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

1 Genomic analysis of 40 prophages located in the genomes of 16 carbapenemaseproducing clinical strains of Klebsiellapneumoniae InesBleriot1,2, RocíoTrastoy1,2, LuciaBlasco1,2, FelipeFernández- Cuenca2,3,4, AntónAmbroa1,2, LauraFernández- García1,2, OlgaPacios1,2, ElenaPerez- Nadales4,5, JulianTorre- Cisneros4,5, JesúsOteo- Iglesias2,4,6, FerranNavarro2,7, ElisendaMiró2,7, AlvaroPascual2,3,4, GermanBou1,2,4, LuisMartínez- Martínez2,4,5† and MariaTomas1,2,4,*,† RESEARCH ARTICLE Bleriot etal., Microbial Genomics 2020;6 DOI 10.1099/mgen.0.000369 Received 21 October 2019; Accepted 31 March 2020; Published 29 April 2020 Author affiliations: 1Microbiology Department, Research Institute Biomedical A Coruña (INIBIC), Hospital A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, Spain; 2Study Group on Mechanisms of Action and Resistance to Antimicrobials (GEMARA), Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC), Madrid; 3Clinical Unit for Infectious Diseases, Microbiology and Preventive Medicine, Hospital Universitario Virgen Macarena. Deparment of Microbiology and Medicine, University of Seville, Seville, Spain; 4Spanish Network for the Research in Infectious Diseases, REIPI, Seville, Spain; 5Microbiology Unit, Maimonides Biomedical Research Institute of Cordoba (IMIBIC), University Hospital Reina Sofía, University of Córdoba, Cordoba, Spain; 6Reference and Research Laboratory for Antibiotic Resistance and Health Care Infections, National Centre for Microbiology, Institute of Health Carlos III, Majadahonda, Madrid, Spain; 7Microbiology Department, Sant Pau Hospital, Autonomous University of Barcelona (Bellaterra), Barcelona, Spain. *Correspondence: Maria Tomas, MA. del. mar. tomas. carmona@ sergas. es Keywords: Klebsiella pneumoniae; prophages; bioinformatics; genomic analysis; comparative genomics; phylogeny. Abbreviations: HGT, horizontal gene transfer; MDR, multidrug resistant; MLST, multilocus sequence typing; MVP, microbe versus phage; NCBI, National Center for Biotechnology Information; SEIMC, Spanish Society of Infectious Diseases and Clinical Microbiology; ST, sequence type; TA, toxin– antitoxin; TEM, transmission electron microscopy. †These authors contributed equally to this work Data statement: All supporting data, code and protocols have been provided within the article or through supplementary data files. Supplementary material is available with the online version of this article. 000369 © 2020 The Authors This is an openaccess article distributed under the terms of the Creative Commons Attribution License. Abstract Klebsiella pneumoniae is the clinically most important species within the genus Klebsiella and, as a result of the continuous emergence of multidrug 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 carbapenemaseproducing 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 hostrange 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. DATA SummARy The genome sequences of the temperate bacteriophages (prophages) of the clinical strains of Klebsiella pneumoniae (Spanish Network for Research in Infectious Diseases, REIPI) 2 Bleriot etal., Microbial Genomics 2020;6 Table 1. Characteristics of the K. pneumoniae clinical strains and their origins (all isolated in Spain between November 2013 and April 2014) Strain MLST* Carbapenemase Origin GenBank accession no.† Genome size (bp) G+C (mol%) ST405- OXA48 ST-405 OXA48 Wound WRXJ00000000 5 774 094 57.0 ST15- VIM1 ST-15 VIM1 Blood WRXI00000000 5 122 717 57.0 ST11- OXA245 ST-11 OXA245 Wound WRXH00000000 5 667 817 57.1 ST437- OXA245 ST-437 OXA245 Rectal WRXG00000000 5 653 384 57.2 ST16- OXA48 ST-16 OXA48 Urine WRXF00000000 5 447 971 57.3 ST101- KPC2 ST-101 KPC2 Rectal WRXE00000000 5 523 997 57.2 ST147- VIM1 ST-147 VIM1 Rectal WRXD00000000 5 695 918 56.8 ST11- VIM1 ST-11 VIM1 Respiratory WRXC00000000 5 594 690 57.2 ST846- OXA48 ST-846 OXA48 Sputum WRXB00000000 5 548 215 57.3 ST340- VIM1 ST-340 VIM1 Rectal WRXA00000000 5 539 