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Environmental Bacteria Antibiotic Resistance Profiling
Indonesia (CIMSA-ISMKI) - Universitas Pelita Harapan, Tangerang
Biology Department, Universitas Pelita Harapan, Jl. M. H. Thamrin Boulevard, Lippo Village, Tangerang, Indonesia
Reinhard Pinontoan, Hans Victor
Type of Research Project
- Basic science
What is the background of the project?
Antibiotic resistance is one main problems out of many in the world. One of the concerning etiology is antibiotic exposure to the environment from improper utilization of antibiotics when not indicated. Microorganism such as bacteria from the environment will then evolve the ability to resist antibiotics through spontaneous mutations or acquisition from other bacteria through horizontal gene transfer. Accordingly, to elaborate and understand the source of resistance and its epidemiology, there are strong needs to generate the antibiotic resistance profile and compare the result to the genome of the bacteria. In order to do so, there are multiple techniques including: broth dilution test, antimicrobial gradient method, and last but not least, disk diffusion test which will be used in this experiment.
What is the aim of the project?
Generate antibiotic resistance profile of some bacteria isolated from the environment and develop the associated genome analysis
What techniques and methods are used?
1. Antimicrobial Susceptibility Testing • Disk Diffusion Test – Agar Phase o Prepare the Mueller-Hinton Agar (MHA) from the dehydrated medium using either distilled water / deionized water o Heat with frequent agitation and boil to dissolve the medium completely o Sterilize by autoclaving at 121 OC for 15 minutes o Check the pH after sterilization, which should be 7.2 – 7.4 at room temperature (done by macerating small amount of medium in distilled water / allowing some medium to gel around a pH electrode) o Cool the agar medium to 40 – 50 OC o Pour the agar to sterile glass or plastic petri dish on a flat surface to the depth of ± 4 mm o Allow the agar to solidify o Dry petri dish in an incubator with temperature 30 – 37 OC for ± 30 minutes • Disk Diffusion Test – Microorganism Phase o From a pure bacterial culture, take 4 – 5 colonies with a wire loop o Transfer to 5 mL of Trypticase soy broth or 0.9% saline o Incubate within optimal growth temperature until turbidity exceed 0.5 MacFarland standard o Compare the turbidity with the standard o Reduce the turbidity with sterile saline o Dip sterile cotton swab into the suspension o Inoculate the agar with streaking pattern o Rotate by 60O and repeat 2x o Dry for 3 – 5 minutes • Disk Diffusion Test – Antibiotic Phase o Place antibiotic disk on the agar o Position disks such that the minimum center to center distance is 24 mm and no closer than 10 to 15 mm from the edge of the petri dish (A maximum of six disks may be placed in a 9-cm petri dish and 12 disks on a 150 mm plate. Reduce the number of disks applied per plate if overlapping zones of inhibition are encountered) 2. Bioinformatics analysis Obtained genome sequences will be analyzed (eg. using interpretative standard for pathogens – diameter of inhibition zone comparison to standard) to establish the presence of resistance / susceptibility, find the mechanism of resistance, and the possible etiology
What is the role of the student?
- The student will mainly observe
- The student will observe the practical experiments but will be highly involved in the analysis of the results
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
1. The incoming students shall be under strict observation of the tutor and/or department head 2. The students will perform antibiotic susceptibility testing using the disk diffusion test 3. Students will do Bioinformatics analysis of the obtained genome sequence to establish the presence of resistance / susceptibility, find the mechanism of resistance, and the possible etiology
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Theoritical teaching about the bacteria, disk diffusion test, and antimicrobial resistance will be given by Reinhard Pinontoan and/or Hans Victor
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a presentation - The student will prepare a scientific report
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Basic laboratory tools (ex. Pippete, agar, pH meter, standard beaker, stirrer, etc.) handling skills. Subjects passed: Microbiology. Previous experience with: basic laboratory task
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Graduated students (less than 6 months) - Pre-Medical students from the American-British system - Students in biomedical fields
- Nathan; Carl; and Otto Cars. “Antibiotic Resistance — Problems; Progress; and Prospects.” New England Journal of Medicine; vol. 371; no. 19; 2014; pp. 1761–1763.; doi:10.1056/nejmp1408040
- Tendencia; E. A. (2004). Disk diffusion method. In Laboratory manual of standardized methods for antimicrobial sensitivity tests for bacteria isolated from aquatic animals and environment (pp. 13-29). Tigbauan; Iloilo; Philippines: Aquaculture Department; Southeast Asian Fisheries Development Center.
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