Progress 10/01/10 to 09/30/15
Outputs
Target Audience:Professionals in the field of food microbiology, microbial food safety and medical microbiology (academia and industry). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One undergraduate and one graduate student from the Health Promoting Naturals Laboratory (microbiology component of the project) received their training in the frames of this project. How have the results been disseminated to communities of interest?Manuscript was accepted for publication. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
IMPACT: Our data indicate feasibility of designing natural-derived novel antimicrobials with targeted (pathogen-oriented) activities, safe for humans and for non-targeted microorganisms. This justifies the statement of the study's results contribution to the CHANGE OF KNOWLEDGE. CAms showed antimicrobial activity against a panel of Gram-positive and Gram-negative bacteria. In addition, the CAms were biologically more active against Gram-positive bacteria than Gram-negative bacteria with the MIC values being 2 to 4-fold lower. A hemolysis assay was conducted and selectivity indices (SI) were determined as HC50/MIC using S. aureus and E. coli as representatives for SI calculations of Gram+ and Gram- respectively. In general, all CAms induced negligible hemolysis at their respective MICs. Scanning electron microscopy and transmission electron microscopy indicated significant morphological and ultrastructural alternations in CAms-treated cells in comparison to the control (untreated bacterial cells). When treated with most active CAms, the bacteria showed profound internal damage, aside from cell membranes disruption. All-atom membrane dynamic simulation was applied to elucidate the interactions between CAms and membranes. Preferable interaction pattern for the most promising CAms was clearly observed with negative charge bearing bacterial membrane compared to neutral mammalian membrane. This may be seen as an indication of the CAms safety for eukaryotic organisms.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Zhang Y, Algburi A, Wang N, Kholodovych V, Oh DO, Chikindas M, Uhrich KE. 2016. Self-assembled cationic amphiphiles as antimicrobial peptides mimics: role of hydrophobicity, linkage type, and assembly state. Nanomedicine: Nanotechnology, Biology, and Medicine.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Target audience includes two categories of individuals: 1) professionals (microbial food safety industry specialists and scientists/investigators) and 2) Rutgers Food Science students. Changes/Problems: We expected natural antimicrobial subtilosin to work against biofilms of L. monocytogenes since it kills the pathogen's planktonic cells and was found active against biofilms of a different human pathogen, Gardnerella vaginalis. However, it did not work well against biofilm-associated L. monocytogenes. Therefore, we will replace subtilosin with the GRAS preservative nisin, which is a well-known inhibitor of listeria. What opportunities for training and professional development has the project provided?
Nothing Reported
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? During the next reporting period, our objective are as following: 1. Formulation and fabrication of the selected food-grade biodegradable carriers loaded with combinations of natural antimicrobials acting synergistically against food-borne pathogen L. monocytogenes. 2. Elucidation of antimicrobial effectiveness of these systems.
Impacts
What was accomplished under these goals?
Previously, we reported on the role of controlled release in efficient antimicrobial activity of natural antimicrobials (preservatives) such as nisin. Then, several commercially-available food-grade polymers were identified as possible carriers for natural antimicrobials with an ultimate goal for testing against foodborne pathogens. During this reporting period, several natural and nature-derived antimicrobials were tested for their activity against foodborne pathogen Listeria monocytogenes. Building on our previous study of these substances' activity against planctonic cells of L. monocytogenes, we pursued elucidation of these substances' activity against biofilms as biofilms are usually more tolerant to various stresses thanplanktonic cells, and foodborne pathogens are often establishing their presence in the food products in a form of biofilms. In brief, we found that the bacteriocin subtilosin is not very effective against biofilms of L. monocytogenes, although it is good in controlling the pathogen's free cells. At the same time, e-poly-L-lysine (PL) (FDA GRAS approved antimicrobial) and lauramide arginine ethyl ester (LAE) were particularly effective in controlling biofiolm formation and in killing of biofilm-associated L. monocytogenes. Based on this finding and considering our previous study showing nisin's (FDA GRAS approved preservative) activity L. monocytogenes in combination with various natural antimicrobials, PL, LAE and nisin are selected for the last phase of this project's study, specifically loading into biodegradable polymers for the targeted-release based control of L. monocytogenes.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
M.L. Chikindas. 2014. Probiotics and atimicrobial peptides: the creatures and substances future in the twenty-first century: An opinion letter. Probiotics and Antimicrobial Proteins 6: 69-72.
