Restriction Digest Unable to Identify Plasmid in Antibiotic-Resistant Bacteria Using Gel Electrophoresis


 

Jeannette Kelly

Group: Big Pimpin’


LBS 145

Lab Section Thursday 1

Dr. Luckie

April 29, 2005

 

                                                                                    Fig. 9. Gel Electrophoresis after Restriction Digest


Abstract: 

            The increase of antibiotic-resistant bacteria threatens society (Jumbe, 2003).  Although bacteria forming resistance to antibiotics is a natural biological phenomenon, the misuse of antimicrobial drugs has amplified this process (Aiken, 2000).  Antibiotic- resistant bacteria pose a threat because the antibiotics used to cure infections are no longer effective against these strains.    The Centers for Disease Control and Prevention (CDC) report that one-third of the 150 million prescriptions written for antibiotics each year are unnecessary (Aiken, 2000).  Ironically, this over-prescription caused the bacteria strains to build resistance and become stronger than the antibiotics used to kill them (Jumbe, 2003).  The purpose of this experiment is to locate where antibiotic-resistant bacteria grow, determine their ideal growing conditions, and identify the plasmids contained in the bacteria to determine which plasmids render bacteria resistant to specific antibiotics.
            Thirteen locations in Holmes Hall were swabbed for bacteria including the weight room, elevator buttons, and bathroom door handles.  Locations that are cleaned frequently were selected because antibiotic-resistant bacteria are more likely to thrive in these environments (Ready et al., 2003).   The bacteria were amplified, subjected to amypicillin, kanamycin, and tetracycline to identify resistance, spread on agar plates, and lysed to obtain plasmids.  Restriction enzyme digestion and gel electrophoresis was performed to identify plasmids.  Knowing the make-up of the plasmid can help identify which antibiotics it is resistant to, and prevent doctors from prescribing ineffective drugs.   I expected and found bacteria from elevator buttons to be antibiotic resistant and expected to identify its plasmids but was unsuccessful.



Discussion:

Growing Bacteria:

            Since almost nothing in our environment is sterile, I predicted that the twelve locations swabbed would contain bacteria.  After incubating the 12 tubes of bacteria in LB broth overnight, I analyzed the contents for bacterial growth.  Eleven of the sites (bathroom doors, shoes, elevator buttons, bike room, cafeteria doors, compact room, sewer plates, keyboard, bus handle, locker room, and weight room) had very cloudy solutions, while the sample containing bacteria from the mouth was still clear.  These results do not prove that there are no bacteria in the mouth, but imply that the mouth was not as heavily concentrated as the other 11 locations. Since saliva contains anti-bacterial peptides that kill bacteria that cause food-borne illness, the concentration of bacteria in the mouth should be lower than that in a trash compactor chute (Krha, 2005).   Therefore, I did not use the mouth bacteria any further in the experiment. 

Identification of Resistant Bacteria:

Bacteria found in locations that are cleaned often are more likely to become resistant to the antibiotics in the cleaning products, and since most of the locations swabbed are cleaned, I predicted that the bacteria from each of the 11 locations would be resistant to at least one of the three antibiotics tested (Ready et al., 2003).  My conclusions regarding antibiotic resistance were based on whether the solutions were still cloudy.  Our data showed that the bacteria from all 11 sites (bathroom doors, shoes, elevator buttons, bike room, cafeteria doors, compact room, sewer plates, keyboard, bus handle, locker room, and weight room) grew in the ampycillin antibiotic (Figure 1). Bacteria from 8 sites had kanamycin resistance: the elevator buttons, keyboard, sewer plates, bike room, shoes, compact room, locker room, and weight room (Figure 2), while cultures from only 4 sites showed tetracycline resistance: sewer plates, locker room, keyboard, and weight room (Figure 3).  Bacteria cultured in broth containing antibiotics would only grow if they contained plasmids with genes making the bacteria resistant (Krha, 2005). 

