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 22 tested strains, the method average size of inhibition areas were as following: Betadine 16 to 30 mm (26.4), tincture of iodine . A quantity of 4.5 ml of glucose broth was 20 to 30 mm (25.71), Videne 16 to 30 mm distributed in a series of five tubes. To the first (25.61), Lugol’sPrototheca solution wickerhamii 10 to14 mm (11,33) and tube was added 0.5 ml of the iodinated product potassium iodide 10 to 24 mm (11,14) (Fig. 1). which, after mixing, was passed in succession 0.5 For strain, the ml in the following tubes, in the end removing inhibition areas were the following: Betadine 26 0.5 ml of the tube no. 5. The resulted-1 -2 iodinated-3 -4 mm, tincture of iodine 24 mm, Videne 24 mm, product-5 dilution rate x10 is (10 , 10 , 10 , 10 , Lugol’s solution 10 mm, potassium iodide 14 mm andPrototheca 10 ). Subsequently, zopfii each tube was Protothecainoculated (Fig. 2). There was no presence of drug resistant wickerhamiiwith a fixed amount of 5 µl (∼2100 UFC/1 ml) or resistant colonies in the inhibition zones. The of (strain 22) and inoculations carried out from the inhibition zones (ATCC 16529). Control tubes, in glucoseThe results broth tubesobtained remained at the sterile, test of confirming different without iodized products: 1) tube that allows inoculumthat the induced density effect according was algaecide. to McFarland the development of culture (positive control); 2) standards. non-inoculated tube to check the culture medium (negative control). The aim of this test was to evaluate The tubes were incubated at 37°C and whether the inoculumPrototheca density zopfii is influencing the maintained for 24 hours. To verify the inhibitory Protothecasize of the wickerhamii inhibition areas. Two strains were effect of each tube in the dilution series set, glucose initially tested, (strain 22) and agar plates were streaked using one plate for each (ATCC collection strain). tested strain, and the plates were divided into It was found that the size of the inhibition 5 sectors of a triangular shape corresponding to zones has been less affected by the density of the each dilution. The plates were incubated at 37° C inoculum, the recorded differencesP. zopfii being of 2-4 and examined at 24 and 48 hours, but kept under mm between the smaller densities P.(0.5 wickerhamii to 1) and observation a further 5 days. The control tubes the larger (2, 3, 4 and 5) (Tab. 1, strainP. zopfii 22). checkingEvaluation was done of the separately inhibitory on at differentanother glucosecontact The results were similar for timesagar plate. Prototheca zopfii strain, as well as other 5 strains of Prototheca wickerhamii subsequently tested. . The tested strains were Pictures with the inhibition areas obtained for (22 strain) and (ATCC the platesThe results with the obtained inoculum for of differentdiluted iodinateddensities 16529), and the testing was done to the iodine productare observed below Fig. 3 (a and b). tincture and Betadine. In a 10/110 mm tube, 0.9 ml iodine tincture or Betadine was dispensed, . Algaecide-1 type inhibitory effect was followed by 0.1 ml culture two strains tested found only in 10 dilution. In that dilution tube in glucose broth. Contact times were 5’, 10’, 15’, environment it remained clear, with a light purple 30’Bulletin and UASVM 60’. Veterinary After Medicine the 72end (2) /of 2015 each contact time colour (due to iodine). The other dilutions/tubes, Prototheca 309

