Abstract
Staphylococcus aureus is one of the major pathogens causing bovine intramammary infections (IMIs) and mastitis. Mastitis is the primary cause for the use of antibiotics in dairy farms but therapeutic failure is often observed. One of the reasons for the lack of effectiveness of antibiotic therapy despite the observed susceptibility of bacterial isolates in vitro are bacterial biofilms. In this study, we used chitosan of well-defined molecular weight (0.4–0.6, 1.3, 2.6 and 4.0 kDa) and investigated their antibiofilm and antibacterial activities in in vitro and in vivo models related to S. aureus IMIs. A chitosan of at least 6 units of glucosamine was necessary for maximum antibacterial activity. The 2.6 and 4.0 kDa forms were able to prevent biofilm production by the biofilm hyperproducer strain S. aureus 2117 and a bovine MRSA (methicillin-resistant S. aureus). The intramammary administration of the 2.6 kDa chitosan showed no adverse effects in mice or in cows, as opposed to the slight inflammatory effect observed in mammary glands with the 4.0 kDa derivative. The 2.6 kDa chitosan killed bacteria embedded in pre-established biofilms in a dose-dependent manner with a >3 log10 reduction in CFU at 4 mg/ml. Also, the 2.6 kDa chitosan could prevent the persistence of the internalized MRSA into the mammary epithelial cell line MAC-T. An in vitro checkerboard assay showed that the 2.6 kDa chitosan produced a synergy with the macrolide class of antibiotics (e.g., tilmicosin) and reduced the MIC of both molecules by 2–8 times. Finally, the intramammary administration of the 2.6 kDa chitosan alone (P<0.01) or in combination with tilmicosin (P<0.0001) reduced the colonization of mammary glands in a murine IMI model. Our results suggest that the use of chitosan alone or in combination with a low dose of a macrolide could help reduce antibiotic use in dairy farms.
Highlights
Mastitis is a major disease for dairy cattle
An intramammary infection (IMI) often occurs when bacteria invade the mammary gland through the teat canal causing an inflammatory response, which translates in either subclinical or clinical mastitis [2, 3]
We show here that the combination of chitosan with antibiotics could be useful to enhance the killing of bacteria within preformed biofilms and in a murine model of S. aureus IMI
Summary
Mastitis is a major disease for dairy cattle. Mastitis decreases milk quality and production and reduces profitability because of the cost of treatment and discarded milk due to the risk of antibiotic residues in milk [1]. An intramammary infection (IMI) often occurs when bacteria invade the mammary gland through the teat canal causing an inflammatory response, which translates in either subclinical (visual symptoms are absent) or clinical mastitis (local and/or systemic symptoms are visible) [2, 3]. S. aureus causes subclinical and persistent IMIs that often remain undetected and poorly respond to antibiotic treatment [5, 6]. S. aureus is frequently isolated from clinical cases [5]. The existence of such strains represents a reservoir that causes the recurrence of S. aureus infections in herds [7]. Prevalence values of 3.4 to 8.2% for quarter infections have been observed in several studies where milk samples have been taken randomly from quarters of all cows in herds [8, 9, 10]
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