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Microorganisms 12 (4): 695 (2024)
Bhandare, Sudhakar
Lawal, Opeyemi U.
Colavecchio, Anna
Cadieux, Brigitte
Zahirovich-Jovich, Yella
Zhong, Zeyan
Tompkins, Elizabeth
Amitrano, Margot
Kukavica-Ibrulj, Irena
Boyle, Brian
Wang, Siyun
Levesque, Roger C.
Delaquis, Pascal
Danyluk, Michelle
Goodridge, Lawrence
Bacteriophages (phages) are potential alternatives to chemical antimicrobials against pathogens of public health significance. Understanding the diversity and host specificity of phages is important for developing effective phage biocontrol approaches. Here, we assessed the host range, morphology, and genetic diversity of eight Salmonella enterica phages isolated from a wastewater treatment plant. The host range analysis revealed that six out of eight phages lysed more than 81% of the 43 Salmonella enterica isolates tested. The genomic sequences of all phages were determined. Whole-genome sequencing (WGS) data revealed that phage genome sizes ranged from 41 to 114 kb, with GC contents between 39.9 and 50.0%. Two of the phages SB13 and SB28 represent new species, Epseptimavirus SB13 and genera Macdonaldcampvirus, respectively, as designated by the International Committee for the Taxonomy of Viruses (ICTV) using genome-based taxonomic classification. One phage (SB18) belonged to the Myoviridae morphotype while the remaining phages belonged to the Siphoviridae morphotype. The gene content analyses showed that none of the phages possessed virulence, toxin, antibiotic resistance, type I–VI toxin–antitoxin modules, or lysogeny genes. Three (SB3, SB15, and SB18) out of the eight phages possessed tailspike proteins. Whole-genome-based phylogeny of the eight phages with their 113 homologs revealed three clusters A, B, and C and seven subclusters (A1, A2, A3, B1, B2, C1, and C2). While cluster C1 phages were predominantly isolated from animal sources, cluster B contained phages from both wastewater and animal sources. The broad host range of these phages highlights their potential use for controlling the presence of S. enterica in foods.
Citation: Bhandare, S.; Lawal, O.U.;Colavecchio, A.; Cadieux, B.;Zahirovich-Jovich, Y.; Zhong, Z.;Tompkins, E.; Amitrano, M.;Kukavica-Ibrulj, I.; Boyle, B.; et al.Genomic and Phenotypic Analysis ofSalmonella enterica BacteriophagesIdentifies Two Novel Phage Species.Microorganisms 2024, 12, 695.https://doi.org/10.3390/microorganisms12040695Academic Editors: Lanying Zengand Junjie ZhangReceived: 8 March 2024Revised: 23 March 2024Accepted: 27 March 2024Published: 29 March 2024Copyright: © 2024 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).microorganismsArticleGenomic and Phenotypic Analysis of Salmonella entericaBacteriophages Identifies Two Novel Phage SpeciesSudhakar Bhandare 1,2,† , Opeyemi U. Lawal 3,† , Anna Colavecchio 1, Brigitte Cadieux 1, Yella Zahirovich-Jovich 1,Zeyan Zhong 1, Elizabeth Tompkins 1, Margot Amitrano 1 , Irena Kukavica-Ibrulj 4, Brian Boyle 4 , Siyun Wang 5 ,Roger C. Levesque 4 , Pascal Delaquis 6, Michelle Danyluk 7 and Lawrence Goodridge 3,*1 Food Safety and Quality Program, Department of Food Science and Agricultural Chemistry,McGill University, Montreal, QC H9X 3V9, Canada; vetsudhakar@bristolalumni.org.uk orsudhakar.bhandare@nottingham.ac.uk (S.B.)2 School of Veterinary Medicine and Science, University of Nottingham, Nottingham LE12 5RD, UK3 Canadian Research Institute for Food Safety, Department of Food Science, University of Guelph,Guelph, ON N1G 2W1, Canada; lawal@uoguelph.ca4 Institute for Integrative Systems Biology (IBIS), Laval University, Québec, QC G1V 0A6, Canada;rclevesq@ibis.ulaval.ca (R.C.L.)5 Faculty of Land and Food Systems, University of British Columbia, Vancouver, BC V6T 1Z4, Canada;siyun.wang@ubc.ca6 Agriculture and Agri-Food Canada, Summerland, BC V0H 1Z0, Canada7 Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611, USA* Correspondence: goodridl@uoguelph.ca† These authors contributed equally to this work.Abstract: Bacteriophages (phages) are potential alternatives to chemical antimicrobials againstpathogens of public health significance. Understanding the diversity and host specificity of phages isimportant for developing effective phage biocontrol approaches. Here, we assessed the host range,morphology, and genetic diversity of eight Salmonella enterica phages isolated from a wastewatertreatment plant. The host range analysis revealed that six out of eight phages lysed more than 81%of the 43 Salmonella enterica isolates tested. The genomic sequences of all phages were determined.Whole-genome sequencing (WGS) data revealed that phage genome sizes ranged from 41 to 114 kb,with GC contents between 39.9 and 50.0%. Two of the phages SB13 and SB28 represent new species,Epseptimavirus SB13 and genera Macdonaldcampvirus, respectively, as designated by the InternationalCommittee for the Taxonomy of Viruses (ICTV) using genome-based taxonomic classification. Onephage (SB18) belonged to the Myoviridae morphotype while the remaining phages belonged to theSiphoviridae morphotype. The gene content analyses showed that none of the phages possessedvirulence, toxin, antibiotic resistance, type I–VI toxin–antitoxin modules, or lysogeny genes. Three(SB3, SB15, and SB18) out of the eight phages possessed tailspike proteins. Whole-genome-basedphylogeny of the eight phages with their 113 homologs revealed three clusters A, B, and C andseven subclusters (A1, A2, A3, B1, B2, C1, and C2). While cluster C1 phages were predominantlyisolated from animal sources, cluster B contained phages from both wastewater and animal sources.The broad host range of these phages highlights their potential use for controlling the presence ofS. enterica in foods.Keywords: Salmonella; bacteriophage; diversity; host specificity; comparative phylogenomics;food safety1. IntroductionNon-typhoidal Salmonella (NTS) is the leading cause of foodborne illness in Canada [1].Globally, NTS causes approximately 80 million foodborne-related illnesses and 155,000 deathseach year [2]. Non-typhoidal Salmonellosis is caused by the main species of the genus,Salmonella enterica, which consists of