2016-17 Rapid Ag: Transdisciplinary approach to characterize the diversity of Streptococcus suis, leading to rapid genotyping by Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)
Department of Veterinary Population Medicine
- 2016 Fiscal Year: $93,563
- 2017 Fiscal Year: $55,313
The inability to discriminate and type Streptococcis suis (S. suis) strains has resulted in the lack of appropriate S. suis prevention and control strategies, which can be rectified by understanding the genetic and phenotypic differences between S. suis strains. The proper identification and classification of S. suis isolates will enable veterinarians to make accurate evaluations of isolates associated with clinical disease.
S. suis is a significant pathogen of swine and recently is an emerging zoonotic disease in southeast and east Asia. S. suis causes severe economic losses to the swine industry as it is an important cause of bacterial mortality in nursery pigs and causes a variety of diseases including meningitis, septicemia, and endocarditis in older pigs. S. suis is an encapsulated, gram-positive bacterium, and 35 serotypes have been determined based on capsular polysaccharides (CPS) though some of the serotypes may be a different Streptococcus species.
Between January 2003 to December 2005, 100 S. suis isolates from the United States were serotyped, revealing 15 different serotypes while 3 samples were untypable. Untypeable S. suis strains occur due to loss of their CPS, leading to a need for a new classification scheme to encompass these strains. Loss of the CPS may increase the affinity to mammalian cells, and biofilms can form in a variety of tissues including brain, joints, heart, and lungs 1. Recently, there has been an undeniably increase in clinical disease resulting from S. suis infections in Minnesota.
Rapid and accurate classification is vital to understanding the ecology, prevention, and treatment of S. suis infections. With the recent legislative action to limit antibiotic usage in livestock animals, accurate diagnosis of S. suis strains will lead to strain specific prevention and treatment, ultimately reducing the overall antibiotic usage in swine production. Locally, reducing the impact of S. suis disease will directly benefit the Minnesota pork industry, ranked second in production in the United States, and valued to the state of Minnesota in excess of 7.28 billion dollars in gross income annually (http://www.mnpork.com/minnesota-pork/). In addition, for every dollar generated by the Minnesota pork industry, the state collects $2.80 in economic return. Globally, multiple efforts in clinical microbiology have been proposed to identify and distinguish different clinical S. suis strains to understand their epidemiological implications, including serotyping, pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and multi-locus variable-number tandem-repeat typing (VNTR). Most recently, a whole-genome sequence based typing scheme has been developed as a novel approach to characterize S. suis strains, which determined the minimum core genome genes (MCGG). Using Next Generation Sequencing (NGS) technology, the raw sequence data from a sample is uploaded onto a website, which determines the MCGG group (1-7 or ungroupable). In 2013, 5 S. suis CPS gene clusters (II-IV, and 1-a and –b) were identified by using NGS technology. However, the previously described techniques are very time consuming, and the swine industry needs a rapid methodology to distinguish different S. suis strains. The significant advancements in S. suis molecular research must be combined with traditional microbiology and include another new technology, the Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), which generates results in minutes by analysis of biomolecules to identify bacterial species and possibly genotypes.
The MALDI-TOF MS uses protein differences to identify individual strains of a species by calculating the cross-wise distance values within a dendrogram spectrum. The University of Minnesota Veterinary Diagnostic Laboratory (UMNVDL) recently purchased a MALDI-TOF MS and an Illumina Miseq to further investigate, combat, and solve clinical diseases in animals. However, MALDI-TOF MS optimization to determine S. suis genotypes requires generating a database of known reference strains. Therefore, we propose a transdisciplinary approach using serotyping (classical microbiology), whole-genome sequencing (NGS technology), and laser desorption/ionization (MALDI-TOF MS) to characterize pathogenic S. suis genotypes, ultimately, developing rapid S. suis identification and genotyping via the MALDI-TOF MS to combat the increased disease association with S. suis for the Minnesota pork producers.
The project goal is to use a transdisciplinary approach, using traditional clinical microbiology, NGS, and MALDI-TOF MS, to characterize the diversity of 200 S. suis strains archived at the UMNVDL. This information will be used to determine and understand the complexity of S. suis strains in the United States and to develop a rapid speciation and genotyping system on the MALDI-TOF MS to prevent and control S. suis infections in swine herds. Specific aims are to:
- Serologically characterize 200 S. suis strains.
- Molecularly characterize the S. suis strains using Next Generation Sequencing.
- Utilize Laser desorption/ionization for characterization of the S. suis strains using MALDI-TOF MS.