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The Effect of Iodine Based Products on Unicellular Algae from Genus Prototheca

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The Effect of Iodine Based Products on Unicellular Algae from Genus Prototheca The Effect of Iodine Based Products on Unicellular Algae from Genus Prototheca University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Faculty of Veterinary Medicine, 3-5Sorin Mănăştur RĂPUNTEAN Street, *, 400372, Gheorghe Romania RĂPUNTEAN, Flore CHIRILǍ, Nicodim Iosif FIŢ, George Cosmin NADǍŞ *Corresponding author: [email protected] Bulletin UASVM Veterinary Medicine 72(2) / 2015, Print ISSN 1843-5270; Electronic ISSN 1843-5378 DOI:10.15835/buasvmcn-vm: 11439 Abstract Iodine based products have been and are still used in medicine as disinfectant or antiseptic substances, because of their bactericidal, sporicidal, protocidal and disinfection effect. Algaecide effect has been lessPrototheca studied and represents a strong motivation for this study. This paper is aiming to test the sensitivityPrototheca to zopfii iodine based products of unicellular algae of the genus . Such products might be used for the treatment of diseases involving these pathogens. A total number of twenty-two strains isolated from cows with mastitis or bovine shelters, Protothecawere tested wickerhamii within this study. The algal strains were identified based on morphological characteristics (shape, size, presence of endospores), cultural (liquid and solid media characteristics) and biochemical (fermentation of sugars). ATCC 16529 reference strain was also included in the evaluation. Iodinated products used were represented by: Lugol solution, iodine tincture, betadine, videne and potassium iodide. Determination of the inhibitory effect was measured by diffusion technique in agarose gel and by liquid medium dilution method. For the tincture of iodine and betadine, the inhibitory effect was also appreciated in relation with the time of contact (5, 10, 15, 30 and 60 minutes). Prototheca zopfii By the agar diffusion technique, it was found that inhibition zones varying sizes were correlated with the composition of those products. For the 22 Prototheca tested strains, wickerhamii the size of inhibition areas was within the following values (average diameter): iodine tincture 25.71 mm, betadine 26.4 mm, videne 25.61 mm, Lugol’s solution 11.33 mm and potassium iodide 11.14 mm. For strain inhibition areas had the following values: betadine 26 mm, iodine tincture 24 mm, videne 24 mm, Lugol’s solution 10 mm, potassium iodine 14 mm. In case of different contact times meant to establish the algaecide effect - occurred in the first 5 minutes of contact, for both iodine tincture and betadine. Iodine based products tested showed inhibitory effect on algal strains. The best efficacy was found for betadine and iodine tincture (pharmaceutical/commercial products). The inhibitory effect occurs in the first 5 minutes of contact.Keywords: There was iodine no resistance based products, phenomenon Prototheca, nor unicellularthe presence algae of resistant colonies in the inhibition zones. INTRODUCTION of ethanol and water), Lugol’s solution (iodine Because of its chemical properties, iodine is and potassium iodide in water). Because iodine used in various fields of science and technology, is only slightly soluble in water, the solutions are including medicine. It can be used as the self, prepared in the form of dyes in ethanol. Iodoform, or as I3 anion dissolved in water. Examples of iodclorhidroxichinolina, iodophors, sodium iodide such preparations are: iodine tincture (iodine in and potassium, exercise also bactericidal effects ethanol or iodine and sodium iodide in a mixture by releasing iodine [25]. Iodine can come from Prototheca 307 The Effect of Iodine Based Products on Unicellular Algae from Genus Iodophors containing iodine complexed with a were isolated for several years (2008-2014), surfactant and solubilizing agent, povidone iodine and have been preserved in the collection of is popular (polyvinilpyrolidon and iodine) andet Microbiology Department, Faculty of ProtothecaVeterinary aliodine cadexomer (polysaccharide complex and wickerhamiiMedicine from Cluj-Napoca. The examination iodine) (Cooper, 2004; Bootham, 2009; Sibbald also covered a standardized strain: ., 2011). MATERIALS ATCC 16529.AND METHODS Since the phenomenon of antibiotic resistance Prototheca zopfii has spread and amplified in recent years, alternatives for finding other preparations which ProtothecaWe tested 22 strains isolated exhibit antimicrobial effects have been frequently from milk from cows with morphological mastitis or cattle sheds. approached. Among them preparations extracted strains have been identified based on from plants or based on different chemical several characteristics: (shape, size, elements (Ag, Cu, Au, Zn, Fe, I and others). In the presence of endosporescultural - for which the size and case of iodine and iodine-based preparations there arrangement constitutes an important criterion are mentions that the resistance phenomenon for identification), (growth on glucose is not installed or it is exceptionally uncommon culture medium, liquid and solid, incubated at (Boothman, 2009). 