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APEC Youth Scientist Journal Vol.7 / No.2 ACQUIRING OF BACTERIAL ABR STRENGTH BY VARIOUS
CONCENTRATIONS OF ANTIBIOTICS AND SPREADING
PATHWAY OF ABR TRAIT
1Suffield Academy, 185 North Main Street, Suffield, CT 06078 USA This research was conducted to examine the extent bacteria develop antibiotic resistance (ABR) from contemporary and old bacteria, and understand its underlying mechanisms of the process. Common bacteria and B. cereus, S. aureus, E. coli, and S. gallinarum were cultured with various concentrations of antibiotics and underwent agar diffusion tests to determine resistance. Then ABR and non-ABR bacteria were cultured in a single solution and ABR was tested. Plasmid DNA from ABR bacteria were implanted into non-ABR bacteria and resistance of possibly transformed bacteria was tested. Finally, plasmid DNA were extracted from ABR bacteria and inserted to non-ABR bacteria of the same kind through heat shock transformation and resistance determined. Results showed that common bacteria and B. cereus, S. aureus, E. coli, and S. gallinarum have grown ABR to old antibiotics and are starting to develop it on newer antibiotics. Non-ABR bacteria develop stronger ABR under lower concentration of antibiotics through proximity with ABR bacteria. Also, bacteria transformed with ABR plasmid DNA exhibited an ABR trait. It can be concluded many bacteria have adjusted to antibiotics, and they have grown this ABR through exposure to low concentration of antibiotics, proximity with ABR bacteria, and plasmid DNA transformation from ABR bacteria.

* Corresponded to: Sang On PARK ([email protected])
APEC Youth Scientist Journal Vol.7 / No.2 1. INTRODUCTION
Antibiotics or antimicrobial drugs are used to cure bacterial causing illnesses. Bacteria are single celled organisms, and certain kinds are believed to cause illnesses. Although antibiotics can decimate bacteria, there is a consequence. When bacteria are exposed to antibiotics, initially, the sensitive majority will be killed; however, there will be surviving minority, which had different trait to dead ones. Like Darwin's survival of the fittest theory, these fit bacteria prevail, and the population will be less susceptible to the next exposure. (Bergman, 2015) Swedish research team further found that heavy usage of antibiotics increases the mutation frequency in bacteria. (Gustafsson et al., 2015) This antibiotics resistance nuisance is becoming more serious as antibiotics resistance was found even for ceftaroline, which was introduced in 2010. (Timeline of Antibiotic Resistance, 2015) Bacteria are acquiring stronger resistance to antibiotics as old introduction and long exposure is decreasing antibiotics effectiveness. (Harjvan et al., 2015) Contemporary bacteria have higher resistance than those from the time before antibiotics were introduced. (Houndt & Ochman, 2015) There are several classes of antibiotics: penicillin, cephalosporin, macrolides, fluoroquinolones, sulfonamides, tetracycline, and aminoglycosides. Symptoms are often bacteria specific, so doctors prescribe appropriate antibiotics accordingly. (Stephens, 2015) There are two classes of bacteria: gram-positive and gram-negative. When purple dye is dropped, gram-positive acquire dye's color, but gram-negative are colored red or pink. (A Brief Overview of Classes of Antibiotics, 2015) Like bacteria there are two big classes of antibiotics. One is bacteriostatic agent, which inhibits the growth and reproduction, and another is bactericidal agent, which destroys bacteria. Penicillin, cephalosporin, fluoroquinolones, and aminoglycosides are considered bacterial agents; macrolides, sulfonamides, and tetracycline are considered bacteriostatic agents. Science has discovered means in which bacteria pass down its antibiotics resistance. One way is the inheritance of plasmid DNA, which will enter other bacteria and make changes accordingly. A University of Granda made a hypothesis that with indiscriminate antibiotics use, antibiotics put more stress on bacteria, which will then move more actively and accept more DNA because bacteria usually do not search for new DNA. (New Hypothesis: Why Bacteria are becoming Increasingly