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: This project's outcomes will serve food manufacturing industry and the industry producing food packaging materials. Changes/Problems: There are no major changes per se. However, in addition to already selected natural antimicrobials, we incorporated kojic acid as an interesting candidate for the role of an anti-biofilm agent when used as a part of multiple hurdle approach. This resulted from a new international collaboration initiated last year. What opportunities for training and professional development has the project provided? This project provided an opportunity for training of two graduate (M.S.) students, one rotation (M.S.) student and two undergraduate (B.S.) students. 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? In addition to already collected data, we will study the effect of control-released antimicrobials on the level of intracellular ATP and the DNA/RNA ratio. In brief, knowing the cellular ATP level will provide us with information on the energy level and the cell's ability to perform its energy-dependant physiological and biochemical functions. The RNA/DNA ratio measured in comparison with non-stressed cells will provide with information on efficiency of synthetic processes in the stressed cells with limited/arrested ability to form biofilms.
Impacts
What was accomplished under these goals?
During the reporting period of time, our group studied effect of control-released AM from several polymer matrices. These include release of a bacteriocin subtilosin A from add-mixed formulated PEG-based hydrogels and from nano-fibers, and release of salicilate (SA) from poly-(SA) film. Our study showed bi-modal (first fast, then slow) release of subtilosin A. Previously, we reported on bimodal release kinetics as the most efficient use of bacteriocins for control of the targeted human pathogens. SA release from poly-(SA) followed similar kinetics and the released SA was effective in inhibiting human pathogens such as Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus. Moreover, three to five days exposure to the released SA resulted in significant inhibition of microbial biofilms. To conclude, using different carriers and several nature-derived antimicrobials, we showed the most effective inhibition on human pathogens by their exposure to first high concentration of an antimicrobial, followed by the extended time exposure to a much lower concentration of an antimicrobial. We identified this diffusion kinetics as most efficient for controlling foodborne pathogens by control-released substances. This finding provides important information regarding the most effective preservative-releasing packaging design to assure the most efficient control of microbial microorganisms in packaged food and thus extend the product’s shelf life.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2014
Citation:
T.D. Arthur, V.L. Cavera and M.L. Chikindas. 2014. On bacteriocin delivery systems and potential applications. Accepted for publication in: Future Microbiology. February 2014.
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Our laboratory studies antimicrobial activity of natural and nature-derived substances against various human pathogens. These substances include several food-grade GRAS formulations such as glycerol monolaurate, lauric arginate, and epsilon-poly-L-lysine. However, our major focus is on a novel bacteriocin, subtilosin, which we see as an alternative to the commercially-available nisin since it kills nisin resistant Listeria (Amrouche et al. 2010. Probiotics and Antimicrobial Proteins 2: 250-257).Our previous studies indicated that bacteriocins might act differently on different target organisms. Therefore, prior to study its efficiency against biofilms of foodborne pathogens, we investigated subtilosin's mechanism of action against Listeria monocytogenes. PARTICIPANTS: 1. Ms. Sandra van Kuijk, international visiting M.S. student from Institute of Food Safety, Wageningen University, Wageningen, The Netherlands. 2. Dr. Katia Sutyak Noll, recently graduated with Ph.D. in microbiology, currently employed by Kraft Foods. TARGET AUDIENCES: This study will be of interest for the food safety specialists working on novel approaches in food preservation and for investigators active in basic research on antimicrobial proteins. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In L. monocytogenes, subtilosin caused a partial depletion of the membrane potential, and had a similar minor effect on the delta pH. There was no significant efflux of intracellular ATP. Subtilosin likely acts upon L. monocytogenes Scott A by perturbing the lipid bilayer of the cellular membrane and causing intracellular damage, leading to eventual cell death. Subtilosin's mode of action against L. monocytogenes Scott A differs from the one previously described for another human pathogen, Gardnerella vaginalis (Sutyak Noll et al.2011. Probiotics and Antimicrobial Proteins 3: 41-47).
