Acquisition of a flocculent phenotype in response to in vitro exposure of Klebsiella pneumoniae to the antibiotic cephalothin.

Presenter Information

Sean M. Dorenkott

Faculty Sponsor

Dr. Terri N. Ellis

Faculty Sponsor College

College of Arts and Sciences

Faculty Sponsor Department

Biology

Location

SOARS Virtual Conference

Presentation Website

https://unfsoars.domains.unf.edu/2021/posters/the-acquisition-of-a-flocculent-phenotype-in-response-to-in-vitro-exposure-of-klebsiella-pneumoniae-to-the-antibiotic-cephalothin/

Keywords

SOARS (Conference) (2021 : University of North Florida) – Archives; SOARS (Conference) (2021 : University of North Florida) – Posters; University of North Florida -- Students -- Research – Posters; University of North Florida. Office of Undergraduate Research; University of North Florida. Graduate School; College students – Research -- Florida – Jacksonville – Posters; University of North Florida – Undergraduates -- Research – Posters; University of North Florida. Department of Biology -- Research – Posters; bacterial pathogen -- antibiotic resistance – Posters; Project of Merit Award

Abstract

Klebsiella pneumoniae is a gram-negative bacterial pathogen that is notorious for being the 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 bacterial populations acquire resistance to various antibiotics. Presently, it is the aim of our laboratory to determine if different genomic mutations in K. pneumoniae acquired in response to antibiotic treatment could result in the same endpoint of antibiotic resistance. In our current experiment, five cultures of K. pneumoniae ATCC 43816 were exposed to low but increasing amounts of the antibiotic cephalothin over a 15-day period. After the 15-day experiment, alterations in the morphology of bacterial colonies have been noted. All tested strains have demonstrated a flocculent phenotype, which is a rarely-seen and understudied characteristic of K. pneumoniae. The flocculent phenotype is a type of protective biofilm that, rather than being attached to a surface (bodily or otherwise), is free-floating. This allows these bacterial aggregates (often called “flocs”) to be carried throughout the body by the blood or other fluids in the form of large, difficult-to-treat structures. Here, a novel method of quantifying antibiotic resistance of bacterial cells trapped in flocs is presented. Preliminary data from the current study indicates that flocculent structures provide K. pneumoniae with substantially increased protection from antibiotics.

Comments

Audio Presentation Transcript:

Hi! My name is Sean Dorenkott, and I am a Senior here at UNF majoring in Biology. I’m extremely excited to have the opportunity to virtually tell you about the research I have been involved in this semester. While I have worked in Dr. Ellis’s laboratory since the summer of 2019, it is not until the beginning of this semester that I specifically began to work on elucidating what is known as the flocculent phenotype in Klebsiella pneumoniae. This is type of protective biofilm that, rather than being attached to a surface (bodily or otherwise), is free-floating. But, more on that later. Let me start from the beginning.

First, a little bit of background on our experiment. Our lab studies a species of bacteria called Klebsiella pneumoniae. This bacterial species is rather notorious for causing hospital-acquired infections. This means that people who visit a hospital have a significant chance of picking up a pathogen from this species and thus a picking up a new sickness. As a result of its unwelcomed “stay” in a hospital, K. pneumoniae is also quite good at acquiring resistance to antibiotics. Our laboratory aims to model how antibiotic resistance evolves in K. pneumoniae so we can better understand how to fight this deadly agent.

To do so, we have conducted a largescale experiment. We have exposed five different cultures of K. pneumoniae to low but increasing concentrations of an antibiotic called cephalothin over a 15-day period. After 15 days of treating these bacteria with cephalothin, we noticed that all of the strains became resistant to cephalothin, which is naturally what you would expect to happen. But what we didn’t expect was for our strains to develop a flocculent phenotype. Bacteria quite commonly form what are known as biofilms, which are essentially slimy layers that bacteria form for protection. You are probably quite familiar with biofilms, although you may not know it. Biofilms constitute a lot of the gunk that forms on teeth. So, biofilms are normally sticky layers that form on surfaces. But what isn’t very common is for these biofilms to detach and float around as one large aggregate, and this is exactly what I mean when I say the “flocculent phenotype.” This is a characteristic that our resistant K. pneumoniae strains have acquired, wherein they form large, detached, slimy structures. This is particularly dangerous because since these structures are not attached to anything, they can be carried by fluids, such as blood, around the body. A picture of a culture containing flocs from one of our resistant strains, Day 15A, can be found at the top of the center of the poster. Day 0, our susceptible strain, shows no flocs. However, in response to 15 days of antibiotic treatment, Day 15A has developed a flocculent phenotype, which is visibly seen.

