ELUCIDATION OF ENVIRONMENTAL FACTORS THAT INFLUENCE ATTACHMENT OF FOODBORNE PATHOGENS TO BIOTIC AND ABIOTIC SURFACES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1005408
• Grant No.: 2015-67006-23092
• Project No.: ND05948
• Proposal No.: 2014-05683
• Program Code: A1331
• Project Start Date: Jan 15, 2015
• Project End Date: Jan 14, 2018
• Grant Year: 2015

Project Director
Bergholz, T.

Recipient Organization
NORTH DAKOTA STATE UNIV
(N/A)
FARGO,ND 58105

Performing Department
Veterinary & Microbiological S

Non Technical Summary
Human illnesses linked to consumption of fresh produce have dramatically increased in the last several decades, and produce is now responsible for one quarter of all foodborne outbreaks. Contamination of produce with human pathogens can occur in the pre-harvest environment as well as during post-harvest handling, and bacterial attachment factors can facilitate persistence of pathogens on produce in both of these environments. Pathogen attachment to surfaces, and subsequent biofilm formation, can significantly reduce efficacy of commonly used decontamination treatments, making these pathogens harder to eliminate on produce and in the produce processing environment. A number of attachment factors have been described, though limited information is available regarding the regulation and expression of molecular mechanisms that pathogens can use to attach to different surfaces. Exposure to conditions that would reduce attachment could increase the efficacy of currently used or novel decontamination treatments. In order to develop new methods for controlling foodborne pathogens on fresh produce and in the produce processing environment, further research on the environmental factors that impact attachment of these pathogens is needed. The goal of this proposal is to provide a comprehensive assessment of pre- and post-harvest environmental factors that influence expression of pathogen attachment mechanisms, and thereby attachment of pathogens to different surfaces. These data will critically inform the development of effective control strategies for pathogens both on produce and during post-harvest handling of produce.

Animal Health Component

0%

Research Effort Categories

Basic

75%

Applied

0%

Developmental

25%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	4010	1100	50%
712	4010	1040	50%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology; 1040 - Molecular biology;

Keywords

ehec

listeria monocytogenes

bacterial attachment

biofilms

fresh produce

gene expression

Goals / Objectives
The goal of this project is to provide a comprehensive assessment of pre- and post-harvest environmental factors that influence expression of pathogen attachment mechanisms, and thereby attachment of pathogens to different surfaces. Attaining this goal is necessary for the development of targeted attachment inhibitors to complement existing pathogen control strategies.Objective 1:Determine which environmental conditions influence attachment ofL. monocytogenes, Salmonella,and EHECto fresh produce and stainless steelObjective 2: Determine how environmental factors influence expression of genes encoding proteins involved in attachment ofL. monocytogenes, Salmonella,and EHEC to surfaces.

