Agreement of conventional microbiological and molecular identification of streptococci isolated from bovine milk

Pathogenic streptococci are implicated in clinical and subclinical mastitis. Most laboratories identify streptococci on the basis of microbiological examination, but molecular diagnostic methods have become the gold standard of mastitis diagnosis in the last few years. Therefore, this study aims to determine the agreement of microbiological and molecular identification of streptococci isolates from bovine milk. Milk samples were taken before the evening milking into sterile tubes. Samples were examined bacteriologically by inoculation on aesculin blood agar. Identification of grown colonies was carried out using internationally accepted methodology. Sequencing of the 16S rRNA gene was the reference method used to confirm Streptococcus sp. in all bacterial isolates. In the study, 54 strains of bacteria isolated from milk samples from the udder quarters of dairy cows with subclinical mastitis were examined using molecular methods. By conventional microbiological examination, the strains were identified to the species (Strep. agalactiae, Strep. dysgalactiae and Strep. uberis) or the genus level (Streptococcus spp.) without final identification of the species. On the basis of 16S rRNA analysis, 47 out of 54 examined streptococcal strains were found to belong to the genus Streptococcus sp. Among the streptococci identified, 6 isolates belonged to Strep. agalactiae, ¸8 isolates to Strep. dysgalactiae, 2 isolates to Strep. canis and 31 isolates belonged to Strep. uberis. Among the seven remaining isolates, three were identified as Enterococcus faecalis and four as Lactococcus lactis. Agreement between the identification procedures used was fair, with a Kappa index of 0.2181 (SE=0.0612; Z=3.56; p=0.0002).
Patogeni streptokoki povezuju se sa subkliničkim i kliničkim mastitisima. Većina laboratorija identifikaciju streptokoka temelji na mikrobiološkoj pretrazi, ali posljednjih godina molekularne metode postaju zlatni standard za dijagnostiku mastitisa. Cilj ovog istraživanja bio je odrediti podudarnost rezultata klasične mikrobiološke pretrage i identifikacije streptokoka molekularnim metodama. Uzorci mlijeka uzeti su u sterilne epruvete prije večernje mužnje. Mikrobiološka pretraga provedena je u skladu s opće prihvaćenim međunarodnim preporukama. Primarno izdvajanje i identifikacija streptokoka provedeni su korištenjem podloge eskulin krvni agar. Molekularna identifikacija vrste Streptococcus spp. učinjena je na osnovu 16S rRNA sekvencioniranja. U istraživanju su molekularnim tehnikama pretražena 54 soja bakterija izdvojena iz uzoraka mlijeka pojedinačnih četvrti vimena krava sa subkliničkim mastitisom. Klasičnom mikrobiološkom pretragom izdvojeni sojevi identificirani su do vrste (S. agalactiae, S. dysgalactiae i S. uberis) ili do roda (Streptococcus spp.) bez konačne identifikacije vrste. Metodom identifikacije na osnovi umnažanja gena 16S rRNA, od 54 pretražena bakterijska soja, za njih 47 je potvrđeno da pripadaju rodu Streptococcus spp. i to 6 Streptococcus agalactiae, 8 Streptococcus dysgalactiae, 2 Streptococcus canis i 31 Streptococcus uberis dok su 3 soja pripadnici vrste Enterococcus faecalis, a 4 soja pripadali su vrsti Lactococcus lactis. Podudarnost primijenjenih metoda za identifikaciju streptokoka je blaga, s Kappa indeksom od 0,2181 (SE=0,0612; Z=3,56; p=0,0002).