six subspecies (I, II, IIIa, IIIb, IV, and VI), with subspeciesMicroorganisms 2024, 12, 695. https://doi.org/10.3390/microorganisms12040695 https://www.mdpi.com/journal/microorganismsMicroorganisms 2024, 12, 695 2 of 17I containing more than 1500 serotypes with high genetic diversity [3]. In Canada, there havebeen several large national and international outbreaks of Salmonellosis linked to vegetables,including whole onions, peaches, frozen corn, and cantaloupes, since 2020 [4–7]. Theseoutbreaks have been caused by several S. enterica serotypes (Enteritidis, Newport, Soahanina,Sundsvall, Oranienburg) and have highlighted the need for improved approaches to controlthe presence and growth of diverse S. enterica in fresh and processed fruits and vegetables,which increasingly contribute to the burden of foodborne disease.Bacteriophages (phages) are increasingly being recognized as natural antimicrobialsthat can reduce the growth and survival of foodborne pathogens (including Salmonella)during food production [8–12] because of their ability to kill their host bacteria [13,14] andalso due to the fact that phages can exhibit broad host ranges [15], making them usefulfor controlling diverse bacterial species such as S. enterica [16]. The majority of phagesdescribed in the scientific literature appear to be generally host-specific, infecting a subsetof species, strains, or serotypes due to the specificity of their host receptors [17]. Whilesome phages can infect a broad range of bacteria belonging to different serotypes, species,and/or genera [17,18], information on these phages is limited.While Salmonella phages and their genomic sequences have been well documen-ted [17,19–25], the phenotypic and genotypic diversity of Salmonella enterica means thatthere are likely many additional phages with unique features that remain to be character-ized. Understanding the biological and genomic characteristics of these phages is essen-tial to the development of phage-based antimicrobial methods to control this foodbornepathogen [13,26]. In this study, we determined the host range spectra and performedcomparative genomic and phylogenetic analyses, as well as morphological characterization,of eight S. enterica phages isolated from wastewater obtained from the Jean R. Marcottewastewater treatment plant in Montreal, QC, Canada.2. Materials and Methods2.1. Bacterial Host Strains and Their GrowthNon-typhoidal S. enterica isolates (n = 43) representing 30 serotypes, and commonlyassociated and/or previously implicated in outbreaks involving fresh produce, were ob-tained from the Salmonella foodborne Syst-OMICS database (SALFOS, Laval University,QC, Canada, https://salfos.ibis.ulaval.ca/, assessed on 22 January 2023) (Figure 1). Frozenstocks of the isolates were maintained at −80 ◦C and were revived by streaking them onLuria–Bertani (LB) agar plates followed by overnight incubation at 37 ◦C. For all experi-ments, a single colony of a respective isolate from a fresh LB agar plate was inoculated into10 mL of LB broth and incubated overnight at 37 ◦C with shaking at 150 RPM.2.2. Bacteriophage Isolation and PropagationBacteriophage isolation was carried out using pre-treated sewage sludge samples(1 L) collected from the Jean-R. Marcotte Wastewater Treatment Plant (WWTP) in Montrealby adopting standard enrichment and agar overlay techniques [27] with slight modifi-cations, as described elsewhere [28]. Twelve S. enterica isolates, originally isolated fromfood plants/fresh produce and representing 11 serotypes, were used for phage isolation(Figure 1). These serotypes included Bareilly, Braenderup, Enteriditis, Heidelberg, Infantis,Javiana, Montevideo, Newport, Saintpaul, Thompson, and Typhimurium.A sewage sample (1 L) was transported to the laboratory on ice, where it was clarifiedby centrifugation at 10,000× g for 30 min in 50 mL tubes and filtered through 0.22 µmsyringe filter units (Pall Corporation, Port Washington, NY, United States). A total of 30 mLof filtrate was mixed with 9 mL of 5× Rappaport-Vassiliadis (RV), 1 mL of 1 M CaCl2, and10 mL of an overnight culture of a Salmonella isolate, and the suspension was incubatedin a 250 mL flask at 37 ◦C for 48 h while shaking at 50 RPM. The culture was clarified bycentrifugation at 10,000× g for 10 min and the supernatant was filtered through a 0.22 µmfilter to remove bacterial debris. An aliquot (10 µL) of filtrate was spot-inoculated ona lawn of the Salmonella isolate used for isolation and the plate was incubated at 37 ◦CMicroorganisms 2024, 12, 695 3 of 17overnight. The next day, the lawns were checked for zones of lysis or individual plaques.Plaque purification was carried out multiple times using a streak plating method, aspreviously described [29], to obtain a clonal phage isolate. Phage stocks were concentratedby centrifugation (40,000× g for 2 h at 4 ◦C) and stored at 4 ◦C until further use. For routineuse, phages were propagated by the liquid lysate method [28] where a mid-exponential-phase bacterial host culture was infected with phage and incubated overnight. The nextday, the phage and the bacterial host culture was centrifuged (10,000× g for 10 min) andfiltered through a 0.45 µm syringe filter, followed by determination of the phage titer.Microorganisms 2024, 12, x FOR PEER REVIEW 3 of 18 Figure 1. Maximum likelihood tree of 55 Salmonella strains representing 30 serovars used for phage propagation and host specificity in this study. The Salmonella strains used for propagating the phages in this study are indicated with a red star. Core-genome sequence alignment was generated from draft genomes using MAFFT v7.453 and the tree that was bootstrapped with 1000 replicates for node support was constructed using FastTree v2.1.11. The scale bar at the bottom represents nucleotide substitution per site. 2.2. Bacteriophage Isolation and Propagation Bacteriophage isolation was carried out using pre-treated sewage sludge samples (1 L) collected from the Jean-R. Marcotte Wastewater Treatment Plant (WWTP) in Montreal by adopting standard enrichment and agar overlay techniques [27] with slight modifica-tions, as described elsewhere [28]. Twelve S. enterica