The Effect of T4 Bacteriophage Treatment on Non Resistant Escherichia coli K12 and Ampicillin Resistant Escherichia coli MM294
By Zoe Herbermann and Kyoko Hirose
Abstract:
Since the development of antibiotics, the over-prescription and misuse of the drugs have led to antibiotic resistant bacteria. Now, the global rise of “superbugs” endangers patients because known, successful antibiotic treatments are dwindling and more bacteria are building up multidrug resistance. Bacteriophage therapy offers a potential alternative to antibiotic treatment because these viruses are specific to bacteria and have a different mode of action than traditional antibiotics. In this experiment, the efficacy of T4 bacteriophage therapy was tested against the common antibiotic, ampicillin, in treating both nonresistant (NR) E. coli K12 and ampicillin resistant (AmpR) E. coli MM294. Both LB plate and cell absorption counter results revealed that though T4 phage therapy inhibited some growth in the K12 (NR) colonies, the ampicillin was still the most effective form of treatment because it inhibited more growth and demonstrated less of a tendency to select for resistant bacteria when administered in the proper concentration. However, when the T4 phage was used to treat the MM294 (AmpR) strain, it greatly limited the bacterial growth whereas the ampicillin was ineffective. When the two treatments were used in conjunction, they seemed to inhibit each other’s efficacy. This result suggested that the combination treatment was less effective in treating the resistant MM294 strain than the phage therapy alone. In conclusion, the phage therapy seemed to prohibit the growth of the resistant strain of bacteria better than the ineffective antibiotic. Nevertheless, it is not yet confirmed that this type of treatment could be a viable alternative due to the possible threat of phage resistant bacteria.
Introduction:
Recent scientific studies have shown that bacteriophages can possibly be used to treat bacterial infections instead of antibiotics (1, 2, 10). With the rise of antibiotic resistant bacteria threatening global health and safety, it has become imperative to find alternative treatment solutions. The use of bacteriophages against bacteria could possibly offer a solution to the rise of drug resistant bacteria because it has a different, more specific mode of action than antibiotics (9). Bacteriophages target very specific strains of bacteria by inserting genetic material and taking over the function of the host cell and ultimately causing lysis (9). This unique mode of action allows bacteriophages to be able to infect bacteria strains that may or may not have resistance to antibiotics (9). Phages are also naturally occurring and therefore employ microbial antagonism to kill malignant bacteria (2, 8, 9) . Since there are around 1031 different phages found in nature, there is at least one bacteriophage that infects every strain of bacteria; therefore, it is just a matter of discovering an isolating these phages in order to use them for treatment (2). Though phage therapy has the ability to be an effective alternative to antibiotics, the lack of research on the treatment dissuades people from believing in its efficacy as compared to traditional antibiotics. It is also unclear how the phages’ mode of action will affect the bacteria because the insertion of genetic material may alter the bacteria instead of actually killing the infection. However, with the number of bacteria becoming drug and multidrug resistant, it is imperative that the medical community establish a solution as quickly as possible.
This experiment plans to test the antimicrobial activity of the T4 phage compared to the antimicrobial activity of the commonly used antibiotic, Ampicillin, on E. coli bacteria. These forms of treatments were administered to both non-resistant and amp-resistant strains of E. coli in order to show the effects of bacteriophages on realistic strains of bacteria. In the real world, the number of resistant strains of bacteria is only increasing and constantly poses an immense health risk; bacteria that have developed resistance to normal antibiotic treatment pose such a threat because if they infect an individual, the known and effective treatment available is limited. Therefore, it was essential to test the T4 bacteriophage treatment and discern if bacteriophage treatment is a viable alternative to antibiotic treatment if a certain bacterial infection does not respond to common antibiotic treatment. Additionally, antibiotic treatment was administered in conjunction with T4 phage treatment in order to test if a combination of treatments would result in greater or decreased antimicrobial activity against resistant bacteria. Based on research of several other studies that show the general efficacy of bacteriophage treatment, it was expected that the bacteriophages alone are just as effective, if not more so, than antibiotics in killing both the K12 and resistant MM294 E. coli strains. Additionally, in conjunction with antibiotics, the antimicrobial efficacy of the bacteriophage was expected to increase increase.
