# Isolation of phages infecting the zoonotic pathogen Streptococcus suis reveals novel structural and genomic characteristics

Emmanuel Kuffour Osei, Reuben O'hea, Christian Cambillau, Ankita Athalye, Frank Hille, Charles M.A.P. Franz, Áine O'doherty, Margaret Wilson, Gemma G.R. Murray, Lucy A Weinert, et al.

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## To cite this version:

Emmanuel Kuffour Osei, Reuben O'hea, Christian Cambillau, Ankita Athalye, Frank Hille, et al. "Isolation of phages infecting the zoonotic pathogen Streptococcus suis reveals novel structural and genomic characteristics." Microbiological Research, 2025, 296, pp.128147. 10.1016/j.micres.2025.128147 hal-05004182

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Submitted on 25 Mar 2025

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Isolation of phages infecting the zoonotic pathogen *Streptococcus suis*
reveals novel structural and genomic characteristics

Emmanuel Kuffour Osei<sup>a,b,c</sup>, Reuben O'Hea<sup>a</sup>, Christian Cambillau<sup>a,b,d</sup>, Ankita Athalye<sup>a</sup>, Frank Hille<sup>e</sup>, Charles M.A.P.

Franz<sup>e</sup>, Áine O'Doherty<sup>f</sup>, Margaret Wilson<sup>f</sup>,

Gemma G.R. Murray<sup>g,h</sup>, Lucy A. Weinert<sup>g</sup>, Edgar Garcia Manzanilla<sup>i,k</sup>, Jennifer Mahony<sup>a,b,*</sup>,

John G. Kenny<sup>b,c,j,*</sup>

ARTICLEINFO

Keywords:

Adhesion devices Antimicrobial resistance Bacteriophage

Genomic characterisation Phage therapy *Streptococcus suis*

ABSTRACT

Bacteriophage research has experienced a renaissance in recent years, owing to their therapeutic potential and versatility in biotechnology, particularly in combating antibiotic resistant-bacteria along the farm-to-fork con- tinuum. However, certain pathogens remain underexplored as targets for phage therapy, including the zoonotic pathogen *Streptococcus suis* which causes infections in pigs and humans. Despite global efforts, the genome of only one infective *S. suis* phage has been described. Here, we report the isolation of two phages that infect *S. suis*: Bonnie and Clyde. The phages infect 58 of 100 *S. suis* strains tested, including representatives of seven different serotypes and thirteen known sequence types from diverse geographical origins. Clyde suppressed bacterial growth *in vitro* within two multi-strain mixes designed to simulate a polyclonal *S. suis* infection. Both phages demonstrated stability across various temperatures and pH levels, highlighting their potential to withstand storage conditions and maintain viability in delivery formulations. Genome comparisons revealed that neither phage shares significant nucleotide identity with any cultivated phages in the NCBI database and thereby represent novel species belonging to two distinct novel genera. This study is the first to investigate the adhesion devices of *S. suis* infecting phages. Structure prediction and analysis of adhesion devices with AlphaFold2 revealed two distinct lineages of *S. suis* phages: *Streptococcus thermophilus*-like (Bonnie) and *S. suis*-like (Clyde). The structural similarities between the adhesion devices of Bonnie and *S. thermophilus* phages, despite the lack of nucleotide similarity and differing ecological niches, suggest a common ancestor or convergent evolution, highlighting evolutionary links between pathogenic and non-pathogenic streptococcal species. These findings provide valuable insights into the genetic and phenotypic characteristics of phages that can infect *S. suis*, providing new data for the therapeutic application of phages in a One Health context.

<footnote><sup>a</sup> School of Microbiology, University College Cork, Co., Cork T12 K8AF, Ireland</footnote>

<footnote><sup>b</sup> APC Microbiome Ireland, University College Cork, Co, Cork T12 YT20, Ireland</footnote>

<footnote><sup>c</sup> Food Bioscience, Teagasc Food Research Centre, Moorepark, Co, Cork P61 C996, Ireland</footnote>

<footnote><sup>d</sup> Laboratoire d'Ingénierie des Systèmes Macromoléculaires (LISM), Institut de Microbiologie, Bioénergies et Biotechnologie (IMM), Aix-Marseille Université - CNRS, Marseille UMR 7255, France</footnote>

<footnote><sup>e</sup> Department of Microbiology and Biotechnology, Max Rubner-Institute, Hermann-Weigmann-Str. 1, Kiel 24103, Germany</footnote>

<footnote><sup>f</sup> Central Veterinary Research Laboratory, Backweston, Co, Kildare, Ireland</footnote>

<footnote><sup>g</sup> Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK</footnote>

<footnote><sup>h</sup> Department of Genetics, Evolution and Environment, University College London, Gower Street, London WC1E 6BT, UK</footnote>

<footnote><sup>i</sup> Pig and Poultry Research and Knowledge Transfer Department, Teagasc Animal and Grassland Research and Innovation Centre, Moorepark, Fermoy, Cork, P61 C996, Ireland</footnote>

<footnote><sup>j</sup> VistaMilk SFI Research Centre, Fermoy, Co, Cork P61 C996, Ireland</footnote>

<footnote><sup>k</sup> School of Veterinary Medicine, University College Dublin, Co., Dublin D04 V1W8, Ireland</footnote>

<footnote>* Corresponding authors at: School of Microbiology, University College Cork, Co., Cork T12 K8AF, Ireland.</footnote>

<footnote>E-mail addresses: j.mahony@ucc.ie (J. Mahony), John.Kenny@teagasc.ie (J.G. Kenny).</footnote>
# 1. 1. Introduction

The intensification of livestock farming systems has been predicted to promote the emergence of pathogens from within the microbiota of host populations (Murray et al., 2023). One such bacterium is *Strepto- coccus suis*, a ubiquitous coloniser of the upper porcine respiratory tract. *S. suis* is a secondary pathogen in the porcine respiratory diseases complex, a polymicrobial syndrome that affects the respiratory system of pigs. However, *S. suis* is capable of systemic dissemination resulting in diseases such as meningitis, endocarditis, arthritis, and septicaemia (Assavacheep and Thanawongnuwech, 2022). Furthermore, the threat posed by *S. suis* is not limited to porcine hosts. Less than 15 years after its discovery in pigs in 1954, *S. suis* was implicated in a case of human meningitis in Denmark (Perch et al., 1968). Subsequently, several out- breaks of human infections with similar clinical manifestations in pigs have been reported globally (Rayanakorn et al., 2018). Zoonotic trans- mission of the pathogen to humans occurs via direct contact with infected pigs, or consumption of undercooked pork products.

Although only partially understood, the pathogenicity and virulence of *S. suis* has been linked to over 100 putative virulence factors including suilysin (*sly*), muramidase-release protein (*mrp*), extracellular protein factor (*epf*), and capsular polysaccharides (*cps*) (Segura et al., 2017). CPS is a critical virulence factor involved in evasion of host immune mech- anisms and is also the basis on which the bacterium is classified into 29 serotypes (Tien et al., 2013). Serotype 2 is reported as the most common cause of *S. suis* infection in pigs and humans globally. However, at the regional level, strains of serotypes 1/2, 9, and 1 are the most common agents of *S. suis* infections in South America, Western Europe, and North America, respectively (Segura et al., 2020). Although vaccine candidates exist, efforts to produce a universal vaccine that is cross-protective is confounded by the genetic diversity of the species (Weinert et al., 2015), particularly in the *cps* loci, a common antigenic target for vaccine development. Additionally, the *cps* clusters are prone to recombination, which can result in capsular switching (Okura et al., 2021). This implies that virulent strains can evolve *cps* types not targeted by vaccines.

Antibiotics remain the primary control strategy against the path- ogen, which has contributed to the emergence and dissemination of antibiotic-resistant strains. Previous studies that examined antimicro- bial resistance (AMR) in *S. suis* reported high levels of resistance to drugs including those not used in treating *S. suis* infections (Hadjirin et al., 2021). Furthermore, several studies have reported high carriage of AMR determinants in *S. suis* genomes (Dechêne-Tempier et al., 2024; Li et al., 2024; Uruén et al., 2024). The impending disaster of AMR has led to increased interest in the potential application of bacteriophages

(phages) as an alternative/adjunct to antibiotics. Phages are viruses that can infect and kill bacteria. With an estimated abundance of 10³⁰ to 10³² particles in the biosphere, these ubiquitous biological entities have been isolated from a wide range of environments (Clokie et al., 2011). As the international community moves towards a One Health approach to tackling AMR along the farm-to-fork continuum, coupled with the consumers' growing preference for greener, antibiotic-free, sustainably farmed products, phages have emerged as a promising candidate for controlling bacterial pathogens. Phages target bacteria in a highly host-specific manner and have no reported serious adverse effects on treated subjects. As such, they have been evaluated for their potential use *in vivo* within laboratory settings, agricultural sites, and in medicine for various pathogens (Nick et al., 2022; Pirnay et al., 2024; Thanki et al., 2024, 2023). However, certain bacteria including *Gardnerella vaginalis*, *Clostridioides difficile*, *Porphyromonas gingivalis*, and *S. suis* remain poorly explored as targets for phage therapy, often due to dif- ficulties in isolating virulent phages against them. Despite global efforts, to date, only one “infective” phage (phage SMP) against *S. suis* has been isolated and genome-sequenced (Ma and Lu, 2008) while temperate phages have also been induced from *S. suis* strains (Osei et al., 2022). Despite the setbacks in isolating and characterising virulent phages, (pro)phage-derived lysins have been developed and evaluated for

bactericidal activity against *S. suis*, as with *G. vaginalis* and *C. difficile* (Arroyo-Moreno et al., 2022; Elst et al., 2020; Mondal et al., 2021; Vázquez et al., 2017). Thus, it is imperative to characterise temperate phages and explore their potential use in bacterial control using their derivatives such as phage-derived lysins or engineered phages. Furthermore, it is important to study temperate phages as phage resis- tance mechanisms have been linked to *S. suis* virulence (Atack et al., 2018).

