Monitoramento da dispersão de cepas de Escherichia coli em ambiente de produção leiteira

Abstract

Dairy farming has been of great importance for the world economy and for such reason producers have, more and more, sought improvements in milk quality, focusing on controlling diseases that affect the cattle such as bovine mastitis. Environmental mastitis, which is commonly caused by microorganisms such as Escherichia coli, can generate great impacts on dairy cattle. This bacterial species has genetic heterogeneity and population characterized by genetically diverse strains, besides the capacity to persist in production environment for an extended period. In this context, the present study aims to evaluate the clonal relationships of E. coli in a milk production environment through the molecular typing techniques Pulsed Field Gel Electrophoresis (PFGE) and Multilocus Sequence Typing (MLST). For this purpose, 444 samples distributed between milk, faeces, water and the production chain from a farm located in the municipality of Barra do Piraí, in the State of Rio de Janeiro, Brazil, were subjected to biochemical tests and theMatrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS) technique for bacterial identification. In addition, water samples (well, pond, drinking fountain, faucet and creek) were subjected to the Most Probable Number (MPN) test for total and thermotolerant coliform evaluation, in which they presented potability standards higher than those stipulated by the Ministry of Health. Out of 183 identified enterobacteria, 152 (83%) were confirmed by both methodologies as being E. coli. Out of these, nine representatives had the gyrB gene sequenced for molecular confirmation of the species exhibiting up to 99% maximum identity when the sequences were compared to the NCBI database sequences. Later, all 152 E. coli strains were phenotypically subjected to biofilm production and virulence genes detection, where it was possible to observe that 41.44% (63/152) of strains were biofilm producers, being 37.5% (57/152) weak producers, 1.31% (2/152) of moderate producers and 2.63% (4/152) strong producers. Moreover, 92.1% (140/152) of fimH gene positive strains could be detected, 88.8% (135/152) for the csgA gene, 29.6% (45/152) for the flu gene (which are biofilm-related genes) and 13.1% (20/152) positive for the eaeA gene; 7.2% (11/152) for the LT gene and 2.6% (4/152) for the stxI gene. No strain was positive for the stxII, ST, ial and eagg genes. From these results, 18 profiles were established with the purpose of selecting 30 strains that were processed in the Laboratory of Genetics of the National University of Río Cuarto, Argentina, through the PFGE technique, where it was possible to observe high genetic variability. Strains that presented from 95% to 100% of similarity (n = 10) were sequenced in order to establish a clonal relationship between them through the MLST technique that generated eight different ST types, with ST164 and ST1308 being the ones that established possible clonal relationships between strains. Furthermore, a new type of sequence (ST) was observed, one which should be subjected to a new generation sequencing and then sent to the MLST healer to generate a new ST number and deposited in the schema database. When researching the database as well as the literature, no ST reports (ST5, ST164, ST165 and ST1308) derived from bovine E. coli strains were found in Brazil, leading to the understanding that this work is the very first report in such country.