Publications
- van Kuijk, S., Noll, K. and Chikindas, M.L. (2011). The species-specific mode of action of the antimicrobial peptide subtilosin against Listeria monocytogenes Scott A. Letters in Applied Microbiology 54:52-58.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: New methodological approach for quantification of biofilm formation by foodborne pathogens was evaluated when the biofilms were treated with novel antimicrobial agent, subtilosin, which is active against Gram-positive foodborne pathogens, including nisin-resistant forms. To approach biofilm-related studies, this antimicrobial was first evaluated against planktonic cells of a selected pathogen, alone and in combination with synergistically-acting natural antimicrobials (Amrouche et al. 2010). Biofilms of Staphylococcus aureus, Listeria monocytogenes, Bacillus subtilus, Salmonella, and Pseudomonas aeruginosa were established and tested in newly implemented assay. PARTICIPANTS: Dr. Evgeniy Turovskiy, post-doctoral researcher, Ms. Veronica Cavera, graduate student. TARGET AUDIENCES: Our research is targeted at microbial food safety and quality specialists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
New coupled assay was evaluated where biofilm formation/stability was elucidated with crystal violet staining, while viability of biofilm-associated cells was studied by measuring ATP in biofilm-associated cells, with assistance of the luciferase reporter assay. Next step of our research will be focused at optimization of the coupled assay conditions, visualization of biofilm formation in intact and antimicrobial treated populations of foodborne pathogens.
Publications
- T. Amrouche, K. Sutyak Noll, Y. Wang, Q. Huang, and M.L. Chikindas. 2010. Antibacterial activity of subtilosin alone and combined with curcumin, poly-lysine and zinc lactate against Listeria monocytogenes strains. Probiotics and Antimicrobial Proteins 2: 250-257.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Activities: Several approaches were investigated to identify most promising formulations for controlled delivery of natural antimicrobials. Previously explored poly(anyhydride)ester polymer was studied in comparison with delivery by nano-encapsulation and the delivery by nano-fibers. Nano-encapsulation was considered as a promising approach for formulation of water-insoluble antimicrobials such as curcumin, and nano-fibers composed of lactate were considered as promising and acting synergistically with natural antimicrobials such as nisin. Three modes of controlled delivery were studied against Salmonella and Listeria monocytogenes. Dissemination: These results were disseminated through the undergraduate course of Food Microbiology and the graduate course of Food Biology. Events: Our research results, with emphasis on controlled delivery and the need for formulations base on multiple synergistically-working antimicrobials, were reported at the IFT seminar on June 23, 2010 (Invited speaker, presentation: "Naturally-derived food preservatives: the world is not enough"). Products: One student graduated with M.S. degree in food science and currently employed by the food industry. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Change in knowledge: our research clearly indicated that the substance can deliver the desired activity when properly formulated and used in combination with synergistically acting agents. Specifically, nano-encapsulated curcumin possessed enhanced anti-listeria activity alone and when combined with nisin.Similarly, lactate-based nano-fibers were found as a useful tool for delivery on GRAS antimicrobial peptide nisin.
Publications
- T. Amrouche, K. Sutyak Noll, Y. Wang, Q. Huang, and M.L. Chikindas. 2010. Antibacterial activity of subtilosin alone and combined with curcumin, poly-lysine and zinc lactate against Listeria monocytogenes strains. Probiotics and Antimicrobial Proteins 2: 250-257.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: The effect of a slow-released natural antimicrobial, salicylic acid (SA), was tested on biofilm formation in Salmonella typhimurium MAE52. Glass coverslips coated with poly(anhydride) ester with salicylic acid built into the polymer backbone (SA-PAE) were used to study the release of SA during polymer degradation. S. typhimurium MAE52 was found to follow bimodal pH kinetics when cultured in initially-neutral BHI medium (pH 7.2), with the formation of biofilms occurring after 12 hours of incubation. Continuous release of SA from SA-PAE coverslips resulted in a disruption of the pH profile and prevention of biofilm formation. The controlled release of SA over time influenced cellular functions (i.e. metabolism), reflected in the disruption of the bimodal pH. While future research is necessary to elucidate how cellular regulation is affected during exposure to salicylic acid, the delivery of salicylic acid through a degradable polymer shows great potential in the prevention of biofilm formation. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
