Outbreak Investigation Of Streptococcus Suis By Multiple-Locus Variable Number Of Tandem Repeat Analysis

Streptococcus suis is an important zoonotic agent in humans, often linking to occupational exposure to pig or porcine products, resulting with meningitis, septicemia, permanent hearing loss. A largest recorded outbreak of S.suis serotype 2 infection occurred in 202 villages in Sichuan Province, China, during the period from July to August of 2005. The sequence type 7 (ST7) S. suis was recognized as the causative pathogen for the Sichuan outbreak in 2005 and Jiangsu in 1998. So far, S. suis ST7 has not been isolated in any other country except China. For the reason that S.suis serotype 2 strains involved in two outbreaks of China can not be differentiated by MLST or PFGE method. This indicated a more descreminative method is required to discriminate strains from different origins.In this study we identified and assessed polymorphic tandem repeat loci within the genomes of four S.suis serotype 2 strains and developed a multiple-locus variable number of tandem repeat analysis (MLVA) method. The complete genome sequence of four S. suis serotype 2 strains were analyzed for potential VNTR loci using the Tandem Repeat Finder software (TRF). By assessing the polymorphism of loci, ultimately, 9 VNTR loci were selected to subtype S. suis in MLVA analyses. The markers we selected in MLVA analysis, eight (TR1-8) belongs to moderately diversity markers. The TR9 locus belongs to higher sensitive marker.A total of 199 S. suis isolates were analyzed in this study including 33 serotype reference strains and 166 clinical isolates in serotype 2 (154 from China and 12 from other countries). A total of 85 MLVA types were defined, in which 33 MLVA types were identified for the 33 serotypes reference strains. 85 MLVA types were clustered into two clusters (Cluster-I, II). S.suis Serotype 2 strains were divided into 53 MLVA types. S.suis serotype2 strains of China and some European country clustered together and shared Cluster-II with serotype 14 and 15 strain.The investigation of outbreak-associated ST7 S. suis strain analyzed by MLVA Scheme showed that 144 ST7 strains were discriminated into thirty-five MLVAs, of which 100 ST7 strains isolated in 2005 were divided into thirty MLVAs. Noteworthy, eight isolates from Jiangsu Province in 1998 were divided into five MLVA types, of which four subtypes were also detected in isolates in Sichuan in 2005, which provide molecular clues that the pathogens responsible for these two outbreaks in China were closely associated or from the same origin. In addition, the MLVA subtypes of ST7 strains isolated from Chongqing, Guangdong, Jiangxi, and Jiangsu Province in 2005 were also detected in strains from Sichuan in 2005, which provided evidence that the causative agents of non-Sichuan provinces in 2005 were originated from strains of 2005 Sichuan outbreak. These isolates isolated in 2006-2007 shared some identical MLVA profiles with Chinese major epidemic S. suis serotype 2 strains in 1998 and 2005, which supported the inference that the isolates collected in Sichuan Province in 2006-2007 were derived from some clones with the former outbreaks strains.The MLVA scheme described here has a higher discriminative typing power than pulsedfield gel electrophoresis (PFGE), which suitable in outbreak surveillance and investigation of S. suis to detect the source of infection and transmission route. Such as one strain, isolated from a Jiangxi patient who processed pork transported from Jiangsu Province, had same MLVA subtypes with anther three strains isolated from pork he processed. These data conclusively demonstrates the Jiangxi S. suis was transmitted from Jaingsu Province in 2005 and S.suis can transmitted from one province to another by porcine products communication; The route of some S.suis infections confirmed by MLVA were also proofed by the epidemiology observation.The MLVA developed here shows many advantages have many necessary characteristics of an efficient typing method and represent a promising tool in molecular subtyping of Streptococcus suis /serotype 2 strains. To our knowledge, this is the first report of MLVA method applied to S. suis. It is believed that this technique can be efficiently used in future phylogenetic investigation, forensic tracking, or epidemiological investigations of S. suis infections.