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Genome analysis provides insight into hyper-virulence of Streptococcus suis LSM178, a human strain with a novel sequence type 1005

Streptococcus suis has been well‐recognized as a zoonotic pathogen worldwide, and the diversity and unpredictable adaptive potential of sporadic human strains represent a great risk to the public health. In this study, S. suis LSM178, isolated from a patient in contact with pigs and raw pork, was assessed as a hyper‐virulent strain and interpreted for the virulence based on its genetic information. The strain was more invasive for Caco‐2 cells than two other S. suis strains, SC19 and P1/7. Sequence analysis designated LSM178 with serotype 2 and a novel sequence type 1005. Phylogenetic analysis showed that LSM178 clustered with highly virulent strains including all human strains and epidemic strains. Compared with other strains, these S. suis have the most and the same virulent factors and a type I‐89 K pathogenicity island. Further, groups of genes were identified to distinguish these highly virulent strains from other generally virulent strains, emphasizing the key roles of genes modeling transcription, cell barrier, replication, recombination and repair on virulence regulation. Additionally, LSM178 contains a novel prophage conducive potentially to pathogenicity.


Results
Zebrafish challenge. The newly isolated strain LSM178 was compared for virulence in a zebrafish intraperitoneal infection model with two other well characterised S. suis isolates, SC19 and P1/7. Before 10 h, all three infection groups (LSM178, SC19 and P1/7) of infected zebrafish showed no signs of disease except for a slight decrease in swimming ability. After that, pathological changes appeared in challenged zebrafish such as systemic hemorrhage or abdominal hemorrhage, abdominal swelling and dyspnea. The survival rate did not show significant difference (P = 0.5287) and stabilized at 10% for three groups (Fig. 1A). However, the 50% and 90% lethal time by LSM178 (24-28 h and 34-38 h) appeared always shorter than that by SC19 (26-32 h and 38-48 h) and P1/7 (30-36 h and 36-40 h) in each of 3 independent repeated tests ( Fig. 1A and Supplementary Fig. 1). The main symptoms of the death caused by LSM178 infection are serious abdominal swelling, blood spots and ecchymosis (Fig. 1B), which were similar to those caused by SC19 and P1/7.
Phylogenetic tree analysis. Using the genome sequence of LSM178 and 52 published S. suis complete genomes, a phylogenetic tree was generated with the 51,520 core-genome SNPs from the nonrecombinant regions (Fig. 4). Within the 19 strains clustered with LSM178 (LSM178 clade), 7 out of 12 pig isolates and 6 out of 7 human strains were from China. Among the 53 strains, most ST1 (6/9) and ST7 (9/12) were clustered in LSM178 clade. It was interesting that in LSM178 clade, the STs of human isolates (GZ1, LSM178, LSM102, 05ZYH33, SC84, BM407 and 98HAH33 with ST1, ST1005, ST658, ST945, ST7, ST1 and ST890 respectively) are more diverse than those of pig strains (just including ST1 and ST7).    