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. 2024 May 4;13(1):2352435. doi: 10.1080/22221751.2024.2352435

Streptococcus suis serotype 4: a population with the potential pathogenicity in humans and pigs

Jinlu Zhu a,b,c, Jianping Wang e, Weiming Kang e, Xiyan Zhang e, Anusak Kerdsin g, Huochun Yao a,b,c, Han Zheng e,f,✉, Zongfu Wu a,b,c,d,CONTACT
PMCID: PMC11097711  PMID: 38703011

ABSTRACT

Streptococcus suis is a major bacterial pathogen in pigs and an emerging zoonotic pathogen. Different S. suis serotypes exhibit diverse characteristics in population structure and pathogenicity. Surveillance data highlight the significance of S. suis serotype 4 (SS4) in swine streptococcusis, a pathotype causing human infections. However, except for a few epidemiologic studies, the information on SS4 remains limited. In this study, we investigated the population structure, pathogenicity, and antimicrobial characteristics of SS4 based on 126 isolates, including one from a patient with septicemia. We discovered significant diversities within this population, clustering into six minimum core genome (MCG) groups (1, 2, 3, 4, 7-2, and 7-3) and five lineages. Two main clonal complexes (CCs), CC17 and CC94, belong to MCG groups 1 and 3, respectively. Numerous important putative virulence-associated genes are present in these two MCG groups, and 35.00% (7/20) of pig isolates from CC17, CC94, and CC839 (also belonging to MCG group 3) were highly virulent (mortality rate ≥ 80%) in zebrafish and mice, similar to the human isolate ID36054. Cytotoxicity assays showed that the human and pig isolates of SS4 strains exhibit significant cytotoxicity to human cells. Antimicrobial susceptibility testing showed that 95.83% of strains isolated from our labs were classified as multidrug-resistant. Prophages were identified as the primary vehicle for antibiotic resistance genes. Our study demonstrates the public health threat posed by SS4, expanding the understanding of SS4 population structure and pathogenicity characteristics and providing valuable information for its surveillance and prevention.

KEYWORDS: Streptococcus suis serotype 4, population structure, pathogenicity, antimicrobial susceptibility, prophage, integrative and conjugative elements

Introduction

Streptococcus suis is an important pathogen in the pig industry, causing septicemia, meningitis, and sudden death in pigs, imposing substantial economic losses on the industry [1]. Moreover, S. suis is an emerging zoonotic pathogen that can be transmitted to humans by contact with diseased animals or contaminated raw pork products [2,3]. Indeed, human cases of S. suis have been reported worldwide. Particularly in Vietnam and Thailand, S. suis was responsible for thousands of human disease cases and has been identified as one of the most prevalent causes of adult bacterial meningitis [2,4,5]. Based on the capsular polysaccharide (CPS) antigenicity variation, S. suis can be classified into 29 serotypes (1–19, 21, 23–25, 27–31, 1/2) [6,7]. Also, serotype Chz and 27 novel cps loci (NCLs) have been identified from non-typeable isolates based on differences in the cps gene cluster [8–13]. To date, 11 serotypes have been documented as capable of inducing human infections, consisting of serotypes 1, 2, 4, 5, 7, 9, 14, 16, 21, 24, and 31 [14,15].

Although the distribution of serotypes in clinical cases may vary across geographic locations, S. suis serotype 2 is universally recognized as the most prevalent pathotype in both swine and humans worldwide [1]. Therefore, most studies have focused on S. suis serotype 2. However, different serotypes exhibit diverse characteristics in population structure and pathogenicity. In recent years, we have noticed an increase in the isolation rate of S. suis serotype 4 in pig populations. In addition, the report of the first human case caused by serotype 4 dated back to 1988 in the Netherlands, and the second human case caused by ST94 strain of serotype 4 was reported in 2018 in Thailand [16,17]. Notably, S. suis serotype 4 has been identified as one of the most prevalent serotypes in diseased pigs in Asia, and strains of this serotype have also been detected in healthy and diseased pigs in specific European and North American nations [6,18–21]. In a recent study, Murray et al. identified 10 pathogenic lineages of S. suis based on isolates sampled from healthy and diseased pigs, wild boar, and humans, from Asia, North America, Europe, and Australia [22]. They revealed that 80.36% of S. suis serotype 4 isolates (90/112) belong to these pathogenic lineages [22]. These findings highlight the significance of S. suis serotype 4 in swine streptococcusis. However, except for the information in human case reports and a few epidemiologic studies, the information on S. suis serotype 4 remains exceedingly limited.

In this study, a total of 126 S. suis serotype 4 genomes from eight different countries were analyzed, including one isolate from a patient with septicemia. A systematic bioinformatic analysis was conducted to investigate their population structure, phylogenetic relationship, putative virulence-associated genes, antibiotic resistance genes, and dissemination vehicles of antibiotic resistance genes. Additionally, animal infection experiments, human cell cytotoxicity assays, and antimicrobial susceptibility testing were performed on S. suis serotype 4 strains isolated from pigs and human to assess their pathogenicity and resistance characteristics. This study contributes to our understanding of S. suis serotype 4 and provides valuable information for the surveillance and prevention of this serotype.

Materials and methods

Bacterial strains and culture conditions

To investigate the characteristics of S. suis serotype 4 population, 48 strains isolated from our labs and 78 genomes from NCBI database were used in this study, as shown in Table 1. The 48 strains were collected from diseased and healthy pigs in China between 2014 and 2022. All strains were confirmed to be S. suis by analyzing their 16S rRNA gene sequences [23] and recN gene [24]. Furthermore, these strains were identified as serotype 4 by the PCR method based on the serotype 4 specific wzy gene [25] and the agglutination test using serotype 4 specific serum purchased from Statens Serum Institute (Copenhagen, Denmark). In addition, 78 S. suis serotype 4 genomes downloaded from the NCBI database originated from eight different countries, consisting of 30 from UK, 21 from China, ten from Canada, seven from Thailand, four from the Netherlands, four from the USA, and one each from Denmark and Spain. These genomes harboured S. suis serotype 4 specific wzy gene and were isolated from 1991 to 2019. The human strain ID36054 was isolated from a patient with septicemia reported in 2018 [17,26]. Strains were cultured in Todd-Hewitt broth (THB, Hope Bio-Technology Co., Ltd, China) and plated on THB agar containing 5% sheep blood at 37°C and 5% CO2.

Table 1.

The information of S. suis serotype 4 strains used in this study.

