The bacteriocin from the prophylactic candidate Streptococcus suis 90-1330 is widely distributed across S. suis isolates and appears encoded in an integrative and conjugative element

The Gram-positive α-hemolytic Streptococcus suis is a major pathogen in the swine industry and an emerging zoonotic agent that can cause several systemic issues in both pigs and humans. A total of 35 S. suis serotypes (SS) have been identified and genotyped into > 700 sequence types (ST) by multilocus sequence typing (MLST). Eurasian ST1 isolates are the most virulent of all S. suis SS2 strains while North American ST25 and ST28 strains display moderate to low/no virulence phenotypes, respectively. Notably, S. suis 90–1330 is an avirulent Canadian SS2-ST28 isolate producing a lantibiotic bacteriocin with potential prophylactic applications. To investigate the suitability of this strain for such purposes, we sequenced its complete genome using the Illumina and PacBio platforms. The S. suis 90–1330 bacteriocin was found encoded in a locus cargoed in what appears to be an integrative and conjugative element (ICE). This bacteriocin locus was also found to be widely distributed across several streptococcal species and in a few Staphylococcus aureus strains. Because the locus also confers protection from the bacteriocin, the potential prophylactic benefits of using this strain may prove limited due to the spread of the resistance to its effects. Furthermore, the S. suis 90–1330 genome was found to code for genes involved in blood survival, suggesting that strain may not be a benign as previously thought.


Introduction
The quick rise of antibiotics resistance in the microbial world is problematic to multiple fields, including the food industry.As such, novel antibiotic compounds and alternate strategies to treat infections and prevent growth of pathogenic bacterial species are increasingly being sought after.One promising avenue is the prophylactic use of bacteriocin-producing commensal or neutral species as probiotics to outcompete virulent ones [1].Bacteriocins form a wide range of antimicrobial peptides produced by microorganisms [2].These molecules, frequently short peptides, can affect a narrow to large spectrum of bacteria excluding the species producing the compounds, which are immune to their effects via varying mechanisms [3].Because of the potentially disruptive effects on the microflora of the host, the ideal species for prophylactic use would produce compounds whose spectrum do not disrupt the commensal organisms inhabiting the targeted environment.Recently, a nisin-related lantibiotic bacteriocin with a membrane permeabilization activity has been found in Streptococcus suis strain 90-1330 [4].This lanthionine-containing bacteriocin is a killing peptide active against a number of Grampositive streptococcal and staphylococcal species but shows little to no activity against Gramnegative bacteria [4].
S. suis is a genetically diverse Gram-positive streptococcal species displaying at least 35 distinct serotypes and categorized in over 700 sequence types by multilocus sequence typing (MLST) [5].Of these, serotype 2 (SS2) strains are of particular importance to the swine industry, where they can cause a plethora of severe infections in pigs including endocarditis, meningitis, pneumonia and septicemia [6,7].S. suis SS2 species are also a growing cause of concern in human health and have been associated with several zoonotic infections [7][8][9][10][11][12][13][14][15][16].Not all serotype 2 species display the same levels of virulence, however; sequence type 1 (ST1) isolates are usually the most virulent while sequence types 25 (ST25) and 28 (ST28) show mild to low/no virulence, respectively [17].Notably, the bacteriocin-producing S. suis 90-1330 (aka 1330) is a SS2-ST28 strain that has been established as avirulent in mice and swine [18].Considering this lack of virulence and the antibacterial activity of its bacteriocin against Gram-positive swine pathogens, including S. suis SS2-ST1 strains and staphylococcal species Staphylococcus aureus and Staphylococcus hyicus [4], this bacterium appears to be an excellent candidate for prophylactic use [2] in the swine industry.
Here we sequenced the complete genome of S. suis 90-1330 using the Illumina and PacBio short and long-read platforms, respectively, to investigate its suitability for prophylactic use and ensure that this strain is indeed devoid of toxin-encoding genes or any other component that could potentially cause problems with its usage as a probiotic.We also explored the origin and distribution of the bacteriocin encoded by S. suis 90-1330 across streptococcal species.In the process, we used the underlying genomic data to identify which S. suis strains are the closest to this SS2-ST28 representative.

