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The present research study investigated the susceptibility of common mastitis pathogens—obtained from clinical mastitis cases on 58 Northern German dairy farms—to routinely used antimicrobials. The broth microdilution method was used for detecting the Minimal Inhibitory Concentration (MIC) of Streptococcus agalactiae (n = 51), Streptococcus dysgalactiae (n = 54), Streptococcus uberis (n = 50), Staphylococcus aureus (n = 85), non-aureus staphylococci (n = 88), Escherichia coli (n = 54) and Klebsiella species (n = 52). Streptococci and staphylococci were tested against cefquinome, cefoperazone, cephapirin, penicillin, oxacillin, cloxacillin, amoxicillin/clavulanic acid and cefalexin/kanamycin. Besides cefquinome and amoxicillin/clavulanic acid, Gram-negative pathogens were examined for their susceptibility to marbofloxacin and sulfamethoxazole/trimethoprim. The examined S. dysgalactiae isolates exhibited the comparatively lowest MICs. S. uberis and S. agalactiae were inhibited at higher amoxicillin/clavulanic acid and cephapirin concentration levels, whereas S. uberis isolates additionally exhibited elevated cefquinome MICs. Most Gram-positive mastitis pathogens were inhibited at higher cloxacillin than oxacillin concentrations. The MICs of Gram-negative pathogens were higher than previously reported, whereby 7.4%, 5.6% and 11.1% of E. coli isolates had MICs above the highest concentrations tested for cefquinome, marbofloxacin and sulfamethoxazole/trimethoprim, respectively. Individual isolates showed MICs at comparatively higher concentrations, leading to the hypothesis that a certain amount of mastitis pathogens on German dairy farms might be resistant to frequently used antimicrobials.
The lytic efficacy of bacteriophages against Staphylococcus aureus isolates from bovine milk was investigated in vitro, regarding possible applications in the therapy of udder inflammation caused by bacterial infections (mastitis). The host range of sequenced, lytic bacteriophages was determined against a collection of 92 Staphylococcus (S.) aureus isolates. The isolates originated from quarter foremilk samples of clinical and subclinical mastitis cases. A spot test and a subsequent plaque assay were used to determine the phage host range. According to their host range, propagation and storage properties, three phages, STA1.ST29, EB1.ST11, and EB1.ST27, were selected for preparing a bacteriophage mixture (1:1:1), which was examined for its lytic activity against S. aureus in pasteurized and raw milk. It was found that almost two thirds of the isolates could be lysed by at least one of the tested phages. The bacteriophage mixture was able to reduce the S. aureus germ density in pasteurized milk and its reduction ability was maintained in raw milk, with only a moderate decrease compared to the results in pasteurized milk. The significant reduction ability of the phage mixture in raw milk promotes further in vivo investigation.
The antimicrobial activity of a phagemixture and a lactic acid bacteriumagainst Staphylococcus aureus isolates from bovine origin was investigated in vitro with regard to possible applications in the therapy of udder inflammation (mastitis) caused by bacterial infections. The S. aureus isolates used for inoculation derived from quarter foremilk samples of mastitis cases. For the examination of the antimicrobial activity, the reduction of the S. aureus germ density was determined [log10 cfu/mL]. The phage mixture consisted of the three obligatory lytic and S. aureus-specific phages STA1.ST29, EB1.ST11 and EB1.ST27 (1:1:1). The selected Lactobacillus plantarum strain with proven antimicrobial properties and the phage mixture were tested against S. aureus in milk, both alone and in combination. The application of the lactic acid bacterium showed only a low reduction ability for a 24 h incubation period. The bacteriophage mixture as well as its combination with the lactic acid bacterium showed high antimicrobial activity against S. aureus for a 24 h incubation period at 37 C, with only the phage mixture showing significance.