Lineages Strains Accession number ST CC MCG Country Date Host Isolation Source
Lineage 1 CPD27 SAMN12784764 17 17 1 China 2010 Pig / NCBI
LSSP193 SAMN26554949 17 17 1 China 2017 Pig / NCBI
SC5B93 SAMN14687788 17 17 1 China 2019 Pig / NCBI
ND7 SAMN34237859 17 17 1 China 2014 Diseased pig Lung This study
ND83 SAMN34237860 17 17 1 China 2014 Diseased pig Lung This study
ND84 SAMN34237861 17 17 1 China 2014 Diseased pig Lung This study
ND90 SAMN34237862 17 17 1 China 2014 Diseased pig Lung This study
WUSS270 SAMN34237819 17 17 1 China 2017 Healthy pig Tonsil This study
WUSS304 SAMN34237824 17 17 1 China 2017 Healthy pig Tonsil This study
WUSS388 SAMN34237830 17 17 1 China 2017 Healthy pig Tonsil This study
WUSS406 SAMN34237833 17 17 1 China 2017 Healthy pig Tonsil This study
2018WUSS011 SAMN34237836 17 17 1 China 2018 Healthy pig Tonsil This study
CPD30 SAMN12784768 850 17 1 China 2011 Pig / NCBI
WUSS303 SAMN34237823 850 17 1 China 2017 Healthy pig Tonsil This study
2021WUSS074 SAMN34237846 2224 17 1 China 2021 Healthy pig Tonsil This study
2021WUSS078 SAMN34237850 2224 17 1 China 2021 Healthy pig Tonsil This study
2021WUSS076 SAMN34237848 2224 17 1 China 2021 Healthy pig Tonsil This study
2021WUSS079 SAMN34237851 2224 17 1 China 2021 Healthy pig Tonsil This study
CPD39 SAMN12784777 2235 17 1 China 2010 Pig / NCBI
CPD29 SAMN12784766 2235 17 1 China 2010 Pig / NCBI
LSSP132 SAMN26111036 2236 17 1 China 2017 Pig Brain NCBI
GD-0057 SAMEA3595235 17 17 1 Netherlands 2004 Diseased pig CSF NCBI
GD-0073 SAMEA3595242 17 17 1 Netherlands 2005 Diseased pig CSF NCBI
GD-0098 SAMEA3595254 17 17 1 Netherlands 2006 Diseased pig CSF NCBI
MA6 SAMN21439331 17 17 1 Netherlands 2017 Diseased pig Brain/blood NCBI
MA2 SAMN21439327 17 17 1 Canada 2016 Pig Tonsil NCBI
DB1V3-4A SAMN14932575 17 17 1 Canada 2016 Pig / NCBI
40439 SAMN13975665 17 17 1 USA 2017 Pig / NCBI
40458 SAMN13975647 17 17 1 USA 2016 Pig / NCBI
JT9 SAMN29093732 17 17 1 Spain 2018 Pig Brain NCBI
Lineage 2 SS967 SAMN14933159 23 87 2 UK 2010 Pig / NCBI
SS1048 SAMN14932752 23 87 2 UK 2010 Pig / NCBI
LSS20 SAMN14932632 23 87 2 UK 2010 Pig / NCBI
S14O SAMEA3234013 23 87 2 UK 2010 Diseased pig Lung NCBI
S97A SAMEA3234092 23 87 2 UK 2010 Pig Lung NCBI
LS0M SAMEA3233887 23 87 2 UK 2010 Healthy pig Tonsil NCBI
SS967_2 SAMEA1316593 23 87 2 UK 2012 Pig / NCBI
SS1048_2 SAMEA1316599 23 87 2 UK 2012 Pig / NCBI
LSS20_2 SAMEA1316631 23 87 2 UK 2012 Pig / NCBI
TMW-SS042 SAMN14933224 23 87 2 UK 2013 Pig / NCBI
LSS94 SAMN14932706 862 87 2 UK 2011 Pig / NCBI
LS4P SAMEA3233921 862 87 2 UK 2011 Healthy pig Tonsil NCBI
LSS94_2 SAMEA1316533 862 87 2 UK 2012 Pig / NCBI
  6407a SAMN02905150 54 54 3 Denmark / Pig / NCBI
Lineage 3 942 SAMN08295926 94 94 3 China 2013 Pig Tonsil NCBI
944 SAMN08295927 94 94 3 China 2013 Pig Tonsil NCBI
1044 SAMN08295942 94 94 3 China 2013 Pig Tonsil NCBI
SH1510 SAMN09460428 94 94 3 China 2015 Pig Lung NCBI
LSSP204 SAMN26554953 94 94 3 China 2017 Pig / NCBI
LSSP213 SAMN26554957 94 94 3 China 2018 Pig / NCBI
LSSP237 SAMN28125328 94 94 3 China 2018 Pig / NCBI
ND6 SAMN34237858 94 94 3 China 2014 Diseased pig Lung This study
WUSS026 SAMN34237817 94 94 3 China 2017 Healthy pig Tonsil This study
WUSS273 SAMN34237820 94 94 3 China 2017 Healthy pig Tonsil This study
2021WUSS075 SAMN34237847 94 94 3 China 2021 Healthy pig Tonsil This study
2021WUSS077 SAMN34237849 94 94 3 China 2021 Healthy pig Tonsil This study
2021WUSS080 SAMN34237852 94 94 3 China 2021 Healthy pig Tonsil This study
WUSS326 SAMN34237826 1175 94 3 China 2017 Healthy pig Tonsil This study
WUSS329 SAMN34237827 1175 94 3 China 2017 Healthy pig Tonsil This study
2018WUSS156 SAMN34237840 2220 94 3 China 2018 Diseased pig / This study
SS1042 SAMN14932750 911 94 3 UK 2010 Pig / NCBI
SS1041 SAMN14932749 911 94 3 UK 2010 Pig / NCBI
SS1040 SAMN14932748 911 94 3 UK 2010 Pig / NCBI
S14J SAMEA3234009 911 94 3 UK 2010 Diseased pig Lung NCBI
S14K SAMEA3234010 911 94 3 UK 2010 Diseased pig Lung NCBI
S14L SAMEA3234011 911 94 3 UK 2010 Diseased pig Lung NCBI
SS1042_2 SAMEA1316637 911 94 3 UK 2012 Pig / NCBI
SS1041_2 SAMEA1316538 911 94 3 UK 2012 Pig / NCBI
SS1040_2 SAMEA1316654 911 94 3 UK 2012 Pig / NCBI
TMW-SS070 SAMN14933250 911 94 3 UK 2014 Pig / NCBI
TRG6 SAMN31277175 94 94 3 Thailand 2010 Diseased pig Lung NCBI
ID36054 SAMN31277174 94 94 3 Thailand 2011 Homo sapiens Blood NCBI
ID34693 SAMN31277178 94 94 3 Thailand 2011 Pig Tonsil NCBI
ID34704 SAMN31277179 94 94 3 Thailand 2011 Pig Tonsil NCBI
ID34545 SAMN31277176 1689 94 3 Thailand 2011 Pig Tonsil NCBI
ID34553 SAMN31277177 1689 94 3 Thailand 2011 Pig Tonsil NCBI
ID34572 SAMN31277173 1689 94 3 Thailand 2011 Healthy pig Tonsil NCBI
1602956 SAMN14932473 94 94 3 Canada 2014 Pig / NCBI
1665814 SAMN14932502 1175 94 3 Canada 2014 Pig / NCBI
CPD36 SAMN12784774 485 839 3 China 2013 Pig / NCBI
LSSP145 SAMN26111047 485 839 3 China 2017 Pig Lung NCBI
LSSP144 SAMN26111046 485 839 3 China 2017 Pig Lung NCBI
WUSS346 SAMN34237829 485 839 3 China 2017 Healthy pig Tonsil This study
2018WUSS160 SAMN34237841 485 839 3 China 2018 Diseased pig Spleen This study
MY1C3-3C SAMN14932601 839 839 3 Canada 2016 Pig / NCBI
MA4T3-4C SAMN14932592 839 839 3 Canada 2016 Pig / NCBI
40436 SAMN13975668 839 839 3 USA 2016 Pig / NCBI
D16-010378 SAMN14932573 977 108 3 Canada / Pig / NCBI
1652716 SAMN14932497 977 108 3 Canada 2014 Pig / NCBI
1607743 SAMN14932477 977 108 3 Canada 2014 Pig / NCBI
WUSS390 SAMN34237831 977 108 3 China 2017 Healthy pig Tonsil This study
Lineage 4 LOLA-SS006 SAMN14932618 856 28 4 UK 2010 Pig / NCBI
LL-U SAMEA3233876 856 28 4 UK 2010 Diseased pig Lung NCBI
91-178-2215 SAMN14932532 2233 1372 4 Canada 1991 Pig / NCBI
Lineage 5 WUSS435 SAMN34237834 1067 / 7-2 China 2017 Diseased pig / This study
WUSS436 SAMN34237835 1067 / 7-2 China 2017 Healthy pig Tonsil This study
2019WUSS015 SAMN34237842 2221 / 7-2 China 2019 Healthy pig Tonsil This study
2019WUSS016 SAMN34237843 2221 / 7-2 China 2019 Healthy pig Tonsil This study
2020WUSS060 SAMN34237845 2223 / 7-2 China 2020 Healthy pig Tonsil This study
2022WUSS016 SAMN34237853 2225 / 7-2 China 2021 Healthy pig Tonsil This study
2022WUSS017 SAMN34237854 2226 / 7-2 China 2021 Healthy pig Tonsil This study
WUSS228 SAMN34237818 2237 / 7-2 China 2017 Healthy pig Tonsil This study
WUSS309 SAMN34237825 2237 / 7-2 China 2017 Healthy pig Tonsil This study
WUSS285 SAMN34237821 2238 / 7-2 China 2017 Healthy pig Tonsil This study
WUSS299 SAMN34237822 2238 / 7-2 China 2017 Healthy pig Tonsil This study
WUSS333 SAMN34237828 2238 / 7-2 China 2017 Healthy pig Tonsil This study
WUSS399 SAMN34237832 2239 / 7-2 China 2017 Healthy pig Tonsil This study
CPD3 SAMN12784778 935 / 7-3 China 2014 Pig / NCBI
HA1003 SAMN09460429 1006 / 7-3 China 2010 Healthy pig Tonsil NCBI
1367 SAMN08295994 2229 / 7-3 China 2013 Pig Tonsil NCBI
1369 SAMN08295995 2229 / 7-3 China 2013 Pig Tonsil NCBI
2018WUSS056 SAMN34237837 2218 / 7-3 China 2018 Healthy pig Tonsil This study
2018WUSS108 SAMN34237838 2219 / 7-3 China 2018 Healthy pig Tonsil This study
2018WUSS109 SAMN34237839 2219 / 7-3 China 2018 Healthy pig Tonsil This study
2020WUSS059 SAMN34237844 2222 / 7-3 China 2020 Healthy pig Tonsil This study
2022WUSS018 SAMN31099739 2057 / 7-3 China 2021 Healthy pig Tonsil This study
2022WUSS019 SAMN34237855 2227 / 7-3 China 2021 Healthy pig Tonsil This study
2022WUSS020 SAMN34237856 2057 / 7-3 China 2021 Healthy pig Tonsil This study
2022WUSS056 SAMN34237857 2228 / 7-3 China 2022 Healthy pig Tonsil This study
2022WUSS141 SAMN31099758 2066 / 7-3 China 2022 Healthy pig Tonsil This study
LSS33 SAMN14932646 895 / 7-3 UK 2011 Pig / NCBI
LS3A SAMEA3233909 895 / 7-3 UK 2011 Pig / NCBI
SS1028 SAMEA1316687 908 / 7-3 UK 2012 Pig / NCBI
S12X SAMEA3233997 908 / 7-3 UK 2010 Diseased pig Brain NCBI
270-6A SAMN14933322 2234 / 7-3 UK 2013 Pig / NCBI
32052 SAMN13975691 1209 / 7-3 USA 2015 Pig / NCBI
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/: unassigned.