Bacterial culture and DNA purification
S. suis isolate 90-1330 (aka 1330), originally isolated from pigs, was acquired from Dr. Marcelo Gottschalk, Faculty of Veterinary Medicine, Universite ´de Montre ´al [18].The isolate was cultivated at 37˚C in THB medium (IBI Scientific, Peosta, IA, USA) with low agitation (100 rpm).Total genomic DNA was purified using the MasterPure Gram Positive DNA Purification Kit (Epicentre Biotechnologies, Madison, WI, USA) with a lysis incubation time of 3 hours and additional centrifugation steps to remove protein precipitates.DNA thus harvested was quantified with a Qubit 2.0 fluorometer (Life Technologies, Waltham, MA, USA) and its purity assessed electrophoretically on 0.8% agarose gels and spectrophotometrically for the presence of RNA and protein contaminants with the A260/A230 and A260/A280 ratios, respectively, using a Nanodrop 2000c (Thermo Scientific, Waltham, MA, USA).

Genome sequencing
Genome sequencing was performed using the Pacific Biosciences (PacBio) long read and the Illumina short read platforms.PacBio libraries were prepared and sequenced by the University of Michigan DNA Sequencing Core (Ann Arbor, MI, USA).PacBio SMRTbell libraries were prepared per the PacBio 20 Kb+ protocol with g-TUBE shearing (Covaris, Woburn, Massachusetts, USA) and BluePippin (Sage Science, Beverly, MA, USA) size selection.A total of 108,170 polymerase reads (N50: 19.39 Kb) were sequenced using the P6-C4 chemistry for a total of 1.34 Gbp (420x coverage).Illumina libraries were prepared and sequenced by the Plate-forme d'Analyses Ge ´nomiques de l'Universite ´Laval (Quebec City, QC, Canada).DNA for Illumina sequencing was fragmented with a Covaris sonicator and libraries were generated using the Illumina TruSeq chemistry.A total of 663,635 Paired-Ends (PE) 300 bp-long reads (1,327,270 reads total) were sequenced on the Illumina MiSeq platform for a total of 398 Mbp (185x coverage).

Genome assembly
PacBio reads were processed and assembled with the RS_HGAP_Assembly.3pipeline as implemented in SMRT Analysis 2.3.0 [https://github.com/PacificBiosciences/SMRT-Analysis/wiki/SMRT-Analysis-Software-Installation-v2.3.0].Base calling on the initial genome assembly was verified by mapping the PacBio reads back onto the assembled genome using the RS_Resequencing.1 pipeline from SMRT Portal.Base calling was further verified and corrected by mapping the Illumina reads onto the refined assembly using Geneious R7.1.8(Biomatters, Auckland, New Zealand) as follows: reads were mapped using the built-in Map to Reference function, divergent positions were highlighted with the Find Variations/SNPs tool, and bases were corrected accordingly.The genome was circularized by detecting overlaps with BLASTN homology searches [19] and by adjusting the edges with Consed 29.0 [20].Genome methylation patterns on the final assembly were detected using the RS_Modification_and_-Motif_Analysis pipeline in SMRT Portal.The presence of 5-methylcytosine (5mC) was not assessed due to the small kinetic differences involved between methylated and non-methylated bases, as 5mC residues were not converted to 5-carboxylcytosine (5caC) using Tet1-oxidation prior to sequencing.

Genome annotation and comparative genomic analyses
Transfer and ribosomal RNAs were positioned on the S. suis 90-1330 genome with tRNAscan-SE 1.3.1 [21] and RNAmmer 1.2 [22], respectively.Preliminary protein predictions and annotations were performed with PROKKA 1.11 [23] with products inferred using an E-value threshold of 1e-30 and validated by InterProScan 5.15-54.0[24] analyses, with conflicting results resolved by manual curation against the UniProt database [25].Final annotations in the RefSeq genome were performed with the NCBI Prokaryotic Genome Annotation Pipeline (April 2017 release).ComRS competence genes and promoters were further identified by BLAST homology searches using homologous sequences from other S. suis strains as queries [26,27].GC-skew analyses were performed with GenSkew [http://genskew.csb.univie.ac.at/].KEGG pathway categories for each putative protein were attributed with BlastKOALA [http:// www.kegg.jp/blastkoala/].Physical maps were plotted with Circos [28].Comparative alignments between S. suis 90-1330 and other genomes were performed using the progressive Mauve algorithm as implemented in Mauve 2.4.0 [29].Transposons shared between S. suis 90-1330 and other strains were identified by BLASTP homology searches.Repeated loci were identified using RepeatFinder from the Geneious R9.1.7 package, with ribosomal RNAs filtered out from the results manually.