To the best of our knowledge, this is the first study showing that the addition of control-released salicylic acid (SA) prevents biofilm formation by Salmonella while disrupting the bimodal nature of pH kinetics. During cultivation in BHI broth with no added antimicrobial, the S. typhimurium growth medium reached its lowest pH value of 5.6 after 12 hours of incubation. Subsequently, biofilm formation was observed with a concurrent increase of pH to 8.6 at 48 hours of incubation. A decrease in the medium's glucose concentration was detected along with a pH shift to acidic levels. The major metabolite produced by Salmonella is acetic acid; with glucose depletion, Salmonella switches to an amino acid metabolism which results in a pH increase. These findings indicate that biofilm formation can be attributed to the availability of nutrients, which is reflected in a bimodal behavior of pH. After addition of SA-PAE to inoculated BHI broth, a controlled release of SA into the microbial environment was observed. In the presence of SA, the pH reached its lowest value by hour 12 of incubation and did not change significantly (P<0.05) over 48 hours of total incubation time. Our results suggest that SA induced metabolic changes in the cells, reflected in disruption of the bimodal pH. We determined the minimal inhibitory concentration (MIC) of SA for MAE52 cells grown in BHI broth as 337 μg mL-1. Controlled release of SA from SA-PAE at pH 7.2 did not significantly reduce the cells' viability, although the final concentration of SA at 48 hours of incubation was approximately 2 folds higher than the determined MIC. The slow release of SA influenced cellular metabolism which resulted in prevention of biofilm formation; however, it was not responsible for significant cell death. Previously, we reported similar results in L. monocytogenes, where low concentrations of an antimicrobial peptide, nisin, efficiently controlled microbial growth in comparison to 5-fold higher concentrations slowly released into the microbial environment. Similarly, our Salmonella cells gained tolerance to the continuous release of SA while at the same time became incapable of biofilm formation. To study the effect of cellular metabolism on SA release, SA-PAE coverslips were tested in inoculated BHI broth. In acidic, neutral, and basic pHs, SA release into the culture medium was significantly (P<0.05) higher than into uninoculated medium. The higher concentrations of SA in the supernatant can be attributed to the action of cellular metabolic products capable of enhancing the breakdown of the SA-PAE polymer. Based on the changes observed in microbial physiology, we speculate that cells may consume SA. However, there are no data published on the effect that bacterial metabolism has on environmentally-present SA. The slow, controlled release of SA may have affected the regulation of certain gene(s,) making cells incapable of biofilm formation. These gene(s) may become future targets for a specific antimicrobial design aimed at the prevention of biofilm formation.
Publications
- A.R. Guinta, A.L. Carbone, L.E. Rosenberg, K.E. Uhrich, M. Tabak and M.L. Chikindas. 2009. Slow release of salicylic acid from degrading poly(anhydride ester) polymer disrupts bimodal pH and prevents biofilm formation in Salmonella typhimurium MAE52 In: Biofilms: Formation, Development and Properties. Nova Science Publishers, Inc. [accepted]
- A. Carbone, L. Rosenberg-Minkow, M.L. Chikindas and K. Uhrich. 2009. Polyanhydrides with natural antimicrobials for control of biofilm formation Biomacromolecules Journal. Manuscript ID: bm-2009-01487d. [submitted]
- N. Mazundar, M. Chikindas and K. Uhrich. 2009. Slow release polymer-iodine tablets for disinfection of untreated surface water. Journal of Applied Polymer Science. Manuscript ID APP-2009-06-1765.R1. [accepted]
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Salicylic acid (SA), a natural antimicrobial, was built into a Poly(anhydride-ester) polymer backbone. Salmonella typhimurium MAE52 begins to produce biofilms after reaching a pH of 7.8 or higher. It is believed that at this crucial point in which nascent biofilms begin to form that SA will be released, allowing for their prevention through interruption of a bimodal pH. When SA built into a Poly(anhydride-ester) polymer is added to the culture, the bimodal pH of the environment is disrupted resulting in a final pH of 5.3 , thus preventing the formation of biofilms. As the SA polymer (SAP) is exposed to the culture, is it speculated that the Poly(anhydride-ester) linkage is broken under the influence of cell metabolic products and the medium components, releasing free SA which interacts with cells, changing their metabolism and physiology. Salmonella typhimurium MAE52 was exposed to an initial acidic pH of 6.2 to ascertain the culture's ability to form biofilms in a low pH environment. In the absence of the SAP no biofilms were formed and the final pH remained acidic. With the addition of the SAP to the medium with an initial pH of 6.2, similar results were obtained showing no biofilm