a

Except for strain 6407, the remaining genomes from MCG group 3 were assigned to lineage 3.

Bioinformatic analysis of S. suis serotype 4 genomes

The draft genomes of 48 strains isolated from our labs were sequenced using Illumina NovaSeq PE150 at Beijing Novogene Bioinformatics Technology Co., Ltd (China). The multilocus sequence type (MLST) and the minimum core genome (MCG) groups of 126 S. suis serotype 4 genomes were determined using the PubMLST database [27] and a previously established approach [28], respectively. Additionally, the global optimal eBURST (goeBURST) analysis [29] was employed to classify clonal complexes (CCs). Bowtie 2 was used to identify single-nucleotide polymorphisms (SNPs) within S. suis serotype 4 genomes, using the genome sequence of SC84 (accession number FM252031) as a reference. The mutational SNP sites were selected based on the procedure described in a previous study [28], and the phylogenetic tree was constructed using the maximum likelihood method by FastTree v2.1.10. As an outgroup, Streptococcus pneumoniae ATCC 700669 (NC_011900) was used to root the tree, and tree visualization was completed using tvBOT v2.5.2 [30]. The distribution of 35 putative virulence-associated genes of S. suis, as described in previous reports [31,32] (listed in Table S1), was investigated among the 126 S. suis serotype 4 genomes. Genes with coverage <80% or a nucleotide sequence identity <80% were determined to be absent. Antibiotic resistance gene analysis was performed using ResFinder 4.1 [33]. The whole genome sequencing data was input for ResFinder 4.1, with parameters set at ≥ 80% identity over ≥ 80% coverage of the reference gene. Prophages were predicted using PHASTER [34]. The prophage is incomplete if the score is less than 60, questionable if it is between 70 and 90, and intact if it is greater than 90 [34]. The integrative and conjugative elements (ICEs) analysis based on the major insertion hotspots rplL and rum loci was conducted according to a previous study [35]. The conjugative plasmids carrying antibiotic resistance genes were predicted using VRprofile2 [36] and oriTfinder [37]. The obtained mobile genetic element (MGE) sequences were further annotated using an online RAST server [38], followed by comparison and visualization using the BLASTn programme inserted within Easyfig 2.2.3 software [39].

