Antibacterial activity of ozonized sunflower oil (Oleozon)

L.A. Sechi1 , I. Lezcano2 , N. Nunez2 , M. Espim2 , I. DupreÁ 1 , A. Pinna3 , P. Molicotti1 , G. Fadda4 and S. Zanetti1 1 Dipartimento di Scienze Biomediche, UniversitaÁ degli studi di Sassari, Italy, 2 Centro Nacional de Investigaciones Cienti®cas, Ciudad de l'Habana, Cuba, 3 Istituto di Oftalmologia, UniversitaÁ degli studi di Sassari, Italy, and 4 Istituto di Microbiologia, FacoltaÁ di Medicina e Chirurgia `Agostino Gemelli', UniversitaÁ Cattolica del Sacro Cuore, Rome, Italy 470/7/00: received 26 July 2000 and accepted 25 October 2000 L.A. SECHI, I. LEZCANO, N. NUNEZ, M. ESPIM, I. DUPREÁ , A. PINNA, P. MOLICOTTI, G. FADDA A N D S. Z A N E T TI. 2001.

Aims: To evaluate the antimicrobial effect of the ozonized sun¯ower oil (Oleozon) on different bacterial species isolated from different sites.

Methods and Results: The effect of Oleozon on Mycobacteria, staphylococci, streptococci, enterococci, Pseudomonas and Escherichia coli was tested. The sun¯ower oil was ozonized at the Centro de Investigaciones del Ozone (CENIC, Havana, Cuba) by an ozone generator. MICs were determined by the agar dilution method. For Mycobacteria, the MIC of Oleozon was determined on solid medium by a microdrop agar proportion test. Oleozon showed antimicrobial activity against all strains analysed, with an MIC ranging from 1á18 to 9á5 mg ml)1 .

Conclusions: Oleozon showed a valuable antimicrobial activity against all micro-organisms tested. Results suggest that Mycobacteria are more susceptible to Oleozon than the other bacteria tested.

Significance and Impact of the Study: The wide availability of sun¯ower oil makes Oleozon a competitive antimicrobial agent. These results should prompt the setting up of some clinical trials to compare Oleozon with other antimicrobial agents.

