Staphylococcus (S.) aureus is considered as a major mastitis pathogen, with considerable epidemiological information on such infections while the epidemiology of coagulase-negative staphylococci (CNS) is more controversial. The aim of this study was to use matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology for identification of staphylococci isolated from bovine milk at species level and to characterize them in reference to presentation, somatic cell count (SCC), bacterial shedding (cfu) and antimicrobial resistance patterns. A total of 200 staphylococcal isolates (S. aureus n = 100; CNS n = 100) originating from aseptically collected quarter milk samples from different quarters of dairy cows were included in the study. They originated from cases of clinical (CM) and subclinical mastitis (SCM) or were isolated from milk with SCC ≤ 100,000 cells/mL in pure culture. We found staphylococci predominantly in cases of SCM (n = 120). In low-SCC cows, 12 S. aureus and 32 CNS isolates were detected. Eighteen percent of each were associated with CM. Eleven CNS species were identified, S. chromogenes (n = 26) and S. xylosus (n = 40) predominated. CNS, particularly those in low-SCC cows, showed higher MIC90 (minimal inhibitory concentration) values for penicillin, ampicillin, cefoperazone, pirlimycin and marbofloxacin. Based on the present results, a careful interpretation of laboratory results is recommended to avoid antimicrobial therapy of staphylococci without clinical relevance and to ensure prudent use of antimicrobials.

The present study describes an outbreak of Pseudomonas (P.) aeruginosa mastitis in a 20-cow dairy herd where throughout genotyping of isolates reusable udder towels were identified as the source of infection. Sampling of cows during three herd surveys and bacteriological culturing showed that P. aeruginosa was isolated from nine cows with a total of 13 infected quarters. Mastitis occurred as mild clinical or subclinical infection. P. aeruginosa was additionally isolated from a teat disinfectant solution, containing N-(3-aminopropyl)-N-dodécylpropane-1,3-diamine 1 as active component, and microfiber towels used for pre-milking teat preparation. Disc diffusion antimicrobial resistance testing revealed that all isolates were susceptible to piperacillin, piperacillin-tazobactam, ceftazidime, cefepime, aztreonam, imipenem, meropenem, tobramycin, amikacin, and ciprofloxacin. Thirty-two isolates of milk samples and 22 randomly selected isolates of one udder towel and of the teat disinfectant solution were confirmed as P. aeruginosa with matrix-assisted laser desorption, ionization time-of-flight mass spectrometry (MALDI Tof MS). Isolates were further characterized with rep-PCR and randomly amplified polymorphic DNA (RAPD) as well as with multiple locus variable-number tandem repeat analysis (MLVA). Results obtained in this study suggested that one single strain was responsible for the whole outbreak. The transmission occurred throughout a contaminated teat cleaning solution as a source of infection. The farmer was advised to change udder-preparing routine and to cull infected cows.

Streptococcus uberis, a major mastitis pathogen associated with intramammary infections (IMI), can be found ubiquitously in the cow's environment. Although Strep. uberis is reported to be susceptible to most antimicrobials, in practice poor responses to treatment and recurrent mastitis are observed. This can be explained by reinfection or by persistence of strains. We hypothesized that among a heterogeneous group of Strep. uberis mastitis isolates, some predominant host-adapted clones might be recurrently isolated from IMI. Therefore, the aim of this pilot study was to determine the Strep. uberis genotype variety found among small-scale dairy herds (127 Austrian dairy farms) and compare this with a large-scale herd (a Slovakian dairy farm). We determined the occurrence and strain diversity of Strep. uberis (n = 309) isolates using molecular analysis. Streptococcus uberis isolates from aseptically collected quarter milk samples were genotypically characterized using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. The Strep. uberis strain set covered isolates from 4 Austrian federal areas [Lower Austria (n = 67), Upper Austria (n = 8), Salzburg (n = 51), and Styria (n = 1)] and the Bratislava Region of Slovakia (n = 1). The PFGE analysis resulted in 187 Smal profiles with 151 unique profiles. Simpson's index of diversity was 0.988. Individual cows (n = 17) harbored up to 3 different PFGE types in the udder. Dairy cows shared distinct PFGE types within a farm. Seven PFGE types were widely distributed among Austrian dairy farms. In the Slovakian farm, 10 predominant PFGE types were recurrently isolated from the same quarters; these genotypes were assigned as persisters. We identified novel sequence types (ST) using multilocus sequence typing related to the global clonal complexes ST5 and ST143. We concluded that Strep. uberis IMI are caused by strains with a wide heterogeneity of PFGE types. This large number of unique subtypes indicates a high diversity of Strep. uberis in the environment. In the large herd, molecular epidemiological results revealed that specific strains might be involved in contagious transmission events and potentially lead to persistence.

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%.

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