The Effect of Iodine Based Products on Unicellular Algae from Genus the medium also remained clear, for the first 24 due to dilution of the iodide product resulting in hours, with the presence of reduced sediment, subinhibitory values. which easily disperse when stirring. During the Such results reveal that in order to obtain examination at 48 hours was found a thin pellicle good efficiency in prevention or treatment of on the surface and the deposit became more protothecosis,The inhibitory iodine effect-1 based observed products mustat different not be abundant, meaning that algae multiplied. contactdiluted moretimes than 10 . The inoculations on glucose agar plates made from each-1 dilution (after 48 hours) revealed that . When examining the inoculated only 10 dilutionPrototheca have not developed colonies- plates (glucose agar) after each contact time - 5, (algaecide2 -5 effect). On the other dilutions (10 10, 15, 30 and 60 minutes, it was observed that no to 10 ) typical colony developed; colonies have developed in any of the tested strains at 24 hours they were small (less than 0.5 mm after incubation at 37°C for 24 and 48 hours (Fig. in diameter), circular, opaque, white, at 48- 6 and 7). Algaecide effect manifested itself in the 72 h, they grow in size (2-4 mm), having an first 5 minutes of contact, for both iodine tincture irregular surface, the edges slightly lobed, white. and Betadine. The effect was algaecide, because A good characterization of colonies can be the microorganisms presented no growth in done by examining using the magnifying glass, culture plates even if maintaining the plates for where typical structure and other algae details the following 5 days at laboratory temperature. (cauliflower surface aspect) were revealed and The inoculations made in glucose broth from can differentiate in the situation of contamination the areas corresponding to each contact time, and by other bacteria colonies mainly staphylococci, examined at 24 and 48 hours, remained sterile, micrococci and yeast. confirming the induction of inhibitory effect. The When examining the wet smears (dispersion experiment was repeated for another 5 strains, in a drop of Lügol), made of tubes-2 that Prototheca-5 but the result was the same - total inhibition. dilutions remained viable (10 to 10 ), it was The researches revealed that the iodine based found that the cells have undergone morphological products (Betadine and iodine tincture), have an changes, which reflected in the growth and inhibitory effect - algaecide, which is observed formationP. of zopfii typical coloniesP. wickerhamii on solid culture within the first five minutes of contact with the media. This behaviour was seen in both tested products. The choice of 5 minutes time as first strains ( and ), both for stage of contact between the iodide products and iodine tincture and Betadine (Fig. 4 (a and b), Fig. a suspension of prothotecs, has been based on the 5 (a and-1 b)). This means that at higher dilutions comparison of the sensitivity of bacteria, which are of 10 , algaecide effect is no longer achieved usually inactivated in 15-30 seconds (according to

Fig. 1. Fig. 2. Prototheca zopfii Prototheca wickerhamii Inhibition areas, Inhibition areas, (strain 22) (16529 ATCC) Bulletin UASVM Veterinary Medicine 72 (2) / 2015 310 et al

RĂPUNTEAN

et al. the literature) (23). Knowing that prototecile are about possible toxic effects on cells (Burck, 1988; larger and have thicker cell wall, we considered Drosou , 2003). It is believedet al that cytotoxicity that five minutes can be taken as the minimum effects are reduced and occur only in the event of time necessary to manifest inhibitory effect. In prolonged treatments (Sibbald ., 2011). On the literature,DISCUSSION we found no information in this regard. other hand, it is shown that the iodine preparations (povidone iodine and cadexomer iodine)et al reduced the bacterialet al load, whichet alpromotes epithelial The antimicrobial properties of iodine regeneration and healing (McLure ., 1992; are known for long time, but research on the Lamme ., 1998; Mertz ., 1999). inhibitory effect of iodine and/or various iodine Some products, such as iodins may even based products were amplified as a result of penetrate biofilms,et al. whichIn arevitro formed on the increasing expansion of the phenomenon of different surfaces, including wounds (Bootham, antibiotic resistance. Numerous studies confirmed 2009; Phillips , 2010). , microbicide that iodine based products have bactericidal, effect has been demonstrated by various study sporicidal, cisticid and virulicid effect. Iodineet methodologies. It shows that povidone iodine al.exact mode of action is not fully known, but it is Staphylococcuscauses deadly aureusalterations > 99% of microbial complex (Cooper, 2004; Bootham, 2009; Sibbald cells (methicillin etsusceptible al. and resistant , 2011). strains) within 10 seconds It is believed that the iodine based products of exposure (Lacey , 1993). Iodine is active are able to quickly penetrate the cell wall of Tab. 1. different microorganisms, causing structural and functional changes of the cell membranes and The size of inhibition areas at different the nucleic acids by denaturation of proteins and densities of the inoculum enzymes,et which al. results in rapid death of various microorganisms (Gottardi, 1999; Bootham, 2009; Inhibition areas (ø mm),Videne strain 22 Sibbald , 2011). The activity is immediately, Density Iodine Potassium Lugol sol. Betadine but also over time due to persistence. The efficacy tincture iodide and stability of the aqueous solutions is influenced 0,5 10 24 20 20 12 by the values of pH (Gottardi, 1999). 1 10 22 20 20 12 A broad use of these preparations is mentioned 2 10 22 18 18 12 in the treatment of various types of wounds in et al 3 8 22 18 18 10 which the antibiotic-resistant bacteria frequently colonized (Giacometti ., 2002). Direct 4 8 22 18 16 10 application on wounds caused some controversy 5 6 22 16 16 10

Fig. 3. P. zopfii

Inhibition areas at different inoculum densities ( strain 22) Bulletin UASVM Veterinary Medicine 72 (2) / 2015 Prototheca 311