743 57.2 ST13- OXA48 ST-13 OXA48 Rectal WRWZ00000000 5 585 239 57.0 ST512- KPC3 ST-512 KPC3 Axillary smear WRWY00000000 5 650 413 57.2 ST15- OXA48 ST-15 OXA48 Axillary smear WRWX00000000 5 188 264 57.3 ST11- OXA48 ST-11 OXA48 Urine WRWW00000000 5 509 234 57.3 ST258- KPC3 ST-258 KPC3 Urine WRWV00000000 5 564 025 57.3 ST974- OXA48 ST-974 OXA48 Urine WRWT00000000 5 414 512 57.3 *Institute Pasteur MLST database (https://pubmlst.org/kpneumoniae). †GenBank accession numbers for the genomes of the 16 K. pneumoniae clinical strains, all belonging to BioProject accession number PRJNA565865 (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA565865). have been deposited in GenBank under BioProject accession number PRJNA565865 (http://www. ncbi. nlm. nih. gov/ bioproject/ 565865). The genome and ORF data for the bacteriophages are included in this BioProject record. INTRODuCTION Bacteriophages, i.e. viruses that infect bacteria, are the most abundant biological entities on Earth [1–3]. They are found in all environmental niches colonized by bacteria, with an estimated global population of 1031 viral particles [4, 5]. Bacteriophages have very diverse genomes and have been suggested to represent the largest source of gene diversity in the environment, as highlighted by the large number of novel genes of unknown function revealed by genome and metavirome sequencing [6]. Detailed comparative analysis has been made of the bacteriophages that infect various hosts and environments, including Mycobacterium [7], Acinetobacter [8], Pseudomonas [9], Bacillus [10], Lactococcus [11], marine cyanobacterium [12], Salmonella [13] and Vibrio species [14] and also members of the family Enterobacteriaceae [15]. However, the analysis of the families of all sequenced bacteriophages has illustrated how little of the global phage population has been genomically sampled [16, 17]. With an almost endless supply of diverse phages readily accessible for isolation and analysis, research programmes will continue to play substantial roles in analysing and describing functional proteins in the phages to understand their ecology and the various clinical, industrial and biotechnological applications [18]. Genomic comparative analysis has highlighted the mosaicism present in the genomes of all the dsDNA tailed bacteriophages. The term mosaicism refers to the fact that bacteriophages harbour in their genomes different regions with different evolutionary history due to the phenomenon of horizontal gene transfer (HGT) [16, 17]. The phylogeny and evolutionary relationships between bacteriophages isolated from bacteria Impact Statement Klebsiella pneumoniae is a successful multidrug resistant human pathogen and an important source of hospital infections associated with high morbidity and mortality due to multiple factors. Temperate bacteriophages (prophages), located in the genome of clinical strains of K. pneumoniae, act as mobile elements of horizontal gene transfer, being able to modulate the behaviour of bacteria by providing virulence and phage defence genes. Therefore, the characterization of these prophages can lead to the discovery of new molecular targets for the development of innovative antibacterial treatments. 3 Bleriot etal., Microbial Genomics 2020;6 Table 2. Characteristics of the genome sequences of the 40 prophages found in 16 clinical strains of carbapenemaseproducing K. pneumoniae (BioProject accession number PRJNA565865; https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA565865) Bacteriophage Family Accession no. Genome size (bp) No. of ORFs Hypothetical protein (%) G+C (mol%) tRNA Klebsiella pneumoniae ST405- OXA48 ST405- OXA48phi1.1 Myoviridae MK388859.1 35.232 52 48.1 53.8 0 ST405- OXA48phi1.2 Myoviridae MK416007.1 40.495 67 59.7 52.1 0 ST405- OXA48phi1.3 Siphoviridae MK416008.1 32.010 47 66.0 50.9 0 Klebsiella pneumoniae ST15- VIM1 ST15VIM1phi2 Myoviridae MK448228.1 46.342 73 54.8 53.6 0 Klebsiella pneumoniae ST11- OXA245 ST11- OXA245phi3.1 Myoviridae MK416009.1 