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[Resumo] As infeccións nosocomiais son un importante problema de saúde global, cunha taxa de incidencia que xa alcanza o 7,1% na Unión Europea. Datos do Centro Europeo de Prevención e Control de Enfermidades (ECDC) estiman aproximadamente 33000 mortes anuais en Europa como consecuencia directa dunha infección por bacterias resistentes a antibióticos. Dentro dos patóxenos bacterianos que máis comunmente provocan infeccións difíciles de tratar inclúese Klebsiella pneumoniae. O obxectivo do presente proxecto de doutoramento é o desenvolvemento de tratamentos antiinfecciosos innovadores e efectivos contra K. pneumoniae resistente e persistente a antibióticos, seguindo dúas estratexias complementarias: por unha banda, a innovación no uso de terapias antiinfecciosas, como o uso de bacteriófagos e o reposicionamento de fármacos “non antibióticos” aprobados pola FDA para outras indicacións terapéuticas; por outro, a combinación de ambas estratexias para conseguir un efecto sinérxico. Seguindo estas premisas, nesta tese doutoral explórase a capacidade lítica de dous bacteriófagos contra K. pneumoniae e se reposicionan axentes anticanceríxenos, mucolíticos e antiparasitarios, que son avaliados in vitro e no modelo in vivo de larvas de Galleria mellonella en combinacións sinérxicas, co fin de lanzar luz no tratamento de infeccións provocadas por K. pneumoniae., [Resumen] Las infecciones nosocomiales son un importante problema de salud global, con una tasa de incidencia que ya alcanza el 7,1% en la Unión Europea. Datos del Centro Europeo de Prevención y Control de Enfermedades (ECDC) estiman aproximadamente 33000 muertes anuales en Europa como consecuencia directa de una infección por bacterias resistentes a antibióticos. Dentro de los patógenos bacterianos que más comúnmente provocan infecciones difíciles de tratar se incluye Klebsiella pneumoniae. El objetivo del presente proyecto de doctorado es el desarrollo de tratamientos antiinfecciosos innovadores y efectivos contra K. pneumoniae resistente y persistente a antibióticos, siguiendo dos estrategias complementarias: por un lado, la innovación en el uso de terapias antiinfecciosas, como el uso de bacteriófagos y el reposicionamiento de fármacos “no antibióticos” aprobados por la FDA para otras indicaciones terapéuticas; por otro, la combinación de ambas estrategias para conseguir un efecto sinérgico. Siguiendo estas premisas, en esta tesis doctoral se explora la capacidad lítica de dos bacteriófagos contra K. pneumoniae y se reposicionan agentes anticancerígenos, mucolíticos y antiparasitarios, que son evaluados in vitro y en el modelo in vivo de larvas de Galleria mellonella en combinaciones sinérgicas, con el fin de arrojar luz en el tratamiento de infecciones provocadas por K. pneumoniae., [Abstract] Nosocomial infections are a major global health problem, with an incidence rate already reaching 7.1% in the European Union. Data from the European Centre for Disease Prevention and Control (ECDC) estimate approximately 33,000 deaths per year in Europe as a direct consequence of infections by antibiotic-resistant bacteria. Among the most common bacterial pathogens causing difficult-to-treat infections, Klebsiella pneumoniae is included. This PhD project aims to develop innovative and effective anti-infectious treatments against antibiotic-resistant and persister K. pneumoniae, following two complementary strategies: on the one hand, innovation in the use of antibacterial therapies, such as the use of bacteriophages and the repositioning of "non-antibiotic" drugs approved by the FDA for other therapeutic indications; on the other hand, the combination of both strategies to achieve a synergistic effect. Following these premises, this doctoral thesis explores the lytic capacity of two bacteriophages against K. pneumoniae and repurposed anticancer, mucolytic and antiparasitic agents, which are evaluated in vitro and in the in vivo model of Galleria mellonella larvae in synergistic combinations, to shed some light on the treatment of K. pneumoniae infections.

Phages and bacteria have acquired resistance mechanisms for protection. In this context, the aims of the present study were to analyze the proteins isolated from 21 novel lytic phages of Klebsiella pneumoniae in search of defense mechanisms against bacteria and also to determine the infective capacity of the phages. A proteomic study was also conducted to investigate the defense mechanisms of two clinical isolates of K. pneumoniae infected by phages. For this purpose, the 21 lytic phages were sequenced and de novo assembled. The host range was determined in a collection of 47 clinical isolates of K. pneumoniae, revealing the variable infective capacity of the phages. Genome sequencing showed that all of the phages were lytic phages belonging to the order Caudovirales. Phage sequence analysis revealed that the proteins were organized in functional modules within the genome. Although most of the proteins have unknown functions, multiple proteins were associated with defense mechanisms against bacteria, including the restriction-modification system, the toxin-antitoxin system, evasion of DNA degradation, blocking of host restriction and modification, the orphan CRISPR-Cas system, and the anti-CRISPR system. Proteomic study of the phage-host interactions (i.e., between isolates K3574 and K3320, which have intact CRISPR-Cas systems, and phages vB_KpnS-VAC35 and vB_KpnM-VAC36, respectively) revealed the presence of several defense mechanisms against phage infection (prophage, defense/virulence/resistance, oxidative stress and plasmid proteins) in the bacteria, and of the Acr candidate (anti-CRISPR protein) in the phages.