Animal infection experiments

Animal infection experiments were conducted at the Laboratory Animal Center of Nanjing Agricultural University (Permit number: SYXK (Su) 2021-0086). According to the population structure of S. suis serotype 4, we selected 34 of 48 strains isolated from our labs, along with the human isolate ID36054, for the zebrafish infection experiment. The infection protocol for zebrafish was described in our previous studies [40,41]. Briefly, bacteria were collected during the mid-log phase, washed twice in PBS, and then adjusted to the proper infection concentrations in PBS. Each experimental group comprised 15 zebrafish, with each fish receiving an intraperitoneal injection containing 3 × 106 CFU of S. suis in 20 µL of PBS. The mortality was recorded from 12 h until 96 h after the challenge. Then, strains highly pathogenic to zebrafish (mortality rate ≥ 80%) were chosen for the mouse infection experiment. Six-week-old BALB/c mice were purchased from the Shanghai SLAC Laboratory Animal Co., Ltd (China). Each experimental group comprised 10 mice, and they were intraperitoneally injected with 3 × 108 CFU of S. suis per mouse. After infection, the mortality of the mice was monitored for 10 days. In the animal infection experiments, the highly virulent S. suis serotype 2 strain SC070731 [42], non-virulent S. suis serotype 9 strain SH040917 [43], and an equivalent volume of PBS were included as controls. According to our previous reports [14,40,41], a strain with a mortality rate ≥ 80% in zebrafish and mice was defined as highly virulent. The Log-rank (Mantel–Cox) test was used to compare the survival curves of zebrafish and mice infected with S. suis strains.

Human cell cytotoxicity assays

Human lung adenocarcinoma cells (A549) and human brain microvascular endothelial cells (hBMEC) were used for cell cytotoxicity assays, according to previous reports [26,44–46]. The human isolate ID36054 and four pig isolates 2021WUSS075, 2018WUSS156, ND90, and 2018WUSS160, with a mortality rate of ≥ 80% in zebrafish and mice, were selected for cell cytotoxicity assays. Strains were cultured and collected during the mid-log phase and were prepared at a concentration of 1 × 107 CFU/mL in THB medium. The A549 and hBMEC cell lines were purchased from the Cell Resource Center, IBMS, CAMS/PUMC (Beijing, China) and cultured in F12/DMEM (Gibco, Carlsbad, USA) and DMEM (Hyclone, Beijing, China), respectively, each supplemented with 10% fetal bovine serum, and maintained at 37°C in an atmosphere of 5% CO2. For each experiment, A549 or hBMEC cells were plated into 24-well flat-bottom plates at 3 × 105 cells/well in 1 mL corresponding medium and maintained in 5% CO2 at 37°C for 48 h to allow cell confluence. Then, the cell number reached approximately 1 × 106 cells/well before the infection assays. The medium was changed every 24 h. In the cytotoxicity assay, S. suis (1 × 107 CFU/well) were added to A549 or hBMEC cells at the multiplicity of infection (MOI) of 10, incubated for 3 and 6 h at 37°C with 5% CO2, respectively. Following the manufacturer's instructions, the supernatant was collected to measure lactate dehydrogenase (LDH) release using the LDH Cytotoxicity Assay-Fluorescence Kit (Thermo Fisher). The unpaired t-test was used to compare the percentage of cytotoxicity of S. suis serotype 4 strains. This experiment has been performed with a minimum of three independent biological replicates.

Antimicrobial susceptibility testing

Minimum inhibitory concentrations (MICs) were determined using the broth microdilution method following the guidelines outlined in the Clinical and Laboratory Standards Institute (CLSI) document (M31-A3). The following 23 antibiotics of 11 categories were tested: β-lactam antibiotics (penicillin, cefotaxime, and amoxicillin), rifamycin (rifampin), glycopeptide (vancomycin), quinolones (marbofloxacin and enrofloxacin), oxazolidinone (linezolid), amphenicols (chloramphenicol and florfenicol), macrolides (tilmicosin, azithromycin, and erythromycin), pleuromutilins (valnemulin and tiamulin), aminoglycosides (gentamicin, streptomycin, kanamycin, and spectinomycin), lincosamides (clindamycin and lincomycin), and tetracyclines (doxycycline and tetracycline). These antibiotics were tested at concentrations ranging from 0.5–256 µg/mL, with breakpoints for resistance according to the CLSI document (VET08-ED4) and guidelines provided by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (https://www.eucast.org/), as listed in our previous study [41].

Results

Population structure and phylogenetic relationship of S. suis serotype 4

MLST analysis showed that the S. suis serotype 4 population exhibited significant genetic diversity, with 40 distinct sequence types (STs) found throughout 126 genomes (Table 1). Most prevalent were ST17 (n = 21) and ST94 (n = 18), followed by ST23 (n = 10), ST911 (n = 10), ST485 (n = 5), ST977 (n = 4), ST2224 (n = 4), ST839 (n = 3), ST862 (n = 3), ST1175 (n = 3), ST1689 (n = 3), and ST2238 (n = 3). Each of the remaining ST54, ST850, ST856, ST895, ST908, ST935, ST1006, ST1067, ST1209, ST2057, ST2066, ST2218-2223, ST2225-2229, ST2233-2237, and ST2239 consisted of two or one genomes. As shown in Figure 1, 18 STs (94/126 genomes, 74.60%) belonged to eight CCs, consisting of CC94 (ST94, ST911, ST1175, ST1689, and ST2220), CC17 (ST17, ST850, ST2224, ST2235, and ST2236), CC87 (ST23 and ST862), CC839 (ST839 and ST485), CC108 (ST977), CC28 (ST856), CC54 (ST54), and CC1372 (ST2233). In contrast, the remaining 22 STs (32/126 genomes, 25.40%) did not belong to any CCs. As shown in Figure 2, according to MCG analysis, 126 genomes belonged to six MCG groups, including MCG groups 1, 2, 3, 4, 7-2, and 7-3. Notably, two main CCs, CC17 (30/126 genomes, 23.81%) and CC94 (35/126 genomes, 27.78%), belonged to MCG groups 1 and 3, respectively. Most strains isolated from diseased pigs belonged to these two CCs. Moreover, the strain ID36054, responsible for human infections, also belonged to CC94. Based on an evolutionary tree constructed from SNPs in core genomes, the 126 genomes were divided into five lineages (Figure 2). Lineages 1 and 2 comprised MCG groups 1 and 2, respectively. Except for strain 6407, the remaining genomes from MCG group 3 were assigned to lineage 3. Lineage 4 was composed of MCG group 4. MCG groups 7-2 and 7-3 were collectively categorized as lineage 5.

Figure 1.

Figure 1.

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A goeBURST analysis of STs of the S. suis serotype 4 population. Numbers in circles indicate partial STs in S. suis MLST database. Deep blue circles and red circles indicate STs identified in the S. suis serotype 4 genomes downloaded from the NCBI database and sequenced in this study, respectively. “n” indicates the number of genomes. STs connected by a line mean that they have six identical alleles. Clusters of linked STs correspond to CCs.

Figure 2.

Figure 2.

