It’s S. marcescens, Chemically Mutated S. marcescens

Lab report on experimentation involving chemical mutagenesis of Serratia marcescens, performed in my Molecular Genetics Laboratory.

Introduction
Serratia marcescens lives a double life. On the one hand, it has been studied extensively and serves as a model bacterium in teaching and research laboratories to study bacterial motility and protein secretion, in part due to its great adaptability (Williams et al., 2022). Concerning this series of experiments, its known ability to produce lipase was of great importance. However, this same adaptability makes S. marcescens an opportunistic pathogen. It bides its time in the ICU, making its way into all manners of medical equipment, onto medical professionals, and even surviving in disinfectant solutions until it can find a suitable host, oftentimes someone whose immune system has already been weakened by other infections (Tavares-Carreón et al., 2023). Mutagenesis of S. marcescens is a useful way to investigate genes and their various phenotypes, as well as the biological processes they control, which can be useful in researching the mechanisms by which it survives and searching for novel treatments. Chemical mutagenesis utilizing nitrosoguanidine (NTG) was chosen for this experimentation because of S. marcescens susceptibility to the compound, and the ability to influence mutation rate through the manipulation of reaction conditions.
To determine the ideal reaction conditions for this mutagenesis to occur, spectrophotometry was performed to quantify the increase in cell density over time and establish a growth curve for S. marcescens. This information was used to determine the colony-forming units (CFU) per milliliter of solution at a given optical density (OD) value. Targeting the OD value corresponding to approximately 1x108 cells/mL then facilitated testing of multiple NTG concentrations to determine the best conditions to achieve a 90% kill ratio of S. marcescens. The selected NTG concentration was used on S. marcescens cells, which were plated on media supplemented with Tween80 to monitor the precipitation of the colonies. We postulated that achieving a 90% kill ratio of S. marcescens using NTG would result in the creation of lipA mutants identifiable through a lack of precipitation on Tween80-supplemented media.
Results
In the first experiment, a nutrient medium LB broth was inoculated with S. marcescens and incubated at 30°C for a total of 2.5 hours, with a 1 mL aliquot of the solution removed for spectrophotometric analysis every 30 minutes. Optical density was measured at A595 and can be seen graphed with respect to incubation time in Figure 1. OD is observed to have increased with incubation time. The sampled aliquots were then 10-fold serially diluted, drop-plated, and incubated overnight to facilitate the calculation of colony-forming units (CFU) per milliliter, which was graphed against the measured OD value in Figure 2. The OD value corresponding to approximately 1x108 cells/mL was determined to be 0.355 Au.
To facilitate the second experiment, the procedure for inoculation and incubation of nutrient medium LB broth was repeated until the desired OD value of 0.355 Au was reached, at which point the cells were split into 6 experimental tubes which were exposed to varying concentrations of NTG diluted into EtOH (0, 1.25, 2.5, 5, 10, 20 mg/mL NTG) for a 15 minute incubation period before extensively washing. 10-fold dilutions of each experimental tube were prepared and drop plated, and CFUs/mL were calculated after overnight incubation, summarized in Table 1. It was determined that the desired 90% kill rate was achieved with exposure to the 2.5 mg/mL NTG solution.
In the final experiment, 100 mL of LB broth was inoculated and incubated until it reached the desired OD value. At that point, it was split into 4 experimental tubes: one which was immediately plated, and three which were incubated and washed as per the NTG treatment specifications. Of these three tubes, one received the determined 2.5 mg/mL NTG treatment, one an EtOH treatment, and the last with no additional treatment. After 10-fold dilutions and drop plating, the plates were incubated and CFUs/mL were calculated as seen in Table 2. The percentage kill as a result of NTG treatment was determined to be 78.57%. More plates of S. marcescens NTG mutant colonies were incubated to facilitate the replica plating of 600 colonies onto Tween 80 (1%) so that they could be monitored for precipitation associated with lipase production. After incubation, these plates were observed to show no lipase-negative mutants, or 0%.
Discussion
Mathematically, there is about a 0.02% chance of causing a mutation in the lipA gene which would be experimentally proven by lack of visible precipitation when plated onto Tween80 plates. Taking into account the 600 NTG mutated colonies replica plated at the end of the final experiment, we anticipated 12 lipase-negative colonies but did not observe any.
With each subsequent experiment, we were building on the results of those that preceded it. First concluding that 1x108 cells/mL corresponded to an OD value of 0.355 Au, and then concluding a 90% kill rate could be achieved using 2.5 mg/mL of NTG. Despite achieving a 90% kill due to NTG treatment in the second experiment, the final percent kill of S. marcescens was only 78.57%. In this final experiment, NTG exposure resulted in reduced CFU/mL during the experimentation process, as expected, which indicates that mutagenesis was successful in generating lethal mutations in S. marcescens. It simply was not as successful as had previously been determined.

There were several irregularities in the data throughout the experimentation process which likely contributed to the less-than-anticipated kill percentage after NTG treatment. It is likely the incubated broths in each week’s experiments were not in the same phase of growth, which would lead to a lessened effect of NTG exposure in the final experiment, simply because it can not be expected that the different cultures will act identically weeks apart from each other. Additionally, during the second experiment, the broth was inoculated in two steps, first with 1 mL of S. marcescens and then an additional 9 mL a few minutes later (for a total of 10 mL), due to a miscommunication in the written protocol. Adding 9 mL after the initial 1 mL means that the cells were not in the same growth phase as the other cells in the broth. It can be seen in Table 1 that optical density does not increase linearly, meaning this action cannot easily be mathematically corrected in the subsequent data. Inconsistencies in following the protocol, such as an incubation step being slightly too long or too short, would also result in slightly skewed results. If they occurred in the initial experiments, it would be the protocol for the final experiment was flawed and would be unable to achieve the desired level of mutagenesis. If they occurred in the final experiment, it would result in an unexpected percent kill. It is also possible that lipase-negative mutants were present in the S. marcescens NTG mutants, and they were simply not represented in the colonies replica plated onto Tween80 medium, or that the same colony may have been replica plated onto more than one plate. None of these factors can completely account for the discrepancy in expected versus actual lipase-negative mutants isolated, however together they introduce uncertainty to the data and may have contributed to these unexpected results.

Our hypothesis can be said to be inconclusive. While no lipase-negative mutants were observed, we were also unable to achieve 90% kill using NTG. To properly test the hypothesis, the experimental plan can be repeated, this time taking into account the missteps made the first time so that they may be avoided. An additional step in which colonies are plated onto minimal media supplemented with lipase could be added, as a means to pre-screen colonies so that the chances of isolating lipase-negative mutants is higher. Alternatively, a similarly designed experiment can be performed to induce mutation in another metabolic gene of S. marcescens.

Works Cited
Tavares-Carreón, F., De Anda-Mora, K., Rojas-Barrera, I. C., & Andrade, Á. (2023). Serratia marcescens antibiotic resistance mechanisms of an opportunistic pathogen: a literature review. PeerJ, 11, e14399. https://doi.org/10.7717/peerj.14399
Williams, D. J., Grimont, P. a. D., Cazares, A., Grimont, F., Ageron, E., Pettigrew, K. A., Cazares, D., Njamkepo, E., Weill, F., Heinz, E., Holden, M. T. G., Thomson, N. R., & Coulthurst, S. J. (2022). The genus Serratia revisited by genomics. Nature Communications, 13(1). https://doi.org/10.1038/s41467-022-32929-2