Streptomycin and kanamycin are both antibiotics used to stop bacteria’s process of translation. To understand how bacteria is resistant to these types of antibiotics and others, streptomycin and kanamycin, both aminoglycosidic antibiotics, were tested in this experiment to determine if cross-resistance was a factor in increased resistance. Streptomycin resistant and non-resistant strains of S. marcesens bacteria were plated and were used to determine the resistance of S. marcesens to kanamycin. After testing, the streptomycin resistant plates showed a higher average cell diameter than the non-resistant plates, meaning the zone of inhibition, or susceptibility to the antibiotic, was significantly higher in streptomycin resistant strain.
Key Words- streptomycin, kanamycin, S. marcesens, antibiotic resistance, disc diffusion, cross-resistance, zone of inhibition
Introduction
The organism being used, S. marcesens, has gram-negative like cell walls, meaning their strains appear pinkish red. There are many types of gram-negative bacteria, like E. coli, and these strains can often cause infections in humans if they are spread. (National Institute of Allergy and Infectious Diseases, 2017) The gram-negative bacteria, however, is often antibiotic resistant, meaning they develop mechanisms to fight off certain types of antibiotic designed to destroy them through random mutations.
The antibiotics used in this experiment are streptomycin and kanamycin, which are both aminoglycosidic antibiotics, meaning they contain amino groups bonded to sugars through glycosidic bonds, and stop bacteria from producing proteins necessary for their survival, known as the process of translation. This will kill the bacteria directly.
Different types of antibiotics, like kanamycin and streptomycin, were used in other studies looking at cross-resistance, since these similar drugs were used to treat diseases like tuberculosis. Multi-drug resistant tuberculosis, for instance, was producing an increased amount of resistance, when it was originally treated with kanamycin. (Jugheli et. al., 2009) Drug resistant bacteria were often isolated from different strains of Mycobacterium tuberculosis to test for mutations that occurred with antibiotics similar to kanamycin and streptomycin. (McClatchy et. al., 2005)
Additionally, in other studies, mutations in the bacteria’s tlyA gene were found to create resistance to the streptomycin and kanamycin drugs. (Maus et. al., 2005) This showed that mutations and the cross-resistant patterns were what created different antibiotic resistant variations, which led to an understanding that bacteria that were resistant to one antibiotic would develop the mutations against another antibiotic. (Alangaden et. al., 1998) This was very similar to what was hypothesized in this cross-resistance experiment. Also, other studies have found that strains that were made resistant in vitro to antibiotics like kanamycin were moderately resistant to streptomycin. (Kunin 1958)
In this experiment, two strains were used: streptomycin resistant S. marcesens and non-resistant S. marcesens. The parent culture does not have resistance yet, but the other strain has already developed defense to streptomycin. With this, it can be predicted that streptomycin resistant colonies will display more resistance to kanamycin than the parent colonies that have been exposed to streptomycin. Since they were already resistant to one aminoglycoside, they have a better chance at being resistant to both streptomycin and kanamycin. Colony count through disc diffusion was used to infer an answer. Mutations were associated with a high level of resistance, so that was taken into account here. (Meier 1996)
Methods
The parent culture used was Serratia marcesens, and the strain was D1, which was purchased from Carolina Biological Supply in 2009. The D1 strain was set in a phosphate buffered saline at 3 degrees Celsius. One colony of cells of S. marcesens was plated on agar with no antibiotic, and another colony was plated on agar with nutrient broth. Both were stored overnight at 30 degrees Celsius and shaken. This established the streptomycin resistant S. marcesens, as well as the non-resistant S. marcesens. To determine cross-resistance, nine plates of streptomycin resistant S. marcesens media were used and were plated with 25 mg/mL kanamycin, along with nine plates of non-resistant S. Marcesens media also plated with 25 mg/mL kanamycin. A plate of just streptomycin resistant S. marcesens media and a plate of just non-resistant S. marcesens media were tested for viability of the bacterias. The samples were plated in sealed plastic containers, and closed in an area for a week. The disc diffusion method was used to count the results, this being the average diameter of the control (non-resistant) and treatment (streptomycin resistant) S. marcesens plates. Data was imported into R software, and the Shapiro-Wilk Normality Test was run, which found normal data, so a T-test was then run, and a bar plot was made.
Results
After one week, the 18 different plates of S. marcesens were measured using the disc diffusion method.
Table 1: Measured Diameter of Control and Treatment S. marcesens
Diameter (cm) Bacteria
2.93 C
3.136 C
2.95 C
2.85 C
3.067 C
2.867 C
3.03 C
2.767 C
2.7467 C
3.417 T
3.07 T
3.017 T
3.1 T
3.167 T
3.183 T
3.2 T
3.05 T
3.213 T
Key: C=control (non-resistant S. marcesens plates) and T=treatment (streptomycin resistant S. marcesens plates)
The average diameter of the control cells is 2.927 cm and the average diameter of the treatment cells is 3.157 cm, allowing the conclusion to be drawn that the zone of inhibition is higher in the treatment strain, or that streptomycin-resistant S. marcesens are less resistant to kanamycin. To test the data for normality, the Shapiro-Wilk Normality Test was run. The p-value for the control samples was 0.8364 and the p-value for the treatment samples was 0.2503. Since these values were both above 0.05, the data was normally distributed. Because of its normality, a T-test was run. The p-value of the data after the T-test was run was 0.00413, which was significant, so the null hypothesis was rejected. A bar plot was then made for the normally distributed data.
Graph 1: Mean of diameter in centimeters of both control (non-resistant) and treatment (streptomycin resistant) strains of S. marcesens with respective SD values; p<0.005
Discussion
The results refute the hypothesis that streptomycin resistant colonies would display more resistance to kanamycin than the parent colonies that have been exposed to streptomycin. This was known through the average cell diameter measured through disc diffusion along with the normality test and t-test, because the average cell density of the treatment cells was greater, suggesting a higher zone of inhibition in the treatment strain. Some sources of error could have been if the spreader for the bacteria was not clean enough when it was being plated, which could have affected a cross contamination of non-resistant bacteria with streptomycin resistant bacteria. Additionally, there was not a perfect measurement of cell diameter, which could have changed some of the overall results, and there could have been an incorrect amount of kanamycin put in the cell during testing. After using the Shapiro-Wilk Normality Test, it was seen that the results were normal, and after using the t-test to check for significance, the p-value was significant, so the null hypothesis was rejected. Overall, the results differ from similar antibiotics, because in this case, the susceptibility of streptomycin resistant bacteria to kanamycin was significantly higher.