LSM178, 05ZYH33, 98HAH33, A7, BM407, CS100322, GZ1, JS14,  LSM102, P1/7, S10, SC070731, SC19, SC84, SS12, SS2-1, T15 and ZY05719) (Fig. 5A). Excluding avirulent T15 34 , these strains almost constituted the LSM178 clade (Fig. 4). In pha17802, the overlapped GI (positions 1,866,902-1,904,229) was characteristic to LSM178 in comparison with other 52 S. suis (Fig. 5A). The left part (positions 1,871,862-1,892,563) of the GI is only highly similar to (identity of 90%) a region of S. suis 6407, whereas the right part (positions 1,892,563-1,903,867) together with sequence of positions 903,868-1,906,268 is not homologous with any S. suis but with Streptococcus agalactiae Sag37 (identity of 91%) (Fig. 5B). The prophage comprises 55,121 bp with an average GC content of 41.87%, Figure 3. The circular diagram of the LSM178 genome. From inside to outside, the first circle, the scale of genome; the second circle, GC skew; the third circle, GC content; the fourth and seventh circles, the COG category of coding sequence (CDS) on two strands; the fifth and sixth circles, the position of CDS, tRNA and rRNA on two strands. The image was generated by online website CGView Server BETA (http:// cgview. ca/). ) was found to be highly similar to type I-89 K-PAI of SC19 and both of them are also similar to ICESsu05SC260 belonging to ICESa2603 family (Fig. 5C). The direct repeat sequences indicated the location of the 84 K-PAI just downstream of rplL (04,665). The PAI encodes Comparison of the 84 K-PAI with the 89 K-PAI from SC19. The 84 K-PAI were marked with key genes (black) including virulence-related factors (bold), antibiotic resistance factors (bold), major differential genes and core transposition elements. Green box, GIs; Red box, Tn916. (C) Comparison of the pha17802 with the homologous region located in S. suis 6407 and S. agalactiae Sag37. Genes rather than hypothetical protein-coding ones were given predictable functions (black). Green box, GI. The functional regions were designed with red line. The image of A was generated using BLAST Ring Image Generator v0.95 (BRIG). The images of B and C were generated using BLAST embedded in Easyfig v2.25.  Table 2). Since LSM178 shares these important virulence markers with epidemic strains and almost all human strains, these strains were referred here as the highly virulent strains (HVS) at least for serotype 2 strains from China, those are LSM178, ZY05719, 98HAH33, SC84, 05ZYH33, SS2-1, SC19 and LSM102.

Genomic comparative analysis. Comparison of virulence factors between strains in LSM178 clade and
closely related avirulent T15 identified 87 virulence factors which were shared by T15 and the LSM178 clade strains, except A7, P1/7, S10 and GZ1 (Supplementary Table 2). These four strains had 87 virulence factors, but they share Epf, NadR, RevS and SBP2 instead of Trag, VirB1, VirB4 and VirD4 as in T15. However, there were no virulence factors belonging exclusively to avirulent T15 or all LSM178 clade strains. To globally identify the characteristic virulence factors, the difference was checked between strains in LSM178 clade and closely related avirulent T15. Referring to T15, the numbers of unique genes of each virulent strain were between 318 and 582, and 224 shared genes were found (Fig. 6A). HVSs possess more core genes (332) than other virulent strains (249, called generally virulent strain here) ( Fig. 6B and C). Addition of any one generally virulent strain except BM407 (Vietnam) resulted sharp decreased number of core high virulence genes (Fig. 6D). However, little change was observed when any one highly virulent strain was removed. This supports HVSs as a seperate group in serotype 2 strains. The characteristic genes from various categories encode a wide range of functions (Fig. 6E-G). Compared with generally virulent strains, HVSs show predominant increase on genes with functions of transcription, cell barrier, replication, recombination, repair and mobile elements ( Fig. 6F and G). GIs of LSM178 occupy 10.4% of the genome (Fig. 6H). While there are 3.7% of core virulence genes in GIs of LSM178, 23.5% of core high virulence genes locate in the GIs (Fig. 6H).

