S. marcescens dominating E. coli

               

Hi folks :)

As midnight approaches, I'm asking myself how I putted myself in this situation where I have to write a blogpost, late at night, about some research I have done in Petnica, a serbian scientific center lost in the countryside. And I think that I actually enjoy sharing my experience with you guys but let's get sciency and start to focus on our story.

Introduction to prodigiosin :

I had to find a topic, a scientific question that I wanted to address, to experiment about and it wasn't something easy to find. Looking at some petri dishes that we plated days before, I noticed strong red spreaded over the plate. I was looking at bacteria Serratia marcescens producing pigments and I started to think : Having a synthesis pathway to produce a pigment must be energetically costing. So why would this ability being kept over generations if it's not giving some advantage in counterpart ?

So I started to search in the literature and I found out that this pigment, actually called prodigiosin, has some antifungal and antibacterial properties. I wanted to know more about prodigiosin and if it was responsible for an interferential competition in between S.marcescens and other Enterobacteriaceae. In other words :  Is the production of prodigiosin giving S. marcescens an advantage against other bacteria sharing its ecosystem inducing S. marcescens domination ?

How to crack the solution of the problem :

To find out about prodigiosin effects on other bacteria, I chose to test it on Escherichia coli as they are also Enterobacteriaceae and can be found in our guts just as S. marcescens. In a second time, I needed to have access to prodigiosin since I wanted to measure its specific effect, getting rid off all the biological noise provided by a living S. marcescens proliferating nearby. For the prodigiosin extraction, I decided to sonicate the cell, meaning use ultrasounds to create cavitation bubbles in the liquid breaking/cracking cell membranes, killing the cells and releasing the pigment in the media.

Here are more details about my protocol for interested ones ;)

I had two solutions, one containing prodigiosin (pig) and another without it (Øpig) that I applied on E. coli and Saccharomyces cerevisiae cultures. S. cerevisiae was  my positive control since the antifungal effect is known and admitted. So if no effects on S. cerevisiae were observed, the sonication process might had destroyed the prodigiosin and that’s why no effects would have been observed on E. coli.

Answer me !

About my results, I discovered that the S. marcescens in my solutions pig and Øpig weren’t dead and they started to proliferate on the E. coli and S. cerevisiae cultures. Nevermind, research is apparently never going well at first time when you’re testing new stuffs. However, S. marcescens took over both cultures ! Even if they were weakened by centrifugation and sonication. That’s why I decided to count how many Serratia marcescens I had in my pigmented and nonpigmented solutions.

Here are the results I obtained the day after :

Fig. 2 : 1 stands for E. coli culture and 2 for S. cerevisiae culture. Blue and green dots are the E. coli test and its replicate. Same for red and yellow dots but for S. cerevisiae.

As I observed 24 hours after, the cell concentration was much higher in the cultures where pig solution was applied compared to those of Øpig in both E. coli and S. cerevisiae.

End of my journey… but maybe start of yours ?

I discovered that 2 x 60 seconds of sonication is not enough for killing Serratia marcescens and I want to improve my protocol by sonicating for 7 minutes long as newly found literature adivises. Killing the bacteria and getting the prodigiosin out of the cells would allow more precise testing and better conclusions. We can even think of purifying the prodigiosin but it is much more complex. Overall, results showed that S. marcescens vigour is correlated with the presence of prodigiosin, making it easier to take over competing bacteria. But be conscious that correlation doesn’t mean causality, I need further experiments to conclude so.

    And that was it, I hope this blogpost gave you some ideas or interests about prodigiosin. Since it has some really cool properties, its popularity among searchers is rising. If you want to know more about it and become a prodigious (searcher) in cancer or infection treatment, here are some materials to start with. Enjoy !

• About prodigiosin on wikipedia
• About S. marcescens on wikipedia
• Biosynthesis of Prodigiosin, a Secondary Metabolite of Serratia marcescens
• The Opportunistic Pathogen Serratia marcescens Utilizes Type VI Secretion To Target Bacterial Competitors
• Prodigiosin is a chloride carrier that can function as an anion exchanger
• Antimicrobial activity of prodigiosin isolated from Serratia marcescens UFPEDA 398
• In Vitro Effect of Ultrasound on Bacteria and Suggested Protocol for Sonication and Diagnosis of Prosthetic Infections

Here is the link to my complete report 

By Nikola Zarevski

L2 FDV  Student Bachelor

 

Just how hot are biofilms?

