Progress 10/01/06 to 09/30/11
Outputs
OUTPUTS: This research on basic science behind biofilm production of L. monocytogenes has identified many unique genes that are utilized by this organism for biofilm formation. This knowledge will be the basis for new biofilm control strategies based upon the biological survival mechanisms. A gene that is homologous to a DNA translocase enzyme was shown to be important for biofilm formation in Listeria monocytogenes under both stangent and flow conditions and on multiple surfaces, including stainless steel, the most common surface used in food processing equipment. During the course of analysis of this mutant, we showed that extracellular DNA is very important component of extracellular matix in biofilm production by L. monocytogenes and the enzyme DNase I was shown to be a highly effective agent for biofilm removal. The identification of specific extracellular components will enable us to design biofilm remediation strategies aimed specifically at extracellular DNA. The common chelating agent is very effective at preventing the adhesion and biofilm formation of L. monocytogenes. The identification of specific inhibitory compounds will enable us to design biofilm remediation strategies specifically to inhibit this important foodborne pathogen. This study found that bacterial cells and expolymeric substances were more strongly bound in the interior of the biofilm in comparison to the exterior. In addition, moisture appears to a major factor in the cohesive strength of a biofilm. This is important to understanding the cross contamination potential of biofilms, and drying may be a future strategy for bacterial removal. These results have been disseminated at presentations at the local and national level and through publication in peer reviewed journals. PARTICIPANTS: This project supported the biofilm research of the Lynne McLandsborough (primary investigator) and contributed to the education of Yuhua Chang (PhD student, mutagenesis study), Imelda Tirtajaya (PhD student, mutant characterization), Fujia Zhang (MS student, biofilm cohesiveness), Songsirin Ruengviesh (MS student, mutant characterization)and Nils Fisher (undergraduate researcher). TARGET AUDIENCES: The target audiences for this project are both academic and the food industry. The academic audience has been served by contributing to the greater knowledge in the area of bacterial biofilm production through presentations and publications. The Food Industry has been served by identifying scientifically sound targets that can be used to remediate or prevent biofilm production by Listeria monocytogenes in the food processing environment. This has been shared with the food industry through presentations are national meeting and interactions through the UMass Food Science Strategic Research Alliance with industry. PROJECT MODIFICATIONS: The project was modified to use a mariner-based transposition system in 2008 which allowed us to identify 23 genetic determinants needed for biofilm formation, a significant leap forward for this project. An Autoclave was purchased to advance research under this project during the 2010 calendar year.
Impacts
Listeria monocytogenes contamination is responsible for a large number of Class I recalls of processed foods due to post-processing contamination of this organism surviving on food processing surfaces. The purpose of this project has been to study the genetics of biofilm formation of this important pathogen, and apply this knowledge to remediation strategies. A mariner-based transposition system was used to generate mutants of L. monocytogenes LM21. Twenty-six genes involved in biofilm formation were identified, including genes previously characterized. New genes identified included surface proteins, genes involved in surface modification, and genes of unknown function. Further characterization has focused upon Mutant 55-D1 which has the transposon insertion in a gene that has homology to a DNA translocase enzyme. In the constructed complemented strain (55-D1c), the biofilm production phenotype was restored to close to wild-type level. The DNA translocase mutant also exhibited reduced biofilm formation under flow conditions, indicating this genetic determinant is important for biofilm formation in the food processing environment. The influence EDTA and DNase I upon biofilm formation were evaluated. The chelating agent EDTA did not affect the planktonic growth of L. monocytogenes, however, it significantly inhibited biofilm formation. EDTA appears to function in the early stage by affecting the initial attachment of L. monocytogenes cells to surfaces. The involvement of extracellular DNA in L. monocytogenes biofilm formation was also studied. DNase I did not affect growth; however, the level of biofilm detected was reduced in proportion to increasing levels of the enzyme. The addition of DNase I was very effective in removal of biofilms, indicating that DNA appears to be an important component of the exopolymeric substances (EPS). The biophysics of L. monocytogenes biofilm formation was assessed using the atomic force microscope to measure cohesive forces holding the biofilm together. Raster experiments upon biofilms equilibrated at 33% RH had a greater cohesive energy with each high pressure scan series, indicating that the bacteria at the top of the biofilm where more loosely attached than the cells deeper within the biofilm. Raster experiments of biofilms equilibrated at higher RH had very little height reduction. This study found that bacterial cells and expolymeric substances where more strongly bound in the interior in comparison to the exterior of the biofilm.