Discussion
Generally, S. suis infections in humans are restricted to workers in close contact with pigs or swine byproducts. However, in southeast Asia, the bacterium has been reported to affect the general population 23 . The sporadic human S. suis, particular those with serotype 2, should get attention in epidemiological monitoring because of www.nature.com/scientificreports/ their unpredictable adaptive potential, as demonstrated by LSM178, a serotype 2 strain with a novel ST1005. LSM178 was more efficiently invasive to Caco-2 cells compared with P1/7 and SC19. However, no significant virulence was detected in cytotoxic activity and zebrafish challenge assays which have been used as a model to evaluate the virulence of S. suis 35 . Indeed, virulence of S. suis could not be intensively evaluated and compared in other models 36 . Probably, virulent strains have their own outstanding virulence aspects, which can balance the weak ones and eventually lead to a similar pathogenicity at least under the inoculation. For instance, P1/7 and SC19 were prominent in biofilm formation ( Supplementary Fig. 3) and adhesion respectively. Recent studies suggested that quinolones, beta-lactams, florfenicol and trimethoprim/sulfamethoxazole could still effectively treat clinical S. suis infection [37][38][39] . However, strains resistant to widely used effective beta-lactams have been increasely reported 40,41 . LSM178 showed the sensitivity at least to beta-lactams and quinolones, consistent with the features of resistance genes. The PBPs of LSM178 are entirely the same as those of sensitive A7, but habor substitutions throughout the sequence compared with the resistant R61 ( Supplementary Fig. 4A-E) 40 . Several mutations in the quinolone resistance-determining region (QRDR) of both GyrA and ParC could increase the resistance to fluoroquinolone 42 . However, no amino acid changes were revealed in the QRDRs between LSM178 and four quinolone-sensitive strains (A7, BM407, P1/7 and SC84) 40 (Supplementary Fig. 5). Additionally, it is interesting that the P1/7 without ANT(6)-Ia confered resistance against streptomycin, whereas ANT(6)-Ia containing LSM178 could not 38 . However, ANT(6)-Ia of LSM178 is only 49.5% identical to the functional homologue (UniProtKB-P12055 (AADK_STAAU)) from Staphylococcus aureus 43 (Supplementary Fig. 6).
Several factors are deemed to be important for the pathogenesis, such as CPS, Fbps, enolases, dipeptidylpeptidase DppIV and SrtA 25 , which were all found in LSM178. It has been demonstrated that S. suis serotype 2 virulent strains are able to exacerbate inflammatory activation scavenging bacteria. All the 15 known anti-immunity factors 22 exist in LSM178 and would modulate the immune responses improving its survival at the inflammation area. Although it is difficult to define a virulent strain only by proposed virulence genes, strains isolated from healthy animal are found with less virulence factors (less than 87), such as WUSS351, 0061, 1081, HA1003, TL13, 05HAS68, YB51, LS9N, SRD478, 90-1330 and T15. These S. suis lack at least one of the marker virulence genes (sly, mrp, epf and cps2). Notably, four of those strains contain less than 70 virulent factors (0061, 1081, HA1003 and LS9N). On the contrary, the HVSs contain consistently the most (95/96) virulent factors. However, it should be realized that the combination of various virulence factors may cause pathogenicity despite the number of the virulence factors. For instance, the CZ130302 contains 69 virulence factors but shown to be a virulent strain 11 , and the strains other than T15 isolated from healthy animal are not necessarily the avirulent strains.
It is very clear that intermediately pathogenic strain could evolve to become a highly pathogenic and then epidemic strain 4 . It should be noted that the evolutionary relationships could be lack of reliably based on the raw sequence due to the extensive genetic recombination in S. suis 24 . With the nonrecombinant regions, phylogenetic analysis should show the reliable evolutionary relationships 1 . In LSM178 clade, all 19 strains possessed the serotype 2. However, while 7 human strains have 6 STs, 12 pig strains were just ST1 (4 strains) or ST7 (8 strains). It may imply that while S. suis has been purified to relatively stable genotype in pig, multiple evolutionary directions are in progress to be epidemic during their adaption to human, at least for serotype 2 strains in China.
Type I-89 K-PAI is specific to highly pathogenic S. suis linked to Chinese epidemics 22 and could horizontally transfer among strains 44 . Humans can carry avirulent S. suis without clinical signs 3,45 . However, the 89 K-PAI of human virulent strain should not be obtained from pig strain owning to the transfer, since all strains with 89 K-PAI presented an extremely short evolutionary distance from each other. It suggested that human pathogenic infection was due to the interspecies transmission of a swine-origin strain. The gradual increase of 89 K-PAIs in human strains and decrease in pig strains may suggest that the 89 K-PAI is one of the markers adapting pig strain to human. On the contrary, the existence of PAI would not be conducive to the adaptation of strains in pigs, that might explain the reason why the pig strains lose it more and more rapidly. Under this consideration, the combination of 95 virulence factors may also be regarded as indicative one of the potential for the adaptation to human.