33.326 44 20.5 51.5 0 ST11- OXA245phi3.2 Podoviridae MK416010.1 60.118 72 37.5 55.6 0 Klebsiella pneumoniae ST437- OXA245 ST437- OXA245phi4.1 Myoviridae MK416011.1 39.642 53 26.4 52.8 0 ST437- OXA245phi4.2 Myoviridae MK416012.1 18.281 27 25.9 50.5 0 Klebsiella pneumoniae ST16- OXA48 ST16- OXA48phi5.1 Siphoviridae MK416013.1 57.025 80 50.0 53.6 0 ST16- OXA48phi5.2 Myoviridae MK448230.1 47.305 79 54.8 53.8 4 ST16- OXA48phi5.3 Siphoviridae MK416014.1 29.301 51 49.0 50.2 0 ST16- OXA48phi5.4 Myoviridae MK416015.1 38.322 46 28.3 50.1 0 Klebsiella pneumoniae ST101- KPC2 ST101- KPC2phi6.1 Myoviridae MK448231.1 48.131 75 53.2 52.6 4 ST101- KPC2phi6.2 Myoviridae MK416016.1 11.454 17 11.8 59.0 0 ST101- KPC2phi6.3 Siphoviridae MK416017.1 43.942 62 43.1 52.2 3 Klebsiella pneumoniae ST147- VIM1 ST147- VIM1phi7.1 Myoviridae MK416018.1 34.141 43 20.5 53.0 1 ST147- VIM1phi7.2 Podoviridae MK448232.1 34.200 45 38.6 40.0 0 Klebsiella pneumoniae ST11- VIM1 ST11- VIM1phi8.1 Myoviridae MK448233.1 42.666 67 37.0 50.7 2 ST11- VIM1phi8.2 Podoviridae MK448234.1 48.230 68 41.7 52.9 0 ST11- VIM1phi8.3 Myoviridae MK416019.1 39.953 62 45.1 51.6 0 ST11- VIM1phi8.4 Myoviridae MK416020.1 33.016 43 20.9 51.6 0 Klebsiella pneumoniae ST846- OXA48 ST846- OXA48phi9.1 Siphoviridae MK416021.1 38.370 38 36.8 53.3 0 ST846- OXA48phi9.2 Myoviridae MK416022.1 57.402 91 63.7 51.6 0 Klebsiella pneumoniae ST340- VIM15 ST340- VIM1phi10.1 Myoviridae MK422455.1 36.124 58 58.6 51.7 0 ST340- VIMphi10.2 Myoviridae MK422454.1 33.326 45 24.4 51.5 0 Continued 4 Bleriot etal., Microbial Genomics 2020;6 Bacteriophage Family Accession no. Genome size (bp) No. of ORFs Hypothetical protein (%) G+C (mol%) tRNA Klebsiella pneumoniae ST13- OXA48 ST13- OXA48phi12.1 Myoviridae MK422453.1 39.086 50 19.6 53.5 1 ST13- OXA48phi12.2 Siphoviridae MK422452.1 34.141 51 49.0 50.5 0 ST13- OXA48phi12.3 Siphoviridae MK422451.1 84.199 93 47.7 49.9 0 ST13- OXA48phi12.4 Siphoviridae MK422450.1 59.049 86 57.5 52.1 0 ST13- OXA48phi12.5 Siphoviridae MK714353.1 44.913 72 58.9 51.8 2 Klebsiella pneumoniae ST512- KPC3 ST512- KPC3phi13.1 Myoviridae MK448235.1 42.666 67 47.8 52.9 2 ST512- KPC3phi13.2 Myoviridae MK422449.1 32.302 44 25.0 51.9 0 ST512- KPC3phi13.5 Myoviridae MN166823.1 25.624 45 55.6 52.0 0 ST512- KPC3phi13.6 Myoviridae MK433577.1 39.643 52 28.8 52.8 0 Klebsiella pneumoniae ST15- OXA48 ST15- OXA48phi14 Myoviridae MK433578.1 33.839 47 21.3 52.7 0 Klebsiella pneumoniae ST11- OXA48 ST11- OXA48phi15.1 Myoviridae MK433579.1 36.137 58 55.2 51.7 0 ST11- OXA48phi15.3 Myoviridae MK433580.1 33.326 44 20.5 51.5 0 Klebsiella pneumoniae ST258- KPC3 ST258- KPC3phi16.1 Myoviridae MK433581.1 39.643 53 32.1 52.8 0 ST258- KPC3phi16.2 Myoviridae MK433582.1 33.326 45 22.2 51.5 0 Klebsiella pneumoniae ST974- OXA48 ST974- OXA48phi18 Siphoviridae MK448237.1 51.967 80 54.3 52.9 1 Table 2. Continued have been analysed in several studies [19, 20], confirming their importance. The phenomenon of HGT, as well as bacteriophages themselves, act as drivers of the evolution and diversification of bacteria, allowing them to acquire virulence factors (e.g. diphtheria or botulinum toxins) and/or genes related to metabolism, antibiotic resistance (e.g. β-lactamases) and adaptation to new environmental niches [21–23]. Klebsiella pneumoniae, which belongs to the family Enterobacteriaceae, is a Gramnegative opportunistic bacterial pathogen associated with a wide range of diseases, such as urinary tract infections, pneumonia and septicaemia, as well as infection of wounds and soft tissues [24]. In recent years, different strains of this species have acquired genes for resistance to antibiotics, especially genes that encode enzymes capable of breaking down most β-lactamases [25]. This has led to the generation of multidrug resistant (MDR) pathogens, which constitute a serious publichealth problem [26]. The success of K. pneumoniae as a nosocomial pathogen is due to its intrinsic virulence, attributed to its ability to cause invasive infection via fimbrial adhesins [27] and to the presence of a thick capsule, which acts as a possible antiphagocytic factor [28], and to toxin–antitoxin (TA) systems associated with the stability of mobile elements acquired through HGT, such as vagCD genes, which encode a functional broadspectrum TA system and are conserved on the large multipleantibiotic- resistanceconferring plasmids in this species [29]. In this study, we analysed the genomic characteristics of 40 genomes of prophages identified in 16 clinical isolates of carbapenemproducing K. pneumoniae. Moreover, we used comparative bioinformatic tools and microscopic techniques to investigate the phylogeny and evolutionary relationships of the prophages. mETHODS Origin of the K. pneumoniae genomes In this study, we analysed 16 isolates of K. pneumoniae belonging to different sequence types (STs), previously determined by multilocus sequence typing (MLST), isolated from clinical samples and harbouring the most prevalent carbapenemase genes (OXA 48, VIM and KPC β-lactamases) (Table1). The STs and carbapenemase β-lactamases were previously determined by Esteban- Cantos et al., according 5 Bleriot etal., Microbial Genomics 2020;6 Table 3. Comparison by sequence of prophage genomes and bacterial genomes Prophage size (bp)* Prophage Bacterial strain MPV international cluster‡ Name Genome accession no.† Name Genome accession no.† 33 226 ST11- OXA245phi3.1 MK416009.1 ST11- OXA245 WRXH00000000 4762 ST437- OXA245phi4.2 MK416012.1 ST437- OXA245 WRXG00000000 ST11- VIM1phi8.4 MK416020.1 ST11- VIM1 WRXC00000000 ST340- VIM1phi10.2 MK422454.1 ST340- VIM1 WRXA00000000 ST512- KPC3phi13.2 MK422449.1 ST512- KPC3 WRWY00000000 ST11- OXA48phi15.3 MK433580.1 ST11- OXA48 WRWW00000000 ST258- KPC3phi16.2 MK433582.1 ST258- KPC3 WRWV00000000 36 124 ST340- VIM1phi10.1 MK422455.1 ST340- VIM1 WRXA00000000 4901 ST11- OXA48phi15.1 MK433579.1 ST11- OXA48 WRWW00000000 39 643 ST437- OXA48phi4.1 MK416011.1 ST437- OXA48 WRXG00000000 3499 ST512- KPC3phi13.6 MK433577.1 ST512- KPC3 WRWY00000000 ST258- KPC3phi16.1 MK4333581.1 ST258- KPC3 WRWV00000000 42 666 ST11- VIM1phi8.1 MK448233.1 ST11- VIM1 WRXC00000000 4280 ST512- KPC3phi13.1 MK448235.1 ST512- KPC3 WRWY00000000 *Prophage genomes located in the genomes of different K. pneumoniae clinical strains have been placed into four size groups (33.3, 36.1, 39.6 and 42.6 kb). †Accession numbers of genomes from BioProject accession no. PRJNA565865. ‡MVP international clusters 4762, 4901, 3499 and 4280 from the MVP database (https://mvp.medgenius.info). to the methods proposed by the Pasteur Institute (https:// bigsdb. pasteur. fr/ klebsiella/ klebsiella. html) [30]. The study highlighted that K. pneumoniae ST258 and ST15 are highrisk clones in the worldwide spread of carbapenemases. Genomic DNA was isolated from the strains with a Wizard genomic DNA kit (Promega), following the manufacturer’s protocol for extraction, for subsequent sequencing of the different genomes with the MiSeq system (Illumina). Sequences of 250 bp pairedend reads of each isolate were assembled ‘de novo’ with Velvet v.1.2.10 (https://www. ebi. ac. uk/~ zerbino/ velvet/). All the assembly attributes were included in GenBank BioProject PRJNA565865 (https://www. ncbi. nlm. nih. gov/ bioproject/? term= PRJNA565865). Identification of prophages in the genome of K. pneumoniae isolates Prophages in the genomes of different strains of K. pneumoniae were identified with the phaster (Phage Search Tool –Enhanced Release) bioinformatics tool (http:// phaster. ca/). Only those temperate bacteriophages identified by the program as intact (score >90) were used for analysis in the present study. The comparative genomic analysis of the four K. pneumoniae prophages found in different isolates of K. pneumoniae was carried out with the blast Ring Image Generator (brig) program [31], which uses the sequence similarity between the bacteriophage regions and the sequences of the assembled bacterial genome. Focusing on bacteriophage clusters of the Microbe Versus Phage (MVP) database (http:// mvp. medgenius. info) in K. pneumoniae strains, we searched for possible homologies between these groups and the prophages under study. For this purpose, we used the Position- Specific Iterative Basic Local Alignment Search Tool (psiblast) to analyse the complete prophage genome against the representative sequence (i.e. the longest) of the cluster and applied the following cut off values: query coverage >60 %, identity >89 %. The MVP database can be used to study phage–host relationships [32], as it provides the user with an important list of interactions in addition to international clusters (sets of viral sequences grouped according to their sequence similarities). We searched the database for possible sequence homologies between the 40 prophages under study and the international clusters. We observed homology with 25 international clusters of K. pneumoniae (549, 934, 1013, 1319, 1690, 1920, 2329, 2808, 3207, 3429, 3546, 3969, 4280, 4331, 4575, 4762, 4864, 4868, 4901, 5263, 8737, 9608, 9912, 10255, 11441), by applying the aforementioned cutoff values. Integration of prophages In the final step of the study, the integration sites of the prophages were identified with the Standard Nucleotide 6 Bleriot etal., Microbial Genomics 2020;6 Fig. 1. Comparative genomic analysis of K. pneumoniae temperate bacteriophages displaying the highest sequence identity with cluster 4762 (query cover >99.9 %; identity >99.8 %) from the MVP database (https://mvp.medgenius.info), constructed with the brig program. The sequence (ST11- OXA245phi3.1) is located on the innermost side and is indicated in black. The double ring adjacent to the reference sequence represents the G+C content (black) and the G+C skew (purple and green). The other rings are as indicated in the key. The white parts of the rings represent absent or divergent content relative to the unknown sequence. Most of the prophages were approximately 33.3 kb in size (ST11- VIM1phi8.4, ST340- VIM1phi10.2, ST512- KPC3phi13.2, ST11- OXA48phi15.3 and ST258- KPC3phi16.2). Basic Local Alignment Search Tool (blastn) by searching for nucleotide sequence similarity between the K. pneumoniae prophages and all sequences in the National Center for Biotechnology Information (NCBI) database. Groups of proteins located before the integrase proteins were considered integration sites. Phylogenic relationships among the 40 temperate bacteriophages predicted to belong to the order Caudovirales The dot plot alignment of the nucleotide sequences of the 40 temperate bacteriophages was inferred using Genome Pair Rapid Dotter (gepard) [33], and a fasta file was constructed with all the sequences. Bacteriophages lack a universal marker gene for phylogenetic analysis; however, the use of the terminase large subunit of the tail protein or the major capsid protein is often reported [34]. In the present study, a phylogenetic analysis of the sequences of the terminase large subunit was performed with the ClustalW program (http:// www. ebi. ac. uk/ clustalw/) in mega x software [35]. A tree was generated by multiple alignments, with the neighbourjoining method. The following parameters were applied to produce the tree: (i) the number of differences was established; (ii) gaps/missing data were treated as complete deletions; (iii) the bootstrap consensus tree was inferred from 1000 replicates [36]; and (iv) the condensed tree was displayed with a value of 100 %. Isolation of the bacteriophages and transmission electron microscopy (TEm) studies Mitomycin C was used to induce bacteriophage production following the protocol described by López et al. [8]. For this purpose, 15 ml Luria–Bertani (LB) broth was inoculated with 150 µl overnight culture of strains and incubated with shaking (180 r.p.m.) at 37 °C, until the optical density measured at wavelength 600 nm (OD600) reached 0.5. Mitomycin C was then added (10 µg ml−1) and the culture was allowed to grow, with shaking (180 r.p.m.) at 37 °C, until lysis occurred, i.e. when the