Genetic diversity inferences
Complete and draft S. suis genomes were downloaded from the NCBI GenBank database with the queryNCBI.plcustom Perl script.Pairwise mutation distances using the MinHash dimensionality-reduction technique were calculated with Mash [30] and genetic clusters were plotted with t-Distributed Stochastic Neighbor Embedding (t-SNE [31]) as implemented in the R [32] Rtsne package [33] (https://github.com/jkrijthe/Rtsne)with the run_Mash.pl,Mash-ToDistanceCSV.pl and MashR_plotter.pl custom Perl scripts.Single nucleotide polymorphisms (SNPs) from Fig 1C were inferred using read mapping followed by variant calling.To standardize datasets, synthetic Illumina reads (300 bp paired-ends, insert size 500 bp) were generated for every genome investigated at a sequencing depth of 100X using random subsampling with SSRG.pl 1.5 (https://github.com/PombertLab/SNPs/tree/master/SSRG).Briefly, this Perl script deconstructs a genome (string) into reads (substrings) of specified length in both forward and reverse-complemented orientations using random subsampling, producing synthetic reads akin to reads produced by shotgun sequencing methods.Synthetic reads were mapped against genomes in paired end mode (PE) with Bowtie2 2.3.4.1 [34] and pairwise SNPs were calculated with VarScan2 2.4.3 [35], as implemented in get_SNPs.pl1.8 (https:// github.com/PombertLab/SNPs/tree/master/SSRG)using default parameters.Results from synthetic reads were validated by mapping the S. suis 90-1330 illumina sequencing data against the same genomes.

A normal streptococcal genome
The S. suis 90-1330 complete genome maps as a single circular molecule of 2,146,151 bp with a GC content of 41.1%.The genome encodes a total of 2,192 genes including 2,120, 56 and 12 genes coding for proteins, transfer RNAs and ribosomal RNAs, respectively.A total of 8 copies of the LSU/SSU ribosomal RNA operon are located on the S. suis 90-1330 chromosome.The genome has a gene density of 1.13 gene per kb and an average length of 111.5 bp for the intergenic spacers.No gene was found interrupted by group I or group II bacterial introns.The genome features a single bidirectional origin of replication, located at position 1,057,979 of the assembled chromosome (S1 Fig), as calculated by GC-skew analyses and by identifying the position of the dnaA gene [44].A putative mobile genetic element (MGE) featuring a GC content lower by about 10% was also found inserted in the S. suis 90-1330 chromosome, near one of the two junctions where the GC-skew shifts (S1 Fig) .The chromosome encodes several transposable elements that are commonly found in other streptococcal species, including S. suis ST28 strain 05HAS68 (S2 Table ).Only one type (type II) of clustered regularly interspaced short palindromic repeats (CRISPR) was identified, limited to a single locus composed of eight 36 bp-long repeat units separated by unevenly-sized spacers.This locus-from 189,953 to 190,448 on the chromosome-is found in other Streptococcus species including strains of S. macedonicus and S. thermophilus (from 76% to 89% identity), and displays homology against the genome of Streptococcus phage 128 (E-value: 2E-18) [45].The 90-1330 genome also features a few additional dispersed repeated elements, mostly from gene duplicates, with a total of 104 distinct motifs of a least 100 nt distributed across 210 loci, with 16 and 32 repeats longer than 1,000 and 500 nt, respectively (excluding the ribosomal RNA copies; S3 Table ).Methylation patterns AGCNNNNNGCT and GTAC were found compatible with the type I and type II restriction-modification systems commonly found in streptococcal species ( [46,47]; S1A Fig).
In addition to the chromosome, S. suis strain 90-1330 also contains a 4,984 bp-long circular plasmid (pSS90-1330) encoding a total of 6 genes involved primarily in recombination and replication (S1B Fig) .Of the 2,120 protein coding genes found on the S. suis 90-1330 chromosome, 1,803 were assigned putative functions by the NCBI PGAP pipeline, with 1,161 assigned KEGG orthologs distributed across common streptococcal metabolic pathways (S1 Fig, S4 Table).The S. suis 90-1330 genome further codes for the type III ComRS competence system typically found in S. suis genomes, including the methylase DpnA required to prevent degradation of foreign DNA acquired via competence ( [26,27,46]; S5 Table ), and overall the S. suis 90-1330 gene content was found very similar to other S. suis SS2-ST28 genomes, as expected.Interestingly, however, the genotypes of S. suis SS2-ST28 isolates were found to be nearly identical to those from serotype 3 strains: 1) the SS2-ST28 and SS3-ST35 strains display very high levels of synteny, with only a few chromosomal rearrangements between SS2-ST28 strain 90-1330 and SS3-ST35 strain ST3 (Fig 1A ), 2) they share a very large number of genes (Fig 1B ), and 3) they show very little divergence at the nucleotide level (Fig 1C).This association between SS2-ST28 and SS3-ST35 strains was found compatible with the previously reported phylogenetic analysis of core S. suis genes (see figure S4 from Okura et al. [48]), in which these strains clustered together.Altogether, these observations suggest that serotype conversion occurred during the evolution of these strains.