INTRODUCTION

Ozone is a powerful oxidant, principally applied as a disinfectant of drinking and waste water (Alvarez and 1O'Brien 1982; Vanden Bossche et al. 1994; Gundarova et al. 1996; Legnani et al. 1996; Arana et al. 1999). Recently, ozone in different forms has also been used in a large number of medical indications (Finch et al. 1993; Alvarez 2et al. 1997; Morris and Menendez 1997; Falcon Lincheta 3et al. 1998; Komanapalli and Lau 1998). Ozone damages bacterial nucleic acids (Sawadaishi et al. 1986). Structural analysis of tRNA has shown that degradation occurs preferentially at guanine residues (Shinriki et al. 1981). Ozonolysis of supercoiled DNA has also been demonstrated (Sawadaishi et al. 1986), and both proteins and lipids are important targets in the reactions of ozone with bacterial membranes (Pryor and Uppu 1993). Ozone fractionates proteins at the tryptophan residues despite differences in amino acid and molecular weight (Pryor and Uppu 1993), whereas the reaction with lipids occurs at the carbon±carbon double bonds present in unsaturated fatty acid, producing different toxic products such as hydrogen peroxide, hydroxyhydroperoxides, aldehydes and Criegee ozonides (Pryor and Uppu 1993; Legnani et al. 1996). The overuse of antibiotics in the treatment of infectious diseases, and the appearance of `multi-drug resistant' bacterial strains (resistant to two or more antibiotics), has driven research towards the study of antimicrobial agents from essential oils (Hammer et al. 1999; Cox et al. 2000; Dorman and Deans 2000). Ozone does not contaminate the atmosphere and no bacterial resistance to this substance has been reported so far. Application of this system can be more extensive, ranging from the treatment of deep infections such as those caused by Helicobacter pylori and Staphylococcus aureus (Yamayoshi and Tatsumi 1993; Correspondence to: Dr L.A. Sechi, Dipartimento di Scienze Biomediche, Sezione di Microbiologia Sperimentale e Clinica, UniversitaÁ degli studi di Sassari, Viale S. Pietro 43/B, 07100 Sassari, Italy (e-mail: sechila@ssmain.uniss.it). ã 2001 The Society for Applied Microbiology Journal of Applied Microbiology 2001, 90, 279±284 Lezcano et al. 1998), to infection of the epidermis (Alvarez and O'Brien 1982). Different ozonized solutions have been used successfully against different infections such as otitis, intraocular infections and vaginitis (Finch et al. 1993; Gundarova et al. 1996; Morris and Menendez 1997). Sun¯ower ozonized oil (Oleozon) has remarkable bactericidal properties as reported by Lezcano et al. (1998) in a preliminary study, and acts directly on the pathogenic micro-organism without damaging the human epithelium (Shinriki et al. 1981). The Cuban `Centro de estudio the medicamento CECMED' (Centro para el Control Estatal de la calidad de los Medicamentos, which has similar functions to the Food and Drug Administration of the USA) approved the Registration of Oleozon in 1999 (no. 1498) after the pre-clinical trial of MartõÂnez Sanchez et al. (1997), which tested the dermal toxicity of Oleozon in rabbits and mice (MartõÂnez Sanchez 4et al. 1997). In mice, they established that 2000 mg of Oleozon kg)1 , applied to the epithelium, did not produce toxic effects. All other parameters tested (weight, feeding, pineal re¯ex, motor ability etc.) were similar to the controls. Oleozon was seen to be slightly irritant, but all histological parameters (liver damage, kidney damage, biochemical parameters) were normal. The action of Oleozon on rabbits was comparable with that on mice. The stability of Oleozon at different temperatures was also determined. Oleozon is stable for up to 1 year in the temperature range )10 to +8°C. Moreover, it is stable for up to 6 months at room temperature (27±30°C); after this period, the antimicrobial properties diminish. The pH is also stable for up to 1 year in the temperature range )10 to +8°C. At 30°C, the pH is stable for up to 6 months. The MIC values after this period increase from 2á37 to 19 mg ml)1 . The purpose of this study was to investigate Oleozon activity against different bacteria, such as Mycobacteria and multi-drug-resistant Gram-positive and Gram-negative strains, isolated from different sites (skin, pus, eyes, stools). The safety of Oleozon at the sites from which the test strains were isolated was reported previously (Gundarova et al. 5,61996; Alvarez et al. 1997; MartõÂnez Sanchez et al. 1997; Morris and Menendez 1997; Falcon Lincheta et al. 1998; Molerio et al. 1999).

MATERIALS AND METHODS

Sun¯ower oil ozonization The sunflower oil was ozonized at the Centro de Investigaciones del Ozone (CENIC, Havana, Cuba) by an ozone generator Aqozo Industrial Ozonizer (Ozone Research Center, Cuba). Standardization of the preparation was carried out according to the following parameters: · Peroxide Index (IP), which indicates the quantity of peroxide within the Oleozon. It is de®ned as the quantity of active oxygen per kilogram of Oleozon (mmol kg)1 ) 7 (Molerio et al. 1999). A range of IP between 500 and 800 (mmol kg)1 ) was considered. The best antimicrobial activity was seen with an IP of 650 (mmol kg)1 ). · Acidity Index, which indicates the free fatty acid in the Oleozon. It is de®ned as the number of milligrams of potassium hydroxide that are necessary to neutralize the free fatty acid in 1 milligram of Oleozon (Panreac 1992). In sun¯ower oil, the value must range between 6 and 8 units (Vajdia and Saenz 1976) whereas in Oleozon, it is not above to 25 units (Molerio and Diaz 1999). · Aldehyde concentration. The aldehyde concentration is measured by adding free hydroxylamine to the aldehyde carboxylic group. The results are expressed in mmol g)1 of Oleozon (Molerio and Diaz 1999); the interval must range between 0á4 and 0á9 mmol g)1 . · Iodine Index, which is a measure of the unsaturation rate of sun¯ower oil expressed as the number of grams of iodine that react with 100 grams of sun¯ower oil. In sun¯ower oil, the rate varies between 125 and 135 units (Vajdia and Saenz 1976) whereas in Oleozon, the value is between 50 and 90 units (Molerio and Diaz 1999). · Viscosity, which is a measure of the polymerization by condensation of the peroxides forming in the sun¯ower oil ozonization process. The values are expressed in mPa.s (centipoise or cP value; Institute of Standards and 8 Technology). In order to obtain Oleozon with an IP between 500 and 800, the viscosity must be between 100 9 and 450 mPa.s (Molerio and Diaz 1999).