Procedures:
Materials:
• For the Agar Plate Experiment:
• For the Cell Absorbance Counter Experiment
*Microplate Reader
How it Works: A 96 well plate is used with each well holding 250 µL. In order to produce consistent, accurate results, the same amount of each solution should be pipetted into their respective wells. Once the wells are filled, the plate is placed into the microplate reader and the black plastic covering is pulled over it (The covering prevents outside light from getting in and affecting the results). The machine is then calibrated to A1, so that all of our results have a common comparison point and run. In order to measure the absorbance of each well, the machine flashes a light through each well and a detector measured the amount of light received on the opposite side. Through this method, bacterial growth can be quantified because as more bacteria grows, the solution becomes more opaque. The results are then printed by the machine (3).
Methods:
For Plate Experiment (Procedure Modified from Source 6):
Making Overnight Broths:
In order to run the tests on the bacteria, both MM294 and K12 bacteria colonies had to be grown in a broth culture. In order to do this, a variable amount of about 4 mL of sterile LB broth was poured into two 15 mL plastic test tubes. These test tubes were then labeled for their respective bacterial strains, MM294 or K12. To grow the bacteria in the broth, a sterile inoculating loop was used to collect 1 or 2 distinct bacterial colonies of each strain and then suspend the colonies in their respective test tubes. The two test tubes were then incubated at 37 ºC overnight, so that the bacteria could grow and multiply.
Making Phage Dilutions:
Before treating the bacteria with phage solutions, serial dilutions had to be made in order to find an ideal concentration for the phage treatment and see the effects of concentration on the antimicrobial efficacy of the phage treatment. The three dilutions that were used were non dilute T4 phage solution from Carolina Sciences, 1:10 dilution, and 1:100 dilution. To make the 1:10 dilution, a p20 micropipette was used to pipette 4µL of non dilute phage solution as well as 36µL of distilled water into a 1.5 mL Eppi tube. The dilution solution was mixed by slight shaking. To make the 1:100 dilution, a p20 micropipette was used to pipette 4µL of already made 1:10 phage solution as well as 36µL of distilled water into a 1.5 mL Eppi tube. The dilution solution was mixed by slight shaking.
Testing T4 Phage, Ampicillin, or Combination on the Growth of E. coli MM294 or K12
This experiment was split into two experimental groups: one group testing antimicrobial treatments on the K12 strain and one group testing antimicrobial treatments on the MM294 +pAMP strain. The two strains of E. coli were chosen in order to demonstrate the differences of treatment efficacy between the non-resistant K12 and ampicillin resistant MM294. In order to determine the most efficient treatment method, both strains of bacteria were treated with antibiotics, T4 bacteriophage solution (with varying dilutions), or a combination of the two forms of treatment. To begin creating the experimental and control groups, 500 µL of either the K12 culture broth or the MM294 culture broth was pipetted into a 1.5 mL Eppi tube. In total, 5 tubes contained K12 broth and 8 tubes contained MM294 broth. Each tube was then labeled with their respective strain of bacteria (K12 or MM294), type of T4 phage treatment that it would receive (No Phage, Non Dilute, 1:10, or 1:100), and type of plate on which it would grow (LB or LB/amp). The specific combinations of strain, phage treatment, and ampicillin treatment are illustrated in Figure 1. Group 1 (based on the group labels in Figure 1) is one of the control groups because it exhibited the uninhibited growth of the K12 bacteria without any form of antimicrobial treatment. Therefore, the antimicrobial properties of the ampicillin or phage treatment could be compared to the normal growth of the bacteria. Similarly, group 6 is the other control group because it exhibited the uninhibited growth of the MM294 bacteria without any form of antimicrobial treatment. Therefore, the antimicrobial properties of the ampicillin, phage, or combination treatment could be compared to the normal growth of the bacteria. To administer the phage to the experimental groups, a 1:50 ratio of phage solution to culture broth was used. Therefore, 10µL of the T4 phage solution was pipetted into each corresponding Eppi tube. The specific phage dilution added to each group/tube is listed in Table 1. After the phage was introduced to the bacteria solutions, each Eppi tube sat at room temperature for 10 minutes so that the phage could begin to infect the bacteria. After the 10 minute incubation period, the ampicillin treatment was administered to the corresponding experimental groups by plating the solution on either an LB or LB/Amp plate. The type of plate that each group was plated on is listed in Table 1. To plate the bacteria on their corresponding agar plate, all of the Eppi tube’s contents were poured on to the agar plate. A sterile glass rod was then used to spread the bacteria cultures all over the plate. The rod was sterilized after every plate by dipping it in ethanol and running it through a butane flame to burn off the excess ethanol. After all the culture solutions were spread on their plates, they then sat rightside-up at room temperature for 5 minutes in order to allow the bacteria solution to be absorbed into the agar. After this, all plates were flipped upside-down in order to prevent condensation from impairing the growth of the bacteria. All the plates were then placed in the incubator at 37 ºC overnight. After overnight growth, the approximate number of plaques were observed and recorded for the K12 control and experimental groups (more plaques signifying more bacterial inhibition). The approximate number of colonies were then recorded for the MM294 control and experimental groups (less colonies signifying more bacterial inhibition).