[Resumen] Los bacteriofagos, tambien conocidos como fagos, son virus que infectan a las bacterias. Estos virus de procariotas son considerados como la entidad biologica mas abundante del planeta, estimandose su tamano poblacional en 1031 particulas virales. La resistencia a los antimicrobianos es la causa de que, para un numero creciente de patógenos bacterianos, la tasa de exito de los antibioticos sea dudosa. Por lo tanto, la rápida propagacion de patogenos multirresistentes ha creado un renovado interes en el uso de los fagos como terapia alternativa para este tipo de infecciones. Las bacterias y los fagos estan en un estado permanente de coevolucion, comunmente denominado carrera armamentistica, porque cuando uno desarrolla un mecanismo para evadir al contrario esto provoca que el segundo evolucione para evitar esta defensa. Asi, las bacterias han desarrollado mecanismos de defensa para protegerse de sus depredadores, mientras que los fagos, a su vez, han desarrollado estrategias de defensa para evadir estos sistemas. En este contexto, la presente tesis doctoral se centro en el estudio los mecanismos de interaccion y defensa de Klebsiella pneumoniae frente a la infeccion por fagos. De acuerdo con eso, en el primer capitulo de esta tesis doctoral se procedio al analisis genomico de 40 profagos localizados en los genomas de 16 aislados clinicos de K. pneumoniae portadores de carbapenemasas. En el segundo capitulo se procedio al estudio del papel del sistema toxina-antitoxina de tipo II PemK/PemI de un aislado clinico de K. pneumoniae en la infeccion por fagos liticos. Y, finalmente, en el tercer capitulo se procedio al estudio proteomico de la interaccion entre fagos y su hospedador bacteriano K. pneumoniae., [Abstract] Bacteriophages, also known as "phages", are viruses that infect bacteria. These viruses that target prokaryotes are considered the most abundant biological entity on the Earth, with an estimated population size of 1031 viral particles. Antimicrobial resistance is the cause of dubious success rates of antibiotics against an increasing number of bacterial pathogens. Therefore, the rapid spread of multidrug-resistant pathogens has renewed interest in the use of phages. Bacteria and phages are in a constant state of coevolution known as arms race, as when one develops a mechanism to evade the other, it triggers the evolution of the latter to overcome this defence. Thus, bacteria have developed defense mechanisms to protect themselves from their predators, while phages, in turn, have developed counterdefense strategies to evade these systems. In this context, this doctoral thesis focused on studying the mechanisms of interaction and defense of Klebsiella pneumoniae against phage infection. Accordingly, the first chapter of this doctoral thesis involved genomic analysis of 40 prophages located in the genomes of 16 clinical isolates of carbapenemase-carrying K. pneumoniae. In the second chapter, the role of the type II toxin-antitoxin system PemK/PemI in a clinical isolate of K. pneumoniae in lytic phage infection was studied. Finally, in the third chapter, proteomic analysis was carried out to investigate the interaction between phages and their bacterial host K. pneumoniae., [Resumo] Os bacteriofagos, tamen conecidos como “fagos”, son virus que infectan as bacterias. Estes virus de procariotas son considerados como a entidade bioloxica mais abundante do planeta, estimandose o seu tamano poboacional en 1031 particulas virales. A resistencia aos antimicrobianos e a causa de que para un numero crecente de patoxenos bacterianos, a taxa de exito dos antibioticos sexa dubidosa. Por tanto, a rápida propagacion de patoxenos multirresistentes creou un renovado interese no uso dos fagos. As bacterias e os fagos estan nun estado permanente de coevolucion denominado carreira armamentistica, isto e, cando un desenvolve un mecanismo para evadir ao contrario isto provoca que o segundo evolucione para evitar esta defensa. Asi, as bacterias desenvolveron mecanismos de defensa para protexerse dos seus depredadores, mentres que os fagos a sua vez desenvolveron contra-estratexias de defensa para evadir estes sistemas. Neste contexto, a presente tese doutoral centrouse no estudo dos mecanismos de interaccion e defensa de Klebsiella pneumoniae fronte a infeccion por fagos. Dacordo con iso, no primeiro capitulo desta tese doutoral procedeuse a analise xenomica de 40 profagos localizados nos xenomas de 16 illados clinicos de K. pneumoniae portadores de carbapenemasas. No segundo capitulo procedeuse ao estudo do papel do sistema toxina-antitoxina de tipo II PemK/PemI dun illado clinico de K. pneumoniae na infección por fagos liticos. E, finalmente, no terceiro capitulo procedeuse ao estudo proteómico da interaccion entre fagos e o seu hospedador bacteriano K. pneumoniae.