Bacterial strains Different ATCC strains were tested: Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, E. coli XL1, Pseudomonas aeruginosa ATCC 27853, Enterococcus maleodoratus ATCC 43197, Ent. durans ATCC 19432, Ent. solitarius ATCC 49428, Ent. pseudoavium ATCC 49372, Ent. avium ATCC 14025, Ent. saccharolyticus ATCC 43076, Ent. hirae ATCC 9790, Ent. mundtii ATCC 43186, Ent. faecalis ATCC 35038, Ent. gallinarum ATCC 49573, Ent. casseli¯avus ATCC 25778, Ent. faecium ATCC 1974, Mycobacterium tuberculosis H37Rv and M. smegmatis mc155. Forty Streptococcus pyogenes strains were isolated from the skin of different patients attending the CENIC of Cuba; they were all tested for their susceptibility to Oleozon. Twenty-one Ent. faecium MDR strains, 27 E. coli MDR and other 13 E. coli strains, isolated at the `Agostino Gemelli' Hospital in Rome (Sechi et al. 1998; Zanetti et al. 1998), and 50 strains of Staph. aureus isolated in Cuba, were also tested for their susceptibility to Oleozon. Nineteen Staph. epidermidis MDR strains were collected from ocular isolates of 280 L.A. SECHI ET AL. ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 90, 279±284 patients attending the Institute of Ophthalmology of the University of Sassari (Sechi et al. 1999). Forty clinical Ps. aeruginosa strains isolated in Cuba were also evaluated. Three M. tuberculosis strains, two M. avium strains, two M. chelonae strains, two M. fortuitum strains and one M. neoaurum strain, isolated from different patients with different skin infections, were evaluated for their susceptibility to Oleozon. MIC determinations MICs were determined by the agar dilution method according to the NCCLS (1993) guidelines; the ®nal inoculum was 104 cfu ml)1 . All susceptibility tests were repeated 10 times and were highly reproducible. For these experiments, Mueller Hinton Broth and Mueller Hinton Agar were obtained from Becton Dickinson. Doubling concentrations of Oleozon in the agar medium were used (19, 9á5, 4á75, 2á37 and 1á18 mg ml)1 ). In order to mix the ozonized sun¯ower oil with the agar, 2 ml Tween-80 were added to 100 ml agar medium. The MIC was de®ned as the lowest concentration of Oleozon inhibiting visible bacterial growth after incubation for 20 h at 37°C. For Mycobacteria, the MIC of Oleozon was determined on solid medium by the microdrop agar proportion test. Middlebrook 7H9 broth and 7H10 were used as media (Bactec TB system, Becton Dickinson; Fadda and Roe 1984). A series of dilutions of the different strains of Mycobacteria was prepared in phosphate-buffered saline as a diluent. An aliquot (5 ll) of each dilution was spotted onto plates of 7H10 agar (Becton Dickinson), containing Tween-80 (to enhance oil solubility) and oleic acid albumin dextrose citric acid (OADC) as a supplement, and a series of dilutions from 19 to 1á18 mg ml)1 10 of Oleozon. The plates were incubated at 37°C (5 days for M. smegmatis and M. neoarum, 14 days for M. avium, 21 days for M. tuberculosis) and the number of bacterial colonies were counted. The MIC was de®ned as the lowest concentration resulting in a 99% reduction of the number of colonies on that plate compared with those on the plates used as controls (7H10 alone, and 7H10 plus Tween-80 and non-ozonized sun- ¯ower oil) for each dilution of the tested substance. Quality control of potentially interfering substances For each experiment, two bacterial spreads were performed as controls, one on the agar medium alone, and the second on the agar medium plus non-ozonized sun¯ower oil and Tween-80, for each concentration of ozonized oil. Bacterial growth was not inhibited by non-ozonized sun¯ower oil and Tween-80. Quality control of agar plates In order to verify the accuracy of the susceptibility test, a quality control programme was adopted as recommended by NCCLS (1993). Each batch of agar dilution plates was tested with the reference strain E. coli ATCC 25922 and Kanamycin as an antibiotic. All the MICs obtained were within the expected range (1±4 lg ml)1 ).

