Suscetibilidade in vitro de Prototheca spp., isoladas de leite bovino, aos óleos essenciais, aos peptídeos e fármacos antimicrobianos

Abstract

Alchlorophilic algae of the genus Prototheca are disease-causing agents in humans and animals. The species Prototheca zopfii (genotype 2), with recent reclassification suggested for P. bovis, is cited by several authors as the most pathogenic species for animals, mainly in the form of bovine mastitis. Infections are difficult to control due to the high resistance capacity of the agent, causing damage to the producer and risk to human health. In the search for alternatives to control animal prototecosis, in this study several drugs of different natures were tested. In this context, the objective of this work was to determine the minimum inhibitory concentration in vitro of antibacterial and antifungal, essential oils, monoterpenes, terpenes and antimicrobial peptides, against P. zopfii (genotype 2) isolates. The testing methodology was broth microdilution, according to CLSI document M27-A3. To carry out the tests, Prototheca spp. isolates were obtained from milk samples, from rural properties in the northwest region, in the State of Rio Grande do Sul. The agent was identified by colony morphology observation technique, in culture and sporangia micromorphology, with visualization of the presence of endosporulation. For species identification, the genotype-specific PCR 18S rDNA assay was used. Thirty-two isolates of Prototheca spp. were found, based on genetic analysis, all isolates were characterized as P. zopfii (genotype 2) being a 450 bp fragment from the internal amplification control and specific amplicon of genotype 2 (165 bp), detectable for all of them. In the tests, resistance to all antibacterial drugs and essential oils, monoterpenes and terpenes with almost absent inhibitory activity, were observed. Among antifungals, amphotericin B (MIC 0,5 to 32 μg/mL; geometric mean: 4,0 μg/mL) and terbinafine (MIC 0,5 to 128 μg/mL; geometric mean: 3,1 μg/mL) were the ones that stood out as well as ketoconazole and itraconazole. Antimicrobial peptides are a new class of drugs with antimicrobial activity, where the highlights were for pexiganan with MIC range from 5,0 to 10 μg/mL, in addition to producing algaecide effect with 5,0 to 20 μg/mL. The MIC produced by h-Lf1-11 was 10 to 80 μg/mL, whereas LL-37 had MIC range from 20 to 80 μg/mL and 40 to 160 μg/mL was the MIC of cecropin B. In general, the results showed the presence of the agent in one of the most important milk-producing regions in Brazil, as well as new drug options against P. zopfii (genotype 2).