Genomics of three new bacteriophages useful in the biocontrol of Salmonella

Non-typhoid Salmonella is the principal pathogen related to food-borne diseases throughout the world. Widespread antibiotic resistance has adversely affected human health and has encouraged the search for alternative antimicrobial agents. The advances in bacteriophage therapy highlight their use in controlling a broad spectrum of food-borne pathogens. One requirement for the use of bacteriophages as antibacterials is the characterization of their genomes. In this work, complete genome sequencing and molecular analyses were carried out for three new virulent Salmonella-specific bacteriophages (UAB_Phi20, UAB_Phi78, and UAB_Phi87) able to infect a broad range of Salmonella strains. Sequence analysis of the genomes of UAB_Phi20, UAB_Phi78, and UAB_Phi87 bacteriophages did not evidence the presence of known virulence-associated and antibiotic resistance genes, and potential immunoreactive food allergens. The UAB_Phi20 genome comprised 41,809 base pairs with 80 open reading frames (ORFs); 24 of them with assigned function. Genome sequence showed a high homology of UAB_Phi20 with Salmonella bacteriophage P22 and other P22likeviruses genus of the Podoviridae family, including ST64T and ST104. The DNA of UAB_Phi78 contained 44,110 bp including direct terminal repeats (DTR) of 179 bp and 58 putative ORFs were predicted and 20 were assigned function. This bacteriophage was assigned to the SP6likeviruses genus of the Podoviridae family based on its high similarity not only with SP6 but also with the K1-5, K1E, and K1F bacteriophages, all of which infect Escherichia coli. The UAB_Phi87 genome sequence consisted of 87,669 bp with terminal direct repeats of 608 bp; although 148 ORFs were identified, putative functions could be assigned to only 29 of them. Sequence comparisons revealed the mosaic structure of UAB_Phi87 and its high similarity with bacteriophages Felix O1 and wV8 of E. coli with respect to genetic content and functional organization. Phylogenetic analysis of large terminase subunits confirms their packaging strategies and grouping to the different phage genus type. All these studies are necessary for the development and the use of an efficient cocktail with commercial applications in bacteriophage therapy against Salmonella

Biodistribution of liposome-encapsulated bacteriophages and their transcytosis during oral phage therapy

This study sheds light on the biodistribution of orally administered, liposome-encapsulated bacteriophages, and their transcytosis through intestinal cell layers. Fluorochrome-labeled bacteriophages were used together with a non-invasive imaging methodology in the in vivo visualization of bacteriophages in the stomach and intestinal tract of mice. In those studies, phage encapsulation resulted in a significant increase of the labeled phages in the mouse stomach, even 6 h after their oral administration, and without a decrease in their concentration. By contrast, the visualization of encapsulated and non-encapsulated phages in the intestine were similar. Our in vivo observations were corroborated by culture methods and ex vivo experiments, which also showed that the percentage of encapsulated phages in the stomach remained constant (50%) compared to the amount of initially administered product. However, the use of conventional microbiological methods, which employ bile salts to break down liposomes, prevented the detection of encapsulated phages in the intestine. The ex vivo data showed a higher concentration of non-encapsulated than encapsulated phages in liver, kidney, and even muscle up to 6 h post-administration. Encapsulated bacteriophages were able to reach the liver, spleen, and muscle, with values of 38% ± 6.3%, 68% ± 8.6%, and 47% ± 7.4%, respectively, which persisted over the course of the experiment. Confocal laser scanning microscopy of an in vitro co-culture of human Caco-2/HT29/Raji-B cells revealed that Vybrant-Dil-stained liposomes containing labeled bacteriophages were preferably embedded in cell membranes. No transcytosis of encapsulated phages was detected in this in vitro model, whereas SYBR-gold-labeled non-encapsulated bacteriophages were able to cross the membrane. Our work demonstrates the prolonged persistence of liposome-encapsulated phages in the stomach and their adherence to the intestinal membrane. These observations could explain the greater long-term efficacy of phage therapy using liposome-encapsulated phages