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The phylogenetic tree and information of STs, CCs, MCG groups, and putative virulence-associated genes (VAGs) for the S. suis serotype 4 population. The superscripts “D” and “P” indicate strains originating from diseased pigs and human patients, respectively. The phylogenetic tree was constructed based on the SNPs of the core genome. The S. pneumoniae ATCC 700669 (NC_011900) was used as an outgroup to root the tree.

Distribution of putative virulence-associated genes in S. suis serotype 4

Thirty-five putative virulence-associated genes preferentially present in highly pathogenic S. suis serotype 2 strains [31,32] were analyzed among S. suis serotype 4 genomes. As shown in Figure 2, 18 of 35 putative virulence-associated genes were present in all S. suis serotype 4 genomes. The genes atl_2 (124/126, 98.41%), SSU05_1311 (124/126, 98.41%), and abpb (124/126, 98.41%) were present in most S. suis serotype 4 genomes. The remaining 14 of 35 putative virulence-associated genes showed distribution correlated with the MCG groups. All genomes within MCG group 1 exhibited the genotype of ef+/mrp+/sly+. For MCG groups 2 and 3, all genomes exhibited the genotype of ef-/mrp+/sly+. For MCG group 4, all genomes exhibited the genotype of ef-/mrp+/sly-. However, all three classical virulence marker genes were absent in MCG groups 7-2 and 7-3. Based on variation in the central region of the gene, mrp was classified as EU, NA1, and NA2 subtypes [14]. MCG groups 1 and 2 are all subtype NA2, while MCG groups 3 and 4 are mostly NA1 (Figure 2). Genes dltA, IdeSsuis, igdE, ofs, and sao were widespread in serotype 4 genomes, except for MCG groups 7-2 and 7-3. In contrast, the sbp2 gene was primarily distributed in MCG group 1, and hp0197 and hp0272 were only present in MCG groups 1 and 2. The regulatory genes rgg and revs were predominantly distributed in MCG group 1, and the tran gene was present in MCG groups 1 and 3. Importantly, it should be noted that all 35 putative virulence-associated genes were found to be present in MCG group 1 (Figure 2).

The results of animal infection experiments

To assess the virulence of the S. suis serotype 4 population, we selected 35 representative strains based on their distribution in the phylogenetic tree and their host for the zebrafish infection experiment. One strain ID36054 originated from a patient, eight strains (ND7, ND83, ND84, ND90, ND6, 2018WUSS156, 2018WUSS160, and WUSS435) originated from diseased pigs, and the remaining from healthy pigs (Table 2). As shown in Table 2, the mortality rate of zebrafish infected with nine strains (ID36054, WUSS406, 2018WUSS011, ND84, ND90, 2021WUSS075, 2018WUSS156, WUSS346, and 2018WUSS160) reached or exceeded 80%, classified as highly virulent strains. The survival curves of zebrafish infected with strains ID36054, 2018WUSS011, ND84, 2021WUSS075, WUSS346, and 2018WUSS160 showed no significant difference compared to zebrafish infected with the highly virulent control strain SC070731. Notably, strains WUSS406, 2018WUSS011, 2021WUSS075, and WUSS346 were isolated from healthy pigs. The abovementioned nine strains belonged to CC17 (4/11 representatives, 36.36%), CC94 (3/8 representatives, 37.50%), and CC839 (2/2 representatives, 100.00%), respectively. They belong to MCG groups 1 and 3, and harbour numerous crucial genes associated with virulence (Figure 2). There are also five strains (WUSS270, WUSS388, ND83, WUSS303, and WUSS026) with mortality rates between 50% and 80%, which also belong to CC17 and CC94. The mortality rate of zebrafish infected with the remaining strains, including all representative strains of MCG groups 7-2 and 7-3 (13/13 representatives, 100.00%), was <50%, classified as lowly virulent strains (Table 2). The gross pathology of zebrafish infected with highly virulent, moderately virulent, lowly virulent, and non-virulent strains of S. suis serotype 4 is shown in Figure S1.

Table 2.

The results of the zebrafish infection experiment.

Strains ST CC Hosta Deaths at different post-infection time points Total deaths Mortality rate (%) P value Significanceb
12 h 24 h 36 h 48 h 60 h 72 h 84 h 96 h
WUSS270 17 17 H 0 4 3 1 1 0 0 0 9 60.00 0.0008 ***
WUSS304 17 17 H 0 2 3 0 0 0 0 0 5 33.33 <0.0001 ****
WUSS388 17 17 H 0 7 4 0 0 0 0 0 11 73.33 0.0051 **
WUSS406 17 17 H 0 9 1 0 0 0 1 1 12 80.00 0.0225 *
2018WUSS011 17 17 H 2 8 4 0 0 0 0 0 14 93.33 0.1408 ns
ND7 17 17 D 0 0 0 0 0 0 1 0 1 6.67 <0.0001 ****
ND83 17 17 D 10 0 1 0 0 0 0 0 11 73.33 0.8457 ns
ND84 17 17 D 13 0 0 0 0 0 0 0 13 86.67 0.0921 ns
ND90 17 17 D 0 8 3 0 1 0 0 0 12 80.00 0.0142 *
WUSS303 850 17 H 0 2 5 1 0 0 0 0 8 53.33 0.0001 ***
2021WUSS076 2224 17 H 1 1 4 0 0 1 0 0 7 46.67 0.0002 ***
ID36054 94 94 P 5 2 5 1 1 0 0 0 14 93.33 0.2186 ns
WUSS026 94 94 H 1 3 4 1 1 0 0 0 10 66.67 0.0026 **
WUSS273 94 94 H 0 0 4 0 0 0 0 0 4 26.67 <0.0001 ****
2021WUSS075 94 94 H 6 5 1 2 0 0 0 0 14 93.33 0.8088 ns
2021WUSS077 94 94 H 0 2 0 1 0 0 0 0 3 20.00 <0.0001 ****
ND6 94 94 D 3 0 1 0 0 0 0 0 4 26.67 0.0007 ***
WUSS329 1175 94 H 0 0 3 0 1 0 0 0 4 26.67 <0.0001 ****
2018WUSS156 2220 94 D 0 13 0 0 0 0 0 0 13 86.67 0.0405 *
WUSS346 485 839 H 8 2 4 0 0 0 0 0 14 93.33 0.8375 ns
2018WUSS160 485 839 D 0 15 0 0 0 0 0 0 15 100.00 0.0979 ns
WUSS390 977 108 H 0 9 1 0 0 0 0 0 10 66.67 0.0080 **
WUSS285 2238 / H 2 0 1 0 1 0 0 0 4 26.67 0.0002 ***
WUSS309 2237 / H 2 1 0 0 0 0 0 0 3 20.00 0.0003 ***
WUSS399 2239 / H 0 0 4 0 0 0 0 0 4 26.67 <0.0001 ****
WUSS435 1067 / D 1 0 1 1 0 0 0 0 3 20.00 <0.0001 ****
2018WUSS056 2218 / H 0 0 0 0 0 0 0 0 0 0.00 <0.0001 ****
2018WUSS108 2219 / H 1 2 1 1 1 0 0 0 6 40.00 0.0004 ***
2020WUSS059 2222 / H 0 1 0 1 0 0 0 0 2 13.33 <0.0001 ****
2022WUSS016 2225 / H 4 0 2 0 1 0 0 0 7 46.67 0.0057 **
2022WUSS017 2226 / H 1 3 1 0 0 0 0 0 5 33.33 0.0004 ***
2022WUSS018 2057 / H 0 0 0 0 0 0 0 0 0 0.00 <0.0001 ****
2022WUSS019 2227 / H 0 1 0 1 0 0 1 0 3 20.00 <0.0001 ****
2022WUSS056 2228 / H 0 0 0 0 0 0 0 0 0 0.00 <0.0001 ****
2022WUSS141 2066 / H 0 1 0 1 0 0 0 0 2 13.33 <0.0001 ****
SC070731 7 1 D 5 9 0 0 0 0 0 0 14 93.33 – –
SH040917 / / H 0 0 0 0 0 0 0 0 0 0.00 <0.0001 ****
PBS       0 0 0 0 0 0 0 0 0 0.00 <0.0001 ****
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a