In this study, we describe the isolation of two phages named Bonnie and Clyde, tripling the number of previously characterised phages infecting *S. suis*. The genomes of both phages were sequenced and compared with phage SMP and other close relatives, revealing both phages belong to distinct, novel genera. We also describe the phage structures and adhesion devices using electron microscopy and *in silico* predictions, respectively. Furthermore, we demonstrate that the two phages can infect and lyse *S. suis* strains of various serotypes and sequence types (STs) isolated from different countries.

# 1. 1. Materials and methods

## 1. 1. Bacterial strains

All *Streptococcus suis* strains used in this study are listed in Table S1. *S. suis* 21171_DNR38 and 19867_M106485_R39 are serotype 2 strains isolated from the respiratory tract of a diseased pigs in Denmark and Spain, respectively (Murray et al., 2023). All strains and phages were cultivated in Todd Hewitt broth (THB) (Neogen, #NCM0061B) or agar (1.5 % w/v) at 37°C under microaerophilic conditions. Serotypes of strains were determined using a two-step multiplex PCR (Table S2) (Okura et al., 2020) or *in silico* prediction based on whole genome sequencing (Athey et al., 2016). Multilocus sequence typing was per- formed with PubMLST (Jolley et al., 2018) and new allele sequences and unassigned MLST profiles were submitted to PubMLST for assignment. In total, 26 novel STs have been identified and subsequently added to the *S. suis* PubMLST database.

## 1. 1. *S. suis* phage screening and isolation

Thirty samples including oral fluids from healthy pigs across 20 farms in Ireland, along with post-mortem lung tissue from diseased pigs, were collected and processed. Oral fluids were supplemented with 0.5 M sodium chloride (NaCl) and centrifuged at 5000 g for 15 minutes. Up to 10 g of lung tissue was homogenised in a stomacher for 10 minutes in
90 mL of sterile SM buffer (50 mM Tris-HCl, 0.1 M NaCl, and 8 mM MgSO₄ [pH 7.4]) and centrifuged at 5000 g for 15 minutes. The supernatants from the oral fluids and tissue homogenates were filtered through a 0.45 µm and then a 0.22 µm polyethersulfone membrane filter (Sarstedt), and the resulting filtrates were screened for phages using the double-layer agar (DLA) plaque assay method (1.5 % agar [w/v] overlay and 0.8 % agar [w/v] overlay) (da Silva et al., 2021). A total of 50 strains were used in screening, with serotypes 2, 9, and 14 more highly represented due to their frequent association with infections (Table S1).

## 1. S. suis phage propagations and purification

To obtain a homogeneous phage lysate, five rounds of single plaque purification were performed, all carried out on strain 21171_DNR38. Subsequently, to generate phage lysates, a clonal plaque was used for plaque assays to generate confluent lysis. Then SM buffer was added to twelve such plates and the plates were incubated on a shaker at 100 rpm for 4 hours. The top layer was disrupted, and the resulting slurry was aspirated into a sterile tube. The lysate was collected by centrifugation at 4500 g for 15 minutes and filtered through a 0.45 µm filter.

For liquid propagation, an exponentially growing culture (OD600 of 0.2) supplemented with 40 mM MgCl₂ and 1 mM CaCl₂ was infected with 0.02 volumes of the plaque-purified lysate and incubated overnight. For

subsequent propagations, multiplicities of infection (MOI) of 0.1 was used to achieve high titre phage lysates. The culture was then centri-fuged, filtered, and the titre was estimated using a DLA spot assay method. Briefly, 10 µL of 10-fold serially diluted lysate was spotted on double-layer plates and incubated overnight at 37°C under aerobic conditions. The resulting plaques were counted, and the titre was expressed as PFU/mL.

## 1. Phage DNA extraction and genome sequencing

The Norgen Biotek phage DNA isolation kit was used to extract DNA from the purified phage lysates with some modifications to the manufacturer's instructions (Norgen Biotek Corp., Ontario, Canada). Briefly, 1 mL of the phage lysate (10⁸ PFU/mL) was treated with 20 U of DNase and 10X DNase buffer (#AM2238), and 1 µL of RNase A (#EN0531). The reaction was incubated at 37°C for 70 minutes followed by nuclease inactivation with 20 mM EDTA. Four microliters of proteinase K (20 mg/ mL) and 500 µL of lysis buffer was added and vortexed for 10 seconds. This was incubated in a water bath at 56°C for 30 minutes. The mixture was treated with 320 µL isopropanol (Fisher Bioreagents) and purified on a column according to the manufacturer's instructions. Phage genome libraries were prepared using the Nextera XT Library Preparation Kit and sequenced on an Illumina MiSeq sequencing system, generating 2 × 250 paired-end reads (executed by GenProbio s.r.l, Italy).

### 1. 1. Phage prediction

The genome sequence of the bacterial host strain (19867_M106485_R39, accession no. DARZKS000000000.1) was screened for the presence of prophages using geNomad with the end-to-end command (Camargo et al., 2024). Nucleotide sequences corre-sponding to 'provirus' topology hits were extracted and annotated with pharokka using default parameters (Bouras et al., 2023). The annotated genomes were manually inspected for hallmark genes using keywords such as "terminase", "capsid", "tail" and "holin".

### 1. 1. Phage induction

Phage induction from exponentially growing cultures of 19867_M106485_R39 was performed using mitomycin C (MitC), D-L-threonine coupled with temperature cycling, or UV light exposure.

For UV induction, 38 mL of THB supplemented with 10 mM MgSO₄ was inoculated with 2 mL of overnight culture and grown to an OD600 of 0.2. Subsequently, 6.5 mL aliquots of the culture were transferred into 90 mm petri dishes, achieving a depth of approximately 1 mm. The cultures were irradiated with a germicidal UV-C lamp at 253.7 nm for either 30 seconds or 2 minutes. After irradiation, the cultures were pooled and incubated at 37°C for 2 hours. The cultures were then centrifuged at 4000 xg for 10 minutes at 4°C and filtered through a 0.45 µm filter. An aliquot of the lysate was taken to visualise the presence of plaques. As low levels of induction were expected, the induction lysate was also enriched by adding 50 µL of host (21171_DNR38) culture to 1 mL of lysate, followed by overnight incubation. The propagation mix was filtered and stored at 4°C.

For mitomycin C induction, MitC was added to a 10 mL exponential-phase (OD600nm of 0.2) culture at a final concentration of 1.5 µg/mL and incubated for 15 hours. The induction mix was centrifuged, filtered, and enriched as described above.

By serendipity, an unintended prophage induction was observed when a culture was moved from 37 to 4 °C incubation overnight. Consequently, we attempted inducing the same prophage using controlled rapid temperature cycling and D-L-threonine supplementation. Briefly, 9.5 mL of THB supplemented with 40 mM D-L-threonine and 10 mM MgSO₄ was inoculated with 500 µL of overnight culture and incubated at 37°C for 3 hours, moved to 56°C for 2 minutes and then incubated at 4°C overnight. The following day, the culture was removed

from 4°C and immediately incubated at 37°C for 3 hours, followed by 1 hour at 4°C. The culture was then centrifuged, filtered, and enriched. All induction lysates were screened for plaque formation on 21171_DNR38 using the DLA spot assay as previously described. Further confirmation of successful induction was determined by amplifying the gene that encodes the terminase large subunit of the predicted prophage from the 19867_M106485_R39 strain using specific PCR primers (Table 1). The induction mixture was treated with DNase and RNase to remove host and unpackaged phage nucleic acid fragments followed by inactivation of the nucleases as described above. Universal 16S rRNA gene primer pair was used as control to detect chromosomal, non-phage bacterial gene (Okura et al., 2020). Lysates from all three methods of induction were subjected to this PCR using PCR Master Mix 2X (#K0172, ThermoFisher Scientific) according to manufacturer's instructions (Table S3, Table 1).
# 1. 1. Genome assembly and annotation

Phage genome *de novo* assembly was carried out using the SPAdes-based genome assembler Shovill (Seemann, 2024). The -trim flag and -depth 100 flag were invoked to remove Illumina adaptors and reads were subsampled to a predicted 100x coverage. The assembled contigs were checked for completeness using CheckV1.0.1 (Nayfach et al., 2021). Genome annotation was performed with Pharokka v1.7.3 (Bouras et al., 2023) and RAST pipeline (Aziz et al., 2008). The pharokka pipeline uses PHANOTATE and Prodigal for gene prediction and assigns functional annotations using MMseqs2 by matching predicted coding sequence (CDS) to the PHROGs, VFDB and CARD databases. The -dnaapler command was used to reorient phage genomes to begin with the large terminase subunit-encoding gene. To improve annotations, the genbank (.gbk) output from pharokka was set as input for phold, a tool which utilises foldseek and colabfold to predict structural homology (Bouras, 2024). Lifestyle of phages was predicted with PhageTYP (Shang et al., 2023). Circular genome maps of phages were constructed with phold plot command. Auxilliary metabolic genes (AMGs) encoded in phage genomes were screened using DRAM-v and VIBRANT (Kieft et al., 2020; Shaffer et al., 2020). An *in silico* screen of anti-viral defense systems was performed using DefenseFinder (Tesson et al., 2022). Protein sequences of phage endolysins were extracted and concatenated for alignment with Clustal Omega (Sievers and Higgins, 2017). Seqvisr (vragh, 2022) was used to visualise amino acid similarity using SMP endolysin as reference.

# 1. 1. Phylogeny and comparative analysis of S. suis (pro)phages

Genomes of (pro)phages that share significant nucleotide homology with the phages isolated in this study were identified with BLASTN using the core nucleotide database in two searches. For both searches, only sequences with 50% query coverage were considered in subsequent analyses. In the first search, the taxid 1307 was excluded to filter out bacterial (i.e. S. suis) hits. This returned few viral (phage) sequences that met the criteria. The search was repeated without the filter and top hits were bacterial (Table 2). The regions in the bacterial genomes (prophages) that share significant homology with the phages were extracted with geNomad as described above. A viral proteomic tree was generated in VipTree using the "with reference" setting (Nishimura et al., 2017). The tree was constructed based on tBLASTx computations of genome-wide similarities. Intergenomic similarities among the phages and their closest relatives were estimated using VIRIDIC. Phag- eGCN and taxmyPHAGE pipelines were used to predict phage taxonomic classification (Millard et al., 2024; Shang et al., 2023). VICTOR (http://ggdc.dsmz.de/victor.php) was used to generate a phylogenomic tree. It estimated the nucleotide pairwise similarity using the Genome-BLAST Distance Phylogeny (GBDP) method. The computed intergenomic distances were used to infer a balanced minimum evolution tree with branch support via FASTME including subtree pruning.