Publications
- L. McLandsborough. Biofilm formation by Listeria monocytogenes. 2009. Invited Preentation. The 44th Annual Region I Meeting of the American Society of Microbiology Cromwell, CT, Oct. 22.
- L. McLandsborough 2009. Listeria monocytogenes biofilm formation: remediation and transfer. Invited presentation. US Military Academy Fifth Annual Microbiology Symposium. West Point, NY.
- Chang, Y., W. Gu, N. Fisher, L. McLandsborough. 2009. Listeria monocytogenes Biofilm Formation: Identification of Genes that Code for Biofilm Phenotypes by mariner-based Transposon Mutagenesis. American Society of Microbiologists Annual Meeting. Philadelphia, PA 2009. P-129.
- Yuhua Chang, Weimin Gu and Lynne McLandsborough. 2010 Disruption of Listeria monocytogenes lmo1386, a Putative DNA Translocase Gene, Affects Biofilm Formation on Abiotic Surfaces. American Society of Microbiologists Annual Meeting 2010. San Diego MA. P-2294
- Zhang, F and L. McLandsborough. 2011. Measurement of Listeria monocytogenes biofilm cohesive energy using atomic force microscopy. 2011 International Association of Food Protection Annual Meeting. Milwaukee, WI. P3-44.
- Fujia Zhang. 2010. High-density polyethylene to cold smoked salmon and Listeria monocytogenes biofilm cohesive energy investigation. MS Thesis, Department of Food Science, University of Massachusetts, Amherst. December 2010.
- Yuhua Chang, Weimin Gu, Nils Ficher, and Lynne McLandsborough. 2012. Identification of genes involved in Listeria monocytogenes biofilm formation by mariner-based transposon mutagenesis Accepted in Applied Microbiology and Biotechnology
- Yuhua Chang, Weimin Gu an and Lynne McLandsborough. 2012. Low concentrations of ethylenediaminetetraacetic acid (EDTA) affects biofilm formation of Listeria monocytogenes by inhibiting its initial adherence. Food Microbiol. 29:10-17
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: The most recent estimates indicate that there are 81.9 million instances of food borne illnesses annually, at a cost that has been estimated to be $152 billion dollars each year. Listeria monocytogenes contamination is responsible for the majority of Class I recalls of processed foods due to post-processing contamination of this organism surviving on food processing surfaces. The purpose of this project has been to study and characterize the genetics of biofilm formation of this important pathogen, so that biofilm remediation strategies can be designed with a broad scientific basis. Research funded by this project identified 26 genes that when interrupted contribute to the reduced biofilm formation (RBF) phenotype. When RBF mutants were created using mutagenesis, 5of the RBF were identified from the same operon region (lmo2445, lmo 2444 and lmo2556). In order to identify the contribution of each genetic determinant to biofilm formation, we have been working on constructing in-frame deletion mutants of which will be added to L. monocytogenes LM21 using allelic exchange. We are also using 2-D gel electrophoresis to characterize the surface proteins present in the wild type and some of the identified RBF mutants. During characterization of RBF, we determined that the common chelating agent EDTA (Ethylenediametetraacetic acid), reduced the level of biofilm formation in microtiter plate assays. EDTA at concentration of down to 0.01 mM significantly efficiently inhibited biofilm formation and concentrations as high as 0.1 mM did not affect the planktonic growth of L. monocytogenes. To determine at which stage EDTA influences biofilm production, 0.1 mM EDTA was added different time internals during growth. Adding EDTA at time zero had the greatest inhibitory effect against the biofilm formation, while the addition of EDTA after 4 hours had little or no biofilm inhibitory effects. These data indicates that EDTA functions in the early stage by affecting the initial attachment of L. monocytogenes cells to surfaces. The addition of Mg2+, Mn+2 or Fe3+ in the presence of EDTA did not restore the biofilm formation, suggesting the biofilm inhibition was not likely due to the depletion of cations from growth medium. The zeta potential measurement found no differences in the cell surface charge between cells treated with or without EDTA. Our results indicate that EDTA acts either directly or indirectly to modify the way bacteria cells interact with surfaces, but the mechanisms remain unclear. The results were presented at an oral presentation and poster at the 2010 American Society of Microbiologists Annual Meeting in San Diego, CA, and a poster presentation at the International Association of Food Protection Meeting in Anaheim, CA. PARTICIPANTS: Lynne McLandsborough (PI) Directed research on the project Yuhua Chang (PhD student) Performed mariner mutagenesis and characterization of mutants. Imelda Tirtajaya (PhD student) Performed research to create a series of in-frame deletion mutants to further characterize RBF muants. Songsiring Ruengvisesh (MS student) Working to develop and efficient cell surface protein extraction method in order to further characterize RBF mutants. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: An Autoclave was purchased to advance research under this project during the 2010 calendar year.
