Progress 11/01/17 to 10/31/20
Outputs
Target Audience:The food industry will recieve beneficial feedback from this research. Food security is a topic of major concern as human population is estimated to increase over the next decades. A substantial percentage of the food produced globally and in the United States is wasted at distribution, retail and consumer level. Microbial spoilage represents an important source of food waste. As microorganisms multiply, they synthesize and release small molecules called quorum sensing mediators. Quorum sensing is defined as the cell-to-cell communication within bacterial populations mediated by autoinducers. This project is based on the concept that growth of spoilage microorganisms in food systems is accompanied by an increase in the concentration of autoinducers, which in turn can be quantified. The main goal of this research study is to identify autoinducers present in packaged foods and design a biosensor array that can monitor microbial development. Three research objectives have been identified to achieve this goal: (1) Construct bioreporters capable to produce a measurable response in the presence of quorum sensing autoinducers (2) Identify and characterize quorum sensing mediators released in situ by the local microbiota in packaged food samples, and (3) Test the biosensors response in shelf life studies of packaged foods and model bacterial growth based on biosensor array response. Outcomes from proposed research objectives have the potential to offer important and novel insight into communication, interaction and physiological processes of the food microbiota. Results from the proposed research will serve as the foundation for biosensors and ultimately intelligent packaging to effectively monitor changes in food microflora and improve food quality and safety. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project allowed research training for a postdoctoral fellow in packaging, microbial and molecular biology techniques. Also, the postdoc gained substantial professional development in project management. How have the results been disseminated to communities of interest?A paper for publication is in process. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Construct bioreporters capable to produce a measurable response in the presence of quorum-sensing inducing molecules. Using standard molecular cloning techniques, bioreporters were constructed to respond to changes in the microbial population in packaged meats based on the accumulation of the small signaling molecules. Bioreporters were constructed to detect (i) autoinducer 2 (AI-2, 12 genetic constructs), (ii) acyl homoserine lactones (long and short chain AHLs, 10 genetic constructs) (iii) autoinducer 1 type transcriptional regulator (AI-1, 5 genetic constructs and, (iv) three peptide signaling sensors in gram-positives (based on Com, Agr, and Fsr systems). Reporters were engineered by fusing their responsive genetic element to a promoterless beta galactosidase gene. Experiments involving a different reporter gene such as bioluminescence and fluorescence were not successful (long persistence of the response signal was recorded after a minimal inducer exposure and the bioluminescence bioreporter had a high background signal). Results obtained based on beta-galactosidase activity assays were used to construct standard dose-response curves, where the bioreporters were exposed to predetermined concentrations of autoinducers. For most reporters, the expression of the signal was time-dependent and also dose dependent. Individual reporters were exposed to their inducer concentrations of 0, 0.1, 0.5, 1.0, 5.0, 10.0, 50.0 and 100 µM of AHLs, and 0, 1, 2, 5, 10, 100, 200, 500, 1000, 2000 and 5000 µM of AI-2. There were no false positives or false negatives observed for the analytical figures of merit for the constructed bioreporters. Linear regression analysis of the dose-response curves indicated that the data fit a linear model (R2≥0.90 for AHLs and R2≥0.85 for AI-2). The lower limit of detection was calculated from the model and found to be ranging from 0.1 to 0.4 µM for AHLs sensors. The model calculated a wider range for the AI-2 sensors, three of them had a lower detection limit of 2 µM but the rest ranged from 200 to 350 µM. The working range of the sensors were between 0.1-10 µM and 2 µM to 1mM for AHLs and AI-2, respectively. There was minimal response when sensors were exposed to inducers from a different class of signaling molecule. However, three AI-2 biosensors showed a cross-response in the presence of a different AI-2 inducer. In general, the minimal time required to elicit a response after exposure to the inducer was at least 20 minutes. Peptide-based inducers were not synthesized for gram-positive biosensors, these constructed were evaluated only for their response to food extracts and in the food challenge study. 