Figure 1. List of experimental combinations of E. coli strain and type of treatment. These flowcharts are first divided by strain of bacteria: K12 or MM294. The MM294 is split into two groups in this figure: groups plated on LB plates and groups plated on LB/Amp plates. The resistance of each strain is also noted (NR-non resistant; AmpR-Ampicillin Resistant). Then each combination of antimicrobial treatment is listed below the corresponding strain. The control groups are highlighted in red. The ampicillin treatment groups are highlighted in blue. The T4 phage treatment groups are highlighted in yellow. The combination groups (phage + ampicillin) are highlighted in green.
Table 1. Illustration of strain of bacteria, bacterial volumes, concentration of phage, volume of phage, and type of agar plate used for each experimental or control group. Each group number illustrated in figure 1 is listed below. The strain of bacteria used in each group is listed in the second column, as well as the volume of bacteria culture broth listed in the third column. The type of phage dilution (Non Dilute, 1:10, or 1:100) that each group received is then listed in the fourth column. If a group did not receive phage treatment, then it is noted as “no phage.” The volume of T4 phage solution used is then listed in the fifth column. Finally, the type of agar plate used for each group is listed in the fifth column. A LB plate signifies that the group was plated on a normal LB nutrient agar plate without antibiotic treatment. A LB/amp plate signifies that the group was plated on a LB nutrient agar plate with ampicillin embedded in the plate.
# | Strain of bacteria | Amount of Bacteria Culture Broth | Type of Phage Dilution | Amount of Phage Solution | Type of Plate Used |
1 | K12 | 500µL | No phage | 0µL | LB |
2 | K12 | 500µL | No phage | 0µL | LB/Amp |
3 | K12 | 500µL | Non Dilute | 10µL | LB |
4 | K12 | 500µL | 1:10 Dilution | 10µL | LB |
5 | K12 | 500µL | 1:100 Dilution | 10µL | LB |
6 | MM294 | 500µL | No phage | 0µL | LB |
7 | MM294 | 500µL | Non Dilute | 10µL | LB |
8 | MM294 | 500µL | 1:10 Dilution | 10µL | LB |
9 | MM294 | 500µL | 1:100 Dilution | 10µL | LB |
10 | MM294 | 500µL | No phage | 0µL | LB/Amp |
11 | MM294 | 500µL | Non Dilute | 10µL | LB/Amp |
12 | MM294 | 500µL | 1:10 Dilution | 10µL | LB/Amp |
13 | MM294 | 500µL | 1:100 Dilution | 10µL | LB/Amp |
For Cell Counter Experiment:
Making Overnight Broths
In order to use the cell plate counter, overnight cultures were made for each experimental and control group. To begin making the culture broths, an LB base had to be used to growth the bacteria. The first control group contained pure LB broth because it demonstrated the normal absorbance of pure LB without any bacterial growth. To make this group, a variable volume of sterile LB broth was poured into a 15mL test tube. The test tube was then labeled and set aside. The next two control groups were the pure K12 and pure MM294 cultures because they demonstrated the uninhibited growth and absorbance rate of the bacteria without any forms of treatment. To make these two cultures, 5mL of sterile LB broth was pipetted into two 15mL test tubes. Each tube was then labeled “K12” or “MM294” in order to distinguish which strain would grow in which tube. Then, a sterile inoculating loop was used to pick up 1-2 distinct colonies from the K12 colony plates. The loop with the colonies was then dipped into the K12 LB tube and swirled around in order to suspend the colonies into the nutrient broth. Once the loop suspended all of the colonies, the loop was disposed of. These steps were repeated in the same fashion except by using the MM294 colonies and the MM294 tube. To make the antibiotic experimental groups, the antibiotic was administered by using a 1:20 antibiotic to culture ratio. In order to achieve this ratio, 4.75 mL (4,750µL) of sterile LB broth was pipetted into two 15mL test tubes. One tube was labeled “K12/amp” and the other was labeled “MM294/amp,” and each tube then received 1-2 colonies of the corresponding E. coli strain via sterile inoculating loop and the same method mentioned before. After the colonies were added to the LB broth, 0.5mL (500µL) of ampicillin was pipetted into both test tubes in order to treat the colonies with the antibiotic. Then to make the phage experimental groups, the bacteriophage treatment was administered by using the same 1:50 phage to culture ratio that was used in the plate experiment. The specific combinations of E. coli strain and phage dilution is listed in figure 2. In order to achieve a 1:50 phage to culture ratio, 4.9mL (4,900µL) of sterile LB broth was pipetted into a 15mL test tube for each group. Each tube was labeled according to their strain of bacteria and type of phage dilution (see figure 2 for all combinations). Then, a sterile inoculating loop was used to pick up 1-2 colonies of the correct strain and suspend the colonies in the correct 15mL test tube (see figure 2). After the bacteria colonies were suspended in each of the LB solutions, 0.1mL (100µL) of the corresponding phage dilution was pipetted into the correct test tube (see figure 2). After all 11 test tubes were prepared, they were all transferred to an incubator set to 37ºC and kept incubating overnight.
