Biofilm-producing ability and efficiency of sanitizing agents against Prototheca zopfii isolates from bovine subclinical mastitis

The objectives of the present study were to evaluate (1) the capacity of the microalga Prototheca zopfii isolated from subclinical bovine mastitis cases to form biofilms; and (2) the resistance of these isolates to sanitizing agents. Ten isolates of P. zopfii from cows with subclinical mastitis (somatic cell count >200 × 103 cells/mL), distributed in 5 dairy farms, were evaluated for their capacity to form biofilms in polystyrene microplate assays and stainless steel coupons, at 25°C and 37°C ± 1°C. Prototheca zopfii were isolated from milk samples via microbiological culture and analyzed by 18S rRNA gene sequencing. Biofilm formation on the coupons was observed by scanning electron microscopy. The resistance to sanitizing agents was assessed using the biofilm-forming P. zopfii isolates in stainless steel coupon assays, which were subjected to 3 sanitizers: peracetic acid, sodium hypochlorite, and iodine solution. To evaluate resistance to the sanitizers, the minimum inhibitory concentration (MIC) technique was performed using decreasing concentrations of the sanitizing agents (20, 10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078, 0.039, and 0.019 g/L). After inoculating the isolates, all concentrations were evaluated at 3 distinct incubation periods (24, 48, and 72 h) to assess the effect of incubation time on the MIC. Using the polystyrene microplate assays, 1 isolate showed weak biofilm production, 5 moderate, and 4 strong, when incubated at 25°C ± 1. For isolates incubated at 37°C ± 1, 6 showed weak biofilm production and 4 moderate. All P. zopfii isolates (n = 10) had the capacity to form biofilms on stainless steel coupons. The longer the incubation period of the P. zopfii isolates at different dilutions, the greater the concentrations of sanitizer needed to prevent growth of the microalgae under the tested conditions. We detected a significant effect of sanitizer and time of incubation (24, 48, and 72 h) on MIC values against P. zopfii isolates. The isolates were sensitive in vitro to peracetic acid (MIC90 ≥0.019 g/L), sodium hypochlorite (MIC90 ≥0.312 g/L), and iodine solution (MIC90 ≥0.625 g/L), after 24 h of incubation (where MIC90 = concentration needed to inhibit 90% of isolates). Of the tested sanitizers, peracetic acid had the greatest efficiency against P. zopfii. We conclude that P. zopfii isolates are capable of biofilm production, which may contribute to their persistence in a milking and dairy environment.