Impacts
The results from this project have shown a common chelating agent is very effective at preventing the adhesion and biofilm formation of L. monocytogenes. The identification of specific inhibitory compounds will enable us to design biofilm remediation strategies specifically to the inhibit this important foodborne pathogen.
Publications
- Y. Chang. 2010. Characterization of Listeria monocytogenes biofilm formation: a molecular approach by target gene knockout and mariner-based transposon mutagenesis.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Listeria monocytogenes contamination is responsible for the majority of Class I recalls of processed foods due to post-processing contamination of this organism surviving on food processing surfaces. A total of 42 reduced surface growth mutants of L. monocytogenes LM21 were identified. After excluding the sibling mutants, we have identified 26 genes that when interrupted contribute to the reduced biofilm formation (RBF) phenotype. Further characterization has focused upon Mutant 55-D1 which has the transposon insertion in a gene that has homology to a DNA translocase enzyme. Using an integration vector, the open reading frame was cloned and transferred into mutant 55-D1 using conjugation and confirmed using PCR. In the constructed complemented strain (55-D1c), the biofilm production phenotype was restored to close to wild-type level, confirming the involvement of the DNA translocase-like open reading frame in biofilm production of L. monocytogenes. To determine if the DNA translocase enzyme is needed for biofilm formation under flow conditions, mutant 55-D1 and wildtype were grown on stainless steel slides in a drip-flow biofilm reactor. In this system, cell numbers of mutant 55-D1 were 2.5 log CFU/cm^2 lower than the wild-type strain. Confocal microscopy showed that the biofilms of the mutant consisted of individual adherent cells, while the wild-type strain consisted of larger microcolonies. This confirmed that the interrupted DNA translocase gene in 55-D1 is essential for biofilm growth on stainless steel under flow conditions. The involvement of extracellular DNA in L. monocytogenes biofilm formation was studied. Wild-type LM21 was grown in PVC microtiter plates in the presence the enzyme DNase I. The enzyme did not affect growth; however, the level of biofilm detected was reduced in proportion to increasing levels of DNase I. In another series of experiments, DNase I was added to wild-type growth at various times. The addition of DNase I was able to greatly reduce the level of attached stained cells even when the enzyme was added after 66 h of incubation, indicating that DNA appears to be an important component of the exopolymeric substances (EPS) holding the L. monocytogenes cells to the surfaces. Biofilm levels of DNA translocase mutant 55-D1 were consistently lower than wild-type, however when grown in the presence of DNase I, stained surface growth levels were reduced, indicating that DNA may still be an important component of EPS in the 55-D1. Confocal microscopy using the BacLight DNA staining system with showed similar levels of extracellular DNA in both wild-type and mutant, indicating that even though the mutant lacks a DNA translocase gene, extracellular DNA is still present. This may be due to autolysis or an alternative DNA translocase system within L. monocytogenes. The results were presented at a oral presentation and poster at the 2009 American Society of Microbiologists Annual Meeting in Philadelphia, PA, and talks at the 44th Annual American Society for Microbiology Region I Meeting, in Cromwell CT and at the 5th Annual Microbiology Symposium at the United States Military Academy in West Point NY. PARTICIPANTS: This project supported the work of the PI, Lynne McLandsborough and two PhD students, Yuhua Chang, who generated the mutant library and performed the DNase I studies, and Imelda Tirtajaya who is starting analysis of other mutants within the library. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The results have shown that a gene that is homologous to a DNA translocase enzyme is important for biofilm formation in Listeria monocytogenes under both stangent and flow conditions and on multiple surfaces, including stainless steel, the most common surface used in food processing equipment. During the course of analysis of this mutant, we showed that extracellular DNA is very important component of extracellular matix in biofilm production by L. monocytogenes. The identification of specific extracellular components will enable us to design biofilm remediation strategies aims specifically at extracellular DNA.