The S. suis 90-1330 suicin appears encoded in an integrative and conjugative element
The S. suis 90-1330 bacteriocin AN924_RS09965 (aka suicin [4]) is encoded in a locus displaying an unexpectedly low GC content that contrasts sharply with the remainder of the chromosome (see Fig 2 and note on S1 Fig) .In addition to the suicin cluster, this low GC content region contains a gene coding for a conjugative relaxase (AN924_RS09970) and a plasmid mobilization relaxosome protein (AN924_RS09975), both involved in plasmid conjugation [49].The locus is flanked on either side by a pseudogenized transposase (AN924_RS10000) and a site-specific integrase (AN924_RS09905).The C-terminal segment of the predicted integrase sequence is homologous (E-value; 1.65e-38) to tyrosine-based site-specific phage recombinases.However, no other viral gene-including those coding for the viral capsid or tail components and necessary for the lytic cycle-was found in the corresponding locus.Genome-wide searches for the presence of phage sequences in S. suis 90-1330 revealed four incomplete and one questionable phages with GC contents averaging that of the chromosome (S1 File), none overlapping with the bacteriocin-containing locus.
Besides the abovementioned relaxase (AN924_RS09970) and relaxosome protein (AN924_RS09975), further investigation of the genomic surroundings of the bacteriocin-containing locus revealed the presence of genes coding for a Type IV coupling protein (T4CP; AN924_RS10200), for Type IV secretion system (T4SS) proteins, including a Virb4 ATPase (AN924_RS10180) and a murine hydrolase (AN924_RS10175), and for a surface adhesin (AN924_RS10155; Fig 2 ; S6 Table) common to integrative and conjugative elements (ICE) [50].In total, 13 genes from the Firmicutes, Actinobacteria, Tenericutes and Archaea (FATA) and Conjugation (CONJ) sets of hidden Markov models [42] were identified by TXSScan/ CONJscan searches (S6 Table ).BlastP searches performed in parallel using Tn5252 transposon sequences from Streptococcus pneumoniae [40,41]   those from several ICESa2603 family members present in S. suis and Streptococcus agalactiae [51] were found located respectively 45 bp upstream of the 3'end of the integrase, from 1,980,062 to 1,980,076, and downstream of the 3' end of the gene coding for ribosomal protein L7/L12 (AN924_RS10245), from 2,051,047 to 2,051,061.Notably, the macrolide and tetracycline resistance genes ermB and tetO were also found encoded in some of these ICESa2603 family members.
To assess the functionality of the putative S. suis 90-1330 ICE, PCR were performed to investigate the presence of the excised circular form of the ICE (S2 Fig) .Results showed the presence of this circular form, confirming the ability of the ICE to adopt the extrachromosomal form that is required for conjugation.This circular form was mainly detected in stationary phase cells while it was barely detected in cells in the logarithmic growth phase (results not shown).A PCR product corresponding to the junction of the genes contiguous to the ICE-Sa2603-like direct repeats was also detected confirming the absence of the chromosome-integrated ICE in a portion of the cells.This type of population heterogeneity has been previously reported for other ICE (e.g.ICESsuCZ130302 [52]).Altogether, these results strongly suggest that the bacteriocin cluster is located in an integrative and conjugative element.
Recently, Pan and colleagues characterized an ICE in S. suis strain CZ130302 (ICES-suCZ130302) featuring a bacteriocin [52].Comparisons between the S. suis 90-1330 suicin locus and that from ICESsuCZ130302 revealed that the encoded products are nearly identical in sequence (S7 Table ), with the bacteriocins differing in sequence by a single amino acid.Further comparisons between the S. suis 90-1330 putative ICE and ICESsuCZ130302 revealed that they are also very similar in structure, with most of the differences located in the cargo area of the ICE (S3 Fig).

