Different Levels Of Antibiotic Resistance In Some Native Michigan Birds Gut Bacteria
Introduction
Antibiotics are chemical compounds that are very toxic to microbes than they are to human cells. Each of them targets a specific part of the cell of the microbe such as cell structure or cell pathway; therefore, they are selective toxics. For example, some antibiotics target the protein synthesis and eventually stop the process by binding to the 70S prokaryotic ribosomes. If the same group of the antibiotics are used in eukaryotic cells which have larger 80S ribosome, they would have less effect. This is an evidence supports the fact that the antibiotics are selective toxics. Each microbial cell has a resistance to antibiotics, this resistance could be measured using Kirby-Baurer assay test. Antibiotics are always challenging the researchers and people who work in public health field because by the time they make the normal bacteria presents in human and other animals more resistant.
Because antibiotics are used for treating bacterial and microbe’s infections in humans and animals, many researchers are working on enhancing and developing their effectiveness. Antibiotics often have microbial origin. In general, animals play an important role in spreading of some antibiotic resistance gens. Wild birds would be a good example of animals spreading the antibiotic-resistant bacteria. Because birds usually migrate for long distances they can carry bacteria and spread antibiotic-resistant genes to different destinations. E coli is the most common organism used in labs for research purposes because it is cheap, easy to get, and has rapid growth. A study was conducted on Escherichia coli isolates from Arctic birds which tested using 17 different antibiotics, this study shows that almost about 8% of E. coli showed resistance to at least 1 of the total 17 antibiotics.
Each environment has its own components such as water, soil, air, ecosystem, and climate features. There is a significant pollution levels in urban areas due to different sources such as motor traffic, smoke, noise, and urban rapid population growth. Compared to urban lands, rural areas have less pollution as there is less traffic, noise, and smoke. Because urban and rural lands have different levels of pollution, the components of their ecosystem would be different. As a result, the antibiotic-resistant gens may have differences between the rural and urban lands. Wild birds living close to urban lands and cities would be good antibiotic resistant bacteria reservoirs. A study was conducted on E. coli in a rural land in republic of Czech has clearly shown the antibiotic resistant in rural areas. The researchers collected 183 rectal samples from calves, 95 rectal samples from cows, 33 rectal samples from young bulls, and 54 cloacal samples from house sparrows. These samples were isolated and tested for antibiotic resistant. The researchers have found that 9% of the E coli isolates from house sparrows were antimicrobial resistant. This is a very significant percentage compared to 3% resistant E coli isolated from cows and 0% antimicrobial resistant E coli isolated from young bulls. Another study was conducted in Hamilton, Ontario (urban area) on E coli, 462 samples were isolated and tested for antibiotic resistance. Most of resistant species come from bird feces and wastewater sources.
Like other vertebrates, birds have a lot of bacteria in their guts as concentrated as 1011 CFU/g in the hindgut. In summer 2014, a study was conducted on four species of Michigan wild bird which are American Robin, Song Sparrows, Black-Capped Chickadess, and Gary Catbirds. Amoxicillin, tetracycline, and ciprofloxacin were used to test the following hypothesis “Some Michigan wild birds spread antibiotic resistant bacteria”. 148 strains of antibiotic resistant bacteria were identified. This project aims to re-testify the previous hypothesis and to identify two unknown antibiotic resistant bacteria collected from some Michigan wild birds gut using different antibiotics from the ones were used back in 2014. The expected outcome of this study is to identify the unknown antibiotic resistant bacteria collected from Michigan wild birds.
Methods
Section/Isolation
Two unknown (#2 and #21) of antibiotic resistant bacteria were assigned to me. To study and examine these two unknowns, a separation process needs to be done in advance to get visible colonies from the unknowns. To do this, two TSA plates were labeled correctly (name, date, organism name, media used, and temperature) and the two unknowns were streaked according to the procedure on page 28-29 of the lab manual. After this, the TSA plates were put in 37C temperature for 48 hours in order to give enough time to bacteria species to divide.
