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

Abstract

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>200×10(3) 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.

In vitro photoinactivation of bovine mastitis related pathogens

“…The alga P. zopfii (wild-type strain) was used in this experiment because of its clinical importance, not only in Brazil but worldwide. The occurrence of mastitis caused by the alga has been described in several countries with no responsive treatment [25,26].…”

Mastitis associated with Prototheca zopfii - an emerging health and economic problem on dairy farms

“…Namely, if only one quarter is infected, dilution with milk from healthy, uninfected quarters will decrease the pathogen count in composite milk sample below the limit of detection (24). Intermittent shedding the organism by infected cows calls for regular repeated testing (10,24). Advanced stages of the disease are characterised by highly elevated numbers of algae in milk of infected cows, and laboratory diagnosis is quite simple to establish.…”

“…1). The reproduction cycle takes place at higher temperatures, with the lowest temperature limit estimated at about 25C (10,26).…”

“…Throughout the past few years, substantial increase in incidence of P. zopfii mastitis has been reported all over the world (3,9,10,16,26,29,31). Since 2006, P. zopfii has been isolated from milk on four dairy farms in the regions of Vojvodina and Central Serbia; however, its much wider prevalence is suspected in some regions of Serbia even though it has not yet been isolated.…”

“…Identification of relevant risk factors is of major importance in the control of the disease; however, causal relationships between various factors in each individual case are still difficult to establish. Poor hygiene practices, excessive antibiotic use, and inadequate milking hygiene are most commonly considered the major risk factors for mastitis development (10,13,17,24,26,31).…”