Tools for Molecular Epidemiology of Streptococcus suis

Diseases caused by Streptococcus suis are a significant economic and welfare concern in pigs as well as in humans. Several molecular methods have been applied to investigate S. suis strain diversity and identify phylogenetic groups. Multilocus sequence typing (MLST), commonly used to differentiate between S. suis strains, has been instrumental in identifying that the species is genetically highly diverse. Recent advances in whole-genome analysis have resulted in schemes permitting the classification of S. suis populations as pathogenic or non-pathogenic, or disease-associated or non-disease associated. Here, we review these and other molecular approaches that can be used for surveillance, outbreak tracking, preventative health management, effective treatment and control, as well as vaccine development, including PCR based-assays that are easy to apply in modest diagnostic settings and which allow for the rapid screening of a large number of isolates at relatively low cost, granting the identification of several major clonal complexes of the S. suis population.


Introduction
Streptococcus suis is a pathogen of pigs that can cause infections in humans who are in close contact with infected animals and/or contaminated pork-derived products, as well as those who have consumed raw pork products [1]. S. suis can cause meningitis, septicemia, endocarditis, and arthritis in humans [2][3][4]. Human S. suis cases have been reported in most countries of Western Europe, North and South America, Africa, Australia, New Zealand, India, Japan, and several other East and Southeast Asian countries, particularly China, Vietnam, and Thailand [1,3,4]. Indeed, S. suis has been shown to be responsible for thousands of human cases, of which 90.2% were in Asia, 8.5% in Europe, and 1.3% in other parts of the world [4].
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans Pathogens 2020, 9,81 4 of 10 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening of a large number of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans Pathogens 2020, 9,81 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening o of isolates at a relatively modest cost (summarized in Table 2). A first study reported that a variant of the serum opacity factor (ofs) can serve as to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, were included in CC27 with the less-stringent group definition (5 alleles sharing) u [39]. However, since type-3 ofs was present in both CC94 and CC27, these two C resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associat correlated with CCs of S. suis [40]. The results showed that such gene profiling co screening method for potentially hazardous S. suis groups. For example, genotyp associated gene profile was strongly associated with CC1, genotype B was associate CC27, and genotype C was related to CC104. Despite its enhanced discriminatory cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessa gene profiles for each genotype.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study agreement of RAPD patterns among the three primers used with the specific patte each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC16.
A last report used random amplification of polymorphic DNA (RAPD) an intergenic spacer polymerase chain reaction-restriction fragment length polymo PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n Pathogens 2020, 9,81 important S. suis CCs (PCR-CC) were developed allowing for the ra of isolates at a relatively modest cost (summarized in Table 2). A first study reported that a variant of the serum opacity factor ( to determine CCs of S. suis [39]. The report showed that a type-1 CC1, while type-3 ofs and type-4 ofs were associated with CC27. Imp were included in CC27 with the less-stringent group definition (5 a [39]. However, since type-3 ofs was present in both CC94 and CC resolved. Similarly, CC25 and CC104 could not be differentiated fro A second study described a PCR assay to determine how correlated with CCs of S. suis [40]. The results showed that such g screening method for potentially hazardous S. suis groups. For ex associated gene profile was strongly associated with CC1, genotype CC27, and genotype C was related to CC104. Despite its enhanced cannot distinguish between CC25 and CC27, and multiple PCR react gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to ide to human infections in a single reaction [41]. Of the 7 sequences e (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muram occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pe for PCR with the srtBCD primers described by Takamatsu et al. (2 study differentiated between CC1, CC25, CC28, CC104, CC221/23 assay could not detect CC16, CC20, and CC94, and the report did no related to pig infections. A fourth report by Maneerat et al. (2013) demonstrated that R OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [4 agreement of RAPD patterns among the three primers used with t each primer. Nonetheless, this study did not evaluate other S. sui CC16.