Primer sequences and PCR conditions for phage detection.

<table><thead><tr><th>Primer ID</th><th>Sequence 5' to 3'</th><th>Target (bp)</th><th>An<br>neal<br>lin<br>g/ti<br>me</th><th>Extension/time</th></tr></thead><tbody><tr><td>TerF</td><td>ACGGCTA<br>TGCTAT<br>CCACGG</td><td>Terminase</td><td>56°<br>C/<br>30<br>sec<br>on<br>ds</td><td>72°C /90 seconds</td></tr><tr><td>TerR<br>16SF<br>seconds<br>16SR</td><td>GCCGTGATGATTTCGCTGAC<br>GAGTTTGATCCTGGCTCAG<br>AGAAAGGAGGTGATCCAGCC</td><td>(988)<br>16S rRNA<br>(1542 bp)</td><td></td><td>56°C/ 30 seconds<br>72°C /90</td></tr></tbody></table>

Primer sequence and cycling conditions for detection of prophage and chromosomal DNA. 16SF and 16SR target non-phage DNA of bacterial chromosome. Further details about reagent concentrations and cycling details are provided in Table S3.

# Table 2

Summary of sources of (pro)phage sequences used in phylogenomic and comparative analyses.

<table><thead><tr><th>Genome ID</th><th>Sequence origin</th><th>Accession No.</th><th></th><th></th></tr></thead><tbody><tr><td>NLS50</td><td>bacterial</td><td>CP134488.1</td><td></td><td></td></tr><tr><td>SC183</td><td>bacterial</td><td>CP071305.1</td><td></td><td></td></tr><tr><td>HA1003</td><td>bacterial</td><td>CP030125.1</td><td></td><td></td></tr><tr><td>HN105</td><td>bacterial</td><td>CP029398.1</td><td></td><td></td></tr><tr><td>M104170_C2</td><td>bacterial</td><td>CP134469.1</td><td></td><td></td></tr><tr><td>YZDH1</td><td>bacterial</td><td>CP065430.1</td><td></td><td></td></tr><tr><td>YSJ17</td><td>bacterial</td><td>CP032064.1</td><td></td><td></td></tr><tr><td>HN38</td><td>bacterial</td><td>CP116604.1</td><td></td><td></td></tr><tr><td>ISU2912</td><td>bacterial</td><td>CP017785.1</td><td></td><td></td></tr><tr><td>MA8</td><td>bacterial</td><td>CP085085.1</td><td></td><td></td></tr><tr><td>90-1330</td><td>bacterial</td><td>CP012731.1</td><td></td><td></td></tr><tr><td>ISU2660</td><td>bacterial</td><td>CP031379.1</td><td></td><td></td></tr><tr><td>DNR43</td><td>bacterial</td><td>CP102143.1 Streptococcus phage Javan597</td><td>prophage MK448818.1</td><td></td></tr><tr><td>Streptococcus phage Javan584</td><td>prophage</td><td>MK448990.1 Streptococcus phage Javan565</td><td>prophage MK448806.1</td><td></td></tr><tr><td>Streptococcus phage Javan577</td><td>prophage</td><td>MK448812.1 Streptococcus phage phiZJ20091101-3</td><td>prophage KX077894.1</td><td></td></tr><tr><td>Streptococcus phage Javan580</td><td>prophage</td><td>MK448989.1 Streptococcus phage Javan548</td><td>prophage MK448981.1</td><td></td></tr></tbody></table>
<table><tr><td>Streptococcus phage phi20c</td><td>prophage</td><td>KC348598.1 Streptococcus phage Javan551</td><td>prophage MK448803.1</td></tr><tr><td>Streptococcus phage phi7917</td><td>prophage</td><td>KC348601.1 Streptococcus phage Javan578</td><td>prophage MK448988.1</td></tr><tr><td>Streptococcus phage phi891591</td><td>prophage</td><td>KC348602.1 Streptococcus phage Javan583</td><td>prophage MK448814.1</td></tr><tr><td>Streptococcus phage Javan566</td><td>prophage</td><td>MK448985.1 Streptococcus phage Javan589</td><td>prophage MK448816.1</td></tr><tr><td>Streptococcus phage SMP cultivated phage EF116926</td><td></td><td></td><td></td></tr></table>

List of genomes identified through BLASTn searches against the core nucleotide database using nucleotide sequences of phages isolated in this study. Results are derived from two distinct searches: (1) excluding taxid 1307 (prophage or cultivated phage) and (2) without filtering to include bacterial genomes. Only sequences with ≥ 50 % query coverage were retained for analysis.

and regrafting postprocessing for the D4 formula. Branch support was inferred from 100 pseudo-bootstrap replicates each. VirClust was used for orthologous genes prediction, virus clustering and the estimation of core proteins (Moraru, 2023).

## 1. 1. Host range analysis, efficiency of plating, and phage killing assay

To determine the host range of the two *S. suis* phages, 10 µL of lysate was spotted onto a double-layer agar lawn of each of the 100 host strains (Table S1). The plates were incubated overnight at 37°C. Host sensitivity was scored as complete/turbid lysis ( ) or no lysis (-). Host sensitivity was confirmed by determining the efficiency of plating (EOP). Diluted phages were spotted on strains shown to be sensitive to a phage, and the plaques counted. The results were expressed as the titre of phages on the test strain relative to the titre on the original host strain.

The *in vitro* lytic activity of phages on sensitive strains was assessed by infecting exponentially growing host cells with phages at MOI of 0.1, 1, 10 and 100. Phages were serially diluted in a 96-well plate and equilibrated by incubating the plate at 37°C for 30 minutes. The phage dilutions were then inoculated with bacteria to a final concentration of

10⁷ CFU/mL. Prior to each reading, the plate was shaken for 5 seconds, and the absorbance at OD600nm was measured every 10 minutes over 24 hours using a microplate reader (Biotek Synergy HT Plate Reader, USA). Wells containing only phage suspension or only bacterial culture served as controls. The results are presented as the average of three independent replicates.

## 1. 1. Thermal and pH stability

The temperature stability of *S. suis* phages was evaluated by incubating phage lysates at 4, 37, 40, 45, 50, 60, or 70°C for 1 hour, followed by estimating the titres using DLA spot assays. For pH stability, the pH of SM buffer was adjusted with either NaOH or HCl to obtain a pH of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. Phage pH stability was assessed by adding 50 µL of 10⁸ PFU/mL phage to 450 µL of pH-adjusted SM buffer. The mixture was incubated at 37°C for 1 hour, 2 hours or 24 hours, and the titres were estimated using the DLA method.

## 1. 1. One-step growth curve

The latent period and burst size of the phages were determined in a one-step growth experiment as described by Kropinski (Kropinski, 2018). Briefly, a 10 mL exponential-phase host culture was centrifuged and resuspended in 900 µL of THB. Phage lysate was added at a final concentration of MOI 0.01 and allowed to adsorb for 15 minutes at 37°C. Unadsorbed phages were removed by centrifugation, and the pellet was resuspended in 10 mL prewarmed medium. The mixture was incubated at 37°C for 2 hours, with 200 µL aliquots taken every 10 minutes, centrifuged and the supernatant diluted to estimate phage titres. The calculated PFU/mL was plotted against time and burst size and latent period were estimated from the graph (Kropinski, 2018).

## 1. 1. Transmission electron microscopy

Two litres of phage lysate of Bonnie and Clyde was centrifuged to remove debris, filtered, treated with 10 % (w/v) polyethylene glycol 8000, and incubated at 4°C overnight. The phage particles were harvested by centrifugation (10,000 g, 15 min) and resuspended in 4 mL of SM buffer. An equal volume of chloroform was used to extract phages from the PEG suspension three times. Purification of phage lysate was performed by loading the phage on a discontinuous cesium chloride (CsCl) gradient (5 M and 3 M) followed by centrifugation at 13,170 g for 2.4 hours at 4°C (Beckman Coulter Optima L-90K series). Phage bands were extracted and dialysed against SM buffer (50 mM Tris-HCl, 0.1 M NaCl, and 8 mM MgSO4 [pH 7.5]). Phages were pipetted on 100-mesh copper grids coated with carbon and allowed to adsorb for 20 minutes. Subsequently, the grids were washed twice with deionised water and negatively stained with 2 % uranyl acetate for approximately 20 seconds. Electron micrographs were generated on a Talos L120C transmission electron microscope using a 4 k x 4 k Ceta camera set to an acceleration voltage of 80 kV. Phage size was measured using the TEM analysis software Velox v3.9.0 (all equipment from ThermoFisher Scientific, Eindhoven, Netherlands) and the mean and standard deviation of at least 10 measured phage particles were calculated. The TEM images were manually improved using GIMP v2.10.32 for contrast and