culture appeared clear (after approximately 2–4 h). The lysates were centrifuged at 3500 r.p.m. for 10 min, and the supernatant was filtered through a 0.22 nm filter (Millipore Express PES membrane; Merck). NaCl was added (to a final concentration of 0.5 M), and the suspensions were then mixed and left on ice for 1 h. The suspensions were centrifuged at 3500 r.p.m. for 40 min at 4 °C, and the supernatants were transferred to sterile tubes. PEG 6000 (10 %, w/v) was added 7 Bleriot etal., Microbial Genomics 2020;6 Fig. 2. Comparative genomic analysis of K. pneumoniae temperate bacteriophages displaying the highest sequence identity with cluster 4901 (% query cover >98.9; % identity >99.9) of the MVP database [https://mvp.medgenius.info], constructed with the BRIG program. The sequence (ST340- VIM1phi10.1) is located on the innermost side and is indicated in black. The double ring adjacent to the reference sequence represents the GC content (black) and the GC skew (purple and green). The white part of the rings represents absent or divergent content relative to the unknown sequence. All prophages were of size 36.1 kb (ST11- OXA48phi15.1). and dissolved by rocking the tubes at room temperature for 1 h and incubating them overnight at 4 °C. Bacteriophages were then precipitated at 3500 r.p.m. for 40 min at 4 °C and resuspended in SM buffer (0.1 M NaCl, 1 mM MgSO4, 0.2 M Tris- HCl, pH 7.5) [37]. Finally, the samples were stored at 4 °C until processed for TEM on a JEOL JEM-1011 electron microscope. Induced bacteriophage mix spot test Spot tests were carried out using the mixed induced samples of bacteriophages obtained from the purified PEG preparation used for TEM. All of them contained representative members of each cluster (A, B, C, D and E). The spot test method was a modified version of the protocol described by Raya and Hebert [38]. When the OD600 reached 0.6 nm, 200 µl culture from the 16 K. pneumoniae isolates was mixed with 4.5 ml soft agar (0.5 % NaCl, 1 % tryptone and 0.4 % agar) and poured onto TA agar plates (0.5 % NaCl, 1 % tryptone and 1.5 % agar). Once the soft medium had solidified, 15 µl bacteriophage mix solutions were added to the TA agar plates. A negative control consisting of SM buffer was included for each plate. ORF annotation The putative functions of the ORFs were determined by sequence identity with the Rapid Annotation by Subsystem Technology (rast) server (http:// rast. nmpdr. org/) and the blastprotein tool psiblast developed by the NCBI (https:// blast. ncbi. nlm. nih. gov/ Blast. cgi). In addition, we used the HHpred tool in the MPI informatics toolkit (https:// toolkit. tuebingen. mpg. de/# hhpred/) [39], which predicts functions through protein structure with a model accuracy that is competitive with that of the best servers in CASP8. The cutoff E value for annotation of proteins was <10−5 and all ORFs with E values >105 were annotated as hypothetical proteins (Critical Assessment of Technique for Protein Structure Prediction) [40]. 8 Bleriot etal., Microbial Genomics 2020;6 Fig. 3. Comparative genomic analysis of K. pneumoniae temperate bacteriophages displaying the highest sequence identity with cluster 3499 (% query cover >92; % identity>99.9) of the MVP database [https://mvp.medgenius.info], constructed with the BRIG program. The sequence (ST258- KPC3phi16.1) is located on the innermost side and is indicated in black. The double ring adjacent to the reference sequence represents the GC content (black) and the GC skew (purple and green). The whitepart of the rings represents absent or divergent content relative to the unknown sequence. All prophages wereof size 39.6 kb (ST437- OXA245phi4.1 and ST512- KPC3phi13.6). RESuLTS Predicted prophage genomes Wholegenome sequencing of the 16 carbapenemaseproducing clinical strains of K. pneumoniae (Table2) and the use of the phaster bioinformatics tool (http:// phaster. ca/) revealed the presence of a total of 40 prophages considered intact (score >90), all belonging to the order