more Resistant to Antibiotics, 2015) Report on 2007 Science magazine says that a research team has found stealth gene like Sfh protein. Sfh protein is believed to transmit plasmid DNA without changing bacteria's fitness much because bacteria had serious change in fitness when plasmid DNA was transmitted without Sfh protein. (Doyle et al, 2015) APEC Youth Scientist Journal Vol.7 / No.2 Howard Hughes medical Institute discovered that antibiotic resistant gene could be transmitted by cell-cell contact and integrating into a chromosome. (Beaber et al., 2015) Finally, research team from Uppsala University discovered that plasmid DNA that contains antibiotics resistance can be strengthened by exposure to very low level of antibiotics and heavy metal. (Gullberg et al., 2015) Based on these researches five experiments were conducted. First two were to test ABR trend in common bacteria and stored specific bacteria. Common bacteria from soil were done agar diffusion test with antibiotics, and stored specific bacteria were done the same agar diffusion test with same antibiotics. Certain bacteria and antibiotics were selected for further experiments to figure out mechanisms for ABR spread, for bacteria with strong ABR to certain antibiotics would be inappropriate for the purpose. Next three experiments were conducted to propose three possible mechanisms for ABR development. The first one was exposing bacteria to different concentrations of antibiotics and doing agar diffusion test with 0.001x cef. to find how concentration of antibiotics could play role in ABR development. The second was cultivating ABR bacteria and non-ABR bacteria in same nutrient broth to find possible directional shift in both or one regarding ABR development. The last was extracting plasmid DNA from ABR bacteria and implanting it on non-ABR bacteria to ensure whether transformation by plasmid DNA could help the ABR development. 2. MATERIALS AND METHODS
Experiment 1: Status of ABR to various antibiotics on different types of soil bacteria
Bacteria samples were made by 0.1 g of three different soil samples, labeled 1 through 3, from different flowerpot mixed with 0.9 mL distilled water in respective 1.5 mL micro tube. Components were then mixed by vortex mixer and waited for soil to suspend. Mixture was streaked with 10 micro liter loop on NA. The solution was streaked on the half of the NA, and dish was turned 90 degrees. The solution was again streaked half of NA from the perspective, but only upper half was overlapping with previous streaking. Streaking continued from the second step until NA was completely covered with solution. NA plates were put in incubator with 37 degree Celsius for 24 hours. Cultivated bacteria were done agar diffusion test by poking 7 circles in the middle of NA with micropipette white tip, picked up by tong heated by alcohol lamp. Dropping 10 microliters of prepared antibiotics on each circle and incubating ends the test. Cefazedone and kanamycin were diluted to 0.1x. Using APEC Youth Scientist Journal Vol.7 / No.2 micropipettes, 0.9 mL of distilled water was put in a tube and 0.1 mL of 1x antibiotics were Experiment 2: Acquiring of ABR on bacteria exposed to various antibiotics
8 petri dishes were prepared for each bacterium: Escherichia coli, Salmonella gallinarum, Bacillus cereus, and Staphylococcus aureus. 10 micro liters of each bacterial solution were dropped on all 8 petri dishes and were spread 30 times with a spreader. A circle was poked on each petri dish and all different eight antibiotics were dropped 10 micro liters on different bacteria petri dish respectively. Experiment 3: Different degree of bacterial ABR strength by various concentrations of
antibiotics
Three selected bacteria that are thought to not have resistance to two chosen antibiotics were done agar diffusion test with different concentrations for cefazedone and lincomycin: 0.1x, 0.01x, 0.001, and 0.0001x. Mixing 100 microliters of 1x, 0.1x, 0.01, and 0.001x with 900 microliters of nutrient broth in a tube. Bacteria were exposed to the antibiotics by dropping 10 microliters of bacterial solution into a tube for every bacterium. After 24 hours of incubation agar diffusion test were done for each bacterium with 5 microliters of 0.001x cefazedone. Experiment 4: Antagonistic effects between ABR acquired bacteria and normal bacteria