Biodistribución de bacteriófagos en mamíferos tras terapia fágica oral y emergencia de resistencia bacteriana a los fagos

La aplicación terapéutica de bacteriófagos líticos como alternativa a los antimicrobianos tradicionales ha resurgido en los últimos años. Sin embargo, existen algunos aspectos asociados a la terapia fágica que necesitan estudiarse con más profundidad para permitir su definitiva implementación. El presente trabajo se centra en el estudio de dos aspectos de especial relevancia: i) la emergencia de la resistencia a los bacteriófagos en diferentes condiciones de aplicación y ii) la biodistribución y transcitosis de bacteriófagos encapsulados en liposomas tras su administración por vía oral en un modelo murino. Para el estudio de la emergencia de la resistencia bacteriana a bacteriófagos se utilizó el modelo de Salmonella/cóctel de bacteriófagos integrado por UAB_Phi20, UAB_Phi78 y UAB_Phi87, cuyos receptores se localizaban en el LPS. Se evidenció que la emergencia de resistencia difería en función del ámbito de aplicación. Así, el 92 % de los aislados de cultivos in vitro de S. Typhimurium infectados con el cóctel de bacteriófagos fueron resistentes a los 3 fagos, siendo la causa probable de la resistencia la pérdida de la proteína RfaJ, implicada en la síntesis del LPS. Análogamente, en lonchas de jamón cocido contaminadas con S. Typhimurium y tratadas con el cóctel fágico, el 1,4 % de los aislados fueron resistentes a los tres fagos, mostrando también una mutación en el gen rfaJ; mientras que el 1,8 % sólo presentó resistencia a los fagos UAB_Phi20 y UAB_Phi78, debido a cambios en el gen rfc, el cual codifica la polimerasa del antígeno O. Finalmente, en los estudios de terapia fágica en el modelo de pollo de engorde, no se encontró ningún clon resistente al fago UAB_Phi78 aislado de animales contaminados con S. Typhimurium y tratados con el cóctel. Sin embargo, el 8,5 % de los aislados del grupo control fueron resistentes a los tres fagos, no habiéndose podido determinar la causa de dicha resistencia. Por otro lado, el 1,3 % y el 3,3 % de los clones de los animales del grupo control y tratamiento, respectivamente, mostraron resistencia al bacteriófago UAB_Phi78, el cual ejerció un efecto bactericida, pero no bacteriolítico y sin producción de progenie fágica y resistencia asociada a UAB_Phi87. Aunque la causa de dicha resistencia no se ha podido identificar, todo sugiere la implicación de un mecanismo basado en una infección abortiva. En conclusión, este estudio revela que tanto la frecuencia de la emergencia de resistencia como los mecanismos causantes de la misma son diferentes in vitro, en alimentos e in vivo. Además, demuestra la necesidad de abordar los estudios de resistencia de un modo más amplio y de utilizar cócteles que contengan bacteriófagos que reconozcan diferentes receptores para disminuir el desarrollo de resistencias. En el estudio de biodistribución, se administró oralmente el fago UAB_Phi20 encapsulado en liposomas y marcado con el fluorocromo VTS-750 a ratones y visualizándolos mediante el sistema IVIS Spectrum. Esta metodología combinada con métodos de cultivo ex vivo, demostró que la encapsulación daba lugar a un aumento significativo de fagos encapsulados en el estómago 6 h después de su administración. Igualmente, los bacteriófagos encapsulados en liposomas también se encontraron en el bazo, hígado y músculo. Por otro lado, la aplicación de CLSM en cultivos in vitro de células intestinales humanas (Caco-2/HT29/Raji-B) reveló que los liposomas se adhieren a las membranas de dichas células pudiendo permanecer en su interior. En cambio, los bacteriófagos no encapsulados son capaces de translocar la barrera intestinal. Así, la prolongada persistencia de los fagos encapsulados en el estómago y su adherencia a la membrana intestinal podría explicar la mayor eficacia en el tiempo de la terapia fágica oral empleando bacteriófagos encapsulados en liposomas.Interest in the therapeutic application of virulent bacteriophages as an alternative to traditional antimicrobials has re-emerged in recent years. However, more detailed studies of several aspects associated with phage therapy are needed to allow its definitive implementation. The aim of the research presented in this dissertation was to study i) the emergence of resistance to bacteriophages under different conditions of their application and ii) the biodistribution and transcytosis of liposome-encapsulated bacteriophages after oral administration in a murine model. The study of bacteriophage resistance made use of the model Salmonella/bacteriophage cocktail composed of the phages UAB_Phi20, UAB_Phi78, and UAB_Phi87, whose receptors involving the LPS. The results showed that the emergence of resistance differed depending on the scope of the application. Thus, 92 % of the isolates recovered from in vitro cultures of S. Typhimurium infected with the bacteriophage cocktail were resistant to the three bacteriophages. This form of resistance was probably due to the loss of the RfaJ protein, involved in the synthesis of Salmonella LPS. Similarly, in slices of cooked ham contaminated with S. Typhimurium and treated with the phage cocktail, 1.4 % of the clones were resistant to the three bacteriophages, also showing mutations in the rfaJ gene; meanwhile, only 1.8 % of the clones were resistant to UAB_Phi20 and UAB_Phi78 due to changes in the rfc gene, encoding the O-antigen polymerase. Finally, in studies of phage therapy in a broiler chicken model, no resistance to UAB_Phi78 was found in isolates from animals contaminated with S. Typhimurium and treated with the phage cocktail. However, 8.5 % of the isolates in the control group were resistant to the three phages, not having been determined the mechanisms of this resistance. In addition, 1.3 % and 3.3 % of the clones in the animals of the control and treated group, respectively, were resistant to bacteriophage UAB_Phi78, which exerted a bactericidal but not a bacteriolytic effect and did not result in the production of phage progeny, and additionally showed resistance to UAB_Phi87. The cause of this resistance was also unclear but the evidence suggested a mechanism based on an abortive infection. In conclusion, this study showed that both the frequency of the emergence of resistance and the mechanisms involved differ in vitro, in food, and in vivo. It, therefore, demonstrates the need for a broader approach to resistance studies and the use of cocktails containing bacteriophages that by recognizing different host receptors reduce the development of resistance. The biodistribution studies were performed by orally administering to mice the liposome-encapsulated phage UAB_Phi20 labeled with VTS-750 fluorochrome and then visualizing the phages by the IVIS Spectrum methodology. Using this method together with ex vivo cultures, we were able to show that encapsulation resulted in a significant accumulation of encapsulated phages in the mouse stomach, even 6 h after phage administration. Similarly, liposome-encapsulated phages were found in spleen, liver, and muscle. By contrast, in CLSM study of in vitro cultures of human intestinal cells (Caco-2/HT29/Raji-B), liposomes were seen adhered to the cell membrane or embedded in this. However, non-encapsulated bacteriophages were able to translocate across the intestinal barrier. Taken together, these results could explain the greater efficacy over time of oral phage therapy using liposome-encapsulated bacteriophages

