Results and Conclusion

We first grew the MRSA in liquid LB broth, and then placed the MRSA in solid LB plate with a variety of antibiotics, and observed its growth. This is the resulting growth of the MRSA.

Top petri dishes are the resulting growth of MRSA from patient 7, and the bottom petri dishes are the resulting growth of MRSA from patient 8. The antibiotics used are tetracycline, kanamycin, gentamycin, and ampicillin, left to right.

The results show that MRSA has resistance to gentamycin, ampicillin, and kanamycin, but slight or no resistance to tetracycline.

Then we wanted to transform the E. coli with the DNA from the MRSA and test its resistance, but first, we had to test the original E. coli in the drugs, as without that, we wouldn’t know if the drug resistance was from the MRSA DNA, or from the E. coli. We placed the E. coli on a LB plate with a variety of antibiotics and observed its growth. This will be the control of this section of the experiment. This is the resulting growth of the E. coli. The results show that the E. coli has no resistance to gentamycin, kanamycin, ampicillin, and tetracycline.

Growth of E. coli 24 hours it was placed in different antibiotics (top left: kanamycin, top right: gentamycin, bottom left: ampicillin, bottom right: tetracycline.) with 1/1000 concentrations.

Then to test the drug resistance of the transformed E. coli, we placed the E. coli in a LB plate and observed its growth. This is the resulting growth of the transformed E. coli.

E. coli transformed with the DNA of the MRSA from patient 7 grown in ampicillin survived in ampicillin

E. coli transformed with the DNA of the MRSA from patient 7 grown in kanamycin survived in ampicillin

E. coli transformed with the DNA of the MRSA from patient 7 grown in tetracycline survived in ampicillin

E. coli transformed with the DNA of the MRSA from patient 7 grown in gentamycin survived in ampicillin

E. coli transformed with the DNA of the MRSA from patient 8 grown in gentamycin survived in kanamycin

E. coli transformed with the DNA of the MRSA from patient 8 grown in gentamycin survived in gentamycin

E. coli transformed with the DNA of the MRSA from patient 8 grown in kanamycin survived in gentamycin

E. coli transformed with the DNA of the MRSA from patient 8 grown in kanamycin survived in kanamycin

Then we wanted to analyze the DNA. To do that, we preformed PCR with a variety of primers and a variety of DNA. PCR includes florescent dye that activates when it attaches to DNA, so the PCR machine can measure the progress of PCR by measuring the fluorescence. These are the resulting graphs.

PCR Program

PCR lane placements

The amplification chart from the PCR process. The top graph represents lanes 2-4, and the bottom graph represents lanes 6-8

The graph is the amplification chart of the DNA. The x-axis is the number of cycles the DNA went through, and the y-axis is the amount of DNA measured by the fluorescence level. A successful PCR starts to grow exponentially at about 20-25 cycles, while flat lining curves represent DNA that is not copied very much.

The melt curves and melt peaks. The top graphs represent lanes 2-4, and the bottom graphs represent lanes 6-8. The left graphs are the melt curves, and the right graphs are melt peaks

The graphs are the melt curves and the melt peaks. The x-axis is the temperature, and the y axis is the florescence level which represents the number of associated double strand DNA (as opposed to disassociated single strand DNA). By taking the first derivate of the melt curve, we can get the melt peak chart. Melt curves with steep slopes mean there are more DNA there to be disassociated, which means PCR was successful. Since the melt peaks are the first derivation of melt curves, high melt peaks mean steep slopes, and therefore successful PCR. We decided to conduct gel electrophoresis on the DNA with the highest melt peaks.

Then we analyzed the amplified DNA using gel electrophoresis. The primers used in PCR copies the DNA at different sites, so if the bands from DNA amplified using the same primer results in similar bands, that most likely means that the section of DNA is included in both of the stands of DNA. This most likely means that the section of DNA that is shared in the DNA segments with multidrug resistance could code for multidrug resistance.

The results show that wells 2-8 has about 300 base pairs, which means that most likely the type V primers were active in PCR, and the section of DNA between V-F and V-R with about 300 base pairs were multiplied in PCR of 7A, 7G, 7K, 7T, 8A, 8G, and 8K. This most likely means that 7A, 7G, 7K, 7T, 8A, 8G, and 8K all included the section of 300 base pairs that is between V-F and V-R. So, this 300 base pair long section of DNA has a possibility of coding for multidrug resistance. The results also show that wells 9-11 has about 1,000 and 1,600 base pairs, which means that most likely the primers ccrAB-α3 and ccrAB-α4 were active in PCR, and the section of DNA between ccrAB-α3 and ccrAB-α4 with about 1000 base pairs and the section of DNA between ccrAB-α3 and ccrAB-α4 with about 1,600 base pairs were multiplied in PCR of 7K, 8G, and 8K. This most likely means that 7K, 8G, and 8K all included the sections of 1,000 and 1,600 base pairs that is between ccrAB-α3 and ccrAB-α4. So, this 1,000 base pair long section of DNA and the 1,600 base pair long section of DNA has a possibility of coding for multidrug resistance.

The results show that wells 12-17 has about 150 base pairs, which means that most likely the primers MecA147 or the primers mecI were active in PCR, and the section of DNA between MecA147-F and MecA147-R or the section of DNA between mecI-F and mecI-R with about 150 base pairs were multiplied in PCR of 7GA, 7KA, 7TA, 8GK, 8GG, and 8KK. This most likely means that 7GA, 7KA, 7TA, 8GK, 8GG, and 8KK all included the section of 150 base pairs that is between MecA147-F and MecA147-R or the section of 150 base pairs between mecI-F and mecI-R. So, this 150 base pair long section of DNA has a possibility of coding for multidrug resistance.

The investigation showed that MRSA showed resistance to some of the antibiotics, and some of the transformed E. coli showed antibiotic resistance. That most likely means that the DNA that codes for antibiotic resistance was successfully transported into the E. coli that survived the antibiotics. By running PCR, we were able to amplify some of the DNA in the drug resistant MRSA and transformed E. coli. Since the section of DNA that codes for the multidrug resistance is most likely shared in all the bacterium that survived the antibiotics, by analyzing the DNA through gel electrophoresis, we were able to get an idea about which section of DNA was shared. If possible, the next step forward would have been to analyze the sequence of the section of DNA, but we were not able to precede that far.