PROJECT OF MERIT
Montserrat Roberts, Fjona Dulli, Sean Dorenkott, Olivia Wright, and Terri N. Ellis
Dr. Terri N. Ellis | College of Arts and Sciences | Department of Biology
Klebsiella pneumoniae is a gram-negative nosocomial pathogen and causative agent of many hospital acquired infections. K. pneumoniae infections have become increasingly of interest due to the rise of hypervirulent variants and multidrug resistant strains. Modeling how antibiotic resistance evolves in K. pneumoniae will allow us to better understand exactly how the bacterium acquires resistance to various antibiotics. A previous experiment in our lab exposed a strain of K. pneumoniae to low but increasing concentrations of the antibiotic cephalothin. As a result, the strain evolved to be mucoid with elongated cellular morphology and resistant to multiple antibiotics. This study aimed to repeat the same experimental approach with multiple cultures, to determine if different genomic mutations could result in the same endpoint of antibiotic resistance. Five cultures of K. pneumoniae 43816 were exposed to increasing amounts of the antibiotic cephalothin over a 14-day period. After the 14 day experiment, cultures were assayed for changes in antibiotic susceptibility, colony, and cellular-level morphology. Preliminary results indicate evolved resistance to cephalothin and tetracycline, but not kanamycin. Further, alterations in the colony morphology have been noted with a mix of small and large colony phenotypes. This variation of colony morphology in the adapted population may indicate different genetic mutations that correspond to these large and small colony variants. Current work is determining the relationship between colony morphology and antibiotic resistance.
Klebsiella pneumoniae is a gram-negative pathogen and causative agent of many hospital acquired infections. With many of these infections becoming resistant to antibiotics, it’s important to investigate how antibiotic resistance evolves.
In this study, five cultures of Klebsiella pneumoniae were exposed to increasing concentrations of cephalothin over a 14-day period. We then observed changes in antibiotic susceptibility, colony morphology changes, and biofilm formation.
So far, we’ve observed resistance to the antibiotics cephalothin and tetracycline, but not kanamycin. Additionally, we’ve observed some phenotypic changes. In one of the replicates that was treated with cephalothin for 14 days we observed 2 different phenotypes that were not present at the beginning of the experiment in the susceptible strain. In addition, the ability to form biofilms was also observed and compared between replicates. An increase in biofilm formation was observed as Klebsiella pneumoniae developed antibiotic resistance.
Our goal in this experiment was to characterize the various changes in Klebsiella pneumoniae in response to developing in vitro cephalothin resistance. Additionally, we determined how cephalothin resistance impacted Klebsiella pneumoniae’s susceptibility to other antibiotics. Finally, we investigated the phenotypic changes and the ability of each replicate to form biofilms.
6 replicates of Klebsiella pneumoniae were grown in six separate test tubes–five if these replicates were exposed to increasing levels of antibiotic cephalothin. We labeled these 5 treated replicates as A, B, C, D, and E. The sixth replicate was not exposed to any antibiotic over the 14 days and was labeled U for untreated. In increments of 12 hours, the 6 separate cultures were given fresh media. The 5 treated cultures were given increasing concentrations of antibiotic every 12 hours. At each time point of 12 hours, samples of the individual 6 replicates were frozen. We continued this process every 12 hours for 14 days.
We then examined phenotypic changes. Colonies of replicate A displayed large and small colony variants after 14 days of growth. These colony variants were not present on Day 0 of the experiment. We then isolated the small and large colony variants onto their own agar plates. Isolation of individual colonies gave rise to their respective phenotypes. In addition, Culturing individual colonies in LB media showcased further phenotypic differences. The large colony variants appeared to have large aggregates in culture whereas small colony variants did not.
We then examined the ability of our isolates to produce biofilms.
A Biofilm is a multicellular community of bacteria that can adhere to surfaces. They contain a protective layer, which can make it difficult for antibiotics to reach them.
An overnight culture of klebsiella pneumoniae was grown in lb broth. Each culture was diluted and added to a microtiter plate where it was grown overnight. The planktonic bacteria was removed and the remaining bacteria was stained with crystal violet. The absorbance of each sample was then read at 570 nm.
All isolates were found capable of forming biofilms. The day zero isolate had the weakest presence of biofilm and was statistically different when compared to all other samples.
On day 0, all isolates began their journey as susceptible to the antibiotic cephalothin. After the 14-day experiment, we examined the susceptibility of our newly adapted strain to different antibiotics. The antibiotics tested include cephalothin, tetracycline, kanamycin, and amikacin. We examined the minimum concentrations needed of each antibiotic to kill our 7 isolates from day 14 and day 0.
Overall, we found that induced cephalothin-resistant K. pneumoniae strains showed increased resistance to cephalothin, mild resistance to tetracycline, and no change in resistance to kanamycin. A phenotypic change in colony morphology was observed in one treated replicate. For Day 14A, plates contained both small and large colonies. Individual colonies were isolated and showed lower breakpoints for cephalothin. However, once colonies were recombined, resistance was restored and even amplified. Each replicate was able to produce biofilm. Day 0 was found to be the weakest biofilm producer and all Day 14 isolates saw a significant increase in biofilm production.
Our future research will focus on Whole-genome sequencing, we wish to identify the genetic mutations responsible for the evolution of antibiotic resistance and the differences between small and large colony variants. We plan on testing to see if there is a relationship between the biofilm capabilities of small and large variants of the 14-day A isolate. Another direction we intend to follow is discovering where within the 14 day experiment the morphology changes arose. We also aim to assess if there is a relationship between colony morphology, biofilm formation, and antibiotic resistance.
We would like to thank UNF’s Transformational Learning Opportunity grant for funding this research as well as all of those who have participated in this experiment. And we would also like to thank you for your time and attention.