<table><caption>**Table 3**
Host range of Bonnie and Clyde.</caption><thead><tr><td>Strain</td><td>Bonnie</td><td>Clyde</td><td>Serotype</td><td>ST</td><td>Year</td><td>Pathotype</td><td>Country</td></tr></thead><tbody><tr><td>94_8576_4887</td><td>-</td><td>+</td><td>14</td><td>28</td><td>1994</td><td>Respiratory</td><td>Canada</td></tr></tbody></table>
<table><tr><td>89_5046</td><td>-</td><td>+</td><td>2</td></tr><tr><td>89_6891_2</td><td>-</td><td>+</td><td>2</td></tr><tr><td>90_2741_7</td><td>-</td><td>+</td><td>2</td></tr><tr><td>1602951</td><td>-</td><td>+</td><td>2</td></tr><tr><td>1596201</td><td>-</td><td>+</td><td>14</td></tr><tr><td>1463646</td><td>-</td><td>+</td><td>14</td></tr><tr><td>1667796</td><td>-</td><td>+</td><td>2</td></tr><tr><td>1093404</td><td>-</td><td>+</td><td>27</td></tr><tr><td>21137_DNR4</td><td>-</td><td>+</td><td>2</td></tr><tr><td>21136_DNR3</td><td>-</td><td>+</td><td>2</td></tr><tr><td>21135_DNR2</td><td>-</td><td>+</td><td>2</td></tr><tr><td>21169_DNR36</td><td>+</td><td>+</td><td>2</td></tr><tr><td>21171_DNR38</td><td>+</td><td>+</td><td>2</td></tr><tr><td>21172_DNR39</td><td>+</td><td>+</td><td>2</td></tr><tr><td>21173_DNR40</td><td>-</td><td>+</td><td>2</td></tr><tr><td>21176_DNR43</td><td>-</td><td>+</td><td>2</td></tr><tr><td>D1</td><td>-</td><td>+</td><td>2</td></tr><tr><td>D3</td><td>+</td><td>+</td><td>2</td></tr><tr><td>D5</td><td>-</td><td>+</td><td>2</td></tr><tr><td>D6</td><td>+</td><td>+</td><td>2</td></tr><tr><td>D7</td><td>+</td><td>+</td><td>2</td></tr><tr><td>D8</td><td>-</td><td>+</td><td>2</td></tr><tr><td>D10</td><td>-</td><td>+</td><td>2</td></tr><tr><td>D14</td><td>-</td><td>+</td><td>2</td></tr><tr><td>D16</td><td>-</td><td>+</td><td>14</td></tr><tr><td>D17</td><td>+</td><td>+</td><td>2</td></tr><tr><td>D19</td><td>-</td><td>+</td><td>2</td></tr><tr><td>D23</td><td>-</td><td>+</td><td>14</td></tr><tr><td>D24</td><td>-</td><td>+</td><td>2</td></tr><tr><td>D29</td><td>-</td><td>+</td><td>7</td></tr><tr><td>D31</td><td>-</td><td>+</td><td>4</td></tr><tr><td>D32</td><td>+</td><td>+</td><td>2</td></tr><tr><td>D35</td><td>-</td><td>+</td><td>2</td></tr><tr><td>D42</td><td>-</td><td>+</td><td>16</td></tr><tr><td>D46</td><td>+</td><td>+</td><td>2</td></tr><tr><td>D49</td><td>-</td><td>+</td><td>16</td></tr><tr><td>D52</td><td>+</td><td>+</td><td>2</td></tr><tr><td>D57</td><td>-</td><td>+</td><td>2</td></tr><tr><td>D58</td><td>+</td><td>+</td><td>14</td></tr><tr><td>D68</td><td>+</td><td>+</td><td>2</td></tr><tr><td>D71</td><td>+</td><td>+</td><td>14</td></tr><tr><td>D75</td><td>-</td><td>+</td><td>9</td></tr><tr><td>D78</td><td>-</td><td>+</td><td>2</td></tr><tr><td>D79</td><td>-</td><td>+</td><td>2</td></tr><tr><td>D84</td><td>-</td><td>+</td><td>2</td></tr><tr><td>D93</td><td>-</td><td>+</td><td>2</td></tr><tr><td>D94</td><td>-</td><td>+</td><td>14</td></tr><tr><td>19858_M105281_R30</td><td>-</td><td>+</td><td>7</td></tr><tr><td>19867_M106485_R39</td><td>+</td><td>+</td><td>2</td></tr><tr><td>19779_M101513_S1</td><td>-</td><td>+</td><td>2</td></tr><tr><td>19797_M104300_S19</td><td>-</td><td>+</td><td>2</td></tr><tr><td>19798_M104300_S20</td><td>-</td><td>+</td><td>2</td></tr><tr><td>19802_M105040_S24</td><td>-</td><td>+</td><td>2</td></tr><tr><td>19803_M105040_S25</td><td>-</td><td>+</td><td>2</td></tr><tr><td>19806_M105244_S28</td><td>-</td><td>+</td><td>2</td></tr><tr><td>19807_M105244_S29</td><td>-</td><td>+</td><td>2</td></tr><tr><td>19785_M102095_S7</td><td>+</td><td>+</td><td>2</td></tr></table>

brightness adjustment.

<table><tr><td>Systemic</td><td>Canada</td></tr><tr><td>Respiratory</td><td>Canada</td></tr><tr><td>Systemic</td><td>Canada</td></tr><tr><td>Respiratory</td><td>Canada</td></tr><tr><td>Systemic</td><td>Canada</td></tr><tr><td>Systemic</td><td>Canada</td></tr><tr><td>Systemic</td><td>Canada</td></tr><tr><td>Respiratory</td><td>Denmark</td></tr><tr><td>Respiratory</td><td>Denmark</td></tr><tr><td>Respiratory</td><td>Denmark</td></tr><tr><td>Respiratory</td><td>Denmark</td></tr><tr><td>Respiratory</td><td>Denmark</td></tr><tr><td>Respiratory</td><td>Denmark</td></tr><tr><td>Unknown</td><td>Ireland</td></tr><tr><td>Respiratory</td><td>Ireland</td></tr><tr><td>Systemic</td><td>Ireland</td></tr><tr><td>Systemic</td><td>Ireland</td></tr><tr><td>Unknown</td><td>Ireland</td></tr><tr><td>Respiratory</td><td>Ireland</td></tr><tr><td>Systemic</td><td>Ireland</td></tr><tr><td>Systemic</td><td>Ireland</td></tr><tr><td>Unknown</td><td>Ireland</td></tr><tr><td>Respiratory</td><td>Ireland</td></tr><tr><td>Respiratory</td><td>Ireland</td></tr><tr><td>Unknown</td><td>Ireland</td></tr><tr><td>Unknown</td><td>Ireland</td></tr><tr><td>Respiratory</td><td>Ireland</td></tr><tr><td>Respiratory</td><td>Ireland</td></tr><tr><td>Unknown</td><td>Ireland</td></tr><tr><td>Unknown</td><td>Ireland</td></tr><tr><td>Respiratory</td><td>Ireland</td></tr><tr><td>Respiratory</td><td>Ireland</td></tr><tr><td>Respiratory</td><td>Ireland</td></tr><tr><td>Systemic</td><td>Ireland</td></tr><tr><td>Systemic</td><td>Ireland</td></tr><tr><td>Systemic</td><td>Ireland</td></tr><tr><td>Systemic</td><td>Ireland</td></tr><tr><td>Systemic</td><td>Ireland</td></tr><tr><td>Systemic</td><td>Ireland</td></tr><tr><td>Systemic</td><td>Ireland</td></tr><tr><td>Systemic</td><td>Ireland</td></tr><tr><td>Systemic</td><td>Ireland</td></tr><tr><td>Systemic</td><td>Spain</td></tr><tr><td>Respiratory</td><td>Spain</td></tr><tr><td>Systemic</td><td>Spain</td></tr><tr><td>Systemic</td><td>Spain</td></tr><tr><td>Systemic</td><td>Spain</td></tr><tr><td>Systemic</td><td>Spain</td></tr><tr><td>Systemic</td><td>Spain</td></tr><tr><td>Systemic</td><td>Spain</td></tr><tr><td>Systemic</td><td>Spain</td></tr></table>
Structures of the adhesion device proteins (Dit, Tal and/or RBP) of Bonnie and Clyde were predicted using AlphaFold2 as well as those of SMP using AlphaFold3 via google servers at https://golgi.sandbox.googLe.com (Abramson et al., 2024; Jumper et al., 2021). Stretches of Tal with overlapping segments were predicted to allow the assembly of full-length multimers using Coot (Emsley et al., 2010). pLDDT and PAE values are reported in Fig. S1. The final predicted domain structures were submitted to the Foldseek server to identify closest structural homologs in the PDB (van Kempen et al., 2024). Sequence alignments were

performed with Multalin (Corpet, 1988). Visual representation of the structures were prepared with ChimeraX (Pettersen et al., 2004).

## 1. 1. Statistical analysis

All quantitative datasets generated from in vitro experiments were analysed by estimating mean and standard error of the mean using independent biological replicates, each conducted on different days with different bacterial cultures and fresh phage lysates. GraphPad Prism 10.2.0 (USA) was used for the statistical analysis. Two-way analysis of variance (ANOVA) with Dunnett's multiple comparisons test was used to evaluate temperature stability data at a statistical significance level of 0.05. The statistical significance of pH stability data was estimated using

Kruskal-Wallis test. For phage *in vitro* lytic activity, one-way ANOVA with Tukey's post-hoc test was performed to determine statistically significant differences. The comparisons were made between the test MOIs and the no-phage control.

## 1. 1. Results

### 1. 1. Isolation of S. suis infecting phages, Bonnie and Clyde

Initially, one phage was isolated from a post-mortem lung sample. This phage was initially plaque-purified on strain 19867_M106485_R39 and subsequently sequenced. However, sequencing results revealed the presence of two contigs in the supposed single phage lysate with contig coverages of 67.3 and 36.9 and sizes of 36,310 bp and 34,743 bp, respectively. CheckV and pharokka annotations confirmed that the contigs represented two distinct phages. To investigate the source of the two phages, we first screened the genome of the propagation host for prophages using geNomad. Two "provirus" topology hits were predicted: a 34 kb prophage and a 13 kb region (Table S4). Nucleotide BLAST (core nucleotide database) results of the 34 kb prophage region showed a 100% match to the second contig. This indicated that the 34 kb prophage was induced from the propagation host. To isolate the two phages individually, a suitable host strain that would minimise the risk of induction was needed. Accordingly, we screened the genomes of susceptible hosts to identify strains without prophages or with prophage-elements that lack hallmark genes including capsid and tail proteins. Strain 21171_DNR38 was found to harbour a 13 kb provirus that encodes no structural genes and was therefore used for further plaque purification. Following 22 successive passages on strain 21171_DNR38, clonal plaques corresponding to the first phage of 36 kb (vB_SsuS-Bonnie hereinafter referred to as Bonnie) were obtained. This was initially verified by PCR using primer pairs that targeted genes encoded by either of the two co-propagated phages (Table S3). The second phage was (re)isolated by inducing it from its host (19867_M106485_R39) through UV-light exposure, MitC induction, or temperature cycling. Lysates emanating from all three methods produced translucent plaques, however, following enrichment, clear spots and transparent plaques were observed (Fig. 1A). Furthermore, PCR was used for the preliminary identification of the phage in the induction lysate, matching it to both the predicted prophage and the second phage/contig (Fig. 1B), vB_SsuS-Clyde (hereinafter referred to as Clyde). The names were inspired by Bonnie Parker and Clyde Barrow, reflecting how inseparable the phages were on strain 19867_M106485_R39. All three methods for inducing phage Clyde were successful; however, UV

induction-derived lysate was used for subsequent experiments based on the higher titres achieved after the enrichment.