A last report used random amplification of polymorphic DN intergenic spacer polymerase chain reaction-restriction fragment PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isola CC16 Pathogens 2020, 9, 81 4 of 10 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening of a large number of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans Pathogens 2020, 9, 81 4 of 10 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening of a large number of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans Pathogens 2020, 9, 81 4 of 10 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening of a large number of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans Pathogens 2020, 9,81 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening o of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods w CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, were included in CC27 with the less-stringent group definition (5 alleles sharing) u [39]. However, since type-3 ofs was present in both CC94 and CC27, these two C resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associat correlated with CCs of S. suis [40]. The results showed that such gene profiling co screening method for potentially hazardous S. suis groups. For example, genotyp associated gene profile was strongly associated with CC1, genotype B was associate CC27, and genotype C was related to CC104. Despite its enhanced discriminatory cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessa gene profiles for each genotype.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study agreement of RAPD patterns among the three primers used with the specific patte each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC16.
A last report used random amplification of polymorphic DNA (RAPD) an intergenic spacer polymerase chain reaction-restriction fragment length polymo PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n Pathogens 2020, 9,81 important S. suis CCs (PCR-CC) were developed allowing for the ra of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. * CC20.
A first study reported that a variant of the serum opacity factor ( to determine CCs of S. suis [39]. The report showed that a type-1 CC1, while type-3 ofs and type-4 ofs were associated with CC27. Imp were included in CC27 with the less-stringent group definition (5 a [39]. However, since type-3 ofs was present in both CC94 and CC resolved. Similarly, CC25 and CC104 could not be differentiated fro A second study described a PCR assay to determine how correlated with CCs of S. suis [40]. The results showed that such g screening method for potentially hazardous S. suis groups. For ex associated gene profile was strongly associated with CC1, genotype CC27, and genotype C was related to CC104. Despite its enhanced cannot distinguish between CC25 and CC27, and multiple PCR react gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to ide to human infections in a single reaction [41]. Of the 7 sequences e (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muram occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pe for PCR with the srtBCD primers described by Takamatsu et al. (2 study differentiated between CC1, CC25, CC28, CC104, CC221/23 assay could not detect CC16, CC20, and CC94, and the report did no related to pig infections. A fourth report by Maneerat et al. (2013) demonstrated that R OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [4 agreement of RAPD patterns among the three primers used with t each primer. Nonetheless, this study did not evaluate other S. sui CC16.
A last report used random amplification of polymorphic DN intergenic spacer polymerase chain reaction-restriction fragment PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isola CC20 ** Pathogens 2020, 9, 81 4 of 10 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening of a large number of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans Pathogens 2020, 9, 81 4 of 10 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening of a large number of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans Pathogens 2020, 9, 81 4 of 10 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening of a large number of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans Pathogens 2020, 9,81 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening o of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods w CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, were included in CC27 with the less-stringent group definition (5 alleles sharing) u [39]. However, since type-3 ofs was present in both CC94 and CC27, these two C resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associat correlated with CCs of S. suis [40]. The results showed that such gene profiling co screening method for potentially hazardous S. suis groups. For example, genotyp associated gene profile was strongly associated with CC1, genotype B was associate CC27, and genotype C was related to CC104. Despite its enhanced discriminatory cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessa gene profiles for each genotype.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study agreement of RAPD patterns among the three primers used with the specific patte each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC16.
A last report used random amplification of polymorphic DNA (RAPD) an intergenic spacer polymerase chain reaction-restriction fragment length polymo PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n Pathogens 2020, 9,81 important S. suis CCs (PCR-CC) were developed allowing for the ra of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. * CC20.
A first study reported that a variant of the serum opacity factor ( to determine CCs of S. suis [39]. The report showed that a type-1 CC1, while type-3 ofs and type-4 ofs were associated with CC27. Imp were included in CC27 with the less-stringent group definition (5 a [39]. However, since type-3 ofs was present in both CC94 and CC resolved. Similarly, CC25 and CC104 could not be differentiated fro A second study described a PCR assay to determine how correlated with CCs of S. suis [40]. The results showed that such g screening method for potentially hazardous S. suis groups. For ex associated gene profile was strongly associated with CC1, genotype CC27, and genotype C was related to CC104. Despite its enhanced cannot distinguish between CC25 and CC27, and multiple PCR react gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to ide to human infections in a single reaction [41]. Of the 7 sequences e (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muram occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pe for PCR with the srtBCD primers described by Takamatsu et al. (2 study differentiated between CC1, CC25, CC28, CC104, CC221/23 assay could not detect CC16, CC20, and CC94, and the report did no related to pig infections. A fourth report by Maneerat et al. (2013) demonstrated that R OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [4 agreement of RAPD patterns among the three primers used with t each primer. Nonetheless, this study did not evaluate other S. sui CC16.
