Detection of Streptococcus suis using the optimized real-time polymerase chain reaction protocol
AbstractThe article presents the results of studies on the detection of Streptococcus suis by real-time polymerase chain reaction. Isolation and species identification of the studied isolates of streptococci was carried out according to morphological, cultural, biochemical and biological properties by conventional methods. The study of cultural characteristics of growth was carried out using conventional bacteriological methods on the brain heart infusion broth (BHI) and BHI agar with the addition of 5% sheep blood (blood BHI agar). To confirm biochemical properties as a confirmatory method, API 20 STREP test kit (bioMerieux, France) was used. In addition, to differentiate S. suis from the non-pathogenic species of streptococci, the hemolysis test was used. As a result of the studies, it was found that the use of the real-time PCR (polymerase chain reaction) method makes it possible to detect S. suis in an amount of 1 x 104 genome copies in the sample. All described validation parameters for the qualitative detection of S. suis DNA by real-time PCR meet international requirements, which guarantees accurate and reliable results. In Ukraine only a diagnostic test kit for convential PCR has been developed for the detection of swine streptococcosis. This approach is more time consuming and complex in comparison with the real-time PCR approach. We recommend that diagnostic laboratories implement this method in their practice. This will increase the number of effective diagnostic tools available to veterinarians on pig farms when they order laboratory tests. The high analytical sensitivity limit of a test is an essential parameter when screening is the focus, and obtaining false negative results causes a risk of the development of infection process among pig populations within infected herds. Our study showed that microbiological diagnostic methods to determine morphological and cultural properties can identify S. suis at the genus level. Determination of biochemical properties using the API 20 STREP test kit can be used to identify S. suis 1 and 2 serotypes. The conventional method and real-time PCR have 100% specificity and can be used to identify S. suis of different serotypes. Real-time PCR is a 2 to 4 times more sensitive limit than conventional PCR depending on the serotype being studied, and can be used to more accurately identify streptococcal DNA. It was found that the use of the real-time PCR method makes it possible to detect S. suis in an amount of 1 x 104 copies of the genome in the sample. Additionally, it was found that all the studied validation parameters of the qualitative method for determining S. suis DNA by real-time PCR meet international requirements, which guarantees accurate and reliable results.
Arai, S., Kim, H., Watanabe, T., Tohya, M., Suzuki, E., Ishida-Kuroki, K., Maruyama, F., Murase, K., Nakagawa, I., & Sekizaki, T. (2018). Assessment of pig saliva as a Streptococcus suis reservoir and potential source of infection on farms by use of a novel quantitative polymerase chain reaction assay. American Journal of Veterinary Research, 79(9), 941–948.
Bleuzé, M., Gottschalk, M., & Segura, M. (2021). Neutrophils in Streptococcus suis infection: From host defense to pathology. Microorganisms, 9(11), 2392.
Chen, L., Song, Y., Wei, Z., He, H., Zhang, A., & Jin, M. (2013). Antimicrobial susceptibility, tetracycline and erythromycin resistance genes, and multilocus sequence typing of Streptococcus suis isolates from diseased pigs in China. The Journal of Veterinary Medical Science, 75(5), 583–587.
Dekker, N., Daemen, I., Verstappen, K., de Greeff, A., Smith, H., & Duim, B. (2016). Simultaneous quantification and differentiation of Streptococcus suis serotypes 2 and 9 by quantitative real-time PCR, evaluated in tonsillar and nasal samples of pigs. Pathogens, 5(3), 46.
Devriese, L. A., Ceyssens, K., Hommez, J., Kilpper-Bälz, R., & Schleifer, K. H. (1991). Characteristics of different Streptococcus suis ecovars and description of a simplified identification method. Veterinary Microbiology, 26, 141–150.
Dutkiewicz, J., Zając, V., & Sroka, J. (2018). Streptococcus suis: A re-emerging pathogen associated with occupational exposure to pigs or pork products. Part II – Pathogenesis. Annals of Agricultural and Environmental Medicine, 25(1), 186–203.
Ferreira, M. B., de-Paris, F., Paiva, R. M., & Nunes, L. S. (2018). Assessment of conventional PCR and real-time PCR compared to the gold standard method for screening Streptococcus agalactiae in pregnant women. The Brazilian Journal of Infectious Diseases, 22(6), 449–454.