Bacteria Growth on Agar Plates:  

Next each resistant bacterium was spread on to agar plates which contained antibiotic.  Bacteria from sites mixed with ampycillin were added to plates containing ampycillin and so on.  Since the bacteria grew under antibiotics while incubating, I predicted that all these sites would grow when spread out on the plates, since resistant bacteria should be still resistant when additional antibiotic is added.  However, the bacteria that grew colonies resisting ampycillin were from: the compact room, keyboard, bus handles, locker room, bathroom door handles, sewer plates, and the elevator buttons (see two examples in Figures 4, 5, and 6), while the bacteria resisting kanamycin were from:  the weight room, locker room, shoes, bike room keyboard, sewer plates, compact room, and elevator buttons (Figure 6).  No bacteria subjected to tetracycline on the agar plates displayed growth.  The fact that the bacteria resistant during incubation did not grow colonies on the agar plates can possibly be accounted for by the error that can occur when spreading the bacteria on the agar.  Not following the precise technique to spread the colonies can lead to no colonies growing, therefore, human error could account for these results (Krha, 2005).   It also suggests that tetracycline is not used as a common cleaning product, since these bacteria have not built up resistance for it.

Harvest, Lysis, mini-Prep, Restriction Digest, and Gel Electrophoresis:

After the colonies grew and a single colony of each bacterium was isolated, harvested and lysed, I focused the experiment on the bacteria resistant to ampycillin found on the elevator buttons. Lysis is done to remove the plasma membrane and DNA, leaving only the plasmid (Krha, 2005).  I predicted that all of the bacteria would display plasmids after digestion and gel electrophoresis, because all resistant bacteria have plasmids that give them those resistant qualities (Kapil, 2005).  After the first mini-prep gel electrophoresis was run for about a twenty minutes, the process was stopped and the gel was examined under a UV light.  The plasmids should have migrated across the gel to the location at which they were cut.  Orange clear bands represented these locations (Krha, 2005).  However, no orange bands were present, suggesting that no DNA was present.  This can be explained by the difficulty of the lysis process.  During multiple steps throughout the lysis process, solutions were centrifuged into tiny pellets, and the excess liquid was removed.  These tiny pellets were easily broken and dislodged and could easily have been sucked up in the solution as waste, therefore removing the plasmid from the solution being tested (Krha, 2005).  In most cases the pellet of plasmid could not be seen and could have easily been sucked up as waste.  After the second lysis of the elevator button bacteria resistant to ampycillin, the mini prep gel electrophoresis produced orange bands suggesting that plasmids were present (Figure 8).   However, the orange traces were hard to identify and were possibly just smears, which does not imply plasmids or DNA (Krha, 2005). After the restriction digest was performed with EcoR1 and HindIII, no orange bands were present (Figure 9).  This may be accounted for by the fact that the DNA was accidentally digested for a whole day, possibly causing it to denature, or may indicate that there was no DNA present in the mini-prep, since only orange smears were present (Krha, 2005).   This also could imply that our restriction enzymes were not suitable or that the amounts were not favorable.  After the same bacteria were lysed, with a successful mini prep of orange bands (Figure 8), a digestion using PstI and HindIII took place for 17 hours.  No bands showed up after the gel electrophoresis (Figure 9).  A possible explanation is that these restriction enzymes were cutting the DNA into pieces so small that they were running right off the gel, which would indicate that the restriction enzyme combination may not be suitable.  Another explanation is that the mini-prep was actually not successful, since the orange bands were not distinct, but smeared.  Again, the bacteria was lysed, showed bands for the mini-prep (Figure 8), and two different digests were run; one digest with HindIII and a digest with HindIII and PstI.  This digestion was conducted for about 4 hours because I hypothesized that in previous trials the plasmids were being digested for too long.  Again, no orange bands were present after the gel electrophoresis, suggesting that either no plasmids were found in the mini prep, just smears of orange, or the enzymes were cutting the sequence into small pieces that slid of the gel (Figure 9).  Considering that all four digestions were unsuccessful in producing orange bands on electrophoresis to identify the plasmid and that all four digestions used a combination of the same two restriction enzymes, HindIII and PstI, these restrictions enzymes were probably not suitable for cutting the DNA from the elevator bacteria.  Results could be skewed by high risk of contamination from the lab.  

            Since no plasmids were displayed on the gel electrophoresis, no plasmid could be identified.  Time ran short in the lab so no more trials could be run.  These results suggest that the bacteria from the Holmes Hall elevator buttons were resistant to ampycillin, but no plasmid could be identified to support my hypothesis that all antibiotic resistant bacteria contain plasmids with genes giving them the resistant abilities.