[Resumen] El vermellógeno es una sal catiónica de piridinio derivada del viológeno, donde las unidades piridínicas se unen mediante un enlace hidrazona. Lo cual permite que el compuesto responda al pH, debido a la acidez del NH, y evitar los problemas de citotoxicidad de la respuesta redox del viológeno. Se sintetizaron dos derivados del vermellógeno, uno de cadena larga y otro de cadena corta. Para ello se siguió la misma metodología en ambos casos, a partir de reactivos comerciales, mediante reacciones SN2 y SNAr, se obtuvieron primero los dos precursores necesarios. Después, a través de una reacción de síntesis de hidrazonas, se obtuvo el producto final, siendo purificado por HPLC o metátesis. A continuación, se caracterizaron estructuralmente con las técnicas espectroscópicas habituales, RMN 13C y 1H monodimensional y bidimensional, en su forma protonada y desprotonada en medio orgánico. Después, se realizaron estudios en medio acuoso, donde se determinó el pKa por potenciometría y UV-VIS obteniendo valores similares a los ya reportados para el vermellógeno. Se observó tendencia a la agregación en el derivado de cadena larga, pudiendo determinar la concentración de agregación crítica (cac) mediante DLS. En medio básico se obtuvo la cac a concentraciones menores que en medio ácido, comprobando así su dependencia con el pH., [Resumo] O vermellóxeno é unha sal catiónica de piridinio, derivada do violóxeno, onde as unidades piridínicas únense mediante un enlace hidrazona. O cal permite que o composto responda ao pH, debido a acidez do NH, e evite os problemas de citotoxicidade da resposta redox do violóxeno. Sintetizáronse dous derivados do vermellóxeno, un de cadea longa e outro de cadea curta. Para iso, seguiuse a mesma metodoloxía en ambos os dous casos, a partir de reactivos comerciais, mediante reaccións SN2 e SNAr, obtivéronse primeiro os dous precursores necesarios. Despois, a través de una reacción de síntese de hidrazonas, obtívose o produto final, sendo purificado por HPLC o metátesis. A continuación, caracterizáronse estruturalmente coas técnicas habituais, RMN 13C y 1H monodimensional e bidimensional, na súa forma protonada e desprotonada en medio orgánico. Despois, realizáronse estudios en medio acuoso, onde se determinou o pKa por potenciometría e UV-VIS, obtendo valores similares aos xa reportados para o vermellóxeno. Observouse tendencia á agregación no derivado de cadea larga, podendo determinar a concentración de agregación crítica (cac) mediante DLS. En medio básico obtívose a cac a concentracións menores que en medio ácido, comprobando así a súa dependencia co pH., [Abstract] The red thread is a pyridinium based cationic salt, derived from the viologen, in which the pyridinic unities are linked through a hydrazone bond. This result in the pH responsiveness of the compound, due to the NH acidity, and avoids the cytotoxicity problems of the redox response of the the viologen. Two derivatives of the red thread were synthesized, one with a longer chain and another with a shorter chain. To that end the same methodology was followed in both cases, starting from commercial reactives, through SN2 and SNAr reactions, two precursor compounds were obtained. Finally, by a hydrazone synthesis reaction, the final product was obtained, then purified by HPLC or metathesis. Next, the products were structurally characterized using the usual spectroscopic techniques, RMN 13C and 1H monodimensional and bidimensional, in their protonated and deprotonated form in organic medium. Then studies in aqueous medium were realized, in which the pKa was determined by potentiometry and UV-VIS, obtaining similar values to those already reported for the read thread. A tendency to aggregation was observed in the long chain derivative, in which the critical aggregation concentration (cac) could be determined by DLS. In basic medium the cac was obtained in lower concentrations than in acidic medium, thus proving the pH dependency.

A partir de la convocatoria formulada por la Universidad Nacional de Mar del Plata, para presentar trabajos de investigación en la VI Edición de las Jornadas Investigar UNMDP 2023, los integrantes del "Grupo Turismo y Territorio Espacios Naturales y Culturales" participaron con una actividad lúdico-académica, titulada "¿A dónde viajamos hoy? Reconocimiento de destinos argentinos con alta especialización turística". El presente recurso de aprendizaje aspira divulgar la actividad presentada en las Jornadas, integrando las funciones docentes desempeñadas en la asignatura "Espacios Turísticos Americanos" con las funciones de investigación desarrolladas en el proyecto en curso titulado "Aportes de la Geografía del Turismo para la comprensión del proceso de turistificación del partido de General Pueyrredon". Se describe la fundamentación teórica de la propuesta, la relación entre redes sociales y sector académico, la planificación de la actividad desarrollada y la apreciación sobre los resultados de su aplicación. Constituye una alternativa innovadora para la difusión de los resultados de la investigación en curso, y al mismo tiempo, una instancia de articulación con la sociedad, potenciando los aportes de la institución al desarrollo de la ciencia.

