Antibiotic Resistance of Bacterial Agents of Disease - Research Proposal Example

APPENDIX 3 APPLICATION FOR BIOL5292M RESEARCH CONTRACT (GRANT PROPOSAL/WORK PLAN Details of Principal Applicant Sur Fore Position Held Telephone Fax E-mail Main contact address if different from company address 2. Project title (not exceeding 120 characters)* Investigations into the mutability of Staphylococcus aureus biofilms: effect of antioxidants 3. Abstract of proposed research project (not exceeding 750 characters)* The project aims to study the effect of antioxidants on the mutational frequency and development of antibiotic resistance of planktonic and biofilm cultures of Staphylococcus aureus mutT strains. Bacteria will be grown in media with different antioxidants namely ascorbic acid, tannic acid, taurine, epigallocatechin and vanillin. After exposure to the antioxidant, the bacteria will be plated to selective nutrient agar plates laced with the antibiotics, fusidic acid and mupirocin. The number of antibiotic-resistant colonies that have survived will be counted and adjusted to the viable cell count, which has been simultaneously determined. The mutational frequencies will be the number of cells expressed as a fraction of the viable cell count. 4. Project length Duration of Project (months) 5 months Proposed Start Date 2 March 2009 5. Consumables required for the project Table 1. Materials and cost of materials that are needed in the conducting the project activities. Item Cost £ Petri dishes 48 Falcon tubes (VWR) 200 Mueller Hinton broth and agar (Oxoid) 200 Brain Heart Infusion Broth (Oxoid) 200 Carbonate-Bicarbonate buffer capsules (Sigma) 50 Citrate buffer ingredients ( Sodium citrate – Sigma and Citric acid – Fisher) 60 Cellulose (Biochemika) 40 Cellulose ester disks 0.22um (Millipore) 115 Antioxidants L-ascorbic acid (Fisher) 14 Tannic acid (Alfa Aesar) 35 Taurine (MP Biomedicals) 12 Epigallocatechin (Sigma) 37 Vanillin (Sigma) 14 Fusidic acid (donated by Leo Pharma) - Mupirocin (donated by Pliva Pharma) - Ethanol (Fisher) 60 Dimethyl sulphoxide (VWR) 90 Phosphate buffered saline (Oxoid) 10 Total Annual Costs 1,185 6. Equipment required to undertake project (no equipment costing over £1000) 7. Description of the research to be conducted The prevalence of increased antibiotic resistance of bacterial agents of disease is a serious clinical concern because it limits and reduces the efficiency of the treatment options that are available. The mechanism of development of antibiotic resistance has been attributed to bacteria’s capacity to mutate. Mutation alters existing resistance determinants to antimicrobial agents and produces different drug targets that have decreased antibiotic affinity. Thus, mutators that have high mutation frequencies could have important roles in developing antibiotic resistance (Chopra, O’Neill, & Miller, 2003). It is believed that bacteria mutate as part of its endogenous survival mechanism. In E. coli, defects in genes in pathways that are involved in DNA repair and error correction systems, and prevention of incorporation of oxidized guanines, lead to the development of permanent mutator phenotypes (Miller, ONeill, & Chopra, 2002). High frequencies of mutations can be toxic to bacterial cultures but there are reports of naturally occurring organisms that have 1000-fold greater than normal mutation frequencies. Agents that can damage the DNA include ionizing radiation and reactive oxygen species that cause strand breaks and removal of amine and purine groups in the DNA. Oxidation also produces 2’-deoxy-7, 8-dihydro-8-oxoguanosine, (or 8-oxo-dG) from guanine. 8-oxo-dG will pair with adenine which resulting in transversion (Shibutani, Takeshita, & Grollman, 1991). MutT, MutM and MutY are bacterial enzymes that eliminate 8-oxo-dG or reduce its effects. Of the three enzymes, it is the loss of mutT activity that has the largest effect on increasing mutational frequencies (Miller J. , 1996). Due to the importance of the problem of antibiotic resistance, there are studies that have evaluated the antimutagenic properties of antioxidants. While the antioxidants do not directly affect the mutator strains physiologically, it is purported that these could inactivate and prevent oxidative stress from increasing mutational frequency. The possibility of the occurrence of this antioxidant mechanism was tested by growing planktonic E. coli mutS and mutT strains with antioxidants, and thereafter determining the mutational frequencies of the cultures. It was found that the antioxidants significantly reduced mutation frequencies in mutT (Chopra, O’Neill, & Miller, 2003). Although this result has no practical significance yet, because there are no reports of naturally occurring mutT strains, follow-up studies are deemed