Viral Evolution and Immune Responses

Antiviral responses are activated rapidly after viral infection in order to control and prevent dissemination of the virus. Different pathways are activated in the immune system, including innate and adaptive responses. On the other hand, viruses have evolved specifi c strategies to evade these responses. Due to the high viral evolutionary rates, escape variants can emerge and spread fast in the population. The co-evolution between viruses and their host is a constant arms race, and is of special interest to understand the viral escape mechanisms that may guide the future development of antiviral treatments and vaccines

Virus-Host Coevolution as a Tool for Controlling Bacterial Resistance to Phage Therapy

Bacterial resistance to antibiotics is a global public health concern. New treatments are needed to combat resistant strains, among which phage therapy is a promising option. Probably the main advantages of phage therapy are its high specificity as well as rapid viral adaptability, which in principle allows using phage evolution to overcome resistance. Here, we have performed serial coevolution passages between Escherichia coli and its phage T7 to investigate the ability of coevolved phages to reduce the emergence of resistances. We find that the initial bacterial population is less likely to undergo resistance when challenged with experimentally coevolved phages than when challenged with the wild-type phage. Hence, our findings suggest that coevolved phage preparations could be used to increase the efficacy of phage therapy

Efectos mutacionales, evolución y adaptación en bacteriófagos de RNA y DNA de cadena sencilla

Las dificultades que plantean los virus para su control se deben, en gran medida, a su rápida evolución. Factores responsables de esta rápida evolución son su rápida tasa de replicación, sus elevados tamaños poblacionales y su elevada variabilidad genética. Estos dos últimos factores aumentan la efectividad de la selección natural, permitiendo a los virus escapar no sólo a las defensas del hospedador, sino también a los fármacos antivirales o de las vacunas. La mutación es la fuente última de variación genética. Por ello, la tasa de mutación es un parámetro fundamental para entender la evolución. No obstante, la relación entre tasa de mutación y adaptación no es inmediata. Por un lado, la producción de variación genética es indispensable para la adaptación, pero por otro lado, las mutaciones son más a menudo deletéreas que beneficiosas, de modo que, en promedio, tienden a reducir la eficacia biológica de las poblaciones (carga genética. La teoría predice que, como consecuencia del balance entre adaptación y carga genética, debe existir un valor intermedio para la tasa de mutación que maximiza el éxito evolutivo. Los virus de ARN presentan las mayores tasas de mutación por sitio nucleotídico descritas, órdenes de magnitud superiores a las de los virus de ADN y los demás genomas de ADN. Aún así, las diferencias en tasas de evolución en la naturaleza son a veces similares entre virus de ARN y ADN, y algunas propiedades de los genomas virales pueden ser debidas no a su material genético, sino al hecho de presentar pequeños genomas, por los que virus de ARN y ADN de cadena sencilla pueden compartir características, y en este caso, la evolución experimental es una herramienta muy útil para poder comparar directamente distintas especies virales. El objetivo central de la tesis es comparar las propiedades evolutivas de virus de ARN y ADN de cadena sencilla. Para ello se han escogido seis especies virales, tres de ARN (Qβ, MS2 y SP), y tres de ADN (ФX174, G4 y f1), todos ellos de cadena sencilla; con el fin de determinar si los efectos mutacionales difieren entre estos dos tipos de virus, si los virus de ARN se adaptan más rápido que los virus de ADN, si las tasas de mutación virales son óptimas para la adaptación, y qué ocurre cuando se aumenta artificialmente la tasa de mutación de virus de ARN y ADN de cadena sencilla.133 p

Vigilancia de COVID 19 en aguas residuales

Utilidad de las aguas residuales como herramienta de vigilancia • Seguimiento temporal • Detección temprana? • Nuevos brotes? • Transmisión comunitaria? • ¿Grandes ciudades? ¿Pueblos? ¿Edificios? • ¿Residencias de ancianos o de estudiantes, colegios, prisiones? ¿Hoteles, cruceros? • Correlación con casos clínicos • Modelización • Predicción • Países no desarrollados • Falta de red de saneamiento • Puesta a punto de metología para futuras pandemiasS

Bacteriófagos para el control de la bacteria fitopatógena Xylella fastidiosa

Xylella fastidiosa (Xf) es una bacteria patógena que habita en el xilema de la planta hospedadora y el tracto digestivo de insectos que actúan como vectores. Afecta a un elevado número de especies vegetales, siendo una de las principales bacterias de cuarentena en la Unión Europea. Puesto que actualmente no existen medidas terapéuticas eficaces y los antibióticos están prohibidos en Europa, el control de Xf se basa principalmente en la erradicación de las plantas infectadas y el uso de compuestos químicos contra los vectores, pero estas medidas no son suficientes

Inference of the life cycle of environmental phages from genomic signature distances to their hosts

