Staphylococcus aureus causes intramammary infections (IMIs), which are refractory to antibiotic treatment and frequently result in chronic mastitis. IMIs are the leading cause of conventional antibiotic use in dairy farms. Phage therapy represents an alternative to antibiotics to help better manage mastitis in cows, reducing the global spread of resistance. A mouse mastitis model of S. aureus IMI was used to study the efficacy of a new cocktail of five lytic S. aureus-specific phages (StaphLyse™), administered either via the intramammary (IMAM) route or intravenously (IV). The StaphLyse™ phage cocktail was stable in milk for up to one day at 37 °C and up to one week at 4 °C. The phage cocktail was bactericidal in vitro against S. aureus in a dose-dependent manner. A single IMAM injection of this cocktail given 8 h after infection reduced the bacterial load in the mammary glands of lactating mice infected with S. aureus, and as expected, a two-dose regimen was more effective. Prophylactic use (4 h pre-challenge) of the phage cocktail was also effective, reducing S. aureus levels by 4 log10 CFU per gram of mammary gland. These results suggest that phage therapy may be a viable alternative to traditional antibiotics for the control of S. aureus IMIs.

Staphylococcus aureus is one of the main pathogens leading to both clinical and subclinical bovine mastitis in dairy cattle. Prediction of disease evolution based on the characteristics of Staph. aureus isolates that cause intramammary infections and understanding the host-pathogen interactions may improve management of mastitis in dairy herds. For this study, several strains were selected from each of the 6 major Canadian spa types associated with mastitis (t267, t359, t529, t605, t2445, and t13401). Adherence to host cells and intracellular persistence of these strains were studied using a bovine mammary gland epithelial cell line (MAC-T). Additionally, relative virulence and host response (cytokines production) were also studied in vivo using a mouse model of mastitis. Whole-genome sequencing was performed on all strains and associations between clonal complex, sequence type, and presence of certain virulence factors were also investigated. Results show that spa type t2445 was correlated with persistence in MAC-T cells. Strains from spa t359 and t529 showed better ability to colonize mouse mammary glands. The exception was strain sa3154 (spa t529), which showed less colonization of glands compared with other t359 and t529 strains but possessed the highest number of superantigen genes including tst. All strains possessed hemolysins, but spa types t529 and t2445 showed the largest diameter of β-hemolysis on blood agar plates. Although several spa types possessed 2 or 3 serine-aspartate rich proteins (Sdr) believed to be involved in many pathogenic processes, most t529 strains expressed only an allelic variant of sdrE. The spa types t605 (positive for the biofilm associated protein gene; bap+) and t13401 (bap-), that produced the largest amounts of biofilm in vitro, were the least virulent in vivo. Finally, strains from spa type t529 (ST151) elicited a cytokine expression profile (TNF-α, IL-1β and IL-12) that suggests a potential for severe inflammation. This study suggests that determination of the spa type may help predict the severity of the disease and the ability of the immune system to eliminate intramammary infections caused by Staph. aureus.

Staphylococcus aureus intramammary infections often leads to clinical and subclinical mastitis in dairy cattle. Prediction of disease evolution and treatment efficacy based on the characteristics of disease-causing strains of S. aureus would significantly improve management of dairy herds. To study the impact of biofilm production and the influence of genetic lineage, we selected S. aureus isolates from the most prevalent Canadian spa types associated with bovine mastitis. Antimicrobial susceptibility in planktonic growth and for bacteria embedded in biofilm was compared. PCR was used to detect the bap gene responsible for atypical biofilm formation. All Canadian spa types from dairy cattle were susceptible to the 8 antimicrobial agents tested. Only strain sa3493 from spa type t267 showed a resistance to pirlimycin. However, bacteria producing larger amounts of biofilms better survived the bactericidal action of antimicrobial agents even when exposed to concentrations 64 folds higher than the minimal inhibitory concentration determined for planktonic cultures. Pirlimycin was more effective on bacteria producing low to moderate levels of biofilm compared with vancomycin or ceftiofur. Antimicrobial agents did not affect the viability of spa types t13401 and t605 that were high biofilm producers. While both these spa types produced high amounts of biofilm, only t605 possessed the bap gene. We also found a close relationship between DIM at sampling and the presence of spa type t605 isolates. These results suggest that detection of S. aureus spa type may help predict the effectiveness of antimicrobial therapy and that some spa types are more likely to be retrieved toward the end of the lactation.