isolates, originally isolated from food plants/fresh produce and representing 11 serotypes, were used for phage isolation (Figure 1). These serotypes included Bareilly, Braenderup, Enteriditis, Heidelberg, Infantis, Javi-ana, Montevideo, Newport, Saintpaul, Thompson, and Typhimurium. A sewage sample (1 L) was transported to the laboratory on ice, where it was clarified by centrifugation at 10,000× g for 30 min in 50 mL tubes and filtered through 0.22 µm syringe filter units (Pall Corporation, Port Washington, NY, United States). A total of 30 mL of filtrate was mixed with 9 mL of 5× Rappaport-Vassiliadis (RV), 1 mL of 1 M CaCl2, and 10 mL of an overnight culture of a Salmonella isolate, and the suspension was incu-bated in a 250 mL flask at 37 °C for 48 h while shaking at 50 RPM. The culture was clarified by centrifugation at 10,000× g for 10 min and the supernatant was filtered through a 0.22 µm filter to remove bacterial debris. An aliquot (10 µL) of filtrate was spot-inoculated on a lawn of the Salmonella isolate used for isolation and the plate was incubated at 37 °C overnight. The next day, the lawns were checked for zones of lysis or individual plaques. Plaque purification was carried out multiple times using a streak plating method, as pre-viously described [29], to obtain a clonal phage isolate. Phage stocks were concentrated by centrifugation (40,000× g for 2 h at 4 °C) and stored at 4 °C until further use. For routine i re 1. Maximum likelihood tree of 55 Salmonella strains presenting 30 se ovars used for phageation t s ifi it i this study. The Salmonella strains used for propag ting the phagesin this study are indicated with a red star. Core-genome sequence alignment was generated fromdraft genomes using MAFFT v7.453 and the tree that was bootstrapped with 1000 replicates for nodesupport was constructed using FastTree v2.1.11. The scale bar at the bottom represents nucleotidesubstitution per site.2.3. Host Range ProfilesHost ranges for each of the isolated phages were determined using the agar overlaymethod. Following the preparation of agar overlays (as described above), the lawns(prepared for each of the 43 Salmonella isolates to be tested) were spot-inoculated withaliquots (10 µL) of each phage, with titers of 108 plaque-forming units (PFUs)/mL. Thespots were allowed to dry before being incubated overnight at 37 ◦C. The scoring for lysiswas completed as reported elsewhere [30,31], where 0 indicated no lysis and +3 indicatedcomplete clear lysis.2.4. Phage DNA Isolation, Sequencing, and AnnotationGenomic DNA from the Salmonella phages was extracted using the Wizard DNAClean-Up system (A7280; Promega, Madison, WI, USA) following the modified PromegaWizard method, as described by the Center for Phage Technology, Texas A&M University,Microorganisms 2024, 12, 695 4 of 17USA [32]. Extracted DNA was purified by ethanol precipitation [33] and whole-genomesequencing (WGS) was performed on an Illumina MiSeq platform (Illumina, San Diego,CA, USA) with 300 bp paired-end libraries and 30× coverage. Raw sequence reads wereassembled using the A5 pipeline [34] and genome annotation was completed using theBacterial and Viral Bioinformatics Resource Center (formerly PATRIC) [35,36]. Annotationswere manually curated and the coding sequences (CDSs) were used to interrogate theNCBI database using BLASTP (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins(accessed on 6 January 2024)) [37]. An HHpred search of the Pfams database was used toidentify conserved protein motifs [38]. To assign a protein to a gene sequence, at least 90%identity was sought in BLASTP searches for protein motifs [39]. Based on the presence orabsence of a gene encoding integrase, phages were putatively classified as temperate orvirulent, respectively [26].2.5. Phylogenetic and Comparative Genomic AnalysesThe whole-genome alignments of the phages reported in this study and 113 homologsthat were extracted from NCBI were generated using MAFFT v7.453 [40]. Maximum likeli-hood trees were constructed using IQtree v2.2 (https://github.com/Cibiv/IQ-TREE) [41]and visualized using Microreact (https://microreact.org/ (accessed on 6 January 2024)) [42].Phages were assessed for genes encoding antimicrobial resistance, virulence, and type I-VItoxin–antitoxin modules using CARD [43], VFDB [44,45], and TADB [46], respectively. Like-wise, phage genome sequences were screened for the presence of tailspike proteins usingan in-house manually curated custom database. Whole-genome comparisons and theirvisual representations were carried out using EasyFig (https://github.com/mjsull/Easyfig)and iTOL (https://itol.embl.de) [47].2.6. Electron Microscopic Imaging of the PhagesPhages were purified for electron microscopy by equilibrium density gradient centrifu-gation through CsCl at ≈22,000 RCF for 24 h in a Beckman Ultra centrifuge (TL100) [48].Post centrifugation, the residual CsCl was removed from the phage fraction by centrifuging500 µL of the supernatant fluid through an Amicon Ultra-0.5 30 kDa MWCO centrifugalfilter unit (Millipore Ltd., Burlington, MA, USA). Following purification, transmissionelectron microscopy was conducted at the Imaging—Microscopy Platform of the Instituteof Integrative Biology and Systems (IBIS), Laval University, Quebec City, QC, Canada.3. Results3.1. Phage Isolation and Biological Characterization by Host Range ProfileEight phages (Table 1) were isolated using S. enterica isolates representing serotypes com-monly associated with fresh produce outbreaks. The host range profile of the isolated phageswas determined using 43 different Salmonella isolates from 30 serotypes (Figures 1 and 2).Salmonella isolates representing the top eight plant-associated salmonellosis-causing serotypeswere given importance due to their frequent implication in produce-associated outbreaks,which account for the majority of foodborne outbreaks [49–52]. The top eight serotypes wereidentified as serotypes Newport, Javiana, Enteriditis, Typhimurium, Thompson, Heidelberg,Saintpaul, and Poona (Figure 2). One additional serotype, Litchfield, was also included be-cause it has been implicated in numerous outbreaks associated