The Effect of Iodine Based Products on Unicellular Algae from Genus

et against many pathogenic bacteria, most being al.localized in different tissueset andal organs, with killed in 15-30 seconds of contact [24]. systemic forms described the cows (Taniyama It is importantS. aureus to note that iodine preparations , 1994) and dogs (Souza ., 2009). Illness do not develop resistance phenomena. However, in occurs more frequently in patients showing the case of strains isolated from mastitic immunodeficiencies or other chronic diseases that etcow al milk, a variation of the iodine minimumin vitro are subject to abusive treatment with antibiotics. inhibitory concentration was described (Azizoglu The greatest risk is observed in dairy cows with ., 2013), and even the induction of mastitis that can evolve as an epidemic outbreak. resistance etby al repeated passages on culture media Following the multiplication and development containing subinhibitory doses ofPrototheca antimicrobials of granulomas, normal tissue architecture and (El Behiry ., 2012). function is impaired, even affecting vital organs. Unicellular algae of the genus have As a result of increasing frequency of cases become increasingly involved in the production of and the antibioticPrototheca inefficiency, papers that have human and animal diseases, in both warm-blooded described the effect of iodine and iodinein vitro based and cold-blooded animals. The disease, called productsP. zopfiion were published, being protothecosis is manifested by the formation of underlined their algaecide effect. An study granulomatous lesions with chronic evolution, on 27 strains isolated from cow’s milk

Fig. 4. Prototheca zopfii

(strain 22)

Fig. 5. Prototheca wickerhamii

(strain 16529 ATCC)Bulletin UASVM Veterinary Medicine 72 (2) / 2015 312 et al

RĂPUNTEAN shows that iodine preparations showed algaecide Iodine and iodine based products (inorganic effect at low concentrations (0.15652-0.625%) and organic), having a high antimicrobial potential and recommendset al its use for routine hygiene (as and broad spectrum, may find Protothecause in the treatment pre- and post-dipping) in cattle mastitis controlP. of many diseases, including those caused by zopfii(Salerno ., 2010). The algaecide effect of unicellularCONCLUSIONS algae of the genus . povidone iodine was also highlightedet al on 20 strains, both biotypeP. zopfii1 (10 strains) and Prototheca biotype 2 (10 strains) (Sobukawa ., 2011). By agar diffusimetrical technique it was In another study on 15 strains tested established that all tested strains were for sensitivity to iodine and chlorhexidine using sensitive to iodine products, the induced effect dilution method on Sabouraud dextrose broth, being algaecide. was demonstrated that both drugs have algaecide Testing on different inoculum densities, effect, with minimum microbicide concentrationet al. allowed to conclude that the inhibitory effect (MMC):0.3125%-1.25% for iodine and 0.0048 occurred regardless of density, with small - 0.0195% for chlorhexidine (Krukowski , differences in the size of the inhibition zones. 2013).

Fig. 6. Prototheca wickerhamii

(strain 16529 ATCC) algaecide effect for different contact times (no colonies developed)

Fig. 7. Prototheca zopfii

(strain 22) Bulletin UASVM Veterinary Medicine algaecide 72 (2) / 2015 effect for different contact times (no colonies developed) Prototheca 313