Biodistribución de bacteriófagos en mamíferos tras terapia fágica oral y emergencia de resistencia bacteriana a los fagos

La aplicación terapéutica de bacteriófagos líticos como alternativa a los antimicrobianos tradicionales ha resurgido en los últimos años. Sin embargo, existen algunos aspectos asociados a la terapia fágica que necesitan estudiarse con más profundidad para permitir su definitiva implementación. El presente trabajo se centra en el estudio de dos aspectos de especial relevancia: i) la emergencia de la resistencia a los bacteriófagos en diferentes condiciones de aplicación y ii) la biodistribución y transcitosis de bacteriófagos encapsulados en liposomas tras su administración por vía oral en un modelo murino. Para el estudio de la emergencia de la resistencia bacteriana a bacteriófagos se utilizó el modelo de Salmonella/cóctel de bacteriófagos integrado por UAB_Phi20, UAB_Phi78 y UAB_Phi87, cuyos receptores se localizaban en el LPS. Se evidenció que la emergencia de resistencia difería en función del ámbito de aplicación. Así, el 92 % de los aislados de cultivos in vitro de S. Typhimurium infectados con el cóctel de bacteriófagos fueron resistentes a los 3 fagos, siendo la causa probable de la resistencia la pérdida de la proteína RfaJ, implicada en la síntesis del LPS. Análogamente, en lonchas de jamón cocido contaminadas con S. Typhimurium y tratadas con el cóctel fágico, el 1,4 % de los aislados fueron resistentes a los tres fagos, mostrando también una mutación en el gen rfaJ; mientras que el 1,8 % sólo presentó resistencia a los fagos UAB_Phi20 y UAB_Phi78, debido a cambios en el gen rfc, el cual codifica la polimerasa del antígeno O. Finalmente, en los estudios de terapia fágica en el modelo de pollo de engorde, no se encontró ningún clon resistente al fago UAB_Phi78 aislado de animales contaminados con S. Typhimurium y tratados con el cóctel. Sin embargo, el 8,5 % de los aislados del grupo control fueron resistentes a los tres fagos, no habiéndose podido determinar la causa de dicha resistencia. Por otro lado, el 1,3 % y el 3,3 % de los clones de los animales del grupo control y tratamiento, respectivamente, mostraron resistencia al bacteriófago UAB_Phi78, el cual ejerció un efecto bactericida, pero no bacteriolítico y sin producción de progenie fágica y resistencia asociada a UAB_Phi87. Aunque la causa de dicha resistencia no se ha podido identificar, todo sugiere la implicación de un mecanismo basado en una infección abortiva. En conclusión, este estudio revela que tanto la frecuencia de la emergencia de resistencia como los mecanismos causantes de la misma son diferentes in vitro, en alimentos e in vivo. Además, demuestra la necesidad de abordar los estudios de resistencia de un modo más amplio y de utilizar cócteles que contengan bacteriófagos que reconozcan diferentes receptores para disminuir el desarrollo de resistencias. En el estudio de biodistribución, se administró oralmente el fago UAB_Phi20 encapsulado en liposomas y marcado con el fluorocromo VTS-750 a ratones y visualizándolos mediante el sistema IVIS Spectrum. Esta metodología combinada con métodos de cultivo ex vivo, demostró que la encapsulación daba lugar a un aumento significativo de fagos encapsulados en el estómago 6 h después de su administración. Igualmente, los bacteriófagos encapsulados en liposomas también se encontraron en el bazo, hígado y músculo. Por otro lado, la aplicación de CLSM en cultivos in vitro de células intestinales humanas (Caco-2/HT29/Raji-B) reveló que los liposomas se adhieren a las membranas de dichas células pudiendo permanecer en su interior. En cambio, los bacteriófagos no encapsulados son capaces de translocar la barrera intestinal. Así, la prolongada persistencia de los