A last report used random amplification of polymorphic DN intergenic spacer polymerase chain reaction-restriction fragment PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isola CC25 Pathogens 2020, 9, 81 4 of 10 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening of a large number of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans Pathogens 2020, 9, 81 4 of 10 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening of a large number of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans * Pathogens 2020, 9, 81 4 of 10 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening of a large number of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans * Pathogens 2020, 9,81 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening o of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods w CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, were included in CC27 with the less-stringent group definition (5 alleles sharing) u [39]. However, since type-3 ofs was present in both CC94 and CC27, these two C resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associat correlated with CCs of S. suis [40]. The results showed that such gene profiling co screening method for potentially hazardous S. suis groups. For example, genotyp associated gene profile was strongly associated with CC1, genotype B was associate CC27, and genotype C was related to CC104. Despite its enhanced discriminatory cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessa gene profiles for each genotype.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study agreement of RAPD patterns among the three primers used with the specific patte each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC16.
A last report used random amplification of polymorphic DNA (RAPD) an intergenic spacer polymerase chain reaction-restriction fragment length polymo PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n Pathogens 2020, 9,81 important S. suis CCs (PCR-CC) were developed allowing for the ra of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** CC20.
A first study reported that a variant of the serum opacity factor ( to determine CCs of S. suis [39]. The report showed that a type-1 o CC1, while type-3 ofs and type-4 ofs were associated with CC27. Impo were included in CC27 with the less-stringent group definition (5 al [39]. However, since type-3 ofs was present in both CC94 and CC resolved. Similarly, CC25 and CC104 could not be differentiated from A second study described a PCR assay to determine how correlated with CCs of S. suis [40]. The results showed that such ge screening method for potentially hazardous S. suis groups. For exa associated gene profile was strongly associated with CC1, genotype CC27, and genotype C was related to CC104. Despite its enhanced d cannot distinguish between CC25 and CC27, and multiple PCR react gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to iden to human infections in a single reaction [41]. Of the 7 sequences en (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 murami occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep for PCR with the srtBCD primers described by Takamatsu et al. (2 study differentiated between CC1, CC25, CC28, CC104, CC221/234 assay could not detect CC16, CC20, and CC94, and the report did no related to pig infections. A fourth report by Maneerat et al. (2013) demonstrated that RA OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [4 agreement of RAPD patterns among the three primers used with th each primer. Nonetheless, this study did not evaluate other S. suis CC16.
A last report used random amplification of polymorphic DN intergenic spacer polymerase chain reaction-restriction fragment PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolat * CC28 Pathogens 2020, 9, 81 4 of 10 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening of a large number of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans Pathogens 2020, 9, 81 4 of 10 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening of a large number of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans * Pathogens 2020, 9, 81 4 of 10 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening of a large number of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans * Pathogens 2020, 9,81 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening o of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods w CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, were included in CC27 with the less-stringent group definition (5 alleles sharing) u [39]. However, since type-3 ofs was present in both CC94 and CC27, these two C resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associat correlated with CCs of S. suis [40]. The results showed that such gene profiling co screening method for potentially hazardous S. suis groups. For example, genotyp associated gene profile was strongly associated with CC1, genotype B was associate CC27, and genotype C was related to CC104. Despite its enhanced discriminatory cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessa gene profiles for each genotype.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study agreement of RAPD patterns among the three primers used with the specific patte each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC16.
A last report used random amplification of polymorphic DNA (RAPD) an intergenic spacer polymerase chain reaction-restriction fragment length polymo PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n Pathogens 2020, 9,81 important S. suis CCs (PCR-CC) were developed allowing for the ra of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** CC20.