### 1. 1. Bonnie and Clyde are temperate phages

Genomic DNA was extracted from purified lysates of Bonnie and Clyde, sequenced, and assembled into genomes of 36,160 bp and 34,734 bp, with average GC content of 40.51 and 41.87%, respectively, similar to that of the propagation host (41.44%). Bonnie has a coding density of 96.74%, encoding 72 coding sequences (CDS) of which 44 could not be assigned a function by pharokka. Clyde has coding density of 95.07%, which encodes 64 CDS with 41 of unknown function. CDS annotations improved following application of the structural homology tool, PHOLD. The number of proteins with assigned function improved from 28 to 35 and 23-33 for Bonnie and Clyde, respectively.

The CDSs with assigned functions could be grouped into structural (tail and capsid), lysis, integration & excision, nucleotide metabolism, and transcriptional regulation. Neither phage genome was predicted to encode tRNAs, AMR genes, toxins, or virulence factors. However, integrases, repressors, and other genes involved in lysogeny-lysis decision making were identified in both genomes. The lifestyle of the phages was further confirmed as temperate based on PhageTYP predictions (Table S5). Moreover, DefenseFinder identified Retron_VII_2_DUF3800, one of two genes involved in Retron Type VII anti-viral system in Clyde implying a role in superinfection immunity. A DNA methyltransferase involved in the cytosine and methionine metabolism pathways was detected within the nucleotide metabolism module of Bonnie using the VIBRANT pipeline. No AMGs were identified in Clyde.

### 1. 1. Phylogeny and comparative analysis of Bonnie and Clyde

A viral proteomic tree was constructed using the nucleotide sequences of both phages in ViPtree. The resulting tree included 1083 dsDNA viruses including Bonnie and Clyde (Fig. 3). Bonnie clustered closely with *Streptococcus* prophage phiD12 and four phages infecting *S. pneumonia* and *S. oralis*. Although distant, Clyde clustered with the *S. suis* phage SMP but more closely with *S. suis*-infected prophage phi20c. Other members within the same clade include phages that infect
*Lactococcus lactis*, *S. parauberis*, *S. mitis* and *S. pyogenes*. The genome size of all the related phages (Fig. 3) ranged from 31–49 kb. Taxmyphage and PhageGCN placed Bonnie and Clyde in the class level (Caudoviricetes). Neither phage could be classified into any genus or species recognised by the International Committee on Taxonomy of Viruses (ICTV).

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Confirmation of induction of phage Clyde by three methods. (A) Spot assays showing filtered induction lysates on sensitive strain 21171_DNR38 before (I, III, V) and after enrichment (II, IV, VI) on overlay plates for UV, MitC, and temperature cycling prophage induction, respectively. (B) PCR products of terminase (T) and 16S rRNA (16S) gene amplified from filtered, nuclease-treated lysates induced by UV, MitC, and temperature-dependent threonine induction. A crude induction mixture (non-filtered and non-nuclease treated) and a culture of host strain 19867_M106485_R39 were used as positive controls. Negative controls included strain 21171_DNR38, which does not harbour the expected prophage, and water. M1 and M2 indicate 100 bp and 1 kb DNA ladders, respectively.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Circular genome map of *S. suis* phages generated with PHOLD. (A) Bonnie and (B) Clyde. CDSs are represented as arrows indicated in the direction they are encoded. Arrows are colour-coded based on function. Nucleotide sequences of Bonnie and Clyde are available on GenBank under accession numbers PQ720431 and PQ720432, respectively.</figcaption></figure>

Furthermore, searches against the NCBI core nucleotide database using BLASTn did not identify any cultured phage with significant homology to Bonnie and Clyde. However, some deposited prophage sequences and regions in bacterial genomes (*S. suis*) shared some nucleotide
similarity with Bonnie and Clyde (with at least 50 % query coverage). Then together with the deposited prophage sequences and phage SMP, the intergenomic similarities were estimated in VIRIDIC. Three redundant prophages extracted from strains ISU2660, 90–1330, and DNR43 were subsequently removed as they were identical to a prophage in strain MA8 resulting in a total of 28 (pro)phages. The closest relative of Clyde is *Streptococcus* phage phi20c, a prophage from *S. suis* strain 8067, with a 52.4 % intergenomic similarity. Bonnie shared

75.8 % similarity with a predicted prophage from strain NLS50, which was isolated from a pig in the Netherlands in 2017 (Fig. 4A). The closest
deposited prophage sequence to Bonnie is *Streptococcus* phage Javan597, which shares 61.8 % intergenomic similarity. Furthermore, Clyde shares
only 0.4 % intergenomic similarity with Bonnie and 16.5 % with phage SMP which is higher than Bonnie shares with SMP (4.4 %).

species (Table S6). As predicted above, Bonnie and NLS50_prophage fall into two different species within a genus cluster (8). However, Clyde falls
within a genus cluster (4) with its closest relative, phage phi20c, as well as *Streptococcus* phage Javan548 and Javan551. Phage SMP is the sole
representative of its genus and species clusters (Fig. 4B).

The genome composition of Bonnie and Clyde and their closest rel- atives was analysed, including a predicted prophage (NLS50_prophage) and a deposited prophage (Javan597) for Bonnie, as well as phi20c for Clyde, with SMP used as a reference. Analysis with VirClust predicted 196 protein clusters among the six phages and each phage encoded 6–22 unique proteins (Table S7 and S8), however, no protein clusters were shared by all the six phages. One gene and three other genes were shared by five and four phages, respectively. Apart from Clyde, the other five phages encoded a conserved single-stranded DNA binding protein (SSB) with an average amino acid similarity of 84.8 %. Other genes shared by four of the six phages included N-acetylmuramoyl-L-alanine amidase (84.3 %), tail tape measure protein (58.8 %), and an unknown protein (38.7 %) (Table S9). Comparative genome alignment revealed

Thus, based on ICTV genus (≥ 70 % nucleotide identity) and species
(≥ 95 % nucleotide identity) delineation for dsDNA bacterial and
high level of synteny particularly in the gene order of the head &
packaging, as well as the tail modules. While this synteny was observed

archaeal viruses, phage Clyde represents a member of a novel species in a novel genus. Similarly, phage Bonnie belongs to a different novel species within a novel genus that includes the uncultivated prophage from strain NLS50. Additional phylogenomic analysis was conducted using VICTOR to generate a tree from the nucleotide sequence of the 28 (pro)phages to infer evolutionary relationships among the phages. This comparison yielded eight genus clusters spread across twenty-two

in all six phages, the average homology in these modules is low and conserved only among closest relatives (Fig. 5).

Apart from using whole phages in bacterial control, phage lysins have been characterised and purified for use against pathogens. The bactericidal activity of *S. suis* (pro)phage-derived lysins such as LySMP, Csl2, Ly7917, PlySs2 and PlySs9, as well as holins like HoISMP, has been experimentally validated both *in vitro* and *in vivo* against *S. suis* and other

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Viral proteomic tree of Bonnie and Clyde with related phages generated in ViPtree. Inner and outer rings represent ICTV virus family and host group. An expanded view of the tree shows Bonnie and Clyde (red) and the virulent phage SMP (green) with other closely related phages. The log scale bar (with dashed lines) represents the genomic similarity scores (SG) computed through normalised tBLASTx scores.</figcaption></figure>
pathogens in previous studies (Elst et al., 2020; Ji et al., 2015; Meng et al., 2011; Shi et al., 2012; Vázquez et al., 2017). The protein sequence of LySMP (SMP) endolysin (481 amino acids) was used as reference for comparative analysis with lysins encoded by Bonnie, NLS50, Clyde, phi20c, and Javan597. These five phages encode lysins ranging from 247 to 468 amino acids in length and share between 19.3 % and 79.3 % amino acid similarity with the SMP endolysin (Figs. 6A and 6B). Pair-wise amino acid identity matrix is supplied in Table S10.

## 1. 1. Protein structure predictions and topological model assembly

The means by which *S. suis* phages recognises and bind to their host is currently unknown. To this end, we sought to predict the structure of the tail-associated proteins of Bonnie and Clyde including the predicted distal tail protein (Dit), tail-associated lysin (Tal), and, when present, the receptor-binding protein (RBP). We then compared the structure pre-dictions of the adhesion machineries of Bonnie and Clyde, with those of previously isolated *S. suis* phage SMP, as well as the well characterised tail structure of *Streptococcus thermophilus* (St) phage STP1 (Goulet et al., 2022) for which the host recognition and binding is described. In lambdoid tailed phages, the genes encoding the adhesion machinery are located between those encoding the tail tape measure protein (TMP) and the holin and lysin (Goulet et al., 2020). The adhesion machineries typically comprise three main proteins: the Dit, the Tal, and, frequently, an RBP, which may occur in variable order (Goulet et al., 2020). In St phages including STP1, these three genes are arranged sequentially in the Dit, Tal, and RBP order (Goulet et al., 2022; Lavelle et al., 2020). In Bonnie, all three genes were identified, whereas for Clyde and SMP, only the genes encoding Dit and Tal were present. Structural predictions were therefore made for the Dit and Tal gene products and their complexes, as well as for the RBP of Bonnie. Dit proteins can be divided into two domains corresponding to the N-and C-terminal regions of the polypeptide chain. The N-terminal domain, known as the belt, comprises two β-sheets, a β-hairpin, and an α-helix (Veesler et al., 2010; Veesler and Cambillau, 2011). The C-terminal domain called the galectin, is a two β-sheet structure similar to a galectin domain but lacking its saccharide-binding residues. The Dit protein of some phages, including that of phage Lambda, lack a galectin domain, while in others, such as phage T5, it is replaced by the OB-fold domain (Flayhan et al., 2014). Dits often contain carbohydrate-binding module (CBM) insertions within the galectin domain and are referred to as “evolved Dits” (Dieterle et al., 2017, 2016). Evolved Dit proteins have been observed, as seen in the ~500 amino acid-long Dit proteins of St phages, which also contain a CBM inserted within the galectin domain (Goulet et al., 2022; Lavelle et al., 2020). In contrast to STP1 and other St phages, the Dits of Bonnie, Clyde, and SMP are not evolved and belong to the classical Dit family. As such, these Dits are unlikely to participate in host binding (Fig. 7A).