with melons [53]. The broadesthost range was exhibited by phages SB3 and SB6, followed by SB9. Phages SB3 and SB6 lysed88.3% (n = 38/43) isolates, while phage SB9 lysed 86% (n = 37/43) isolates, phage SB10 lysed76.7% (n = 33/43), phages SB13 and SB18 lysed 83.7% (n = 36/43) isolates, phage SB28 lysed81.3% (n = 35/43), and phage SB15 showed the lowest lysis percentage of 67.4% (n = 29/43)isolates. Five of eight (62.5% (SB3, SB6, SB9, SB10, SB13)) phages isolated in this study lysedthe top eight serotypes and Litchfield (Figure 2).Microorganisms 2024, 12, 695 5 of 17Table 1. Summary of Salmonella enterica bacteriophages isolated in this study.PhageSalmonella HostStrain Used forIsolation *Morphotype New Classification Old ClassificationGenBankAccessionNumberGenome Length(Kb)No. of CodingGenes No. of tRNA G+C (%)SB3 S. enterica ser.Enteritidis (S7) SiphovirusGenus: Jerseyvirus;Species: JerseyvirusAG11Genus:Jerseyvirus;Species: Salmonellavirus AG11MK578530 41.15 63 0 50.0SB6 S. enterica ser. Javiana(S1297) SiphovirusGenus:EpseptimavirusGenus:Haartmanvirus MK809530 112.31 180 16 39.9SB9 S. enterica ser.Saint-Paul (S1326) SiphovirusGenus:EpseptimavirusGenus:Tequintavirus MK867835 113.98 180 20 39.9SB10 S. enterica ser.Typhimurium (S1295) SiphovirusGenus:Epseptimavirus;Species:EpseptimavirusfuchurGenus:Tequintavirus;Species: Salmonellavirus fuchurMK947458 111.35 183 21 40.1SB13 S. enterica ser.Typhimurium (S580) SiphovirusGenus:Epseptimavirus;Species:EpseptimavirusSB13.Genus:Tequintavirus MK947459 112.51 175 15 39.9SB15 S. enterica ser.Braenderup (S3) SiphovirusGenus: Jerseyvirus;Species: JerseyvirusAG11Genus:Jerseyvirus;Species: Salmonellavirus AG11MK759883 41.44 73 0 50.0SB18 S. enterica ser. Infantis(S43) MyovirusGenus: Ounavirinae;Species:Kolesnikvirus Ea214Genus:Ounavirinae;Species:KolesnikvirusMK759884 85.31 118 17 43.8SB28 S. Typhi ser. T42 DEF472 (S203) SiphovirusGenus: Macdonald-campvirus; Species:Macdonaldcam-pvirus SB28Was not classifiedbeyondSiphoviridae familyMK947460 45.13 73 0 46.2* Numbers in parenthesis indicate Salfos number.Microorganisms 2024, 12, 695 6 of 17Microorganisms 2024, 12, x FOR PEER REVIEW 6 of 18 virus; Species: Macdonaldcampvirus SB28 Siphoviridae family * Numbers in parenthesis indicate Salfos number. Figure 2. Heat map of the lytic spectra of isolated phages with scoring of lysis, where 0 indicates no lysis and 1 to 3 indicate clearing (3 indicates complete clear lysis). Figure 2. Heat map of the lytic spectra of isolated phages with scoring of lysis, where 0 indicates nolysis and 1 to 3 indicate clearing (3 indicates complete clear lysis).3.2. Comparative Phylogenomic Analysis of Phage UnderstudyTo ascertain the suitability of the isolated phages for biocontrol purposes, the phageswere sequenced. Raw reads were assembled into draft genomes that were functionallyannotated as described above (see Section 2).Microorganisms 2024, 12, 695 7 of 173.2.1. Comparative Gene Content Analysis of Phages under StudyThe genome size of the phages ranged from 41 to 114 kb, while their GC contentswere between 39.9 and 50.0% (Table 1). Whole-genome-based phylogenetic analysis of theeight phages revealed two clusters (cluster A and B) and two singletons (Figure 3). PhagesSB28, SB9, SB10, and SB13 were nested together in cluster A, SB15 and SB3 were clusteredin cluster B, and SB6 and SB18 were singletons. The homology of phage genomes withpreviously identified phages was determined using a comparative genomic approach withBlastN. Phages in cluster A were heterogenous in terms of their genome size, number oftRNAs, and coding sequences (Table 1). Phages SB9 and SB10 were similar to Salmonellaphage 116 (accession number: NC_048007.1; 99.58% identity and 88% coverage) andSalmonella phage fuchur (accession number: NC_048869.1; 97% identity and 96% coverage),which were both isolated from wastewater in Denmark [54] (Figure 4A). Phages SB13and SB28, respectively, had 99% (coverage: 92%) and 96% (coverage: 79%) nucleotidesimilarity to Escherichia phage Supergirl (accession number: MZ501105.1), which wasisolated from a sewage plant in Switzerland (Figure 4B) [55], and Salmonella phage E1(accession number: NC_010495.1), which was isolated from an unknown source in theUnited Kingdom (Figure 4C) [56].Microorganisms 2024, 12, x FOR PEER REVIEW 7 of 18 3.2. Comparative Phylogenomic Analysis of Phage Understudy To ascertain the suitability of the isolated phages for biocontrol purposes, the phages were sequenced. Raw reads were assembled into draft genomes that were functionally annotated as described above (see Section 2). 3.2.1. Comparative Gene Cont t Analysis of Phage under Study The genome size of the phages ranged from 41 to 114 kb, while their GC cont nts were between 39.9 and 50.0% (Table 1). Whole-genome-based phylogenetic analysis of the eight phages revealed two clusters (cluster A and B) and two singletons (Figure 3). Phages SB28, SB9, SB10, and SB13 were nested together in cluster A, SB15 and SB3 were clustered in cluster B, and SB6 and SB18 were singletons. The homology of phage genomes with previously identified phages was determined using a comparative genomic approach with BlastN. Phages in cluster A were heterogenous in terms of their genome size, number of tRNAs, and coding sequences (Table 1). Phages SB9 and SB10 were similar to Salmonella phage 116 (accession number: NC_048007.1; 99.58% identity and 88% coverage) and Salmonella phage fuchur (accession number: NC_048869.1; 97% identity and 96% coverage), which were both isolated from wastewater in Denmark [54] (Figure 4A). Phages SB13 and SB28, respectively, had 99% (coverage: 92%) and 96% (coverage: 79%) nucleotide similarity to Escherichia phage Supergirl (accession number: MZ501105.1), which was isolated from a sewage plant in Switzerland (Figure 4B) [55], and Salmonella phage E1 (accession number: NC_010495.1), which was isolated from an unknown source in the United Kingdom (Figure 4C) [56]. Figure 3. Maximum likelihood phylogenetic tree showing the relatedness of the phages under study. The phage genomes’ alignment was generated using MAFFT v7.453 and the tree that was bootstrapped with 1000 