H indicates healthy pig, D indicates diseased pig, and P indicates human patient.

b

The survival outcome of serotype 4 strains for zebrafish was compared with that of the highly pathogenic strain SC070731 using the Log-rank (Mantel-Cox) test. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and “ns” indicates no significant difference.

/: unassigned.

The mouse infection experiment was performed further to validate the virulence of S. suis serotype 4 strains. Nine strains with ≥ 80% mortality to zebrafish and two strains with no mortality to zebrafish were selected for BALB/c mice infection. As shown in Table 3, the mortality rate of mice infected with eight strains with ≥ 80% mortality to zebrafish was also ≥ 80%. The survival curves of mice infected with strains ID36054, WUSS406, ND90, 2021WUSS075, 2018WUSS156, WUSS346, and 2018WUSS160 showed no significant difference compared to mice infected with the highly virulent control strain SC070731. Strain ND84, with 86.67% mortality to zebrafish, exhibited 60% mortality to mice. Two strains with no mortality to zebrafish also showed no mortality to mice. The results of mice and zebrafish infection experiments were consistent. In summary, 35.00% (7/20) of representative pig isolates of CC17, CC94, and CC839 were highly virulent in zebrafish and mice (mortality rate ≥ 80%), similar to the human isolate ID36054 (belonging to CC94) (Table 3). Thus, S. suis serotype 4 strains of CC17 (ST17), CC94 (ST94, ST2220), and CC839 (ST485) are potentially pathogenic.

Table 3.

The results of the BALB/c mice infection experiment.

Strains Mortality rate (%)a CC Deaths at different post-infection time points Total deaths Mortality rate (%)b P value Significancec
1 d 2 d 3 d 4 d 5 d 6 d 7 d 8 d 9 d 10 d
WUSS406 80.00 17 10 0 0 0 0 0 0 0 0 0 10 100.00 0.3173 ns
2018WUSS011 93.33 17 5 0 0 0 0 3 0 0 0 0 8 80.00 0.0111 *
ND84 86.67 17 6 0 0 0 0 0 0 0 0 0 6 60.00 0.0317 *
ND90 80.00 17 7 1 0 0 0 0 0 0 0 0 8 80.00 0.1512 ns
ID36054 93.33 94 9 1 0 0 0 0 0 0 0 0 10 100.00 >0.9999 ns
2021WUSS075 93.33 94 9 1 0 0 0 0 0 0 0 0 10 100.00 >0.9999 ns
2018WUSS156 86.67 94 6 2 0 0 0 0 0 0 0 0 8 80.00 0.0863 ns
WUSS346 93.33 839 10 0 0 0 0 0 0 0 0 0 10 100.00 0.3173 ns
2018WUSS160 100.00 839 9 0 0 0 0 0 0 0 0 0 9 90.00 0.5567 ns
2022WUSS018 0.00 / 0 0 0 0 0 0 0 0 0 0 0 0.00 <0.0001 ****
2022WUSS056 0.00 / 0 0 0 0 0 0 0 0 0 0 0 0.00 <0.0001 ****
SC070731 93.33 1 9 1 0 0 0 0 0 0 0 0 10 100.00 - -
PBS 0.00   0 0 0 0 0 0 0 0 0 0 0 0.00 <0.0001 ****
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a

Mortality rate of each strain in zebrafish.

b

Mortality rate of each strain in BALB/c mice.

c

The survival outcome of serotype 4 strains for mice was compared with that of the highly pathogenic strain SC070731 using the Log-rank (Mantel-Cox) test. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and “ns” indicates no significant difference.

/: unassigned.

Cell cytotoxicity

To evaluate the cell cytotoxicity of highly virulent S. suis serotype 4 strains on human cells, the human isolate ID36054 and four pig isolates belonging to CC17, CC94, and CC839 were selected for cell cytotoxicity assays, and the mortality rate of these strains in both zebrafish and mice is ≥ 80%. As shown in Figure 3(A), the human strain ID36054 exhibited significant cytotoxicity to A549 at 3 h incubation with 93.04% cytotoxicity, and the pig isolates showed 88.67%−100.00% cytotoxicity. As shown in Figure 3(B), the human strain ID36054 exhibited significant cytotoxicity to hBMEC at 6 h incubation with 66.42% cytotoxicity, and the pig isolates showed 45.25%−67.41% cytotoxicity. These data indicate that the human and pig isolates of S. suis serotype 4 exhibit significant cytotoxicity to human cells.

Figure 3.

Figure 3.

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Human cell cytotoxicity assays of S. suis serotype 4 highly virulent strains. Cell cytotoxicity of S. suis strains on A549 (A) for 3 h and hBMEC (B) for 6 h. The percentage of cytotoxicity of S. suis serotype 4 strains was compared with that of the human reference strain ID36054 using an unpaired t-test. A summary of the p-values is provided, with an asterisk indicating a significant difference (p < 0.05) and “ns” denoting no significant difference.