Mucins are important glycoproteins that form a protective layer throughout the gastrointestinal and respiratory tracts. There is scientific evidence of increase in phage-resistance in the presence of mucin for some bacterial pathogens. Manipulation in mucin composition may ultimately influence the effectiveness of phage therapy. In this work, two clinical strains of K. pneumoniae (K3574 and K3325), were exposed to the lytic bacteriophage vB_KpnS-VAC35 in the presence and absence of mucin on a long-term co-evolution assay, in an attempt to mimic in vitro the exposure to mucins that bacteria and their phages face in vivo. Enumerations of the bacterial and phage counts at regular time intervals were conducted, and extraction of the genomic DNA of co-evolved bacteria to the phage, the mucin and both was performed. We determined the frequency of phage-resistant mutants in the presence and absence of mucin and including a mucolytic agent (N-acetyl L-cysteine, NAC), and sequenced them using Nanopore. We phenotypically demonstrated that the presence of mucin induces the emergence of bacterial resistance against lytic phages, effectively decreased in the presence of NAC. In addition, the genomic analysis revealed some of the genes relevant to the development of phage resistance in long-term co-evolution, with a special focus on the mucoid environment. Genes involved in the metabolism of carbohydrates were mutated in the presence of mucin. In conclusion, the use of mucolytic agents prior to the administration of lytic phages could be an interesting therapeutic option when addressing K. pneumoniae infections in environments where mucin is overproduced.

Carbapenem-resistant pathogens have been recognized as a health concern as they are both difficult to treat and detect in clinical microbiology laboratories. Researchers are making great efforts to develop highly specific, sensitive, accurate, and rapid diagnostic techniques, required to prevent the spread of these microorganisms and improve the prognosis of patients. In this context, CRISPR-Cas systems are proposed as promising tools for the development of diagnostic methods due to their high specificity; the Cas13a endonuclease can discriminate single nucleotide changes and displays collateral cleavage activity against single-stranded RNA molecules when activated. This technology is usually combined with isothermal pre-amplification reactions in order to increase its sensitivity. We have developed a new LAMP-CRISPR-Cas13a-based assay for the detection of OXA-48 and GES carbapenemases in clinical samples without the need for nucleic acid purification and concentration. To evaluate the assay, we used 68 OXA-48-like-producing Klebsiella pneumoniae clinical isolates as well as 64 Enterobacter cloacae complex GES-6, 14 Pseudomonas aeruginosa GES-5, 9 Serratia marcescens GES-6, 5 P. aeruginosa GES-6, and 3 P. aeruginosa (GES-15, GES-27, and GES-40) and 1 K. pneumoniae GES-2 isolates. The assay, which takes less than 2 h and costs approximately 10 € per reaction, exhibited 100% specificity and sensitivity (99% confidence interval [CI]) for both OXA-48 and all GES carbapenemases. IMPORTANCE Carbapenems are one of the last-resort antibiotics for defense against multidrug-resistant pathogens. Multiple nucleic acid amplification methods, including multiplex PCR, multiplex loop-mediated isothermal amplification (LAMP) and multiplex RPAs, can achieve rapid, accurate, and simultaneous detection of several resistance genes to carbapenems in a single reaction. However, these assays need thermal cycling steps and specialized instruments, giving them limited application in the fiel

Clinical case of a patient with a Pseudomonas aeruginosa multidrug-resistant prosthetic vascular graft infection which was treated with a cocktail of phages (PT07, 14/01, and PNM) in combination with ceftazidime-avibactam (CZA). After the application of the phage treatment and in absence of antimicrobial therapy, a new P. aeruginosa bloodstream infection (BSI) with a septic residual limb metastasis occurred, now involving a wild-type strain being susceptible to ß-lactams and quinolones. Clinical strains were analyzed by microbiology and whole genome sequencing techniques. In relation with phage administration, the clinical isolates of P. aeruginosa before phage therapy (HE2011471) and post phage therapy (HE2105886) showed a clonal relationship but with important genomic changes which could be involved in the resistance to this therapy. Finally, phenotypic studies showed a decrease in Minimum Inhibitory Concentration (MIC) to ß-lactams and quinolones as well as an increase of the biofilm production and phage resistant mutants in the clinical isolate of P. aeruginosa post phage therapy.

La Organización Mundial del Turismo (OMT) realiza una regionalización del mundo en base a los continentes, con modificaciones en referencia a los patrones culturales similares, tales como, la religión, las etnias, las costumbres y las prácticas sociales de países que incluso se encuentren en distintos continentes geográficos. A partir de ello, publica anualmente el documento Panorama del Turismo Internacional, con información por región de los flujos internacionales principales. Un análisis a escala menor necesita una detallada documentación estadística de cada país en particular. En este documento se presenta el resultado sintético de los flujos internacionales, durante el período 2013-2021, con base en la variable llegada de turistas internacionales (% de crecimiento/decrecimiento en relación al año anterior) y se identifican los principales destinos, para las siguientes regiones presentadas por la OMT: Américas: 1) América del Norte, El Caribe, América Central y América del Sur. 2) África: África, exceptuada Libia y Egipto. Presenta las siguientes subdivisiones relativas: África del Norte, África Central, África Occidental, África Oriental y África Meridional. 3) Europa: Europa, incluyendo Siberia, Turquía, Kazajstán, Armenia, Georgia. Presenta las siguientes subdivisiones relativas: Europa del Norte, Europa Occidental, Europa Meridional y Europa Central/Oriental. 4) Oriente Medio: península Arábiga más Libia y Egipto. 5) Asia y el Pacífico: Asia, exceptuada Siberia y Península Arábiga, más Oceanía.