important because they give insights into possible pathways that can be expanded to understanding antibiotic resistance in other economically important pathogenic bacteria like Staphylococcus, Streptococcus and Bacillus. Likewise, it is also interesting to study the response of bacterial biofilms to antioxidants. Possibly the interactions that are present in biofilms will affect antibiotic resistance on a different level. By their very nature, biofilms have an inherent tolerance to antibiotic agents and, through horizontal gene transfer, can spread antibiotic resistance genes (Fux, Costerton, Stewart, & Stoodley, 2005). This research project, therefore, aims to study the effect of different antioxidants on the mutational frequency and the development of antibiotic resistance of Staphylococcus aureus mutT strains grown in planktonic and biofilm cultures. Detailed plan of research Staphylococcus aureus mutT strains will be obtained through the laboratory of Dr. Ian Chopra. The bacteria will be cultured at 37 °C in Mueller Hinton Agar (MHA), which will be purchased from Oxoid (Basingstoke, UK). Planktonic cultures will be grown in Mueller Hinton broth (MHB). All chemicals, reagents, antioxidants, and antibiotics will be purchased as listed in Table 1. The minimum inhibitory concentrations (MIC) of the antibiotics fusidic acid and mupirocin will be determined using the agar dilution method, before doing the main experiments. Antibiotics will be diluted serially and mixed with MHA after media sterilization, and before pouring to Petri plates. S. aureus mutT inocula of 104 cfu/spot will be applied and allowed to grow. MIC is defined as the lowest antibiotic concentration preventing visible bacterial growth after 18 h of incubation at 37°C. Selective MHA plates will be prepared by adding 4x MIC of the antibiotics to the culture media. Antioxidant enriched- MH broth will be prepared in sufficient amounts, and 10 ml will dispensed into 12 separate culture flasks. Twelve flasks will allow for duplication of cultures and blanks. A flask will contain 1 mg antioxidant/ml broth (but for epigallocatechin 0.1mg/ml). The control flask will have only MHB and no antioxidant. To prepare the planktonic cultures, an ml of overnight culture of bacteria will be grown in 10 ml of antioxidant enriched-MHB for 24 hours at 37°C, with shaking. After this period, 200 ul bacterial culture will be plated to selective MHA containing either fusidic acid or mupirocin. After 48 hours, the colony growth on selective media, and viable cells will be counted. S. aureus biofilms will be created using a Sorbarod apparatus as described previously (Muli & Struthers, 1998; Budhani & Struthers, 1997). Cellulose Sorbarods (10 mm) in diameter and 20 mm in length) will be placed in 10 cm of silicone tubing with an internal diameter of 10 mm. These will be connected by tubing to a supply of broth. The effluent end of the Sorbarod will be attached to 150 ml sterile glass bottles to enable the collection of effluent fluid. Twelve individual Sorbarods will be prepared in order to have sufficient number for duplicate trials for each antioxidant, and an antioxidant-free control. The Sorbarod biofilms shall be inoculated with 3 ml of an overnight broth culture of S. aureus. The entire set-up will be placed inside an incubator at 37°C. MHB will be delivered to each biofilm at the rate of 0.1 ml/min by means of a 12-channel peristaltic pump. The effluent will be collected downstream of the biofilm. After 24 hours, the individual biofilms will be exposed for another 24 h to a single concentration of an antioxidant, which will be applied in brain heart infusion (BHI) broth, to be delivered to the biofilms at the same flow rate of 0.1 ml/min. After 24 hours, bacteria will be harvested from Sorbarod filters by splitting the filters to release the fibres, and then vigorously vortexing for 30 s, sonication, and further vortexing (Driffield, Miller, Bostock, & ONeill, 2008). Mutation frequencies for resistance to fusidic acid and mupirocin will be determined for planktonic and biofilm cultures. Two hundred microliters of bacterial cells recovered from biofilms and planktonic cultures will be immediately plated to MHA selection plates containing 4x MIC to determine the mutational frequency. To determine the viable counts, aliquots of diluted culture will be plated to non-selective MHA. Colony counts will be done after 24 h of incubation at 37°C on non-selective media and after 48 h of incubation on selective media (Driffield, Miller, Bostock, & ONeill, 2008). The effect of antioxidants on the mutational frequencies will be expressed as the number of the number of antibiotic-resistant mutants recovered as a fraction of the viable count. All experiments will be done twice for validation