replicates for node support was constructed using IQtree v2.2. The scale bar at the bottom shows nucleotide substitution per site. Phages in the light green rectangle belonged to cluster A; those within the blue rectangle were designated as cluster B phages, and the others were singletons. The gray and white boxes depict the presence or absence of tailspike proteins in the phages. Phages SB3 and SB15 (cluster B) had a comparable genome size (41 kB), number of tRNAs, and GC content, and shared 99% nucleotide similarity between them. They also had >95% nucleotide identity (coverage: 97%) with Salmonella phage vB_SenS_AG11 (accession number: NC_041991.1), which was isolated from sewage in Guelph, Canada, in 2007 [57,58] (Figure 4D). The singletons, Phage SB6 and SB18, respectively, had 99.35 and Figure 3. Maximum likelihood phylogenetic tree showing the relatedness of the phages understudy. The phage genomes’ alignment was generated using MAFFT v7.453 and the tree that wasbootstrapped with 1000 replicates for node support was constructed using IQtree v2.2. The scale barat the bottom shows nucleotide substitution per site. Phages in the light green rectangle belongedto cluster A; those within the blue rectangle were designated as cluster B hages, and the otherswere singletons. The gray and white boxes depict the presence or absence of tailspike proteins inthe phages.Phages SB3 and SB15 (cluster B) had a comparable genome siz (41 kB), numberof tRNAs, and GC content, nd shared 99% nucleotide similarity between them. Theyalso had >95% nucleotide identity (coverage: 97%) ith Salmonella phage vB_SenS_AG11(accession number: NC_041991.1), which was isolated from sew ge in Guelph, Canada,in 2007 [57,58] (Figure 4D). The singletons, Phage SB6 and SB18, respectively, had 99.35and 98% nucleotide sequence similarity to Salmonella phage oselot (accession number:NC_048871.1; 95% coverage), recovered from wastewater in Denmark [59], and Erwiniaphage phiEa21-4 (accession number: NC_011811.1; 98% coverage), which was isolatedin soil beneath a pear tree with active blight in Canada [60,61] (Figure 4E,F). Collectively,given the broad host ranges of the phages isolated in this study, these results confirm theMicroorganisms 2024, 12, 695 8 of 17utility of wastewater as a rich source from which to isolate diverse and broad-host-rangephages [62,63].Microorganisms 2024, 12, x FOR PEER REVIEW 8 of 18 98% nucleotide sequence similarity to Salmonella phage oselot (accession number: NC_048871.1; 95% coverage), recovered from wastewater in Denmark [59], and Erwinia phage phiEa21-4 (accession number: NC_011811.1; 98% coverage), which was isolated in soil beneath a pear tree with active blight in Canada [60,61] (Figure 4E,F). Collectively, given the broad host ranges of the phages isolated in this study, these results confirm the utility of wastewater as a ric e from which to isolate diverse and broad-host-range phages [62,63]. Figure 4. Homology and gene synteny comparison of phages isolated in this study with previously sequenced phages. BlastN comparisons of the phages in this study with their closest references. (A) SB9 and SB10 with Salmonella phage fuchur; (B) SB13 with Escherichia phage Supergirl; (C) SB28 with the reference Salmonella phage E1; (D) SB3 and SB15 with Salmonella phage vB_SenS_AG11; (E) SB6 with Salmonella phage oselot, and (F) SB18 with Erwinia phage phiEa21-4. The direction of arrows indicates the DNA strand direction. Figures were generated using EasyFig. The genomes of the phages isolated in this study were screened for the presence of lysogeny, antimicrobial resistance, virulence, or toxin–antitoxin-related genes. Of note, Figure 4. Homol gy and gene s t comparison of phages isolated in this study with previouslysequenced phages. BlastN comparisons of the phages in this study with their closest references.(A) SB9 and SB10 with Salmonella phage fuchur; (B) SB13 with Escherichia phage Supergirl; (C) SB28with the reference Salmonella phage E1; (D) SB3 and SB15 with Salmonella phage vB_SenS_AG11;(E) SB6 with Salmonella phage oselot, and (F) SB18 with Erwi ia phage phiEa21-4. The direction ofarrows indicates the DNA strand direction. Figures were generated using EasyFig.The genomes of the phages isolated in this study were screened for the presence oflysogeny, antimicrobial resistance, virulence, or toxin–antitoxin-related genes. Of note,genes encoding virulence, type I-VI toxin–antitoxin modules, antimicrobial resistance,or lysogeny were not detected in any of the phages. Given the broad host range of theisolated phages, an assessment of the genes encoding bacterial recognition proteins wasconducted. Three (SB3, SB15, and SB18) out of the eight phages possessed tailspike proteins(Figure 3). The tailspike proteins from phages SB3 and SB15 were identical (100% nucleotidesequence similarity and coverage) and shared 93% nucleotide sequence similarity withthe Salmonella phage vB_SenS_AG11 tailspike protein (accession number: AF012431.1).Although annotation of the phage genome predicted that these tailspike proteins belongto the Salmonella phage P22-like tailspike protein, comparative genomic analysis revealedMicroorganisms 2024, 12, 695 9 of 17that they shared only 66% nucleotide similarities with the Salmonella phage P22 tailspikeprotein (accession number: NP_059644.1). Phages SB10, SB13, and SB18 had a bulb-likestructure at the base of the tail, typical of phages with tailspike proteins, but there wereno genes annotated as having a tailspike protein in their genome. More so, BLASTingthese phages’ genomes (SB10, SB13, and SB18) against a manually curated custom databasecontaining 8077 unduplicated tailspike proteins did not yield any significant hits. However,phage SB18 contained a gene annotated as a baseplate protein that had 97% nucleotidesimilarity to the baseplate spike protein of Erwinia phage phiEa21-4 (accession number:YP_004327040.1).3.2.2. Assessing the Genetic Relatedness of Phages under Study with Other SalmonellaPhage GenomesPhylogenetic analysis of the phages showed high genetic relatedness with otherphages. For example, based on genome comparisons with previously identified