Identification of Plasmid from Antibiotic Resistant Bacteria by Amplification, Restriction Enzyme Digestion and Gel Electrophoresis

April 29, 2005

Abstract

Antibiotic resistant bacteria have become an increasing problem for public health. One way bacteria acquire antibiotic resistance is to obtain plasmid(s) containing antibiotic resistant gene(s) (Krha et al., 2005). The purpose of this study is to investigate if there are antibiotic resistant bacteria in Holmes Hall of Michigan State University, what antibiotic the bacteria are resistant to, and to identify the plasmid(s) they carry. Since antibiotic resistant bacteria are often found in bathrooms because they are frequently cleaned with antibiotics (Stiffler, 1974), bacteria were collected from bathroom sink faucets, door handles, and the corner of showers. They were grown in LB broth and then inoculated on LB agar plates containing kanamycin, tetracycline, or ampicillin to find bacteria resistant to these antibiotics. Bacteria colonies were found on kanamycin plates with bacteria from the shower and sink faucets, on ampicillin plates with bacteria from the shower, and on tetracycline plates with bacteria from doorknobs. These bacteria colonies were amplified by growing in LB broth containing the corresponding antibiotics. The plasmids in the bacteria were purified using the minipreparation method and analyzed by restriction enzyme digestion and agarose gel electrophoresis. Identification of the plasmids by restriction enzyme digestion and comparison with known plasmids failed because no DNA bands were present after restriction enzyme digestion. This maybe due to the low concentration of plasmid DNA in the sample for restriction enzyme digestion. Our results support the hypothesis that there are antibiotic resistant bacteria in Holmes Hall and some of them carry plasmid(s) with antibiotic resistant genes.

Introduction

At the beginning of the last century, Alexander Fleming found the first antibiotic — penicillin (Rodriguez-Saiz, et al., 2005). Since then, many antibiotics were found. These antibiotics can kill the invading bacteria or make them feeble so that our immune system can easily eradicate them (Reddy, 2004). Because of this, antibiotics have been used to treat infectious diseases and have saved countless lives. However, after several decades of extensive use of antibiotics in both human and veterinary medicine, antibiotics have become much less effective, making it difficult to treat infectious diseases. Thus, new antibiotics have to be developed to treat infectious diseases (Anonymous-1, 2004).

The decrease in the effectiveness of antibiotics is caused by bacteria resistant to antibiotics. These bacteria have developed the ability to escape the effects of antibiotics or antibiotics resistance as a result of natural selection. When a large population of bacteria is exposed to antibiotics, the effects of antibiotics eliminate most of the bacteria, but changes take place in some bacteria allowing them to survive. The bacteria that survived will grow and pass the ability of antibiotic resistance to other bacteria (Anonymous-2, 2004).

The mechanisms through which bacteria develop antibiotic resistance are not fully understood. Bacteria susceptible to antibiotics need to have either genetic mutation or antibiotic resistant genes to become antibiotics resistant (Levy and Marshall, 2004). Mutation usually changes the property of the targets of antibiotics. For example, fluroquinolones bind to DNA gyrase and block the DNA replication of bacteria. But mutation of the gyrase gene can reduce the affinity of the antibiotic to the enzyme, giving the bacteria tolerance to fluroquinolones. Efflux is another mechanism in which the antibiotic inside the bacteria is pumped out by an efflux pump, a channel that actively exports the antibiotics. Bacteria can also develop antibiotic resistance by having an enzyme that can destroy or modify antibiotics so that they become harmless to bacteria. In order to do this, the bacteria can acquire genetic materials that enable the bacteria to be antibiotic resistant through gene transfer. Gene transfer occurs through transformation, transduction and conjugation. In transformation, bacteria take DNA released by other cells. In transduction, the antibiotic resistance gene is transferred from one bacterium to another by phage. In conjugation, a bacterium takes a circular plasmid DNA, which contains the antibiotic resistance gene, from other bacteria through a bridge-like structure formed between two cells. Conjugation is known to be one of the most important mechanisms that bacteria acquire antibiotics resistance. (Anonymous-2, 2004)

Since the Michigan State campus is a heavily populated area and antibiotics have been used on the campus, I hypothesized that the antibiotic resistant bacteria could exist in the dormitory, especially in the bathroom where it is most possibly contaminated by human wastes and where antibiotics are heavily used (Stiffler, 1974). The purpose of our study is to investigate if antibiotic resistant bacteria exists on the campus, what antibiotics the bacteria are resistant to, and which plasmid(s) the bacteria carry. Expriments were performed to search for plasmid carrying bacteria in the bathrooms in Holmes Hall. Bacteria from different places of the bathroom: sink faucets, door handles, and the corner of showers were collected and grown in LB broth, then spread on antibiotic containing LB agar plates. The colonies on the plates were then amplified in LB broth with different antibiotics such as ampicillin, kanamycin, and tetracycline (Krha et al., 2005). Through this procedure, I expect to find several strains of bacteria that are resistant to kanamycin, ampicillin and tetracycline. The plasmids in the bacteria were isolated and purified through the minipreparation method (Krha et al., 2005). I expect to find the DNA fingerprints of these isolated plamids by performing restriction enzyme digestion and agarose gel electrophoresis. And finally I plan to identify the plasmids by performing gel electrophoresis with the restriction enzyme digested plasmids, a DNA ladder and known plasmids (Krha et al., 2005). The known plasmids are pAMP, pBR322, pKAN, pUC19, pGPS3 and pBLU. I expect to find a strain of E. faceium, which has been found to be resistant to kanamycin (Coleri, 2004).

Zhao, Z.  2002.  Lab protocol. Dr. Friedman's lab, Department of 

            Pathology, Wayne State University, Detroit.