Caudovirales. Most were members of the family Myoviridae (27 prophages), and the others belonged to the families Siphoviridae (10 prophages) and Podoviridae (3 prophages). The families were assigned by sequence homologies with the most common bacteriophage indicated by phaster in the Virus- Host Database (https://www. genome. jp/ virushostdb/). After annotation of the prophage genomes (see the Supplementary Material, available with the online version of this article) with rast, blastp and HHpred, it was evident that four of them (33.3, 36.1, 39.6 and 42.6 kb) were represented in different strains of K. pneumoniae (in seven, two, three and two strains, respectively) (Table3). Comparative genomic analysis of these prophages was performed with the brig program according to their sequence similarities (Figs1–4). Comparison of the sequences with different prophage sequences and clusters from the MVP database (see Table3, Figs1–4) showed that the 33.3 kb prophage genome sequences derived from different K. pneumoniae isolates were identical in multiple prophages (ST11- OXA245phi3.1, ST340- VIM1phi10.2, ST11- OXA48phi15.3 and ST258- KPC3phi16.2). The genome sequences also displayed a 100 % sequence identity with MVP database cluster 4762, which has the highest host range (67 bacterial strains) of all phage clusters included in the MVP database and is also known to infect K. pneumoniae, among other hosts (Fig.1). Moreover, the 33.3 kb genomes displayed only partial sequence identity with genomes of the other two prophages (32.302 kb ST512- KPC3phi13.2 and 33.016 kb ST11- VIM1phi8.4), possibly due to their shorter size. We believe that prophage ST437- OXA245phi4.2, also belonging to the 33.3 kb cluster, is an incomplete prophage, as its genome is about half the size of that of the 33.3 kb genomes (18.281 kb versus 33.326 kb). The sequence of the 36.1 kb prophage genome was identical in two prophages (ST340- VIM1phi10.1 and ST11- OXA48phi15.1) and it was also identical to cluster 4901 (which showed a host range of 57 bacterial strains) (Fig.2). The sequence of the 39.6 kb prophage was identical to the genomes of three prophages (ST437- OXA48phi4.1, ST512- KPC3phi13.6 and ST258- KPC3phi16.1). In addition, cluster 3499 (which showed a 19 strain host range) had partial sequence identity with the three prophages (Fig.3). Finally, the sequence of the 42.6 kb temperate bacteriophage (prophage) (ST512- KPC3phi13.1) had an identical sequence in the prophage (ST11- VIM1phi8.1) and the cluster 4280 (Fig.4). 9 Bleriot etal., Microbial Genomics 2020;6 Fig. 4. Comparative genomic analysis of K. pneumoniae temperate bacteriophages displaying the highest sequence identity with cluster 4280 (query cover >98.9 %; identity >99.9 %) from the MVP database (https://mvp.medgenius.info) analysed with the brig program. The sequence (ST512- KPC3phi13.1) is located on the innermost side and is indicated in black. The double ring adjacent to the reference sequence represents the G+C content (black) and the G+C skew (purple and green). The other rings are as indicated in the key. The white parts of the rings represent absent or divergent content relative to the unknown sequence. All prophages were of size 42.6 kb (ST11- VIM1phi8.1). Integration sites of prophages In all prophages, the attachment sites (attL and attR) were identified by phaster. Moreover, by studying the integration sites of the prophages, we observed that these were integrated at various sites, as detailed in the following text. (i) Fourteen prophages were integrated before or after intact host tRNA (ST405- OXA48phi1.2, ST405- OXA48phi1.3, ST15- VIM1phi2, ST437- OXA245phi4.1, ST101- KPC2phi6.1, ST101- KPC2phi6.2, ST147- VIM1phi7.2, ST11- VIM1phi8.1, ST13- OXA48phi12.1, ST13- OXA48phi12.2, ST13- OXA48phi12.5, ST512- KPC3phi13.1, ST512- KPC3phi13.6 and ST258- KPC3phi16.1), with tRNA- arg being the most common host tRNA present before prophages in six cases. (ii) Seven prophages were integrated between the TerT transcriptional