Salmonellaphages deposited in public databases, the phages were clustered into three main clustersdesignated as A, B, and C, which were further grouped into seven subclusters (A1, A2, B1,B2, C1, C2, and C3) (Figure 5). The singleton phages SB6 and SB18 (Figure 3) were placedin different subclusters of cluster C. Phage SB6 clustered with Bacteriophage T5-like insubcluster C1, whereas SB18 was found in subcluster C2 along with Erwinia phages phiEa21-4 and Salmonella phage ST-3, among others. Phage SB28 with Salmonella phage E1 formed adistinct subcluster (A1), while phages SB9, SB10, and SB13 were found in subcluster A2with other Salmonella phages (Figure 5). Conversely, phages SB3 and SB15 were clusteredin subcluster B1 together with Salmonella phage vB_SenS_AG11. In subcluster A2, phageSB9 was clustered with SB10 and SB13 along with Salmonella phages S116 and fuchur andEscherichia phage Supergirl, respectively (Figure 5).Of note, the phages that were isolated from animals/animal products were predomi-nantly found in subcluster C1. Relative to the other clusters, cluster B contained phagesisolated from more diverse sources and associated with the presence of tailspike proteins.For the phages with Salmonella as the host, serotype Typhi was enriched in cluster A(Figure 5). Overall, these results reiterate the high genetic diversity that has been observedamong S. enterica phages [25].3.3. Salmonella Phages under Study Are Morphologically and Taxonomically DifferentPhage morphological characterization was performed using transmission electron mi-croscopy (TEM). Based on the TEM imaging, all of the phages belonged to the Siphoviridaemorphotype except SB18, which belonged to the Myoviridae morphotype (Table 1). Siphoviri-dae morphotypes have long flexible tails, while Myoviridae morphotypes are defined by arigid contractile tail (ICTV) [64,65]. Images of phages representative of clusters A, B, and Care shown in Figure 6A–C, respectively.In silico analysis of the isolated phages and their comparison with existing homologsallowed for taxonomical associations. With recent changes to the taxonomic classification ofphages, major families such as Siphoviridae, Podoviridae, and Myoviridae have been abolishedby the International Committee on Taxonomy of Viruses (ICTV [64,65] (https://ictv.global/vmr/current, accessed on 6 January 2024)). Phylogenomic analysis was employed to classifythe phages into classes and genera based on the new ICTV classification schema [66]. All ofthe phages were classified into the class Caudoviricetes. The phages SB6, SB9, SB10, and SB13were classified into the family Demerecviridae, being part of the subfamily Markadamsvirinaeand the genus Epseptimavirus, and species-level classification was given only for SB10and SB13 viz. Epseptimavirus fuchur and Epseptimavirus SB13, respectively. Phage SB18belonged to the same family as phages SB6, SB10, and SB13 but was classified as beingpart of the subfamily Ounavirinae and the genus Kolesnikvirus. Phage SB28 represents anovel virus that was recently classified by ICTV as Macdonaldcampvirus at the genus leveland as Macdonaldcampvirus SB28 at the species level. Phages SB3 and SB15 were classifiedMicroorganisms 2024, 12, 695 10 of 17as being part of the subfamily Guernseyvirinae and Jerseyvirus AG11 at the genus level(https://viralzone.expasy.org/6319, accessed on 6 January 2024).Microorganisms 2024, 12, x FOR PEER REVIEW 10 of 18 Figure 5. Maximum likelihood phylogenetic tree showing the relatedness of phages under study,with previously identified Salmonella phages having a coverage of ≥80% and nucleotide homologyof ≥95%. The phage genomes’ alignment was generated using MAFFT v7.453 and the maximumlikelihood tree that was bootstrapped with 1000 replicates for node support was constructed usingIQtree v2.2. The first three blocks show the source, host, and serovars (for the Salmonella host) of thephages, while the other blocks indicate the presence and absence of tailspike proteins. The scale barat the bottom indicates the nucleotide differences amongst them. The phages under study are labeledin blue.Microorganisms 2024, 12, 695 11 of 17Microorganisms 2024, 12, x FOR PEER REVIEW 11 of 18 Figure 5. Maximum likelihood phylogenetic tree showing the relatedness of phages under study, with previously identified Salmonella phages having a coverage of ≥80% and nucleotide homology of ≥95%. The phage genomes’ alignment was generated using MAFFT v7.453 and the maximum likelihood tree that was bootstrapped with 1000 replicates for node support was constructed using IQtree v2.2. The first three blocks show the source, host, and serovars (for the Salmonella host) of the phages, while the other blocks indicate the presence and absence of tailspike proteins. The scale bar at the bottom indicates the nucleotide differences amongst them. The phages under study are labeled in blue. 3.3. Salmonella Phages under Study Are Morphologically and Taxonomically Different Phage morphological characterization was performed using transmission electron microscopy (TEM). Based on the TEM imaging, all of the phages belonged to the Siphoviridae morphotype except SB18, which belonged to the Myoviridae morphotype (Table 1). Siphoviridae morphotypes have long flexible tails, while Myoviridae morphotypes are defined by a rigid contractile tail (ICTV) [64,65]. Images of phages representative of clusters A, B, and C are shown in Figure 6A–C, respectively. Figure 6. Transmission electron microscopy (TEM) images of representative phages from different clusters. (A) Cluster A phages: subcluster A1: SB28; subcluster A2: SB9, SB10, and SB13. (B) Cluster B phages: SB3 and SB15. (C) Cluster C phages: subcluster C1: SB6; subcluster C2: SB18. In silico analysis of the isolated phages and their comparison with existing homologs allowed for taxonomical associations. With recent changes to the taxonomic classification of phages, major families such as Siphoviridae, Podoviridae, and Myoviridae have been abolished by the International Committee on Taxonomy of Viruses (ICTV [64,65] (https://ictv.global/vmr/current, accessed on 6 January 2024)). Phylogenomic analysis