Figure 2. List of control and experimental groups containing bacteria. This figure includes the combinations of LB, bacteria, and type of treatment. The one control group that is not illustrated is the pure LB because it does not contain any bacteria. The other two control groups, LB+K12 and LB+MM294, are highlighted in red. The experimental ampicillin treatment groups are highlighted in blue. The experimental T4 bacteriophage treatment groups are highlighted in yellow.
Using Cell Absorbance Counter
In order to use the cell absorbance counter, a 96 well plate was used. Each plate is 8 rows lettered A through H and 12 columns numbered 1 through 12. A p200 pipette was used to fill the wells as follows:
Group | Volume of Solution | Rows | Column |
Plain LB | 100µL | A-D | 1 |
K12 | 100µL | A-D | 2 |
K12/amp | 100µL | A-D | 3 |
K12/non-dilute | 100µL | A-D | 4 |
K12/1:10 | 100µL | A-D | 5 |
K12/1:100 | 100µL | A-D | 6 |
MM294 | 100µL | A-D | 7 |
MM294/amp | 100µL | A-D | 8 |
MM294/non-dilute | 100µL | A-D | 9 |
MM294/1:10 | 100µL | A-D | 10 |
MM294/1:100 | 100µL | A-D | 11 |
Each control and experimental group was pipetted into four wells to increase the validity and consistency of the results. The plate was then placed into the machine and run. (for more information on how the cell absorbance counter works see under the materials section) Results were then printed and recorded, and each group’s four entries were averaged by using the following formula:
Avg. OD Reading for Columnn =
The average OD reading for each group was then recorded (see results section). The bacterial broth cultures were then placed back in the incubator in order for the process to be repeated for the next four days. This experiment was continued over multiple days to observe trends in the bacterial growth and observe the potential for antibiotic or phage resistance in both strains of bacteria.
Results:
Plate Results:
The overall results of the plate test showed that the ampicillin more easily killed the non-resistant K12 than all three of the phage dilutions did. For the ampicillin resistant MM294 strain, exclusive ampicillin treatment did not effectively kill any bacteria, whereas all three of the phage dilutions effectively decreased the growth of the MM294 from a lawn to small, disparate colonies. However, when the ampicillin was administered in conjunction with all three of the phage dilution, more colonies grew than the groups that only received phage treatment without ampicillin.
The results of the agar plates are divided into two categories: K12 groups and the M294 groups. In the K12 group, the K12/no phage/LB control group exhibited a complete lawn over the agar plate; no bacterial death was observed and therefore 0 plaques were recorded. In the K12/non dilute phage/LB experimental group, a lawn-type growth was observed and approximately 1692 total plaques were recorded. In the K12/1:10 phage/LB experimental group, a similar lawn-type growth was observed and 130 total plaques were recorded. In the K12/1:100 phage/LB experimental group, a similar lawn-type growth was observed and 12 total plaques were recorded. Finally, the K12/LB/amp experimental exhibited no growth, therefore there were not distinct plaques in a lawn of bacteria, but instead no living bacteria whatsoever.