2) Identify and characterize autoinducer quorum sensing molecules (AHLs, AI, oligopeptides) released in situ by the local microbiota in select packaged food samples. In order to obtain information on the background microorganisms that can cause spoilage in packaged meats, microbial microflora of packaged beef and poultry were identified. Colonies from total microbial count were pooled together and the genomic DNA was extracted. The 16S rDNA sequences of the genomic DNA were amplified with universal primers 27F and 1497R. 16S rRNA is a highly conserved gene typically used for taxonomic identification. In an attempt to broadly characterize spoilage microflora, meat extracts were pooled together from 5 beef samples and then 5 poultry samples. The analysis identified Pseudomonas spp, Serratia spp, as well as Brochothrix spp, Achromobacter, Streptococcus and Enterococcus as predominant species in the packed beef samples (not vacuum packed). Poultry samples revealed contamination by mostly Hafnia, Serratia, Brochothrix spp, other Enterobacteriaceae, and Lactobacillus. To test the response in situ and to identify potential microbial strains for spoilage detection, individual bioreporters placed in an array format were exposed to extracts from the meat and poultry samples. Dose-response calibration curves with the inducers were also performed as controls. Beta-galactosidase assays with food extracts were performed by the same methodology as in Objective 1, except that filtered food extracts were used to induce bioreporters. The influence of the food matrix on the inducer availability for quantification was tested by spiking food filtrate with known concentrations of the purified inducers. In the same experiments, to determine the total plate count number, meat extracts were diluted and plated on appropriate media. At least three sets of experiments were performed to verify reproducibility, both with standard solutions and in food matrices. An agar lawn diffusion assay was used to identify quorum sensing producing microbial strains. Meat isolates were colony replica plated on the top of agar lawn petri plates seeded with a bioreporter indicator for AHL-hexanoyl homoserine lactone and AI-2 as 4-hydroxy-5-methyl 3-furanone. The reporters had a fluorescence reporter gene expressed instead of the beta galactosidase. The meat-isolated strains producing strong QS molecules induced the fluorescence in the agar lawn which could be visualized with a UV transilluminator. Several strains were isolated as strong QS-producing bacteria including Pseudomonas, Halvia and Enterobacteriaceae. These results prove that biosensing systems could be employed to detect spoilage in food matrices. Analytical characteristics of tests of the spiked food samples with purified inducers were similar to those seen when modelling standard curves with prepared standards solutions. AHLs were detected in the spiked foods in the range of range of 1 to 10 µM and 2 to 1000 µm added AHL and AI-2, respectively. Plate counts revealed that meat extracts contained 7 × 105and 4 × 107CFU/ml meat extract. These results indicated that some of purified indicators were not available to the inducers, due to the interaction with the food matrix. 3)Test the biosensors' response (in an array format) in shelf-life studies of packaged foods and model bacterial growth based on array response. Two sets of packaged and packaged beef and poultry were prepared and stored, one in refrigeration whereas a second set was stored in abused conditions in a humidity cabinet at room temperature and 90% relative humidity. Samples (2x2 cm2 in petri dishes) were removed and analyzed over time by total plate count and exposed to bioreporters placed in 48-well microtiter plates. The microtiter plates containing filtered meat extract were analyzed for beta-galactosidase activity. Overall, the array of bioreporters detected AHLs and AI-2 when cell numbers reached approximately 106 CFU/g of meat. Data collected indicates that these compounds-AHL and AI-2 are present in spoiling meat and they can be used to predict shelf-life.
