1. Abstract Allicin is recognised as the main bioactive agent from Allium sativum or garlic. This compound is highly active but generally unstable. Using a cold aqueous extraction method, we have obtained a novel extract of allicin (AB1000) that we have reported is stable and highly active in vitro against methicillin resistant Staphylococcus aureus (MRSA). Due to national publicity of AB1000, patients with long standing unresolved MRSA infections requested this agent for treatment. MRSA is commonly related to delayed closure for many chronic and acute wounds. This is associated with high levels of bacteria in tissues but they can also through toxin secretion. These toxins can cause local necrosis and disrupt the delicate balance of critical mediators such as cytokines and proteases necessary for healing progression. We present initial findings from three patients who have completed a course of treatment. These courses consisted of capsules (450mg, 3 per day ); spraying liquid AB1000 (1000 ug ml-1) onto the affected areas once per day and applying AB1000 Cream (500 ug ml-1) to the infected area once daily. Patients were screened, nasal and wound for MRSA prior and during treatment. All patients were nose and wound swab MRSA positive prior to treatment. All were over 60 years of age and had either major surgery or long term skin infections leading to the formation of ulcers infected by MRSA. Two of the MRSA infections were community acquired and one hospital acquired. The strains isolated from each patient were tested in vitro against AB1000 and all were susceptible. Patients reported an improvement in their condition after 2 and 6 weeks treatment and the infections resolved in 3 to 4 months. Although the timescales required for treatment may be longer than those normally required using antibiotics, the initial relief from weeping ulcers and pain was much quicker. It should be noted these the patients had been receiving unsuccessful treatment with antibiotics for months or years prior to treatment with AB1000. A possible reason for the initial relief from symptoms could relate to the reported activity of garlic extracts to neutralise bacterial exoenzymes in vitro. This could account for the findings that patients got relief from their symptoms before the MRSA were fully removed from the lesion site. MRSA is still a major cause of nosocomial infections. There is a need for new topical and systemic antibiotics that may help in controlling and treating MRSA infections (1). MRSA is often linked to delayed closure for chronic and acute wounds associated with high levels of bacteria in tissues and toxin secretion by the bacteria. Patients report weeping lesions for months or years despite treatment with conventional antimicrobial agents. We have previously reported the in vitro effectiveness of a novel stabilised form of allicin (AB1000) against MRSA (2). The main antimicrobial effect of allicin may be due to its reaction with the thiol-containing enzymes. In this study we investigate the use of AB1000 in neutralizing microbial enzymes in an attempt to explain the results from volunteer studies in people with long-term chronic MRSA infections. Background Enzyme studies: The effect of AB1000 at different concentrations on the in vitro activity of microbial alcohol dehydrogenase (ADH), a thiol containing enzyme important in alcohol metabolism, was examined. A range of concentrations of AB1000 were pre-incubated with ADH and the ability of the enzyme to catalyze the transfer of a hydride from the hydroxyl carbon of either ethanol or allyl alcohol to NAD. This was determined using spectroscopy (340nm-measuring the conversion of NAD+ to NADH) Volunteer Studies: People who had chronic weeping lesions infected with MRSA volunteered to use topical and systemic treatment with AB1000 cream, liquid and capsules. These patients had long term MRSA infections and had been unsuccessfully treated with antibiotics. Volunteers were given full information on AB1000 and were asked to discuss the treatment with their clinician prior to taking part in the study. Volunteers were supplied with: Liquid AB1000 spray (1000 mg/l) to be used in the morning spraying onto the infected areas. AB1000 cream (500 mg/l) to be applied once per day in the evening onto the infected areas. AB1000 capsules (450mg), to be used 3 times per day ; Patients were screened, nasal and wound for MRSA prior and during treatment. Patients tested their skin for sensitivity prior to use and were told to stop use if at anytime there was any sign of irritation related to the use of AB1000. All were over 60 years of age and had either major surgery or long term skin infections leading to the formation of ulcers infected by MRSA. MethodsResults Enzyme Studies: After 60 minutes pre-incubation with 250ug/ml of allicin the activity of ADH was reduced by 31% using ethanol as a substrate and by 36% with allyl alcohol. We also demonstrated that ADH activity was related to the concentration of allicin used in pre-treatment ( Figure 1) Figure 1: Activity of ADH after treatment with AB1000 Volunteer Studies: The initial results from volunteer studies showed all patients were nose and wound swab MRSA positive prior to treatment. Two of the MRSA infections were acquired in nursing homes and one was hospital acquired. The strains isolated from each patient were tested in vitro against AB1000 and all were susceptible. Patients reported an improvement in their condition after 2 and 6 weeks treatment. This included reduction in pain, redness, irritation and swelling. (Figure 2). Infections resolved in 3 to 4 months (Figure 3). Conclusions Treatment of chronic MRSA infections using a novel aqueous extract of Allicin (AB1000) Ronald R. Cutler 1, Peter D. Josling 2 and Norman J. Bennett 2 (1.) School of Health and Bioscience, University of East London, UK. (2.) Allicin International, Rye, UK. References 1.Schmitz, F and Jones ME. Antibiotics for treatment of infections caused by MRSA and the elimination of MRSA carriage. What are the choices? Int. J. Antimicrob. Agents, 1997; 9: 1-19. 2.Cutler, RR, Wilson,P. Antibacterial activity of a new, stable, aqueous extract of allicin against methicillin-resistant Staphylococcus aureus. Brit. Jour. Biomed. Sci. 2004, 61:71-74 Figure 2: Pictures of leg wounds before and after 2 months treatment. The left picture shows a red inflamed weeping lesion (before allicin treatment). The right shows the inflammation has reduced and the lesion is healing. Figure 3: Picture on the left shows weeping inflamed lesions (before allicin treatment). The picture on the right shows that the lesions have dried and are healing after 3 months treat- ment. Patient is MRSA free. Patients reported an improvement in their condition after 2 to 6 weeks treatment and the infections resolved in 3 to 4 months. Although the timescales required for treatment are longer than those normally required using antibiotics, the initial relief from weeping ulcers and pain happened in weeks. It should be noted these the patients had been receiving unsuccessful treatment with antibiotics for months or years prior to treatment with AB1000. One possible reason for the initial relief from symptoms could relate to ability of Allicin to neutralise thiol-containing enzymes, demonstrated here. The potential to reduce the activity of extra-cellular virulence factors could account for the findings that patients got relief from their symptoms before the MRSA were fully removed from the lesion site.