formation and a final pH of 5.1. To characterize the ability of Salmonella typhimurium MAE52 to form biofilm in the presence of an environmental alkaline pH, the cells were grown in the medium with an initial pH of 8.5. In this case, in the absence of the polymer there was a bimodal pH with a final pH of 8.8 which resulted in partial biofilm production. Similarly, the addition of the SAP resulted in the production of fragile biofilms and a final pH of 8.5. These data indicate that biofilm formation depends on the environmental pH. The type of nutritional media used can have an effect on the final pH of a culture due to metabolism of different substances in its composition. It is expected that metabolism of a high protein medium will result in a final alkaline pH and metabolism of a high sugar medium will results in a final acidic pH. PARTICIPANTS: Jason Turovskiy, Ph.D. student, is being involved in this project for the whole period of time. Allison Guinta, M.S. student, is being involved in this project for the last 12 months. TARGET AUDIENCES: As previously, the target audience is the industry, academia and regulatory bodies food safety specialists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Microbial biofilms are much more resistant to stresses and mechanical removal than planktonic (free) cells. Therefore, prevention of biofilm formation is of a great importance for food industry and agriculture. The salicylic acid (SA) based polymer (SAP) is a prominent antimicrobial applicable for the use in foods because of its inherent safety. Alkaline pH seems to be a major contributing factor in the formation of biofilms in Salmonella typhimurium, allowing the cells to undergo essential physiological changes that permit biofilm formation. Under neutral conditions SA appears to prevent the cells from attaining the most crucial bimodal pH, thus preventing the formation of biofilms. It can be concluded that SA provides a stress to cells, preventing them from undergoing physiological and metabolic changes which will allow for biofilm formation.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Control of bacterial biofilms has always been a major challenge in food industry and medicine as these microbial formations are more resistant to the antimicrobials than the free cells. Quorum sensing (QS) has a critical role in biofilm formation and is involved in some of the antimicrobial resistances. We investigated salicylic acid as a prominent biofilm antagonist with potential food applications. The Salmonella enterica-formed biofilms were targeted using a unique salicylic acid-based polymer coating. S. enterica was chosen instead of Listeria monocytogenes as the model organism for this particular study because the chosen strain is capable of forming biofilms at the air/liquid interface which eliminates many essential variables associated with the attachment surfaces. The polymer coating allowed for the gradual release of salicylic acid, which at certain concentrations interfered with Salmonella's ability to form biofilms. These data imply that salicylic acid does not
interfere with the attachment of cells to the solid surface as has been previously suggested. We speculate that this naturally occurring compound may interfere with the QS, a hypothesis that is currently being investigated.
PARTICIPANTS: Jason Turovskiy, Ph.D. student, is being involved in this project for the whole period of time.
TARGET AUDIENCES: As previously, the target audience is the industry, academia and regulatory bodies food safety specialists.
PROJECT MODIFICATIONS: In order to model microbial behavior in response to the selected safe antimicrobial, we conducted our studies on Salmonella instead of using Listeria monocytogenes. The obtained information will allow us to progress with our research on other biofilm-forming microroganisms, including L. monocytogenes, which is our primary target.
Impacts
Antimicrobials which interfere with the 'communal properties' of the bacteria are less prone to select for adaptive mutations occurring in a target microorganism. Since biofilms are resistant to stresses and mechanical removal, treatments that specifically prevent formation of biofilms are of great interest. The salicylic acid-based polymer evaluated in this study, makes a prominent 'communal interrupter' applicable for the use in foods because of its inherent safety.
Publications
- Y. Turovskiy, L. Rosenberg, and M. L. Chikindas. Quorum sensing is not involved in Listeria monocytogenes' adaptive responses to food preservatives lactic acid and nisin. 2007. Journal of Food Safety 27: 386-399.
- Y. Turovskiy, D. Kashtanov, B. Paskhover and M. L. Chikindas. 2007. Quorum sensing: fact, fiction and everything in between. Advances in Applied Microbiology 62: 191-234.
- L.E. Rosenberg, A.L. Carbone, U. Romling, K.E. Uhrich, and M.L. Chikindas. 2007. Salicylic acid-based poly(anhydride-esters) for control of biofilm formation in Salmonella enterica serovar Typhimurium. Accepted for publication in: Journal of Applied Microbiology.