The Tals of siphophages are trimeric and consist of an N-terminal structural domain of ~350–400 amino acids (Mahony et al., 2023; Sciara et al., 2010; Veesler and Cambillau, 2011). In many phages, this domain is followed by an extension that is believed to play a role in cell wall polysaccharide/peptidoglycan degradation, as seen in *L. lactis* P335 phage TP901–1 (Mahony et al., 2023; Stockdale et al., 2013), or in host binding as in the *Bacillus subtilis* phage SPP1 (São-José et al., 2006). The Tal extension of STP1 is 1092 amino acids long, containing an N-terminal structural domain and two CBMs in the extension (Fig. 7A, STP1). Bonnie possesses a trimeric Tal of 990 amino acids, with a classical N-terminal structural domain (residues 1–384). Unlike STP1, which possesses an Ig-like domain, Bonnie presents a tandem of small β-stranded domains (Goulet et al., 2022)(Fig. 7A, Bonnie). This is followed by a triple β-stranded domain, forming a distinct small module, though no relevant similar structure was identified by Foldseek in the PDB. Short collagen linkers connect to a domain similar to the three β-domains described in the St phages’ Tals adjacent to a CBM. Foldseek identified this CBM as a member of the CBM X-2 family, according to the

A

<figure><img src="image_6.png" /></figure>

B

<figure><img src="image_7.png" /></figure>
**Fig. 4.** Phylogenomic analysis of Bonnie and Clyde. (A) Heatmap showing intergenomic similarities among Bonnie and Clyde, and closely related (pro)phages. (B) Phylogenomic GBDP tree of isolated phages inferred using formula D4 in VICTOR. Numbers above nodes (given that branch support exceeds ≥50%; nodes without annotated values indicate branches with lower support) represent pseudo-bootstrap support values from 100 replications. Tree was rooted at midpoint and the branch lengths are scaled using the GBDP distance formula *d*ₐ. The scale bar (0.02) represents normalised dissimilarity between genomes. Coloured annotations on the right indicate the taxonomic clustering of phages based on ICTV cut-offs (Table S6). For each taxonomic rank, phages of the same taxon are assigned the same colour and/or shape. GC content is represented by gradient-coloured squares from ~40% (min; grey) to ~44% (max; blue) Genome length is represented by black horizontal bars.

CAZy classification. After the CBM, a helical triplex abuts a β-prism as observed for STP1 (Goulet et al., 2022).

In addition to Dit and Tal, Bonnie possesses RBPs. Structural pre- dictions of the RBPs of Bonnie and STP1 as monomers identified a linear assembly of β-stranded domains forming a β-sandwich along with a complex C-terminal domain (Fig. 8A and B). Attempting to predict the full-length RBPs as a trimer revealed that the four β-sandwiches do not pack together, while the C-terminal domain forms a compact trimer (Fig. 8C and D; Fig. S1). The structure includes a small β-prism composed of 3 × 4 β-strands, followed by three packed β-sheets and terminated in three packed β-sandwiches. This last domain resembles the RBP heads of lactococcal phages, and Foldseek identified hits with RBP of the lacto- coccal phages TP901–1 and p2 (Dunne et al., 2018; Goulet et al., 2020; Spinelli et al., 2006a, 2006b). While the RBP heads of STP1 and Bonnie exhibit similar folds, differences in the length of their loops and their sequences suggest they may target distinct polysaccharidic receptors (Fig. S2A).

Following the Dit hexamer, Clyde's Tal N-terminal structural domain is immediately followed by a long triple helix and a CBM, which was identified by Foldseek as belonging to the CAZy CBM X-2 family (Fig. 7, Clyde). From this CBM, a long triple helix abuts another CBM, which is identified as belonging to the CAZy CBM22-1 family. Immediately following this, a large β-prism domain is observed, continuing into a complex triplex domain formed of β-turns and α-helices, which abuts a complex C-terminal domain (Fig. 7, Clyde). This C-terminal domain forms an intertwined β-prism, comprising 14 β-strands of variable length, decorated with long loops, and culminating in a C-terminal segment of two α-helices per monomer, forming a trimeric bundle of six α-helices (Fig. 7D and E). Foldseek retrieved a weak hit with the fibre tip of *Bdellovibrio bacteriovorus* H100 (Prob. 0.21; E-value 9.8 10⁻¹; PDB ID: 8ond) (Caulton et al., 2024). The *B. bacteriovorus* H100 fibre trimeric

domain exhibits a large crevice at the interface between its monomers, a feature also observed in *Salmonella* phage P22, where it is filled by an O-antigen oligosaccharide (PDB ID: 30th0) (Andres et al., 2013). Notably, a similar crevice is also observed in Clyde's Tal C-terminal domains (Fig. 7F), strongly suggesting that it may act as a binding domain for polysaccharidic receptors.

SMP exhibits very similar features when compared to Clyde, with the main difference being the shorter length of the second triplex helix and the presence of only one CBM, which is structurally similar to the CBM1 of Clyde (Fig. 7, SMP). The C-terminus of the Tal is highly superim- posable to that of Clyde, as both Tal sequences share 98.4% identity in their last 250 residues (Fig. 7E, Fig. S2B)

## 1. 1. Plaque and virion morphology of Bonnie and Clyde

Both phages were propagated on strain 21171_DNR38 in THB. Bonnie produced clear, medium-sized plaques with frayed edges that measured 1.02 0.05 mm in diameter and Clyde produced clear pinhead plaques measuring 0.29 0.06 mm (Figs. 9A and 9C). TEM analysis revealed both phages exhibit a long, flexible, non-contractile tail and an icosahedral capsid. Clyde has a shorter tail compared to Bonnie, however, no obvious structures such as central fibre were observed at the base of either phage (Figs. 9B and 9D).

## 1. 1. Host spectrum of Bonnie and Clyde

The susceptibility of 100 pathogenic *S. suis* strains to phages Bonnie and Clyde was evaluated by plaque assays. Bonnie produced plaques or clear zones on 15 % of tested strains while Clyde formed plaques/clear zones on 58 %. Collectively, the phages could infect 58 of the 100 strains tested. These susceptible strains include seven serotypes and thirteen
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Comparative linear alignment displaying genomic features of isolated phages and their closest relatives. SMP is used as a reference. Arrows indicate position and direction of CDSs in genomes of the (pro)phages with colour-coding representing functional categories. Shaded regions between genomes represent levels of similarity computed with tBLASTx. Dot plot of pairwise genome alignments is shown on the far left, with a corresponding colour scale for both dot plot and alignments displayed in the top left. Both dot plot and alignments were generated in ViPTree using concatenated nucleotide sequence of the (pro)phages.</figcaption></figure>

known *S. suis* sequence types. Neither Bonnie nor Clyde could infect strains of other species (*S. thermophilus*, *L. cremoris*, and *E. coli*; Table S1). The EOP relative to strain 21171_DNR38 for 29 susceptible strains that formed plaques was tested. The EOP ranged from 0.001 to 1.2 (10⁵ to 10⁸ PFU/mL). Both Bonnie and Clyde produced plaques at a comparable efficiency on strains D71 and 21169_DNR36.

Host range of Bonnie and Clyde determined using spot assay. A total of 100 strains were tested (Table S1) but only strains susceptible to at least one of the phages are presented here. Susceptibility is scored as + (plaques/clear zone) or - (no plaques/clear zone). Abbreviations: NF, not found (sequence type undetermined). Pathotype: "Respiratory" refers to strains recovered from the respiratory tract of diseased pigs; "Systemic" refers to strains isolated from sites outside the respiratory tract; "Unknown" refers to strains of unspecified origin.

## 1. 1. *In vitro* lytic activity of Bonnie and Clyde

The changes in bacterial density in response to phage exposure was monitored over 24 hours. On strain 21171_DNR38, the bactericidal activity of Clyde was generally proportional to the MOI in a dose-dependent manner. All MOIs tested showed a statistically significant difference compared to the no-phage group (*p* < 0.0001). However, at higher MOIs (100 and 10), regrowth of resistant populations was observed at 8 hours and 20 hours post-infection, respectively (Fig. 11B). In the case of Bonnie, highest inhibition was observed at MOI 10 (highest MOI tested) and MOI 0.001 (*p* < 0.0001). Near the 6-hour time point, an exponential increase in bacterial density, comparable to the no phage control was recorded at MOI 1, 0.1 and 0.01 (Fig. 11A).

Upon infection with a cocktail of Bonnie and Clyde, 21171_DNR38 growth was inhibited, particularly at MOIs 10 and 1 for up to 6 hours, following which bacterial density increased exponentially. Based on Clyde's lytic activity at MOI 10, two different mixed-strain cultures were exposed to Clyde at this MOI. The growth of both multi-strain mix A (21171_DNR38, 19867_M106485_R39, DNR36, D71, and M105040_S24) and multi-strain mix B (21171_DNR38, D94, D8, D52, and D75) was significantly suppressed (*p* < 0.0001), although not completely.

## 1. 1. *One step growth curve and burst size*
One-step growth experiments determined Clyde had an approximate latent period of 55 minutes and a burst size of 35 PFU per infected cell. The latent period of Bonnie was estimated to be 55 minutes with a smaller burst size of 27 PFU per cell (Figs. 11E and 11F).