in co-culture
ABR bacteria and non-ABR bacteria were mixed in NB. ABR bacteria were scratched from experiment 3 samples, specifically ones close to the clear zone. For non-ABR bacteria stored bacterial solution was used. The NB solution was incubated for 24 hours. There were three solutions: E. coli and S. gallinarum solutions with lincomycin resistant bacteria mixed and cefazedone resistant S. gallinarum mixed with stored S. gallinarum. Then, the solution was done agar diffusion test with 0.001x cefazedone and incubated for 24 hours. Experiment 5: Possibility of ABR spread by plasmid DNA transfection
Before mixing ABR bacteria from experiment 3 and bacterial solution without ABR volume for each solution had to be calculated so that the absorbance is 0.1 compared to nutrient broth. After mixing appropriately the solution was incubated for 24 hours. Then, 20 or 30, depending on clarity, microliters of solution were dropped and spread on NA. NA was APEC Youth Scientist Journal Vol.7 / No.2 poked with a circle in the middle and 5 microliters of 0.001x cefazedone was dropped. NA are incubated for 24 hours again. - Plasmid DNA isolation; Both no ABR bacterial solution and ABR bacterial
solutions were centrifuged at 13000 rpm for a minute. The remaining liquid is disposed. 250 microliters of buffer 1 was added to remainders and mixed by vortex machine. 250 microliters of buffer 2 was added and mixed by inverting the micro tube 4 times. 350 microliters of buffer 3 were added and mixed by inverting 4 times. The tube is centrifuged at 13000 rpm for 19 minutes. Cleared lysate was transferred to DNA binding column tube and centrifuged at 13000 rpm for a minute. Liquid was disposed and 700 microliters of buffer 4 was added to DNA binding column tube. The tube was centrifuged at 13000 rpm for a min. Liquid was again disposed and the filter was dried of residual ethanol by centrifugation at 13000 rpm for a minute. The filter as moved to 1.5 mL micro centrifuge tube, and 100 microliters of buffer 5 or distilled water were added to DNA binding filter column and centrifuged at 13000 rpm for a minute for elution. DNA is ready. - Heat shock transformation; To combine DNA, 2 mL of bacteria were put in ice for
10 minutes first. Then, bacteria were centrifuged at 4500 rpm for 10 minutes. Liquid formed will be disposed, and 1 mL of 0.1M CaCl2, made by mixing 0.50 g of CaCl2 and 45 mL of distilled water, was dropped in and mixed by inverting. The bacteria are then put in ice for 15 minutes. Then, bacteria are centrifuged at 3500 rpm for 10 minutes. Liquid is disposed again, and 200 mL of previous CaCl2 solution was added. Again, bacteria are put in ice for 30 minutes, 100 microliters of plasmid DNA are dropped in. The tube is put in 42 degree Celsius for 2 minutes and at 0 degree Celsius for 2 minutes. 9 mL of nutrient broth was put in, and bacteria were incubated. After planting plasmid DNA of ABR bacteria to no ABR bacteria and incubating for 24 hours the solution was dropped 20 to 30 microliters, depending on the clarity, on NA and did diffusion test with 0.001x cefazedone. APEC Youth Scientist Journal Vol.7 / No.2 3. RESULTS
Experiment 1: Status of ABR to various antibiotics on different types of soil bacteria
Different bacteria from each soil sample were done agar diffusion test to observe how much ABR did common bacteria grown. Classifying of bacteria was based on the size and color of soil bacterial colonies within the same soil sample. While most or all bacteria had resistance to relatively old antibiotics like ampicillin, methicillin, chloramphenicol, and penicillin, which means they had clear zone diameter of only initial hole for agar diffusion test, only 3 had resistance to cefazedone. All others had increase in clear zone diameter, ranging from 1.3 cm in soil sample 2 bacteria d1 to 4.8 cm increase in bacteria e from soil sample 3. Additionally, only 5 had resistance for kanamycin, and only 1 had resistance for lincomycin. The most resistant without ABR was soil sample 1 bacteria b2 with diameter of 0.8 cm, and the least resistant was soil sample 1 bacteria c2 