The opportunistic pathogen P. aeruginosa is the leading cause of morbidity and mortality in cystic fibrosis patients. During chronic infection of the cystic fibrosis lung, P. aeruginosa undergoes conversion to a mucoid phenotype, constitutively producing the exopolysaccharide alginate, composed of the uronic acids D-mannuronate and L-guluronate. This alginate production contributes significantly to virulence in the cystic fibrosis lung. Evidence suggests that the acetylation state of the mannuronate component of the alginate influences the ability of components of the immune system to phagocytose the organism. To garner new and relevant information regarding the mechanism of alginate acetylation in Pseudomonas aeruginosa, a variety of approaches were undertaken. Analysis of the alginate produced by algX, algG, and algK alginate biosynthesis mutants revealed that the small oligouronides they produced were unacetylated. This strongly supports the hypothesis that the mannuronates are acetylated in periplasm, and that a polymer of at least some specific size is required. While three alginate biosynthesis gene products (AlgI, AlgJ, and AlgF) have been shown to be involved in alginate acetylation, another gene in the cluster, algX, shares 30% identity with one of them and thus generates speculation as to its potential involvement in the process. To test this possibility, an algX mutant was complemented with a plasmid carrying a mutation at a conserved residue shown to be required for alginate acetylation in the homologous protein. Analysis of alginate from this construct suggested that AlgX is not involved in alginate acetylation. To determine if changes in levels of alginate acetylation are accomplished at the transcriptional level, transcript levels of several alginate biosynthesis genes in different media were determined by real-time PCR. As qRT-PCR had not been previously performed on any of the alginate biosynthesis genes, this yielded important information about the transcription of the operon. In addition, beta-galactosidase assays on upstream regions of several biosynthesis genes identified two previously unrecognized promoters, one upstream of algG and one upstream of algI. The remaining approach was to examine protein interactions of AlgF, the protein product of one of the three acetylation genes. 2-D redox SDS-PAGE gels indicated that disulfide bonding may be important for interactions with this protein. While mass spectrometry was unable to identify the binding partners of AlgF, efforts are ongoing to create a mutation in the P. aeruginosa genome that changes the cysteine residue in AlgF to a serine residue. This would be a definitive method for determining the importance of disulfide bonding in AlgF.

Magíster en Bioquímica área de Especialización en Bioquímica ambiental / Memoria para optar al Título de Bioquímico / Los problemas ambientales y la disminución de las reservas de petróleo han reiniciado la búsqueda de combustibles alternativos a los combustibles fósiles. El etanol se presenta como una alternativa sustentable para resolver esta problemática. En la naturaleza existen microorganismos silvestres productores de etanol, tales como la bacteria Zymomonas mobilis y la levadura Saccharomyces cerevisiae. Si bien estos dos microorganismos son altamente eficientes en la generación de etanol, sólo son capaces de utilizar azúcares de 6 carbonos, limitando la producción industrial de etanol solo a materias primas que contengan hexosas. En los residuos lignocelulósicos se encuentra una gran reserva de hidratos de carbono almacenada como polímeros de glucosa (celulosa) o azúcares de 5 carbonos como xilosa y arabinosa. Para el aprovechamiento de pentosas disponibles en esta materia prima se han modificado estos microorganismos, mediante la introducción de los genes de las enzimas necesarias para metabolizar arabinosa y xilosa o se han incorporado los genes de las enzimas de la ruta etanológica de Z. mobilis, piruvato descarboxilasa (pdc) y alcohol deshidrogenasa II (adhB) a bacterias capaces de utilizar pentosas, como Escherichia coli, Klebsiella oxycota, Lactobacillus plantarum, Lactococcus lactis y Bacillus subtilis. El género Pseudomonas, principalmente Pseudomonas aeruginosa, posee muchas de las características necesarias para la producción industrial de etanol, es una versátil bacteria Gram negativo, capaz de adaptarse y sobrevivir bajo amplio rango de condiciones ambientales y al igual que Z. mobilis asimila azúcares por la ruta de Entner-Doudoroff, además posee una toleracia a etanol mayor que bacterias E. coli recombinantes utilizadas en la producción de etanol. El objetivo general de este trabajo es la construcción de un operón productor de etanol para la expresión de los genes pdc y adhB de la ruta etanologénica de Z. mobilis en P. aeruginosa PAO1. El diseño del operón artificial incluyó los siguientes elementos genéticos: el promotor