Gram-staining technique
The first step in the process of identifying the bacteria is gram morphology (whether a bacterium is gram-negative or gram-positive). A gram-positive bacteria cell wall has a very thick layer of peptidoglycan (about 90% of the total components of the cell wall) and small pores, while a gram-negative bacterium has a thin layer of peptidoglycan (about 10% to 15% of the total components of the cell wall) and larger pores. Because of these differences in the structure, gram-positive and gram-negative appear differently under the microscope. A gram stain test was conducted on the unknowns as described in page 88-89 on the lab manual. PCRTwo micro centrifuge tubes were labeled with our unknown numbers. Two DNA templates for the isolated colonies were prepared, 10 uL sterile water were added to each tube, two disposable plastic loops were used to transfer the colonies from the agar plated to the micro tubes, each tube was mixed thoroughly. 1uL of the previous dilution mixture was transferred to the PCR tubes and PCR mix (0. 5uL of forward primer 8F, 0. 5uL reverse primer 1492R, 1 uL DNA template, 10. 5 uL sterile water, and 12. 5uL mean green master mix) was added to previous tube. Everything was centrifuged for 3 to 5 seconds.
Gel electrophoresis
PCR samples were run on agarose gel to confirm that the 16s rRNA gene was amplifies correctly. A dye was added to visualize how far the samples have traveled through the gel. To do the gel electrophoresis step, 3uL were added to each PCR in a 0. 7 agarose gel, the gel was run for one hour in at 80V in SB buffer, after this the gel was stained with Ethidium Bromide for fifteen minutes and visualized with a kodak gel logic 1500 imaging system. The gel electropolish was completed by the teaching assistant (PCR handout).
DNA purification
Two microcentrifuge tubes were labeled with our unknowns. 125uL of buffer were mixed with 25uL PCR using pipette, the mixture was transferred to a spin column, centrifuged at 10000 x g for 30 seconds. After this step, 200uL of buffer was added to the column and the centrifuge step was repeated for 30 seconds. The flow was discarded, and the remaining components were centrifuged for 30 seconds to get rid of ethanol in the column. The contents were transferred to a new microcentrifuge tube, 20uL of water was added, the column was left to sit for one minute and centrifuged at 10000 for 30 seconds.
DNA preparation for sequencing
A DNA sequence for each of our unknown organisms was obtained from the previous PCR test. The Basic Local Alignment Search Tool searches for similarities between our unknown sequences and other sequences stored in huge database. First, the BLAST website was accessed, then “Nucleotides BLASR” section was clicked, the unknown sequence was copied to the “Search” box, “Blast” box was clicked to start searching for similarities. The closest and most similar unknown was selected. This procedure has repeated twice since we have two unknowns.
Section two
In order to test the response of our unknown’s bacteria strains, a Kirby-Bauer assay test was used for this purpose. After the unknown strains were spread on the plate, paper disks that have the antibiotics are applied to the surface of the plate, and then stored at 37C for 48 hours. This test was repeated for the second unknowns. On of unknowns (#21), did not work in the temperature so the procedure was repeated, and the plate was stored at the room temperature for another 48 hours. The antibiotics used in this test were Sulfadiazine, Doxycycline, Ampicillin, and Vancomycin.
Sulfadiazine cn denature the hydrogen bonds between Nitrogen bases in the DNA
Doxycycline is one of the tetracycline family which blocks aminoacyl-tRNA binding site on the ribosomal acceptor. Ampicillin can bind to PBPs proteins inside the bacterial wall and inhibit the cell wall synthesis process which leads to lysis of the cells. Vancomycin binds to the cell wall precursor terminating in Lys-Ala-Ala, which alters the permeability of the cell wall and RNA synthesis.
Results
Unknow #2 and Unknown #21 were bacterial unknowns from some Michigan wild birds. The first test was bacterial growth in glucose nutrient broth, and both unknowns formed a small pellet in the bottom of the test tube (sediment). The second test were the gram stain test, and both are gram-negative. Then, a gel electrophoresis was used to separate DNA fragments based on their size, because DNA is negatively charged, small DNA fragments move away from the positive side faster than the big fragments. Both of unknowns were well separated and stopped at around 1500 bp which means both of our unknowns’ DNA have close masses. The data from PCR was entered to BLAST data base to determine the identity of our unknowns. According to BLAST tool, unknown #2 is Bacillus Cereus with 100% match, and unknown #21 is Providencia Sp with 100% match. Both match percentages are 100% which indicates to us that the PCR results are identical to the real species in BLAST database.
Based on the antibiotic sensitivity test we preformed, both of our bacteria species shows a resistivity to at least one antibiotic. Bacillus Cereus showed a resistivity to Ampicillin and Providencia Sp showed a resistivity to Doxycycline, Ampicillin, and Vancomycin. This data confirms our hypothesis we proposed for our research “Some Michigan wild birds spread antibiotic resistant bacteria” and identifies two resistant bacteria species isolated form some Michigan wild birds. Our data confirms that Michigan birds spread some resistant bacteria while they migrate or move from one place to another.