A first study reported that a variant of the serum opacity factor ( to determine CCs of S. suis [39]. The report showed that a type-1 o CC1, while type-3 ofs and type-4 ofs were associated with CC27. Impo were included in CC27 with the less-stringent group definition (5 al [39]. However, since type-3 ofs was present in both CC94 and CC resolved. Similarly, CC25 and CC104 could not be differentiated from A second study described a PCR assay to determine how correlated with CCs of S. suis [40]. The results showed that such ge screening method for potentially hazardous S. suis groups. For exa associated gene profile was strongly associated with CC1, genotype CC27, and genotype C was related to CC104. Despite its enhanced d cannot distinguish between CC25 and CC27, and multiple PCR react gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to iden to human infections in a single reaction [41]. Of the 7 sequences en (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 murami occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep for PCR with the srtBCD primers described by Takamatsu et al. (2 study differentiated between CC1, CC25, CC28, CC104, CC221/234 assay could not detect CC16, CC20, and CC94, and the report did no related to pig infections. A fourth report by Maneerat et al. (2013) demonstrated that RA OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [4 agreement of RAPD patterns among the three primers used with th each primer. Nonetheless, this study did not evaluate other S. suis CC16.
A last report used random amplification of polymorphic DN intergenic spacer polymerase chain reaction-restriction fragment PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolat * CC94 Pathogens 2020, 9, 81 4 of 10 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening of a large number of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans Pathogens 2020, 9, 81 4 of 10 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening of a large number of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans * Pathogens 2020, 9, 81 4 of 10 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening of a large number of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans Pathogens 2020, 9,81 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening o of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods w CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, were included in CC27 with the less-stringent group definition (5 alleles sharing) u [39]. However, since type-3 ofs was present in both CC94 and CC27, these two C resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associat correlated with CCs of S. suis [40]. The results showed that such gene profiling co screening method for potentially hazardous S. suis groups. For example, genotyp associated gene profile was strongly associated with CC1, genotype B was associate CC27, and genotype C was related to CC104. Despite its enhanced discriminatory cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessa gene profiles for each genotype.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study agreement of RAPD patterns among the three primers used with the specific patte each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC16.
A last report used random amplification of polymorphic DNA (RAPD) an intergenic spacer polymerase chain reaction-restriction fragment length polymo PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n Pathogens 2020, 9,81 important S. suis CCs (PCR-CC) were developed allowing for the ra of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. * CC20.
A first study reported that a variant of the serum opacity factor ( to determine CCs of S. suis [39]. The report showed that a type-1 CC1, while type-3 ofs and type-4 ofs were associated with CC27. Imp were included in CC27 with the less-stringent group definition (5 a [39]. However, since type-3 ofs was present in both CC94 and CC resolved. Similarly, CC25 and CC104 could not be differentiated fro A second study described a PCR assay to determine how correlated with CCs of S. suis [40]. The results showed that such g screening method for potentially hazardous S. suis groups. For ex associated gene profile was strongly associated with CC1, genotype CC27, and genotype C was related to CC104. Despite its enhanced cannot distinguish between CC25 and CC27, and multiple PCR react gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to ide to human infections in a single reaction [41]. Of the 7 sequences e (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muram occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pe for PCR with the srtBCD primers described by Takamatsu et al. (2 study differentiated between CC1, CC25, CC28, CC104, CC221/23 assay could not detect CC16, CC20, and CC94, and the report did no related to pig infections. A fourth report by Maneerat et al. (2013) demonstrated that R OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [4 agreement of RAPD patterns among the three primers used with t each primer. Nonetheless, this study did not evaluate other S. sui CC16.
A last report used random amplification of polymorphic DN intergenic spacer polymerase chain reaction-restriction fragment PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isola  Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans Pathogens 2020, 9,81 4 of 10 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening of a large number of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans * Pathogens 2020, 9, 81 4 of 10 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening of a large number of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans Pathogens 2020, 9,81 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening o of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods w CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, were included in CC27 with the less-stringent group definition (5 alleles sharing) u [39]. However, since type-3 ofs was present in both CC94 and CC27, these two C resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associat correlated with CCs of S. suis [40]. The results showed that such gene profiling co screening method for potentially hazardous S. suis groups. For example, genotyp associated gene profile was strongly associated with CC1, genotype B was associate CC27, and genotype C was related to CC104. Despite its enhanced discriminatory cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessa gene profiles for each genotype.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study agreement of RAPD patterns among the three primers used with the specific patte each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC16.
A last report used random amplification of polymorphic DNA (RAPD) an intergenic spacer polymerase chain reaction-restriction fragment length polymo PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n Pathogens 2020, 9,81 important S. suis CCs (PCR-CC) were developed allowing for the ra of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** CC20.