fagos encapsulados en el estómago y su adherencia a la membrana intestinal podría explicar la mayor eficacia en el tiempo de la terapia fágica oral empleando bacteriófagos encapsulados en liposomas.Interest in the therapeutic application of virulent bacteriophages as an alternative to traditional antimicrobials has re-emerged in recent years. However, more detailed studies of several aspects associated with phage therapy are needed to allow its definitive implementation. The aim of the research presented in this dissertation was to study i) the emergence of resistance to bacteriophages under different conditions of their application and ii) the biodistribution and transcytosis of liposome-encapsulated bacteriophages after oral administration in a murine model. The study of bacteriophage resistance made use of the model Salmonella/bacteriophage cocktail composed of the phages UAB_Phi20, UAB_Phi78, and UAB_Phi87, whose receptors involving the LPS. The results showed that the emergence of resistance differed depending on the scope of the application. Thus, 92 % of the isolates recovered from in vitro cultures of S. Typhimurium infected with the bacteriophage cocktail were resistant to the three bacteriophages. This form of resistance was probably due to the loss of the RfaJ protein, involved in the synthesis of Salmonella LPS. Similarly, in slices of cooked ham contaminated with S. Typhimurium and treated with the phage cocktail, 1.4 % of the clones were resistant to the three bacteriophages, also showing mutations in the rfaJ gene; meanwhile, only 1.8 % of the clones were resistant to UAB_Phi20 and UAB_Phi78 due to changes in the rfc gene, encoding the O-antigen polymerase. Finally, in studies of phage therapy in a broiler chicken model, no resistance to UAB_Phi78 was found in isolates from animals contaminated with S. Typhimurium and treated with the phage cocktail. However, 8.5 % of the isolates in the control group were resistant to the three phages, not having been determined the mechanisms of this resistance. In addition, 1.3 % and 3.3 % of the clones in the animals of the control and treated group, respectively, were resistant to bacteriophage UAB_Phi78, which exerted a bactericidal but not a bacteriolytic effect and did not result in the production of phage progeny, and additionally showed resistance to UAB_Phi87. The cause of this resistance was also unclear but the evidence suggested a mechanism based on an abortive infection. In conclusion, this study showed that both the frequency of the emergence of resistance and the mechanisms involved differ in vitro, in food, and in vivo. It, therefore, demonstrates the need for a broader approach to resistance studies and the use of cocktails containing bacteriophages that by recognizing different host receptors reduce the development of resistance. The biodistribution studies were performed by orally administering to mice the liposome-encapsulated phage UAB_Phi20 labeled with VTS-750 fluorochrome and then visualizing the phages by the IVIS Spectrum methodology. Using this method together with ex vivo cultures, we were able to show that encapsulation resulted in a significant accumulation of encapsulated phages in the mouse stomach, even 6 h after phage administration. Similarly, liposome-encapsulated phages were found in spleen, liver, and muscle. By contrast, in CLSM study of in vitro cultures of human intestinal cells (Caco-2/HT29/Raji-B), liposomes were seen adhered to the cell membrane or embedded in this. However, non-encapsulated bacteriophages were able to translocate across the intestinal barrier. Taken together, these results could explain the greater efficacy over time of oral phage therapy using liposome-encapsulated bacteriophages