In the MM294 group, the MM294/no phage/LB control group exhibited a complete lawn over the agar plate; no bacterial death was observed and therefore the number of specific colonies was unquantifiable and instead recorded as a “lawn.” In the MM294/non dilute phage/LB experimental group, 29 small white bacteria colonies were observed and recorded. In the MM294/1:10 phage/LB experimental group, 52 small white bacteria colonies were observed and recorded. In the MM294/1:100 phage/LB experimental group, 53 small white bacteria colonies were observed and recorded. The MM294/no phage/LB/amp experimental group exhibited a complete lawn over the agar plate; no bacterial death was observed and therefore the number of specific colonies was unquantifiable and instead recorded as a “lawn.” In the MM294/non dilute phage/LB/amp experimental group, 229 large/medium white bacteria colonies were observed and recorded. In the MM294/1:10 phage/LB/amp experimental group, 414 large/medium white bacteria colonies were observed and recorded. Finally, in the MM294/1:100 phage/LB/amp experimental group, 201 large/medium white bacteria colonies were observed and recorded.
Photo Set 1. K12 colony plate results. In this photoset, the results of the K12 colony plates are displayed. The top row contains the results of the K12/no phage/LB control group, the K12/non dilute phage/LB group, and the K12/1:10 dilute phage/LB group. The second row contains the results of the K12/1:100 dilute phage/LB group and the K12/no phage/LB/Amp group.
Table 2. The Number of Plaques in K12 Control and Experimental Plate Colonies. In this table, the results of the K12 control and experimental groups are recorded. The control group that received no phage or ampicillin treatment is noted by “(C).” The number of plaques recorded corresponds with the number of plaques/holes in the lawn of bacteria. For the ampicillin group, there was no bacterial group and therefore there was not a quantifiable number of plaques to be recorded.
K12 | No Phage (C) | Non Dilute | 1: 10 | 1: 100 | Ampicillin |
Number of Plaques | 0 (Lawn) | 1692 | 130 | 12 | No Growth |
Figure 3. The Effect of T4 Bacteriophage and Ampicillin Treatment on the Growth of Non-resistant E. coli K12. In this figure, the number of plaques in each K12 group is presented on a bar graph. The no phage/no amp control group is noted with a “(C).” The specific number of plaques recorded in each group is also displayed in the figure.Though the Ampicillin bar only extends to the top of the graph which only signifies 4000 plaques, the “number of plaques” was actually infinite because there was no growth on the plate and therefore an infinite number of holes in the non existent lawn of bacteria.
Photoset 2. MM294 colony plate results. In this photoset, the results of the MM294 colony plates are shown. The first row contains the results for the MM294/no phage/LB control group, the MM294/non-dilute phage/LB group, and the MM294/1:10 phage dilution/LB group. The second row contains the MM294/1:100 phage dilution/LB group, the MM294/no phage/LB/amp group, and the MM294/non-dilute phage/LB/amp group. The third row contains the MM294/1:10 phage dilution/LB/amp group and the MM294/1:100 phage dilution/LB/amp group.
Table 3. The Number of Plaques in MM294 Control and Experimental Plate Colonies.. In this table, the results of the MM294 control and experimental groups are recorded. The control group that received no phage or ampicillin treatment is noted by a “(C).” The number of colonies corresponds with the number of distinct colonies observed on each plate. For both the control and ampicillin group, there was not a quantifiable number of colonies, so the growth was recorded as “lawn.”
MM294 | No Phage (C) | Non Dilute Phage | 1:10 Phage | 1:100 Phage | Ampicillin | Amp + ND | Amp + 1:10 | Amp + 1:100 |
Number of Colonies | Lawn | 29 | 52 | 53 | Lawn | 229 | 414 | 201 |
Graph 2. The Effect of T4 Bacteriophage Treatment and Ampicillin Treatment on the Growth of Ampicillin Resistant E. coli MM294. In this figure, the number of colonies recorded in each MM294 group is presented in a bar graph. The no phage/no amp control groups is noted by a “(C).” The specific number of colonies recorded in each group is also displayed in the figure. Though the No Phage (C) and Ampicillin groups extend to the top of the graph which only signifies 1000 colonies, the number of colonies was actually unquantifiable and nearly infinite because a complete lawn of bacteria was observed on both plates.
Cell Plate Counter Results:
Written Results:
In this experiment, the absorbance or optical density of several bacteria cultures were observed and recorded. The overall results of the K12 colonies show that ampicillin treatment was the most effective treatment with no observable increase of absorbance over the course of 4 days. The overall results for the MM294 show that the ampicillin treatment did not inhibit the growth of the bacteria because the absorbance of the MM294/amp group was extremely similar to the controlled bacteria group that received no treatment. The MM294 groups treated with the various phage dilutions exhibited only a slight increase of absorbance over the course of three days. However, after day 3, there was a stark decrease of absorbance, suggesting that the MM294 bacteria began to die and therefore the data for that day does not accurately show the general trend.