The bacteriocin-containing locus is found across several divergent S. suis isolates
Initially thought to be rare [4], the suicin encoded in S. suis 90-1330 (AN924_RS11115) was found at 100% amino acid identity in several strains of S. suis, S. agalactiae, Streptococcus pneumoniae and other streptococcal species, in seven Staphylococcus aureus isolates, and in one strain (F0221F 224) of Enterococcus hirae (Fig 3).When lowering the similarity threshold to 85%, the bacteriocin was also found in streptococcal species Streptococcus pseudopneumoniae, Streptococcus thoraltensis and Streptococcus equinus (93, 91 and 85% aa identity, respectively).Expanding the search to all genes present in the suicin-containing locus and those contained in the S. suis 90-1330 putative ICE, a total of 43 genes were found distributed across most of the bacteriocin-containing strains, including ICESsuCZ130302-encoding S. suis strain CZ130302 (Fig 3).These genes include the T4SS and conjugative DNA processing proteins identified by TXSScan/CONJscan searches, suggesting that this bacteriocin locus is common in streptococcal ICEs.In most loci, the suicin-containing segments were also found to display lowered GC contents compared to the rest of their surroundings (averaging 31.1% vs. 41.1%,respectively).Many strains like S. suis JS14, S. suis LS9N or S. agalactiae C001 feature a different integrase/recombinase than that of S. suis 90-1330 and the S. suis 90-1330 suicin locus was found to be closer to that of Streptococcus pasteurianus ATCC 43144 (99% identity over 100% of its sequence) than to that of the other S. suis strains.Interestingly, the inner section of the S. suis 90-1330 putative ICE appears unique to this genome (Fig 3), with homology searches returning matches covering at best 58% of the region in Streptococcus parauberis.The inner segment of the putative ICE element in S. suis 90-1330 is flanked by a phage terminase and a recombinase, which suggests that the region was added by recombination.
To map the distribution of the bacteriocin-containing locus across S. suis strains in a phylogenetic framework, we performed genetic distances estimations and plotted their presence on a dimensionality reduction graph.The bacteriocin-containing strains were found scattered across several distinct clusters (I, II, III, IV, V, VIII, XII and XIV; Fig 4), including the highly divergent clusters XII and XIV, suggesting a wide distribution across sequenced S. suis isolates.