Publications
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Progress 11/01/17 to 10/31/18
Outputs
Target Audience:The food industry will recieve beneficial feedback from this research. Food security is a topic of major concern as human population is estimated to increase over the next decades. A substantial percentage of the food produced globally and in the United States is wasted at distribution, retail and consumer level. Microbial spoilage represents an important source of food waste. As microorganisms multiply, they synthesize and release small molecules called quorum sensing mediators. Quorum sensing is defined as the cell-to-cell communication within bacterial populations mediated by autoinducers. This project is based on the concept that growth of spoilage microorganisms in food systems is accompanied by an increase in the concentration of autoinducers, which in turn can be quantified. The main goal of this research study is to identify autoinducers present in packaged foods and design a biosensor array that can monitor microbial development. Three research objectives have been identified to achieve this goal: (1) Construct bioreporters capable to produce a measurable response in the presence of quorum sensing autoinducers (2) Identify and characterize quorum sensing mediators released in situ by the local microbiota in packaged food samples, and (3) Test the biosensors response in shelf life studies of packaged foods and model bacterial growth based on biosensor array response. Outcomes from proposed research objectives have the potential to offer important and novel insight into communication, interaction and physiological processes of the food microbiota. Results from the proposed research will serve as the foundation for biosensors and ultimately intelligent packaging to effectively monitor changes in food microflora and improve food quality and safety. Changes/Problems:Change in the work plan as mentioned previously. What opportunities for training and professional development has the project provided?A postdoctoral fellow was trained in molecular food microbiology obtained in depth knowledge regarding microbial interactions in complex food systems. The findings can be applied not only in reducing microbial spoilage in packaged foods but also interfering with biofilm formation pathways. How have the results been disseminated to communities of interest?Study is not yet completed. In the end, the research will be published in the appropriate journal and also disseminated through poster and oral presentations for shareholdersat industry and scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?Part of Objective 1 regarding Gram-positive quorum-sensing. We have isolated microbial cells (Enterococci and Streptococci) from packaged foods through co-culture assays that cause instant clumping to other cells. Specific microbial interactions are being assessed by microbial growth, turbidity assays and identification of the responsible compound. Part of Objective 1: Gram-positive bioreporters. A different strategy is employed: instead of cloning genetic elements in vectors, the lacZ cassette is inserted as gene fusion (shuttle vector constructed) in the cluster of genes of interest (e.g. comCDE for the competence stimulating peptide). These mutants will be tested by the same beta-galactosidase assay as described in the project, but experiments require more time since it involves homologous gene recombination. We are also working on transposon mutagenesis to identify new pathways for autoinducers. Part of Objective 1: Synthesize autoinducers and confirm activity for bioreporters. These are small peptides to be synthesized de novo and we will assess their biological activity on the microflora from packaged foods. Part of Objective 2: Complete standard curves and their response to autoinducers for the remaining Gram-positive bioreporter strains. Objective 3: Shelf-life study 1 for packaged foods. Packaged meat, poultry and fish (90 samples per set) will be stored in their normal and temperature abuse conditions (1 set each condition). Samples will be removed at select intervals and exposed to bioreporters in array format and compare the reporter response side-by-side by plate count. The bioreporter arrays will include Gram-negative and Gram-positive bacteria. Beta-galactosidase assay will be performed to determine standard curves for bioreporters Objective 3: Shelf-life study 2 for packaged foods. Packaged meat, poultry and fish (90 samples per set) will be stored in their normal and temperature abuse conditions (1 set per condition). Samples will be removed at select intervals and exposed to bioreporters in array format and compare the reporter response side-by-side by plate count. Beta-galactosidase assay will be performed to determine standard curves for bioreporters.
Impacts
What was accomplished under these goals?