A first study reported that a variant of the serum opacity factor ( to determine CCs of S. suis [39]. The report showed that a type-1 o CC1, while type-3 ofs and type-4 ofs were associated with CC27. Impo were included in CC27 with the less-stringent group definition (5 al [39]. However, since type-3 ofs was present in both CC94 and CC resolved. Similarly, CC25 and CC104 could not be differentiated from A second study described a PCR assay to determine how correlated with CCs of S. suis [40]. The results showed that such ge screening method for potentially hazardous S. suis groups. For exa associated gene profile was strongly associated with CC1, genotype CC27, and genotype C was related to CC104. Despite its enhanced d cannot distinguish between CC25 and CC27, and multiple PCR react gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to iden to human infections in a single reaction [41]. Of the 7 sequences en (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 murami occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep for PCR with the srtBCD primers described by Takamatsu et al. (2 study differentiated between CC1, CC25, CC28, CC104, CC221/234 assay could not detect CC16, CC20, and CC94, and the report did no related to pig infections. A fourth report by Maneerat et al. (2013) demonstrated that RA OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [4 agreement of RAPD patterns among the three primers used with th each primer. Nonetheless, this study did not evaluate other S. suis CC16.
A last report used random amplification of polymorphic DN intergenic spacer polymerase chain reaction-restriction fragment PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolat * CC233/379 Pathogens 2020, 9, 81 4 of 10 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening of a large number of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans Pathogens 2020, 9,81 4 of 10 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening of a large number of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans Pathogens 2020, 9,81 4 of 10 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening of a large number of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans Pathogens 2020, 9,81 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening o of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods w CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, were included in CC27 with the less-stringent group definition (5 alleles sharing) u [39]. However, since type-3 ofs was present in both CC94 and CC27, these two C resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associat correlated with CCs of S. suis [40]. The results showed that such gene profiling co screening method for potentially hazardous S. suis groups. For example, genotyp associated gene profile was strongly associated with CC1, genotype B was associate CC27, and genotype C was related to CC104. Despite its enhanced discriminatory cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessa gene profiles for each genotype.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study agreement of RAPD patterns among the three primers used with the specific patte each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC16.
A last report used random amplification of polymorphic DNA (RAPD) an intergenic spacer polymerase chain reaction-restriction fragment length polymo PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n Pathogens 2020, 9,81 important S. suis CCs (PCR-CC) were developed allowing for the ra of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** CC20.
A first study reported that a variant of the serum opacity factor ( to determine CCs of S. suis [39]. The report showed that a type-1 o CC1, while type-3 ofs and type-4 ofs were associated with CC27. Impo were included in CC27 with the less-stringent group definition (5 al [39]. However, since type-3 ofs was present in both CC94 and CC resolved. Similarly, CC25 and CC104 could not be differentiated from A second study described a PCR assay to determine how correlated with CCs of S. suis [40]. The results showed that such ge screening method for potentially hazardous S. suis groups. For exa associated gene profile was strongly associated with CC1, genotype CC27, and genotype C was related to CC104. Despite its enhanced d cannot distinguish between CC25 and CC27, and multiple PCR react gene profiles for each genotype. A third study reported a multiplex PCR that can be used to iden to human infections in a single reaction [41]. Of the 7 sequences en (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 murami occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep for PCR with the srtBCD primers described by Takamatsu  important S. suis CCs (PCR-CC) were developed allowing for the rapid screening of a large number of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S Pathogens 2020, 9, 81 4 of 10 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening of a large number of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans Pathogens 2020, 9, 81 4 of 10 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening of a large number of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilusassociated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans Pathogens 2020, 9,81 important S. suis CCs (PCR-CC) were developed allowing for the rapid screening o of isolates at a relatively modest cost (summarized in Table 2).
Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods w CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, were included in CC27 with the less-stringent group definition (5 alleles sharing) u [39]. However, since type-3 ofs was present in both CC94 and CC27, these two C resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associat correlated with CCs of S. suis [40]. The results showed that such gene profiling co screening method for potentially hazardous S. suis groups. For example, genotyp associated gene profile was strongly associated with CC1, genotype B was associate CC27, and genotype C was related to CC104. Despite its enhanced discriminatory cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessa gene profiles for each genotype.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study agreement of RAPD patterns among the three primers used with the specific patte each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC16.
