Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Angel Li BSCI105 (0101) 7/6/06 Kanamycin Found to Inhibit the Growth of the Washington 1 Strain of Escherichia coli ABSTRACT: The emergence of a new strain of Escherichia coli (E. coli) containing the Washington 1 plasmid has been causing severe diarrhea in patients and was found to be resistant to many of the usual antibiotic treatments. The hypothesis was that the Washington 1 plasmid contained genes coding for the resistance of certain antibiotics. In order to find an appropriate treatment for the new strain, transformation was performed to incorporate the Washington 1 plasmid into laboratory strains of E. coli, and these cells, along with non-plasmid cells, were then cultured and treated with ampicillin and kanamycin. The results of the positive and negative controls were as expected, and kanamycin, but not ampicillin, was shown to be effective in inhibiting E. coli growth. However, further testing on the actual Washington 1 strain must be conducted to validate the use of kanamycin as an effective treatment for patients. INTRODUCTION: Escherichia coli, or E. coli, is a bacterium that is prevalent in the intestinal tract of humans as well as other warm-blooded animals (Feng, 2006). E. coli accounts for about 0.1% of the total bacteria within an average adult’s intestines, but its presence is essential for a properly functioning digestive system (Brown, 2006). The bacteria help to digest food by converting glucose into necessary macromolecules and by supplying certain vitamins that the human body can absorb (Todar, 2006). However, each organism hosts a different strain of E. coli and these bacteria have localized functions, so it is the emergence of an unfamiliar strain or colonization in an incorrect region of the body that leads to illness (Brown, 2006). These different strains can be transmitted through various means, but fecal contamination is by far the most common method (Todar, 2006). Different strains of bacteria come about by the acquisition and incorporation of extra genetic information, often in the form of plasmids through a process called transformation (Brown, 2006). A plasmid is a piece of DNA, similar to a parasite, which enters the cell and uses its resources to replicate (Campbell, 2002). Oftentimes, the cell can benefit from the incorporation of plasmids because the foreign DNA may contain genes coding from certain antibiotic resistance (Wassenaar, 2006). These genes can either be translated into antibiotic-destroying proteins or enzymes that act to cut and inactivate antibiotics, which are drugs used specifically to kill bacteria, through the modification of the target site or the creation of an alternate metabolic pathway (Tenover, 2006). E. coli can easily acquire drug resistance because plasmids are readily passed between bacteria (Gross, 2006). It is extremely important to be prudent in the use of antibiotics because over-prescription can select for resistance and lead to the proliferation of harmful and difficult to treat bacteria (Murchan, 2006). It was recently observed that a new strain of E. coli has been plaguing hospitals by causing severe diarrhea in patients. Labeled Washington 1, this E. coli was found to be resistant to the usual antibiotics, penicillin, chloramphenicol, and neomycin, and has therefore been difficult to treat. Furthermore, this new strain cannot be grown under normal laboratory conditions, but its plasmid can be extracted from the cells. Based on the known information, it was hypothesized that the Washington 1 plasmid confers resistance to certain antibiotics. Therefore, in order to discover an antibiotic that will be effective in treating the E. coli, it was necessary to culture plasmid-incorporated laboratory E. coli and treat those cells with different antibiotics – in this case, ampicillin and kanamycin – to see whether either drug will inhibit bacterial growth (Gross, 2006). METHODS: Knowing that the Washington 1 strain of E. coli is resistant to penicillin, chloramphenicol, and neomycin, it was necessary to treat the bacteria with different antibiotics in order to determine which antibiotics would be effective in killing this particular strain. However, it was found that the Washington 1 strain cannot be cultured in a laboratory setting. Therefore, the experiment first required the incorporation of the Washington 1 plasmid into a laboratory strain of E. coli. To perform this transformation, competent E. coli cells treated with CaCl2 were separated into 2 tubes of 100 µl each. The cells were allowed to just thaw and were then put on ice immediately to stabilize the phospholipids membrane. Ten µl of plasmid was added to one of the tubes and the other tube was used as the non-plasmid control. The plasmid-containing tube was swirled and placed on ice for 30 more minutes. After 30 minutes, both tubes were placed in a 42º water bath for 90 seconds in order to heat shock the cells and allow the plasmids to be properly incorporated. They were then placed on ice for one to two minutes to restabilize the cells and their membranes. For the purpose of diluting the CaCl2 and providing food for the cells, 800 µl of growth media was added to each tube. Following that, the tubes were incubated in a 37º (normal body temperature) water bath for 20 minutes to allow the genes on the plasmid to be transcribed and translated into the necessary drug resistance proteins. Once incubation was completed, the cells were plated onto Petri dishes along with the antibiotics they were treated with. Three plates were each added with 200 µl of plasmid-containing cells and three different plates with 200 µl each of non-plasmid cells. Each set of 3 plates included one containing no antibiotics, one containing ampicillin, and