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Progress 01/01/06 to 12/31/06
Outputs
Recent reports indicate the presence of AI-2 in foods such as frozen fish, tomatoes, cantaloupes, tofu and milk; it is unclear what effect this molecule may have on virulence and survivability of foodborne pathogens. Quorum sensing has been implicated in stress responses of certain bacteria and this particular study had investigated AI-2-mediated quorum sensing as one of the mechanisms through which the general stress response in Listeria monocytogenes can be triggered. In particular, the ability of L. monocytogenes to acquire resistance to the food preservatives nisin and lactic acid was examined. The indirect approach comprised of the two-stage experimental design was utilized in this study, because of the complications related to the direct approach involving the mutagenesis of the gene coding for AI-2 synthase. In the first stage the production of AI-2 by L. monocytogenes in response to the selected stresses was assessed. Our data indicates that the levels of AI-2
produced by the pathogen are not affected by the presence of nisin and lactic acid. The second set of experiments has also indicated that pre-exposure to extracellular AI-2 does not improve the survivability of the cells in the presence of these two antimicrobials. All together, the results indicate that AI-2 present in foods does not induce adaptive response in L. monocytogenes to the common food preservatives, nisin and lactic acid. In addition, we did not find any evidence for the quorum sensing, in general, being involved in the general stress response of the pathogen.
Impacts
The understanding of cellular responses such as quorum sensing, which may be triggered in L. monocytogenes by the foods, is essential for the design of effective food preservation methods. The fact that AI-2 is not involved in the L. monocytogenes adaptive stress responses, allows to eliminate this variable from a rather complex model of L. monocytogenes interaction with the foods.
Publications
- Y. Turovskiy and M. L. Chikindas. Autoinducer-2 bioassay: qualitative, not quantitative method influenced by glucose. 2006. Journal of Microbiological Methods. 66: 497-503.
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Progress 01/01/05 to 12/31/05
Outputs
Autoinducer-2 (AI-2) is a cell-to-cell signaling molecule which is thought to be utilized for quorum sensing processes by a variety of prokaryotic species. This molecule is usually detected using a so-called autoinducer bioassay, which relies on the ability of a Vibrio harveyi reporter strain to produce light in response to AI-2. This method was utilized in a number of studies in an attempt to characterize the mode of AI-2 production in various microorganisms. For example, Cloak et al.(2002) monitored AI-2 production by various food pathogens in milk, chicken broth, and apple juice at three different temperatures and did not observe AI-2 production by microorganisms in apple juice. Lu et al. (2004) used the AI-2 bioassay to detect the possible AI-2-like activity which may be associated with various foods and found that some foods can indeed induce bioluminescence in BB170, presumably because of molecules in the food which mimic AI-2 structurally. Nealson et al (1972)
showed that even a small quantity of glucose inhibits bioluminescence in Vibrio harveyi species. It was proposed that this inhibition happens on the transcriptional level of the luciferase biosynthesis in Vibrio harveyi, through a catabolite repression mechanism. This finding is largely disregarded when Vibrio harveyi is utilized for the detection of AI-2 (DeKeersmaecker and Vanderleyden, 2003). In our study we further investigate the validity of the Autoinducer-2 bioassay, and also demonstrate how data can be misinterpreted if the pitfalls of this assay are overlooked. Our data suggest that glucose in concentrations bellow those required for the inhibition of the bioluminescence can actually cause the false detection of AI-2 presence. Our results also suggest that even if all the limitations of this bioassay are considered, the large standard deviation of the method allows only for the qualitative and not quantitative interpretation of the obtained results.
Impacts
It is a well-documented fact that microorganisms communicate with each other about approaching stresses such as changes in the environment, including food preservatives. Initially, such communication was attributed to the ability of bacteria to sense their number (hence the term was coined: quorum sensing) and the specific small size substances produced by communicating bacteria were called autoinducers since they trigger the activity of many genes including the genes responsible for microbial pathogenicity. One of these autoinducers, AI-2 was reported as being produced by foodborne pathogens in milk, chicken broth, and in some other foods, hence making researchers assume that some foods can induce microbial communication and preparedness for incoming stresses. Now that the study of quorum sensing in bacteria receives such tremendous attention and the role of cell-to-cell communication and the ability to tolerate stresses is broadly acknowledged, it is of outmost
importance to have reliable and reproducible methods at hand to study these processes. The reported effort is dedicated to the largely overlooked yet highly important issue of how some essential factors (nutrients, growth phase etc.) may influence the results and their interpretation. We believe these data will be considered as an important message that deserves the attention of researchers studying quorum sensing in bacteria and its role in microbial food safety.
Publications
- Manuscript entitled "Autoinducer-2 bioassay is a qualitative, not quantitative method influenced by glucose" (2006) was submitted for publication in the Journal of Microbiological Methods. Ms. Ref. No.: MIMET-D-06-00024.
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