Antimicrobial activities of polyhexamethylene biguanide against biofilm-producing Prototheca bovis causing bovine mastitis

Prototheca spp. is a frequent cause of bovine mastitis and is highly resistant to commonly used disinfectants. This study aimed to: (1) evaluate the antimicrobial activity of polyhexamethylene biguanide (PHMB) against mastitis-causing Prototheca spp., and (2) evaluate the biofilm production ability of Prototheca spp. A total of 85 Prototheca bovis and 2 Prototheca blaschkeae isolates from bovine mastitis cases were submitted to biofilm production assays and antimicrobial susceptibility tests against PHMB and disinfectants commonly used in dairy herds (chlorhexidine digluconate, povidone-iodine, sodium dichloroisocyanurate, and sodium hypochlorite). The minimal inhibitory concentration (MIC) and minimal algicidal concentration (MAC) were determined by microdilution assays. We observed that PHMB (MIC₉₀: ≥2 µg/mL and MAC₉₀: ≥4 µg/mL) and chlorhexidine gluconate (MIC₉₀ and MAC₉₀: ≥2 µg/mL) presented the highest antimicrobial activity against P. bovis isolates, followed by sodium dichloroisocyanurate (MIC₉₀ and MAC₉₀: ≥1,400 µg/mL), sodium hypochlorite (MIC₉₀ and MAC₉₀: ≥2,800 µg/mL), and povidone-iodine (MIC₉₀ and MAC₉₀: ≥3,200 µg/mL). Concerning P. blaschkeae isolates, PHMB (MIC and MAC ≥1 µg/mL) and chlorhexidine gluconate (MIC and MAC ≥1 µg/mL) were the disinfectants that presented the lowest concentration values required to inhibit the isolates. Regarding biofilms formation, 63.5% (n = 54/85) of the P. bovis isolates were classified as strong, 28.3% (n = 24/85) moderate, and 8.2% (n = 7/85) weak biofilm producers. In contrast, the P. blaschkeae isolates were classified as weak and moderate biofilm producers. These findings suggest that PHMB has the potential to be used for teat and milking-equipment disinfection for the prevention of mastitis-causing Prototheca spp. in dairy herds.