Just how hot are biofilms?

Have you ever wondered the extent of biofilm protection? Well I have! For a week, I had the opportunity of working on the influence of temperature on biofilms, and if the later were really efficient when it came to protecting cells from a heat shock.

A biofilm is a clog of cells, linked together by a matrix called Extracellular Polymeric Substance (EPS). When bacteria are close enough, they send each other a signal, which tells them whether or not there is enough of them to form bonds. When a bacterium receives enough signals, it will modify its structure and create carbon and / or sugar bonds with each other. This EPS is able to provide protection from the outside environment, such as heat shock.

P.aeruginosa is perfect to work with as they are obligate aerobes, meaning that they need oxygen to live. This way, all the bacteria would come up to the surface to breathe, thereby all being at the same place at the same time. This way the bacteria will receive enough signals to form biofilms.

The first step for answering my hypothesis was to prepare my tubes. In order to do so, I put 1,5 mL of a mixture of a special growing media called LB, and P.aeruginosa, a bacteria that can easily develop biofilm, in 20 different tubes. Half of these tubes went into an incubator, and the other half in a thermo shaker (an incubator that moves non stop). 24 hours later, I took 10 tubes out (5 of each) and exposed 4 to 50°C for 5 minutes. I then vortexed the 10 tubes and plated them at 10^-4, 10^-5 and 10-6. Finally, I waited overnight for the colonies to develop, and counted them.

This graph represents the results I got. As you can maybe tell, they are not what I had expected… Once the plates had grown colonies, I was able to count them and therby determine the living cells ratio. First I had to count all the colonies. I obtained the following results: 63 and 49 for the plates that were in the incubator and that underwent heat shock (6,3 x 10^7 and 4,9 x 10^7 live cells); 123 and 98 for the samples that were in the incubator but didn’t go through heat shock (1,23 x 10^8 and 9,8 x 10^7 live cells). Regarding the samples that were in the thermo-shaker (and thereby had developed biofilms), the plates had 51 and 49 colonies for heat shock (5,1 x 10^7 and 4,9 x 10^7 live cells) and 80 and 93 (8,0 x 10^7 and 9,3 x 10^7 live cells) for no heat shock. What these results are showing is that in the samples without biofilms, the cells survived better to heat shock. When we do the ratio, we obtain 0,51 for the cells that were protected by a biofilm, and 0,58 for the cells that weren't.

However, these results cannot approve nor disapprove any hypothesis, as they are a single data. Moreover, when I looked at the samples under the microscopes, all the bacteria were alive and moving. This might suggest that the heat shock was not high enough. In order to answer my hypothesis, there are many options that could be furthered. For example, I could try putting the solution into test tubes instead of eppendorfs, or why not directly into petri dishes, thereby creating more interface with oxygen and easing the process of biofilm creation. Finally, as biofilms are supposed to protect cells from the outside environment, we could try to determine the limit of the protection by establishing the breaking point of the biofilm, meaning the point where we wouldn’t need to vortex the solution as the heat shock would have broken the biofilm itself.

If you want to know more, you can check these links :

            https://en.wikipedia.org/wiki/Biofilm#Properties

https://repositorium.sdum.uminho.pt/bitstream/1822/26324/1/PDF2.pdf

            http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4568995/

Special thanks to Petunia center, the CRI and Paris Descartes

Daphné Guénée 

@daphne_guenee

A mysterious wikipedia quote : does phosphate have an influence on prodigiosin secretion by S. marcescens ?

Picture from upload.wikimedia.org : Serratia marcescens

 

Wikipedia, a large and free dictionary, enriched by everyone is full of surprise. To find an idea about my project on microbiology in the Petnica Science Center, I did some research about one bacteria available in the lab : Serratia marcescens. The latter produce a red pigment that is also a secondary metabolite. To put word differently, it plays a role in the development of the bacteria but it isn't a vital one. During my research on wikipedia, it says that a “low quantity of phosphate” increase the concentration of pigment. This point was interesting me to create my project. Thus, I looked at the article related to this information. However, it isn't available on Internet. After more researches, I found other references to this article and also some old articles that made some similar experimentations.