A last report used random amplification of polymorphic DNA (RAPD) an intergenic spacer polymerase chain reaction-restriction fragment length polymo Pathogens 2020, 9,81 important S. suis CCs (PCR-CC) were developed allowing for the ra of isolates at a relatively modest cost (summarized in Table 2). A first study reported that a variant of the serum opacity factor ( to determine CCs of S. suis [39]. The report showed that a type-1 CC1, while type-3 ofs and type-4 ofs were associated with CC27. Imp were included in CC27 with the less-stringent group definition (5 a [39]. However, since type-3 ofs was present in both CC94 and CC resolved. Similarly, CC25 and CC104 could not be differentiated fro A second study described a PCR assay to determine how correlated with CCs of S. suis [40]. The results showed that such g screening method for potentially hazardous S. suis groups. For ex associated gene profile was strongly associated with CC1, genotype CC27, and genotype C was related to CC104. Despite its enhanced cannot distinguish between CC25 and CC27, and multiple PCR react gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to ide to human infections in a single reaction [41]. Of the 7 sequences e (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muram occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pe for PCR with the srtBCD primers described by Takamatsu et al. (2 study differentiated between CC1, CC25, CC28, CC104, CC221/23 assay could not detect CC16, CC20, and CC94, and the report did no related to pig infections. A fourth report by Maneerat et al. (2013) demonstrated that R OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [4 agreement of RAPD patterns among the three primers used with t each primer. Nonetheless, this study did not evaluate other S. sui CC16.
A last report used random amplification of polymorphic DN intergenic spacer polymerase chain reaction-restriction fragment Note: * reveal the same profile; thus, could not be differentiated for each. ** PCR methods were not applied to CC20.
A first study reported that a variant of the serum opacity factor (ofs) can serve as a genetic marker to determine CCs of S. suis [39]. The report showed that a type-1 ofs was strongly associated with CC1, while type-3 ofs and type-4 ofs were associated with CC27. Importantly, CC25, CC28, and CC104 were included in CC27 with the less-stringent group definition (5 alleles sharing) used in that study [39]. However, since type-3 ofs was present in both CC94 and CC27, these two CCs could not be resolved. Similarly, CC25 and CC104 could not be differentiated from CC27.
A second study described a PCR assay to determine how pilus-associated gene profiles correlated with CCs of S. suis [40]. The results showed that such gene profiling could be used as a screening method for potentially hazardous S. suis groups. For example, genotype A of the pilus-associated gene profile was strongly associated with CC1, genotype B was associated with CC25 and CC27, and genotype C was related to CC104. Despite its enhanced discriminatory power, this assay cannot distinguish between CC25 and CC27, and multiple PCR reactions are necessary to identify the gene profiles for each genotype.
A third study reported a multiplex PCR that can be used to identify S. suis CCs that are relevant to human infections in a single reaction [41]. Of the 7 sequences encoded-2 hypothetical proteins (hp1 and hp2), 1 ribonuclease G (ribG), 1 peptidase (pep), 1 muramidase-like protein (mp), 1 zonula occludens toxin (zot), and 1 collagen adhesion (col)-only hp1, mp, pep, and col were selected as targets for PCR with the srtBCD primers described by Takamatsu et al. (2009). The test developed in that study differentiated between CC1, CC25, CC28, CC104, CC221/234, and CC233/379. However, the assay could not detect CC16, CC20, and CC94, and the report did not evaluate assays on CCs mostly related to pig infections.
A fourth report by Maneerat et al. (2013) demonstrated that RAPD using OPB-07, OPB-10, and OPB-17 could distinguish CC1, CC104, CC233/379, and ST336 [42]. This study revealed a high agreement of RAPD patterns among the three primers used with the specific pattern of each CC in each primer. Nonetheless, this study did not evaluate other S. suis CCs, such as CC25, CC28, and CC16.