The results are divided into the absorbance readings for the K12 cultures and the absorbance readings for the MM294 cultures, but the same procedure was followed for both sets of cultures. For both, the A1 well was filled with plain LB broth in order to get a constant point of comparison. On K12 Day 0, all of the wells had an absorbance of 0.032. After the overnight incubation, the Day 1 readings were taken. Uninhibited, the K12 bacteria had an absorbance rate of 0.500. The blank LB and the K12/amp cultures both exhibited practically no growth with absorbance readings of 0.036 and 0.045, respectively. The non-dilute phage also showed very little growth at 0.071. The 1:100 phage concentration allowed more bacterial growth than the non-dilute at 0.110. The results for the 1:10 phage dilution were inconclusive and therefore excluded. By Day 2, there was noticeably more bacterial growth in the cultures treated with phage dilutions. The non-dilute culture increased in opacity by 0.032, indicating some resistance to the treatment. The 1:100 dilution increased in opacity by 0.156, mimicking the results shown in the non-dilute culture. This trend would continue throughout the entire testing period. The ampicillin-treated culture and blank LB culture stayed relatively constant throughout the testing period.
The initial MM294 readings indicated an absorbance rate of 0.032 throughout all of the cultures. The Day 1 readings showed a significant amount of bacterial growth within the MM294 and MM294/amp cultures, as both were growing relatively uninhibited. The absorbance rates for these cultures were 0.332 and 0.327 respectively. The blank LB stayed constant with virtually no bacterial growth at 0.036, 0.035, 0.023, and 0.035 throughout the trial period. The phage dilutions had Day 1 absorbance rates of 0.066, 0.065, and 0.065 for the non-dilute, 1:10 dilution, and 1:100 dilution. On Day 2, the absorbance rate of the non-dilute phage culture increased by 0.082 while the absorbance rates for the 1:10 and 1:100 dilution only increased by 0.005 and 0.012. On Day 3, the non-dilute phage culture showed a dip in its absorbance rate, dropping down to 0.135. The 1:10 and 1:100 phage dilutions exhibited increased absorbance rates of 0.149 and 0.141 The results for the MM294, and MM294/amp cultures stayed constant through Day 3. The Day 4 results for the controls, the MM294 culture and the MM294/amp, indicated a drop in the absorbance rates from 0.503 and 0.480 on Day 3 to 0.112 and 0.168 on Day 4. For the non-dilute, 1:10, and 1:100 phage dilutions, there was relatively no change in their respective absorbance rates with each dilution changing 0.006, 0.009, and 0.034 from their Day 3 readings.
Table 6. Average absorbance reading for K12 cultures over the span of 4 days. For each group, there were four absorbance readings and the average of those four readings are displayed below. The wells in which each group was placed in is denoted next to the name of the group. For the K12/1:10 group, the readings made on day 1 and day 3 are excluded because there was no conclusive mean and therefore no data could be recorded.
Day 0 | Day 1 | Day 2 | Day 3 | Day 4 | |
Blank LB (A1-D1) | 0.032 | 0.036 | 0.035 | 0.023 | 0.035 |
K12 (A2-D2) | 0.032 | 0.500 | 0.513 | 0.486 | 0.605 |
K12/Amp (A3-D3) | 0.032 | 0.045 | 0.037 | 0.048 | 0.049 |
K12/Non Dilute (A4-D4) | 0.032 | 0.071 | 0.103 | 0.105 | 0.18 |
K12/1:10 (A5-D5) | 0.032 | x | 0.089 | x | 0.098 |
K12/1:100 (A6-D6) | 0.032 | 0.110 | 0.266 | 0.223 | 0.101 |
Graph 4. The Effect of T4 Bacteriophage Treatment and Antibiotic Treatment on the Absorbance Rate of E. Coli K12 Broth Culture. This graph depicts the average absorbance of each group in nanometers over the time cultured in days. There is no line for the K12/1:10 phage dilution because our results were not continuous.
Table 7. Average absorbance reading for MM294 cultures over the span of 4 days. For each group, there were four absorbance readings and the average of those four readings is displayed below. The wells in which each group was placed in is denoted next to the name of the group.