one containing kanamycin. Under a hood, the bacteria were spread across the surface of each of the plates using sterile cotton swabs. The plates were then turned upside down, sealed with Para film, and placed in a 37º incubator for 24 hours. After 24 hours, the plates were examined for growth and/or contamination in order to determine whether the antibiotics were effective against the Washington 1 strain (Gross, 2006). RESULTS: After 24 hours of incubation, the 6 plates were observed for growth. Colonization was apparent in both of the positive controls containing no antibiotics, representing cell viability. There was no growth in either of the negative controls containing non-plasmid cells and antibiotics, eliminating the possibility of contamination having occurred. As for the experimental plates, there was growth in the plasmid-containing cells treated with ampicillin and no growth in the cells treated with kanamycin, as shown in Table 1. Table 1: Growth of E. coli bacteria with and without Washington 1 plasmid under different antibiotics No Plasmid Plasmid No antibiotics Growth Growth Kanamycin No Growth No Growth Ampicillin No Growth Growth Half of the E. coli cells were treated with the Washington 1 plasmid while the other half were not. The two plates with no antibiotics acted as positive controls to check for cell viability and the two plates containing antibiotics and non-plasmid cells acted as negative controls to check for contamination. Antibiotic effectiveness was tested with the two experimental plates containing Washington 1 E. coli and antibiotics. DISCUSSION: Based on the growth patterns observed in this experiment, it can be concluded that the antibiotic kanamycin can be a possible treatment for the Washington 1 strain of E. coli. As expected, growth was observed for the positive controls, signifying that the cells used were viable. Also expected was the lack of colonization in the negative controls. This ruled out any possible occurrence of cross contamination, environmental contamination, or spontaneous mutation and helped to validate the results obtained from the experimental plates. The plasmid-containing cells treated with ampicillin still grew and colonized, signifying that the Washington 1 plasmid most likely contained genes coding for the resistance of ampicillin, which was not unexpected since it was hypothesized that the plasmid conferred resistance to certain antibiotics not limited to the ones already known. However, there was no growth in the cells treated with kanamycin, meaning that the isolated plasmid may not contain kanamycin-resistant genes and the antibiotic could be a possible treatment for the patients suffering from E. coli infection. The testing of specific antibiotics against bacterial strains is necessary in order to prevent the further spread of multi-drug resistance. Exposing patients to a plethora of antibiotics can actually lead to the elimination of useful bacteria while promoting the growth of harmful, resistant bacteria. Same is true of using an incorrect antibiotic. Although bacterial cells are quite different from human cells and the antibiotic will have little or no effect on the rest of the body, it will kill all susceptible bacteria that it comes in contact with. This selects specifically for the antibiotic-resistant bacteria, which can then reproduce rapidly without having to compete with other bacteria for space and resources. These bacteria can then confer their resistance upon other bacteria through the transmittance of DNA, causing those bacteria to be immune to antibiotics that they might not even have previously come in contact with. The result is a limited availability of future bacterial treatments (Irwin, 2006). The next step would be to further test the effectiveness of kanamycin against the actual Washington 1 strain of E. coli. A possible hypothesis could be that kanamycin is an effective antibiotic treatment against E. coli infections caused by the Washington 1 strain. Since bacteria acts differently in different environments, as do antibiotics, the usefulness of kanamycin must be tested against the actual strain versus the laboratory engineered cells in this study. This might even call for the use of kanamycin in treating select patients to determine whether individuals respond similarly to the antibiotic. REFERENCES: Brown, J.C. What the Heck is an E. coli ? 5 July 2006 < http://www.people.ku.edu/~jbrown/ecoli.html> Campbell, N.A. and Reece, J.B. 2002. The Structure, Function, and Reproduction of Prokaryotes. In: Biology. Pearson Education, Inc., San Francisco, pp528-530. Feng, P., Weagant, S.D., and Grant, M.A. Enumeration of Escherichia coli and the Coliform Bacteria. U.S. Food & Drug Administration. 5 July 2006 < http://www.cfsan.fda.gov/~ebam/bam-4.html> Gross, P. and Lanford, P.J. 2006. Demonstrating Drug Resistance in Bacteria. In: Lanford, P.J. (ed.), Introduction to Experimental Biology. Plymouth, MI: HaydenMcNeil Publishing, Inc., pp95-98. Irwin, K., Smith, D.R., Ebako, G.M., Ensley, S., Griffin, D.D., and Wohlers, A. Guidelines for the Prudent Use of Antibiotics in Food Animals. University of Nebraska – Lincoln. 5 July 2006. < http://www.ianrpubs.unl.edu/epublic/pages/publicationD.jsp?publicationId=427> Murchan, S and Cunney, R. 2006. Rise in antimicrobial resistance in invasive isolates of Escherichia coli and Enterococcus faecium in Ireland. Euro Surveill 11(4):E060413.3. Tenover, F.C. 2006. Mechanisms of antimicrobial resistance in bacteria. Am J Infect Control 34(5 Suppl):S3-S10. Todar, K. Pathogenic E. coli. University of Wisconsin. 5 July 2006 < http://www.textbookofbacteriology.net/e.coli.html> Wassenaar, T. Plasmid DNA Function. 5 July 2006 < http://www.newton.dep.anl.gov/askasci/mole00/mole00391.htm>