Identificação polifásica e detecção simultânea por PCR Multiplex de patógenos presentes em tambaqui (Colossoma Macropomum) oriundo de diferentes sistemas de manejo

Abstract

The Amazon region stands out in fish farming with the production of native species, especially the tambaqui (Colossoma macropomum), the species with the highest Brazilian production. Fast and accurate methods for pathogens detection in fish can help in microbiological quality control, promoting food safety and fish farming performance in the country. Therefore, the objective of this work was to identify the bacterial isolates of Tambaqui obtained from two different management systems, using polyphasic analysis. Furthermore, a multiplex Polymerase Chain Reaction method was adapted for the simultaneous detection of Escherichia coli, Salmonella spp., and Staphylococcus aureus species in fish musculature samples. First, 470 isolates (181 from net cages and 289 from excavated ponds) from swab samples of skin, musculature, and water from tambaqui culture were morphologically and biochemically characterized, being grouped according to their phenotypic characteristics. Among the isolates, 85 were characterized and grouped into 12 distinct groups: five groups belonging to the genus Salmonella spp., two groups of Escherichia spp, two of Staphylococcus spp., and three characteristic groups of the Enterobacteriaceae family. From each group, representative isolates were confirmed by sequencing the 16S-23S rRNA region as belonging to the species Salmonella tiphymurium, E. coli, S. aureus, Enterobacter cloacae, Enterobacter asburie, and Pseudomonas aeruginosa, with the similarity between 77-100%. DNA from 30 tambaqui musculature samples from both management systems was extracted and submitted to multiplex PCR, using specific primers for the phoA, invA, and nuc genes to detect the pathogens E. coli, Salmonella spp. and S. aureus, respectively, simultaneously in a single reaction. In the reaction, Salmonella spp. was detected in five samples, two from net cages and three from excavated ponds. E. coli was detected in two excavated pond samples. S. aureus was not detected in any of the samples. The results obtained in the polyphasic identification of the isolates and the multiplex PCR analysis of the fish musculature samples were correlated. Therefore, the proposed methodology has the potential to be used as an effective method of simultaneous detection of pathogens, which can positively impact the production chain of native fish, and provide quickly and efficiently information on the microbiological quality of fish.