## 1. 1. Stability of Bonnie and Clyde under different pH and temperature conditions

The stability of phages under different thermal and pH conditions is crucial for storage and downstream processing. To evaluate this, the stability of Bonnie and Clyde was assessed by incubating aliquots at various temperatures and pH levels (Figs. 12A and 12B). For Clyde, titres remained stable at 37, 40 and 50°C for up to 120 minutes, showing no significant difference compared to storage temperature (4°C) (*p* > 0.05). However, at 60°C phage titres dropped below detection limits within

<figure><img src="image_9.png" /><figcaption>B</figcaption></figure>

<table><thead><tr><td>Phage</td><td>Lysin ID</td><td>Lysin length (aa)</td><td>Lysin type</td><td>% ID to LySMP</td><td>No of TMD</td><td>Holin Class</td></tr></thead><tbody><tr><td>Bonnie</td><td>Phage lysin, N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28)</td><td>476</td><td>Amidase</td><td>79.07</td><td>3</td><td>I</td></tr><tr><td>NLS50_prophage</td><td>Phage lysin, N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28)</td><td>476</td><td>Amidase</td><td>79.28</td><td>3</td><td>I</td></tr><tr><td>Javan597</td><td>Phage lysin, N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28)</td><td>468</td><td>Amidase</td><td>73.33</td><td>2</td><td>II</td></tr><tr><td>Clyde</td><td>PlySs2 family phage lysin</td><td>245</td><td>CHAP</td><td>20.67</td><td>3</td><td>I</td></tr><tr><td>phi20c</td><td>Phage peptidoglycan hydrolase</td><td>247</td><td>CHAP</td><td>19.32</td><td>1</td><td>III</td></tr><tr><td>SMP</td><td>Phage lysin, N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28)</td><td>481</td><td>Amidase</td><td>100</td><td>3</td><td>I</td></tr></tbody></table>

**Fig. 6.** Phylogenetic tree of endolysins of the six (pro)phages. (A) Neighbour-joining tree based on amino acid sequence of endolysins constructed with Clustal Omega. Alignment was visualised with seqvisr tool using experimentally validated phage SMP endolysin (LySMP) as reference. (B) Predicted holin classes and endolysins. Holin transmembrane domains (TMDs) were predicted with DeepTMHMM. Lysin type is based on catalytic domains; N-terminal N-acetylmuramoyl-L-alanine amidase (amidase) and N-terminal cysteine/histidine-dependent amidohydrolase/peptidase (CHAP). Abbreviation: “aa” represents length in amino acids.

30 minutes. Titres of Bonnie did not significantly decrease at 37 or 40°C following exposure for up to 120 minutes compared to 4°C (*p* > 0.05). However, at 50°C, titres gradually declined, with a 3.2 log PFU/mL loss from the initial 8.6 log PFU/mL after 120 minutes. At 60°C, titres significantly dropped to 2.9 log PFU/mL by 30 minutes and by 90 minutes no plaques were detected. Phage stability was lost at 70°C for both phages. When incubated in pH-adjusted SM buffer (2,3,4,5,6,7,8,9,10,11,12 and 13) for 1, 2 or 24 hours, no significant change in viability was recorded across time points (Figs. 12C and 12D). There was complete inactivation of phage particles at pHs 2 and 13. However, both phages were stable from pH 4 to pH 10.

## 1. 1. Discussion

In this study, we report the isolation of two temperate phages that infect *S. suis*: Bonnie, isolated from pig tissue, and Clyde, obtained through prophage induction. Despite extensive screening of diverse samples, only Bonnie was isolated during the screening of pig-derived samples by plaque assay. Although the previously isolated *S. suis* phage SMP has been described as “lytic” and “virulent”, in our study, PhaTYP analysis predicted its lifestyle as temperate (Table S5). There are currently several bacteria for which virulent phages have not been reported, including Legionella pneumophila, Clostridioides difficile, Bifi- dobacterium species, and *S. suis* (Deecker et al., 2021; Mavrich et al., 2018; Nale et al., 2016). A potential reason for the difficulty in isolating phages against *S. suis* is due to the high level of phase variation in the defence mechanisms of the bacterium (Atack et al., 2018; Roodsant et al., 2024; Tram et al., 2021). This would mean that in a culture derived from a single colony, phase variation rapidly produces geno- typically and phenotypically diverse subpopulations, some of which cannot be infected by phage leading to their growth in a plaque assay, and a failure to observe plaques. We made a similar observation when
Bonnie was passaged on 19867_M106485_R39—the initial isolation host—where plaque formation was inconsistent based on the version of bacterial stock used. Modulation of bacterial susceptibility to phage through phase variation has been described in other species including *Campylobacter jejuni*, *C. difficile*, and *Haemophilus influenzae* (Cayrou et al., 2021; Sekulovic et al., 2015; Turkington et al., 2019). In the case of *H. influenzae*, phage extinction was observed when the resistant subpopulation exceeded 34% (Turkington et al., 2019). Future work will use 19867_M106485_R39 to investigate the mediation of phage susceptibility by phase-variable genes.

While MitC is routinely used to induce prophages in bacteria, previous attempts to induce prophages in *S. suis* lysogens harbouring full-length prophages were largely unsuccessful, with only two prophages induced from fifty-six isolates (Tang et al., 2013). All three methods used in this study successfully induced the expected prophage (Clyde) from 19867_M106485_R39; however, the resulting plaques were turbid and of low titres. Spiking the filtered induction lysates with 21171_DNR38 improved plaque formation and enabled subsequent single-plaque purification. As previously demonstrated in other bacteria (Alexeeva et al., 2018; Bruneaux et al., 2022; Schuster et al., 1973; Zeng et al., 2016), temperature cycling was shown to trigger prophage induction in *S. suis*. Genomic analysis revealed that Bonnie and Clyde shared only 4.4% and 16.5% nucleotide similarity with phage SMP, respectively. A search could not identify any closely related cultivated phage. Consequently, Bonnie and Clyde were compared to SMP, 15 publicly available *S. suis* prophages, and ten prophages predicted from publicly available host genomes, based on nucleotide similarity estimated using BLASTn. This comparison confirmed that Bonnie and Clyde are two novel species that belong to two distinct novel genera. Orthologous proteins from six (pro)phages, including Bonnie and Clyde, their closely related phages, and SMP as reference, were grouped into protein clusters. While no core clusters were identified, all (pro)phages, except Clyde, encode a

<figure><img src="image_10.png" /><figcaption>**Fig. 7.** Structural representation of the Dits and Tals from *S. thermophilus* phage STP1 and *S. suis* phages Bonnie, Clyde and SMP. (A) Surface representation of the complexes formed by hexameric Dits and trimeric Tals. CBMs are annotated with their closest structural homologues. (B) Ribbon view of the Tal C-termini of STP1 and Bonnie. (C) Ribbon view of the superimposition of the Tal C-termini of STP1 (blue) and Bonnie</figcaption></figure>
(yellow). (D) Ribbon view of the Tal C-termini of Clyde and SMP.

(E) Ribbon view of the superimposition of the Tal C-termini of SMP (blue) and Clyde (yellow). (F) Close-up surface view of the Tal C-terminus of Clyde. The crevice and putative receptor binding site is boxed (white).

conserved SSB protein, a protein previously identified in seven of twelve

S. suis (pro)phages (Tang et al., 2013). The SSB bind to single-stranded DNA with high affinity to protect against degradation and formation of secondary structure during phage DNA replication (Gascón et al., 2000). In its absence, phages co-opt host SSB for viral replication. The use of SSB by phages is so common that they have recently been shown to activate defence systems such as Hna, Retron-Eco8, Hachiman, and AbpAB (Sasaki et al., 2023; Sather et al., 2023). The previously char- acterised PlySs2, which has a CHAP domain shares 90.2 % protein identity with the endolysin of Clyde (Elst et al., 2020). In contrast, the endolysin encoded by Bonnie, and the other S. suis phages is a N-ace- tylmuramoyl-L-alanine amidase. Lysins and holins of S. suis phages have previously been purified and tested both in vitro and in vivo. The

endolysins of Bonnie and Clyde could further be developed and evalu- ated for their specific antimicrobial activity against S. suis .

Structural predictions of the adhesion device proteins of Bonnie and Clyde revealed two distinct lineages of S. suis phages: St-like, exempli-
fied by STP1 (Goulet et al., 2022), and S. suis -like, similar to SMP. Bonnie's structure aligns with the St phage-like lineage, possessing an RBP,
while Clyde and SMP are specific to phages infecting S. suis . The

S. suis lineage phages lack RBPs but display a Tal C-terminal domain that may function in receptor-binding. Regarding the Tal C-terminus of Clyde (or SMP), it is noteworthy that Foldseek returned many hits from AlphaFold database (AFDB) consisting of Tal C-termini from S. suis and other streptococcal phages, but not from St phages (Fig. S3A). Similarly, despite the overall similarity of Bonnie's RBP C-terminus to those St

<figure><img src="image_11.png" /><figcaption>**Fig. 8.** Structural representation of the RBPs from *S. thermophilus* phage STP1 and *S. suis* phage Bonnie. Ribbon representation of the RBP monomer from STP1 (A) and Bonnie (B), rainbow coloured from its N- (blue) to C-terminus (red). (C) Ribbon representations of the RBP C-terminal trimer assemblies from STP1 and Bonnie.</figcaption></figure>

phages, submitting Bonnie's RBP C-terminus to Foldseek also returned hits in the AFDB from Tals of S. suis and other streptococcal phages, but not
from St phages (Fig. S3B). These observations suggest that, from the perspective of adhesion device, two very different families of phages can
specifically infect S. suis. Furthermore, despite the lack of nucleotide similarity and their different ecological niches, the structural similarities
between the adhesion devices of Bonnie and St phages, might point to two possible evolutionary scenarios. The structural similarities could

arise from a common ancestral gene, connecting *S. suis* and *S. thermophilus* through divergent evolution, as observed in the struc- turally similar RBP (Spinelli et al., 2006b) and capsid proteins (Baker et al., 2005) shared by some mammalian viruses and phages. Alterna- tively, the observed similarities may be the result of convergent evolu- tion, where these similar structures have evolved independently to adapt for adhesion to host surfaces. Such convergence has been described between the baseplates of coliphage T4 and lactococcal phage p2 (Sciara et al., 2010), as well as the Dits of phages infecting *S. aureus*, *Bacillus subtilis*, and *L. lactococcus* (Kizziah et al., 2020). Both scenarios highlight potential complex evolutionary links between pathogenic and non-pathogenic streptococcal species.