In published S. suis genomes, many prophages were held as remnants 46,47 . A few intact prophages was described and one of them was reported to be induced to lyse S. suis 48 . The two prophages in LSM178 could not be induced to form plaques on several S. suis strains. The reason might be that improper induction method was adopted or that the phages produced are defective on infection even for the almost intact pha17802 48 . Alternatively, the lysis spectrum of the phages is very narrow, or no phages were induced at all, which could be supported by the facts that pha17801 contains only integrase and pha17802 does not include excisionase. Genomic comparative analysis suggested that the pha17802 may integrate two elements horizontally transferred from S. suis 6407 and S. agalactiae Sag37 respectively. The unique gene fragment from S. agalactiae Sag37 has not been found in any other S. suis genome published so far. The empty target site might accommodate potentially unpredictable integration of other mobile genetic elements. Prophages make up a platform for the dissemination of virulence determinants between intra-and inter-species, contributing to the evolution of pathogenic bacteria 30,49 . For pha17802, except the hicAB located in the arm homologous to 6407, no known genes contributing to virulence were identified. There was no evidence demonstrating the contribution of the two transferring elements to virulence. However, the rarity of pha17802 suggested that it might provide an advantage under certain circumstances. At least, the lysogenic state would increase the survival in the environment by resisting to infection of similar viruses. Furthermore, it may increase the fitness of the bacteria by modulating host metabolism 48 .
While the core virulence genes were identified through the comparation between avirulent T15 and virulent strains in LSM178 clade, they should be responsible for the enhanced pathogenicity. Moreover, some genes could be used to make a distinction between HVSs and other general virulent strains. Particularly, it should emphasize the roles of genes with functions of transcription, cell barrier, replication, recombination and repair in virulence enhancement, since their number increased obviously in HVSs core genes. Thus, the importance of the mobilizable elements is beyond all doubt, because it is the carrier responsible for genetic differences 50  www.nature.com/scientificreports/ make up a considerable part of the genome for LSM178 (10.4%). And, higher proportion of virulence-enhancing genes are dispersed in GIs, for example that GIs of LSM178 are colonized with 3.7% of core general virulence genes but with 23.5% of core high virulence genes. However, there are less GIs in LSM178 (number of 23 and total length of 21, 5087 bp) than T15 (29 and 31, 4245 bp) (Supplementary Fig. 7B). These suggested that fusion of specific PAIs increased the virulence. The non-existence of CRISPRs defending against foreign invading elements should be one of the reasons interpreting the rich GIs 51 . In addition, assay showed that there are more core avirulence genes (535) (Supplementary Fig. 7A) than core virulence genes (224). Probably, both the gain of virulence genes and loss of avirulence genes contribute to the increased virulence. Other potential difference, such as SNPs and patches of insertion and deletion, deserve also to be explored and should not be ignored. These differences are small but numerous and they would definitely interpret the change of strain virulence through just affecting the genes expression or protein activity 52 .

Materials and methods
Strains and antimicrobial susceptibility testing. S. suis LSM178 was isolated at 2016 from a patient with clinical symptoms of fever, nausea, and general malaise. The patient had been in contact with pigs and handled raw pork before admission. The S. suis SC19 and S. suis P1/7 were stored in our laboratory. Antimicrobial susceptibility was tested by E-test (AB Biodisk, Sweden) with Streptococcus pneumoniae ATCC49619 as a control. All protocols was approved by committee of State Key Laboratory of Agriculture Microbiolgy and the ethics committee of Huazhong Agricultural University. Experiments were performed in ABSL 3 laboratory, Huazhong Agricultural University.
