Proteomic Study of the Interactions between Phages and the Bacterial Host Klebsiella Pneumoniae

Proteomic Study of the Interactions between Phages and the Bacterial Host Klebsiella pneumoniae Inés Bleriot,aLucia Blasco,aOlga Pacios,aLaura Fernández-García,aMaría López,aConcha Ortiz-Cartagena,a Antonio Barrio-Pujante,aFelipe Fernández-Cuenca,b,e Álvaro Pascual,b,e Luis Martínez-Martínez,c,e Jesús Oteo-Iglesias,d,e María Tomás, a on behalf of the GEMARA (SEIMC) a Microbiology Translational and Multidisciplinary (MicroTM)-Research Institute Biomedical A Coruña (INIBIC) and Microbiology Department of Hospital A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, Spain b Clinical Unit of Infectious Diseases and Microbiology, Hospital Universitario Virgen Macarena, Institute of Biomedicine of Seville (University Hospital Virgen Macarena/ CSIC/University of Seville), Seville, Spain c Clinical Unit of Microbiology, Reina Sofía University Hospital, Department of Agricultural Chemistry, Edaphology and Microbiology, University of Cordoba, Maimonides Biomedical Research Institute (IMIBIC), Cordoba, Spain d Reference and Research Laboratory for Antibiotic Resistance and Health Care Infections, National Centre for Microbiology, Institute of Health Carlos III, Majadahonda, Madrid, Spain e CIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain ABSTRACT 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. IMPORTANCE Researchers, including microbiologists and infectious disease specialists, require more knowledge about the interactions between phages and their bacterial hosts and about their defense mechanisms. In this study, we analyzed the molecular mechanisms of viral and bacterial defense in phages infecting clinical isolates of K. pneumoniae. Viral defense mechanisms included restriction-modification system evasion, the toxin-antitoxin (TA) system, DNA degradation evasion, blocking of host restriction and modification, and resistance to the abortive infection system, anti-CRISPR and CRISPR-Cas systems. Regarding bacterial defense mechanisms, proteomic analysis revealed expression of proteins involved in the prophage (FtsH protease modulator), plasmid (cupin phosphomannose isomerase protein), defense/virulence/resistance (porins, efflux pumps, lipopolysaccharide, pilus elements, quorum network proteins, TA systems, and methyltransferases), oxidative stress mechanisms, and Acr candidates (anti-CRISPR protein). The findings reveal Editor JJ Miranda, Barnard College, Columbia University Copyright © 2023 Bleriot et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to María Tomás, [email protected]. The authors declare no conflict of interest. Received 3 October 2022 Accepted 8 February 2023 Published 6 March 2023 March/April 2023 Volume 11 Issue 2 10.1128/spectrum.03974-22 1 RESEARCH ARTICLE Downloaded from https://journals.asm.org/journal/spectrum on 05 February 2025 by 150.214.182.235. some important molecular mechanisms involved in the phage-host bacterial interactions; however, further study in this field is required to improve the efficacy of phage therapy. KEYWORDS Klebsiella pneumoniae, lytic phage, phage-host interaction, defense mechanism, prophage, plasmid, Klebsiella, bacteriophage evolution, bacteriophage, virus-host interactions Bacteriophages, or phages, are natural predators of bacteria. Phages are the most abundant and ubiquitous biological entities on Earth, accounting for an estimated total of 10 31 viral particles (1, 2). In the current context of increasing antibiotic resistance, the emergence of alternative therapies is welcome. Thus, lytic phages are currently considered one of the best options for treating infections caused by multidrug-resistant (MDR) bacteria (3, 4), as demonstrated in clinical trials conducted to date (5–10). In general, phage therapy has some advantages over the use of conventional antibiotics, such as low toxicity and high host specificity (3, 11). Another characteristic of phages that makes them good candidates for therapy is their ability to adapt to changes in the bacterial host, which has resulted from the coevolution of both types of organisms (12). Bacteria have developed mechanisms to prevent phage infection at almost all stages of the viral replication cycle (13, 14). First, bacteria can prevent phage attachment by mutating or altering their surface receptors, by producing inhibitors that outcompete the phage for receptors, or by producing polysaccharides that physically mask phage receptors (13). In order to prevent injection of phage DNA into the cytoplasm, the bacteria then use the superinfection exclusion system, which is characterized by proteins that block the entry of phage DNA into host cells (15). If the phage nevertheless manages to enter the cell, the bacteria employ other defense systems such as the abortive infection system (Abi) to interrupt phage development at any stage (replication, transcription, or translation) (16), the bacteriophage exclusion system (17) or the defense island system associated with restriction-modification (18) to interrupt replication. Bacteria can also use their toxin-antitoxin (TA) systems, which often lead to reduced bacterial metabolism and phage inhibition (19–21), when the antitoxin is degraded under stress conditions by the protease system (22). Bacteria can also employ other types of systems to cleave phage DNA, such as the restriction-modification (RM) system and a cognate DNA methylase, which modifies and protects the host DNA (23). They can also employ the clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins (CRISPR-Cas) system, an adaptive immune system (24) characterized by the acquisition of spacer sequences, which are small fragments of foreign nucleic acids