Biofilms are a structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface. These structures and the organisms that cause them can pose a very serious problem if they colonize on medical devices. This is because biofilms have the ability to communicate within the colony and with other organisms that might attach to the surface, acting like a community working together. Biofilms allow the organism to be resistant to harsh and unfavorable conditions allowing them to survive longer and spread. Several genes in Escherichia coli (E. coli) have been associated with biofilm formation by that organism. Many of those genes encode surface appendages such as flagella, fimbriae, and pili. We created mutations in genes encoding curli (csgA) and fimbriae (fimA) with the aim of comparing their ability to form biofilms. The respective genes were selected on kanamycin- containing agar and disrupted with a kanamycin resistance gene. Biofilm formation in nutrient- rich medium and nutrient-poor medium is currently in progress, and the ability of the mutant E. coli strains to form biofilms will be compared with that of the parent wild type strain using a crystal violet microplate assay

Snyder, Nicole

Willaert, Sean

English

Institutional Repository for Minnesota State University, Mankato

Journal of Undergraduate Researchat Minnesota State University,MankatoVolume 14 Article 92014Biofilm Formation by Escherichia coli csgA andfimA mutantsNicole SnyderMinnesota State University, MankatoSean WillaertMinnesota State University, MankatoFollow this and additional works at: http://cornerstone.lib.mnsu.edu/jurPart of the Genetics CommonsThis Article is brought to you for free and open access by the Undergraduate Research Center at Cornerstone: A Collection of Scholarly and CreativeWorks for Minnesota State University, Mankato. It has been accepted for inclusion in Journal of Undergraduate Research at Minnesota State University,Mankato by an authorized administrator of Cornerstone: A Collection of Scholarly and Creative Works for Minnesota State University, Mankato.Recommended CitationSnyder, Nicole and Willaert, Sean (2014) "Biofilm Formation by Escherichia coli csgA and fimA mutants," Journal of UndergraduateResearch at Minnesota State University, Mankato: Vol. 14, Article 9.Available at: http://cornerstone.lib.mnsu.edu/jur/vol14/iss1/9Student Agreement:I am submitting my research article to be published in the JUR (The Journal of Undergraduate Researchat Minnesota State University, Mankato), an electronic journal of the Minnesota State UniversityUndergraduate Research Center.I/We certify have followed the accepted standards of scientific, creative, and academic honesty andethics.I understand that my article submission will be blind-reviewed by faculty reviewers who will recommendacceptance for publication; acceptance with revisions; or reject for publication.I understand that as author, I retain the right to present any part of the research in any form in otherpublications.The JUR has the right to reproduce and reprint published submissions for instructional or promotionalpurposes.For complete details, see Journal of Undergraduate Research at Minnesota State University, Mankato policiespage.Mentor Agreement:I have reviewed the submission, and I support its inclusion in the JUR (The Journal of UndergraduateResearch at Minnesota State University, Mankato). I understand that I will be acknowledged as thefaculty mentor for the student author(s). To the best of my knowledge, the student has followed theaccepted standards of scientific, creative, and academic honesty and ethics.           Nicole Snyder and Sean Willaert and Dr. Timothy Secott (Faculty Mentor) Department of Biological Sciences Minnesota State University, Mankato  Biofilm Formation by Escherichia coli csgA and fimA mutants 1Snyder and Willaert: Biofilm Formation by Escherichia coli csgA and fimA mutantsPublished by Cornerstone: A Collection of Scholarly and Creative Works for Minnesota State University, Mankato, 2014 Abstract  Biofilms are a structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface. These structures and the organisms that cause them can pose a very serious problem if they colonize on medical devices. This is because biofilms have the ability to communicate within the colony and with other organisms that might attach to the surface, acting like a community working together. Biofilms allow the organism to be resistant to harsh and unfavorable conditions allowing them to survive longer and spread. Several genes in Escherichia coli (E. coli) have been associated with biofilm formation by that organism. Many of those genes encode surface appendages such as flagella, fimbriae,   and pili. We created mutations in genes encoding curli (csgA) and fimbriae (fimA) with the aim  of comparing their ability to form biofilms. The respective genes were selected on kanamycin- containing agar and disrupted with a kanamycin resistance gene. Biofilm formation in nutrient- rich medium and nutrient-poor medium is currently in progress, and the ability of the mutant E. coli strains to form biofilms will be compared with that of the parent wild type strain using a crystal violet microplate assay. 