent a vancomicina (ERV), són un problema creixent en els pacients hospitalitzats, que són resistents a la majoria d'antibiòtics disponibles per la qual cosa es necessita estratègies alternatives per a combatre aquests patògens. Les infeccions causades per ERV solen començar amb la colonització del tracte intestinal, un pas crucial que es veu afectat per la presència de la microbiota. No obstant això, els antibiòtics alteren la microbiota i això promou la colonització de ERV. Una vegada que el patogen ha colonitzat l'intestí, aconsegueix nivells molt alts podent disseminar a altres òrgans i pacients. Malgrat la seua importància, se sap molt poc sobre els gens que codifica ERV per a colonitzar l'intestí i sobre el mecanisme pel qual la microbiota suprimeix la seua colonització intestinal. En primer lloc hem utilitzat una metodologia prèviament descrita (Zhang et al., 2017, BMC Genomics), basada en la generació d'una llibreria de mutants per transposició junt amb seqüenciació massiva, amb la finalitat d'identificar els gens codificats per ERV necessaris per a la colonització de l'intestí en ratolins. A més a més, hem realitzat anàlisi metatranscriptòmics per a identificar aquells gens més expressats. L'anàlisi ha identificat gens quina interrupció redueix significativament la colonització intestinal en l'intestí gros. Els gens que més van afectar la colonització codifiquen proteïnes relacionades amb l'absorció o el transport de diversos nutrients com els carbohidrats (subunitat EIIAB del transportador PTS de manosa, el regulador transcripcional de la família LacI, àcid N-acetilmuràmic 6-fosfat eterasa) o ions (proteïna transportadora dependent d'ATP (ABC) i proteïnes del grup [Fe-S]). El paper d'aquests gens en la colonització s'ha confirmat mitjançant experiments de mutagènesis directa i de competició amb el cep salvatge. A més, aquests gens afecten la colonització intestinal amb diferents antibiòtics (clindamicina i vancomicina). Per a identificar el mecanisme molecular pel qual cada gen afecta a la colonització, hem realitzat experiments in vitro i ex viu a més de l'anàlisi transcriptòmic. Els experiments in vitro confirmen que les proteïnes del grup [Fe-S] estan involucrades en el transport d'ions de ferro, principalment Fe3+. D'altra banda, els gens de la subunitat EIIAB del transportador PTS de manosa i de l'àcid N-acetilmuràmic 6-fosfat eterasa són necessaris per a la utilització de la manosa i l'àcid N-acetilmuràmic, respectivament, sucres que solen estar presents en l'intestí. També confirmem que el regulador transcripcional de la família LacI és un repressor que afecta proteïnes transportadores ABC, probablement implicades en l'absorció de carbohidrats. A més a més, alguns d'aquests gens estan codificats principalment per ceps clínics de E. faecium i en menor mesura per ceps comensals. En segon lloc, estudiem els mecanismes de protecció d'un consorci de cinc bacteris comensals, que adès s'havia demostrat que disminuïen la colonització intestinal per ERV en ratolins. Amb l'ús de transcriptòmica, metabolòmica i els assajos in vivo observem que el consorci bacterià inhibeix el creixement de ERV mitjançant la reducció de nutrients, concretament fructosa. Finalment, l'anàlisi ARN-Seq in vivo de cada aïllat en combinació amb els assajos ex viu i in vivo van demostrar que un sol bacteri (Olsenella sp.) proporciona protecció. En conjunt, els resultats obtinguts han identificat la funció de gens específics requerits per ERV per a colonitzar l'intestí. A més, hem identificat un mecanisme mitjançant el qual la microbiota confereix protecció. Aquests resultats podrien conduir a nous enfocaments terapèutics per a previndre les infeccions causades per aquest patogen multiresistent als antibiòtics. / [EN] Multidrug-resistant bacteria, such as vancomycin-resistant-Enterococcus (VRE), are an increasing problem in hospitalized patients. Some VRE strains can be resistant to most available antibiotics, thus, alternative strategies to antibiotics are urgently needed to combat these challenging pathogens. Infections caused by VRE frequently start by colonization of the intestinal tract, a crucial step that is impaired by the presence of the intestinal microbiota. Administration of antibiotics disrupts the microbiota, which promotes VRE intestinal colonization. Once VRE has colonized the gut, it reaches very high levels, which promotes its dissemination to other organs and its transfer to other patients. Despite the relevance of VRE gut colonization, very little is known about the genes encoded by this pathogen to colonize the gut and about the mechanisms by which the microbiota suppresses VRE gut colonization. In this thesis, we have utilized a previously described methodology (Zhang et al., 2017, BMC Genomics), based on the generation of a transposon mutant library coupled with high-throughput sequencing, in order to identify VRE encoded genes required for colonization of the mouse intestinal tract. In addition, we have performed metatranscriptomic analysis in mice to identify VRE genes specifically expressed in the gut. Our analysis has identified genes whose disruption significantly reduces VRE gut colonization in the large intestine. The genes that most affected VRE gut colonization encoded for proteins related to the uptake or transport of diverse nutrients such as carbohydrates (PTS mannose transporter subunit EIIAB, LacI family DNA-binding transcriptional regulator, N-acetylmuramic acid 6-phosphate etherase) or ions (phosphate ABC transporter ATP-binding protein and proteins from [Fe-S] cluster). The role of these genes in gut colonization has been confirmed through targeted mutagenesis and competition experiments against a wild type strain. Moreover, these genes affect gut colonization under different antibiotic treatments (clindamycin and vancomycin). To elucidate the mechanism by which each gene influences gut colonization, we have performed in