of phage or foreign DNA, between the repeats of the CRISPR locus (25). Phages have, in turn, developed counterstrategies to evade bacterial defense mechanisms (26). For example, for successful adsorption, phages can modify their receptorbinding proteins by acquiring mutations to obtain new receptors (27). In turn, they are also able to acquire enzymes such as depolymerase to access masked receptors (28, 29) in a way that allows them to interact with a surface component expressed by the host at that time (30). However, when a phage genome manages to enter the cell, it can still face the myriad intracellular antiviral barriers described above. Phages can respond to these by promoting the mutation of specific genes to prevent activation of the bacterial Abi system (31). They can also evade bacterial RM systems by reducing the number of restriction sites in their genome (32), modifying bases in their genome (33), coinjecting protein (for instance, DarA and DarB in the phage P1) with the genome to bind directly to the phage DNA and mask restriction sites (34), stimulating the action of modification enzymes and degrading an RM cofactor. Phages can also sequester host antitoxins via a protein that probably inhibits Lon protease activity to avoid the deleterious action of the toxins of the TA systems (35). To circumvent the effect of bacterial TA systems, one phage, T4, encodes its own antitoxin protein (Dmd) that functionally replaces the unstable antitoxin of the host, thereby promoting phage propagation (36). Finally, Molecular Response to Phages in the Host Klebsiella Microbiology Spectrum March/April 2023 Volume 11 Issue 2 10.1128/spectrum.03974-22 2 Downloaded from https://journals.asm.org/journal/spectrum on 05 February 2025 by 150.214.182.235. phages have developed mechanisms to evade the bacterial CRISPR-Cas system. For instance, through a single-nucleotide substitution or a complete deletion in the protospacer region or in the conserved protospacer-adjacent motif (37). Phages have also developed anti-CRISPR systems, which basically consist of Acr proteins (typically small proteins of 80 to 150 amino acids) that inhibit bacterial CRISPR-Cas activity by binding directly to, and thus inactivating, the Cas protein, so that phages can successfully replicate in the bacterial host (38). A better understanding of phage-host interaction could lead to the development of more successful therapeutic applications for phages. In this context, the aims of the present study were to analyze the proteins isolated from 21 novel lytic phages of K. pneumoniae in search of defense mechanisms against bacteria and also to determine their infective capacity. In addition, the other aim of this work was to investigate the defense mechanisms of bacteria in response to phage infection. RESULTS Isolation, propagation, and electron microscopic analysis of phages. The 21 phages examined in this study, named according to accepted practices (39) (Table 1), were obtained from wastewater samples. However, to facilitate reading this article, we will incorporate the data previously found in these studies in order to enable comparison between the phages. Thus, transmission electron microscopy (TEM) studies revealed, according to the morphology, that 15 phages have long and flexible tails (vB_KpnS-VAC2, vB_KpnS-VAC4, vB_KpnS-VAC5, vB_KpnS-VAC6, vB_KpnS-VAC7, vB_KpnS-VAC8, vB_KpnS- VAC10, and vB_KpnS-VAC11 [20]; and vB_KpnS-VAC35, vB_KpnS-VAC51, vB_KpnS-VAC70, vB_KpnS-VAC110, vB_KpnS-VAC111, vB_KpnS-VAC112, and vB_KpnS-VAC113). Moreover, three (vB_KpnM-VAC13 [40], vB_KpnM-VAC36, and vB_KpnM-VAC66 [41]) present icosahedral capsid and a rigid, contractile tails. Finally, three phages have small and noncontractile tails (vB_KpnP-VAC1 [20], vB_KpnP-VAC25, and vB_KpnP-VAC71) (Fig. 1B). Phage genome annotation. (i) Phage genome analysis. The phage genome sequencing revealed that all phages under study, available from the GenBank BioProject PRJNA739095 (Table 1), were lytic Caudovirales phages, i.e., dsDNA tailed phages, lacking lysogenic genes such as integrase, recombinase, and excisionase. More specifically, TABLE 1 Characteristics of 21 K. pneumoniae lytic phages a Phage Natural host GenBank accession no. Family Genus Genome size (bp) %G+C Source or referenceBiosample Nucleotide vB_KpnP-VAC1 ATCC 10031 SAMN19773206 MZ428229.1 Autographiviridae Teetrevirus 39,371 50.75 20 vB_KpnS-VAC2 ATCC 10031 SAMN19773207 MZ428221.1 Drexlerviridae Webervirus 51,784 47.86 20 vB_KpnS-VAC4 ATCC 10031 SAMN19773215 MZ428222.1 Drexlerviridae Webervirus 45,558 51.11 20 vB_KpnS-VAC5 ATCC 10031 SAMN19773216 MZ428223.1 Drexlerviridae Webervirus 49,636 50.46 20 vB_KpnS-VAC6 ATCC 10031 SAMN19773219 MZ428224.1 Drexlerviridae Webervirus 51,554 51.63 20 vB_KpnS-VAC7 ATCC 10031 SAMN19773224 MZ428225.1 Drexlerviridae Webervirus 49,684 51.25 20 vB_KpnS-VAC8 ATCC 10031 SAMN19773221 MZ428226.1 Drexlerviridae Webervirus 48,933 50.52 20 vB_KpnS-VAC10 ATCC 10031 SAMN19773232 MZ428227.1 Drexlerviridae Webervirus 48,935 50.65 20 vB_KpnS-VAC11 ATCC 10031 SAMN19773540 MZ428228.1 Drexlerviridae Webervirus 48,826 50.83 20 vB_KpnM-VAC13 ATCC 10031 SAMN22059222 MZ322895.1 Myoviridae Slopekvirus 174,826 41.93 40 vB_KpnP-VAC25 K3579 SAMN20298869 MZ571827.1 Autographiviridae Drulisivirus 43,777 53.76 This study vB_KpnS-VAC35 K3574 SAMN20298871 MZ571828.1 Demerecviridae Sugarlandvirus 112,862 45.44 This study vB_KpnM-VAC36 K3573 SAMN20298872 MZ571829.1 Myoviridae Marfavirus 169,970 40.90 This study vB_KpnS-VAC51 K3325 SAMN23489160 MZ571830.1 Demerecviridae Sugarlandvirus 113,149 45.40 This study vB_KpnM-VAC66 K3320 SAMN22059211 MZ612130.1 Myoviridae Slopekvirus 178,532 41.72 41 vB_KpnS-VAC70 K3318 SAMN20298916 MZ571831.1 Drexlerviridae Webervirus 49,631 