Introduction

Streptococcus suis (S. suis) is an important zoonotic pathogen, with pigs being the primary source of infection. In humans, S. suis can cause various serious illnesses1. Severe cases of S. suis infection can result in fatality. Since its recognition as a zoonotic pathogen of significant occupational origin in 1968, S. suis has been frequently detected worldwide2. Among the 1642 human cases of S. suis infections reported globally between 2002 and 2013, 90.2% were recorded in Asia, while 8.5% occurred in Europe and 1.3% in other parts of the world3. Three outbreaks of S. suis in China, in Jiangsu (1998), Sichuan (2005) and Guangxi (2016), caused multiple infections and deaths, resulting in serious local economic losses4.

S. suis can be classified into 29 serotypes based on their capsular antigens podococcal antigens5. Serotypes 2, 4, 7, and 9 are frequently isolated from both healthy and diseased animals6,7. Among human pathogenic isolates, 74.7% were serotype 2, which is the most prevalent and virulent serotype across different geographic regions8. Subsequently, S. suis serotype 14 has also been isolated from human isolates on several occasions9. Bacterial virulence has been described as the ability to invade and replicate in the host and to evade the host immune system10. Previous research has identified virulence factors of S. suis involved in processes such as adhesion, invasion, immune evasion, and inflammatory injury11. MRP, EPF and SLY genes play an important role in Streptococcus suis infection12,13,14,15. The detection of these genes can provide further insights into the pathogenicity of the strain.

Currently, the prevention and treatment of streptococcal infections in swine by both humans and veterinarians rely on the effective use of antibiotics16. However, the global overuse of antibiotics had resulted in the development of antimicrobial resistance in bacteria, leading to an alarming increase in multidrug resistance17. Many countries in the Americas, Asia and Europe have reported an increase in antimicrobial resistance cases of S. suis isolated from both swine and humans18,19. Additionally, S. suis can serve as a reservoir for antibiotic resistance genes, which can be horizontally transferred to other streptococcal pathogens such as Streptococcus pyogenes, Streptococcus pneumoniae, and Streptococcus lactis20. High resistance to tetracycline, macrolides and lincosamides antibiotics has been reported in both human and swine isolates of S. suis17,21. Some studies have shown that the antibiotic resistance genes tet(O), tet(M) are genes associated with resistance to tetracycline. The subsequent increasing trend of macrolide resistance in S. suis has also been widely reported22. Therefore, monitoring the susceptibility patterns of bacterial pathogens to antibiotic drugs is important to provide evidence-based guidance for further optimization of effective antimicrobial treatment regimens and tracking the emergence of antibiotic drug resistance.

In this study, lung tissue samples and nasal swab samples were collected from pigs in 18 cities in East China. The objective of this study was to determine the prevalence of S. suis in East China and to assess its virulence and drug resistance properties. The continuous monitoring of Streptococcus suis in East China offers a valuable reference for the prevention and control of Streptococcus suis.

Result

Isolation and identification of S. suis

A total of 154 strains of S. suis were isolated from 1642 lung tissue and nasal swabs samples from 18 cities in eastern China, which were finally determined by PCR amplification of the 16S gene. A total of 86 strains were isolated from lung of 801 healthy pigs and 57 strains from nasal swabs of 390 diseased-pigs and 451 healthy pigs. According to data from the Shandong Provincial Bureau of Statistics website (http://tjj.shandong.gov.cn/), it is evident that Linyi had the highest hog population in eastern China in 2023, followed by Weifang, Dezhou, Heze, and Yantai. Conversely, the cities of Rizhao, Dongying, Weihai, Zibo, Xuzhou, Xinyi and Zaozhuang recorded the lowest hog numbers during the same period. It was found that there was a positive correlation between the number of pigs slaughtered and the detection rate of S. suis. In the cities with more pig herds, more streptococci were isolated, such as Weifang and Yantai, where more than 25 strains of Streptococcus suis were isolated, while in Rizhao, Dongying, Weihai, Xinyi, Zibo and Zaozhuang, which are the cities with fewer pig herds, S. suis was not even isolated.

Determination of serotypes

Based on the analysis of PCR results, as shown in Fig. 1, the most frequently identified serotype among the 143 S. suis was type 2, with a total of 98 strains. Type 5 was the second most prevalent, with 12 strains detected. Furthermore, 6 strain each of type 4, type 19 and type 21 were identified. However, no highly pathogenic strains of type 4, 7, and 14 were isolated. This trend aligned with a previous study conducted in northeastern China23, where type 2 was found to be the prevailing serotype of S. suis, occasionally accompanied by other serotypes.

Fig. 1
figure 1

Bar graph on the serotype distribution of 143 strains of Streptococcus suis.