vitro and ex vivo experiments besides transcriptomic analysis. In vitro experiments confirm that proteins from [Fe-S] cluster are involved in the transport of different forms of iron ions, mostly Fe3+. On the other hand, the PTS mannose transporter subunit EIIAB and N-acetylmuramic acid 6-phosphate etherase genes are required for the utilization of mannose and N-acetyl-muramic acid, respectively, sugars that are usually present in the intestinal environment. We have also confirmed that LacI family DNA-binding transcriptional regulator is a repressor that affects the expression of genes encoding for an ABC transporter probably involved in the uptake of carbohydrates. Furthermore, we have confirmed that some of these genes are encoded mainly by E. faecium clinical strains but not or to a lower extent by commensal strains. Secondly, we studied the mechanisms of protection of a consortium of five commensals bacteria, previously shown to restrict VRE gut colonization in mice. Functional transcriptomics in combination with targeted metabolomics and in vivo assays performed in this thesis indicated that the bacterial consortium inhibits VRE growth through nutrient depletion, specifically by reducing the levels of fructose. Finally, in vivo RNA-Seq analysis of each bacterial isolate of the consortium in combination with ex vivo and in vivo assays demonstrated that a single bacterium (Olsenella sp.) could recapitulate the protective effect. Altogether, the results obtained have identified the function of specific genes required by VRE to colonize the gut. In addition, we have identified a specific mechanism by which the microbiota confers protection against VRE colonization. These results could lead to novel therapeutic approaches to prevent infections caused by this pathogen. / Flor Duro, A. (2021). Characterization of Genes and Functions Required by Multidrug-resistant Enterococci to Colonize the Intestine [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/166494 / TESIS

Antibiotikaresistenz/-toleranz und Evasion des menschlichen Immunsystem sind wesentliche Probleme persistierender chronischer Infektionen, die im Zusammenhang mit Biofilmen stehen. Eine Notwendigkeit alternativer Behandlungen liegt daher nahe. Für diese Studie wurden zehn ayurvedische Pflanzen ausgewählt, die die Produktion von Curli-Fasern und/oder pEtN-Cellulose in E. coli K-12 Makrokolonie-Biofilmen eindeutig hemmten. Eine Reihe molekularer Reporter wurde verwendet, um die molekularen Ziele im Modellorganismus E. coli zu identifizieren. Eine Kombination von mikrobiologischen, molekularbiologischen und enzymatischen Methoden und Experimenten wurde dann verwendet, um die Aktivitäten der Pflanzenextrakte weiter zu charakterisieren. Um ihre Wirkung auf Biofilme eines breiteren Spektrums von Bakterien zu testen, wurden einige relevante gramnegative Pathogene (EAEC, UPEC, P. aeruginosa) und grampositive Bakterien (B. subtilis, S. aureus) als Makrokolonie-Biofilme sowie als submerse Biofilme in Gegenwart der Pflanzenextrakte inkubiert. Die wichtigsten Ergebnisse dieser Studie sind, dass es kein „Allheilmittel“ gibt, das effektiv gegen verschiedene Biofilmstrukturen wirken kann. Es konnte gezeigt werden, dass fast alle Pflanzenextrakte die CsgA Amyloidogenese hemmen. Drei der zehn Pflanzenextrakte beeinflussten die Curli- und pEtN-Cellulose-Gene signifikant, indem sie csgB und dgcC über den Regulator CsgD herunterregulierten. Darüber hinaus wurde festgestellt, dass ein Extrakt die Expression flagellarer Gene in E. coli hochreguliert - eine neue Anti-Biofilm Strategie. Überraschenderweise wurde auch festgestellt, dass ein Pflanzenextrakt, das die Biofilmbildung des Kommensalen E. coli K-12 hemmt, während es die Biofilmbildung von UPEC fördert. Daher können Anti-Biofilm-Effekte stammspezifisch sein. Eine Strategie, bei der verschiedene Pflanzenextrakte kombiniert werden, könnte gegen Biofilme wirken, die aus mehreren Arten bestehen, erfordert jedoch weitere Forschung. / Antibiotic resistance/tolerance and evasion from the human immune system are major causes of concern associated with biofilm-related persistent chronic infections. So, the need for an alternative source of treatment is obvious. In this study, 10 Ayurvedic plants were selected as they clearly inhibited the production of curli fiber and pEtN-cellulose or of curli fibers only in E. coli K-12 macrocolony biofilms. A series of molecular reporters were used to determine the molecular targets using E. coli as model bacteria. A combination of microbiological, molecular biological, and enzymatic assays and experiments were then used to further characterize the activities of the plant extracts. To test anti-biofilm effects on a wider range of bacteria, some relevant Gram-negative pathogens (EAEC, UPEC, P. aeruginosa) and Gram-positive bacteria (B. subtilis, S. aureus) were grown in macrocolony biofilms and submerged biofilms in the presence of active plant extracts. The major findings of this study are that there is not one single “magic bullet” that can effectively work against the diverse biofilm compositions and structures. Nearly all plant extracts were found to inhibit CsgA amyloidogenesis. Three of the ten plant extracts affected the curli and pEtN-cellulose genes significantly by downregulating csgB and dgcC via the CsgD regulator. In addition, one extract was found to upregulate flagellar gene expression in E. coli - this is a new anti-biofilm strategy that had not considered before. Surprising, it was also noticed that one plant extract, which inhibits biofilm formation by commensal E. coli K-12, promotes biofilm formation by UPEC. Thus, anti-biofilm effects can be strain-specific because of the diversity of composition of the matrix within the same bacterial species. A strategy of combining different plant extracts may work to deal with biofilms involving multiple species, but requires more research and understanding.