50.70 This study vB_KpnP-VAC71 K3318 SAMN20298917 MZ571832.1 Autographiviridae Przondovirus 40,388 52.98 This study vB_KpnS-VAC110 K2691 SAMN24377650 OM032871.1 Drexlerviridae Webervirus 45,195 50.44 This study vB_KpnS-VAC111 K2691 SAMN20298918 ON881905.1 Drexlerviridae Webervirus 50,559 50.50 This study vB_KpnS-VAC112 K2691 SAMN20298919 MZ571833.1 Drexlerviridae Webervirus 49,068 50.20 This study vB_KpnS-VAC113 K2691 SAMN20298921 MZ571834.1 Drexlerviridae Webervirus 49,568 50.46 This study a Phages selected for phage interaction studies are indicated in boldface. Molecular Response to Phages in the Host Klebsiella Microbiology Spectrum March/April 2023 Volume 11 Issue 2 10.1128/spectrum.03974-22 3 Downloaded from https://journals.asm.org/journal/spectrum on 05 February 2025 by 150.214.182.235. FIG 1 (A) Phylogenetic analysis of 21 phages performed with the nucleotide sequence of the large terminase subunit of each phage. (B) TEM images showing the structure of the 21 phages under study. All belong to the order Caudovirales. vB_KpnS-VAC2, vB_KpnS-VAC4, vB_KpnS-VAC5, vB_KpnS-VAC6, vB_KpnS-VAC7, vB_KpnS-VAC8, vB_KpnS-VAC10, vB_KpnS-VAC11, vB_KpnS-VAC35, vB_KpnS-VAC70, vB_KpnS- VAC110, vB_KpnS-VAC111, vB_KpnS-VAC112, and vB_KpnS-VAC113 are characterized by large and flexible tails. On the other hand, phages vB_KpnP-VAC1, vB_KpnP-VAC25, and vB_KpnP-VAC71 are characterized by a short tail. Finally, phages vB_KpnM-VAC13, vB_KpnM-VAC36, and vB_KpnM-VAC66 are characterized by an icosahedral capsid and a rigid, contractile tail. The TEM scale bar represents 50 or 100 nm, depending on the phage. Molecular Response to Phages in the Host Klebsiella Microbiology Spectrum March/April 2023 Volume 11 Issue 2 10.1128/spectrum.03974-22 4 Downloaded from https://journals.asm.org/journal/spectrum on 05 February 2025 by 150.214.182.235. 61.90% of the phages belonged to the Drexerviridae family (13 of 21 phages), 14.29% belonged to the Autographiviridae family (3 of 21 phages), 14.29% belonged to the Myoviridae family (3 of 21 phages), and 9.52% belonged to the Demerecviridae family (2 of 21 phages). The genomes ranged from 39,371 bp in the case of phage vB_KpnP- VAC1, a member of the family Autographiviridae and the genus Teetrevirus,to 178,532 bp in the case of phage vB_KpnM-VAC66, a member of the family Myoviridae, and the genus Slopekvirus. The guanine-cytosine content ranged from 40.90% in the case of vB_KpnM-VAC36 to 53.76% in the case of vB_KpnP-VAC25. The genomic study revealed that the structure of the phage genomes varied depending on the type of phage. In the case of the members of the Drexerviridae (vB_KpnS-VAC2-11, vB_KpnS- VAC70, and vB_KpnS-VAC110-113), Autographiviridae (vB_KpnP-VAC1, vB_KpnP-VAC25, and vB_KpnP-VAC71), and Demerecviridae (vB_KpnS-VAC35 and vB_KpnS-VAC51) families, the genome was organized in functional modules of genes related to structure, packaging, lysis, transcription, and regulation. In contrast, for members of the Myoviridae (vB_KpnM- VAC13, vB_KpnM-VAC36, and vB_KpnM-VAC66) family, which are “larger phages”(.100 bp), no lysis-specific blocks were distinguished, and structural and morphogenesis-related proteins were repeated in several blocks throughout the genome. Considering the lysis genes, all phages had endolysins and holins, proteins which are responsible for the degradation of the bacterial cell wall during infection of the host. However, a difference was observed in terms of spanin, a protein involved in the lysis process in Gram-negative hosts, depending on the family to which the phage belongs: the Drexerviridae family had a unimolecular spanin (U-spanin), while the Autographiviridae,Demerecviridae,andMyoviridae families had a heterodimer molecule of spanin (I-spanin and O-spanin). Regarding the depolymerase genes, generally related to the tail receptor, five of the phages (23.81%) had one depolymerase (vB_KpnS- VAC4, vB_KpnS-VAC7, vB_KpnS-VAC10, vB_KpnS-VAC11, and vB_KpnS-VAC70), while two of the phages (9.52%) (vB_KpnS-VAC2 and vB_KpnS-VAC6) had two different depolymerase genes. It has been observed that the “larger phages”(vB_KpnM-VAC13, vB_KpnS-VAC35, vB_KpnM-VAC36, vB_KpnS-VAC51, and vB_KpnM-VAC66) had numerous tRNA genes (1, 21, 7, 22, and 1, respectively). The presence of HNH homing endonucleases has been observed in the genomes of three phages (vB_KpnP-VAC1, vB_KpnM-VAC13, and vB_KpnM-VAC66). The phage vB_KpnM-VAC66 contains the highest number of HNH homing endonucleases. (ii) Genetic defense mechanisms of phages. An in-depth study of the phage genomes with different bioinformatic tools revealed the presence of defense mechanisms (Table 2): (i) 35 RM system evasion mechanisms located in 16 phages, (ii) six TA systems located in two phages (vB_KpnM-VAC13 and vB_KpnM-VAC66), (iii) one DNA degradation evasion located in phage vB_KpnP-VAC1, (iv) four blocking RM of host bacteria located in phage vB_KpnM-VAC36, (v) seven genes that confer resistance to the Abi system of host bacteria located in three phages (vB_KpnM-VAC13, vB_KpnM- VAC36, and vB_KpnM-VAC66), and, finally, (vi) two possible orphan CRISPR-Cas system were located in the phages vB_KpnS-VAC35 and vB_KpnS-VAC51. In addition, almost all phages possessed a possible anti-CRISPR system, composed by Acr and Aca protein, except for phages vB_KpnS-VAC112 and vB_KpnS-VAC113 (see Table S1 in the supplemental material). An inhibitor of the TA system (protein ID QZE51102.1) was also found in the genome of the phage vB_KpnP-VAC1. (iii) Phylogenetic analysis of phages. Phylogenetic analysis was performed by aligning the nucleotide sequence of the large subunit terminase of each phage with MAFF server, followed by the elaboration of the phylogenetic tree with RAxMLHPC- PTHREADS-AVX2 version 