Análise de polimorfismos em genes relacionados à virulência e resistência de Streptococcus suis em uma população suína.

Background: Streptococcus suis is a bacterium that causes several diseases in swines and can infect humans; in addition, it represents an important risk factor for professionals from this area. In cases of outbreak, despite some animals may remain as asymptomatic disease carriers, mortality can vary between 4 and 14%. Virulence of this bacterium may be associated with different serotypes exist, as well as with cellular proteins present in the capsule, or by mutations in the genomes of different serotypes, or even between samples of the same serotype. The strains of serotype 2 are considered to be the most virulent, although they can have moderate virulence, or even being less virulent. The objective of this work is to analyze genetic variability in S. suis samples. Materials, Methods & Results: Forty S. suis samples provided by the Laboratório Microvet (Minas Gerais, Brazil) and isolated between 1995 and 2003 on a farm located in Ponte Nova, MG, have been used. The lyophilized samples were reactivated in BHI, for 24 h, at 37°C; after that, they were cultivated and isolated in Todd Hewitt nutrient medium plus agar-agar and 10% defibrinated sheep blood and kept at 37°C, for 18 h. Serotyping of samples was conducted by Microvet and the data was compiled for comparison with the results. Genomic DNA was extracted from 3x10 cells, using DNAzol kit (Invitrogen), under the protocol proposed by the manufacturer, followed by PCR individually for each primer pair - dpr and aroA, using 200 µM dNTP, 3.4 mM MgCl2, 2 U of Taq DNA polymerase (Invitrogen), 1.6x amplification buffer and 0.20 µM of the primers in a final volume of 25 µL. The annealing temperature was 59°C, for aroA gene, and 60°C, for dpr gene. The amplified fragments of genes were digested with 0.2 µL of the restriction enzymes Alu I, AflIII, EcoRI and MPS I, at 37°C, for 2 h. All products were analyzed by separation on 3% agarose gel. Eight in the forty samples analyzed showed polymorphisms in the dpr gene for cleavage site of Alu I enzyme, thus generating samples with two and three DNA fragments. Analysis with the other endonucleases does not show differences; besides, no cleavage has occurred in the fragment. All samples analyzed for the aroA gene were observed to be identical for the cleavage sites of the used enzymes. During the analysis of serotypes, 39 (97.5%) samples were identified as type 2 and one (2.5%) as type 3. Discussion: The dpr gene showed different patterns between the assessed S. suis colonies, what indicates the existence of polymorphisms in the restriction sites. These data partially corroborate those already reported in the literature, where the percentage of amino acid polymorphisms was 17.9% in the dpr gene. In this study, changes identified in the dpr gene are not conclusive; still, modifications in nucleotide bases of certain genes may be related to expressions of differential proteins, associated with pathogenicity and virulence of pathogens. Even with the use of antibiotics and hygienic-sanitary control measures, records of difficult-to-control outbreaks are constant in the swine populations from which the samples were isolated. The sample of serotype 3 was collected from healthy animal, whereas the serotype 2 samples were collected from sick animals. These factors reinforce the idea that alterations in genomes of bacteria may be establishing new mechanisms to escape, hence favoring the survival of pathogens. [ABSTRACT FROM AUTHOR]