del operón inducible arabinosa (PBAD), sitio de unión a ribosoma (RBS), gen pdc, gen adhB y un terminador transcripcional para P. aeruginosa. Las primeras etapas para la construcción del operon pet, contemplaron la realización de 2 construcciones genéticas mediante PCR. La primera comprende la mínima región codificante del gen pdc y una región adaptadora de 33 pb (adp), la segunda corresponde a la unión de adp2 (secuencia que contiene una zona complementaria a adp y un sitio de unión a ribosoma) con la mínima región codificante del gen adhB y un terminador transcripcional específico para Pseudomonas. Los análisis de restricción, PCR y secuenciación mostraron la obtención de un fragmento 1740 pb correspondiente al producto pdc-adp, un fragmento de 1247 pb para el producto adp2- adhB-term. Para los productos de PCR de la fusión de las construcciones pdc-adp con adp2- adhB-term se obtuvieron amplicones inespecíficos de tamaños superiores e inferiores al tamaño teórico esperado (3 Kb). De manera alternativa para la obtención de la construcción que contenga los genes pdc y adhB con la secuencia adaptadora y de unión a ribosoma, se realizaron reacciones de ligación de ambos fragmentos utilizando la enzima T4 DNA ligasa, las cuales fueron clonadas en el vector pSC-B. De todos los clones analizados solo uno contenía la construcción esperada (plasmidio pCL27-B), pero los análisis de PCR revelaron que los genes no se encontraban en la orientación adecuada, por lo tanto no fue posible obtener la construcción del fragmento pdc-adp-RBS-adhB, construcción clave en el término de la construcción del operón pet. / Environmental problems and declining oil reserves have resumed the search for alternative fuels to fossil fuels. Ethanol is presented as a sustainable alternative to solve this problem. In nature there are wild ethanol producing microorganisms, such as bacteria Zymomonas mobilis and yeast Saccharomyces cerevisiae. While these two microorganisms are highly efficient in the generation of ethanol, are capable of using only 6 carbon sugars, limiting the industrial production of ethanol only to raw material containing hexoses. In lignocellulosic waste there is a large reservoir of stored carbohydrates like glucose polymers (cellulose) or 5-carbon sugars such as xylose and arabinose. For the utilization of available pentoses in this raw material, such microorganisms have been modified by the introduction of genes of the necessary enzymes to metabolize arabinose and xylose or enzymes genes have been incorporated of etanologic pathway from Z. mobilis, pyruvate decarboxylase (pdc) and alcohol deshidrogenasa II (adhB) to bacteria capable to use pentoses, such as Escherichia coli, Klebsiella oxycota, Lactobacillus plantarum, Lactococcus lactis and Bacillus subtilis. The genus Pseudomonas, mainly Pseudomonas aeruginosa, has many of the features needed for industrial production of ethanol, is a versatile Gram negative bacteria, able to adapt and survive under wide range of environmental conditions and like Z. mobilis assimilated sugars by the Entner-Doudoroff pathway, and in addition has ethanol higher tolerance than recombined E. coli bacteria used in the production of ethanol. The overall aim of this work is the construction of an ethanol producer operon for the expression of pdc and adhB genes from the Z. mobilis etanologic pathway on P. aeruginosa PAO1. The artificial operon design included the following genetic elements: the arabinose inducible operon promoter (PBAD), ribosome binding site (RBS), pdc gene, adhB gene and a transcriptional terminator to P. aeruginosa. The first step to construct the pet operon, contemplated 2 genetic constructs by PCR. The first involves a pdc gene minimum coding region and a adapter region of 33 bp (adp), the second corresponds to the union of adp2 (sequence that contains a complementary zone to adp and a ribosome binding site) with adhB gene minimal encoding region and a specific transcriptional terminator to Pseudomonas. The restriction analysis, PCR and sequencing showed the obtention of a 1740 pb fragment corresponding to the pdc-adp product, a 1247 pb fragment for the product adp2-adhB-term. For the PCR products of pdc-adp and adp2-adhB-term construction fusion, non specific amplicons were obtained in higher and lower sizes than the theoretical size expected (3 Kb). Alternatively for the obtention of the construction which have the pdc and adhB genes with the adapter sequence and ribosome binding, ligation reactions were performed of both fragments using T4 DNA ligase enzyme, which were cloned on the vector pSC- B. Of all the clones analyzed just one contained the expected construction (plasmid pCL27-B), but PCR analysis revealed that genes were not in the proper orientation, therefore it was not possible to obtain the construction of fragment pdc-adp-RBS-adhB, key construction in the completion of the pet operon construction.

