Week 17 (02/13/23 - 02/17/23): Moving Forward with New Species of Deinococcus

 Introduction:

 

The goals for this week were to finish with our MIC testing on D. aquaticus, as well as beginning our antibiotic testing with three new species of Deinococcus that were plated last week. The species chosen for our first part of antibiotic testing were D. grandis, D. roseus, and D. indicus. These species were chosen because they are fast growing, as well as being easier species to start testing on. The goal is to work on the easier species while we work to master our protocols and technique, then move on to the more difficult species as our project progresses. D. grandis and D. indicus are gram negative rods that grow in TGY. Like many of the Deinococcus species, they often stain gram positive due to their thick cell wall. Despite their appearance, they are classified as gram negative because their structure closely resembles gram negative bacteria. However, D. roseus is considered to be gram positive (Asker et al., 2008).

 

The plan is to test 3 different antibiotics at a time on one species to first identify what the bacterium is sensitive to. The overall goal of this entire project is to determine if there are any similarities between the different species of Deinococcus and the antibiotics they respond to. There are many antibiotics available in the lab, leading us to choosing 3 antibiotics each with a different mechanism of action: a cell wall synthesis inhibitor, a protein synthesis inhibitor, and DNA synthesis inhibitor. It will be interesting to see if all of the species we test on respond to the same antibiotics, or antibiotics with the same mechanism of action. Methicillin is the cell wall synthesis inhibitor chosen. This antibiotic is very dangerous to handle and is not used in medical practice anymore due to the antibiotic resistance, like methicillin-resistant Staphylococcus aureus (MRSA). Kanamycin is the second antibiotic chosen; it is a protein synthesis inhibitor that was selected because it is what D. aquaticus responded to best in our antibiotic testing last semester. Finally, ofloxacin was chosen as the DNA synthesis inhibitor because it has a high success rate of inhibiting growth in gram negative bacteria.

 

 

Methods:

 

MIC testing with Tetracycline: 

We started with a 500 ml flask containing 150 ml of TGY with 0.5% soft agar. The TGY was heated on a hot plate and set aside to cool when all of the media was liquified. After the media in the flask passed the baby bottle test, 2.5 ml of this TGY was added into 12 test tubes and was autoclaved for 30 minutes.

 

Gram staining was performed on D. aquaticus to confirm there was no contamination in the sample, and the sample was read on the nanodrop to determine the OD600 value. Gram staining results are shown in figure 3, OD values are shown in figure 4. The original OD value was 2.76, which was too high to use in our experiment. To obtain a lower OD value, 200 ul of the D. aquaticus sample was mixed with 200 ul of TGY. The sample was measured again on the nanodrop to confirm we had a sufficient amount of bacterial cells in our sample. 

 

The 12 test tubes each containing 2.5 ml of solidified TGY were heated in a beaker filled with water over a hot plate. Once the media in the test tubes had completely liquified, the tubes were removed from the beaker and set aside to cool. Then TGY was removed from each tube; this was done to have the total volume in each test tube at 2.5 ml after the tetracycline was added. Specific amounts of TGY removed from each test tube are shown in figure 1. Once all the test tubes were cooled enough to pass the baby bottle test, a serial dilution was performed. The 0.05 mg/ml stock solution of tetracycline was mixed on the vortexer at level 6 for 6 seconds, and 50 ul of tetracycline was removed from this solution and added to test tube 1. In between each serial dilution, each tube was mixed on the vortexer at level 6 for 6 seconds after tetracycline was added. This was done to ensure that the antibiotic is being distributed throughout the media, allowing us to have a more accurate estimate of the concentration of tetracycline in each tube. Continuing on with the dilution: 1250 ul was removed from test tube 1 and added to test tube 2, 500 ul was removed from test tube 2 and added to test tube 3, 250 ul was removed from test tube 3 and added to test tube 4, and 250 ul was removed from test tube 4 and added to test tube 5. No antibiotics were added to test tube 6 and 7. 25.25 ul of D. aquaticus was added to test tubes 1-7, and all the tubes were mixed on the vortexer. Before the contents in all 7 test tubes solidified, the tubes were poured onto their designated plate and the plate was swirled gently to create a lawn of liquid. The plates were left out to dry for 10 minutes and placed in the 30^o C incubator for 48 hours. Diagram of the MIC testing is shown in figure 1.  

Figure 1: Illustration of the entire MIC procedure performed on D. aquaticus with tetracycline. There were 5 different antibiotic concentrations tested in duplicates (tubes 1-5), totaling 10 tubes. Additionally, there was 1 sample taken with a tetracycline concentration of 0 ng/ml, and there was 1 negative control containing only 1.5% TGY and 0.5% TGY. Tetracycline concentrations are highlighted in green in the top part of the diagram and in purple in the bottom part of the diagram.

 

Antibiotic testing on D. grandis, D. roseus, and D. indicus:

200 ml and 400 ml of TGY were prepared and autoclaved, with intentions to use in antibiotic testing with the three new species of Deinococcus. Gram staining was performed on D. grandis, D. roseus, and D. indicus to confirm the samples were not contaminated. Stock solutions of methicillin, kanamycin, and ofloxacin were made at a concentration of 2 ug/ml. TGY was added to 21 test tubes and the tubes were autoclaved for 30 minutes. Amounts of TGY added to each test tube are shown in figure 2. 

Figure 2: Outline for antibiotic testing on the new species of Deinococcus.

Results:

Figure 3: Gram staining results for 4 different Deinococcus species, viewed under the 100X oil immersion lens. The species are as follows: a is D. roseus, b is D. grandis, c is D. indicus, and d is D. aquaticus.

 

Date	Species	A600	Correction	λ	A(λ)
02/15	D. roseus	1.04	0.10	600	1.04
02/15	D. grandis	1.89	0.10	600	1.89
02/15	D. indicus	0.64	0.10	600	0.64
02/14	D. aquaticus	0.76	0.10	600	0.76

Figure 4: OD600 values from the 4 Deinococcus species listed.

Figure 5: Plating results from the MIC procedure performed on D. aquaticus with different of tetracycline. The concentrations of tetracycline are as follows (from left to right): 1000 ng/ml, 500 ng/ml, 100 ng/ml, 10 ng/ml and 1 ng/ml. The negative control and the 0 ng/ml concentration of tetracycline are not pictured, however, they produced the expected result.

Discussion:

 

The gram staining and OD values of all of our bacterias looked good, the OD value of D. indicus was the only one that needed improvement so this bacteria was placed back into the 30^o C incubator. Based on our results from the MIC test, the MIC is either at 1000 ng/ml, or it is higher. It is hard to tell because one of our 1000 ng/ml plates had no growth, and the other one did have growth. Moving forward, we will test D. aquaticus with larger concentrations of tetracycline to see if 1000 ng/ml is at the perfect concentration to inhibit growth, or if more antibiotic is needed. Further antibiotic testing will be done on the new species of Deinococcus, and 3 additional species will be prepared next week so we can continue moving forward with antibiotic classification.

References:

Asker, D., Awad, T. S., Beppu, T., & Ueda, K. (2008). Deinococcus misasensis and Deinococcus roseus, novel members of the genus Deinococcus, isolated from a radioactive site in Japan. Systematic and Applied Microbiology, 31(1), 43–49. https://doi.org/10.1016/j.syapm.2007.10.002