with diameter of 1.8 cm. The least resistant to lincomycin was soil sample 1 bacteria a with 4.7 cm diameter and most resistant was soil sample 2 bacteria c2 with diameter of 0.8 cm. Antibiotics with low resistance were introduced later than those with high resistance. Penicillin and Chloramphenicol were introduced in between 1940 and 1950. Methicillin and Ampicillin were introduced around 1960. Cefazedone, kanamycin, and lincomycin were introduced after 2000. The conclusion can be drawn that common bacteria like those in the soil have developed resistance to old antibiotics over time, but did not form resistance to newer antibiotics. They are growing ABR, however, as evident in some bacteria having strong or ABR to modern antibiotics. APEC Youth Scientist Journal Vol.7 / No.2 Table 1: Diameter of clear zone to various antibiotics on soil bacteria Experiment 2: Acquiring of ABR on bacteria exposed to various antibiotics
Four bacteria, E. coli, S. gallinarum, B. cereus, and S. aureus, were done agar diffusion test with seven different antibiotics to figure which bacteria has ABR to which antibiotics thus selecting appropriate bacteria and antibiotics for experiment 3. Resistance was measured in diameter of the clear zone. While S. gallinarum did not have resistance for all antibiotics, E. coli, B. cereus, and S. aureus all had resistance for all antibiotics except cefazedon, kanamycin, and lincomycin. All four bacteria had resistance to cefazedone. E. coli had 5 cm clear zone. S. gallinarum had 3.4 cm and B. cereus had 5.4 cm. S. aureus had 8.5 cm clear zone. Bacteria for next experiment were selected; they were all bacteria except S. aureus because it had the lowest resistance to antibiotics that all bacteria did not have resistance to. This is shown in kanamycin and lincomycin as well because S. aureus had 1.8 cm clear zone and 5.4 clear zone respectively. E. coli had 1.1 cm and 3 cm; S. gallinarum had

APEC Youth Scientist Journal Vol.7 / No.2 1.1 cm and 4.4 cm; B cereus had 1 cm and 3.7 cm respectively. Antibiotics were selected cefazedone and lincomycin because even though bacteria did not form complete resistance against kanamycin, they had the smallest clear zone with kanamycin, suggesting that they formed quite robust but not complete resistance. Conclusion can be drawn that E. coli, B. cereus, and S. aureus are appropriate bacteria, and cefazedone and lincomycin, which bacteria not yet have resistance but have smaller clear zone, are appropriate antibiotics for next experiment. Table 2: diameter of bacterial clear zone according to various antibiotics Figure 1: Clear zones of four bacteria with different antibiotics Experiment 3: Different degree of bacterial ABR strength by various concentrations of
antibiotics
To figure out whether bacteria exposed to cefazedone acquires antibiotics resistance to cefazedone, three kinds of bacteria, E. coli, S. gallinarum, and B. cereus, were cultivated with various concentrations of antibiotics and did agar diffusion test to cefazedone S. gallinarum had identical size from 0.1x cefazedone to 0.001x cefazedone of 8.5 cm, but on 0.0001x cefazedone clear zone diameter of 8.5 cm, nearly a diameter petri dish, dropped to 5 cm. B. cereus, like S. gallinarum, had similar size up to 0.001x cefazedone of 7.1 cm, 6.4 cm, APEC Youth Scientist Journal Vol.7 / No.2 and 7.3 cm, but at 0.0001x cefazedone, its clear zone decreased from 7.3 cm to 5.8 cm. E. coli had different pattern. As the concentration got dimmer the size of clear zone dwindled. From 0.1x cefazedone to 0.01x cefazedone, diameter dropped from 8.5 cm to 6.6 cm. Transition from 0.01x to 0.001x did not affect the diameter, but diameter dropped from 6.6 cm to 4 cm as concentration dropped from 0.001x cefazedone to 0.0001x cefazedone. All three kinds of bacteria exposed to cefazedone had decrease in clear zone size at the lowest concentration of cefazedone. With the exception of S. gallinarum two bacteria showed drop in diameter at first change in concentration of cefazedone. On second change of concentration E. coli and S. gallinarum maintained same diameter while B. cereus had increased diameter from 6.4 cm to 7.3 cm. Therefore, result can be concluded that low concentration