Project Methods
Assay conditions. In the pre- and post-harvest environment, any pathogens present on fresh produce will experience a variety of stresses related to nutrient levels, moisture, temperature and sanitizer exposure, all of which are known to impact subsequent survival and persistence in the food supply. Each stress condition will be generated in phosphate buffered saline (PBS) to increase consistency and reduce interference of the stress with components of a rich medium. These stresses include: nutrient limitation (PBS alone), low temperature (15°C, 10°C, and 5°C), sub-lethal level of chlorine (25 ppm), low pH (lactate at pH 5.5), sub-lethal level of sanitizer (18 ppm benzalkonium chloride), peroxide (5 mM H2O2), lettuce leaf exudates, and leafy green lysates (lettuce lysate and spinach lysate). The in vitro test environments will be incubated at 20°C, with the exception of the low temperature stress conditions.Attachment assays. Attachment assays will be conducted on two different biotic surfaces (baby romaine lettuce leaves and baby spinach leaves) and on an abiotic surface, stainless steel. Strains will be grown to stationary phase and washed cell pellets will be diluted to 104 CFU/mL in PBS, or PBS + stress condition, and placed into a 50mL tube. The surface to attach to will be added to the liquid (a lettuce or spinach leaf, or a stainless steel coupon), the tube sealed and placed on a rocking platform and incubated at 20°C for the length of time shown to induce or repress expression of attachment genes. Following incubation, leaves/coupons will be removed, and non-adherent cells will be removed by washing each surface three times in PBS. Attached cells will be removed by placing the washed surface into 10mL PBS with 6-mm glass beads and vortexing for 1 minute, then plating the cell suspension onto selective media using the Spiral Plater. Colonies will be enumerated with a Q-count following overnight incubation at 37°C. Attachment assays will be conducted in triplicate for each bacterial strain under each stress condition. Differences in attachment between stressed and non-stressed samples will be determined by ANOVA.Reporter fusion construction. Single copy chromosomal reporter fusions will be constructed in each strain to assay the promoter activity of selected genes involved in attachment of pathogens to biotic or abiotic surfaces. We will create reporter fusions for promoters of genes known to be involved in attachment (Table 1) and then monitor promoter activity under stress conditions representing those found in the pre- and post-harvest environment. To create the reporter fusions in L. monocytogenes, we will use pMJG2, a derivative of pPL2 with β-glucuronidase (GUS) as the reporter protein. This vector integrates into the tRNAArg - attBB' site on the chromosome, and is designed to be used in both serotype 1/2 and 4b strains. Integration into the chromosome alleviates the need for antibiotic selection to maintain the reporter fusion plasmid. For EHEC and Salmonella, we will utilize pMCI002 and RedET recombinationto generate single copy chromosomal β-galactosidase reporter fusions. As positive controls, reporter fusions will also be constructed from promoters of genes known to be induced under each environmental stress, such as SigB for L. monocytogenes and RpoS for EHEC and Salmonella. Reporter fusions will be constructed in multiple strains of the same serotype as differences in expression of attachment factors have been observed for strains of L. monocytogenes as well as EHEC O157:H7.Measuring promoter activity. Changes in expression of each attachment protein under environmental stress will be assessed by measuring either GUS (L. monocytogenes) or β-galactosidase (EHEC and Salmonella) activity of each reporter fusion strain. Prior to GUS measurements, cells will be lysed with CellLytic B reagent (Sigma-Aldrich). The enzymatic reaction will be initiated by addition of 0.4-mg/ml 4-methylumbelliferyl-β-D-glucuronide to the lysate. The reaction will be stopped after 10 min by addition of Na2CO3. Fluorescence will be measured at 460 nm using a Biotek plate reader. A standard curve of at least 5 different concentrations of 4-methylumbelliferone (MU) will be included with every plate, and the concentration of liberated MU in each sample will be calculated using the standard curve. The GUS activity for each sample will be reported as nM MU/log CFU/min. To measure β-galactosidase activity, the OD600 of each culture will be determined prior to lysis with toluene. The colorimetric substrate ortho-nitrophenyl-β-D-galactoside (ONPG) is then added to a final concentration of 1mg/ml. After development, the reaction is terminated by the addition of Na2CO3 and cell debris is removed by centrifugation. The absorbance at 420 nm (measuring the cleavage product o-nitrophenol) is used to calculate Miller units (1,000 x A420)/(reaction time in minutes x OD600). At least three independent replicates of each reporter fusion strain will be assayed, in duplicate (technical replicates), for each stress condition. Activation of a specific attachment gene by a particular stress will be determined based on a significant difference in nM MU/log CFU/min or in Miller units compared to the control condition (without environmental stress) using ANOVA. A significant difference in reporter fusion activity between the control and at least one point in time during exposure to the stress will indicate modulation of expression of the attachment gene by the stress.