Biodistribución de bacteriófagos en mamíferos tras terapia fágica oral y emergencia de resistencia bacteriana a los fagos /

Departament responsable de la tesi: Departament de Genètica i de Microbiologia.La aplicación terapéutica de bacteriófagos líticos como alternativa a los antimicrobianos tradicionales ha resurgido en los últimos años. Sin embargo, existen algunos aspectos asociados a la terapia fágica que necesitan estudiarse con más profundidad para permitir su definitiva implementación. El presente trabajo se centra en el estudio de dos aspectos de especial relevancia: i) la emergencia de la resistencia a los bacteriófagos en diferentes condiciones de aplicación y ii) la biodistribución y transcitosis de bacteriófagos encapsulados en liposomas tras su administración por vía oral en un modelo murino. Para el estudio de la emergencia de la resistencia bacteriana a bacteriófagos se utilizó el modelo de Salmonella/cóctel de bacteriófagos integrado por UAB_Phi20, UAB_Phi78 y UAB_Phi87, cuyos receptores se localizaban en el LPS. Se evidenció que la emergencia de resistencia difería en función del ámbito de aplicación. Así, el 92 % de los aislados de cultivos in vitro de S. Typhimurium infectados con el cóctel de bacteriófagos fueron resistentes a los 3 fagos, siendo la causa probable de la resistencia la pérdida de la proteína RfaJ, implicada en la síntesis del LPS. Análogamente, en lonchas de jamón cocido contaminadas con S. Typhimurium y tratadas con el cóctel fágico, el 1,4 % de los aislados fueron resistentes a los tres fagos, mostrando también una mutación en el gen rfaJ; mientras que el 1,8 % sólo presentó resistencia a los fagos UAB_Phi20 y UAB_Phi78, debido a cambios en el gen rfc, el cual codifica la polimerasa del antígeno O. Finalmente, en los estudios de terapia fágica en el modelo de pollo de engorde, no se encontró ningún clon resistente al fago UAB_Phi78 aislado de animales contaminados con S. Typhimurium y tratados con el cóctel. Sin embargo, el 8,5 % de los aislados del grupo control fueron resistentes a los tres fagos, no habiéndose podido determinar la causa de dicha resistencia. Por otro lado, el 1,3 % y el 3,3 % de los clones de los animales del grupo control y tratamiento, respectivamente, mostraron resistencia al bacteriófago UAB_Phi78, el cual ejerció un efecto bactericida, pero no bacteriolítico y sin producción de progenie fágica y resistencia asociada a UAB_Phi87. Aunque la causa de dicha resistencia no se ha podido identificar, todo sugiere la implicación de un mecanismo basado en una infección abortiva. En conclusión, este estudio revela que tanto la frecuencia de la emergencia de resistencia como los mecanismos causantes de la misma son diferentes in vitro, en alimentos e in vivo. Además, demuestra la necesidad de abordar los estudios de resistencia de un modo más amplio y de utilizar cócteles que contengan bacteriófagos que reconozcan diferentes receptores para disminuir el desarrollo de resistencias. En el estudio de biodistribución, se administró oralmente el fago UAB_Phi20 encapsulado en liposomas y marcado con el fluorocromo VTS-750 a ratones y visualizándolos mediante el sistema IVIS Spectrum. Esta metodología combinada con métodos de cultivo ex vivo, demostró que la encapsulación daba lugar a un aumento significativo de fagos encapsulados en el estómago 6 h después de su administración. Igualmente, los bacteriófagos encapsulados en liposomas también se encontraron en el bazo, hígado y músculo. Por otro lado, la aplicación de CLSM en cultivos in vitro de células intestinales humanas (Caco-2/HT29/Raji-B) reveló que los liposomas se adhieren a las membranas de dichas células pudiendo permanecer en su interior. En cambio, los bacteriófagos no encapsulados son capaces de translocar la barrera