Susceptibility of Prototheca zopfii strains isolated from cows with mastitis to chlorhexidine and iodine

* Correspondence: [email protected] Protothecosis is an infection reported in humans and in many other animal species caused by achlorophyllous algae of the genus Prototheca. These algae are primitive unicellular organisms considered to be chlorophylllacking mutants that have adopted a heterotrophic mode of nutrition. These organisms are widely disseminated in different environments of high humidity (1,2). The predominant form of protothecosis in cattle has been bovine mastitis, most commonly induced by Prototheca zopfii. Although bovine mastitis associated with Prototheca is considered a rare pathology, its prevalence is increasing. This disease is responsible for heavy economic losses due to dramatic loss in milk yield and its quality, as well as culling of the infected cows. The pathogens are isolated from a variety of environmental sources including drinking water, sewage, liquid manure, feces from both healthy animals and those infected by bovine protothecal mastitis, barn walls and floors, as well as milking machine liners and milking cups (2–4). Chlorhexidine represents one of the most important products in dairy practice, used as a postdipping antiseptic. This antimicrobial shows a high spectrum of action. Moreover, 2% chlorhexidine solution is recommended to promote the definitive drying of the mammary gland (5). Moreover, iodine is recognized as an efficient antiseptic. Iodine has high cell penetrating power, causing protein precipitation and oxidation of essential enzymes. In dairy farms, iodine is generally indicated in preand postdipping for the control of bovine mastitis (6). The aim of the present study was to evaluate the effect of chlorhexidine and iodine on P. zopfii strains isolated from bovine milk. For this study, 15 P. zopfii isolates previously obtained from clinical and subclinical bovine mastitis were revived by subculture on Sabouraud 4% dextrose agar (bioMérieux, Poland) and incubated for 48 to 72 h at 37 °C. Identification was performed using routine culture, macroand microscopic morphological characterization, and API 20CAUX (bioMérieux, Poland) methods (7–10). The test based on the susceptibility of each species of algae to clotrimazole according to Casal and Gutierrez (11) was also used. This test was used to distinguish between P. zopfii and P. wickerhamii. The in vitro susceptibility tests to chlorhexidine and iodine of the strains of Prototheca zopfii for the determination of their minimal microbicidal concentrations (MMCs) were performed using the tube dilution method in Sabouraud dextrose broth and an evaluation of colony growth after plating in Sabouraud dextrose agar. The studies were carried out under the strict methodological protocol reported by Melville et al. (5). Based upon morphologic features, resistance to clotrimazole, and the ability to assimilate glucose and glycerol, all of the isolates were identified as P. zopfii. In Abstract: Bovine mastitis caused by Prototheca spp. can assume high significance because of economic losses and the potential risk to public health. The aim of our study was to evaluate the effect of chlorhexidine and iodine on Prototheca zopfii strains isolated from bovine milk. For this study, 15 Prototheca zopfii isolates previously obtained from cows with clinical and subclinical mastitis in Poland were used. The in vitro susceptibility tests to chlorhexidine and iodine of the strains of Prototheca zopfii for the determination of their minimal microbicidal concentrations (MMCs) were performed using the tube dilution method in Sabouraud dextrose broth and the evaluation of colony growth after plating in Sabouraud dextrose agar. In this study, the MMC was 0.3125%–1.25% for iodine and 0.0048%–0.0195% for chlorhexidine.