Since their discovery, toxin-antitoxin (TA) systems have captivated the attention of many scientists. Recent studies have demonstrated that TA systems play a key role in phage inhibition. The aim of the present study was to investigate the role of the PemIK (PemK/PemI) type II TA system in phage inhibition by its intrinsic expression in clinical strains of Klebsiella pneumoniae carrying the lncL plasmid, which harbours the carbapenemase OXA-48 and the PemK/PemI TA system. Furthermore, induced expression of the system in an IPTG-inducible plasmid in a reference strain of K. pneumoniae ATCC10031 was also studied. The results showed that induced expression of the whole TA system did not inhibit phage infection, whereas overexpression of the pemK toxin prevented early infection. To investigate the molecular mechanism involved in the PemK toxin-mediated inhibition of phage infection, assays measuring metabolic activity and viability were performed, revealing that overexpression of the PemK toxin led to dormancy of the bacteria. Thus, we demonstrate that the PemK/PemI TA system plays a role in phage infection and that the action of the free toxin induces a dormant state in the cells, resulting in inhibition of phage infections.

Since their discovery, toxin-antitoxin (TA) systems have captivated the attention of many scientists. Recent studies have demonstrated that TA systems play a key role in phage inhibition. The aim of the present study was to investigate the role of the PemIK (PemK/PemI) type II TA system in phage inhibition by its intrinsic expression in clinical strains of Klebsiella pneumoniae carrying the lncL plasmid, which harbours the carbapenemase OXA-48 and the PemK/PemI TA system. Furthermore, induced expression of the system in an IPTG-inducible plasmid in a reference strain of K. pneumoniae ATCC10031 was also studied. The results showed that induced expression of the whole TA system did not inhibit phage infection, whereas overexpression of the pemK toxin prevented early infection. To investigate the molecular mechanism involved in the PemK toxin-mediated inhibition of phage infection, assays measuring metabolic activity and viability were performed, revealing that overexpression of the PemK toxin led to dormancy of the bacteria. Thus, we demonstrate that the PemK/PemI TA system plays a role in phage infection and that the action of the free toxin induces a dormant state in the cells, resulting in inhibition of phage infections.

Klebsiella pneumoniae is a human pathogen that worsens the prognosis of many immunocompromised patients. Here, we annotated and compared the genomes of two lytic phages that infect clinical strains of K. pneumoniae (vB_KpnM-VAC13 and vB_KpnM-VAC66) and phenotypically characterized vB_KpnM-VAC66 (time of adsorption of 12 min, burst size of 31.49 ± 0.61 PFU/infected cell, and a host range of 20.8% of the tested strains). Transmission electronic microscopy showed that vB_KpnM-VAC66 belongs to the Myoviridae family. The genomic analysis of the phage vB_KpnM-VAC66 revealed that its genome encoded 289 proteins. When compared to the genome of vB_KpnM-VAC13, they showed a nucleotide similarity of 97.56%, with a 93% of query cover, and the phylogenetic study performed with other Tevenvirinae phages showed a close common ancestor. However, there were 21 coding sequences which differed. Interestingly, the main differences were that vB_KpnM-VAC66 encoded 10 more homing endonucleases than vB_KpnM-VAC13, and that the nucleotidic and amino-acid sequences of the L-shaped tail fiber protein were highly dissimilar, leading to different three-dimensional protein predictions. Both phages differed significantly in their host range. These viruses may be useful in the development of alternative therapies to antibiotics or as a co-therapy increasing its antimicrobial potential, especially when addressing multidrug resistant (MDR) pathogens.