Distribution and dissemination vehicles of antibiotic resistance genes in S. suis serotype 4

The distribution of antibiotic resistance genes in 126 S. suis serotype 4 genomes was investigated. We identified twenty-two distinct antibiotic resistance genes, which showed ≥ 95% identity in nucleotide sequence across over 90% coverage of the reference gene. They were classified into six categories: tetracyclines, macrolides-lincosamides-streptogramin B (MLSB), aminoglycosides, lincosamides, oxazolidinones, and chloramphenicol (Figure 4). The distribution of tetracycline resistance genes was found to be highest at a rate of 81.75% (103/126), corresponding to the phenotypes of resistance to doxycycline and tetracycline, with tet(O) being the predominant gene (77/126, 61.11%), followed by tet(M) (22/126, 17.46%), tet(40) (7/126, 5.56%), tet(O/W/32/O) (6/126, 4.76%), and tet(L) (3/126, 2.38%). Ninety-two genomes (73.02%) possess the MLSB resistance gene erm(B), corresponding to the phenotypes of resistance to erythromycin, azithromycin, lincomycin, and clindamycin. Other lincosamides resistance genes lsaE (20/126, 15.87%), lnuB (20/126, 15.87%), and lnuC (2/126, 1.59%) were also found in 22 genomes (17.46%). Nine types of aminoglycoside resistant genes were found among 53 genomes (42.06%), consisting of ant(6)-Ia (51/126, 40.48%), aac(6′)-aph(2″) (36/126, 28.57%), aph(3′)-III (6/126, 4.76%), as well as one each of ant(6)-Ib, aph(2″)-Ia, aph(2″)-Ic, aph(3′)-IIa, aph(4)-Ia, and aac(3)-IV. The florfenicol and linezolid resistance gene optrA (12/126, 9.52%), chloramphenicol resistance gene cat (8/126, 6.35%), and efflux pump gene mef(A) (6/126, 4.76%) were also found in the S. suis serotype 4 genomes.

Figure 4.

Figure 4.

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The distribution of antibiotic resistance genes (ARGs), prophages, and ICEs for S. suis serotype 4 genomes. Color-filled square boxes on the periphery indicate the presence of antibiotic resistance genes-associated prophages and ICEs, and unfilled boxes indicate their absence.

Our investigation successfully identified an intact prophage with a score of 100 in one genome. Moreover, questionable prophages with predicted scores of 90 and 80 were present in seven and fifteen genomes, respectively (Figure 4). Integration analysis revealed that these prophages were primarily inserted into the rum locus. However, prophages ΦSsuWUSS303 and ΦSsuCPD30 were inserted into the comEC locus instead. It is worth mentioning that the MLSB resistance gene erm(B), aminoglycosides resistance genes ant(6)-Ia and aac(6′)-aph(2″), tetracyclines resistance genes tet(O), tet(O/W/32/O), and tet(40), and other resistance genes optrA, mef(A), lsaE, lnuB, lnuC, and cat were detected within prophages (Figure 5(A)). In addition, nine different ICEs were distributed in 12 genomes, containing five ICEs inserted into the rplL locus and four ICEs inserted into the rum locus (Figure 4). These ICEs predominantly carried erm(B) and tet(O) genes (Figure 5(B)). We utilized online analysis platforms to detect conjugative plasmids carrying antibiotic resistance genes in the strains studied. The results indicate that no conjugative plasmids carrying antibiotic resistance genes were found. This discovery implies that prophages were the primary vehicle of antibiotic resistance genes and played a crucial role in disseminating MLSB, tetracyclines, aminoglycosides, and linezolid resistance genes. Additionally, the presence of ICEs further facilitated the dissemination of MLSB and tetracycline resistance genes. We also observed that genomes harbouring prophages associated with antibiotic resistance genes were exclusively isolated from China, whereas those harbouring ICEs associated with antibiotic resistance genes were discovered mainly in other nations (Figure 4).

Figure 5.

Figure 5.

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Genetic context of antibiotic resistance genes-associated prophages and ICEs in S. suis serotype 4 genomes. The direction of transcription for each gene is denoted by arrows, and distinct colours represent the various genes. (A) Prophages with scores of 100, 90, and 80 by PHASTER analysis. (B) ICE structure predicted by ICEfinder software. T4SS, type IV secretion system; T4CP, type IV coupling protein.

Antimicrobial susceptibility profiles of S. suis serotype 4 strains

We detected the antibiotic resistance phenotypes of 48 S. suis serotype 4 strains isolated from our labs using the broth microdilution method. The MIC values of 23 antimicrobials tested for 48 strains are listed in Table S2. As shown in Figure 6(A), all strains were resistant to lincomycin and clindamycin, and most strains were also resistant to erythromycin (97.92%), azithromycin (97.92%), doxycycline (93.75%), and tetracycline (87.50%). The presence of the genes erm(B) (89.58%), lsaE (20.83%), and lnuB (20.83%) conferred resistance to lincosamides and macrolides, and the presence of the genes tet(O) (60.42%), tet(M) (33.33%), and tet(O/W/32/O) (8.33%) conferred resistance to tetracyclines (Figure 4, Table S2). The resistance rate for kanamycin, streptomycin, gentamicin, tilmicosin, tiamulin, spectinomycin, florfenicol, and valnemulin was 64.58%, 62.50%, 60.42%, 35.42%, 31.25%, 25.00%, 22.92%, and 18.75%, respectively. The carriage of the genes aac(6′)-aph(2″) (64.58%) and aph(3′)-III (6.25%) led to high-level resistance to gentamicin and kanamycin, while the presence of the gene ant(6)-Ia (72.92%) resulted in high-level resistance to streptomycin (Figure 4, Table S2). All S. suis serotype 4 strains displayed susceptibility to amoxicillin, cefotaxime, and vancomycin. There was a low resistance rate to enrofloxacin (14.58%), marbofloxacin (14.58%), penicillin (10.42%), linezolid (10.42%), chloramphenicol (8.33%), and rifampin (4.17%). It is worth noting that 95.83% (46/48) of the strains were resistant to ≥3 classes of antimicrobial agents (Figure 6(B)) and were classified as multidrug-resistant. Most strains were resistant to four or five classes of antimicrobial agents.

Figure 6.

Figure 6.

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The antimicrobial susceptibility profiles in S. suis serotype 4 strains isolated from our labs. (A) The resistance rates of 48 strains to 23 antibiotics. PEN, Penicillin; AMO, Amoxicillin; CTX, Cefotaxime; RIF, Rifampin; VAN, Vancomycin; LNZ, Linezolid; ENR, Enrofloxacin; MAR, Marbofloxacin; CHL, Chloramphenicol; FLO, Florfenicol; LIN, Lincomycin; CLI, Clindamycin; TIA, Tiamulin; VAL, Valnemulin; GEN, Gentamicin; KAN, Kanamycin; STR, Streptomycin; SPE, Spectinomycin; TIM, Tilmicosin; ERY, Erythromycin; AZM, Azithromycin; DOX, Doxycycline; TET, Tetracycline. (B) The resistance rates of 48 strains to 11 categories of antibiotics.

Discussion

Serotyping is vital for gaining insights into bacterial epidemiology, such as its prevalence across different geographic regions and transmission dynamics. Identifying specific serotypes or STs linked to outbreaks offer invaluable information for epidemiologists to comprehend disease spread and implement targeted control measures. Currently available vaccines against S. suis infection are mainly bacterins, which are supposed to confer serotype-specific protection. Thus, serotyping helps to develop serotype-specific vaccines based on epidemiological data.