Day 0 | Day 1 | Day 2 | Day 3 | Day 4 | |
Blank LB (A1-D1) | 0.032 | 0.036 | 0.035 | 0.023 | 0.035 |
MM294 (A7-D7) | 0.032 | 0.332 | 0.466 | 0.503 | 0.112 |
MM294/Amp (A8-D8) | 0.032 | 0.327 | 0.377 | 0.48 | 0.168 |
MM294/Non Dilute (A9-D9) | 0.032 | 0.066 | 0.148 | 0.135 | 0.121 |
MM294/1:10 (A10-D10) | 0.032 | 0.065 | 0.070 | 0.149 | 0.14 |
MM294/1:100 (A11-D11) | 0.032 | 0.065 | 0.077 | 0.141 | 0.175 |
Graph 5. The Effect of T4 Bacteriophage Treatment and Antibiotic Treatment on the Absorbance Rate of E. Coli MM294 Broth Culture. This graph depicts the average absorbance of each group in nanometers over the time cultured in days.
Discussion:
Through this investigation, the antimicrobial efficacy of the T4 phage was compared to the efficacy of the common antibiotic ampicillin. In this experiment two strains of E. coli were used as host organisms: E. coli K12 and E. coli MM294, which contained the pAmp plasmid. Two different strains were used to show the differences in antibiotic and phage efficacy against both non resistant and antibiotic resistant bacteria. The inclusion of the E. coli MM294 was meant to replicate an incurable, resistant bacteria that could very well be encountered in the real world. Initially, this experiment was designed to use the same strain of E. coli (K12) for both resistant and nonresistant groups of bacteria by using rapid colony transformation to create a strain of the K12 bacteria that received the pAMP plasmid and was resistant to ampicillin while keeping some colonies nonresistant. This method would have been ideal because it would eliminate the numerous variables that arise when comparing two different strains of bacteria (K12 and MM294); instead, the two groups of bacteria, non-resistant and amp. resistant, would essentially be identical except for the one variable of the pAMP plasmid. However, attempted rapid colony transformation and competent cell transformation resulted in inadequate growth of transformed colonies, and therefore a resistant version of the K12 strain was not able to be used. Though it would have been ideal to use the same strain of bacteria in both resistant and nonresistant groups, E. coli MM294 is a derivative of E. coli K12, thus making the MM294 strain susceptible to the same phage treatment a comparable strain to the K12. In order to test the efficacy of both antibiotic and phage treatment, each of the two strains was treated with either Ampicillin, a dilution of the phage solution (non dilute, 1:10, or 1:100), or a combination of the two treatments. By comparing the amount of bacterial growth, the relative efficacy of each form of treatment was determined.
The K12 results indicated that Ampicillin was the most effective in the treatment of non-resistant bacteria. In the plate experiments, the ampicillin was clearly the most deadly. There was no growth on the plate, giving it a one hundred percent efficacy. The phage dilutions, on the other hand, were not quite as successful. The non-dilute phage treatment produced a lawn with extensive plaques, but did not eliminate all of the bacteria and each dilution had progressively fewer plaques. Though the dilutions seemed to work because the number of plaques decreased approximately by ten fold with each dilution, even the most concentrated phage treatment resulted in significantly more bacterial growth than the ampicillin treatment. This indicates that, while a potential option, phage therapy is not as well suited to non-resistant bacteria strains. Therefore, without the presence of antibiotic resistance, the antibiotics designed to treat the bacterial infections are, in fact, more effective than alternative phage treatment.
The K12 cell counter results showed data that mirrored the findings in the plate experiment. Ampicillin proved to be the best method of treatment here as well because it showed very little deviation from the control of pure LB broth. This indicated that the ampicillin was killing all of the K12 bacteria. More importantly, though, the absorbance rates for the ampicillin culture were constant over the course of multiple days showing that there was no developing antibiotic resistance among the bacteria because there was no additional bacterial growth over the span of the four days. This is significant because it demonstrates that proper antibiotic treatment administered in the correct concentration can not only effectively kill the bacteria, but also will not produce a significant amount of resistant bacteria.