The S. suis 90-1330 genome codes for genes involved in blood survival
To investigate the suitability of S. suis 90-1330 as a prophylactic strain for the swine industry, we searched for the presence of genes coding for virulence factors and for products involved in antimicrobial resistance.
Because there is no clear consensus on what causes pathogenicity in S. suis strains, we searched the S. suis 90-1330 genome for all putative virulence factors catalogued and discussed by Segura et al. [36] using a permissive cut-off of 1E-10 (see S8 Table ).Unsurprisingly, the S. suis 90-1330 genome codes for capsular polysaccharides (CPS) genes (e.g.cpsC, AN924_RS02950; cpsE, AN924_RS02940) and for capsule regulatory genes (e.g.ccpA, AN924_RS01545), as expected from a serotypable strain.In contrast, of the three genes commonly used as multiplex PCR markers to predict S. suis virulence [5]-sly (hemolysin suilysin; a thiol-activated toxin [53]), epf (extracellular factor) and mrp (muramidase-released protein)-only mrp (whose product is involved in epithelial cell adherence [54,55]) was found encoded in the genome.This result was expected based on previous PCR-multiplex virulence inquiries [4] and genomic investigations of various ST28 [56] and ST25 [57] S. suis strains.Expanding the search to genes coding for products potentially involved in bacteremia and/or enhancing S. suis survival in blood revealed the presence of yqfA (AN924_RS10290) coding for hemolysin III, of fbpA (AN924_RS01115) whose product's N-terminus shows strong homology (9.6E-151) with fibronectin-binding protein A (FbpA) in InterProScan/PFAM searches, and of iga (AN924_RS10410), coding for a proteinase potentially involved in host immune evasion [36].However, the latter was found upon further inspection to be pseudogenized, with read-mapping over the corresponding locus confirming that the gene is indeed broken and not the result of a sequencing error.Overall, only 24 out of the 140 genes searched against the S. suis 90-1330 genome were not found at the 1E-10 threshold (S8 Table ).Additional searches against the virulence factor database (VFDB; http:// www.mgc.ac.cn/VFs/main.htm)revealed the presence of more CPS genes and of a gene coding for a TlyC-like (CDD E-value 1e-130) hemolysin C family transporter (AN924_RS07935) potentially involved in virulence (S9 Table ).These searches also returned a putative match with a cytolysin activator (cylA; E-value: 8.00E-31) but not for a cytolysin, and upon further inspection the match was found to be spurious, as the corresponding gene (AN924_RS09965) is located on the bacteriocin locus [4] and codes for a lantiobiotic-specific protease (E-value 2.81e-114).

Discussion
In healthy microbiotas, the presence of commensal and/or neutral microorganisms outcompete pathogenic species for the limited pool of nutrients, resources available and adherence sites, providing a helpful barrier against diseases.This helpful association between host and commensal/ neutral microorganisms forms the basis behind the prophylactic use of probiotics.Intuitively, prospective probiotics should be devoid of toxicity and side effects while providing clear positive benefits to the health of the individual(s).Recently, strain 90-1330 from the low virulence/nonvirulent Streptococcus suis serotype 2 -sequence type 28 was shown to express a bacteriocin with a membrane permeabilization effect that inhibits not only the growth of virulent serotype 2 strains of S. suis, but also of other Gram positive swine pathogens-including species hyicus and aureus from the genus Staphylococcus [4].This suggested that its use as a probiotic could serve as a robust preventive method in the swine industry.Here we determined the complete sequence of the S. suis 90-1330 genome to investigate its suitability as a probiotic.However, our results suggest that the use of this strain for prophylactic purposes may not yield the expected benefits and that despite is apparent lack of virulence [4,18], S. suis 90-1330 may not be entirely harmless.