Pseudomonas aeruginosa ist ein gramnegatives Bakterium, welches insbesondere bei abgeschwächter Immunabwehr schwere Infektionen auslösen kann. Es besitzt eine hohe intrinsische Resistenz gegenüber Antibiotika, so dass nur eine begrenzte Anzahl von Antimikrobiotika wie beispielsweise Aminoglykoside für die Behandlung zur Verfügung steht. Unter Antibiotikatherapie wird zudem eine schnelle Resistenzentwicklung beobachtet, daher ist die Weiterentwicklung und Optimierung der Therapieoptionen von großer Bedeutung. Die vorliegende Arbeit untersucht den Einfluss von Colistin auf die Aminoglykosidresistenz bei Pseudomonas aeruginosa. 25 Bakterienstämme, die zu Beginn gegenüber Amikacin, Tobramycin und Gentamicin resistent waren, wurden Colistin ausgesetzt. Mithilfe des Epsilometertests wurde die minimale Hemmkonzentration (MHK) der Antibiotika für die zu untersuchenden Bakterienstämme vor und nach Colistineinfluss bestimmt. Es konnte ein signifikanter Rückgang der minimalen Hemmkonzentration nach Colistineinwirkung dokumentiert werden. Zu den Hauptresistenzmechanismen von Pseudomonas aeruginosa gehören die Effluxpumpen, welche die Antibiotika aus dem Bakterium ausschleusen. Für den Transport von Aminoglykosiden ist die MexXY-Pumpe verantwortlich, die in dieser Arbeit weiteruntersucht wurde. Durch eine quantitative Echtzeit-PCR unter Nutzung des Fluoreszenz-Resonanz-Energie-Transfers (FRET) wurde die Expression der Pumpe vor und nach Colistin verglichen. Es konnte nachgewiesen werden, dass durch Colistin die Expression reduziert wurde. Ein linearer Zusammenhang zwischen MHK-Veränderung und mexXY-Expressionslevel wurde anhand der Untersuchungsergebnisse nicht ermittelt. Es ist dementsprechend davon auszugehen, dass andere Resistenzmechanismen ebenfalls durch Colistin beeinflusst werden und so die MHK-Reduktion erklären können.

Identifica la prevalencia de genes que codifican carbapenemasas de tipo metalo β-lactamasas (MBL) en aislados clínicos de P. aeruginosa. Analiza 76 aislados clínicos de P. aeruginosa resistentes a Ceftazidima y “no sensibles” (intermedio o resistentes) a Imipinem y/o Meropem colectados en el Hospital Militar Central de enero a setiembre del 2016. Muestras de secreciones respiratorias, heridas, orinas y hemocultivos de pacientes hospitalizados son procesadas en el Laboratorio de Microbiología. Determina la sensibilidad antimicrobiana por el método de disco de difusión según los criterios del Clinical and Laboratory Standards Institute. Realiza la detección fenotípica de MBL por el test de sinergia de doble disco con imipenem, meropenem y EDTA. La detección genotípica se realiza amplificando por PCR multiplex, los genes blaIMP y blaVIM, mientras que para el gen blaNDM se hizo PCR convencional. Obtiene fenotípicamente 25 de 76 pruebas positivas para MBL, 24(31.58%) de las cuales se confirmaron genéticamente, encontrando el gen blaIMP (23/24, 95.83%) y el gen blaVIM (1/24, 4.17%). La sensibilidad de la prueba fenotípica es del 100% y la especificidad del 98%. Concluye que el 31.58% de los aislamientos clínicos de P. aeruginosa recuperados de pacientes hospitalizados en el HMC, presentan MBL, siendo el gen blaIMP el más prevalente. / Tesis

Publicación a texto completo no autorizada por el autor / Optimiza la producción de ramnolípidos a escala piloto con Pseudomonas aeruginosa 6K-11 en cultivos sumergidos por lotes. Con la finalidad de establecer los parámetros óptimos para la aireación, agitación y concentración de nitrógeno en la producción de ramnolipídos a escala piloto, se evaluaron dos niveles para cada factor: 0.25 vvm y 0.5 vvm (aireación), 50 RPM y 70 RPM (agitación) y 2.45 g/L y 4.89 g/L (concentración de NaNO3). Con estos tres factores y dos niveles para cada uno se evaluó la producción de ramnolípidos a escala piloto con el diseño experimental de Taguchi L4 [2]3 o arreglo L4. / Tesis

Bacteriophages (phages) are obligate intracellular parasites of bacteria that usually kill the bacterial host. Bacteriophage therapy is a recently revived approach for treating bacterial infection that relies on the traits of the phage lytic cycle. A lot of attention has been given to phage therapy with new research being published weekly and international conferences organised every year, bringing together the academic and industrial phage communities. However, despite this huge effort and considerable scientific interest there is still a great lack of understanding on how to use phage effectively and overcome the many obstacles in the near future. One of the main triggers for such interest was the increasing evidence