Development of a Two-Step Multiplex PCR Assay for Typing of Capsular Polysaccharide Synthesis Gene Clusters of Streptococcus suis

Abstract

iWe developed a practical and easy two-step multiplex PCR assay to aid in serotyping of Streptococcus suis. The assay accurately typed almost all of the serotype reference strains and field isolates of various serotypes and also identified the genotypes of capsular polysaccharide synthesis gene clusters of some serologically nontypeable strains. Streptococcus suis is an important zoonotic pathogen that causes meningitis, septicemia, endocarditis, and other diseases in pigs and humans. S. suis strains have been classified into 35 serotypes (serotypes 1 to 34 and 1/2, which reacts with both serotype 1 and 2 typing sera) (1-4) on the basis of antigenic differences in their capsular polysaccharide (CP) (5). Serotyping of S. suis is one of the most useful methods to understand the epidemiology of a particular outbreak and monitor the prevalence of potentially hazardous strains. However, serotyping with all 35 typing antisera is time-consuming, and preparing the antisera is not easy due to the high cost and labor associated with its production. Additionally, cross-reactions in coagglutination tests with typing antisera and the presence of autoagglutinating strains increase the difficulty of serotyping in some cases. Therefore, the development of more practical and easier serotyping methods is desired.CP synthesis (cps) genes are clustered on a single locus of the chromosome in S. suis (6, 7). We recently sequenced and analyzed the cps gene clusters of all 35 serotype reference strains (8) and reported that 31 (serotypes 3 to 13 and 15 to 34) possessed serotype-specific genes, while the cps gene clusters of serotypes 1 and 14 and serotypes 2 and 1/2 were almost identical in each pair (8). Liu et al. (9) recently developed multiplex PCR assays to target serotype-specific cps genes for the molecular serotyping of S. suis. In their method, the reference strains of 33 serotypes (serotypes 1 to 31, 33, and 1/2) were sorted into their respective serotypes by three multiplex PCR assays, although serotypes 1 and 1/2 were not distinguished from serotypes 14 and 2, respectively (9). In addition, 84 isolates from a patient and clinically healthy pigs, which covered 20 serotypes, were correctly assigned serotypes predicted by coagglutination tests, except for those of a new serotype (serotype 21/29) (9). However, these methods have not yet been validated using field isolates from diseased pigs as well as nontypeable ones by the coagglutination test. Moreover, serotypes 32 and 34 were not included as targets for typing, because the reference strains of these serotypes have been reclassified as Streptococcus orisratti (10). In addition to the serotype 32 and 34 reference strains, those of serotypes 20, 22, 26, and 33 were also recently suggested to be removed from the taxon of S. suis (11). However, whether all isolates of these serotypes actually belong to a species that is different from S. suis remains unclear. From a diagnostic point of view, the isolates of these serotypes recovered from diseased pigs are still identified as S....

Novel Capsular Polysaccharide Loci and New Diagnostic Tools for High-Throughput Capsular Gene Typing in Streptococcus suis

“…CPSs of all serotypes are thought to be synthesized by the Wzx/Wzy pathway. Based on serotype-specific wzy genes, 3 multiplex capsular gene typing systems have been developed to identify S. suis serotypes (13)(14)(15). These systems allow the simultaneous detection of multiple nucleic acid sequences in a single reaction and can greatly reduce the time, cost, and work associated with conventional serotyping technologies.…”

Eight Novel Capsular Polysaccharide Synthesis Gene Loci Identified in Nontypeable Streptococcus suis Isolates

“…The wzy gene has been used as a marker for molecular serotyping of S. suis (11,12,26,34). When the 78 wzy sequences were compared with reference wzy sequences, they were clustered away from the reference sequences as 8 novel clusters.…”

“…Chromosomal DNA was prepared from all isolates using the method described previously (11). Strains were tested as No capsule 2011 Beijing 6 ST305 KM972281 YS8 NCL1-6 Polyagglutinating No capsule 2011 Beijing 6 ST305 KM972290 YS21 NCL1-3 Nonagglutinating 40-60 2011 Sichuan Province 6 ST310 KM972259 YS22 NCL1-3 Nonagglutinating 40-60 2011 Sichuan Province 6 ST310 KM972260 YS23 NCL2-2 Polyagglutinating 30-50 2011 Sichuan Province 6 ST328 KM972261 YS27 NCL3-1 Polyagglutinating 20-40 2011 Beijing 6 ST312 KM972262 YS33 NCL4 Autoagglutinating No capsule 2011 Beijing 7-3 ST423 KM972263 YS34 NCL4 Polyagglutinating No capsule 2011 Beijing 7-3 ST314 KM972264 YS35 NCL5 Polyagglutinating 40-60 2011 Beijing 7-2 ST315 KM972265 YS37 NCL4 Autoagglutinating 20-40 2011 Beijing 7-3 ST339 KM972266 YS38 nonserotypeable by both coagglutination and PCR testing (11,12). Serum for the seroagglutination test was purchased from the Statens Serum Institut (Copenhagen, Denmark).…”

“…Nontypeable S. suis strains from healthy pigs have been reported in many studies (12,13,(15)(16)(17)(18). The nontypeability of some of these strains is likely due to nonfunctional cps loci which produce an acapsular phenotype.…”

“…Hence, the serotype-specific wzy gene is an ideal target to discriminate the cps loci for molecular serotyping. Recently, two multiplex PCR (mPCR) assays based on the wzy gene have been developed to identify S. suis serotypes (11,12). Both methods greatly facilitate the serotype discrimination of S. suis isolates.…”