Human whole blood resistance assay. Blood assays were conducted according to an approval issued by the Medical Ethics Committee of the Huazhong Agriculture University (Wuhan, China). Strain suspension (100 μL, 5.0 × 10 7 CFU/ml) was transferred into 900 μL of fresh human whole blood followed by incubation at 37℃. Samples were withdrawn every 1 h and diluted to incubate on TSA solid medium (containing 10% fresh FBS) at 37 ℃ overnight to count colony-forming units (CFU). Growth index (%) = (CFU at a certain time point -CFUoriginal inoculum ) / CFU original inoculum × 100%.
Zebrafish challenge. Zebrafish were fed as previously described 53 . Inoculum was collected at the end of the logarithm period, cleaned twice with phosphate buffered saline (PBS), and adjusted to the appropriate dose (2.5 × 10 9 CFU/ml) 35 . Adult zebrafish were infected by intraperitoneal inoculation with 20 μL of bacterial solution per tail. Each group contained 10 zebrafish. The symptoms of zebrafish were recorded every 2 h. Euthanasia of zebrafish was conducted at 60 h after challenge using Tris-buffered tricaine at a concentration of 320 μg/ml.

Biofilm formation assay.
Strains (20 μL, 5.0 × 10 7 CFU/ml) were inoculated into 2 mL TSB medium (containing 10% fresh FBS) and cultured in a 24-well cell plate at 37℃ for 3 days, and the un-inoculated medium was used as the control. After that, the strains were washed twice with sterilized PBS, and fixed with 500 μL methanol for 30 min to attach the strains to the wall. Then, methanol was removed and the plate was air-dried at room temperature. Next, 500 μL of 0.1% crystal violet dye solution was added in and removed out until 30 min later. After drying at 56℃, 500 μL of 33% acetic acid solution was added and placed on a shaker for 30 min to release the crystal violet bound to the biofilm. The released solution (200 μL) from each well was measured at a wavelength of 600 nm.
Cell experiments. Strains at log phase was used in the expriments. For cytotoxic assay, Caco-2 cells (1 × 10 4 ) 54 in 96 well plate was used to detect cytotoxicity of the strains (2 × 10 5 CFU) with lactate dehydrogenase kit (Beyotime, Beijing, China). The percentage of cytotoxicity was calculated referring to the protocol of the kit: cytotoxicity (%) = (LDH release from infected cells -spontaneous release of LDH from uninfected cells) / (maximum LDH release from cell lysate-spontaneous release of LDH from uninfected cells) × 100%. For cell adhesion and invasion, single layer Caco-2 cells in the 24 well culture plate were inoculated with 500 μL bacterial suspension (1 × 10 6 CFU) for 3 h. After washing to remove unadhesive strain, cells were then treated by trypsin digestion for 2 h. In invasion, extracellular bacteria were treated with gentamicin (100 μg/ml) and penicillin G (5 μg/ml) before trypsin treatment. The digested cells were lysed using 1% saponin and the lysis was inoculated on THB plate. The rate of adhesion (Ra) and invasion (Ri) was expressed as (CFU determined from plate / CFU original inoculum ) × 100%. The relative invasion rate was expressed as Ri strain / Ri P1/7 × 100%.