A mobile bacteriocin locus?
One major argument against the use of an unmodified S. suis 90-1330 strain as a probiotic is the fact that the bacteriocin locus was found encoded within what appears to be an integrative and conjugative element (ICE) shared between different streptococcal species.Mobile genetic elements are common in bacterial genomes and are drivers of genetic diversity between and within lineages [61].Although derelict/incomplete ICEs are no longer capable of mobility, complete ones can propagate to other loci upon the proper stimuli.The S. suis 90-1330 bacteriocin locus contained in the putative ICE codes not only for the suicin itself but also provides protection against its effects [4,62].One can easily envision a scenario wherein a mobile bacteriocin-containing ICE could integrate their genetic material in other microorganisms, including the very pathogens whose growth we are trying to inhibit, under sublethal bacteriocin concentrations.This would render the bacteriocin ineffective against them, and while it is not known exactly how prevalent or efficient this process would be, over time there is a non-negligible probability that an active ICE could integrate itself into the genomes of the very organisms that are we are trying to get rid of.Given that a near identical bacteriocin locus was found in a mobile ICE from S. suis strain CZ1303002 [52], a potent pathogen shown to cause severe meningitis [63], this scenario likely has occurred in the not so distant past.Furthermore, because Pan and colleagues [52] successfully transconjugated the S. suis CZ1303002 ICE from suicin+ to suicin-species, it appears that suicin-species can indeed survive long enough to acquire the proper resistance mechanisms under the right conditions.
Here, although the mobility of the S. suis 90-1330 suicin-containing putative ICE has not been tested in vitro, the combined presence of the ICESa2603-like direct repeats, of a putative origin of transfer oriT, of an integrase together with the conjugative DNA processing and Type IV secretion system proteins required for the ICE propagation to ectopic sites and recipient cells [61] (Fig 2), together with the detected presence of both integrated and excised forms of this element in S. suis 90-1330 (S2 Fig), strongly suggests that this element is genuine.Clearly, more experiments are required to confirm that this element is mobile.However, considering that the suicin-containing locus from the S. suis 90-1330 putative ICE also has been found in the mobile ICESsuCZ130302 from S. suis CZ1303002 [52] and in several other bacterial species also harboring ICE-related genes (Fig 3), it would be prudent to consider the S. suis 90-1330 ICE as capable of mobility unless demonstrated otherwise.
But even if the bacteriocin-containing locus in S. suis 90-1330 is no longer mobile, streptococcal species are naturally competent [64], and this strain is no exception.All the genes involved in the ComRS competence system and in the methylation of foreign DNA acquired by competence were found in the S. suis genome, which provides this strain with the opportunity to acquire genetic material from bacteria that it kills with its bacteriocin, and one could envision that by killing pathogenic strains, S. suis 90-1330 may acquire/reacquire virulence genes.Considering all the above, engineering the S. suis 90-1330 strain to ensure that the locus is no longer mobile and to knockout the streptococcal competence system prior to its use as a prophylactic appears an essential step to minimize undesirable outcomes.

Would using S. suis 90-1330 as a prophylactic provide tangible benefits to the host?
Intuitively, effectiveness and the absence of side effects are desirable traits of any treatment.One of the major draws of using the S. suis 90-1330 bacteriocin as a substitute for traditional antibiotics resides in its relatively narrow spectrum, which reduces the risk of disturbing the healthy microflora [2].However, the wide distribution of the S. suis 90-1330 bacteriocin cluster across several streptococcal strains and species (Figs 3 and 4) raises doubt about the overall effectiveness of using this strain as a prophylactic.Because the bacteriocin locus also confers protection against its effect, many strains are already protected against this bacteriocin, including several pathogens.But even for other streptococcal pathogens lacking this locus, because they are naturally competent, merely knocking out mobility of the S. suis 90-1330 bacteriocincontaining locus may not be sufficient to prevent its propagation to other organisms.Any genetic material left in the environment by S. suis 90-1330 could be integrated by pathogens and, therefore, decoupling the bacteriocin from its protectin would help minimize the risks of propagation by preventing the simultaneous acquisition of both genes.Clearly, knocking out the protectin gene from the S. suis 90-1330 genome would render this strain susceptible to its effect, and is therefore not desirable.Considering these issues, administering the bacteriocin alone via other distribution mechanisms may prove more effective than using S. suis 90-1330 as a probiotic.

Is S. suis 90-1330 truly harmless?
Using S. suis 90-1330 and the bacteriocin it produces for prophylactic purposes appeared a very interesting prospect given its apparent lack of virulence [18].However, our results raise doubts as to whether this strain is indeed devoid of toxicity.While we did not identify the suilysin typically associated with S. suis virulence, we identified several genes whose products are involved in blood survival.Indeed, the presence of genes coding for hemolysin III, for a TlyClike hemolysin transporter and for platelet-binding protein FbpA all support the ability of this strain to survive if not thrive in the bloodstream.Complementation studies involving TlyC turned a nonhemolytic bacteria into an hemolytic one [65] and FbpA is considered an important factor in infective endocarditis, with the S. suis FbpA featuring both the N-terminal fibrinogen binding domain and the C-terminal DUF814 domain found in Enterococcus faecalis homologs shown to bind to immobilized fibronectin [66].While previous infection experiments performed on mice and pigs with S. suis 90-1330 did not lead to infection [18], one of the three intravenous replicates performed on pigs led to a transient lameness suggesting that this strain might not be entirely benign.The presence of several CPS genes in this serotypable strain also suggests that it may be able to avoid phagocytosis to some extent, and the conditions performed to test the virulence of S. suis 90-1330 cannot, by their nature, reproduce all the possible conditions found in the environment.As discussed recently by Segura et al. [36], S. suis infections usually originate from the respiratory tract and the intravenous method sidesteps potentially critical early infection stages of this pathogen.But even if S. suis 90-1330 is indeed avirulent, synergies between different strains have been observed in blood infections [47].Those infections included other supposedly low virulence SS2-ST28 strains, such that under the right conditions, it would be reasonable to expect virulence from the 90-1330 strain.

