Identification of Mycobacterium smegmatis in Bovine Mastitis

Abstract

Background: Rapidly growing mycobacteria (RGM) are ubiquitous in the environment, can be isolated from soil and wa­ter, and demonstrate visible growth on culture media within seven days. Mycobacterium smegmatis is an acid-alcohol fast bacterium, which belong to RGM group. The diagnosis of M. smegmatis infections may be quite difficult by conventional methods; therefore, biochemistry associated to nucleic acid-based approaches provided fast and accurate identification. Although this specie may be associated to animals and humans infections, there is few cases description. Nontuberculous mycobacterial bovine mastitis is uncommon, and bovine mastitis by M. smegmatis has been reported but non-confirmed case once in the past. This paper reports M. smegmatis recovered from a cattle with relapsing pyogranulomatous mastitis.Case: Milk samples from an adult Holstein cow showing relapsing pyogranulomatous mastitis history and by pronounced glandular hardening were cultivated and analyzed accordingly to standard milk cultivation protocols. The animal had been subject to several intramammary and parenteral antibiotic therapies protocols without adequate response. After 48 h incubation, a slow and sparse growth of slightly pigmented, shiny and smooth colonies was observed on the blood agar plate. The bacterium isolated was named as strain 55/08. The morphological and biochemical profile were tested, and the ability of the isolate to grow at Lowenstein-Jensen slants was confirmed. The isolated have showed positive reaction to catalase, glucose, sucrose, mannitol and nitrate. The pigment formation was observed for 14 days incubation, and the colonies produce pigment after prolonged time. Gram and Ziehl-Neelsen staining revealed poorly pigmented, irregular, slender Gram-positive and acid fast rods. The staining and biochemical profile showed closed isolated relationship to M. smegmatis. A discriminatory identification based in the 16S rRNA gene sequence analysis was performed. The total DNA from the strain 55/08 was extracted and the partial 16S rRNA sequence was amplified, using prokaryotic universal primer pairs and the extract DNA as template, by PCR assay following the purification and sequencing of the amplicons. A total of 1,443 nucleotides form consensus sequence were alignment to M. smegmatis and other mycobacteria 16S rRNA avail­able sequences. The sequence analysis confirmed the M. smegmatis identification as etiological agent of bovine relapsing pyogranulomatous mastitis. M. smegmatis strain 55/08 partial 16S rRNA gene sequence was submitted to GenBank. The phylogenetic relationship of the strain 55/08 with other mycobacteria was performed in order to confirm the identification of the isolate as M. smegmatis. Discussion: Nontuberculous mycobacteria are uncommon causes of bovine mastitis. Some old reports have described M. smegmatis as etiological agent of mastitis, but without definitive diagnostic. M. smegmatis mammary quarter introduction may be related to the repeated intramammary treatment protocols, because this mycobacteria is related to environmental infections. The relapsing pyogranulomatous mastitis infection could be associated to other bacteria species. However, the phenotypic and molecular characterization which was performed demonstrated the accurate identification of the isolated as M. smegmatis. Milk contaminated by M. smegmatis may be a potential infection source for human and other animal species. This report reinforces the need to optimize quality programs and laboratorial diagnosis to further the accurate microorganism identification in milk samples.Keywords: RGM mycobacteria, relapsing mastitis, M. smegmatis, molecular identification.