of the initial results. Justification of resources requested The resources requested are all necessary for the successful conduct of the research. Five antioxidants will be used because it is presumed that different antioxidants have different effects on the mutational frequency. Significance of the research for the UK (health & wealth of the UK) Staphylococcus aureus is a dangerous pathogen in humans. S. aureus is the causative agent for many dangerous health conditions like boils, abscesses, sties, food poisoning, pneumonia, deep abscesses and meningitis. There are steady increases in the community and hospital acquired staphylococcal infections with treatment becoming more difficult due to the emergence of multi-antibiotic resistant strains (Lowy, 1998). Knowing the role of mutations on the development of antibiotic resistance is already a big step in medicine, but no means to reduce and control mutation have been identified. The observation that mutational frequency can be reduced by the introduction of antioxidants to bacteria offers hope that natural products can help in indirectly reducing antibiotic resistance. Research towards validation of antioxidant effects and understanding the mechanisms of action will lead to the design of more effective drugs to fight microbial diseases. This will therefore contribute indirectly towards the reduction in the prevalence of infectious bacterial diseases, design of better drugs leading to a healthier population in the country. Dissemination of knowledge and route of exploitation or translation of research outcomes into relevant sectors of industry/society Knowledge derived from the study can be disseminated through articles in scientific journals and news reports. It is hoped that this preliminary study will continue on to larger studies towards understanding the many facets of antibiotic resistance. 8. Project objectives The main objective of the project is to understand the effect of antioxidants on the development of antibiotic resistance in planktonic bacteria and bacterial biofilms culture of S. aureus mutT strains. Project plan provide details of the main milestones and target dates for the proposed project. You may wish to include a Gantt chart or similar to illustrate the project plan. Milestone No. Target Date Title 1 March 10, 2009 Completed purchase of materials required for project 2 March 17, 2009 Flasks, plates sterilized, and bacterial cultures prepared 3 March 31, 2009 Subcultures prepared; MIC determined 4 April 14, 2009 Preliminary trials for setting up Sorbarod filter apparatus 5 April 24, 2009 Initial trial for planktonic bacterial cultures finished 6 April 31, 2009 Second run on planktonic bacterial cultures finished 7 May 15, 2009 Initial run on Sorbarod filters finished 8 May 30, 2009 Second run on Sorbarod filters finished 9 July 17, 2009 Trials with questionable results will have been repeated 10 August 10, 2009 Report submission 11. Health and Safety i) I have completed and submitted appropriate risk assessment forms to the laboratory I am working in: Yes No  ii) I have completed and submitted the safety induction form to the School Safety Officer : Yes No  Signed: Date: References Budhani, R & Struthers, J 1997, ‘The use of Sorbarod biofilms to study the antimicrobial susceptibility of a strain of Streptococcus pneumoniae’, Journal of Antimicrobial Chemotherapy, vol. 40, 601-602. Chopra, I, O’Neill, A & Miller, K 2003, ‘The role of mutators in the emergence of antibiotic-resistant bacteria’, Drug Resistance Updates, vol. 6, pp. 137-145. Driffield, K, Miller, K, Bostock, J & ONeill, A 2008, ‘Increased mutability of Pseudomonans aeruginosa in biofilms’, Journal of Antimicrobial Chemotherapy, vol.61, pp. 1053-1056. Fux, C, Costerton, J, Stewart, P & Stoodley, P 2005, ‘Survival strategies of infectious biofilms’, Trends in Microbiology, vol. 13, no. 1, pp. 34-40. Lowy, FD 1998, ‘Staphylococcus aureus infections’, New England Journal of Medicine, vol. 339, no. 8, pp. 520-535. Miller, J 1996, ‘Spontaneous mutators in bacteria: insights into pathways of mutagenesis and repair’, Annual Review of Microbiology, vol. 50, pp. 626-643. Miller, K, ONeill, A & Chopra, I 2002, ‘Response of Escherichia coli hypermutators to selection pressure with antimicrobial agents from different classes’, Journal of Antimicrobial Chemotherapy, vol. 49, pp. 925-934. Muli, F & Struthers, J 1998, ‘Use of a continuous-culture biofilm system to study the antimicrobial susceptibilities of Gardnerella vaginalis and Lactobacillus acidophilus’, Antimicrobial Agents and Chemotherapy, vol. 42, no.6, pp. 1428-1432. Shibutani, S, Takeshita, M & Grollman, A 1991, ‘Insertion of specific bases during DNA-synthesis past the oxidation-damaged base 8-oxo-dg’, Nature, vol. 349, pp. 431-434. Read More