Conclusions
The intent for this study was to look for the suitability of using S. suis 90-1330 as a prophylactic strain in the swine industry.However, based on our results, we cannot recommend its use as is without further engineering given the apparent mobility of its lantibiotic bacteriocin locus cargoed in what is likely an integrative and conjugative element.But even if this element is no longer mobile, given the wide distribution of this bacteriocin and its resistance mechanism across several streptococcal species, the use of this S. suis isolate as a probiotic may provide only limited protection against virulent strains.Furthermore, based on its genetic paraphernalia, the S. suis 90-1330 strain may not be as benign as previously thought and further testing and/or engineering would be required to ensure the safety of the animals subjected to potential prophylactic treatments involving this strain.Considering all the above, administering the bacteriocin directly as food preservative/supplement using alternate mechanisms that are not reliant on probiotics may prove a better approach to minimize undesirable outcomes, and further work will be required to determine if using this bacteriocin in such a fashion would indeed be effective.

Fig 2 .
Fig 2. Structure of the putative suicin-containing ICE in S. suis 90-1330.A zoom-in of the low-GC locus surrounding the bacteriocin and the predicted map of the excised ICE are shown on the top and bottom of the figure, respectively.Genes and their directionality are indicated by arrows and color-coded by their products: orange-integrase/recombinase; light-purple-suicin-related proteins; deep-blue-conjugative DNA processing proteins; deep-purple-Type IV coupling protein; red-Type IV secretion channel proteins; light green-surface adhesin; yellow-DNA methyltransferases; light-blue-antibiotic resistance proteins; deepgray-replication initiator protein; deep-green-TXSScan FATA_gbs matches (E-value cutoff 1e-40); light-gray-miscellaneous.The GC content of the zoom-in is shown in the histogram above the schema (sliding window 100 nt, step 100 nt).oriT (origin of transfer); DR (ICESa2603-like direct repeat).https://doi.org/10.1371/journal.pone.0216002.g002

Fig 3 .
Fig 3. Distribution of the S. suis 90-1330 suicin-containing locus in other bacterial species.Locus tags/gene names from S. suis 90-1330 are indicated on top (pseudogenes are not shown); genes from Lebel et al. [4] are highlighted in magenta (sslA; bacteriocin).ICE-related genes identified by TXSScan/CONJscan searches and by Tn5252 BLASTP homology searches are highlighted in blue.The internal cargo-like segment of S. suis 90-1330 is delineated by black bars.Gene copy numbers were inferred by TBLASTN searches with E-value and identity cutoffs of 1e-10 and 75%, respectively.Strains with identical contents were collapsed into single rows to increase readability.S. suis 90-1330 and CZ130302 from S3 Fig are highlighted in orange.https://doi.org/10.1371/journal.pone.0216002.g003

Fig 4 .
Fig 4. Distribution of the bacteriocin locus across complete and draft S. suis genomes (847 total).MASH pairwise genetic distances were clustered by similarity using t-Distributed Stochastic Neighbor Embedding (t-SNE [31]).Bacteriocin-containing clusters are indicated by asterisks ( � ).Nodes identified by roman numerals (I-XX) were color-coded by proximity.Complete genomes are indicated by black circles and labelled using the corresponding color palette.Note that because of the multidimensional reduction, the relative distances between the nodes are not quite to scale on the two-dimensional plane.https://doi.org/10.1371/journal.pone.0216002.g004