Esta ponencia propone demostrar el uso de conceptos y teorías provenientes de la Geografía en la formación del Técnico y Licenciado en Turismo, que se constituyen en parte de la alfabetización académica del especialista en turismo. Se realiza un análisis del perfil de los egresados y de los planes de estudio de las carreras: Técnico Superior en Turismo, formación superior de carácter provincial (provincia de Buenos Aires), Técnico Universitario en Turismo y Licenciado en Turismo, de carácter universitario y nacional (Universidad Nacional de Mar del Plata). Por otra parte, se realizan encuestas semiestructuradas a docentes del nivel universitario y superior que desarrollan clases en dichas carreras, cuyas variables tuvieron en cuenta: campos de las Geografía, contenidos temáticos y abordaje pedagógico. Los resultados dan cuenta de que hay una inclinación por las teorías y concepto provenientes de la Geografía Física, en sus campos Geomorfología, Hidrología, Climatología y Biogeografía, quedando relegada la formación en Geografía Social, Económica o Política. La escala de análisis regional cobra importancia, así como el concepto de Espacio Turístico. Sin embargo, a partir del Aprendizaje Basado en Proyectos que los estudiantes tienen la posibilidad de seleccionar problematizaciones de tipo ambientales, económicas, demográficas o políticas en el abordaje del espacio turístico., Fil: Padilla, Noelia Aymara. Universidad Nacional de Mar del Plata. Facultad de Ciencias Económicas y Sociales; Argentina., Fil: Babini, Natalia. Universidad Nacional de Mar del Plata. Facultad de Ciencias Económicas y Sociales; Argentina., Fil: Blasco, Lucía. Universidad Nacional de Mar del Plata. Facultad de Ciencias Económicas y Sociales; Argentina.

[Abstract] To optimize phage therapy, we need to understand how bacteria evolve against phage attacks. One of the main problems of phage therapy is the appearance of bacterial resistance variants. The use of genomics to track antimicrobial resistance is increasingly developed and used in clinical laboratories. For that reason, it is important to consider, in an emerging future with phage therapy, to detect and avoid phage-resistant strains that can be overcome by the analysis of metadata provided by whole-genome sequencing. Here, we identified genes associated with phage resistance in 18 Acinetobacter baumannii clinical strains belonging to the ST-2 clonal complex during a decade (Ab2000 vs. 2010): 9 from 2000 to 9 from 2010. The presence of genes putatively associated with phage resistance was detected. Genes detected were associated with an abortive infection system, restriction–modification system, genes predicted to be associated with defense systems but with unknown function, and CRISPR-Cas system. Between 118 and 171 genes were found in the 18 clinical strains. On average, 26% of these genes were detected inside genomic islands in the 2000 strains and 32% in the 2010 strains. Furthermore, 38 potential CRISPR arrays in 17 of 18 of the strains were found, as well as 705 proteins associated with CRISPR-Cas systems. A moderately higher presence of these genes in the strains of 2010 in comparison with those of 2000 was found, especially those related to the restriction–modification system and CRISPR-Cas system. The presence of these genes in genomic islands at a higher rate in the strains of 2010 compared with those of 2000 was also detected. Whole-genome sequencing and bioinformatics could be powerful tools to avoid drawbacks when a personalized therapy is applied. In this study, it allows us to take care of the phage resistance in A. baumannii clinical strains to prevent a failure in possible phage therapy.

[Abstract] Since their discovery, toxin-antitoxin (TA) systems have captivated the attention of many scientists. Recent studies have demonstrated that TA systems play a key role in phage inhibition. The aim of the present study was to investigate the role of the PemIK (PemK/PemI) type II TA system in phage inhibition by its intrinsic expression in clinical strains of Klebsiella pneumoniae carrying the lncL plasmid, which harbours the carbapenemase OXA-48 and the PemK/PemI TA system. Furthermore, induced expression of the system in an IPTG-inducible plasmid in a reference strain of K. pneumoniae ATCC10031 was also studied. The results showed that induced expression of the whole TA system did not inhibit phage infection, whereas overexpression of the pemK toxin prevented early infection. To investigate the molecular mechanism involved in the PemK toxin-mediated inhibition of phage infection, assays measuring metabolic activity and viability were performed, revealing that overexpression of the PemK toxin led to dormancy of the bacteria. Thus, we demonstrate that the PemK/PemI TA system plays a role in phage infection and that the action of the free toxin induces a dormant state in the cells, resulting in inhibition of phage infections.

Klebsiella pneumoniae is a human pathogen that worsens the prognosis of many immuno compromised patients. Here, we annotated and compared the genomes of two lytic phages that infect clinical strains of K. pneumoniae (vB_KpnM-VAC13 and vB_KpnM-VAC66) and phenotypically char acterized vB_KpnM-VAC66 (time of adsorption of 12 min, burst size of 31.49 ± 0.61 PFU/infected cell, and a host range of 20.8% of the tested strains). Transmission electronic microscopy showed that vB_KpnM-VAC66 belongs to the Myoviridae family. The genomic analysis of the phage vB_KpnM VAC66 revealed that its genome encoded 289 proteins. When compared to the genome of vB_KpnM VAC13, they showed a nucleotide similarity of 97.56%, with a 93% of query cover, and the phylo genetic study performed with other Tevenvirinae phages showed a close common ancestor. How ever, there were 21 coding sequences which differed. Interestingly, the main differences were that vB_KpnM-VAC66 encoded 10 more homing endonucleases than vB_KpnM-VAC13, and that the nu cleotidic and amino-acid sequences of the L-shaped tail fiber protein were highly dissimilar, leading to different three-dimensional protein predictions. Both phages differed significantly in their host range. These viruses may be useful in the development of alternative therapies to antibiotics or as a co-therapy increasing its antimicrobial potential, especially when addressing multidrug resistant (MDR) pathogens.