of antibiotic resistance among human bacterial pathogens, which were once efficiently eliminated by drugs but are now causing alarmingly high levels of morbidity and mortality. Also, bacteria when causing a disease are able to produce highly protective biofilm communities. Biofilms are major causes of impairment of wound healing and two of the most common and aggressive wound pathogens are Staphylococcus aureus (Gram-positive) and Pseudomonas aeruginosa (Gram-negative), both displaying a large repertoire of virulence factors and reduced susceptibility to antibiotics. This work reports and explores the use of phages to target both S. aureus and P. aeruginosa pathogen biofilm producers. Firstly, isolation of promising phage candidates was performed and cocktails were established. Two phages (DRA88 and phage K) formed the cocktail to target S. aureus and six phages (DL52, DL54, DL60, DL62, DL64 and DL68) formed a cocktail to target P. aeruginosa. A thorough characterisation of each of the selected phages was performed, including their range of host infectivity and their genome sequences were analysed. The phage’s ability to infect and kill planktonic cultures was successfully studied and afterwards such ability was assayed on biofilms using an in vitro static biofilm system (microtitre-plate), followed by an in vitro dynamic biofilm system (The Modified Robbins Device). Both cocktails were shown to be effective in reducing and dispersing biofilms formed by the clinical strains showing them to be promising not only to combat topical bacterial infections (related to biofilm production), but also to control biofilms produced on the surfaces of medical devices, such as catheters. Finally, the phage cocktail’s ability to treat systemic infections caused by the two pathogens was assessed in an in vivo G. mellonella infection model. In the case of the P. aeruginosa infection, although the phages were not able to fully treat the larvae, the cocktail allowed a delay of larval death, caused by the infection. For the S. aureus infection, the cocktail did not show the same trend, but most likely the high bacterial cell numbers involved in the experiment interfered with a successful study on the phage cocktail. The phage mixture may form the basis of an effective treatment for infections caused by S. aureus and P. aeruginosa biofilms.

Many bacteria secrete secondary metabolites, whose production is decoupled from active growth in laboratory cultures. Historically, the advantages of secondary metabolite production have mostly been explored in the context of cellular interactions, such as antibiotic effects on competing organisms, damage caused to host tissues during infection, or cell density-dependent signaling. However, recent studies in the opportunistic pathogen Pseudomonas aeruginosa have brought into focus the physiological effects of secondary metabolites on their producer and their implications for multicellular behavior. P. aeruginosa produces antibiotics called phenazines, which can act as mediators to transfer reducing power to an extracellular oxidant and thereby support bacterial survival when oxygen is not accessible. In the crowded environments of biofilms, communities of bacteria surrounded by self-made matrices, this property of phenazines could support energy generation for cells in anoxic subzones. As biofilm formation is a hallmark of P. aeruginosa colonization at various infection sites within the body, I was motivated to investigate the regulation of phenazine production at the level of synthesis and transport, the distribution of phenazines in P. aeruginosa biofilms, and the effects of individual phenazines on P. aeruginosa gene expression and colony biofilm morphogenesis. As part of this work, a novel electrochemical device was developed that enables direct detection of phenazines released from intact colony biofilms. Application of this device and other electrochemical techniques enabled detection of the reactive phenazine intermediate 5-Me-PCA, which was found to be the primary phenazine affecting P. aeruginosa colony morphogenesis. The production of this phenazine was found to be sufficient for activation of the redox-active transcription factor SoxR and full induction of the RND efflux pump MexGHI-OpmD. Finally, results described in this thesis show that 5-Me-PCA is transported by MexGHI-OpmD, constituting a unique demonstration of the self-protective role of an efflux pump in a gram-negative antibiotic-producing bacterium. These findings raise broad questions about the effects of individual phenazines on biofilm