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Survey of Plasmids Collected from Antibiotic Resistant Enteric Department of Biology, Carroll College, 100 N. East Ave., Waukesha, WI 53186 Abstract:
Materials and Methods:
Bacterial antibiotic resistance is becoming increasingly problematic. Antibiotics are being prescribed more and more frequently at doctors’ offices, vets, and even in the agricultural industries. The effectiveness of antibiotics is •Water samples were collected along four Milwaukee Lake Michigan beaches being severely hampered due to increased resistance to these drugs, but and plated onto tetracycline embedded LB plates. increasing the use of theses drugs is causing more resistance to be expressed. •A total of 35 strains grew up on the tet plates, indicating their resistance. To break this cycle, more knowledge must be gained about how a bacterium attains its resistance. Since resistance is usually conferred by plasmidal DNA, •Strains were named and stored in a -80ºC freezer. studying plasmids of resistant bacteria is a good way to understand antibiotic resistance. In this study, 31 strains of tetracycline resistant enteric bacteria were collected, and through a lysing and collection process, the plasmids To characterize the plasmids, they must first be removed. were removed and run through a gel. Plasmid length was then recorded. This process allows for further testing and analysis. In so doing this, we •Freezer isolates were plated onto tetracycline embedded plates. can further our knowledge of bacterial resistance and plasmidal flow through •Single colonies of bacteria were cultured in tubes with tetracycline overnight to attain appropriate amounts of bacteria, but more importantly, •The bacteria were centrifuged, reconstituted, and then lysed to release Introduction:
• Plasmids were then filtered out and stored in a microcentrifuge tube in a As bacteria become increasingly problematic to humans, humans strive to find ways to control bacteria. The problem is, the presence of an antimicrobial agent inherently selects for types of bacteria that tend to be more resistant as well as selecting for more resistance in these types of bacteria. Decades of •Plasmids were run through gel electrophoresis to characterize their relative use of antibiotics in the medical, veterinary, and agricultural fields have resulted in multiple strains of resistant pathogenic bacteria, as well as increased amounts of resistant bacteria in the wild. According to McDermott, Walker, and White (2003) there are a few different ways bacteria can acquire To see whether or not the plasmid confers resistance, the plasmid has to resistance: the cell wall permeability can change restricting the chemical be observed to protect bacteria with known antibiotic sensitivity. agent’s target; the bacterium can “learn” to expel the agent; a random Competent Hb 101 E. coli is known to not withstand the presence of mutation can create new chemical and metabolic pathways; the utilization of new enzymatic and metabolic pathways can alter the chemical agent’s effectiveness; and the acquisition of new metabolic or enzymatic activities • Competent Hb 101 cells along with previously extracted plasmids from an outside source can allow the bacterium to avoid the effects of were mixed together in ideal conditions to allow for the most effective the chemical agent. Such an acquisition could involve transferable extrachromosomal DNA pieces known as plasmids. Plasmids can be Isolates Tested
• Bacteria was then plated on antibiotic infused plates so that only the obtained through the active transmission of DNA from one bacterium bacteria that took up the plasmid, and only under the condition that to another know as conjugation, or another process known as transformation the plasmid actually confers resistance, will then yield bacterial colonies where the bacterium absorbs the DNA from its surrounding environment. Depending on the bacterial strain and the condition it’s in, a bacterium could survive with or without a plasmid. It is not uncommon to find a single •A test of the newly transformed bacteria was performed to determine bacterium containing multiple copies of a single plasmid as well as multiple what other, if any, antibiotics the plasmid(s) confer resistance to.
different plasmids. Interestingly, multiple resistance genes can accumulate on a single plasmid. Knowing this, knowing that a bacterium can have more than one plasmid, and knowing that a bacterium can pass its plasmids on to other bacteria, studying plasmids would seem to be a great way of studying antibiotic resistance. A qualitative study has been proposed to characterize the plasmids of 35 resistant enteric bacterial strains collected from the shores of Lake Michigan to facilitate more quantitative studies to file://C:\Documents and Settings\tserio\Desktop\RyanBlack2005_files\slide0001.htm

Source: http://www.carrollu.edu/programs/biology/images/RyanBlack2005.pdf