Che Lagarto es una cadena privada de hostels que tiene más de veinte años de historia y experiencia en el mercado Sudamericano. Ha estado presente en diversas ciudades de Argentina, Brasil, Chile, Uruguay y Perú, desde 1997. El hostel es un establecimiento hotelero que provee servicios de alojamiento, gastronomía y entretenimiento destinados al público joven. En el marco de la Unidad 3. Caribe y América Latina, de la asignatura Espacios Turísticos Americanos, se elabora este documento para las carreras de Licenciado en Turismo y Técnico Universitario en Turismo, con el objetivo de identificar los destinos turísticos de Brasil vinculados con una forma de alojamiento atractiva y accesible para el segmento de los estudiantes de la asignatura. De manera introductoria, el texto comienza con una definición del concepto "hostel", y a manera de estudio de caso se selecciona la empresa Che Lagarto representativa en el mercado sudamericano de esta modalidad, analizando su origen, significado del logotipo y diferentes localizaciones a lo largo de su historia. Finalmente se realiza una descripción de la oferta turística de cada uno de los destinos en Brasil donde la empresa tiene unidades de alojamiento: Río de Janeiro, Isla Grande, Paraty, Búzios, Foz de Iguazú, Bonito, Morro de São Paulo, Itacaré y Porto de Galinhas. Algunos destinos se especializan en el Turismo de Sol y Playa, en cambio otros son sitio Patrimonio de la Humanidad, combinando escenarios para prácticas de Turismo Alternativo.

El Caribe es una de las regiones receptoras de turismo a nivel mundial más frecuentadas y específicamente el área de las Antillas, es el principal espacio del mundo por densidad de Cruceros. Por tal motivo, se consideró interesante abordar la región a través de la modalidad de Turismo de Cruceros. En el marco de la Unidad 3. Caribe y América Latina, de la asignatura Espacios Turísticos Americanos, se elabora este documento para los estudiantes de las carreras de Licenciado en Turismo y Técnico Universitario en Turismo, con la intención de transmitir la experiencia vivida, a partir de la realización de un viaje en crucero por destinos de América Central. Se comienza con una breve definición del concepto y referencias históricas, para luego explicar los diferentes tipos de cabinas, el procedimiento de Check in que comienza en el puerto y finaliza en el interior de la nave, una descripción de la vida a bordo de un crucero, relatando tanto aspectos vinculados con la seguridad del crucerista, así como la gran variedad de actividades recreativas a bordo y en los puertos. Luego se presenta una referencia de la Geografía de los Cruceros Marítimos, y a continuación se describe la experiencia realizada del recorrido por las Antillas y el Caribe Sur, incluyendo una descripción de la oferta turística de cada uno de los puertos visitados. El valor del documento se centra en la posibilidad de transmitir la experiencia vivida, información que surge de la propia observación y participación de un crucero. Por tanto, constituye una fuente de información única y oportuna, que permitirá conocer la vida que propicia un viaje en crucero.

[Abstract] Klebsiella pneumoniae is a human pathogen that worsens the prognosis of many immunocompromised patients. Here, we annotated and compared the genomes of two lytic phages that infect clinical strains of K. pneumoniae (vB_KpnM-VAC13 and vB_KpnM-VAC66) and phenotypically characterized vB_KpnM-VAC66 (time of adsorption of 12 min, burst size of 31.49 ± 0.61 PFU/infected cell, and a host range of 20.8% of the tested strains). Transmission electronic microscopy showed that vB_KpnM-VAC66 belongs to the Myoviridae family. The genomic analysis of the phage vB_KpnM-VAC66 revealed that its genome encoded 289 proteins. When compared to the genome of vB_KpnM-VAC13, they showed a nucleotide similarity of 97.56%, with a 93% of query cover, and the phylogenetic study performed with other Tevenvirinae phages showed a close common ancestor. However, there were 21 coding sequences which differed. Interestingly, the main differences were that vB_KpnM-VAC66 encoded 10 more homing endonucleases than vB_KpnM-VAC13, and that the nucleotidic and amino-acid sequences of the L-shaped tail fiber protein were highly dissimilar, leading to different three-dimensional protein predictions. Both phages differed significantly in their host range. These viruses may be useful in the development of alternative therapies to antibiotics or as a co-therapy increasing its antimicrobial potential, especially when addressing multidrug resistant (MDR) pathogens.

[Abstract] One of the biggest threats we face globally is the emergence of antimicrobial-resistant (AMR) bacteria, which runs in parallel with the lack in the development of new antimicrobials. Among these AMR bacteria pathogens belonging to the ESKAPE group can be highlighted (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) due to their profile of drug resistance and virulence. Therefore, innovative lines of treatment must be developed for these bacteria. In this review, we summarize the different strategies for the treatment and study of molecular mechanisms of AMR in the ESKAPE pathogens based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins’ technologies: loss of plasmid or cellular viability, random mutation or gene deletion as well directed mutations that lead to a gene’s loss of function.