cell physiology and have implications for the contributions of individual phenazines to virulence and survival during infection. The technology developed also has potential applications in novel diagnostic and therapeutic approaches. Chapters 1-3 introduce and highlight advances made in understanding secondary metabolite production, with a focus on P. aeruginosa. Chapter 1 provides an introduction to antibiotic production, the concept of self-resistance and other physiological effects of antibiotics in their producers, and infections caused by P. aeruginosa. Chapter 2 reviews recent studies that have brought into focus the physiological effects of secondary metabolites on their producers and their implications for multicellular behavior. Chapter 3 provides an overview of our current understanding of the regulation of phenazine production in pseudomonads and other bacterial species. Chapter 4 describes the development of an integrated circuit-based platform for detection of redox-active metabolites released from multicellular samples, and demonstrates its application to mapping phenazines released from P. aeruginosa biofilms. The study described in Chapter 5 investigates the role of the P. aeruginosa SoxR regulon, which is induced by phenazines, in phenazine transport and shows that the understudied reactive phenazine 5-methylphenazine-1-carboxylic acid (5-Me-PCA) is transported by the RND efflux pump MexGHI-OpmD and is required for wild-type biofilm formation. Chapter 6 describes the development of an assay for 5-Me-PCA production and studies exploring the role of the regulator PsrA in controlling phenazine biosynthesis. Chapter 7 provides an overview of the findings and open questions to be explored in future research. The P. aeruginosa genome contains two nearly identical operons that encode biosynthetic enzymes for the production of phenazine-1-carboxylic acid, the precursor to all of the other phenazines. The study described in Appendix A characterizes the respective contributions of these operons to phenazine production in shaken liquid cultures and biofilms. Appendix B presents evidence that electron acceptor availability influences, and is influenced by, the morphogenesis of P. aeruginosa colony biofilms. Finally, Appendix C describes a screen for commercially available compounds that inhibit production of the phenazine pyocyanin by P. aeruginosa. Together, these findings reveal the unique physiological roles of specific phenazine-related genetic loci and regulatory proteins and of 5-Me-PCA, a phenazine that was previously overlooked due to the technical challenges associated with its detection. They have also uncovered novel aspects of phenazine production in both shaken liquid cultures and biofilms relevant for the development of therapeutics.

A bacterial biofilm is a community of sessile cells encased in a matrix composed of polysaccharides, proteins, and extracellular DNA that develops according to a reproducible morphogenic program. This morphogenic program is deeply influenced by prevailing redox conditions within the biofilm, which are established by a gradient of terminal electron acceptor through the depth of the biofilm. Terminal electron acceptor limitation leads to redox stress, measured as an elevated ratio of reduced to oxidized forms of the metabolic cofactor nicotinamide adenine dinucleotide, NAD(H). In biofilms of the gram-negative bacterium Pseudomonas aeruginosa, redox stress is relieved by the presence of diffusible redox-cycling molecules, phenazines, that are able to act as an electrical conduit between intracellular NADH and oxygen in the aerobic zone of the biofilm. This is most apparent in the dramatically hyperspread and hyperwrinkled morphologies observed in colony biofilms unable to produce phenazines. However, the ability of phenazines to act as a biologically relevant redox couple between the reducing equivalents of metabolism and atmospheric oxygen also renders them toxic to producing cells. In order to avoid phenazine toxicity, P. aeruginosa encodes self-resistance mechanisms under the control of the redox-sensitive transcription factor SoxR. Two components of the SoxR regulon, the efflux pump MexGHI-OpmD and the monooxygenase PumA, are known to be major contributors to survival in the presence of toxic concentrations of phenazines. This work further details the role of the small protein MexG (Chapter 3) and PumA in phenazine resistance (Chapter 4), and presents an electrochemical platform for studying the effects of a phenazine redox gradient in biofilm morphogenesis (Chapter 5).

