Study of the Lysogenic Phages and Their Potential Applications in Clinical Strains of Multi-Drug Resistant Bacteria

Programa Oficial de Doutoramento en Bioloxía Celular e Molecular . 5004V01[Abstract] Bacteria with multiple resistance entail a global threat. In recent years, phage therapy has been widely reconsidered as an alternative to antibiotics. In particular, lytic phages have been shown to have great potential for treating infections with multi-resistant bacteria. In this thesis, we present the utility and study of lysogenic phages in clinical strains of multi-resistant bacteria. Concerning the potential of lysogenic phages in phage therapy, we have developed the strategy of transforming a lysogenic phage into a lytic one, Ab105-2φΔCI, and characterized its microbial activity. We also have purified and assayed the antimicrobial activity of two endolysins ElyA1 and ElyA2, from two prophages of a clinical strain of A. baumannii. Both alternatives have been shown to be effective in combination with antibiotics. In relation to the study of lysogenic phages in clinical strains, we pointed out the problem of the possible appearance of bacterial resistance against phages and the importance of searching and characterizing these resistance systems by searching in silico for phage resistance mechanisms in clinical strains of A. baumannii. We also identified 4 complete prophages in clinical strains of P. aeruginosa, 2 of them were newly identified: a Siphovirus phage, AUS531phi, and a filamentous Inovirus phage, pf8. Furthermore, we characterized a gene that increases the ability of one of them, the bci gene in AUS531phi, to infect the bacteria by the regulation of the Quorum system.[Resumo] As bacterias con múltiples resistencias supoñen unha ameaza a nivel global. Nos últimos anos, a terapia de fagos reconsiderouse amplamente como unha alternativa aos antibióticos. Especialmente, demostrouse que os fagos líticos teñen un gran potencial para tratar infeccións con bacterias multirresistentes. Nesta tese presentamos a utilidade e o estudo dos fagos lisoxénicos en cepas clínicas de bacterias con múltiple resistencia aos antibióticos. En relación ao potencial dos fagos lisoxénicos en terapia de fagos, desenvolvemos a estratexia de converter un fago lisoxénico nun lítico, Ab105-2φΔCI, e caracterizar a súa actividade microbiana. Tamén purificamos e caracterizamos a actividade microbiana de dúas endolisinas, ElyA1 e ElyA2 de dous profagos dunha cepa clínica de A. baumannii. Ambas alternativas demostraron ser efectivas en combinación con antibióticos. En relación ao estudo dos fagos lisoxénicos en cepas clínicas, sinalamos a problemática da posible aparición de resistencia contra fagos e a importancia de buscalas e caracterizalas buscando in silico mecanismos de resistencia contra fagos en cepas clínicas de A. baumannii. Tamén localizamos 4 profagos completos en cepas clínicas de P. aeruginosa, 2 deles foron novamente identificados: un fago do tipo Siphovirus, AUS531phi, e outro filamentoso do tipo Inovirus, pf8. Ademais, caracterizamos un xene que incrementa a habilidade dun deles, o xene bci en AUS531phi, para infectar a bacteria mediante a regulación do Quorum Sensing.[Resumen] Las bacterias con múltiples resistencias suponen una amenaza a nivel global. En los últimos años, la terapia de fagos se ha reconsiderado ampliamente como una alternativa a los antibióticos. Especialmente, se ha demostrado que los fagos líticos poseen un gran potencial para tratar infecciones con bacterias multirresistentes. En esta tesis presentamos la utilidad y estudio de los fagos lisogénicos en cepas clínicas de bacterias multirresistentes. En relación al potencial de los fagos lisogénicos en terapia de fagos hemos desarrollado la estrategia de convertir un fago lisogénico en uno lítico, Ab105-2φΔCI, y caracterizar su actividad microbiana. También purificamos y caracterizamos la actividad microbiana de dos endolisinas, ElyA1 y ElyA2, de dos profagos de una cepa clínica de A. baumannii. Ambas alternativas han demostrado ser efectivas en combinación con antibióticos. En relación al estudio de fagos lisogénicos en cepas clínicas, hemos señalado la problemática de la posible aparición de resistencia contra fagos y la importancia de buscarlas y caracterizarlas buscando in silico mecanismos de resistencia contra fagos en cepas clínicas de A. baumannii. También localizamos 4 profagos completos en cepas clínicas de P. aeruginosa, 2 de ellos fueron nuevamente identificados: un fago del tipo Siphovirus, AUS531phi, y otro filamentoso del tipo Inovirus, pf8. Además, también caracterizamos un gen que incrementa la habilidad de uno de ellos, el gen bci en AUS531phi, para infectar a la bacteria mediante la regulación del Quorum Sensing

The role of PemIK (PemK/PemI) type II TA system from Klebsiella pneumoniae clinical strains in lytic phage infection

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

Genomic Analysis of Molecular Bacterial Mechanisms of Resistance to Phage Infection

[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.This study was funded by grants PI16/01163 and PI19/00878 awarded to MT 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 of evaluation and Promotion of Research-European Regional Development Fund “A way of Making Europe” and Instituto de Salud Carlos III FEDER. MT was financially supported by the Miguel Servet Research Programme (SERGAS and ISCIII

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

Adaptation of clinical isolates of Klebsiella pneumoniae to the combination of niclosamide with the efflux pump inhibitor phenyl-arginine-β-naphthylamide (PaβN): co-resistance to antimicrobials