2Journal of Undergraduate Research at Minnesota State University, Mankato, Vol. 14 [2014], Art. 9http://cornerstone.lib.mnsu.edu/jur/vol14/iss1/9 Introduction   Biofilms are structured communities of bacteria that live in a self-produced polymeric matrix that adhere to a living or nonliving surface. By doing so, they can allow the organisms to be more resistant to harsh conditions, enabling them to survive longer and spread. These structures and the organisms that are able to form them can cause serious problems if they colonize on certain objects, such as medical devices. Escherichia coli (E. coli) is one of these organisms. Several genes in E. coli have been associated with biofilm formation, including the genes for curli (csgA) and fimbriae (fimA). Mutations in these two genes were created and the organism grown in two different types of media to: 1) compare the genes effect on biofilm formation, and 2) determine the effect on nutrient availability has on biofilm formation. The hypotheses of the experiment were that E. coli carrying mutations in csgA and fimA will not be able to form biofilm as effectively as the wild type E. coli and that organisms grown in nutrient rich environments will produce more robust biofilms than organisms grown in nutrient poor environments. Methods  Formation of Mutants  The organisms used in the experiment were isogenic, meaning that the only difference in the strains were the mutations we introduced. To obtain the mutants, a single-step inactivation procedure was followed (1). A strain of Escherichia coli BW 25113 carrying the plasmid PKD- 4, which contained a kanamycin resistance gene, was obtained and the plasmid isolated. Gene disruption cassettes were formed by Polymerase Chain Reaction (PCR) using primers designed to amplify the kanamycin resistance cassette and introduce sequences from csgA (primers CsgA- 3Snyder and Willaert: Biofilm Formation by Escherichia coli csgA and fimA mutantsPublished by Cornerstone: A Collection of Scholarly and Creative Works for Minnesota State University, Mankato, 2014  1 and -2) and fimA (primers fimA-1 and -2). The PCR product was purified and electroporated into wild E. coli containing the helper plasmid pKD64, which encoded ampicillin resistance and a bacteriophage recombinase complex. Transformants were obtained by plating the sample onto nutrient agar containing kanamycin and ampicillin to select for the strains containing both the PCR product and the helper plasmid. Confirmation of the target genes was verified by PCR to confirm that the mutations were in the desired location using specific primers. Biofilm Assay  To do the biofilm assay, the procedure suggested by George O’Toole (2) was followed. Once the mutants were confirmed, overnight cultures of the organisms (both mutant csgA and fimA strains as well as the wild type) were prepared in either LB (nutrient-rich broth) or SMB (nutrient-poor) broth. The optical densities of the cultures were adjusted to OD550 nm= 1.0 and the cultures were seeded onto microtiter plates to test for their ability to form biofilms. The biofilms formed were stained with 0.1% crystal violet and then the biofilms were quantified by dissolving in 30% acetic acid and measuring the absorbance at 550 nm with a microplate spectrophotometer. Results  Based on the data collected in the assay, it was shown that when comparing the strains of mutant E. coli with the wild type, the amount of biofilm formed by the mutant types was less than the amount formed by the wild strain. For each environment (csgA in LB broth or fimA in SMB for example) there were 192 replicates taken. These values were then analyzed using various statistical tests and then put into graphs for a more visual representation. In Figures 1 and 2, it is shown that the wild type strains, which did not have the resistance gene formed greater 4Journal of Undergraduate Research at Minnesota State University, Mankato, Vol. 14 [2014], Art. 9http://cornerstone.lib.mnsu.edu/jur/vol14/iss1/9 amounts of biofilm than the mutant types, which did have the resistance gene. Meaning the wild type strain produced more biofilm formation in the individual wells of the microtiter plates leading to greater statistical data. This supports one of the hypotheses for the experiment, that E. coli with the mutations in either csgA or fimA are not able to form as robust biofilms as the wild type E. coli.                   Fig. 1 Fig. 2  Another variable in biofilm formation tested in this experiment was to determine how the amount of nutrients present as the organism is growing has an effect on the amount of  biofilm formed. It was hypothesized that the organisms grown in nutrient rich environments (where LB media was used) would produce a greater amount of biofilm than organisms grown in nutrient poor environments (where SMB media was used). Figures 3 and 4 show the data collected displayed in the form of bar graphs. The patterned columns represent the wild strains while the non-patterned columns represent the mutant strains. Figure 3 is the data from the assays with the csgA mutant grown in either LB or SMB media. When simply comparing the media, the organisms grown in the SMB media formed 0.8 0.6 b a a 0.6 0.4 c 0.4 c c 0.2 0.2 b b 0.0 0.0 A nm 5 5 0 n A m 5Snyder and Willaert: Biofilm Formation by Escherichia coli csgA and fimA mutantsPublished by Cornerstone: A Collection of Scholarly and Creative Works for Minnesota State University, Mankato, 2014 greater amounts of biofilm. This did not support the hypothesis that organisms grown in LB would form more biofilm than the organisms grown in SMB. When looking at the difference between the wild strain and mutant strain on this graph, support of the first hypothesis was reinforced. The wild strain of E. coli formed over twice the amount of biofilm as was seen in  the mutant strain. The same observations were seen in the assays run with the fimA mutant (data shown in Figure 4).       0.6  0. 