was employed to classify the phages into classes and genera based on the new ICTV classification schema [66]. All of the phages were classified into the class Caudoviricetes. The phages SB6, SB9, SB10, and SB13 were classified into the family Demerecviridae, being part of the subfamily Markadamsvirinae and the genus Epseptimavirus, and species-level classification was given only for SB10 and SB13 viz. Epseptimavirus fuchur and Epseptimavirus SB13, respectively. Phage SB18 belonged to the same family as phages SB6, SB10, and SB13 but was classified as being part of the subfamily Ounavirinae and the genus Kolesnikvirus. Phage SB28 represents a novel virus that was recently classified by ICTV as Macdonaldcampvirus at the genus level and as Macdonaldcampvirus SB28 at the species level. Phages SB3 and SB15 were classified as being part of the subfamily Guernseyvirinae and Figure 6. Transmission electron microscopy (TEM) images of representative phages from differentclusters. (A) Cluster A phages: subcluster A1: SB28; subcluster A2: SB9, SB10, and SB13. (B) ClusterB phages: SB3 and SB15. (C) Cluster C phages: subcluster C1: SB6; subcluster C2: SB18.4. DiscussionSalmonella enterica is a major foodborne pathogen of global importance and its ge-nomic diversity has been widely studied [67–72]. Subspecies I contains more than 1500of the total 2600+ serotypes in the species and is of utmost importance with respect tohuman infections [73]. Many studies have reported on the isolation and characterizationof Salmonella p ages, but these studies have reported on the isolation of phages fromonly a few of the 1500 serotypes from subspecies I, with a specific focus on th serotypesmost commonly implic ted in hum n diseas [74]. In this study, we isolated Salmonellaenter ca phages from wastewater and assessed their diversity and host spe ificity usinga combination of microscopic, biolo ical, and genomic approaches. Ph ge isolation wasconducted on a panel of 12 S. enterica isola es representing 11 serotypes (Figure 1). Theis ated phages w re then charac erized using a p nel of 43 highly diverse S. enterica iso-lates representing 30 serotypes (Figures 1 and 2) that re commonly associated with freshproduce outbreaks [75–77]. There are only a few reports of phages isolated f om many ofthe serotypes chosen in this study, allowing for an assessment of Salmonella phage diversityfrom food-plant-associated serotypes.Wastewater is reported to be a rich source of phages, containing a vast diversity ofboth temperate and virulent phages that infect a wide array of host bacteria, includingSalmonella [63,78]. In this study, eight S. enterica phages were isolated from the Jean-R.Marcotte WWTP in Montreal, which is the third largest WWTP in North America andprovides wastewater treatment for the entire island of Montreal [79]. Montreal (population:4.34 million) is a large urban and diverse city with inhabitants from more than 50 nationali-ties [80] and a rich history of gastronomy, meaning that a wide variety of North Americanand ethnic foods are consumed within the city as a whole. Therefore, the size of the Jean-R.Marcotte WWTP and the diversity of wastewater treated there made it an ideal location forthe isolation of a diverse group of S. enterica phages.Our results indicate that the phages isolated in this study had broad host ranges, asthe majority of the Salmonella isolates used in this study were lysed by the phages. Thebroadest host range phages (SB3, SB6, and SB9) lysed more than 85% of the isolates usedfor lytic spectra testing, which suggests the presence of one or more conserved receptorsused by these phages to infect S. enterica and indicates that these phages could be goodcandidates for a phage-based control strategy for reducing the microbial contaminationof food plant produce. In an earlier study, we demonstrated that phages SB3 and SB6Microorganisms 2024, 12, 695 12 of 17successfully reduced S. enterica populations on lettuce and cantaloupe tissues [12]. Futurestudies will focus on the elucidation of the bacterial receptor/s used by phages SB3, SB6,and SB9 to better assess their potential for use in controlling foodborne contamination dueto common and rare Salmonella serotypes. In addition, further studies on the potential ofother phages isolated in this study to reduce S. enterica on food matrices are required tofully assess their biocontrol efficacy.Whole-genome-based phylogeny of the phages sequenced in this study revealedthat two of them were unique, while the remaining six were closely related to previouslysequenced phages. The genome size and GC and gene contents of the phages were het-erogenous. Comparative genomic analysis showed that, when compared to 113 phagesfrom public databases, the eight phages from this study were grouped into three differentclusters: cluster A (SB28, SB9, SB10, and SB13), cluster B (SB3 and SB15), and cluster C (SB6,SB18) (Figure 5). Phages are known to be heterogenous and have been recognized as oneof the major drivers of diversity, evolution, and adaption of their hosts in different envi-ronmental matrices, including wastewater [17,19–25,81,82]. Salmonella Typhi phages wereenriched in cluster A, whereas phages that were isolated from animals/animal productswere enriched in cluster C. This diversity is reflected in the host- and/or source-associatedclustering of the phages in the phylogeny [20].The taxonomic classification of phages is based on electron microscopy and whole-genome sequencing [83]. The great majority of the phages sequenced in this study belongedto the Siphoviridae morphotype, while one (SB18) belonged to the Myoviridae morphotype.Two phages (SB13 and SB28) differed significantly from the previously sequenced phagegenomes and represented novel species. Based on this observation, the bacterial andarchaeal viruses subcommittee (BAVS) of ICTV has created two new species viz. Epsep-timavirus SB13 and Macdonaldcampvirus SB28 [84]. The novel genus Macdonaldcampvirushas one more species Macdonaldcampvirus ViIIE1 attributed to Salmonella phage Vi II-E1(AM491472.1). Knowledge regarding classification aids in the design of phage cocktails