In the recent study by Murray et al., ten pathogenic lineages of S. suis were identified, among which disease-associated serotypes 1, 1/2, 2, 3, 4, 5, 6, 7, 8, 9, and 14 were found to be prevalent [22]. However, different S. suis serotypes exhibit diverse population structure and pathogenicity characteristics. Therefore, for the pathogenicity of S. suis, it is important to not only focus on disease-associated serotypes but also ST [1,6]. S. suis serotype 2 is the most prevalent pathotype in both swine and humans worldwide [1]. The common pathogenic ST of S. suis serotype 2 are ST1, ST7, ST20, ST25, ST28, and ST104. Notably, ST1 strains belonging to MCG group 1 are globally distributed and demonstrate the highest pathogenicity in humans and pigs [6]. Besides this serotype, S. suis serotype 9 has emerged as the predominant serotype among diseased pigs in Western Europe; ST16 strains showed a zoonotic potential [1,47,48]. S. suis serotype 7, a non-negligible pathotype with the most predominant STs being ST29, ST373, and ST94, prevalent in Europe, China, and North America, respectively; ST373 strains were responsible for septicemia in humans and widely prevalent in China [14]. S. suis serotype 8, as one of the main pathogenic serotypes causing clinical diseases in pigs, was frequently isolated from clinical cases in Asia, North America, South America, and Europe [1,49,50]. Among the isolates of serotype 8 in China, the predominant STs included ST308, ST198, and ST1241, with the ST1241 strains showing significant pathogenicity in zebrafish and mice [40]. The main purpose of population structure analysis for different S. suis serotypes is to identify the high-pathogenic sub-lineages from the whole population and provide important information to precisely prevent the infection caused by the high-pathogenic sub-lineage strains.

Between 2002 and 2013, in China and South Korea, S. suis serotype 4 ranked as the third most predominant serotype isolated from infected pigs, accounting for 5.6% [1]. In Canada, there was an increasing trend in the proportion of S. suis serotype 4 among isolates from diseased pigs between 2009 and 2011, rising from 3.7% to 5.9% [18]. In Germany, S. suis serotype 4 accounted for 10% and 10.3% of S. suis isolates identified between 1996–2004 and 2015–2016, respectively [20]. In this study, the results of animal infection experiments showed that among the representative strains of CC17 (ST17), CC94 (ST94, ST2220), and CC839 (ST485), 66.67% (14/21) of the strains exhibited a mortality rate exceeding 50% in zebrafish (Table 2). Therefore, S. suis serotype 4 is a non-negligible pathotype.

In a recent study, Hatrongjit et al. analyzed the genome of seven serotype 4 strains belonging to CC94 and group 3 [26]; they performed cytotoxicity assays using human cell lines and demonstrated that CC94 serotype 4 strains are potentially virulent [26]. However, the virulence of those strains was not validated in animal models, and only seven strains were analyzed in their study. In the present study, we investigated the population structure and pathogenicity of the S. suis serotype 4 population based on 126 isolates from eight different countries. Within the S. suis serotype 4 population, CC17 and CC94, which belong to MCG group 1 and MCG group 3, respectively, exhibited the highest proportions. Chen et al. reported seven MCG groups among S. suis population; MCG group 1 included all the highly virulent isolates of ST1, the epidemic isolates of ST7, and all isolates from human infections and outbreaks [28]. Notably, S. suis serotype 4 strain ID36054 causing human infection belongs to CC94. In this study, the results of animal infection experiments showed that 35.00% (7/20) of pig isolates from CC17, CC94, and CC839 (also belonging to MCG group 3) were highly virulent in zebrafish and mice, similar to the human isolate ID36054. We found a correlation between the virulence phenotype and the distribution of putative virulence-related genes. These highly virulent strains harbour numerous crucial genes associated with virulence, particularly including mrp, sly, IdeSsuis, igdE, sbp2, hp0197, hp0272, rgg, and tran which have been identified as potential zoonotic virulence factors in a recent study [51]. In addition, we found that the genotype of S. suis classical virulence markers in highly virulent strains was ef+/mrpNA2/sly+ or ef-/mrpNA1/sly+. Notably, the genotype of ef+/mrpNA2/sly+ was prevalent in human infection strains [52,53]. In contrast, all strains from MCG groups 7-2 and 7-3 were lowly virulent in zebrafish. Compared to the highly virulent strains, they lack several crucial virulence genes, including ef, mrp, sly, ofs, sao, dltA, IdeSsuis, igdE, revS, rgg, and tran. Furthermore, cell cytotoxicity assays confirmed that the human and pig isolates of S. suis serotype 4 exhibit significant cytotoxicity to human cells A549 and hBMEC. Thus, S. suis serotype 4 strains of CC17, CC94, and CC839 exhibit significant threat to humans and pigs and should be monitored.

Previous studies have shown S. suis to be a reservoir of clinically significant antibiotic resistance genes for major streptococcal pathogens [35,54,55]. In this study, we observed that 95.83% S. suis serotype 4 strains isolated from our labs were multidrug-resistant. The presence of erm(B), tet genes, ant(6)-Ia, aac(6′)-aph(2″), and optrA were the main reasons for the prevalent resistance phenotypes to macrolides, lincosamides, tetracyclines, aminoglycosides, and linezolid in serotype 4 strains. Prophages were identified as their primary vehicle, and ICEs further facilitated the dissemination of erm(B) and tet(O). In Streptococcus species, the dissemination of antibiotic resistance genes is primarily facilitated through the horizontal gene transfer of ICEs and prophages [35,56–60]. However, major vehicles may differ for S. suis serotypes. For example, prophages were the primary vehicle of antibiotic resistance genes in serotype 31 strains [41]. In serotype 7 and 8 strains, antibiotic resistance genes were mainly disseminated by integrative and mobilizable elements (IMEs) [40]. We found that S. suis serotype 4 genomes containing prophages associated with antibiotic resistance genes were mainly isolated from China, while those containing ICEs associated with antibiotic resistance genes were mainly discovered in other countries, suggests that the predominant vehicles for the spread of antibiotic resistance genes may vary by nation or region.

In conclusion, S. suis serotype 4 exhibited a distinct population structure. 61.90% of strains (78/126) were clustered into MCG groups 1 and 3, with potential pathogenicity in humans and pigs, particularly strains belonging to CC17, CC94, and CC839. S. suis serotype 4 strains showed multidrug-resistant, with prophages crucial in disseminating antibiotic resistance genes. Our study expands the understanding of the S. suis serotype 4 population and provides valuable information for the surveillance and prevention of this serotype.

Supplementary Material

Supplemental Material
TEMI_A_2352435_SM3774.docx (45.9KB, docx)

Funding Statement

This work was supported by grants from the National Key Research and Development Program of China [2021YFD1800402], the National Natural Science Foundation of China [32172859], Open Project Program of Jiangsu Key Laboratory of Zoonosis [R2103], and Open Project Program of Engineering Research Center for the Prevention and Control of Animal Original Zoonosis, Fujian Province University [2021ZW001].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Ethical approval

Animal infection experiments were conducted at the Laboratory Animal Center of Nanjing Agricultural University (Permit number: SYXK (Su) 2021-0086). This study and the application of the animal experiments were reviewed and approved by the Experimental Animal Welfare and Ethics Committee of Nanjing Agricultural University and were performed according to Animal Welfare Agency Guidelines.

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