The Effect of Iodine Based Products on Unicellular Algae from Genus

12. McLure AR,Staphylococcus Gordon J (1992). aureus. In vitro evaluation of Algaecide effect was also demonstrated by povidone-iodine and chlorhexidine against methicillin dilution method in liquid culture medium (dilution-1 resistance J Hosp Infect 21(4):291- rate x10), stating that it occurred only at 10 294. dilution, and never-2 produced-5 at higher dilutions, 13. Mertz PM, Oliviera-Gandia MF, Davis SC (1999). The respectively 10 to 10 . evaluation of a cadexomer iodine wound dressing on In relation to the contact time, set for iodine methicillin resistance (MRSA) in acute wounds. Dermatol Surg 25(2):89-93. tincture and betadine, it can be shown that the 14. Phillips PL, Wolcott RD, Fletcher J, Schultz GS (2010). algaecideREFERENCES effect is fast, within 5 min of contact. Biofilms Made Easy. Wounds International 1(13). Avaiable from: http://www.woundsinternational.com. Prototheca Staphylococcus aureus: 15. Răpuntean S (2002). Studiu asupra algelor unicelulare din 1. Azizoglu RO, Lyman R, Anderson KL (2013). Bovine genul : aspect epidemiologice, morfoculturale, dose response to iodine and biochimice, antigenice și semnificația etiopatogenetică. chlorhexidine and effect of iodine challenge on antibiotic Teză de doctorat, USAMV, Cluj-Napoca. susceptibility. J Dairy Sci 96(2):993-999. 16. Salerno T, Ribeiro MG, Lagnoni H, Siqueira AK, Costa E, 2. Burck RI (1988). Povidone-iodine solution in wound In vitro Melville PA, Bueno VF, Yamamura AA Roesler U, daSilva AV treatment. Phys Ther 78(2):212-8. Prototheca zopfii (2010). algaecide effect of sodium hypochlorite 3. Buyukmihci N, Rubi LF, DePaoli A (1975). Protothecosis and iodine based antiseptics of strains with ocular involvement in a dog. JAVMA 167(2):158-161. isolated from bovine milk. Res Vet Sci 88(2):211-213. 4. Cooper R (2004). A review of the evidence for the use 17. Sibbald RG, Leaper DJ, Queen D (2011). Iodine Made Easy. to topical antimicrobial agents in wound care. World Wounds International 2(2):1- 4. Wide Wounds, Centre for Biomedical Sciences School od In vitro 18. Sobukawa H, Watanabe M, Kano R, Ito T, Onozaki M, Applied Sciences, University of Welles Institute Cardiff. Prototheca Hasegawa A, Kamata H (2011). Algaecide Effect http://worldwidewounds.com/2004/february/Cooper/ of Disinfectants on Genotypes 1 and 2. J Vet Topical-Antimicrobial-Agents.html Med Sci 73(11):1527-1599. 5. Drosou A, Falabella A, Krisner RS (2003). Antiseptics on 19. Souza NL, Estrela-Lima A, Moreira LTE, Ribeiro LGR et al wounds: an area of controversy. Wounds 15(5):149- (2009). Systemic canine protothecosis. Braz J Vet Pathol 166. Staphylococcus aureus 2(2):102-106. 6. El Behiry A, Schlenker G, Szabo I, Roesler U (2012). In 20. Taniyama H, Okamoto F, Kurosawa T, Furuoka H, vitro susceptibility of strains Prototheca zopfii Kaji Y, Okada H, Matsukawa K (1994). Diseminated isolated from cows with subclinical mastitis to different Protothecosis caused by in a cow. Vet antimicrobial agents. J Vet Sci 13(2):153-161. Path 31(1): 123- 126. 7. Giacometti A, Cirioni O, Greganti G, Fineo A, Ghiselli R, Del 21. Wirth FA, Passalacqua JA, Kao G (1999). Disseminated Pretem S, et al (2002). Antiseptic compounds still active cutaneous protothecosis in an immunocompromised against bacterial strains isolated from still surgical wound host: a case report and literature rewiev. Cutis 63(3):185- infectious increasing antibiotic resistance. Eur. J Clin 188. Microbiol Infect Dis 21(7):553-556. 22. Woolorich A, Koestenblatt E, Don P, Szaniawski W (1994). 8. Gottardi W (1999). Iodine and disinfection: theoretical Cutaneous prototecosis and AIDS. J Amer Acad Dermatol study on mode de action, efficiency, stability, and analytical 31(5/II):920-924. aspects in the aqueous system. Arch Pharm (Weinheim) 332:151- 157. 23. ***The End of Antibiotics and the Rise of Iodine as Prototheca zopfii an Effective Alternative. http://www.naturalnews. 9. Krukowski H, Lisowski A, Nowakowicz-Debek B, Wlazło L com/022800_antibiotic_infection.htm (2013). Susceptibility of strains isolated from cows mastitis to chlorhexidine and iodine. Turk J Vet 24. *** Iod. Wikipedia. http://en.wikipedia.org/wiki/iodine. Anim Sci 37:106-108. 25. ***Iodine Disinfection in the use of individual Water 10. Staphylococcus Lacey RW, Catto aureus. A (1993). Action of povidone-iodine Purification Devices. www.phc.amedd.army.mil/ against methicilin-sensitive and - resistant cultures of 26. ***Clinical and Laboratory Standard Institute (2013). Postgrad Med J 69(3):S78-83. Performance Standards for Antimicrobial Disk and 11. Lamme EN, Gustafsson TO, Middelkoop E (1998). Dilution Susceptibility Test for Bacteria Isolated from Cadexomer-iodine ointment show stimulation of Animals. Vet 01-A4. epidermal regeneration in experimental full-thickness wounds. Arch Dermatol Res 290(1-2):18-24.

Bulletin UASVM Veterinary Medicine 72 (2) / 2015