intestinal. Así, la prolongada persistencia de los fagos encapsulados en el estómago y su adherencia a la membrana intestinal podría explicar la mayor eficacia en el tiempo de la terapia fágica oral empleando bacteriófagos encapsulados en liposomas.Interest in the therapeutic application of virulent bacteriophages as an alternative to traditional antimicrobials has re-emerged in recent years. However, more detailed studies of several aspects associated with phage therapy are needed to allow its definitive implementation. The aim of the research presented in this dissertation was to study i) the emergence of resistance to bacteriophages under different conditions of their application and ii) the biodistribution and transcytosis of liposome-encapsulated bacteriophages after oral administration in a murine model. The study of bacteriophage resistance made use of the model Salmonella/bacteriophage cocktail composed of the phages UAB_Phi20, UAB_Phi78, and UAB_Phi87, whose receptors involving the LPS. The results showed that the emergence of resistance differed depending on the scope of the application. Thus, 92 % of the isolates recovered from in vitro cultures of S. Typhimurium infected with the bacteriophage cocktail were resistant to the three bacteriophages. This form of resistance was probably due to the loss of the RfaJ protein, involved in the synthesis of Salmonella LPS. Similarly, in slices of cooked ham contaminated with S. Typhimurium and treated with the phage cocktail, 1.4 % of the clones were resistant to the three bacteriophages, also showing mutations in the rfaJ gene; meanwhile, only 1.8 % of the clones were resistant to UAB_Phi20 and UAB_Phi78 due to changes in the rfc gene, encoding the O-antigen polymerase. Finally, in studies of phage therapy in a broiler chicken model, no resistance to UAB_Phi78 was found in isolates from animals contaminated with S. Typhimurium and treated with the phage cocktail. However, 8.5 % of the isolates in the control group were resistant to the three phages, not having been determined the mechanisms of this resistance. In addition, 1.3 % and 3.3 % of the clones in the animals of the control and treated group, respectively, were resistant to bacteriophage UAB_Phi78, which exerted a bactericidal but not a bacteriolytic effect and did not result in the production of phage progeny, and additionally showed resistance to UAB_Phi87. The cause of this resistance was also unclear but the evidence suggested a mechanism based on an abortive infection. In conclusion, this study showed that both the frequency of the emergence of resistance and the mechanisms involved differ in vitro, in food, and in vivo. It, therefore, demonstrates the need for a broader approach to resistance studies and the use of cocktails containing bacteriophages that by recognizing different host receptors reduce the development of resistance. The biodistribution studies were performed by orally administering to mice the liposome-encapsulated phage UAB_Phi20 labeled with VTS-750 fluorochrome and then visualizing the phages by the IVIS Spectrum methodology. Using this method together with ex vivo cultures, we were able to show that encapsulation resulted in a significant accumulation of encapsulated phages in the mouse stomach, even 6 h after phage administration. Similarly, liposome-encapsulated phages were found in spleen, liver, and muscle. By contrast, in CLSM study of in vitro cultures of human intestinal cells (Caco-2/HT29/Raji-B), liposomes were seen adhered to the cell membrane or embedded in this. However, non-encapsulated bacteriophages were able to translocate across the intestinal barrier. Taken together, these results could explain the greater efficacy over time of oral phage therapy using liposome-encapsulated bacteriophages