A last report used random amplification of polymorphic DNA (RAPD) and 16S-23S rDNA intergenic spacer polymerase chain reaction-restriction fragment length polymorphism (16S-23S PCR-RFLP) to determine clonal complexes (CCs) of 684 S. suis isolates from pigs (n=72) and humans (n=612) [43]. The study also revealed that RAPD was better at distinguishing the major S. suis CCs than the PCR-RFLP method: the assay was capable of simultaneously distinguishing CC1, CC16, CC25, CC28, CC104, CC221/234, and CC233/379. RAPD with the OPB-10 primer clearly distinguished each CC in the study, and no cross-patterns were found. Finally, the study also showed that neither the OPB-06 nor OPB-11 primer could distinguish CC104 from CC233/379 isolates. The report showed that PCR-RFLP and DNA sequencing using the 16S-23S rDNA intergenic spacer could not clearly differentiate among most CCs [43]. Both techniques could classify S. suis isolates into four clusters: cluster 1 consisting of CC25, CC28, CC104, and CC233/379; cluster 2 consisting of CC221/234; cluster 3 consisting of CC16 (ST16); and cluster 4 consisting of CC1.
In addition to these PCR-based techniques, MLVA is a technique with high discriminatory power that has also been used to investigate S. suis. While PFGE could not differentiate between isolates, one MLVA scheme using nine loci (TR1-TR9) was successfully used to link and to sub-type S. suis ST7 isolates from two outbreaks that occurred in China in 1998 and 2005 [27]. Additional advantages of MLVA are that it is relatively easy to perform, rapid and reliable, and that it permits high-throughput screenings. MLVA also allows the comparison of results between laboratories and is useful in case of outbreak investigations.

Whole-Genome Sequencing Approaches
WGS approaches have increasingly been used to investigate S. suis isolates, including molecular determination of serotype [44], characterization of outbreaks [45,46], evaluation of S. suis reinfection [47], and to determine the population structure of S. suis isolates of serotype 2 belonging to ST25 and ST28 [48,49], as well as of serotype 9 [50]. WGS-based bacterial typing strategies commonly use one of two approaches: SNP (sequence)-based, or MLST (allele)-based. The SNP approach compares single nucleotide differences between isolates in comparison to a reference genome and is particularly useful to determine the clonal relationship between highly similar isolates. The MLST approach is an extension of conventional 7-gene MLST that expands the range of genes to the genome scale and can be roughly divided into core-genome-based MLST (cgMLST) and whole-genome-based MLST (wgMLST). Allele-based comparisons can be conducted using analysis tools available on the internet, such as BacWGSTdb (http://bacdb.org/BacWGSTdb/) [51], pubMLST (https://pubmlst.org/ssuis/), or the Center for Genomic Epidemiology (https://cge.cbs.dtu.dk/services/MLST/) [52].
The definition of virulence in S. suis remains controversial, and WGS-based approaches have played an important role in the efforts to identify virulence markers permitting to differentiate between commensal organisms and those prone to cause disease. A comparative genome hybridization (CGH) was used to analyze 55 S. suis isolates from different serotypes, recovered from different hosts, different clinical sources, and different geographical locations [53]. Clustering of CGH data divided S. suis isolates into 2 clusters. Cluster A exclusively contained virulent CC1 isolates of serotypes 1 and 2 isolates [53]. Cluster B, on the other hand, contained mainly a more divergent and heterogeneous group of serotype 7 and 9 isolates [53]. Another study used CGH on 39 isolates of different serotypes, sources, geographic locations, isolation years, and STs. This study revealed that the S. suis strains tested could be classified into three groups of differing levels of virulence: (i) epidemic and highly virulent (E/HV group), which included ST1, ST6, ST7, and ST11 isolates; (ii) virulent (V group), containing ST81, ST13, ST56, ST87, ST308, ST54, and ST53 isolates; and (iii) intermediately or weakly virulent (I/WV group), composed of isolates belonging to several STs that were all recovered from non-human sources [28].
The genomes of 375 S. suis isolates with detailed clinical phenotypes from pigs and humans from the United Kingdom and Vietnam were analyzed using a Bayesian Analysis of Population Structure (BAPS) [54]. The study showed clear genetic differences between systemic, respiratory, and non-clinical (carriage) S. suis isolates. Interestingly, systemic isolates had a smaller genome than respiratory and carriage isolates, and they tended to have an over-representation of virulence factors and a larger number of genes involved in defense functions [54]. Willemse et al. (2016) used BAPS to investigate the whole genomes of 98 S. suis isolates from human patients and pigs with invasive disease in the Netherlands, and 18 genomic complete and available S. suis sequences. The BAPS-based population