4        0.2  0.0    Fig. 3 Fig. 4    Discussion  Our hypotheses were that mutant E. coli would not be able to form biofilms as well as wild type E. coli and that organisms grown in nutrient-rich environments will produce more biofilm growth than organisms frown in nutrient poor environments. More robust biofilms were produced by the wild type E. coli in both LB and SMB, which supported our first hypothesis. However, our second hypothesis was not supported because the mutant strains produced more biofilm in SMB than in LB in the csgA and the opposite for the fimA strain. One reason for the discrepancy is simply there could have been a small amount of contamination. One of the 0.8 a 0.6 b b 0.4   0.2  c 0.0 A550 nm A550 nm   b a     a  a  6Journal of Undergraduate Research at Minnesota State University, Mankato, Vol. 14 [2014], Art. 9http://cornerstone.lib.mnsu.edu/jur/vol14/iss1/9  reasons an organism forms a biofilm is to help it survive difficult conditions, and it can be argued that in LB, conditions become stressful only as the organisms begin to reach stationary phase. In contrast, nutrient limitations present in cultures in SMB would be expected to be comparatively stressful throughout the organisms’ time in culture. Consequently, one might expect an increase in biofilm production for cells grown in minimal media due to the unfavorable conditions. Further research should be attempted to test this hypothesis, as well as the ability of csgA-fimA double mutants to form biofilms. Our work shows that csgA and fimA are both resposible for the formation of biofilms. With this information we maybe able to produce products such as catheters which are resistant to either of these genes. Doing so would help reduce the formation of biofilms and directly reducing the occurrence of infections. With further research into a double gene knock out we could possible discover yet another gene that may be responsible for the production of biofilms. Again knowing which genes are responsible for the formation of biofilms would allow us to produce products that are resistant to biofilms or even slow the formation. 7Snyder and Willaert: Biofilm Formation by Escherichia coli csgA and fimA mutantsPublished by Cornerstone: A Collection of Scholarly and Creative Works for Minnesota State University, Mankato, 2014  References  1. Datsenko, K.A., and B.L. Wanner. 2000. One-step inactivation of chromosomal genes in  Escherichia coli K-12 using PCR products. Proc Nat Acad Sci USA 12: 6640-6645.  2. O’Toole, G.A. 2011. Microtiter dish biofilm formation assay. J. Vis. Exp. 47: e2437. 8Journal of Undergraduate Research at Minnesota State University, Mankato, Vol. 14 [2014], Art. 9http://cornerstone.lib.mnsu.edu/jur/vol14/iss1/9  Professional Biographies  Nicole Snyder  I am from Tracy, Minnesota. I am currently an undergraduate student at Minnesota State University, Mankato majoring in Microbiology. I will be graduating in May 2014 with a BS in Microbiology. I presented this research with my partner at the 2014 Minnesota Undergraduate Scholars Conference and the 2014 Undergraduate Research Symposium. It is my goal to acquire a job in quality assurance or research. Sean Willaert  I am from Mankato, Minnesota. I am an undergraduate student at Minnesota State University, Mankato. I will graduate in the spring 2016 with a degree in Human Biology. I presented this research with my partner at the 2014 Minnesota Undergraduate Scholars Conference and the 2014 Undergraduate Research Symposium. I would like to attend medical school and pursue a career in the medical field. Dr. Timothy Secott  Dr. Secott received his BS in Biology from Millersville University in Pennsylvania.  After working as a research technician in the Department of Microbiology at Penn State University’s Hershey Medical Center, he supervised the activities of the Bacteriology and Molecular Diagnostic Sections of the Pennsylvania State Veterinary Laboratory for more than 10 years before starting PhD studies at Purdue University. He joined the Department of Biological Sciences faculty at Minnesota State University, Mankato in 2003.      9Snyder and Willaert: Biofilm Formation by Escherichia coli csgA and fimA mutantsPublished by Cornerstone: A Collection of Scholarly and Creative Works for Minnesota State University, Mankato, 2014

Biofilm Formation by Escherichia coli csgA and fimA mutants

Biofilms are a structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface. These structures and the organisms that cause them can pose a very serious problem if they colonize on medical devices. This is because biofilms have the ability to communicate within the colony and with other organisms that might attach to the surface, acting like a community working together. Biofilms allow the organism to be resistant to harsh and unfavorable conditions allowing them to survive longer and spread. Several genes in Escherichia coli have been associated with biofilm formation by that organism. Many of those genes encode surface appendages such as flagella, fimbriae, and pili. We created mutations in genes encoding curli (csgA) and fimbriae (fimA) with the aim of comparing their ability to form biofilms. The respective genes were disrupted with a kanamycin resistance gene and selected on kanamycin-containing agar. Biofilm formation in nutrient-rich medium and minimal medium is currently in progress, and the ability of the mutant E. coli strains to form biofilms will be compared with that of the parent wild type strain using a crystal violet microplate assay

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Biofilm Formation by Escherichia coli csgA and fimA mutants

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