8.2.12 (42) under the GTRGAMMA model and 100 bootstrap replicates. This analysis revealed that phages were clustered in the following families: (i) Drexlerviridae (vB_KpnS-VAC2, vB_KpnS-VAC4, vB_KpnS-VAC5, vB_KpnS-VAC6, vB_KpnS- VAC7, vB_KpnS-VAC8, vB_KpnS-VAC10, vB_KpnS-VAC11, vB_KpnS-VAC70, vB_KpnS-VAC110, vB_KpnS-VAC111, vB_KpnS-VAC112, and vB_KpnS-VAC113), (ii) Autographiviridae (vB_KpnP- VAC1, vB_KpnP-VAC25, and vB_KpnP-VAC71), (iii) Demerecviridae (vB_KpnS-VAC35 and vB_KpnS-VAC51), and (iv) Myoviridae (vB_KpnM-VAC36, vB_KpnM-VAC13, and vB_KpnM- VAC66). It is worth mentioning that in the case of Autographiviridae,phagevB_KpnP-VAC1 Molecular Response to Phages in the Host Klebsiella Microbiology Spectrum March/April 2023 Volume 11 Issue 2 10.1128/spectrum.03974-22 5 Downloaded from https://journals.asm.org/journal/spectrum on 05 February 2025 by 150.214.182.235. TABLE 2 Phage defense mechanisms against bacteria detected in the phages under study a Phage E value Description Accession no. Evasion of the RM system vB_kpnS-VAC2 1.00E–177 DNA adenine methyltransferase QZE50413.1 4.00E–166 DNA methylase QZE50429.1 2.00E–47 DNA cytosine methylase QZE50430.1 4.00E–47 DNA cytosine methyltransferase QZE50431.1 vB_KpnS-VAC4 1.00E–171 Cytosine DNA methylase QZE50543.1 3.00E–180 DNA adenine methyltransferase QZE50559.1 vB_KpnS-VAC5 3.00E–179 DNA adenine methyltransferase QZE50572.1 4.00E–170 DNA cytosine methyltransferase QZE50589.1 vB_KpnS-VAC6 2.00E–180 DNA adenine methyltransferase QZE50665.1 9.00E–178 DNA methylase QZE50681.1 vB_KpnS_VAC7 8.00E–171 DNA cytosine methyltransferase QZE50785.1 3.00E–180 DNA adenine methyltransferase QZE50801.1 vB_KpnS-VAC8 2.00E–170 DNA cytosine methyltransferase QZE50869.1 3.00E–179 DNA adenine methyltransferase QZE50885.1 vB_knpS-VAC10 2.00E–41 Methyltransferase type 11 QZE50901.1 3.00E–179 DNA adenine methyltransferase QZE50946.1 vB_KpnS-VAC11 1.00E–22 DNA adenine methyltransferase QZE50991.1 8.00E–123 DNA adenine methyltransferase QZE50992.1 2.00E–173 DNA cytosine methylase QZE51007.1 vB_KpnM_VAC13 0.00E100 Putative methyl transferase QWY13741.1 0.00E100 Cytosine-specific methyltransferase QWY13835.1 vB_KpnM-VAC66 0.00E100 DNA adenine methylase QYC51093.1 0.00E100 Putative methyl transferase QYC51156.1 vB_KpnS-VAC70 3.00E–180 DNA adenine methyltransferase UEW68160.1 4.00E–169 DNA cytosine methyltransferase UEW68177.1 vB_KpnS-VAC110 2.10E–164 Cytosine DNA methylase UKL59162.1 3.90E–162 DNA adenine methyltransferase UKL59178.1 vB_KpnS-VAC111 7.00E–179 DNA adenine methyltransferase UEP19890.1 3.00E–164 Cytosine DNA methylase UEP19925.1 vB_KpnS-VAC112 2.00E–41 Methyltransferase type 11 UEW68274.1 4.00E–169 DNA cytosine methyltransferase UEW68302.1 2.00E–180 DNA adenine methyltransferase UEW68318.1 vB_KpnS-VAC113 5.00E–171 Cytosine DNA methylase UEP19765.1 3.00E–179 DNA N-6-adenine-methyltransferase UEP19781.1 TA system vB_KpnM_VAC13 0.00E100 RNA ligase/RnlB antitoxin QWY13674.1 3.10E–05 DUF1778 domain-containing protein; TA toxin, antitoxin, N-acetyltransferase QWY13811.1 1.50E–13 RNase III inhibitor; macrodomain, toxin-antitoxin QWY13854.1 vB_KpnM-VAC66 0.00E100 RNA ligase/RnlB antitoxin QYC51086.1 3.10E–05 DUF1778 domain-containing protein; TA toxin, antitoxin, N-acetyltransferase QYC51228.1 1.50E–13 RNase III inhibitor; macrodomain, toxin-antitoxin QYC51272.1 Evasion of DNA degradation vB_KpnP-VAC1 2.00E–30 Inhibitor of recBCD nuclease QZE51097.1 Block RM of host bacteria vB_KpnM-VAC36 4.00E–159 Anti-restriction nuclease UEP19370.1 4.00E–41 Putative anti-restriction nuclease UEP19372.1 1.00E–87 Anti-restriction nuclease UEP19373.1 0.00E100 Anti-restriction endonuclease UEP19375.1 (Continued on next page) Molecular Response to Phages in the Host Klebsiella Microbiology Spectrum March/April 2023 Volume 11 Issue 2 10.1128/spectrum.03974-22 6 Downloaded from https://journals.asm.org/journal/spectrum on 05 February 2025 by 150.214.182.235. was phylogenetically more similar to vB_KpnP-VAC71 than to vB_KpnP-VAC25. Moreover, in thecaseoftheMyoviridae, phage vB_KpnM-VAC36, belonging to the genus Marfavirus,was phylogenetically more distant than phages vB_KpnM-VAC13 and vB_KpnM-VAC66, which are very similar to each other, as demonstrated in a previous study conducted by our research group, both belonging to the Slopekvirus genus(41)(Fig.1A). (iv) Genomic comparison. In the case of the Drexlerviridae family, the results show that phages vB_KpnS-VAC110 and vB_KpnS-VAC113 are very similar (query, 97%; identity, 99.83%), as are phages vB_KpnS-VAC7 and vB_KpnS-VAC4 (query, 91%; identity, 98.93%) and phages vB_KpnS-VAC111 and vB_KpnS-VAC112 (query, 90%; identity, 99.55%). However, phages vB_KpnS-VAC70, vB_KpnS-VAC2, vB_KpnS-VAC6, and vB_KpnS-VAC111 show a low degree of similarity (query, .75%; identity, .82%). In the Autographiviridae family, a low degree of similarity between all phages was observed (query, .22%; identity, .76%). In the Demerecviridae family, partial similarity between phages vB_KpnS-VAC35 and vB_KpnS-VAC51 (query, 93%; identity, 95.37%) was observed. Finally, in the Myoviridae family, the phage vB_KpnM-VAC36 member of the Marfavirus genus was very different from the other two phages, member of the Slopekvirus family, vB_KpnM-VAC13 (query, 0%; identity, 80.75%) and vB_KpnM-VAC66 (query, 3%; identity, 85.05%), which are very similar (query, 95%; identity, 97.56%), as previously demonstrated in a study carried out by our research group (41) (Fig. 2). Host range assay. The phage infectivity assay was performed in the collection of 47 K. pneumoniae clinical strains (Table 3) by the spot test technique (Fig. 3A). The criteria used to determine the phage infectivity were the presence of clear spots (infection), the presence of turbid spots (low infection or resistance), and the lack of spots (no infection). The results showed a high variability of infectivity between the phages (Fig. 3B). Phage vB_KpnP-VAC1 had the lowest host range, infecting only the strain K2986, while phage vB_KpnM-VAC13 presented the highest range of activity, infecting 27 strains. Phages vB_KpnS-VAC35 and vB_KpnM-VAC36 also exhibited a high host range, infecting both 17 and 18 strains, respectively. Therefore, the following experiments focused on these two phages (vB_KpnS-VAC35 and vB_KpnM-VAC36). Bacterial genome analysis. The genomes of twenty-seven clinical isolates infected by the vB_KpnS-VAC35 and vB_KpnM-VAC36 phages, according to the host range assay, were analyzed for the presence of CRISPR-Cas systems. The result revealed the presence of CRISPR-Cas systems in 14 strains (Fig. 3B). However, we proceeded to a more detailed study of K. pneumoniae clinical strains K3574 (SAMEA3649560) and K3320 (SAMEA3649520), as they were the ones best infected by phages vB_KpnS- VAC35 and vB_KpnM-VAC36, respectively. The result of this study revealed that both strains had an intact type I-E CRISPR-Cas in their genome, with 35 spacers in the case of K3574 and with 11 spacers in the case of the strain K3320 (Fig. 4A and B). In turn, five plasmids located in five different contigs were found in the strain K3574, while TABLE 2 (Continued) Phage E value Description Accession no. Resistance to Abi system of host bacteria vB_KpnM-VAC13 0.00E100 RIIB protector from prophage-induced early lysis QWY13711.1 0.00E100 RIIA-RIIB membrane associated protein/rIIA lysis inhibitor QWY13712.1 vB_KpnM-VAC36 3.00E100 RIIA lysis inhibitor UEP19351.1 0.00E100 RIIA membrane-associated protein UEP19352.1 0.00E100 RIIB lysis inhibitor UEP19353.1 vB_KpnM-VAC66 0.00E100 RIIB protein QYC51123.1 0.00E100 RIIA protector from prophage-induced early lysis QYC51124.1 CRISPR-Cas system vB_KpnS-VAC35 Orphan CRISPR array, 94; no. of spacers, 1 ND vB_KpnS-VAC51 CRISPR-CAS length, 219; DR length, 25; no. of spacers, 3 ND a The clinical strains selected for phage interaction studies are indicated in boldface. Molecular Response to Phages in the Host Klebsiella Microbiology Spectrum March/April 2023 Volume 11 Issue 2 10.1128/spectrum.03974-22 7 Downloaded from https://journals.asm.org/journal/spectrum on 05 February 2025 by 150.214.182.235. strain K3320 had four plasmids located in three different contigs (Table 4). On the other hand, only strain K3574 exhibited an RM system, which was type II and functioned as a methyltransferase (Table 4). Finally, five prophages (two intact and three questionable) were detected in strain K3574, and seven prophages (three intact, two incomplete, and two questionable) were detected in strain K3320. However, only the data of the prophages considered intact are shown in Table 5. Characterization of phages vB_KpnS-VAC35 and vB_KpnM-VAC36. (i) Phage adsorption. Adsorption of phages vB_KpnS-VAC35 and vB_KpnM-VAC36 (Fig. 5A and B) to the bacterial surface receptor was studied with the previously selected strains K3574 and K3320, at a multiplicity of infection (MOI) of 0.01. Phage vB_KpnS-VAC35 showed a high percentage of adsorption, with 91.28% of phage adsorbed in the strain K3574 after 5 min, while phage vB_KpnM-VAC36 showed slight adsorption in the strain K3320, with 39.02% of phage adsorbed after 2 min (Fig. 5C and D). (ii) One-step growth curve assay. The latent period, determined by the one-step growth curve indicating the time taken for a phage particle to reproduce inside an infected host cell, and the burst size, defined as the number of viral particles released in each infection cycle per cell, were 10 min and 45.52 PFU/mL, respectively, for phage vB_KpnS-VAC35 in strain K3574 and 8 min and 2.71 PFU/mL for the phage vB_KpnM- VAC36 in strain K3320 at an MOI of 0.01 (Fig. 5E and F). (iii) Phage kill curve. The infectivity assay in liquid medium to determine the infection curve for phages vB_KpnS-VAC35 and vB_KpnM-VAC36 at an MOI of 1 in the FIG 2 Graphic comparison of the homology of the 21 phages, grouped according to their families and in the same order as in the phylogenetic tree. The schematic representation was conducted with VipTree (https://www.genome.jp/viptree/, accessed in June 2022). Molecular Response to Phages in the Host Klebsiella Microbiology Spectrum March/April 2023 Volume 11 Issue 2 10.1128/spectrum.03974-22 8 Downloaded from https://journals.asm.org/journal/spectrum on 05 February 2025 by 150.214.182.235. selected clinical strains: K3574 and K3320, showed that both phages yielded successful infection, with optical density at 600 nm (OD 600 ) values of 0.155 60.05 and 0.05 6 0.01, respectively, reached after 1 h 30 min of infection (Fig. 6A and B). We monitored the CFU/mL and observed that, for phage vB_KpnS-VAC35 in clinical strain K3574, the count reached 1.15 10 4 61.34 10 4 CFU/mL. However, we observed a slight increase in the number of CFU/mL after 3 h of phage infection; it reached 2.35 10 4 6 2.12 10 4 CFU/mL. The same phenomenon appears in the case of the phage vB_KpnM-VAC36, where we observed a slight decrease in CFU/mL counts, which reached a value of 5.0 10 4 62.83 10 4 after 1 h 30 min of phage infection and an TABLE 3 Characteristics of 47 clinical K. pneumoniae strains Strain MLST a Carbapenemase(s) b Capsular type(s) c Origin GenBank accession no. Source or reference K2535 ST15 SHV-28, SHV106 KL112 Blood SAMEA3538911 This study K2551 ST15 OXA-48, OXA-1, TEM-1B, CTX-M-15 KL112 Blood SAMEA3538915 This study K2597 ST15 OXA-48, OXA-1, TEM-1B, CTX-M-15 KL112 Blood SAMEA3538926 This study K2691 ST11 CTX-M-15 KL24 Blood SAMEA3538940 40 K2707 ST11 KPC-2 KL13 Blood SAMEA3538945 40 K2715 ST45 SHV-1 KL24 Blood