1. Susceptibility of Drug Resistant Acinetobacter baumanii (DRAB) to a stabilized aqueous allicin extract from garlic (AB1000 ). Researchers’/Presenters’ Names Institution/Organization/Company Susceptibility of Drug Resistant Acinetobacter baumanii (DRAB) to a stabilized aqueous allicin extract from garlic (AB1000 ). Cutler RR 1, Wilson P 2, Thota MR 1, Vivekananthn S 1, Bennett NJ 3, and Josling, PD 3 1School of Health and Bioscience, University of East London, UK, 2Dept of Medical Microbiology, The Royal London and St Bartholomew’s Hospitals, London, UK, 3 Allicin International, Rye, UK Methods Abstract Introduction Results Conclusions References 1.Davis KA, Moran KA, McAllister CK, Gray PJ. (2005) Multidrug-resistant Acinetobacter extremity infections in soldiers. Emerg Infect Dis. 11(8):1218-24 2.Cutler, RR., Wilson,P. (2004) Antibacterial activity of a new, stable, aqueous extract of allicin against methicillin- resistant Staphylococcus aureus. Brit.J.Biomed.Sci.:61: 71-74 3.Cutler RR, Josling PD and Bennett NJ (2005) Treatment of chronic MRSA infections using a novel aqueous extract of Allicin (AB1000). Clinical Microbiology and Infection. 11: suppl2. p515 Objectives: DRAB is a nosocomial pathogen. Globally many Intensive Care Units have their own endemic strain. Some patients become colonised with the organism, with no adverse effect, whereas others have life-threatening infections. DRAB can be resistant to many antimicrobial agents and treatment of infections is increasingly difficult due to the dwindling choice of active agents. There is an urgent need for new agents active against DRAB and our objective was to contribute to this search. Methods: The antimicrobial activity of a novel aqueous allicin extract (AB1000) was tested against 11 clinical isolates of DRAB. The allicin content of AB1000 was confirmed using HPLC. Strains were screened for activity using agar diffusion methods, MICs and MBCs were carried out and growth (using spectroscopy at 490nm) and killing curves (using viable counts) for selected organisms were determined. Results: In agar diffusion tests using AB1000 concentrations ranging from 125 to 1000mg/l were made up in aqueous solution. Zone diameters on Oxoid Muller Hinton agar ranged from 14mm to 31mm, slightly but not significantly smaller than the zones found with Oxoid Isosensitest agar of 20mm to 34mm. At 500mg/l the zone sizes for the 11 strains tested varied from 22-29mm with an average zone diameter of 26.3 mm and a mode diameter of 27mm. Minimum inhibitory concentrations varied from 15 mg/l to 62.5mg l -1 with a mode concentration of 62.5 mg l -1. Minimum bactericidal concentrations varied from 62.5 to 125 mg l -1. In growth curves, when compared to the allicin free control, growth at sub-inhibitory concentrations was delayed by 1-2hrs at 15 mg l-1 and by 3-4hrs at 31mg l -1. At 62.5 mg l -1 growth was completely inhibited. In killing curves using AB1000 at a concentration of 500mg l -1, growth was reduced by 30% in 1 hour and by 99.9997% at six hours. This represented a reduction in cfu ml -1 from Log 7.3 to Log 3.8 cfu ml -1. At 24hrs no growth was detected. Conclusion: Allicin (AB1000) was shown to be bactericidal against DRAB at pharmacokinetically achievable concentrations. Killing curve data shows antibacterial activity begins within the first hour of contact. We have also demonstrated that sub-inhibitory concentrations as low as 15mg l -1 can reduce growth. Allicin has previously be shown to be active against multiply drug resistant organisms. Extracts of garlic have also been demonstrated to act as quorum sensing inhibitors reducing the formation of bacterial biofilms. We have shown that our aqueous allicin extract is active in vitro against a range of clinical isolates of DRAB. Allicin can inhibit the growth of DRAB at concentrations as little as 31 to 62mg l -1. We have also shown Allicin can be bactericidal against DRAB Further work is being carried out to determine if allicin inhibits the formation of biofilms by DRAB and how allicin can work in combination with other antibiotics against this multiply – drug resistant organism. The antimicrobial activity of a novel aqueous allicin extract (AB1000) was tested against 11 isolates of DRAB isolated from patients at the Royal London Hospital. Allicin was extracted using a patented cold aqueous extraction method. The allicin content of AB1000 was confirmed using HPLC (Fig 1). Strains were screened for activity using agar diffusion methods on 3 media, isosensitest, nutrient and Muller- hinton (all Oxoid Ltd,UK). Individual plates were lawned with each test organism using the BSAC standard method, 6mm holes were cut in the centre of the plate, 100ul of each dilution of allicin liquid was added to the hole and plates incubated at 37 0 C overnight. Inhibition zones sizes were measured the next day. Growth curves, MICs and MBCs were carried out. Growth was determined using spectroscopy (at 490nm) and killing curves were determined (using viable counts and 500mg ml-1 allicin). Figure 2: Growth curves for LH3 (left) and LH6 (right) showing the effects of different concentrations of allicin Figure 1: HPLC of allicin extract Agar Dilution Tests : Zone sizes were greatest using iso- sensitest agar and least using Nutrient agar. At 500mg l -1 (ppm) the average zone size for all 11 strains was 26.3mm (+/- 3mm)on iso-sensitest agar. Acinetobacter an important nosocomial pathogen. It can grow at various temperatures and pHs, and carries resistance to many antibiotics. Acinetobacter is common in the environment, in soil and water and in addition found on the skin and distal urethra of healthy people. It is also part of the normal skin flora. Acinetobacter has a very high level of resistance to antimicrobials, and relatively few antibiotics are active against it. There is a strain of highly resistant Acinetobacter that is causing wound infections in injured troops in Iraq 1. The respiratory tract is the most common site of infection. Acinetobacter can cause a nosocomial pneumonia, which is often associated with ventilator use. Other predisposing factors for Acinetobacter pneumonia include intensive care residence, intubation, tracheostomy, antibiotic therapy, surgery, and underlying pulmonary disease. Allicin is the main biologically active agent produced in garlic. It is normally very unstable however this aqueous extract is stable for up to 2yrs as a powder. We have previously demonstrated that it can be active against multiply drug resistant bacteria such as MRSA 2, 3 and this study shows its activity against DRAB Figure 1 shows demonstrates the purity of the extract over 98% pure allicin. Figure2 shows that in liquid culture, grow is inhibited at 62.5mg l -1. but below that some strains grow more slowly at 31 or 15 mg l -1. Using 500 15 mg l-1 of allicin (Fig 3) DRAB showed reduced growth (30%) after 1hr. There was no growth at 24hrs. Figure 3: Comparative Killing curves against 4 strains of DRAB (162)