During the transition period, the incidence of diseases increases due to a negative energy balance that affects metabolic and immune status. Limiting milk production at the beginning of lactation by milking once a day or by incompletely milking twice a day improves the metabolic and immune status of cows. Previous studies have shown that milk production is highly responsive to additional MP. Therefore, reducing MP intake could provide an alternative strategy. This study evaluated whether reducing the MP supply during the transition period could limit milk yield temporarily and thereby improved the cows' metabolic and immune status. A first group of cows (n = 11, 80% MP) was fed a diet formulated to supply 80% of their MP needs during the treatment period (from 10 d before expected calving to 14 DMI) while they received a diet formulated to supply 100% of their MP needs before treatment (adaptation, d28 to d11 before expected calving) and after (follow-up, 15 to 28 DMI) the treatment period. A second group of cows (n = 12, 100% MP) was fed a diet formulated to supply 100% of their MP needs during the 3 periods . Blood samples were taken on average at d 26 ± 3.6; d 19 ± 3.7; d 12 ± 3.6; d 9 ± 3.6 before calving and at 2, 5, 7, 10, 14, 21, and 28 DMI to assess the metabolic and immune status. Milk samples were collected at the same frequency to assess milk composition. The milk production of the 80% MP cows was numerically smaller than that of the 100% MP cows (2.4 kg/d) from d 0 to 14, but not after the MP restriction ended. The 80% MP cows had numerically more plasma BHB during the treatment period than the 100% MP cows (694.6 vs 574.0 ± 389.8 µM, respectively). The 80% MP cows had significantly less glucose on d 7 and d 10 than the 100% MP cows (3.14 vs 3.44 ± 0.06 and 3.15 vs 3.42 ± 0.07 mM, respectively). Throughout the 3 experimental periods, no differences were observed for nonesterified fatty acids or for some of the markers of the mobilization of muscle reserves. However, the concentration in 3-methyl-His was numerically greater for the 100% MP cows during treatment and was greater during the follow-up period. The PMN's oxidative burst of the 80% MP cows was numerically greater during treatment and on d 21. The treatment did not alter the proliferation of peripheral blood mononuclear cells or PMN phagocytosis capacity. In conclusion, a 20% reduction in MP supply during the transition period did decrease milk production, but this decrease was not sufficient to improve energy balance and to reduce metabolic and immune disturbances.

Staphylococcus aureus is one of the major pathogens causing bovine mastitis, and antibiotic treatment is most often inefficient due to its virulence and antibiotic-resistance attributes. The development of new antibiotics for veterinary use should account for the One Health concept, in which humans, animals, and environmental wellbeing are all interconnected. S. aureus can infect cattle and humans alike and antibiotic resistance can impact both if the same classes of antibiotics are used. New effective antibiotic classes against S. aureus are thus needed in dairy farms. We previously described PC1 as a novel antibiotic, which binds the S. aureus guanine riboswitch and interrupts transcription of essential GMP synthesis genes. However, chemical instability of PC1 hindered its development, evaluation, and commercialization. Novel PC1 analogs with improved stability have now been rationally designed and synthesized, and their in vitro and in vivo activities have been evaluated. One of these novel compounds, PC206, remains stable in solution and demonstrates specific narrow-spectrum activity against S. aureus. It is active against biofilm-embedded S. aureus, its cytotoxicity profile is adequate, and in vivo tests in mice and cows show that it is effective and well tolerated. PC206 and structural analogs represent a promising new antibiotic class to treat S. aureus-induced bovine mastitis.