Feuerschuette, O. M., Serratine, A. C., Bazzo, M. L., Martins, T. R., Silveira, S. K., & da Silva, R. M. (2012). Performance of real-time PCR in the detection of Streptococcus agalactiae in the anogenital tract of pregnant women. Archives of Gynecology and Obstetrics, 286(6), 1437–1442.
Gottschalk, M., Higgins, R., Jacques, M., Beaudoin, M., & Henrichsen, J. (1991). Characterization of six new capsular types (23 through 28) of Streptococcus suis. Journal of Clinical Microbiology, 29(11), 2590–2594.
Goyette-Desjardins, G., Auger, J. P., Xu, J., Segura, M., & Gottschalk, M. (2014). Streptococcus suis, an important pig pathogen and emerging zoonotic agent – an update on the worldwide distribution based on serotyping and sequence typing. Emerging Microbes and Infections, 3(6), 45.
Groves, M. D., Jordan, D., Chapman, T. A., & Jassim, R. A. (2015). Multilocus sequence typing of Australian Streptococcus suis type 2 by MALDI-TOF mass spectrometry analysis of PCR amplicons. Veterinary Microbiology, 177, 394–397.
Hlebowicz, M., Jakubowski, P., & Smiatacz, T. (2019). Streptococcus suis meningitis: Epidemiology, clinical presentation and treatment. Vector Borne and Zoonotic Diseases, 19(8), 557–562.
Huang, W., Chen, Y., Li, Q., Jiang, H., Lv, Q., Zheng, Y., Han, X., Kong, D., Liu, P., & Jiang, Y. (2021). LytR plays a role in normal septum formation and contributes to full virulence in Streptococcus suis. Veterinary Microbiology, 254, 109003.
Ishida, S., Tien, H. T., Osawa, R., Tohya, M., Nomoto, R., Kawamura, Y., Takahashi, T., Kikuchi, N., Kikuchi, K., & Sekizaki, T. (2014). Development of an appropriate PCR system for the reclassification of Streptococcus suis. Journal of Microbiological Methods, 107, 66–70.
Jiang, X., Zhu, L., & Zhan, D. (2022). Development of a recombinase polymerase amplification assay for rapid detection of Streptococcus suis type 2 in nasopharyngeal swab samples. Diagnostic Microbiology and Infectious Disease, 102(2), 115594.
Kerdsin, A., Dejsirilert, S., Akeda, Y., Sekizaki, T., Hamada, S., Gottschalk, M., & Oishi, K. (2012). Fifteen Streptococcus suis serotypes identified by multiplex PCR. Journal of Medical Microbiology, 61(12), 1669–1672.
Liang, Z., Wu, H., Bian, C., Chen, H., Shen, Y., Gao, X., Ma, J., Yao, H., Wang, L., & Wu, Z. (2022). The antimicrobial systems of Streptococcus suis promote niche competition in pig tonsils. Virulence, 13(1), 781–793.
Okwumabua, O., O’Connor, M., & Shull, E. (2003). A polymerase chain reaction (PCR) assay specific for Streptococcus suis based on the gene encoding the glutamate dehydrogenase. FEMS Мicrobiology Letters, 218(1), 79–84.
Ouattara, M., Tamboura, M., Kambire, D., Sanou, M., Ouattara, K., Congo, M., Kaboré, A., Sanou, S., Kabré, E., Sharpley, S., Tran, T., Schwartz, S., Ouangraoua, S., Ouedraogo, A. S., Sangaré, L., Ouedraogo-Traore, R., Whitney, C. G., & Beall, B. (2020). Identification of Streptococcus suis meningitis by direct triplex real-time PCR, Burkina Faso. Emerging Infectious Diseases, 26(9), 2223–2226.
Pérez-Sancho, M., Vela, A. I., García-Seco, T., Gottschalk, M., Domínguez, L., & Fernández-Garayzábal, J. F. (2015). Assessment of MALDI-TOF MS as alternative tool for Streptococcus suis identification. Frontiers in Public Health, 3, 202.
Segura, M., Fittipaldi, N., Calzas, C., & Gottschalk, M. (2017). Critical Streptococcus suis virulence factors: Are they all really critical? Trends in Microbiology, 25(7), 585–599.