Klebsiella pneumoniae is the clinically most important species within the genus Klebsiella and, as a result of the continuous emergence of multi-drug resistant (MDR) strains, the cause of severe nosocomial infections. The decline in the effectiveness of antibiotic treatments for infections caused by MDR bacteria has generated particular interest in the study of bacteriophages. In this study, we characterized a total of 40 temperate bacteriophages (prophages) with a genome range of 11.454-84.199 kb, predicted from 16 carbapenemase-producing clinical strains of K. pneumoniae belonging to different sequence types, previously identified by multilocus sequence typing. These prophages were grouped into the three families in the order Caudovirales (27 prophages belonging to the family Myoviridae, 10 prophages belonging to the family Siphoviridae and 3 prophages belonging to the family Podoviridae). Genomic comparison of the 40 prophage genomes led to the identification of four prophages isolated from different strains and of genome sizes of around 33.3, 36.1, 39.6 and 42.6 kb. These prophages showed sequence similarities (query cover >90 %, identity >99.9 %) with international Microbe Versus Phage (MVP) (http://mvp.medgenius.info/home) clusters 4762, 4901, 3499 and 4280, respectively. Phylogenetic analysis revealed the evolutionary proximity among the members of the four groups of the most frequently identified prophages in the bacterial genomes studied (33.3, 36.1, 39.6 and 42.6 kb), with bootstrap values of 100 %. This allowed the prophages to be classified into three clusters: A, B and C. Interestingly, these temperate bacteriophages did not infect the highest number of strains as indicated by a host-range assay, these results could be explained by the development of superinfection exclusion mechanisms. In addition, bioinformatic analysis of the 40 identified prophages revealed the presence of 2363 proteins. In total, 59.7 % of the proteins identified had a predicted fu

Klebsiella pneumoniae is the clinically most important species within the genus Klebsiella and, as a result of the continuous emergence of multi-drug resistant (MDR) strains, the cause of severe nosocomial infections. The decline in the effectiveness of antibiotic treatments for infections caused by MDR bacteria has generated particular interest in the study of bacteriophages. In this study, we characterized a total of 40 temperate bacteriophages (prophages) with a genome range of 11.454–84.199 kb, predicted from 16 carbapenemase-producing clinical strains of K. pneumoniae belonging to different sequence types, previously identified by multilocus sequence typing. These prophages were grouped into the three families in the order Caudovirales (27 prophages belonging to the family Myoviridae, 10 prophages belonging to the family Siphoviridae and 3 prophages belonging to the family Podoviridae). Genomic comparison of the 40 prophage genomes led to the identification of four prophages isolated from different strains and of genome sizes of around 33.3, 36.1, 39.6 and 42.6 kb. These prophages showed sequence similarities (query cover >90 %, identity >99.9 %) with international Microbe Versus Phage (MVP) (http://mvp.medgenius.info/home) clusters 4762, 4901, 3499 and 4280, respectively. Phylogenetic analysis revealed the evolutionary proximity among the members of the four groups of the most frequently identified prophages in the bacterial genomes studied (33.3, 36.1, 39.6 and 42.6 kb), with bootstrap values of 100 %. This allowed the prophages to be classified into three clusters: A, B and C. Interestingly, these temperate bacteriophages did not infect the highest number of strains as indicated by a host-range assay, these results could be explained by the development of superinfection exclusion mechanisms. In addition, bioinformatic analysis of the 40 identified prophages revealed the presence of 2363 proteins. In total, 59.7 % of the proteins identified had a predicted fu

Acinetobacter spp. are found in all environments on Earth due to their extraordinary capacity to survive in the presence of physical and chemical stressors. In this study, we analyzed global gene expression in airborne Acinetobacter sp. strain 5-2Ac02 isolated from hospital environment in response to quorum network modulators and found that they induced the expression of genes of the acetoin/butanediol catabolism, volatile compounds shown to mediate interkingdom interactions. Interestingly, the acoN gene, annotated as a putative transcriptional regulator, was truncated in the downstream regulatory region of the induced acetoin/butanediol cluster in Acinetobacter sp. strain 5-2Ac02, and its functioning as a negative regulator of this cluster integrating quorum signals was confirmed in Acinetobacter baumannii ATCC 17978. Moreover, we show that the acetoin catabolism is also induced by light and provide insights into the light transduction mechanism by showing that the photoreceptor BlsA interacts with and antagonizes the functioning of AcoN in A. baumannii, integrating also a temperature signal. The data support a model in which BlsA interacts with and likely sequesters AcoN at this condition, relieving acetoin catabolic genes from repression, and leading to better growth under blue light. This photoregulation depends on temperature, occurring at 23°C but not at 30°C. BlsA is thus a dual regulator, modulating different transcriptional regulators in the dark but also under blue light, representing thus a novel concept. The overall data show that quorum modulators as well as light regulate the acetoin catabolic cluster, providing a better understanding of environmental as well as clinical bacteria.