Detection and characterization of bovine rumen microorganismsresistant to sodium fluoroacetate

ABSTRACT

Background: Poisoning of animals due to toxic plants is found in Brazil and other countries. One of the known toxic plantsin Brazil, with the active ingredient sodium fluoroacetate (SF), is Palicourea marcgravii. Dehalogenases that inactivatethe fluor-carbon bonds are enzymes found in microorganisms and may prevent intoxication. This study evaluated the occurrence of rumen microorganisms naturally resistant to SF.Materials, Methods & Results: Two samples of rumen fluid of cattle from the Experimental Farm of Federal University ofMato Grosso fed with Brachiaria sp. were obtained via fistula in flasks. An aliquot of 2 mL was placed in a microtube andcentrifuged at 9000 g for 1 min. Then, the sample was inoculated into 2 tubes, one containing 100 µL of clarified rumenfluid in 2 mL of modified liquid culture medium (0.1% ammonium sulfate, 0.1% potassium phosphate monobasic, 0.05%sodium phosphate dibasic, 0.01% magnesium sulfate, 0.01% yeast extract, pH 7.0) and 0.4% of SF and the other samplecontaining 2 mL of liquid culture medium and 100 µL of clarified rumen fluid. The 2 samples were incubated at 40°C for24 h. Dilutions were performed under the same conditions every 24 h until the attainment of microorganisms resistant toSF, and the finaldilution containing 50 µL of each sample was plated in the middle containing SF (0.4%) and incubated at40°C for 24 h for the isolation of bacteria. The bacterial colonies resistant to SF were identified by morphological methods, stained, and subjected to DNA extraction sequencing using the universal primers 27f and 1492r (16S rDNA) for theidentification of the bacterial genus using Blast DNA identity analysis. These bacteria were cultured with and without SF(0.4%), and the presence of fluoride ions was detected by an ion-selective electrode (fluoride) during incubation for 0, 30,60, 90, and 120 min. Two resistant microorganisms were isolated, one was a Gram-positive coccus and the other was aGram-positive rod...