SAMEA3538948 This study K2783 ST11 KPC-2 KL13 Blood SAMEA3538957 This study K2791 ST11 CTX-M-15 KL24 Blood SAMEA3538958 This study K2982 ST605 ND KL58 Blood SAMEA3649451 This study K2983 ST2449 ND KL5 Blood SAMEA3649452 75 K2984 ST405 CTX-M-15, SHV-76, TEM-1B KL151/K1151 Blood SAMEA3649453 40 K2986 ST307 CTX-M-15 KL102 Blood SAMEA3649454 40 K2989 ST661 OXA-1, SHV-27 KL24 Blood SAMEA3649457 40 K2990 ST107 SHV-1 KL142 Blood SAMEA3649458 This study K3318 ST15 OXA-1, CTM-X-15, OXA-48, TEM-1B KL112 Blood SAMEA3649518 This study K3320 d ST163 SHV-16 KL139 Blood SAMEA3649520 This study K3321 ST466 SHV-33 KL22/K37 Blood SAMEA3649521 This study K3322 ST35 SHV-1 KL22/K37 Blood SAMEA3649522 This study K3323 ST3645 ND KL126 Blood SAMEA3649523 This study K3324 ST542 SHV-1 KL8 Blood SAMEA3649524 This study K3325 ST42 ND KL64 Blood SAMEA3649525 40 K3416 ST483 SHV-27, VIM-1 KL110 Blood SAMEA3649537 This study K3509 ST35 SHV-1 KL22/K37 Blood SAMEA3649551 This study K3571 ST33 SHV-108 KL13 Blood SAMEA3649557 This study K3573 ST37 ND KL15/K51/K52 Blood SAMEA3649559 This study K3574 ST3647 ND KL30 Blood SAMEA3649560 This study K3575 ST14 SHV-1 KL2 Blood SAMEA3649561 This study K3579 ST16 CTX-M-15, OXA-1 KL51 Blood SAMEA3649562 This study K3667 ST326 ND KL25 Blood SAMEA3649564 This study K3668 ST405 OXA-48, OXA-1, SHV-1 KL151/K1151 Blood SAMEA3649629 This study K3669 ST258 KPC-3 KL107/K81 Blood SAMEA3649638 This study ST405-OXA48 ST405 OXA-48 KL151/K1151 Wound WRXJ00000000 2 ST15-VIM1 ST15 VIM-1 KL24 Blood WRXI00000000 2 ST11-OXA245 ST11 OXA-245 KL24 Wound WRXH00000000 2 ST437-OXA245 ST437 OXA-245 KL36 Rectal WRXG00000000 2 ST16-OXA48 ST16 OXA-48 KL51 Urine WRXF00000000 2 ST101-KPC2 ST101 KPC-2 KL17 Rectal WRXE00000000 2 ST147-VIM1 ST147 VIM-1 KL64 Rectal WRXD00000000 2 ST11-VIM1 ST11 VIM-1 KL24 Respiratory WRXC00000000 2 ST846-OXA48 ST846 OXA-48 KL110 Sputum WRXB00000000 2 ST340-VIM1 ST340 VIM-1 KL15 Rectal WRXA00000000 2 ST13-OXA48 ST13 OXA-48 KL30 Rectal WRWZ00000000 2 ST512-KP3 ST512 KPC-3 KL107/K81 Axillary WRWY00000000 2 ST15-OXA48 ST15 OXA-48 KL112 Axillary WRWX00000000 2 ST11-OXA48 ST11 OXA-48 KL24 Urine WRWW00000000 2 ST258-KPC3 ST258 KPC-3 KL107/K81 Urine WRWV00000000 2 ST979-OXA48 ST974 OXA-48 KL38 Urine WRWT00000000 2 a MSLT, multilocus sequence type according to https://bigsdb.pasteur.fr/klebsiella/. b Carbapenemase, as determined by https://bio.tools/resfinder. c Capsular type, as determined by https://github.com/kelwyres/Kaptive-Web. d Clinical strains selected for phage interaction studies are indicated in boldface. Molecular Response to Phages in the Host Klebsiella Microbiology Spectrum March/April 2023 Volume 11 Issue 2 10.1128/spectrum.03974-22 9 Downloaded from https://journals.asm.org/journal/spectrum on 05 February 2025 by 150.214.182.235. processing or due to the inherent proprieties of the bacteria. Therefore, the results revealed the expression of FtsH protease modulator located in prophage of strains K3574 and K3320, which controls the lytic pathway (58), as well as the expression of the cupin protein located in plasmid in the strain K3574, a phosphomannose isomerase involved in lipopolysaccharide (LPS) synthesis, which is an important determinant of pathogenicity and phage susceptibility (59). In addition, proteins related to bacterial defense, resistance, and virulence and also to oxidative stress mechanisms (60) have been observed. The difference in expression compared to the control without phage infection of porins, efflux pumps, LPS, and pilus elements, previously described in the literatureasphagereceptors(61,62),wasalsoobserved.Moreover,proteinsinvolvedinthe quorum network were observed, e.g., the LuxS that synthesizes AI-2 molecules, or the presence of the CsrA regulator in the strain K3320 infected by phage vB_KpnM-VAC36 (63, 64). Indeed, previous studies have associated the quorum network with phage infection (65– 67). In addition, a type II RelE/ParE TA system was expressed in strain K3320. This is a very interesting finding, since phage vB_KpnM-VAC36 did not successfully infect strain K3320. The fundamental role played by TA systems in the inhibition of phage infection has recently been demonstrated (20, 68–70). Interestingly, an inhibitor of the TA system (protein ID QZE51102.1)wasfoundinthegenomeofthephagevB_KpnP-VAC1.Thistypeof gene, previously only described in one E. coli phage (35), may play a role in phage defense against bacteria. The methyltransferases, other important proteins that play a key role in phage infection (71), were found in both K3574 and K3320 strains infected by phages. In addition, several Acr candidate proteins were expressed in the infected strains. This is a very interesting finding, because the anti-CRISPR could inhibit the host’s CRISPR-Cas system and thus promote infection (54). Therefore, the difference in expression compared to the control of all mechanisms could be due to the phage-host interaction, with the bacteria trying to use all their defense mechanisms in response to the infection. Conclusion. Phage-host interactions have been examined ever since the discovery of phages a century ago. The present study revealed numerous defense mechanisms both against bacteria by phage (RM system evasion, TA system, DNA degradation evasion, RM block of host, resistance to Abi, anti-CRISPR and CRISPR-Cas system) and against phage infection by bacteria (prophage, plasmid, defense/virulence/resistance, and oxidative stress proteins). However, phage-host bacterial interactions remain poorly understood, and further study is required in order to improve the efficacy of phage therapy. MATERIALS AND METHODS Bacterial strains. A collection of 47 clinical isolates of K. pneumoniae obtained from the Virgen Macarena University Hospital (Seville, Spain) and the National Centers for Microbiology (Carlos III Health Institute, Spain) TABLE 6 Acr candidate protein in phages vB_KpnS-VAC35 and vB_KpnM-VAC36 detected in the proteomic study (NanoUHPLC-Tims-QTOF) a Description Accession no. –10LogP Position(s) K35741vB_KpnS-VAC35 Hypothetical protein KPN4_89 (Acr candidate) [Klebsiella phage KPN4] QEG11275.1 146.67 24497–24667 4-Hydroxy-3-polyprenylbenzoate decarboxylase (Acr candidate) [Klebsiella phage vB_KpnS-VAC35] UEP19035.1 107.65 22800–233162 Hypothetical protein JIPhKp127_0059 (Acr candidate) [Klebsiella phage JIPh_Kp127] QFR57489.1 104.09 23369–23911 6-Phosphofructokinase (Acr candidate) [Klebsiella phage vB_KpnS-VAC35] UEP19028.1 102.81 20466–20858 Hypothetical protein (Acr candidate) [Klebsiella phage vB_KpnS-VAC35] UEP19032.1 86.070 22064–22273 Hypothetical protein (Acr candidate) [Klebsiella phage vB_KpnS-VAC35] UEP19030.1 66.120 21405–21677 Hypothetical protein (Acr candidate) [Klebsiella phage vB_KpnS-VAC35] UEP19036.1 61.440 23155–23382 K3320 1vB_KpnM-VAC36 Hypothetical protein (Acr candidate) [Klebsiella phage vB_KpnM-VAC36] UEP19294.1 78.95 66295–66492 a The protein header information is presented as listed in the NCBI database. The accession number of the protein as seen in the NCBI database is indicated. “–10LogP” indicates the protein confidence score, and “position(s)”indicates localization in the phage genome. Molecular Response to Phages in the Host Klebsiella Microbiology Spectrum March/April 2023 Volume 11 Issue 2 10.1128/spectrum.03974-22 16 Downloaded from https://journals.asm.org/journal/spectrum on 05 February 2025 by 150.214.182.235. was used in this study (Table 3). The sequence type (ST) and the capsular type (K) were determined using the methodsavailableonthePasteurInstitutewebsite(https://bigsdb.pasteur.fr/klebsiella/, accessed between 2018 and the present) and the Kaptive website (https://github.com/kelwyres/Kaptive-Web, accessed in April 2020), respectively. All strains were grown in Luria-Bertani medium (0.5% NaCl, 0.5% yeast extract, 1% tryptone). Isolation, purification, and propagation of lytic phages and TEM. Ten new lytic phages isolated from sewage water samples and twelve lytic phages previously isolated by our research group (20, 40, 41) were used in this study. Isolation, purification, and propagation of the new phages was performed according to the procedures used in the previously cited articles, using strains of K. pneumoniae as a natural host (Table 1). Next, the 10 new lytic phage solutions were negatively stained with 1% aqueous uranyl acetate before being analyzed by TEM in a JEOL JEM-1011 electron microscope. Phage DNA extraction and WGS. The phage DNA of the 10 new lytic phages was isolated with the phenol-chloroform method according to a previously published phage-hunting protocol (https://phagesdb .org/media/workflow/protocols/pdfs/PCI_SDS_DNA_Extraction_2.2013.pdf, accessed on 1 February 2021), and WGS was performed as described by Bleriot et al. (20). Phage genome annotation. (i) Defense mechanisms. All assemblies were initially annotated by sequence homology using Patric 3.6.9 (http://patricbrc.org, accessed on 22 February 2021) and were then manually refined using BLASTX (http://blast.ncbi.nlm.nih.gov, accessed between August and October 2021) and Hhmer (http://hmmer.org, accessed between August and October 2021), as well as the Hhpred tool (https:// toolkit.tuebingen.mpg.de/tools/hhpred, accessed between August and October 2021), which predict functions through protein structure. In addition, to search for phage defense mechanisms against bacteria, the CRISPR Miner 2 (http://www.microbiome-bigdata.com/CRISPRminer/, accessed in March 2022) and PADLOC (https:// padloc.otago.ac.nz/padloc/, accessed in March 2022) tools were used to search for possible CRISPR-Cas systems, as well as the AcrDB tool (https://bcb.unl.edu/AcrFinder/, accessed on October 2021) to search for possible anti-CRIPSR-cas systems with the defect parameter of the website (Aca E value, 0.01; Aca identity, 30%; Aca coverage, 0.8; maximum intergenic distance between genes [bp], 150; operon up/downstream range for MGE- Prophage search [no. of genes], 10). Finally, the family and genus of the different phages were determined by sequence homology with the phage sequences available in the NCBI database. Complete genome sequences were included in the GenBank BioProject PRJNA739095. (ii) Phage phylogenetic analysis and genome comparison. Phylogenetic analysis of the 21 phages was performed using the nucleotide sequence of the large terminase subunit of each phage. Alignment was first performed with MAFF server (https://mafft.cbrc.jp/alignment/server/index.html, accessed on 3 January 2022), and a phylogenetic tree was then constructed using RAxMLHPC-PTHREADS-AVX2 v8.2.12 (42) under the GTRGAMMA model and 100 bootstrap replicates. A graphical representation of the comparison of all phage genomes was then constructed with the VipTree website (https://www.genome.jp/ viptree/, accessed in June 2022) according to the previously established phylogenetic relationship. Host range assay. The phage host spectrum was tested by the spot test technique (72), in a collection of 47 clinical strains of K. pneumoniae. A negative control consisting of SM buffer (0.1 M NaCl, 10 mM MgSO 4 , 20 mM Tris-HCl; pH 7.5) was included in each plate. All determinations were made in triplicate. The criteria used to determine the phage infectivity were the presence of clear spots (infection), the presence of turbid spots (low infection or resistance), and the lack of spots (no infection). Study of bacterial genome. The genome of 27 clinical isolates infected by the vB_KpnS-VAC35 and vB_KpnM-VAC36 phages, according to