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Detection of virulence-related genes

In 143 S. suis strains, a total of 24 virulence genes were detected in all strains. Results were showed in Table 1. Among these genes, pgda, dlta, mann, fbps, orf2 and sspa exhibited high detection rates, which were 67.1%, 65.5%, 73.4%, 76.2%, 78.3%, and 78.3%, respectively. The detection rates of sly, sum, and salkr genes were comparatively lower, at 30.1%, 24.5%, and 18.2%, respectively. Among the 143 strains analyzed, 112 strains were found to have more than 10 virulence-related genes, while only 5 strains (SDFMU-M003, SDFMU-M0016, SDFMU-M021, SDFMU-M041 and SDFMU-M045) exhibited 1 or no virulence-related genes. Notably, strain SDFMU-M011 and SDFMU-M008 demonstrated the highest number of virulence-related genes, with 21 and 22 genes respectively. Compared with a survey of S. suis isolates from Fujian Province, China24, the types of virulence-related genes detected in the two provinces were similar, but S. suis from east China had a higher number of virulence genes.

Table 1 Virulence-related genes test assay of 143 strains of Streptococcus suis.
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Detection of drug-resistance genes

Multiple resistance genes are present in 143 strains of S.suis. These genes encompass aph3 for aminoglycosides, ermB for macrolides, mefA, gyrA, and parC for quinolones, Sul1 and Sul3 for sulfonamides, tetL and tetO for tetracyclines. As illustrated in Fig. 2, the drug resistance gene tetO was identified as the most prevalent among the 143 strains, with a detection rate of 75.5%. This was followed by aph3, which exhibited a detection rate of 52.4%. The detection rates for other resistance genes were as follows: gyrA (30.1%), parC (20.9%), ermB (18.2%), and mefA (15.4%), Sul1 (14.1%), Sul3 (12.6%), tetL (9.7%). No resistance genes for chloramphenicol and β-lactam were detected in 143 streptococci strains. In addition, three S. suis of the isolates did not exhibit any resistance genes. The detection rate of resistance genes of streptococci to tetracyclines was as high as 90.1%.

Fig. 2
figure 2

Bar graph on the drug-resistance gene distribution of 143 strains of Streptococcus suis.

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Antibacterial sensitivity test

The resistance distribution of the 143 strains is shown in Table 2. Among the tested strains, only isolate SDFMU-M001 (serotype 2) demonstrated susceptibility to all antibiotics, whereas all other strains exhibited resistance to at least one antimicrobial drug. Notably, the 56 strains exhibited the highest levels of resistance, demonstrating resistance to more than 10 of the 19 antibiotics tested. This contrasts with the presence of resistance genes, leading to the hypothesis that it could be attributed to undetected resistance genes. Based on the Clinical and Laboratory Standards Institute (CLSI) breakpoints, the resistance rates of polymyxin B, ampicillin, ceftriaxone, kanamycin, cefotaxime, streptomycin, and erythromycin were 87.4%, 76.9%, 54.5%, 52.4%, 48.3%, 48.2% and 45.5%, respectively.

Table 2 Analysis of 19 antibiotic susceptibility assays of 143 strains of Streptococcus suis.
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Discussion

S. suis is a bacterial strain that causes widespread epidemics in pigs. It is also considered as an emerging zoonotic pathogen transmitted through infected pigs and their by-products, posing a potential threat to public health security25. Antibiotics have predominantly been employed for the control and treatment of S. suis. However, the strain has developed resistance to antibiotics, leading to a gradual increase in the rate of multidrug resistance.

The relationship between various virulence factors of Streptococcus suis, such as sly, mrp, and epf, and the virulence of Streptococcus suis has been well-established26. Our study observed that three virulence-related genes, sly, mrp, and epf, were prevalent in eastern China. Notably, all strains exhibited amplification of at least one of these three genes, with the exception of SDFMU-M002, which was sly-/mrp-/epf-. Furthermore, SDFMU-M005 and SDFMU-M012 were found to be sly + /mrp + /epf + . This finding aligns with the epidemiological observations reported in Europe and the United States, where the prevalence and pathogenicity of Eurasian strains of Streptococcus suis have been positively correlated with the sly, mrp, and epf genes27. In the present study, the orf2 gene was detected in 112 out of 143 isolates, exhibiting a high detection rate. In addition to the orf2 gene, the dltA, pgdA, sspA, and fbps genes were also detected at a high rate. These genes are associated with cell wall formation, bacterial adhesion, and evasion of mucosal immune systems28,29,30,31. The distribution of virulence genes was similar in northeast China as well as in Fujian24,25. We hypothesized that Streptococcus suis in East China has significant virulence potential and thus poses a high risk to public health safety.