Srinivasan, V., McGee, L., Njanpop-Lafourcade, B., Moïsi, J., & Beall, B. (2016). Species-specific real-time PCR assay for the detection of Streptococcus suis from clinical specimens. Diagnostic Microbiology and Infectious Disease, 85(2), 131–132.
Sunaga, F., Tsuchiaka, S., Kishimoto, M., Aoki, H., Kakinoki, M., Kure, K., Okumura, H., Okumura, M., Okumura, A., Nagai, M., Omatsu, T., & Mizutani, T. (2020). Development of a one-run real-time PCR detection system for pathogens associated with porcine respiratory diseases. The Journal of Veterinary Medical Science, 82(2), 217–223.
Tarasov, O. A., Zakharova, O. M., & Savcheniuk, M. O. (2021). Metodychni rekomendatsii shchodo typizatsii zbudnyka streptokokozu svyney metodom polimeraznoyi lantsiuhovoyi reaktsiyi [Methodological recommendations for typing of the causative agent of streptococcosis of pigs by the method of polymerase chain reaction]. The Institute of Veterinary Medicine of National Academy of Agrarian Sciences of Ukraine, Kyiv (in Ukrainian).
Tarasov, O. A., Zakharova, O. M., Hudz, N. V., & Savcheniuk, M. O. (2021). Poshyrennia serotypiv Streptococcus suis na terytoriyi Ukrayiny [Dissemination of Streptococcus suis serotypes in Ukraine territory]. Veterinary Biotechnology, 39, 117–127 (in Ukrainian).
Vaillancourt, K., LeBel, G., Frenette, M., Gottschalk, M., & Grenier, D. (2015). Suicin 3908, a new lantibiotic produced by a strain of Streptococcus suis serotype 2 isolated from a healthy carrier pig. PLoS One, 10(2), e0117245.
Van Samkar, A., Brouwer, M. C., Schultsz, C., van der Ende, A., & van de Beek, D. (2015). Streptococcus suis meningitis: A systematic review and meta-analysis. PLoS Neglected Tropical Diseases, 9(10), e0004191.
Wang, J., Zhengl, Y., Pian, Y., Guo, J., Hao, H., & Jiang, Y. (2015). Correlation between type IV secretion system component VirD4 and virulence for Streptococcus suis 2. Wei Sheng Wu Xue Bao, 55(5), 643–649.
Wang, Y., Wang, Y., Sun, L., Grenier, D., & Yi, L. (2018). Streptococcus suis biofilm: Regulation, drug-resistance mechanisms, and disinfection strategies. Applied Microbiology and Biotechnology, 102(21), 9121–9129.
Werinder, A., Aspán, A., & Backhans, A. (2020). Streptococcus suis in Swedish grower pigs: Occurrence, serotypes, and antimicrobial susceptibility. Acta Veterinaria Scandinavica, 62, 36.
Werinder, A., Aspán, A., Söderlund, R., Backhans, A., Sjölund, M., Guss, B., & Jacobson, M. (2021). Whole-genome sequencing evaluation of MALDI-TOF MS as a species identification tool for Streptococcus suis. Journal of Clinical Microbiology, 59(11), e0129721.
Xia, X., Wang, X., Wei, X., Jiang, J., & Hu, J. (2018). Methods for the detection and characterization of Streptococcus suis: From conventional bacterial culture methods to immunosensors. Antonie Van Leeuwenhoek, 111(12), 2233–2247.
Xu, Q., Chen, H., Sun, W., Zhang, Y., Zhu, D., Rai, K. R., Chen, J. L., & Chen, Y. (2021). sRNA23, a novel small RNA, regulates to the pathogenesis of Streptococcus suis serotype 2. Virulence, 12(1), 3045–3061.
Yang, X. P., Fu, J. Y., Yang, R. C., Liu, W. T., Zhang, T., Yang, B., Miao, L., Dou, B. B., Tan, C., Chen, H. C., & Wang, X. R. (2016). EGFR transactivation contributes to neuroinflammation in Streptococcus suis meningitis. Journal of Neuroinflammation, 13(1), 274.
Yi, L., Li, J., Fan, Q., Mao, C., Jin, M., Liu, Y., Sun, L., Grenier, D., & Wang, Y. (2020). The otc gene of Streptococcus suis plays an important role in biofilm formation, adhesion, and virulence in a murine model. Veterinary Microbiology, 251, 108925.
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