The environmental impact of uncultured phages is shaped by their preferred life cycle (lytic or lysogenic). However, our ability to predict it is very limited. We aimed to discriminate between lytic and lysogenic phages by comparing the similarity of their genomic signatures to those of their hosts, reflecting their co-evolution. We tested two approaches: (1) similarities of tetramer relative frequencies, (2) alignment-free comparisons based on exact k = 14 oligonucleotide matches. First, we explored 5126 reference bacterial host strains and 284 associated phages and found an approximate threshold for distinguishing lysogenic and lytic phages using both oligonucleotide-based methods. The analysis of 6482 plasmids revealed the potential for horizontal gene transfer between different host genera and, in some cases, distant bacterial taxa. Subsequently, we experimentally analyzed combinations of 138 Klebsiella pneumoniae strains and their 41 phages and found that the phages with the largest number of interactions with these strains in the laboratory had the shortest genomic distances to K. pneumoniae. We then applied our methods to 24 single-cells from a hot spring biofilm containing 41 uncultured phage-host pairs, and the results were compatible with the lysogenic life cycle of phages detected in this environment. In conclusion, oligonucleotide-based genome analysis methods can be used for predictions of (1) life cycles of environmental phages, (2) phages with the broadest host range in culture collections, and (3) potential horizontal gene transfer by plasmids

Essential topics for the regulatory consideration of phages as clinically valuable therapeutic agents: A perspective from Spain

This article belongs to the Special Issue Bacteriophages-Based Technologies for a One Health Society: Applications in Clinical, Veterinary, and Industrial Settings.Antibiotic resistance is one of the major challenges that humankind shall face in the short term. (Bacterio)phage therapy is a valuable therapeutic alternative to antibiotics and, although the concept is almost as old as the discovery of phages, its wide application was hindered in the West by the discovery and development of antibiotics in the mid-twentieth century. However, research on phage therapy is currently experiencing a renaissance due to the antimicrobial resistance problem. Some countries are already adopting new ad hoc regulations to favor the short-term implantation of phage therapy in clinical practice. In this regard, the Phage Therapy Work Group from FAGOMA (Spanish Network of Bacteriophages and Transducing Elements) recently contacted the Spanish Drugs and Medical Devices Agency (AEMPS) to promote the regulation of phage therapy in Spain. As a result, FAGOMA was asked to provide a general view on key issues regarding phage therapy legislation. This review comes as the culmination of the FAGOMA initiative and aims at appropriately informing the regulatory debate on phage therapy.This work was supported by Grant RED2018-102589-T funded by MCIN/AEI/10.13039/501100011033. R.V. and D.G. were supported by the Research Foundation–Flanders (FWO) under grant [G066919N]. Pe. G. and R.V. were supported by the Spanish Ministry of Science and Innovation (Grant number SAF2017-88664-R), with additional funding provided by the Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), an initiative of the Instituto de Salud Carlos III. P.D.-C. was financially supported by a Ramón y Cajal contract RYC2019-028015-I and project PID2020-112835RA-I00 funded by MCIN/AEI/10.13039/501100011033, ESF Invest in your future, project SEJIGENT/2021/014, funded by Conselleria d’Innovació, Universitats, Ciència i Societat Digital (Generalitat Valenciana), and project AP2020-27 funded by VLC-Biomed (UV-La Fe 2020 Programa Acciones Preparatorias). M.M. was supported by project PID2020-113355GB-I00 of Spanish Ministry of Science and Innovation /Agencia Estatal de Investigación (AEI)/European regional fund (ERF). M.Á.T.-M. was supported by Spanish Ministry of Science and Innovation (Grant number SAF2017- 82251-R).Peer reviewe

Phenotypic and Genomic Comparison of Klebsiella pneumoniae Lytic Phages: vB_KpnM-VAC66 and vB_KpnM-VAC13

[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.This study was funded by grants PI19/00878 awarded to M. Tomás within the State Plan for R+D+I 2013-2016 (National Plan for Scientific Research, Technological Development and Innovation 2008–2011) and co-financed by the ISCIII-Deputy General Directorate for Evaluation and Promotion of Research—European Regional Development Fund “A way of Making Europe” and Instituto de Salud Carlos III FEDER, Spanish Network for the Research in Infectious Diseases (REIPI, RD16/0016/0001, RD16/0016/0006 and RD16/CIII/0004/0002) and by the Study Group on Mechanisms of Action and Resistance to Antimicrobials, GEMARA (SEIMC, http://www.seimc.org/ accessed on 1 February 2021) and project PID2020-112835RA-I00 funded by MCIN/AEI /10.13039/501100011033, and project SEJIGENT/2021/014 funded by Conselleria d’Innovació, Universitats, Ciència i Societat Digital (Generalitat Valenciana). M. Tomás was financially supported by the Miguel Servet Research Programme (SERGAS and ISCIII). O. Pacios, L. Fernández-García and M. López were financially supported by the grants IN606A-2020/035, IN606B-2021/013 and IN606B-2018/008, respectively (GAIN, Xunta de Galicia). I. Bleriot was financially supported by pFIS program (ISCIII, FI20/00302). P. Domingo-Calap was financially supported by a Ramón y Cajal contract RYC2019-028015-I funded by MCIN/AEI /10.13039/501100011033, ESF Invest in your futureGeneralitat Valenciana; SEJIGENT/2021/014Xunta de Galicia; IN606A-2020/035Xunta de Galicia; IN606B-2021/013Xunta de Galicia; IN606B-2018/00