Given that a flocculent phenotype, especially in K. pneumoniae, is relatively uncommon, it is also not well-studied. Thus, it is the aim of my project to characterize these flocculent aggregates, also called flocs, and elucidate how they are tied to antibiotic resistance. To do so, I had to begin by developing a novel method for determining how a flocculent phenotype specifically affects resistance to antibiotics. It is very important in a clinical setting to actually quantify how much antibiotic it takes to kill bacteria. Normally when bacteria grow in a liquid media, they make the liquid media very cloudy. Using a special machine called a spectrophotometer, you can actually measure how “cloudy” a culture is to determine how much bacteria is in a sample. And then, when you have killed the bacteria with an antibiotic, the cloudiness disappears, which can also be measured. But, given that with a flocculent phenotype bacteria form large clumps, we cannot use cloudiness to determine how many living bacteria are present within a floc, since they are all clumped together into one concentrated aggregate. Thus, for my project, I turned to a metabolic dye called resazurin. In the presence of living bacterial cells, this dye irreversibly changes color from blue to pink. We can then use a spectrophotometer to measure how much resazurin was “reduced”, or how much resazurin turned pink. And this directly correlates to how much bacteria are alive.

A graphical diagram of the resazurin test I developed for this project can be found in the top right corner of the poster. I have utilized this test to specifically quantify how much cephalothin it takes to inhibit bacterial growth. We begin by isolating a floc using tweezers, and then washing the floc using a solution similar to saline. This removes any unwanted planktonic cells, as we’re only interested in cells that are actually trapped in the flocs. Then, the floc is added to fresh liquid media, along with a range of different antibiotic concentrations. The bacteria are treated in their antibiotic concentrations for a period of 24 hours, and then resazurin is added. It is originally blue, but in the presence of living cells, turns pink after 10 minutes. Then, a spectrophotometer can be used to quantify how much resazurin turned pink, which correlates to how much bacteria is still alive.

The data I have received from this test indicates that flocculent structures are allowing trapped bacteria to survive in abnormally high levels of antibiotic. That is, bacteria that form these clumps are much more resistant to antibiotic treatment. In the top graph with green bars at the center of the poster, it can be seen that our susceptible strain, Day 0, demonstrates significant growth up to a concentration of cephalothin of about 31.25 μg/mL. Planktonic cells of Day 15A, cells that are not trapped in a floc, can survive to concentrations of 125 μg/mL, which is an at least four-fold increase in the amount of cephalothin needed to inhibit growth compared to Day 0. However, when the bacteria are trapped in flocs, faint signals are seen at 250, 500, and 1000 μg/mL. This indicates that flocculent bacteria are still surviving and growing in these extremely high concentrations of antibiotic.

Overall, my preliminary data suggests that flocculent structures are able to confer additional resistance to antibiotics, specifically cephalothin, but likely others as well. Bacteria present within flocs are able to survive at much higher levels of antibiotic compared to when they are not found in flocs. In the future, I aim to characterize the flocs utilizing microscopy as well as culturing and plating techniques. I would also like to utilize whole-genome sequencing to identify what genetic mutations are responsible for the appearance of the flocculent phenotype. Lastly, and perhaps most importantly, I would like to develop a physical or chemical treatment that can be used to destroy flocculent structures and potentially restore antibiotic susceptibility of bacteria trapped inside of flocs.

Thank you so much for your time! I would now like to acknowledge funding from the UNF Transformational Learning Opportunity Grant that was used to support the present research, as well as Montserrat Roberts, Fjona Dulli, Olivia Wright, Jasmine Anderson, and Michael Durnin for their work and dedication to our 15-day experiment.

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