RESULTS

Antimycobacterial activity Under the test conditions, Oleozon showed an antimycobacterial activity against all strains evaluated (Table 1). The fast-growing strains M. aurum and M. smegmatis mc155 were both susceptible to Oleozon at a concentration of 0á95 mg ml)1 , as well as M. tuberculosis H37Rv. Oleozon was active against M. abscessus at a concentration of 2á37 mg ml)1 . Mycobacterium fortuitum strains showed an MIC ranging from 0á95 to 2á37 mg ml)1 , whereas Oleozon activity on both strains of M. avium analysed generated an MIC of 2á37 mg ml)1 . Mycobacterium chelonae strains were susceptible at 2á37 mg ml)1 Oleozon. Mycobacterium tuberculosis MIC ranged between 0á95 and 2á37 mg ml)1 (Table 1). In particular, the M. tuberculosis strain resistant to ®rst line drugs (Rifampicin and Isoniazide) was susceptible to 2á37 mg ml)1 Oleozon. Enterococci Different Enterococcus ATCC strains were evaluated for their susceptibility to Oleozon (Table 2). Enterococci were susceptible from 2á37 to 9á5 mg ml)1 . Enterococcus durans ATCC 19432, Ent. solitarius 49428, Ent. pseudoavium ATCC 49372, Ent. avium ATCC 14025, Ent. saccharolyticus ATCC 43076, Ent. hirae ATCC 9790, Ent. faecalis ATCC 35038, Ent. faecium ATCC 19474 and Ent. casseli¯avus ATCC 25778 were susceptible at a concentration of 9á5 mg ml)1 Table 1 Susceptibility to Oleozon of different species of Mycobacteria Species (no. of strains) MIC (mg ml)1 ) M. tuberculosis H37 Rv (1) 0á95 M. smegmatis mc155 (1) 0á95 M. abscessus (1) 2á37 M. aurum (1) 0á95 M. avium (2) 2á37 M. fortuitum (2) 0á95±2á37 M. chelonae (2) 2á37 M. tuberculosis (1) 0á95 M. tuberculosis MDR (2) 0á95±2á37