Antibiotics have been utilized in the swine industry for the treatment and prevention of disease for an extended period. However, it is imperative to exercise caution when employing antibiotics to mitigate the selection of resistant Streptococcus suis isolates.Antibiotic susceptibility testing of the isolates revealed that Streptococcus suis demonstrated high resistance to commonly employed antibiotics. All isolates exhibited resistance to at least one class of antibiotics, with high rates of resistance to polymyxin B, ampicillin, ceftriaxone, and kanamycin observed, as reported in previous studies5,16. Contrary to previous studies, which indicated a high rate of resistance to polymyxin B in the strains examined in this study, previous research has demonstrated that globally, the prevalence of resistance to macrolide and tetracycline antibiotics is high, while the prevalence of resistance to β-lactam antibiotics is low32,33,34. This discrepancy could be attributed to the distinct patterns of veterinary drug utilization observed in eastern China, which may have resulted in alterations in the resistance profiles of Streptococcus strains.To further investigate this discrepancy, we conducted a comparative analysis of the identified resistance genes with the results of antimicrobial susceptibility testing. Our findings revealed a disconnection between the resistance genes and the observed outcomes. Specifically, resistance genes associated with β-lactam (pbp2b), amino alcohols (cat1, cat2), and macrolides (ermA, ermB, mefA) were detected at a low rate, or even not detected at all.This may be due to the formation of biofilm that allows the strains to develop resistance25. Of particular concern is the identification of vancomycin resistance (LVR), which is a critical concern due to its role as a last resort antimicrobial agent in the treatment of multidrug-resistant Gram-positive bacterial infections, posing a grave threat to public health.The emergence of LVR isolates of porcine streptococci has been previously documented35,36, underscoring the urgent need for comprehensive monitoring of antibiotic resistance in Streptococcus suis.

Conclusions

This study revealed the widespread presence of Streptococcus suis type 2 in eastern China, aligning with the global trend of streptococcal strains. Both virulence and resistance genes demonstrated high detection rates, underscoring the necessity for close monitoring. However, an inconsistency was observed between the presence of resistance genes and the results of susceptibility testing, which may be attributable to factors such as biofilm formation or the presence of undetected resistance genes.This study provides valuable insights into the prevalence and antimicrobial resistance of Streptococcus suis in East China, and helps to strengthen the strategy of effective prevention and control of Streptococcus suis outbreaks.

Materials and methods

Isolation of S. suis

S. suis were isolated in 2021–2023, from porcine lung tissue and nasal swabs from 58 farms in 18 cities in western China (Table 3). The collection of samples was conducted without the administration of anesthesia or euthanasia of the animals, and lung tissue was obtained from pigs afflicted with the disease. The bacterial isolation method was performed using the protocol as described previously37. Porcine lung tissue was aseptically collected, homogenized with 1 mL of PBS using a tissue homogenizer (MagNA Lyser Instrument, USA), and streaked onto blood agar medium (Detgerm Microbiological Science Ltd., Guangzhou, China). The plates were then incubated at 37 °C for 18–24 h. After incubation, five small grey-white colonies were selected, subcultured onto fresh blood agar plates, and incubated again at 37 °C for 18–24 h. The conserved 16S rRNA gene of S. suis was subsequently detected, and positive isolates were confirmed by ribonucleic acid electrophoresis, revealing the expected bands38.

Table 3 Region of pig sample collection.
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Serotype identification

PCR amplification technique was used to identify the serotype of S. suis38. The PCR primers were synthesized by Shandong Qingdao Dyke Biological Co. Two PCR were used. The first PCR was performed to group the strains into seven cps groups by detecting the cps genes conserved in multiple serotypes. The second PCR was performed for typing, detecting cps genes in each serotype group, which in turn determined the cps type of the strain. The reaction conditions of PCR were as follows: initial denaturation at 95 °C for 5 min; 30 cycles of 95 °C for 30 s, 58 °C (for typing PCR) or 60 °C (for grouping PCR) for 30 s, 72 °C for 1 min, followed by final extension 72 °C for 10 min and holding at 4 °C. Furthermore, agglutination tests were used to confirm streptococcal serotypes using the commercially available Immulex™ S. suis kit (SSI Diagnostica A/S, Denmark)39.

Detection of virulence-related genes and drug resistance genes

The 24 virulence-related genes (Supplementary Table 1) and 16 drug resistance genes (Supplementary Table 2) were determined using PCR technique24,25. PCR primers were synthesized by Qingdao Tsingke Biotech Co., Ltd. The reaction conditions of PCR were as follows: initial denaturation at 95 °C for 5 min; 35 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min, followed by final extension 72 °C for 10 min and holding at 4 °C.

Antibacterial sensitivity test

Antimicrobial susceptibility testing was performed by determining the minimum inhibitory concentration (MIC) of the strains using the Dry Plate Eiken broth microdilution method (Eiken Kagaku, Tochigi, Japan) according to the manufacturer’s instructions. A total of 19 antimicrobial agents were evaluated, including aminoglycosides (gentamicin, streptomycin, neomycin, kanamycin), beta-lactams (penicillin, ceftriaxone, ampicillin, cefotaxime, amoxicillin), macrolides (erythromycin), lincosamides (clindamycin), quinolones (ofloxacin, enrofloxacin, levofloxacin), tetracyclines (tetracycline, doxycycline), phenicols (chloramphenicol, florfenicol), and polypeptides (polymyxin B, vancomycin). MIC breakpoints were determined according to the Clinical and Laboratory Standards Institute (CLSI) 2018 criteria (M100-ED28) for Streptococcus spp. of the viridans group.