La résistance aux antibiotiques est une menace sérieuse pour la santé publique. En effet, si on ne change pas rapidement notre consommation excessive d'antibiotiques, la situation actuelle va se dégrader jusqu'à basculer dans une ère dite "post-antibiotique", dans laquelle plus aucun antibiotique ne sera efficace contre les infections microbiennes. Bien que ce phénomène de résistance apparaît naturellement, l'utilisation abusive d'antibiotiques accélère le processus. De plus, la présence de pathogènes multi-résistants neutralise l'effet des traitements existants et dans le cas de chirurgies courantes (césariennes, transplantations d'organe...), la situation peut rapidement s'aggraver voire devenir mortelle. C'est pourquoi des directives, émanant des autorités sanitaires, doivent être mises en place afin de contrôler l'utilisation des médicaments, et ce, à tous les niveaux de la société, des individus au secteur agricole en passant par les professionnels de santé et les industries pharmaceutiques. Le monde de la recherche scientifique, quant à elle, doit trouver des nouvelles stratégies pour enrayer la propagation de la résistance. Dans ce contexte, cette thèse a pour objectif le développement d'une méthode de prédiction, par calculs d'énergie libre, des mutations de β-lactamases favorables à l'hydrolyse des β-lactames. Ces travaux méthodologiques ont donc conduit au développement : (1) de nouveaux paramètres pour les enzymes à zinc, implémentés dans le champ de force OPLS-AA et validés par des simulations de dynamique moléculaire sur un panel de métalloenzymes représentatives, (2) d'un protocole de paramétrisation de ligands covalents pour étudier le comportement de certains β-lactames dans CMY-136, une nouvelle β-lactamase caractérisée au laboratoire, et (3) d'un protocole de calcul d'énergie libre évalué au moyen de compétitions internationales de prédiction. Ce dernier a ensuite été utilisé pour tenter d'expliquer pourquoi la carbamylation de la sérine catalytique n'a pas lieu dans certaines oxacillinases. Au travers de ces travaux, nous avons pu améliorer significativement notre approche computationnelle et désormais tout est en place pour une exploration exhaustive des mutations possibles dans les β-lactamases. / Antibiotic resistance is a global concern threatening worldwide health. Indeed, if we don't change our overconsumption of antibiotics, the current situation could worsen until a "post-antibiotic" era in which existing treatment would be ineffective against microbial infections. Despite the natural occurrence of antibiotic resistance, the misuse of antibiotics is speeding up the process. Furthermore, presence of multi-resistant pathogens negates the effect of modern treatments and usual surgeries (caesarean sections, organ transplantations...) might be riskier in the future, or even lethal. That's why, common guidelines have to be edicted by health authorities in order to control antibiotic use at every level of society, from individuals to healthcare industry including health professionals and agriculture sector. As for scientific research, new strategies have to be considered in order to limit the spread of antibiotic resistance. In that context, the presented thesis aimed at developing a protocol to predict, by free energy calculations, β-lactamase mutations which could promote the hydolysis of β-lactams antibiotics. In order to achieve that, we developed several methodological approaches including: (1) new parameters for zinc enzymes implemented in OPLS-AA force field and thereafter validated using molecular dynamics simulations of representative zinc-containing metalloenzymes, (2) a protocol to parameterize covalent ligands in order to analyze the dynamical behavior of some β-lactams in CMY-136, a novel β-lactamase recently characterized in our laboratory, and (3) a pmx-based free energy protocol. The latter was also assessed through several international blinded prediction challenges, and finally used to find out why carbamylation of the catalytic serine is not observed in certain OXA enzymes. Throughout this work, we made significant improvements in our protocol, and now everything is in place for an exhaustive prediction of possible mutations in β-lactamases.