Objectives: To search for new means of combatting carbapenemase-producing strains of Klebsiella pneumoniae by repurposing the anti-helminth drug niclosamide as an antimicrobial agent and combining it with the efflux pump inhibitor (EPI) phenyl-arginine-β-naphthylamide (PaβN). Methods: Niclosamide and PaβN MICs were determined for six clinical K. pneumoniae isolates harbouring different carbapenemases by broth microdilution and chequerboard assays. Time-kill curves in the presence of each drug alone and in combination were conducted. The viability of bacterial cells in the presence of repetitive exposures at 8 h to the treatment at the same concentration of niclosamide and/or PaβN (adapted isolates) was determined. The acrAB-tolC genes and their regulators were sequenced and quantitative RT-PCR was performed to assess whether the acrA gene was overexpressed in adapted isolates compared with non-adapted isolates. Finally, the MICs of several antimicrobials were determined for the adapted isolates. Results: Niclosamide and PaβN had synergistic effects on the six isolates in vitro, but adaptation appeared when the treatment was applied to the medium every 8 h, with an increase of 6- to 12-fold in the MIC of PaβN. Sequencing revealed different mutations in the regulators of the tripartite AcrAB-TolC efflux pump (ramR and acrR) that may be responsible for the overexpression of the efflux pump and the adaptation to this combination. Co-resistance to different antimicrobials confirmed the overexpression of the AcrAB-TolC efflux pump. Conclusions: Despite the synergistic effect that preliminary in vitro stages may suggest, the combinations of drugs and EPI may generate adapted phenotypes associated with antimicrobial resistance that must be taken into consideration

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

Combined Use of the Ab105-2φΔCI Lytic Mutant Phage and Different Antibiotics in Clinical Isolates of Multi-Resistant Acinetobacter baumannii

Phage therapy is an abandoned antimicrobial therapy that has been resumed in recent years. In this study, we mutated a lysogenic phage from Acinetobacter baumannii into a lytic phage (Ab105-2phi∆CI) that displayed antimicrobial activity against A. baumannii clinical strain Ab177_GEIH-2000 (isolated in the GEIH-REIPI Spanish Multicenter A. baumannii Study II 2000/2010, Umbrella Genbank Bioproject PRJNA422585, and for which meropenem and imipenem MICs of respectively, 32 μg/mL, and 16 μg/mL were obtained). We observed an in vitro synergistic antimicrobial effect (reduction of 4 log–7 log CFU/mL) between meropenem and the lytic phage in all combinations analyzed (Ab105-2phi∆CI mutant at 0.1, 1 and 10 MOI and meropenem at 1/4 and 1/8 MIC). Moreover, bacterial growth was reduced by 8 log CFU/mL for the combination of imipenem at 1/4 MIC plus lytic phage (Ab105-2phi∆CI mutant) and by 4 log CFU/mL for the combination of imipenem at 1/8 MIC plus lytic phage (Ab105-2phi∆CI mutant) at both MOI 1 and 10. These results were confirmed in an in vivo model (G. mellonella), and the combination of imipenem and mutant Ab105-2phi∆CI was most effective (p < 0.05). This approach could help to reduce the emergence of phage resistant bacteria and restore sensitivity to antibiotics used to combat multi-resistant strains of Acinetobacter baumannii

Viral Related Tools against SARS-CoV-2

At the end of 2019, a new disease appeared and spread all over the world, the COVID-19, produced by the coronavirus SARS-CoV-2. As a consequence of this worldwide health crisis, the scientific community began to redirect their knowledge and resources to fight against it. Here we summarize the recent research on viruses employed as therapy and diagnostic of COVID-19: (i) viral-vector vaccines both in clinical trials and pre-clinical phases; (ii) the use of bacteriophages to find antibodies specific to this virus and some studies of how to use the bacteriophages themselves as a treatment against viral diseases; and finally, (iii) the use of CRISPR-Cas technology both to obtain a fast precise diagnose of the patient and also the possible use of this technology as a cure

Genomic analysis of 40 prophages located in the genomes of 16 carbapenemase-producing clinical strains of Klebsiella pneumoniae

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 function, mainly involving viral structure, transcription, replication and regulation (lysogenic/lysis). Interestingly, some proteins had putative functions associated with bacterial virulence (toxin expression and efflux pump regulators), phage defence profiles such as toxin-antitoxin modules, an anti-CRISPR/Cas9 protein, TerB protein (from terZABCDE operon) and methyltransferase proteins

Relationship between Tolerance and Persistence Mechanisms in Acinetobacter baumannii Strains with AbkAB Toxin-Antitoxin System

The molecular mechanisms of tolerance and persistence associated with several compounds in Acinetobacter baumannii clinical isolates are unknown. Using transcriptomic and phenotypic studies, we found a link between mechanisms of bacterial tolerance to chlorhexidine and the development of persistence in the presence of imipenem in an A. baumannii strain belonging to clinical clone ST-2 (OXA-24 β-lactamase and AbkAB toxin-antitoxin [TA] system carried in a plasmid). Interestingly, the strain A. baumannii ATCC 17978 (AbkAB TA system from plasmid) showed persistence in the presence of imipenem and chlorhexidine.This study was funded by grants PI13/02390 and PI16/01163, awarded to M.T., and by grant PI11-02046, awarded to F.F.-C., 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 of Evaluation and Promotion of Research—European Regional Development Fund “A Way of Making Europe” and the Instituto de Salud Carlos III FEDER, Spanish Network for the Research in Infectious Diseases (REIPI, grants RD12/0015/0010 and RD16/0016/0001), as well as by the Study Group on Mechanisms of Action and Resistance to Antimicrobials, GEMARA (SEIMC). M.T. was financially supported by the Miguel Servet Research Programme (SERGAS and ISCIII). L.F.-G. was financially supported by a predoctoral fellowship from the Xunta de Galicia (GAIN, Axencia de Innovación).Peer reviewe