Genomic analysis. The genomic DNA was extracted using a DNA extraction kit (TaKaRa DNAiso, TaKaRa Biotechnology Co., Ltd., Dalian, China). The genome of LMS178 was sequenced using combined plaforms of Illumina Miniseq and PacBio sequel. Illumina Miniseq generated 4,234,826 reads producing a total of 926,438,020 bp with Q30 of 76.81%. PacBio sequel generated 234,808 reads and a total sequence length of 1,538,801,765 bp with N50 of 9,236 bp. The data from Illumina Miniseq and PacBio sequel were assembled by A5-Miseq v20150522 55 and CANU 56 respectively. After correction of the results using Pilon 57 , the complete circular genome was constructed.  Table 2) and used to detect the potential virulence genes in genome. Of the 96 virulence factors, 84 are from the 2016 study by Willemse et al 58 . Another 12 putative virulence factors were identifed by systematic search using the term 'Streptococcus suis virulence factor' on July 27 th 2017 in NCBI PubMed. TAs were predicted by Rasta 59 and TAfinder 60 . Antibiotic resistance genes were predicted with the comprehensive antibiotic resistance database (CARD) with default settings 61 . The ST was determined using the MLST typing scheme (https:// pubml st. org/ ssuis/). Using BLAST (evalue ≤ 1e-10) 62 , serotyping strategy was executed based on the levels (≥ 80%) of both identity and coverage between WZY amino acid sequence of the LSM178 and 33 standard strains with known serotypes 63 . The sequence of CpsK was used to discriminate the serotype 2 (W161) from 1/2 (C161) for all strains with serotype 2 or 1/2 64,65 . ST complexes were analysed by goeBURST 61 program (http:// goebu rst. phylo viz. net). Prediction of gene islands (GI) and prophages were performed using IslandViewer 4 66 and PHAST 67 respectively. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) were predicted by CRISPR recognition tool (CRT) 68 . Open reading frames (ORFs) were predicted with Glimmer3.0 69 . tRNA and rRNA were predicted with the Aragorn 70 and RNAmmer 71 embedded in Prokka 72 . The proteins in prophage and PAI were integrally annotated by databases of NR 73 , eggNOG 74 , KEGG 75 , Swiss-Prot 76 and GO 77 . Genome mapping with information was generated by CGView 78 . To identify the unique regions in the genome of LSM178, the whole-genome sequence was used as a reference to compare with that of the other 52 strains using BLAST Ring Image Generator (BRIG) 79 . The 84 K-PAI in LSM178, 89 K-PAI in SC 19 and ICESsu05SC260 were compared to determine their similarity using BLAST embedded in Easyfig 80 . Similarly, the characteristics of the prophages in LSM178 were evaluated. The differential genes between two strains were extracted with Roary 81 to create Venn diagrams and their COG functions were classified. Circos 82 was conducted to map the distribution of GIs and interesting genes in genomes of LSM178 and T15. The.
Phylogenetic analysis. Complete genomic sequences of 52 S. suis sequences were downloaded from NCBI database (https:// www. ncbi. nlm. nih. gov/ genome/ genom es/ 199) (Supplementary Table 3). The chromosomal sequences were aligned using Parsnp 83 generating core-genome single-nucleotide polymorphisms (SNPs). Phylogenetic tree based on core SNP of 53 complete S. suis genome (including LSM178) was constructed using maximum-likelihood phylogenetic trees by FastTree embedded in the Parsnp. In sequence alignment, recombinant regions were filtered using Gubbins v2.4.0 84 . The bootstrap value was set at 1000 times. The phylogenetic tree was displayed using the online website iTOL (http:// itol. embl. de/). Statistical analysis. Independent determinations were performed in dupicate or triplicate and experiment was repeated at least 3 times in each group. The significance was analyzed with unpaired student's test using GraphPad Prism 5. The bar represent the mean ± standard error of mean (sem). The P < 0.05 and P < 0.01 were represented as * and ** respectively.

Ethics statement.
This study was carried out in compliance with the ARRIVE guidelines. The study was approved by ethics committee of Huazhong Agricultural University and all experiments were performed in accordance with guidelines of State Key Laboratory of Agriculture Microbiology. The informed consent was obtained from all participants and/or their legal guardians. Statement on Guidelines for Human. All procedures performed in studies involving human participants were in accordance with the ethical standards of national research committee and with the 1975 Helsinki Declaration (or its later amendments). Statement on Guidelines for Animal. All procedures performed in studies involving animal were in accordance with the ethical standards of national institutional guidelines on the care and use of animals.

Data availability
The whole-genome sequence of the LSM178 was deposited in NCBI Genbank (Accession Number CP047248). Accession numbers for raw data from Illumina Miniseq and PacBio sequel are SRR15853891 and SRR15943338 respectively.