Objective 1. In order to identify genetic elements of interest and autoinducers we followed 2 different approaches : (i) a literature search was performed and data already available in the public domain (such as microbial host, type of autoinducer, type of response and genetic information) was used to construct bioreporters and (ii) we isolated microbial species from packed foods (meat and poultry) and we tested their influence on other spoilage microorganisms in co-culture experiments. To construct bioreporters that respond to microbial quorum sensing elements we used a plasmid with a promoterless lacZ gene, pBG . Transcriptional fusions were created by cloning promoters of interest in multiple cloning site of pSF βGal, upstream of lacZ. These cloned elements can be expressed in generic laboratory strains such as E. coli DH5&. Also, it was necessary to co-express in the same cell the promoter regulatory element in addition to promoter fusion. These elements were cloned in compatible plasmids (pGEM T-easy or pCR TOPO) and transformed in the same cell with pBG plasmid. The bioreporters were exposed to known concentrations of their respective autoinducers and the beta-galactosidase activity was quantified for standard dose-response curves. Determination of the beta-galactosidase activity expressed in the E. coli transformants was performed by established protocol. Briefly, beta-galactosidase activity was assayed by measuring hydrolysis of the chromogenic substrate, o-nitrophenyl-ß-D-galactoside (ONPG). The amount of o-nitrophenol formed from ONPG can be measured by determining the absorbance at 420 nm. In the presence of excess ONPG, the amount of o-nitrophenol produced is directly proportional to the amount of beta-galactosidase released by the cell and a function of the time of the reaction and initial cell number. The reaction is stopped by adding sodium carbonate which inactivates beta-galactosidase, stopping the reaction and converts most of the o-nitrophenol to a yellow colored anionic form (determined by absorbance at 420nm). We obtained E. coliwhole cell biosensing systems recognition and regulatory elements derived from thePseudomonas aeruginosa(gram-negative and a common spoilage microorganism) AHL-dependent RhlR/RhlI and LasR/LasI systems. The E coli strains lack therhlIandlasIgenes that code for the AHL synthase enzymes and, thus, exogenous AHL needs to be supplied in order for the sensing cells to synthesize the reporter gene lacZ. When AHL are present in the environment of the sensing cells, they bind to the regulatory proteins (rhlR and lasR) and induce the expression of thelacZ and production of the enzyme in a manner proportional to the concentration of AHLs present. With this strategy we cloned the AHL-responsive sdiA from E. coli and Salmonella spp. Both these gram-negative microorganisms encode the protein SdiA that recognizes to AHL. This is considered to be the LuxI homologue but when we tested in the laboratory our constructs, SidA reacted to both long and short chain AHL including oxoC10. In addition, we created a AI-2 response bioreporter by cloning the promoter for luxP from plasmid pGEX4T1-LuxP and and lsrB reporter system from Salmonella spp. Both luxP and lsrB respond to AI-2 autoinducer. Results obtained based on beta-galactosidase activity were used to construct standard dose-response curves, where the bioreporters were exposed to predetermined concentrations of autoinducers. Typically, based on the beta galactosidase assays, short chain AHLs were detected in the range of 10-9M to 10−5 M, long chain AHL in the range of 10-9 M to 10−6M, and AI-2 in the range of 10−9 M to 10−4 M. There were no false positives or false negatives observed for the analytical figures of merit for the constructed bioreporters. The highest correlation coefficient was R2=0.9930 and the lowest R2=0.8510. Beta-galactosidase assays were performed with bioreporters exposed to food extracts to test sensor response to inducers produced in situ by the food microbiota. Testing was performed on packaged meat, and poultry samples (foods affected most by spoilage). Beta-galactosidase assays with food extracts were performed by the same methodology as in Objective 1, except that filtered food extracts were used to induce bioreporters. To determine the total plate count number, food samples were diluted and plated on appropriate media. In addition, standard curves were performed side-by-side with the chemically purified bioinducers. Bacterial species (biomarkers) were identified by performing beta- galactosidase assays of the bioreporters against individual colonies isolated from the plate count. To identify the isolates, we amplified the 16S rDNA sequences of the genomic DNA using the universal primers 27F and 1497R (ref). 16S rRNA is a highly conserved gene that is used for taxonomic identification. We tested the response in food samples by (i) dose-response curves generated by spiking the food product with a known concentration of the bioinducer and, (ii) by testing the whole cell bioreporter response to the cell extract filtrate of the food product. Analytical characteristics of tests of the food samples were similar to those seen when analyzing standard curves with prepared standards solutions. In this instance, short chain AHL were detected in the range of 10-8 M to 1 × 10−7 M, long chain AHL in the range of 10-8 M to 1 × 10−6 M, and AI-2 in the range of 10−8 M to 10−5 M. Assays performed using filtrated food samples had lower limits of detection and dynamic ranges than the autoinducer spikes samples: 10−4 M for AHLs and 10−6 M for AI-2. Dynamic ranges spanned from 10−5 M to 1 × 10−4 M for AHL and 10−7 M to 10−6 M for AI-2. At least three sets of experiments were performed to verify reproducibility, both with standard solutions and in food matrices. These results prove that biosensing systems could be employed to detect spoilage in food matrices, and the autoinducer that can be detected mostly is AI-2. However, the high microbial load in the food samples correlated more with the presence of gram-positive bacteria in these samples. We concluded that the response we observed from a number of gram-negative microorganisms but in order to develop an early detection bioreporter system we decided to focus more on creating chromosomal lacZ translational fusions in gram-positive bacteria. Gram-positives can respond to AI-2 but their quorum sensing is based on extracellular peptides that interact with a 2-component system spanning the cell membrane. This work is on-going.
Publications
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