37°C; the colonies were small initially, gradually Iodine and iodine based products were increasing in size,biochemical reaching 3-4 mm, white, matte, processed and used as a disinfectant or antiseptic with slightly lobed edges and grainy/cauliflower- in medicine, especially in the preparation of the like surface) and (fermenting glucose, operative field before surgery, known to have glycerol and fructose, variable at other sugars; bactericidal, sporicidal and virulicid activity. The oxidase and catalase positive reaction; indole and 2 effect is especially microbicideGiardia againstCriptosporium bacteria H S negative). Strains are preserved in soft agar.® and viruses, but less intense against some forms of Iodinated products testing were: Lugol, iodine parasitic cysts such as and Videne(SC Hippocrates® SRL, Bucharest), Betadine [26]. Algaecide effect has been less studied,Prototheca good (EGIS Pharmaceuticals PLC, Budapest Hungary), reason for the research within this study. (Adams, Gorforth Leeds, England) and Unicellular algae of the genus 10% potassium iodide (GL Pharma GmbH). began to be increasingly popular, with implications The work technique was based on the 2013 causing fatal chronic granulomatous lesions CLSIThe vet. inhibitorymethod (26) effect with establishedsome modifications by disc localized in different tissues. Illness are seen more diffusionlisted below. method frequently in cows (as mastitis) in dogs and several other species of warm-blooded and poikilothermic . The tested strains were grown in animals but humans too. It is known that the glucose broth and incubated at 37° C for 48 hours. treatment with antibiotics and chemotherapeutics The prepared suspension was used to flood 9 cm is frequently etineffective, al. animal slaughter or diameter Petri dishes containing glucose agar euthanasia is in some cases the final solutionet al. having a thickness of 3 mm. The suspension was (Buyukmihci , 1975). Contaminated milk is evenly spread using a Drigalski spatula, then the a major risk to public health (Krukowski , excess liquid removed. The plates were placed in 2013). Algae are considered as opportunistic thermostat for 30 minutes with the lid half open. pathogens with a low virulence, but can also After this time, in the agarose gel, wells were cut out indicate the pathogenicity ofet the al. host for variouset using a 4 mm diameter mold, according to a radial alforms of immunosuppression such aspects being pattern. This size, considering the thickness of 3 etfound al in natural (Woolorich , 1994; Wirth mm of the agar layer enables a well with a narrow ., 1999) and experimental infections (Răpuntean diameter, which ensures a good distribution of ., 2001). the iodinated product in the surrounding gel. The ProtothecaThe paper zopfii aimed to test the in vitro wells were filled with 20 µl of the tested medium. effectiveness of iodine based products against The plates were placed in a thermostat at 37°C 22 Prototheca wickerhamiistrains isolated from bovine and examined at 24 and 48 hours. mastitis cow milk or in bovine shelters and ATCC The inhibition areas were appreciated, 16529 strain. The strains measuring their diameterBulletin UASVM (mm). Veterinary To Medicine establish 72 (2) / 2015the 308 et al RĂPUNTEAN algaecide effect, inoculations were performed on streaking’s were performed on solid medium glucose broth. Prototheca (glucose agar), previously distributed in Petri In a different working method, from a thick dishes, and divided into 5 triangular sectors. The suspension, prepared from colonies plates were incubated in a thermostat at 37°C. The developed on the agar, dilutions were performed interpretation was made at 24 and 48 hours, with in saline to densities according to McFarland the finalRESULTS examination AND DISCUSSION after 5 days. standards: 0.5; 1; 2; 3; 4 and 5. Each dilution was The results for disc diffusion method test used to inoculate two Petri dishes (1 ml per plate). After homogenization and removal of excess, the Prototheca . plates were placed in a thermostat for the surface Iodine based products revealed inhibitory effect to dry (30 minutes), then cut wells with a diameter against strains. Inhibition areas were of 4 mm filled with 20 µl of each iodinated product. correlated withPrototheca products zopfii composition and the The Establishingplates were incubatedthe efficiency in a ofthermostat iodized products at 37°C ability of iodine release. (tinctureand examined of iodine at 24 andand 48 Betadine)hours. by dilution For the
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