SUSCEPTIBILITY TO OLEOZON OF DIFFERENT BACTERIA

281 ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 90, 279±284 Oleozon; Ent. maleodoratus ATCC 43197 generated an MIC of 4á75 mg ml)1 whereas Ent. mundtii ATCC 43186 and Ent. gallinarum ATCC 49573 generated an MIC of 2á37 mg ml)1 . Oleozon showed good activity against 21 Ent. faecium clinical isolates (Table 3, MIC90s £ 9á5 mg ml)1 ). Three strains were susceptible at 9á5 mg ml)1 , and nine strains showed an MIC of 4á75 mg ml)1 ; six strains generated an MIC of 2á37 mg ml)1 whereas three strains showed an MIC of 1á18 mg ml)1 Oleozon (Table 3). Ten clinical isolates of Ent. faecalis were also evaluated, and Oleozon was active at a concentration of 4á75±9á5 mg ml)1 . Streptococci Forty strains of Strep. pyogenes, isolated from the infected skin of Cuban patients, were evaluated for their susceptibility to Oleozon. The MIC range was from 2á37 to 9á5 mg ml)1 (Table 3). Staphylococci In Table 2, the activity of Oleozon against two Staphylococcus ATCC strains is shown: the MIC for Staph. aureus ATCC 25923 and Staph. epidermidis ATCC 14990 was 9á5 mg ml)1 Oleozon. Nineteen strains of Staph. epidermidis were also evaluated for their susceptibility to Oleozon (Table 3). MICs ranged from 2á37 to 9á5 mg ml)1 . Only four strains were susceptible at 2á37 mg ml)1 Oleozon. These strains were resistant to different drugs (strain 2 was resistant to penicillin, gentamicin and erythromycin; strain 7 was resistant to penicillin, tetracycline and erythromycin; strain 4 was resistant to penicillin and tetracycline; strains 6 and 18 were resistant to penicillin). Escherichia coli The activity of Oleozon against different E. coli strains is shown in Table 2. The MIC of E. coli XL1 was £ 1á18 mg ml)1 , whereas the MIC for E. coli ATCC 25922 was 4á75 mg ml)1 . The MIC for the clinical isolates was 4á75 mg ml)1 , except for three strains which showed an MIC of 2á37 mg ml)1 , and two strains with an MIC £ 1á18 mg ml)1 . Pseudomonas aeruginosa Pseudomonas aeruginosa ATCC 27853 was susceptible to 4á75 mg ml)1 (Table 2). Forty Ps. aeruginosa clinical isolates were also tested for their susceptibility to Oleozon (Table 3). All strains except one were susceptible at 4á75 mg ml)1 , one strain was susceptible at 9á5 mg ml)1 . DISCUSSION Pyogenic skin infections are produced in 90% of cases by Staph. aureus and Strep. pyogenes; Ps. aeruginosa and E. coli can participate as secondary agents (Alvarez et al. 1997; Falcon Lincheta et al. 1998; Neubert et al. 1999). Moreover, Gram-positive bacteria are rapidly becoming the most important pathogens in nosocomial infections. Recently, there has been great concern about multi-drug resistant Staph. aureus, Staph. epidermidis, Ent. faecalis and Ent. faecium strains (Yamayoshi and Tatsumi 1993; Esperson 1998). In this study, the action of Oleozon against different staphylococci, streptococci and enterococci is shown; some Table 3 Susceptibility of different clinical isolates of Enterococcus faecium, Ent. faecalis, Streptococcus pyogenes, Staphylococcus aureus, Staph. epidermidis, Escherichia coli and Pseudomonas aeruginosa MIC (mg ml)1 ) Strains (no. of micro-organisms) Range 50% 90% Ent. faecium (21) 1á18±9.5 4á75 9á5 Ent. faecalis (10) 4á75±9á5 4á75 9á5 Strep. pyogenes (40) 2á37±9á5 4á75 9á5 Staph. aureus (50) 2á37±9á5 4á75 9á5 Staph. epidermidis (19) 2á37±9á5 4á75 9á5 E. coli (40) 1á18±9á5 4á75 9á5 Ps. aeruginosa (40) 4á75±9á5 4á75 4á75 Table 2 Susceptibility to Oleozon of different enterococci, staphylococci, Escherichia coli and Pseudomonas aeruginosa ATCC strains ATCC strains MIC (mg ml)1 ) Ent. hirae ATCC 9790 9á5 Ent. faecalis ATCC 35038 9á5 Ent. faecium ATCC 19474 9á5 Ent. gallinarum ATCC 49573 2á37 Ent. casseli¯avus ATCC 25778 9á5 Ent. maleodoratus ATCC 43197 4á75 Ent. durans ATCC 19432 9á5 Ent. solitarius ATCC 49428 9á5 Ent. pseudoavium ATCC 49372 9á5 Ent. avium ATCC 14025 9á5 Ent. saccharolyticus ATCC 43076 9á5 Ent. mundtii ATCC 43186 2á37 Staph. aureus ATCC 29213 9á5 Staph. epidermidis ATCC 14990 2á37 E. coli ATCC 25922 4á75 E. coli XL1 1á18 Ps. aeruginosa ATCC 27853 4á75 282 L.A. SECHI ET AL. ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 90, 279±284 of the strains were previously characterized by molecular methods (Sechi et al. 1998; Pinna et al. 1999). Different forms of cutaneous tuberculosis are caused by various species of Mycobacteria (M. tuberculosis, M. avium, M. fortuitum, M. chelonae, M. marinum etc.) (Gulisano and 11Mariani 1998; Noguchi et al. 1998; Ena et al. 1999). In this study, in vitro activity of Oleozon against different bacteria (Mycobacteria, streptococci, enteroccoci, staphylococci, E. coli and Ps. aeruginosa) isolated from different sites (mostly skin infections) was evaluated. Oleozon showed a valuable antimicrobial activity against all micro-organisms tested. The activity of Oleozon (MIC range 1á18±9á5 mg ml)1 ) against all tested bacteria, including b-lactam-, vancomicin- and gentamicin-resistant strains, is expressed in mg ml)1 . These concentrations may seem high if compared with the amount of antibiotics, expressed in lg ml)1 , necessary to inhibit bacterial growth. This is due to the dilution of the active compounds in the sun¯ower oil that has not been altered in the ozonization process. For Gram-negative bacteria, the activity of Oleozon was in the range 1á18±4á75 mg ml)1 (except for Ps. aeruginosa strains). Most of the E. coli strains tested showed an MIC of 4á75 mg ml)1 . Oleozon showed the same activity against Ps. aeruginosa ATCC strains. It was very effective against the slow-growing Mycobacteria tested in this study (M. tuberculosis and M. avium) and the fast-growing Mycobacteria (M. aurum and M. smegmatis). It was able to inhibit growth of these bacteria within a narrow range of concentration (0.95±2á37 mg ml)1 ). Some M. tuberculosis multi-drugresistant strains appeared less susceptible, with an MIC of 2á37 mg ml)1 , whereas other M. tuberculosis tested (H37Rv and a clinical isolate) generated an MIC of 0á95 mg ml)1 . The activity of Oleozon against the M. avium, M. fortuitum and M. abscessus strains tested was very ef®cient, with an MIC of 2á37 mg ml)1 . It seems from these preliminary results that Mycobacteria are even more susceptible to Oleozon than the other bacteria tested. This may be explained in part by the composition of their cell wall and the high lipid content, which may facilitate the passage of Oleozon-active compounds into the bacteria. It has been reported that Oleozon is effective in the treatment of different types of skin diseases caused by herpes, Mycobacteria, staphylococci, streptococci etc. (Ena et al. 1999; 12Gulisano and Mariani 1998; Neubert et al. 1999). For 13instance, Morris and Menendez (1997) successfully treated 180 patients affected by Herpes simplex infection in the lips with a twice-daily application of Oleozon. Oleozon has also been applied for the cure of fungal infections; 213 patients with a mean age of 28 years were followed up. Trichophytom rubrum, Candida albicans, Microsporum canis and Trichophytom mentagrophytes were the isolates. Seventy-®ve percent of the patients were cured with topical applications; treatment with the antimycotic compound Nizoral on the same type of infections was effective for 81% of the patients (Falcon Lincheta et al. 1998). Alvarez et al. (1997) reported a successful treatment of a patient with pyoderma by daily topical applications of Oleozon with an I.P. of 650. The results obtained in this study should lead to the setting up of some clinical trials in order to compare the ef®cacy of Oleozon with other antimicrobial agents. The wide availability of sun¯ower oil makes Oleozon a competitive antimicrobial agent. The fact that most MICs were clustered around 2á37 mg ml)1 , and no strains were found with an MIC higher than 9á5 mg ml)1 , may suggest that the activity is due to toxicity rather than to metabolic interruption, as is the case for traditional antimicrobial agents. The fact that Oleozon showed no toxicity to rabbits and mice (Molerio and Diaz 1999), however, is an indication that it has multiple targets (i.e. membrane proteins) but shows no generalized toxicity to the cells.

ACKNOWLEDGEMENTS This work was supported by grant L.R. 19 of the Regione Autonoma della Sardegna. The authors would like to thank Mr D. Delogu and Mr E. Manca for technical help.

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