The Role of PemIK (PemK/PemI) Type II TA System from Klebsiella pneumoniae Clinical Strains in Lytic Phage Infection

[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.This study was funded by grant PI19/00878 awarded to M. Tomás within the State Plan for R+D+I 2013-2016 (National Plan for Scientific Research, Technological Development and Innovation 2008-2011) and co-financed by the ISCIII-Deputy General Directorate for Evaluation and Promotion of Research—European Regional Development Fund "A way of Making Europe" and Instituto de Salud Carlos III FEDER, Spanish Network for the Research in Infectious Diseases (REIPI, RD16/0016/0001, RD16/0016/0006 and RD16/CIII/0004/0002) and by the Study Group on Mechanisms of Action and Resistance to Antimicrobials, GEMARA (SEIMC, http://www.seimc.org/). M. Tomás was financially supported by the Miguel Servet Research Programme (SERGAS and ISCIII). I. Bleriot was financially supported by pFIS program (ISCIII, FI20/00302). O. Pacios and M. López was financially supported by a grant IN606A-2020/035 and IN606B-2018/008, respectively (GAIN, Xunta de Galicia) and M. Gonzalez-Bardanca was financially supported by the Rio Hortega program (ISCIII, CM20/00198)Xunta de Galicia; IN606A-2020/035Xunta de Galicia; IN606B-2018/00

Enhanced antibacterial activity of repurposed mitomycin C and imipenem in combination with the lytic phage vB_KpnM-VAC13 against clinical isolates of Klebsiella pneumoniae

Study Group on Mechanisms of Action and Resistance to Antimicrobials (GEMARA) on behalf of the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC).Klebsiella pneumoniae is an opportunistic Gram-negative pathogen that employs different strategies (resistance and persistence) to counteract antibiotic treatments. This study aimed to search for new means of combatting imipenem-resistant and persister strains of K. pneumoniae by repurposing the anticancer drug mitomycin C as an antimicrobial agent and by combining the drug and the conventional antibiotic imipenem with the lytic phage vB_KpnM-VAC13. Several clinical K. pneumoniae isolates were characterized, and an imipenem-resistant isolate (harboring OXA-245 β-lactamase) and a persister isolate were selected for study. The mitomycin C and imipenem MICs for both isolates were determined by the broth microdilution method. Time-kill curve data were obtained by optical density at 600 nm (OD600) measurement and CFU enumeration in the presence of each drug alone and with the phage. The frequency of occurrence of mutants resistant to each drug and the combinations was also calculated, and the efficacy of the combination treatments was evaluated using an in vivo infection model (Galleria mellonella). The lytic phage vB_KpnM-VAC13 and mitomycin C had synergistic effects on imipenem-resistant and persister isolates, both in vitro and in vivo. The phage-imipenem combination successfully killed the persisters but not the imipenem-resistant isolate harboring OXA-245 β-lactamase. Interestingly, the combinations decreased the emergence of in vitro resistant mutants of both isolates. Combinations of the lytic phage vB_KpnM-VAC13 with mitomycin C and imipenem were effective against the persister K. pneumoniae isolate. The lytic phage-mitomycin C combination was also effective against imipenem-resistant K. pneumoniae strains harboring OXA-245 β-lactamase.This study was funded by grants PI16/01163 and PI19/00878 awarded to M. Tomás within the State Plan for R+D+I 2013–2016 (National Plan for Scientific Research, Technological Development and Innovation 2008–2011) and cofinanced by the ISCIII-Deputy General Directorate for Evaluation and Promotion of Research–European Regional Development Fund and Instituto de Salud Carlos III FEDER, Spanish Network for the Research in Infectious Diseases (REIPI; RD16/0016/0001, RD16/0016/0006, and RD16/CIII/0004/0002) and by the Study Group on Mechanisms of Action and Resistance to Antimicrobials, GEMARA (SEIMC; http://www.seimc.org/). M. Tomás was financially supported by the Miguel Servet Research Program (SERGAS and ISCIII). I. Bleriot was financially supported by pFIS program (ISCIII, FI20/00302). O. Pacios and M. López were financially supported by grants IN606A-2020/035 and IN606B-2018/008, respectively (GAIN, Xunta de Galicia), and P. Domingo-Calap was financially supported by the ESCMID Research Grant 20200063.Peer reviewe