forbiocontrol purposes, as phages with different morphotypes use different host receptors [85]and help to overcome phage resistance [86]. Indeed, phage cocktails containing differentmorphotypes could be more effective in reducing bacterial loads in food products. In a pre-vious study, the inclusion of phages SB3 and SB6 (Siphoviridae morphotype) from this studywith three other phages belonging to the Myoviridae morphotype in a five-phage cocktailwas effective in reducing Salmonella enterica on lettuce and cantaloupe flesh sections [12].One of the safety concerns of using phages as biocontrol in food products is theirpropensity to harbor and/or facilitate horizontal gene transfer of antimicrobial resistancedeterminants in the environment [87–89]. In this study, none of the phages carried genesencoding virulence or antimicrobial resistance. Another concern arises from the pharma-cological limitations of using phages as antimicrobials. For example, there is a significantsize disparity between phage particles and antibiotic and other antimicrobial compounds,with phages being millions of times larger and composed of multiple proteins. This sizediscrepancy restricts dosing options and diminishes uptake and transportation rates [90].To address this limitation, interest is increasingly turning to utilizing phage components asantimicrobials, with a specific focus on using phage lysins that are active against Gram-positive bacteria [91]. These enzymes are not active against Gram-negative bacteria due tothe protective nature of the outer-membrane protein. More recently, several groups havedemonstrated the antimicrobial effects of phage tailspike proteins against Gram-negativebacteria. Phage tailspike proteins are highly thermostable and protease-resistant [92]. Theypossess carbohydrate depolymerase activity and recognize and cleave components of thelipopolysaccharide (LPS) to position the phage toward a secondary membrane receptorduring infection [93]. Ayariga et al. [92] demonstrated that the ε34 phage tailspike proteinhas an enzymatic property as an LPS hydrolase and synergizes with the Vero Cell culturesupernatant in killing Salmonella Newington. Miletic and colleagues [94] expressed thereceptor-binding domain of the phage P22 Gp9 tailspike protein in plant tissue (Nicotianabenthamiana) and demonstrated that upon oral administration of lyophilized leaves ex-Microorganisms 2024, 12, 695 13 of 17pressing Gp9 tailspike protein to newly hatched chickens, Salmonella concentrations werereduced on average by approximately 1-log, relative to controls. This result is underwhelm-ing but promising as each dose contained much less of the Gp9-ELP. Other studies led toreduced Salmonella motility and colonization [94–96]. In this study, three phages possessedtailspike proteins, viz. SB3 (GenBank: QBQ74073.1); SB15 (GenBank: QHI00505.1); andSB18 (GenBank: QHI00609.1). Future work will include the isolation and purification ofthese tailspike proteins and analyze them as potential antimicrobials to control S. entericain foods.5. ConclusionsIn order to study Salmonella enterica bacteriophage diversity and host specificity, weisolated and characterized eight bacteriophages using microscopic, biological, and genomicapproaches. Biological characterization by host range profile revealed that all eight phageswere broad-host-range phages; genomically, none of the phages possessed virulence, toxin,antibiotic resistance, or lysogeny genes, and we could classify them using their physi-cal/morphological characterization. Phages SB3 and SB6 had been previously identifiedand proved to be good biocontrol candidates, owing to their desirable characteristics.Phages SB3, SB6, and SB9 had the broadest host range and could be promising candidatesfor phage-based biocontrol, either alone or in a cocktail. This study reported two newphage species recognized by ICTV, i.e., Epseptimavirus SB13 and Macdonaldcampvirus SB28.Most of the phage genomes had a significant number of hypothetical proteins and this lackof understanding or the unknown functions of these proteins could be limitations to the useof these (and other) phages as biocontrol agents. Nonetheless, our attempt to understandthe diversity and host specificity of the isolated phages could contribute constructively toour understanding of phage biology and help us to better utilize this understanding inthe development of biocontrol strategies in controlling Salmonella worldwide, in variousenvironmental settings.Author Contributions: Conceptualization, L.G.; data curation, S.B. and O.U.L.; formal analysis, S.B.and O.U.L.; funding acquisition, L.G.; investigation, S.B. and O.U.L.; methodology, S.B., O.U.L., A.C.,B.C., Y.Z.-J., Z.Z., E.T., M.A., I.K.-I., B.B., R.C.L., P.D. and L.G.; project administration, L.G.; software,O.U.L.; supervision, L.G.; visualization, S.B. and O.U.L.; writing—original draft, S.B. and O.U.L.;writing—review and editing, S.B., O.U.L., S.W., R.C.L., P.D., M.D. and L.G. All authors have read andagreed to the published version of the manuscript.Funding: This research was funded by a Large-Scale Applied Research Project (LSARP) from GenomeCanada, grant number 8505.Data Availability Statement: Data are contained within the article.Acknowledgments: We thank the researchers who contributed to and/or maintained The SalmonellaFoodborne Syst-OMICS Database and researchers at the Institute for Integrative and Systems Biology,Université Laval.Conflicts of Interest: The authors declare no conflicts of interest.References1. Thomas, M.K.; Murray, R.; Flockhart, L.; Pintar, K.; Fazil, A.; Nesbitt, A.; Marshall, B.; Tataryn, J.; Pollari, F. Estimates of foodborneillness–related hospitalizations and deaths in Canada for 30 specified pathogens and unspecified agents. Foodborne Pathog. Dis.2015, 12, 820–827. [CrossRef] [PubMed]2. 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Salmonella
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diversity
host specificity
comparative phylogenomics
food safety
Genomic and Phenotypic Analysis of Salmonella enterica Bacteriophages Identifies Two Novel Phage Species
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