Progress 01/15/15 to 01/14/18

Outputs
Target Audience:Food safety and microbiology researchers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided training opportunities for two graduate students, Sara Levadney Smith and Autumn Kraft. Sara completed her Master's thesis in spring 2018 on L. monocytogenes attachment and biofilm formation. Both students were able to present their research at regional and national conferences. How have the results been disseminated to communities of interest?Results have been presented via poster and oral presentations at regional and national meetings. Manuscripts are in preparation, but have not been submitted yet. We anticipate two manuscripts will published in peer-reviewed journals describing the EHEC and L. monocytogenes attachment under different environmental conditions. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? For objective 1, to better understand how environmental conditions impact the attachment of EHEC to an abiotic surface, we utilized an attachment assay with strains from the two EHEC serotypes, O157 and O26, on stainless-steel coupons under the following test conditions at room temperature (25°C): phosphate buffered saline (PBS), glucose-defined minimal media (GDMM), 4.5% NaCl, and 4% lettuce lysate. Strains were grown to a concentration of 106-107 CFU/ml in GDMM and the assay was performed in a 6-well plate with a sterile stainless-steel coupon submerged in 7.2 ml of the stress condition and 0.8 ml of the inoculum. At time points of 15, 30, 60, and 90 min post inoculation, samples from the free media in the well, poorly-adhered cells, and adhered cells were collected from each well/coupon, diluted, and plated on LB for enumeration. When comparing the poorly-adhered and adhered counts collected from coupons incubated in GDMM, salt, and lysate to those counts from PBS, we found that adhered cells of the O157 strain grown in 4% lysate had nearly 4 times greater attachment while the O26 strain was about 2 times lower over the 75 min period. However, poorly-adhered cell counts were 1.5 times greater for O26 in lysate when compared to PBS. For the O157 strain, the salt condition lead to 2 times lower attachment of poorly-adhered cells when compared to that of PBS. Attachment, adhered and poorly-adhered, in GDMM was similar to PBS for both strains, and for O26 the attachment in the salt condition was similar to PBS as well. For L. monocytogenes, attachment assays were conducted on stainless steel, incubated at 25°C for 120 min under the following conditions: glucose defined minimal medium (control), 5 ppm chlorine, PBS, 10 mg/mL PEA, 4.5% NaCl, and 2% lettuce lysate. After 120 min aliquots of loosely attached and strongly attached cells were collected, serially diluted to 10-4 and 10-1, respectively, and enumerated on BHI agar. The differences in attachment to stainless steel in GDMM were then compared to the attachment on stainless steel in each test condition and measured in logCFU/cm2. In 5ppm chlorine, a significant reduction in both loosely attached and strongly attached cells to stainless steel compared to the control was observed, by 0.7 ± 0.55 logCFU/cm2 (P = 0.0206) and 0.55 ± 0.44 logCFU/cm2 (P = 0.0223), respectively. Of interest, 2% lettuce lysate also reduced both loosely and strongly attached cells to stainless steel, compared to the control, by 0.49 ± 0.30 logCFU/cm2 (P = 0.0241) and 1.02 ± 0.48 logCFU/cm2 (P = 0.0109). For objective 2, we focused on monitoring expression of lcp (encoding a cellulose binding protein) which has been shown by other researchers to impact attachment of L. monocytogenes to surfaces. Expression was monitored with a GUS reporter fusion. Control strains included reporter fusion for uspA (encoding a general stress response protein) and for uspA in a delta sigB background. The test conditions included 2% lettuce lysate, 4.5% NaCl, 5ppm chlorine, beta-phenylethylamine, phosphate buffered saline (nutrient limitation), and 1% cellulose. Expression was monitored in each condition at both 4ºC and 25ºC. Expression of uspA was as expected - significantly upregulated under osmotic stress, nutrient limitation, and lettuce lysate, indicating activation of the general stress response. In the delta sigB background, uspA expression was not significantly induced under those conditions. These controls indicated the system was working as expected, reporter fusions were correctly constructed, and we were able to assay changes in GUS levels from the fusions. We observed no significant changes in lcp expression under any of the test conditions, indicating that these are not environmental signals for activation of this gene. Not much is known about the regulators that may influence lcp transcription, which would likely be a target of future research.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Pruess, B.M, M. Irsfeld, S. Levadney Smith, J. Murphy, and S.M. Horne. Beta-phenylethylamien and acetoacetic acid and inhibitors of bacterial biofilm. Bacterial Locomotion and Signal Transduction (BLAST) XIII, Tuscon, Arizona, January 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Kraft, A. L. and T. M. Bergholz. Evaluation of the impact of environmental stressors on EHEC attachment gene expression using a reporter fusion construction. North Central Branch American Society for Microbiology Annual Meeting, De Pere, Wisconsin. 2017
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Bergholz, T. M. Using transcriptomics to understand pathogen survival on foods. Food Microbiology Meets Genomics Symposium, American Society for Microbiology Microbe 2017 meeting, New Orleans, Louisiana. 2017
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Levadney Smith, S., T. M. Bergholz, and B. M. Pruess. Congo red screening of curli production in Escherichia coli and Salmonella enterica. North Dakota EPSCoR State Conference, Fargo, North Dakota. 2015.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Kraft, A. L. and T. M. Bergholz. Attachment of EHEC to a Stainless-Steel Surface While Under Different Environmental Conditions. American Society for Microbiology Microbe 2018 meeting, Atlanta, Georgia. 2018