CkP1 bacteriophage, a S16-like myovirus that recognizes Citrobacter koseri lipopolysaccharide through its long tail fibers

Citrobacter koseri is an emerging Gram-negative bacterial pathogen, which causes urinary tract infections. We isolated and characterized a novel S16-like myovirus CKP1 (vB_CkoM_CkP1), infecting C. koseri. CkP1 has a host range covering the whole C. koseri species, i.e., all strains that were tested, but does not infect other species. Its linear 168,463-bp genome contains 291 coding sequences, sharing sequence similarity with the Salmonella phage S16. Based on surface plasmon resonance and recombinant green florescence protein fusions, the tail fiber (gp267) was shown to decorate C. koseri cells, binding with a nanomolar affinity, without the need of accessory proteins. Both phage and the tail fiber specifically bind to bacterial cells by the lipopolysaccharide polymer. We further demonstrate that CkP1 is highly stable towards different environmental conditions of pH and temperatures and is able to control C. koseri cells in urine samples. Altogether, CkP1 features optimal in vitro characteristics to be used both as a control and detection agent towards drug-resistant C. koseri infections. • CkP1 infects all C. koseri strains tested • CkP1 recognizes C. koseri lipopolysaccharide through its long tail fiber • Both phage CkP1 and its tail fiber can be used to treat or detect C. koseri pathogens The online version contains supplementary material available at 10.1007/s00253-023-12547-8

Microencapsulation with alginate/CaCO 3 : A strategy for improved phage therapy

Altres ajuts: La Caixa and the Associació Catalana d'Universitats Públiques 2010ACUP00300Bacteriophages are promising therapeutic agents that can be applied to different stages of the commercial food chain. In this sense, bacteriophages can be orally administered to farm animals to protect them against intestinal pathogens. However, the low pH of the stomach, the activities of bile and intestinal tract enzymes limit the efficacy of the phages. This study demonstrates the utility of an alginate/CaCO encapsulation method suitable for bacteriophages with different morphologies and to yield encapsulation efficacies of ∼100%. For the first time, a cocktail of three alginate/CaCO-encapsulated bacteriophages was administered as oral therapy to commercial broilers infected with Salmonella under farm-like conditions. Encapsulation protects the bacteriophages against their destruction by the gastric juice. Phage release from capsules incubated in simulated intestinal fluid was also demonstrated, whereas encapsulation ensured sufficient intestinal retention of the phages. Moreover, the small size of the capsules (125-150 μm) enables their use in oral therapy and other applications in phage therapy. This study evidenced that a cocktail of the three alginate/CaCO-encapsulated bacteriophages had a greater and more durable efficacy than a cocktail of the corresponding non-encapsulated phages in as therapy in broilers against Salmonella, one of the most common foodborne pathogen

Biodistribution of liposome-encapsulated bacteriophages and their transcytosis during oral phage therapy