of antibiotics can incur stronger ABR. Figure 2: Diameter of clear zone for three bacteria with different concentration of cef. To find whether bacteria exposed to lincomycin will have ABR to different antibiotics like cefazedone, E. coli, S. gallinarum, and B. cereus, previously cultivated with various concentrations of lincomycin, were on agar diffusion test to cefazedone. S. gallinarum's size of clear zone dropped from 8.5 cm to 2.6 cm as the concentration changed from 0.1x lincomycin to 0.01x lincomycin, and the size of clear zone maintained similar diameter for 0.01x lincomycin, 0.001x lincomycin, and 0.0001x lincomycin. of 2.6 cm, 2.2 cm, and 2.4 cm. B cereus had similar pattern of dropping from 6.6 cm to 4.5 cm from 0.1x lincomycin to 0.01x lincomycin and maintained similar clear zone size for 0.01x lincomycin, 0.001x lincomycin, and 0.0001x lincomycin of 4.5 cm, 4.9 cm, and 4.9 cm. E. coli maintained similar clear zone size for 0.1x lincomycin, 0.01x lincomycin, and 0.001x lincomycin of 3.6 cm, 3.2 cm, and 3.1 cm, but as the concentration shifted from 0.001x lincomycin to 0.0001x lincomycin, the diameter of clear zone dropped from 3.1 cm to 1.5 cm. Therefore, the ABR APEC Youth Scientist Journal Vol.7 / No.2 developed at lower concentration of antibiotics is stronger, and despite the discrepancy in kinds of antibiotics, ABR can be acquired to newly contacted antibiotics. Figure 3: Diameter of clear zone with different concentration of lin. Experiment 4: Antagonistic effects between ABR acquired bacteria and normal bacteria
in co-culture
S. gallinarum with ABR to licomycin from experiment 3 was mixed with non ABR S. gallinarum and incubated to observe effect of ABR bacteria have on non ABR bacteria. The diameter of ABR bacteria and non ABR bacteria decreased from 2.4 to 1 and from 5.4 to 3 respectively. Decrease in clear zone means that both grew ABR to cefazedone, and the difference in clear zone decreasing from 3 to 2 concludes that the antagonistic effect of two bacteria was applied. Additionally, control S. gallinarum has dominance over ABR S. gallinarum. Same thing was done except with cefazedone ABR bacteria for this one. Diameters of clear zone decreased from 5 to 0.7 and 5.4 to 3.8 for ABR bacteria and control bacteria respectively. The general increase in ABR was observed, but increase in difference of clear zone from 0.4 to 3.1 shows that ABR bacteria are dominant over control. Same thing was done except with control E. coli and licomycin ABR E. coli. Diameters of clear zone decreased from 1.5 to 1.3 and 5 to 3.2 for ABR bacteria and control respectively. The difference of clear zone decreased from 3.5 to 1.9, so conclusion can be drawn that control has dominance over ABR bacteria in antagonistic effect. APEC Youth Scientist Journal Vol.7 / No.2 Figure 4: Diameters of lin. ABR and control bacteria clear zone before and after co- Figure 5: Diameters of cef. ABR transformed and control bacteria clear zone before and after co-cultivation Figure 6: Diameters of lin. ABR transformed and control bacteria clear zone before and after co-cultivation

APEC Youth Scientist Journal Vol.7 / No.2 Figure 7: Cultivation of control and ABR transformed bacteria Experiment 5: Possibility of ABR spread by plasmid DNA transfection
To confirm that ABR can be spread with plasmid DNA, heat shock transformation was used to implant plasmid DNA from ABR E. coli and S. gallinarum to non ABR E. coli and S. gallinarum respectively. Transformed bacteria were done agar diffusion test. S. gallinarum showed resistance to both lincomycin and cefazedone, so both were used. E. coli had resistance to only lincomycin, so only lincomycin resistant plasmid DNA was used. E. coli's clear zone decreased from 3.4 cm of control to between 2.9 with transformation. S. gallinarum control had 3.3 cm clear zone, but lincomycin plasmid DNA had 3 cm and cefazedone 3.2 cm. The conclusion can be drawn that transformation by antibiotics resistant plasmid DNA helps bacteria to acquire resistance, which is shown by decrease in clear zone. Figure 7: Diameters of control and ABR transformed bacteria in co-cultivation 4. DISCUSSION