Progress 01/15/16 to 01/14/17

Outputs
Target Audience:Food safety and microbiology researchers Changes/Problems:We have experienced some technical difficulties in reporter fusion construction that have led to set backs in our planned timeline. We originally planned to create reporter fusions in multiple strains of Salmonella, it seems unlikely that we will be able to accomplish that in the time remaining. We will still include Salmonella strains in our assessment of attachment, along with L. monocytogenes and E. coli. What opportunities for training and professional development has the project provided?This project has provided a research training opportunity for an undergraduate student in the microbiology program at NDSU. How have the results been disseminated to communities of interest?Through presentations at research conferences. What do you plan to do during the next reporting period to accomplish the goals?We will continue to construct reporter fusions in L. monocytogenes, and continue construction of fusions in E. coli O157:H7 and O26:H11 strains. These newly constructed reporter fusions will be measured under the same set of conditions as already tested for L. monocytogenes. We will also conduct attachment assays for strains of the three pathogens under the stress conditions to determine which stresses influence attachment to lettuce leaves and stainless steel coupons.

Impacts
What was accomplished under these goals? For objective 2, we have successfully constructed reporter fusions that can be used to measure changes in expression of proteins involved in attachment of L. monocytogenes when exposed to different environmental conditions. Reporter fusions have been constructed in 4 different strain backgrounds for Lcp, a cellulose binding surface protein; FlaA, the major protein subunit of the flagella known to be involved in attachment as well as motility, and LapB, a surface protein. As a control, we have constructed a reporter fusion for UspA, a general stress response protein known to be regulated by SigB and induced under stress, such as osmotic stress. This strain will serve as a positive control for stress conditions known to induce the general stress response. We have measured Lcp expression upon exposure to lettuce lysate, 4.5% sodium chloride, 5 ppm chlorine, and minimal medium at both 4°C and 25°C. Expression was measured at multiple intervals over a 2 hour period following initial exposure to each condition. Lettuce lysate was found to increase Lcp expression at 25°C. We have begun construction of reporter fusions in E. coli O157:H7 strains.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Levadney Smith, S., B. M. Pruess, and T. M. Bergholz. Environmental factors influence the expression of Listeria monocytogenes attachment factors. American Society for Microbiology Microbe 2016 Meeting, Boston, Massachusetts.

Progress 01/15/15 to 01/14/16

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided a research training opportunity for an undergraduate student in the microbiology program at NDSU. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We will continue to construct reporter fusions in L. monocytogenes, and begin construction of fusions in E. coli O157:H7 and Salmonella strains. These newly constructed reporter fusions will be measured under the same set of conditions as already tested for L. monocytogenes. We will also conduct attachment assays for strains of the three pathogens under the stress conditions to determine which stresses influence attachment to lettuce leaves and stainless steel coupons.

Impacts
What was accomplished under these goals? For objective 2, we have successfully constructed reporter fusions that can be used to measure changes in expression of proteins involved in attachment of L. monocytogenes when exposed to different environmental conditions. Reporter fusions have been constructed for Lcp, a cellulose binding surface protein; FlaA, the major protein subunit of the flagella known to be involved in attachment as well as motility, and LapB, a surface protein. As a control, we have constructed a reporter fusion for UspA, a general stress response protein known to be regulated by SigB and induced under stress, such as osmotic stress. This strain will serve as a positive control for stress conditions known to induce the general stress response. We have measured Lcp expression upon exposure to lettuce lysate, 4.5% sodium chloride, 5 ppm chlorine, and minimal medium at both 4°C and 25°C. Expression was measured at multiple intervals over a 2 hour period following initial exposure to each condition. Lettuce lysate was found to increase Lcp expression at 25°C.

Publications