the host range assay, were analyzed for the presence of CRISPR-Cas systems by using the CRISPR Miner 2 (http://www.microbiome-bigdata.com/CRISPRminer/, accessed in January 2023). However, we proceeded to a more detailed study of K. pneumoniae clinical strains K3574 (SAMEA3649560) and K3320 (SAMEA3649520), since it was the one showing the best phage infectivity; for this, the genomic annotation of the Rastserver was also studied to check and validate the results obtained with other tools (https://rast.nmpdr.org, accessed in August 2022). Plasmids were then searched for using PlasmidFinder v2.0.1 (20 July 2001) (https://cge.food.dtu.dk/services/PlasmidFinder/, accessed in July 2022), the RM system using the Restriction-ModificationFinder v1.1 (accessed in June 2015) (https://cge.food.dtu .dk/services/Restriction-ModificationFinder/history.php, accessed in July 2022), and prophages using the Phaster tools (http://phaster.ca, accessed in July 2022). Characterization of phages vB_KpnS-VAC35 and vB_KpnM-VAC36. (i) Phage adsorption. Adsorption of phages vB_KpnS-VAC35 and vB_KpnM-VAC36 to the bacterial surface receptors of clinical strains K3574 and K3320, respectively, was determined from the adsorption curve (73) at an MOI of 0.01. The number of phages mixed with bacterial host cells at time zero was considered 100% free of phages. The adsorption curve analysis was performed in triplicate. (ii) One-step growth curve assay. A one-step growth curve of phages vB_KpnS-VAC35 and vB_KpnM-VAC36 at an MOI of 0.01, was constructed in clinical strains K3574 and K3320, respectively, to determine the latent period (L) and the burst size (B), according to the procedure of Bleriot et al. (20). One-step growth curve analysis was performed in triplicate. (iii) Phage kill curve assay in liquid medium. Killing curves were constructed from the selected isolates K3574 and K3320, in accordance with the presence of intact CRISPR-Cas system in their genome. Phages vB_KpnS-VAC35 and vB_KpnM-VAC36 were used to monitor the infection of the strain by optical density measured at a wavelength of 600 nm (OD 600 ) and counts of CFU/mL and PFU/mL. For this purpose, when the strains reached an early logarithmic phase (OD 600 = 0.3 to 0.4), cultures were infected with phages at an MOI of 1. The OD 600 , the CFU/mL, and the PFU/mL were determined every 30 min for 3 h. In all cases, the control was the strain without phage infection. All analyses were performed in triplicate. NanoUHPLC-Tims-QTOF proteomic analysis: interaction between phages (vB_KpnS-VAC35 and vB_KpnM-VAC36) and clinical strains (K3574 and K3320). NanoUHPLC-Tims-QTOF analysis was performed for quantitative study of the protein profile of strain K3574 and K3320 with or without infection Molecular Response to Phages in the Host Klebsiella Microbiology Spectrum March/April 2023 Volume 11 Issue 2 10.1128/spectrum.03974-22 17 Downloaded from https://journals.asm.org/journal/spectrum on 05 February 2025 by 150.214.182.235. with phages vB_KpnS-VAC35 and vB_KpnM-VAC36. When the cultures reached an early logarithmic phase of growth (OD 600 = 0.3 to 0.4), they were infected with phages at an MOI of 1. After 1 h, the cultures of the strains were harvested by centrifugation at 4,302 gfor 20 min at 4°C. The pellets were then stored at 280°C to facilitate cell disruption. The next day, the pellet was resuspended in phosphate-buffered saline and sonicated. Finally, the sonicated pellets were centrifuged at 4,302 gfor 20 min at 4°C, and the flowthrough was analyzed using NanoUHPLC-Tims-QTOF. The equipment, as well as the procedure used, are as described by Bleriot et al. (20). Data availability. Phage genome sequencing revealed all phages under study (available from GenBank BioProject PRJNA739095). SUPPLEMENTAL MATERIAL Supplemental material is available online only. SUPPLEMENTAL FILE 1, XLSX file, 0.1 MB. SUPPLEMENTAL FILE 2, XLSX file, 0.1 MB. SUPPLEMENTAL FILE 3, PDF file, 0.1 MB. ACKNOWLEDGMENTS This study was funded by grant PI19/00878 and PI22/00323 awarded to M.T. within the State Plan for R1D1I 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 “A Way of Making Europe”and Instituto de Salud Carlos III FEDER, Spanish Network for the Research in Infectious Diseases (REIPI; RD16/0016/0006 and RD16/0016/0008), CIBERINFEC (CIBER21/13/00012, CB21/13/00049, CIBER21/13/00084, and CIBER21/13/ 00095), and Personalized Medicine Project (MePRAM; PMP/00092) and also by the Study Group on Mechanisms of Action and Resistance to Antimicrobials, GEMARA (SEIMC; http://www.seimc.org/). M.T. was financially supported by the Miguel Servet Research Program (SERGAS and ISCIII). I.B. was financially supported by pFIS program (ISCIII, FI20/ 00302). O.P., L.F.-G., and M.L. were financially supported by grants IN606A-2020/035, IN606B-2021/013, and IN606C-2022/002, respectively (GAIN; Xunta de Galicia). The authors acknowledge CESGA (www.cesga.es) in Santiago de Compostela, Spain, for providing access to computing facilities and the RIAIDT-USC analytical facilities. Finally, We thank researchers from the Spanish Network of Bacteriophages and Transducer Elements (FAGOMA) for contributing the lytic phages. I.B., L.B., O.P., and L.F.-G. developed the experiments, analyzed the results, and wrote the original manuscript. M.L., C.O.C. and A.B.P. helped to prepare the visual presentation of the results. F.F.C., Á.P., L.M.-M., and J.O.-I. rewrote the manuscript. M.T. financed and directed the experiments and supervised the writing of the original manuscript. We declare that there are no conflicts of interest. REFERENCES 1. Bergh O, Børsheim KY, Bratbak G, Heldal M. 1989. High abundance of viruses found in aquatic environments. 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