Thus, to check if the wikipedia reference has disappeared for good reasons, I chose to work on the impact of phosphate on the prodigiosin secretion by S. marcescens. 

How ? I chose eight different concentrations of phosphate based on the work of K. Bahadur and S. Ranganayaki : 0g, 0.003g, 0.0045g, 0.006g, 0.0075g, 0.009g, 0.0105g, 0.0120g and 0.0135g that I put on 3 mL. I did three replicates for each concentrations. To check if the phosphate have an impact on the pigment, I did a negative control with two tested concentrations without pigment : one at 0.003g and an other at 0.0135g of phosphate. To obtain colony without pigment, I put during one night the tube in an incubator at 44°C while for the other one I put the temperature at 30°C.

Then to estimate the density of pigment, I put all the tube in a spectrophotometer. According to literature, the wavelength for the pigment is 532 nm. However, each tube didn't have the same concentration of cell. It is crucial to have this information because if the number of cell is hight, the quantity of pigment alss and conversely. Thus, I also measured the optical density of the bacteria, with a wavelength at 600nm. Next, I calculated the ratio pigment density per bacteria density to quantify the quantity of pigment produced per cell.

Ratio pigment density per bacteria density according to the concentration, in red the negative controle without pigment and in blue the other tube with pigment

Here the result : in red the negative control and in blue the ratio with pigment. The first point interesting is that without phosphate there are less pigment than cell. Then, the best quantity of phosphate is 0.006g. However, it is interesting to notice that the ratio decrease for 0.009g and increase again at 0.0105g. Thus, ion phosphate could interfere with the bacteria and slow down the production of pigment. 

Furthermore, an unexpected result is showing by this graph : the tube without pigment … produced pigment ! According to literature it is unexpected. However, it is fun to imagine that temperature could have an impact on the activity of the phosphate. To put word differently, at 44 °C the phosphate could help the bacteria to produce pigments while without the bacteria couldn't. Especially, the amount of pigment is higher than at 30 °C. Moreover, a higher quantity of phosphate seems to increase the production of prodigiosin. To test this hypothesis it could be interesting to put 0g of phosphate in one tube without pigment. Indeed, we could know if the presence of pigment is causes by the phosphate or if at 44°C pigment is produce.

Finally, to improve this experimentation, two things could be change. For instance, we can estimate the number of phosphate ion produce. It is interesting because we can know the level of saturation of the bacteria. Furthermore, we could extract the pigment from the bacteria to estimate more exactly the quantity of pigment. In fact, the wavelength of the bacteria and the pigment are close. Indeed, this overlapping created a bias because the bacteria could absorbed a little bit at 532nm.

To put in a nutshell, this humble work seems to be agree with the mysterious article quote by wikipedia. However, it could be interesting to improve it in order to increase our knowledge about the influence of phosphate on the bacteria.

If you want to know more :

- about prodigioson : https://en.wikipedia.org/wiki/Prodigiosin

- about secondary metabolite : https://en.wikipedia.org/wiki/Secondary_metabolite

- about Serratia marcescens : http://bacterioblog.over-blog.com/article-3817367.html

- about some characteristic of our bacteria :

1) J Bacteriol. 1971 May;106(2):438-43.Influence of temperature of incubation and type of growth medium on pigmentation in Serratia marcescens.Williams RP, Gott CL, Qadri SM, Scott RH.

2) Jpn J Microbiol. 1958 Apr;2(2):197-201.A study of the influence of milk, phosphate and calcium carbonate on the formation of 2,3 butanediol in Serratia marcescens cultures.BAHADUR K, RANGANAYAKI S.

- And if you would try to find the mysterious article : M. Todd-Guay and P.H. Demchick. 1995. Role of prodigiosin in phosphate-starved Serratia marcescens. Abstract of the Annual Meeting, American Society for Microbiology.