This study sheds light on the biodistribution of orally administered, liposome-encapsulated bacteriophages, and their transcytosis through intestinal cell layers. Fluorochrome-labeled bacteriophages were used together with a non-invasive imaging methodology in the in vivo visualization of bacteriophages in the stomach and intestinal tract of mice. In those studies, phage encapsulation resulted in a significant increase of the labeled phages in the mouse stomach, even 6 h after their oral administration, and without a decrease in their concentration. By contrast, the visualization of encapsulated and non-encapsulated phages in the intestine were similar. Our in vivo observations were corroborated by culture methods and ex vivo experiments, which also showed that the percentage of encapsulated phages in the stomach remained constant (50%) compared to the amount of initially administered product. However, the use of conventional microbiological methods, which employ bile salts to break down liposomes, prevented the detection of encapsulated phages in the intestine. The ex vivo data showed a higher concentration of non-encapsulated than encapsulated phages in liver, kidney, and even muscle up to 6 h post-administration. Encapsulated bacteriophages were able to reach the liver, spleen, and muscle, with values of 38% ± 6.3%, 68% ± 8.6%, and 47% ± 7.4%, respectively, which persisted over the course of the experiment. Confocal laser scanning microscopy of an in vitro co-culture of human Caco-2/HT29/Raji-B cells revealed that Vybrant-Dil-stained liposomes containing labeled bacteriophages were preferably embedded in cell membranes. No transcytosis of encapsulated phages was detected in this in vitro model, whereas SYBR-gold-labeled non-encapsulated bacteriophages were able to cross the membrane. Our work demonstrates the prolonged persistence of liposome-encapsulated phages in the stomach and their adherence to the intestinal membrane. These observations could explain the greater long-term efficacy of phage therapy using liposome-encapsulated phages

Global variation in postoperative mortality and complications after cancer surgery: a multicentre, prospective cohort study in 82 countries

© 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 licenseBackground: 80% of individuals with cancer will require a surgical procedure, yet little comparative data exist on early outcomes in low-income and middle-income countries (LMICs). We compared postoperative outcomes in breast, colorectal, and gastric cancer surgery in hospitals worldwide, focusing on the effect of disease stage and complications on postoperative mortality. Methods: This was a multicentre, international prospective cohort study of consecutive adult patients undergoing surgery for primary breast, colorectal, or gastric cancer requiring a skin incision done under general or neuraxial anaesthesia. The primary outcome was death or major complication within 30 days of surgery. Multilevel logistic regression determined relationships within three-level nested models of patients within hospitals and countries. Hospital-level infrastructure effects were explored with three-way mediation analyses. This study was registered with ClinicalTrials.gov, NCT03471494. Findings: Between April 1, 2018, and Jan 31, 2019, we enrolled 15 958 patients from 428 hospitals in 82 countries (high income 9106 patients, 31 countries; upper-middle income 2721 patients, 23 countries; or lower-middle income 4131 patients, 28 countries). Patients in LMICs presented with more advanced disease compared with patients in high-income countries. 30-day mortality was higher for gastric cancer in low-income or lower-middle-income countries (adjusted odds ratio 3·72, 95% CI 1·70–8·16) and for colorectal cancer in low-income or lower-middle-income countries (4·59, 2·39–8·80) and upper-middle-income countries (2·06, 1·11–3·83). No difference in 30-day mortality was seen in breast cancer. The proportion of patients who died after a major complication was greatest in low-income or lower-middle-income countries (6·15, 3·26–11·59) and upper-middle-income countries (3·89, 2·08–7·29). Postoperative death after complications was partly explained by patient factors (60%) and partly by hospital or country (40%). The absence of consistently available postoperative care facilities was associated with seven to 10 more deaths per 100 major complications in LMICs. Cancer stage alone explained little of the early variation in mortality or postoperative complications. Interpretation: Higher levels of mortality after cancer surgery in LMICs was not fully explained by later presentation of disease. The capacity to rescue patients from surgical complications is a tangible opportunity for meaningful intervention. Early death after cancer surgery might be reduced by policies focusing on strengthening perioperative care systems to detect and intervene in common complications. Funding: National Institute for Health Research Global Health Research Unit