The K12 groups treated by the phage dilutions all initially started with very low absorbance rates indicating that there was a relatively low amount of bacterial growth due to the presence of the phage. However, the readings of the course of successive days showed that the K12 strain developed a slight resistance to the phage treatment fairly quickly. The absorbance rates increased everyday, but they remained significantly lower than the plain K12 culture, indicating that the treatment was still beneficial. This shows that over time the effectiveness of the phage decreased and that some bacteria could have developed resistance to the phage treatment and begun growing in the culture despite the presence of the phage. Regarding the data for the K12/1:10 group, the average absorbances of day 1 and day 3 are excluded because there was not a conclusive mean between the four readings (A5-D5). On day 1, the absorbance readings ranged from 0.216, 0.307, 0.202, and 0.049. On day 3, the absorbance readings ranged from -0.073, -0.002, and 0.413. These data point showed no conclusive mean and therefore no viable average absorbance could be deduced. There was also the potential problem with the specificity of the phage. As discussed in the introduction, bacteriophages have a very narrow range and therefore only really treat one specific strain of bacteria completely. Therefore, it is potentially possible that the effectiveness of the phage therapy could have been compromised because the T4 wasn't necessarily suited best to the K12 bacteria strain.
The MM294 results, in contrast to the K12 results, proved that phage therapy would be the most effective method in treating antibiotic resistant bacteria strains. In the plate experiment, both the MM294 culture grown on a plain LB plate and the MM294 culture grown on a LB/amp plate showed the same amount of growth with a lawn of bacteria covering the plate. This indicated that ampicillin was not an effective treatment for these bacteria and proved that this particular strain was, in fact, resistant. Looking at the results from phage therapy, there appears to be promise for this method of treatment on antibiotic-resistant bacteria. The non-dilute phage only allowed for the growth twenty-nine small colonies, while both the 1:10 and 1:100 dilutions allowed for 52 and 53 colonies respectively. The low number of colonies that were able to grow shows that phage therapy could be very effective in treating bacterial strains that have developed resistant to commonly effective antibiotics, much like this one.
With the MM294 cell counter results, the potential for phage resistant bacteria was explored. The pure MM294 culture and the MM294/amp culture both had very high absorbance rates and, therefore, had significant bacterial growth, as was expected from the plate experiment. The Day 1 results for the phage dilutions indicated that all the phage dilutions were working with approximately the same efficacy because their absorbances were approximately the same. There was very limited growth of bacteria as was indicated by the minute increase in the absorbance rates as compared to the day 0 readings. However, by Day 2, the absorbance rates for the phages dilutions had doubled for the 1:10 and 1:100 dilutions and had quadrupled for the non-dilute phage group. The absorbance rates continued to increase over the trial period. These results present the possible threat of phage and antibiotic resistant bacteria, eliminating the known ways to kill these strains; if phage therapy was actually used to treat resistant bacteria, these results prove that the issue of resistance is not exclusive to antibiotic treatment, but could also occur using this alternative form.
In order to confirm these findings, more trial runs over a longer period of time were probably necessary. Also, the bacteria would have to be fresher colonies possibly grown in higher volumes because by Day 4, the cultures began dying, as made evident by the stark drop of absorbance from the absorbance of Day 3 to Day 4 in the MM294 and the MM294/amp colonies. Because this experiment was only run over the course of a week, these findings are not definite and conclusive and these trends should continue to be observed in future experiments.
With the combination of Ampicillin and phage treatment, the results showed that these two types of treatment counteracted each other and proved to be less effective than the phage treatment alone, but more effective that the ampicillin treatment on the resistant bacteria. These tests were only run on the antibiotic resistant strain MM294 because the experiment was designed to test the efficacy of the dual treatment against antibiotic resistant bacteria. Compared to the 29, 52, and 53 colonies exhibited by the non dilute, 1:10, and 1:100 phage treatments respectively, the combinations of ND/amp, 1:10/amp, and 1:100/amp grew much more colonies that were also larger in scale (229, 414, and 201 colonies respectively). These results demonstrated that though phage treatment on the resistant MM294 was relatively successful, in conjunction with antibiotics, the efficacy of the phage treatment decreased. Though the amp/phage treatment didn’t exhibit as much of a lawn as the amp. treatment, it was determined that the best form of treatment for resistant strains of bacteria was in fact exclusive phage treatment and not a combination of phage and antibiotics.
In conclusion, these experiments have proved that antibiotic treatment is still much more effective than phage therapy against non resistant bacteria. But, phage therapy still presents a possible solution to the rise of antibiotic resistant bacteria due to the relative effectiveness of the T4 phage as compared to ampicillin treatment against the resistant MM294. Despite the threat of bacteria developing phage-resistance, bacteriophages are naturally occurring and, as a result, are not difficult to develop and researchers would be able to genetically modify the viruses to make them more effective. However, the efficacy of both the antibiotic treatment and the phage treatment are compromised when used in conjunction with one another.
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