regulator intact genes and transporter intact genes (all bacteriophages 33.3 kb: ST11- OXA245phi3.1, ST437- OXA245phi4.2, ST11- VIM1phi8.4, ST340- VIM1phi10.2, ST512- KPC3phi13.2, ST11- OXA48phi15.3 and ST258- KPC3phi16.2), integration of the prophages did not truncate these genes. (iii) Four prophages were integrated next to the host transcriptional regulator (ST405- OXA48phi1.1, ST11- OXA245phi3.2, ST16- OXA48phi5.2 and ST846- OXA48phi9.1). In some cases (ST16- OXA48phi5.2, ST846- OXA48phi9.1), the integration of the prophage involved the disruption of the adjacent gene. (iv) Four prophages were integrated within the SapABCDEF operon that encodes an ATP- binding cassette (ABC) transporter, more precisely between sapB and sapC intact gene (ST16- OXA48phi5.1, ST11- VIM1phi8.2, ST846- OXA48phi9.2 and ST974- OXA48phi18). (v) Five prophages were integrated immediately after an intact protease (ST16- OXA48phi5.3, ST11- VIM1phi8.3, ST340- VIM1phi10.1, ST512- KPC3phi13.5 and ST11- OXA48phi15.1). (vi) Five prophages were integrated next to a protein of unknown function (ST101- KPC2phi6.3, ST147- VIM1phi7.1, ST13- OXA48phi12.3, ST13- OXA48phi12.4 and ST15- OXA48phi14). In the case of ST15- OXA48phi14, the phage integration truncated the protein of unknown function. (vii) One prophage was integrated after a sensor domaincontaining diguanylate cyclase, truncating the latter (ST16- OXA48phi5.4). Phylogenetic study To observe the phylogenetic relationships between the temperate bacteriophages under study and the clusters included in the MVP database, we reconstructed a neighbourjoining tree by the dot plot alignment of nucleotide sequences, constructed from a fasta file with all genomes of the 40 prophages studied with the gepard program. This enabled 16 Bleriot etal., Microbial Genomics 2020;6 reported to be involved in protecting the viral genome against the host restriction enzymes from the bacteria [53, 80]. Conclusion This study characterized 40 prophages (order Caudovirales) in the genomes of 16 clinical strains of K. pneumoniae belonging to different STs (MLST). The analysis revealed the presence of four size groups of prophages (33.3, 36.1, 39.6 and 42.6 kb), whose sequences were similar to those of the international hostbacteriophage clusters registered in the MVP database, 4762, 4901, 3499 and 4280, respectively. Moreover, these 33.3, 36.1, 39.6 and 42.6 kb prophages were included in three main clusters (A, B and C) by phylogenetic relationships. The annotation of prophages has revealed 40.3 % of prophage genomes encoded genes of unknown function. However, this annotation has also revealed that the second most important group of genes constituted the minimum unit of Caudovirales bacteriophages. Interestingly, we observed virulence factors (compounds of secretion systems or regulators) and components of phage defence against bacteria and also against other bacteriophages (TA modules, anti- CRISPR/Cas9, TerB protein and methyltransferase proteins) in prophage genomes. Future lines of research should focus on obtaining more information about genes of unknown function to provide a better understanding of phage genomes for possible therapeutic applications of bacteriophages, such as phagederived protein, as well as for engineering phages with activity against MDR pathogens. Funding information This study was funded by grant PI16/01163 awarded to M.T. 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 ‘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 RD16/0016/0008) and by the Study Group on Mechanisms of Action and Resistance to Antimicrobials (GEMARA) (Spanish Society of Infectious Diseases and Clinical Microbiology – SEIMC; http://www. seimc. org/). R.T. and L.F.-G. were financially supported by grants from the SEIMC and the Deputacion Provincial da Coruña (Xunta de Galicia), respectively. Conflicts of interest The authors declare that there are no conflicts of interest. Data Bibliography 1. 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