Publications
- CHANG, Y., W. Gu, N. Fisher, L. McLandsborough. Listeria monocytogenes Biofilm Formation: Identification of Genes that Code for Biofilm Phenotypes by mariner-based Transposon Mutagenesis. American Society of Microbiologists Annual Meeting. Philadelphia, PA 2009. P-129
- Rodriguez-Lozano, A. and L. McLandsborough. 2009. Biofilm formation by Listeria monocytogenes and transfer to foods. In Biofilms in the food and beverage industries. P. M. Fratamico, B. A. Annous and N. W. Gunther, Editors. Woodhead Publishing Limited, Cambradge. p200-225
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Listeria monocytogenes contamination is responsible for the majority of Class I recalls of processed foods due to post-processing contamination of this organism surviving on food processing surfaces. Although the biofilm formation of L. monocytogenes has been studied, only a few genes have been identified as necessary for biofilm formation to date. Over the past year, a mariner-based transposition system (pAM38) was used to generate mutants of L. monocytogenes LM21. A total of 6500 colonies were screened for reduced surface growth (RSG) characteristics in a microtiter plate assay in modified Welshimer's broth at 32C for 48h. The RSG phenotype was defined as a strain that exhibited normal growth density at 48h, but had a biofilm level three or more standard deviations below the wild type surface growth. A total of 43 RSG phenotype mutants were confirmed and the location the disrupted genes were identified by sequencing PCR reactions performed with a combination of primers specific to the transposon and an arbitrary primer. Thirty different genes involved in biofilm formation were identified, which included genes previously characterized (flagella and the agr quarum-sensing system). New genes identified include surface proteins (LPXTG containing genes), genes involved in surface modification, and genes of unknown function. Currently, we are performing complementation experiments and are continuing characterization of reduced surface growth phenotypes by monitoring biofilm morphology via confocal microscopy and gene expression. PARTICIPANTS: Lynne McLandsborough; PI; Yuhua Chang, PhD student; Nils Fischer, undergraduate researcher TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Previously in this project we used transposon mutagenesis to generate 36 random mutants within L. innocua LCDC 81-861with reduced surface growth (RSG) characteristics in a microtiter plate assay. However, despite years of work, we were only able to identify the location of one of the transposon insertion sites. This past year, the mutagenesis was started from scratch with a new mariner-based transposition system (pAM38) and we were able to identify the location of 42/43 RSG mutants, a significant leap forward for this project.
Impacts
This research on basic science behind biofilm production of L. monocytogenes has identified many unique genes that are utilized by this organism for biofilm formation. This knowledge will be the basis for new biofilm control strategies based upon the biological survival mechanisms
Publications
- No publications reported this period
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: An Honor student performed the research on Alicylobacillus and produced a capstone experience manuscript for UMass Commonwealth College entitled "SPORE AND BIOFILM FORMATION OF ALICYCLOBACILLUS ACIDOTERRESTRIS"
PARTICIPANTS: Lynne McLandsborough; PI; Yuhua Chang, PhD student; Michael Miller, honor thesis
Impacts
The biofilm formation of A. acidoterrestris, a common spoilage organism of apple juice was investigated. Prior to biofilm research, thirteen industrial isolates of Alicyclobacillus were characterized by amplifying 16S rDNA by PCR and sequence analysis. All strains were confirmed to belong to the species A. acidoterrestris by sequencing and guaiacol formation, which is responsible for spoilage flavors tested and all strains tested positive. In a microtiter plate biofilm assay, the 13 strains of A. acidoterrestris were evaluated for biofilm formation in two types of apple juice and defined BAM broth. Of the 13 strains tested, three strains P6, P7 and P8 produced the most biofilm, especially in the BAM broth, as opposed to apple juice. Therefore, one strain was selected (P7) for use in drip flow reactor biofilm studies. The genetics of Listeria innocua biofilm formation was further investigated. We identified lin2619 as a gene that when disrupted with Tn917 decreases the
adherence and biofilm formation of Listeria innocua. In order to determine if gene lin2619 has the same function in Listeria monocytogenes (gene lmo2476) we created interruption and deletion mutants in lmo2476 using a temperature sensitive vector system. Mutants are currently being analyzed.
Publications
- No publications reported this period
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