ABR spread through plasmid DNA; Experiment 5 clearly showed that implanting ABR plasmid DNA can grant the bacteria with ABR trait. This ties closely with transformation of bacteria. The question rises, however. What is the incentive for the bacteria APEC Youth Scientist Journal Vol.7 / No.2 to accept the new DNA. Strengthened ABR due to difference in types or concentrations of antibiotics; Experiment 3 and 4 expressed that ABR can be strengthened by exposure to low concentration of antibiotics or different antibiotics. Low concentration could not be explained in perspective of transformation. Although it is antibiotics, low concentration would not kill as many bacteria as higher concentration. This leads to lesser dead bacteria, which leads to more surviving bacteria. With higher concentration of antibiotics Survival of the Fittest would be the only factor, but besides those with inborn traits, those without traits cannot acquire traits in anyway even with transformation because dead bacteria would not have any useful gene for building defense against the antibiotics. Preexisting ABR on old antibiotics; Experiment 1 and 2 showed that common bacteria in soil and stored bacteria both had ABR to old antibiotics. This relates back to Charles Darwin's Survival of the Fittest theory. When bacteria are exposed to antibiotics, many are decimated, but few mutated bacteria survive. These bacteria inherit traits that make antibiotics less effective, and the population grows with individuals with that trait, resulting in ABR for the majority. Stored bacteria would not have problem developing ABR because their origins are ultimately humans, who used antibiotics. The question is how common bacteria exposed to antibiotics before did. One hypothesis is that disposal of antibiotics. Antibiotics have form of liquid, so they were disposed in sink. This sink system is linked to everywhere, so bacteria in soil first contacted the antibiotics. They formed and grew ABR population spread the ABR through one or more of the methods above. 5. CONCLUSION
Experiment 1 and 2 showed that many bacteria have grown ABR to old antibiotics and are growing ABR to newer ones. Experiment 3 through 5 proposed possible mechanisms for ABR spread. The final conclusion is that ABR has been first introduced to bacteria by exposure to low concentration of antibiotics, and this ABR has been spread by proximity with ABR bacteria and transformation through ABR bacteria's plasmid DNA. This spread was done for long time, which affected common bacteria as well, while bacteria had less time to spread ABR for newer antibiotics, thus showing less ABR than to older

APEC Youth Scientist Journal Vol.7 / No.2 6. REFERENCES
Bergman, J . (2011, May 2). Bacteria and Antibiotics: An Example of Evolution by Gustafsson, I., Sjölund, M., Torell, E., Johannesson, M., Engstrand, L., Cars, O. Andersson, D. (2003). Bacteria with increased mutation frequency and antibiotic resistance are enriched in the commensal flora of patients with high antibiotic usage. Journal of Antimicrobial Chemotherapy, 52, 645-650. Timeline of Antibiotic Resistance. (2015, April 14). Switchyard Media. Retrieved Harjvan, C., Ho, A., and Farooqi, S. (2015, April 14). The Emergence of Novel Pathogens: The Greatest Risk of Hubris in Human Health. PWC. Retrieved from Houndt, T and Ochman, H. (2000) Long-Term Shifts in Patterns of Antibiotic Resistance in Enteric Bacteria. Appl Environ Microbiol, 66(12), 5406–5409. Stephens, E. (April 14, 2015). Types of Antibiotics. Emedicine health. Retrieved A Brief Overview of Classes of Antibiotics. (2015, April 14). Compound Interest. Retrieved from http://www.compoundchem.com/2014/09/08/antibiotics/. New Hypothesis: Why Bacteria are becoming increasingly more Resistant to Antibiotics. (2015, April 14). Phys.org. Retrieved from http://phys.org/news/2013- An H-NS-like Stealth Protein Aids Horizontal DNA Transmission in Bacteria. Science, 315 (5809), 251-252. [10] Beaber, J. W., Hochnut, B. and Waldor, M. K. (2004). SOS response promotes horizontal dissemination of antibiotic resistance genes. Nature, 427, 72-74. [11] L. CLD. I. (2014). Selection of a Multidrug Resistance Plasmid by Sublenthal Levels of Antibiotics and Heavy Metals. Mbio, 5 (5), 01918-14.

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