Bonnie and Clyde have long, non-contractile tails consistent with previously reported S. suis (pro)phages Ss2, and SMP (Harel et al., 2003; Ma and Lu, 2008). Collectively, Bonnie and Clyde infect 58/100 strains tested, with Bonnie displaying a narrower host range (15/100 strains), similar
to SMP. Every strain susceptible to Bonnie was also susceptible to Clyde albeit with varying EOP. This observation is likely driven by transient factors—such as mutations in host receptors and acquisition of anti-phage systems through horizontal gene transfer—shaped by coevolutionary dynamics and evolutionary trade-offs within the same host species rather than fixed intrinsic barriers like non-host resistance due to differences in receptor binding sites observed in host range at higher taxonomic levels (Holtappels et al., 2023). Bonnie and Clyde's differential host range may result from receptor-level adaptations, with Clyde's adhesion device targeting a subset of receptors not recognised by Bonnie's RBP. These specificities align with nested bipartite interaction networks, where specialist phages like Bonnie target subsets of gener-alist phages' hosts (Beckett and Williams, 2013; Gurney et al., 2017). Moreover, anti-phage defence systems may contribute to the observed host range. As we showed previously, *S. suis* genomes encode between one to ten of 20 distinct defence systems that may target different stages of the phage life cycle (Osei et al., 2022). Bonnie-resistant strains may harbour defence systems that are activated by Bonnie-specific triggers such as SSB, while Clyde overcomes these barriers through alternative infection strategies. Future transcriptomic and knockout studies could investigate these dynamics by exploring host responses to Bonnie and Clyde using Clyde-susceptible, Bonnie-resistant strains, as well as bacteriophage-insensitive mutants (BIMs). Bonnie and Clyde were first co-isolated when superinfection of 19867_M106485_R39 by Bonnie triggered the induction of Clyde (prophage). Such induction events increase the likelihood of recombination between the two phages which

<figure><img src="image_12.png" /><figcaption>**Fig. 9.** Plaque and virion morphology of Bonnie and Clyde. (A) plaques of Bonnie and (C) Clyde on double-layer agar plates. Plaque diameters of Bonnie (1.02 ± 0.05 mm) and Clyde (0.28 ± 0.06 mm) were measured with ImageJ software (National Institute of Health, Bethesda, USA). Scale bar represents 1 mm. Repre- sentative electron micrographs of (B) Bonnie and (D) Clyde. Bonnie has a capsid (56.27 ± 2.33 nm in diameter) and a long non-contractile tail of length 186.92 ± 5.92 nm and width 9.38 ± 0.81 nm. Clyde has a capsid that measures 54.22 ± 3.37 nm in length with a short non-contractile tail of 143.01 ± 6.26 nm and 10.2 ± 0.44 nm. Scale bar represents 100 nm.</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>**Fig. 10.** Efficiency of plating. The EOP relative to strain 21171_DNR38 was estimated using spot assays for 29 susceptible strains that formed plaques in host range analysis. The EOP values ranged from 0.001 to 1.2 (10⁵ to 10⁸ PFU/mL) for strains with detectable plaque formation. Strains where no plaques were observed (EOP = 0.00) are indicated by blank white boxes.</figcaption></figure>

may lead to the emergence of new phage genotypes with wider host specificities (Bull et al., 2024; Peters et al., 2020). Additionally, induced phages could infect and lyse cells resistant to the superinfecting phage (Abedon and LeJeune, 2007) thereby potentially improving therapeutic outcomes.

We investigated the *in vitro* lytic activity of the phages against 21171_DNR38 at different MOIs. Clyde generally inhibited 21171_DNR38 growth in an MOI-dependent manner whereas with Bonnie, the highest inhibition was observed at MOI 10 and 0.001. Resistance to Bonnie and Clyde was observed at certain MOIs (Figs. 11A and 11B) by 6 hours and 8 hours, respectively. In the context of phage therapy, BIMs have been shown to display attenuated virulence *in vivo* (Capparelli et al., 2010), facilitate the reversion of antibiotic resistance (Fujiki et al., 2023), and sensitise resistant strains to other infecting phages (Acton et al., 2024). Phenotypic characterisation and *in vivo* experiments could be used to evaluate the fitness trade-offs in Bonnie and Clyde-resistant mutants.

Combining the phages into cocktails did not improve lytic activity beyond that observed with individual phages, as the activity of the cocktail was comparable to Bonnie alone, with resistance emerging by 6 hours post-infection in three of the five MOIs tested. While phage cocktails have been shown to improve bacterial clearance and minimise emergence of phage-resistant populations through synergistic interactions, antagonism is not uncommon, where similar phages compete for the same surface molecules (Thanki et al., 2024; Yoo et al., 2023). Given that Bonnie and Clyde have different adhesion devices, antagonism seem less likely, but our results are more consistent with the findings in *E. coli* infecting-phages, where the efficacy of individual phages did not predict efficacy of cocktails (Niu et al., 2021). When tested *in vitro* against two multi-strain cultures that mimic the polyclonal
<figure><img src="image_14.png" /><figcaption>**Fig. 11.** *In vitro* lytic activity and one-step growth curves. Bacterial killing activity of (A) Bonnie, (B) Clyde and (C) a cocktail of both phages at different MOIs was tested *in vitro*. OD<sub>600</sub> was read every 10 minutes for 24 hours. (D) Lytic activity of Clyde (MOI 10) against two multi-strain cultures was monitored for 24 hours. Multi-strain mix A (21171_DNR38, 19867_M106485_R39, DNR36, D71, and M105040_S24) and Multi-strain mix B (21171_DNR38, D94, D8, D52, and D75). One step growth curves for (E) Bonnie and (F) Clyde at MOI 0.01. The error bars represent the standard error of the mean from independent replicate experiments.</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>**Fig. 12.** Stability of phages under different physicochemical conditions. Stability of Bonnie (A) and Clyde (B) at different temperatures was monitored over 120 minutes. Stability of Bonnie (C) and (D) Clyde was assessed following incubation in different pH-adjusted SM buffer for 1 hour, 2 hours, and 24 hours. Error bars represent the standard error of the mean from independent replicate</figcaption></figure>
experiments.

nature of infection, Clyde significantly inhibited bacterial growth. Each multi-strain group (Fig. 11D) consisted of strains from two or three different serotypes (serotypes 2, 9, and 14), which are among the most commonly implicated agents in human and animal *S. suis* infections globally.

Phage stability is a key factor in their application as therapeutics, particularly as the acidic environment of the mammalian gastrointestinal tract can inactivate free phages when administered orally. Both Bonnie and Clyde were shown to be very stable across different pH and temperature conditions. Downstream processing such as encapsulation, freeze drying, and formulation into inhalable dry powders and steam pellet feed can be used to improve stability in different temperatures and extend shelf-life in phages such as Bonnie and Clyde (Thanki et al., 2022; Zhang et al., 2024).

Overall, the phages described herein will support future investigations into the evolutionary relationships between phages and *S. suis* in the context of host recognition and infection, anti-phage defence mechanisms, and bacterial virulence. Beyond fundamental research, the demonstrated stability and *in vitro* activity of Bonnie and Clyde highlight their potential as antibacterial agents. Although Bonnie and Clyde cannot be used in their current form as temperate phages in phage therapy, engineering obligately virulent mutants, or using their encoded proteins, such as endolysins, presents promising avenues for future research.

## Funding statement

This project (Improved Pig Health through the Novel Application of SynBio in Phage Therapy, 2020US-IRL201) was funded by the Irish Department of Agriculture, Food and the Marine, through the 2020 US-IRL R&D Partnership Call. Collection of Danish and Spanish isolates was funded by an EU Horizon 2020 grant "PIGSs" (727966).

## CRediT authorship contribution statement

**Mahony Jennifer:** Writing – review & editing, Writing – original draft, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.
**Osei Emmanuel Kuffour:** Writing – review & editing, Writing – original draft. Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.
**Manzanilla Edgar Garcia:** Writing – review & editing, Methodology, Funding acquisition.
**Weinert Lucy A.:** Writing – review & editing, Investigation, Data curation.
**Hille Frank:** Visualization, Software, Methodology.
**Athalye Ankita:** Investigation, Data curation.
**Cambillau Christian:** Writing – review & editing, Writing – original draft, Visualization, Software, Methodology, Investigation.
**Kenny John G.:** Writing – review & editing, Writing – original draft, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.
**O’Hea Reuben:** Investigation, Data curation.
**Murray Gemma G. R.:** Writing – review & editing, Data curation.
**Wilson Margaret:** Investigation, Data curation.
**O’Doherty Áine:** Investigation, Data curation.
**Franz Charles M.A.P.:** Visualization, Software.

## Declaration of Competing Interest

The authors declare no conflict of interest.

## Acknowledgement

We would like to thank John Moriarty and the Pathology Division (Department of Agriculture Laboratories, Backweston) for generously providing some of the bacterial strains used in this study, as well as Marcelo Gottschalk and Nahuel Fittipaldi for the Canadian isolates. Transmission electron microscopy was carried out with technical assistance from Morten Bratschke (Max Rubner-Institut). We thank members

of the Mahony Lab for assistance during the CsCl purification. We are grateful to Mário Ornelas for his assistance in sampling. We are grateful for the HPC resources from GENCI-IDRIS [2023-AD010714075; in part] and INRAE MIGALE bioinformatics facility. We acknowledge UCSF ChimeraX which is developed by the Resource for Biocomputing, Visualization and Informatics at the University of California, San Francisco, with support from National Institutes of Health [R01-GM129325]. We are grateful to Gabriele A. Lugli and Marco Ventura (GenProbio s.r.l, Parma, Italy) for carrying out the genome sequencing of the phages. Authors are grateful to Ramya Balasubramanian and other members of Vision 1 Lab at Teagasc Moorepark for their insights and constructive feedback on the analysis and preparation of the manuscript.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.micres.2025.128147.

## Data availability

Data will be made available on request.

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