Bacteria, while traditionally studied in liquid suspensions, are often found in nature as biofilms, aggregates of cells enclosed in self-produced matrices. Cells in biofilms have a fitness advantage over those that are free-living, as the biofilm lifestyle is correlated with increased resistance to various assaults, including antimicrobials, UV exposure, and dehydration. These biofilm-associated characteristics have important clinical implications, as biofilm-based bacterial infections are a major cause of morbidity in immunocompromised individuals. With this increased resiliency, however, comes a major challenge that arises during biofilm growth: the formation of resource gradients. My thesis work focused on one particular gradient, that of oxygen, which is established in biofilms formed by Pseudomonas aeruginosa. This bacterium has multiple mechanisms for coping with limited access to oxygen, including a highly-branched respiratory system for optimal oxygen scavenging and production and utilization of redox-active molecules called phenazines. The purpose of this thesis has been to investigate the different strategies used by P. aeruginosa to deal with the oxygen limitation precipitated by the biofilm lifestyle. In Chapter 1, I will provide the necessary background for understanding the principles of redox balancing, metabolism, respiration, biofilm physiology, and phenazine utilization in P. aeruginosa. The work described in Chapter 2 provides evidence for the formation of a novel terminal oxidase complex that plays a biofilm-specific role in P. aeruginosa growth. The results in this chapter also suggest that specific terminal oxidase complexes differ in the timing of their contributions to biofilm growth and implicate the novel complex in mediating reduction of phenazines in biofilms. Chapter 3 expands upon the principle of metabolic versatility exemplified by the results discussed in Chapter 2. The research presented in this chapter looks at how varying the source of electrons that feed into the respiratory chain influences downstream electron transfer steps, including terminal oxidase activities and phenazine production and utilization. The data presented in Chapters 2 and 3 add to the growing body of evidence that bacterial growth in liquid culture is distinct from that in biofilms and underscores the need for more biofilm-based research that can inform treatment strategies for P. aeruginosa infections. The results described in Chapter 4 take an even broader look at the strategies used by P. aeruginosa to sustain efficient metabolism under conditions of potential stress. An important node of central metabolism is pyruvate, which can be transformed in a number of ways. In this chapter, I will consider two pathways of pyruvate metabolism: fermentation to lactate and carboxylation to oxaloacetate. I will present data indicating that a previously-uncharacterized lactate dehydrogenase contributes to P. aeruginosa growth under specific growth conditions and that pyruvate carboxylation contributes to optimal progress through central metabolic pathways. I will also describe experiments that characterize the contributions of another carboxylase, previously thought to function as the pyruvate carboxylase, to P. aeruginosa’s ability to grow on selected nutrient sources. Finally, I will discuss how redox state informs biofilm formation in a phylogenetically distinct bacterium, Bacillus subtilis, highlighting the universality of redox reactions in driving metabolic processes. In sum, the research presented in this thesis broadens our understanding of the immense respiratory and metabolic flexibility of P. aeruginosa and serves as an important reminder of the discrete factors that govern liquid culture and biofilm growth.

Los biofilms o biopelículas, comunidades complejas de microorganismos, se encuentran colonizando diferentes ambientes bióticos como abióticos; los problemas generados por estos son diversos en diferentes actividades humanas. Ya que las biopelículas son de difícil erradicación, se han planteado diversas formas de control en cada una de las etapas del ciclo de una biopelícula. Una de ellas es la interrupción de las moléculas de señalización del quorum sensing, sistema que regula el proceso de la formación de biopelículas, para tal efecto se están buscando actualmente tanto antagonistas sintéticos como de origen natural. La presente investigación utiliza el Senecio calvus, una planta de uso medicinal, nativa del Perú. Se han expuesto extractos etanólico, butanólico, acuoso, metanólico y de diclorometano además de fracciones cromatográficas de Senecio calvus contra Pseudomonas aeruginosa, una bacteria oportunista muy utilizada debido a que es una bacteria que forma rápidamente biopelículas. Se mezclaron los cultivos de P. aeruginosa en fase de crecimiento junto a los extractos y fracciones de S. calvus para determinar el grado de inhibición de las bioopelículas; para tal efecto se usaron concentraciones subinhibitorias, previa prueba de concentración mínima inhibitoria (CMI). Los resultados arrojaron una inhibición de 92.9 y 76.4% en dos de los extractos y de hasta 88% en las fracciones cromatográficas, lo cual indica que Senecio calvus es un buen candidato para el aislamiento de una molécula inhibidora de biopelículas con potencial patentable. / Tesis