title: Diagnostic accuracy of a standardized scheme for identification of Streptococcus uberis in quarter milk samples: A comparison between conventional bacteriological examination, modified Rambach agar medium culturing, and 16S rRNA gene sequencing.
abstract: Bacteriological examination of milk samples is a prerequisite for pathogen-specific therapy and aids in limiting antimicrobial resistance. The aims of this study were to establish a standardized scheme for reliable Streptococcus uberis identification in routine diagnosis and to evaluate the accuracy of conventional tests and growing patterns of Strep. uberis on a selective medium (modified Rambach agar medium, MRAM) using 16S rRNA gene sequencing analysis as a reference method. We obtained isolates of presumptive Strep. uberis (n = 336) from quarter milk samples of dairy cows with intramammary infections and classified the isolates into 2 clusters using biochemical characterization. In cluster 1 (n = 280), cocci grew as non-hemolytic colonies, hydrolyzing esculin, carrying no Lancefield antigen (A/B/C/D/G) or Christie Atkins Munch-Petersen factor, and their growth was inhibited on an Enterococcus agar. Production of β-d-galactosidase on MRAM was shown by 257 of the cluster 1 isolates (91.79%), and 16S rRNA gene sequencing verified 271 (96.79%) of the isolates to be Strep. uberis. In 264 isolates (94.29%), MRAM agreed with the sequencing results. In cluster 2 (n = 56), isolates showed different characteristics: 37 (66.07%) were β-d-galactosidase-positive, and based on 16S sequencing results, 36 (64.29%) were identified correctly as Strep. uberis using biochemical methods. Identification success in this group differed significantly between routine diagnosis and MRAM application: MRAM agreed with sequencing results in 47 isolates (83.93%). To identify Strep. uberis and differentiate it from other lactic acid bacteria in routine diagnosis, we suggest using catalase reaction, hemolysis, esculin hydrolysis, and growth on enterococci agar. Isolates that show a typical biochemical profile can be identified satisfactorily with these tests. For Strep. uberis isolates with divergent patterns, application of MRAM as a follow-up test increased the diagnostic accuracy to 94.64%.
authors.affiliation: University Clinic for Ruminants, Milk Technology and Food Science, Department of Farm Animal and Public Health in Veterinary Medicine, University of Veterinary Medicine, 1020 Vienna, Austria. Electronic address: Regina.Wald@vetmeduni.ac.at.
authors.forename: Regina
authors.initials: R
authors.lastname: Wald
authors.affiliation: University Clinic for Ruminants, Milk Technology and Food Science, Department of Farm Animal and Public Health in Veterinary Medicine, University of Veterinary Medicine, 1020 Vienna, Austria.
authors.forename: Martina
authors.initials: M
authors.lastname: Baumgartner
authors.affiliation: University Clinic for Ruminants, Milk Technology and Food Science, Department of Farm Animal and Public Health in Veterinary Medicine, University of Veterinary Medicine, 1020 Vienna, Austria.
authors.forename: Verena
authors.initials: V
authors.lastname: Urbantke
authors.affiliation: Institute for Milk Hygiene, Milk Technology and Food Science, Department of Farm Animal and Public Health in Veterinary Medicine, University of Veterinary Medicine, 1020 Vienna, Austria.
authors.forename: Beatrix
authors.initials: B
authors.lastname: Stessl
authors.affiliation: University Clinic for Ruminants, Milk Technology and Food Science, Department of Farm Animal and Public Health in Veterinary Medicine, University of Veterinary Medicine, 1020 Vienna, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Wittek
mesh_terms: D000362:Agar; D000818:Animals; D002417:Cattle; D005260:Female; D008414:Mastitis, Bovine / Q000382:microbiology*; D008892:Milk / Q000382:microbiology; D012336:RNA, Ribosomal, 16S; D013290:Streptococcal Infections / Q000662:veterinary; D013291:Streptococcus / Q000302:isolation & purification*
chemical_list: D012336:RNA, Ribosomal, 16S; D000362:Agar
keywords: Streptococcus uberis; mastitis; modified Rambach agar medium