Detection of bacterial contamination in food matrices by integration of quorum sensing in a paper-strip test.

There are an estimated 48 million cases of foodborne illness in the United States every year. In general, these illnesses are the result of unintentional contamination and improper food handling. Because bacterial contamination plays a major role in food spoilage and, hence, in foodborne illnesses, it is important to design easy, portable methods to detect bacteria in food. Quorum sensing (QS) enables bacteria to communicate with one another and by doing so they can modulate their behavior in a cell-density dependent manner. In bacteria, quorum sensing molecules (QSMs) are known to control several factors such as virulence factor production, antibiotic production, biofilm formation, and gene regulation. Herein, we demonstrate the applicability of whole cell biosensing systems for the early identification of food contamination via detection of QSMs. Additionally, we have developed a portable system for detection of bacterial contamination using microdots of immobilized whole cell-based biosensors on paper that boast nanomolar level detection of QSMs in two different food matrices, namely beef and milk. Limits of detection ranged from 1 × 10-7 M to 1 × 10-9 M with relative standard deviations (RSDs) of 1-16%. This rapid, easy, and portable test could be a useful tool for use in the field and during all stages of food manipulation, i.e., from farms to distribution, storage, sales, and preparation prior to consumption, to ensure that food is free of bacterial contamination.

Transcriptional regulatory proteins as biosensing tools.

We have developed sensing systems employing different classes of transcriptional regulatory proteins genetically and chemically modified to incorporate a fluorescent reporter molecule for detection of arsenic, hydroxylated polychlorinated biphenyls (OH-PCBs), and cyclic AMP (cAMP). These are the first examples of optical sensing systems based on transcriptional regulatory proteins.

Whole-Cell Biosensors as Tools for the Detection of Quorum-Sensing Molecules: Uses in Diagnostics and the Investigation of the Quorum-Sensing Mechanism.

Genetically engineered bacterial whole-cell biosensors are powerful tools that take advantage of bacterial proteins and pathways to allow for detection of a specific analyte. These biosensors have been employed for a broad range of applications, including the detection of bacterial quorum-sensing molecules (QSMs). Bacterial QSMs are the small molecules bacteria use for population density-dependent communication, a process referred to as quorum sensing (QS). Various research groups have investigated the presence of QSMs, including N-acyl homoserine lactones (AHLs) and autoinducer-2 (AI-2), in physiological samples in attempts to enhance our knowledge of the role of bacteria and QS in disease states. Continued studies in these fields may allow for improved patient care and therapeutics based upon QSMs. Furthermore, bacterial whole-cell biosensors have elucidated the roles of some antibiotics as QS agonists and antagonists. Graphical Abstract.

Bacterial autoinducer-2 detection via an engineered quorum sensing protein.

Autoinducer-2 (AI-2) is a Quorum Sensing (QS) molecule utilized by bacteria in interspecies communication. More recently, it is identified to be vital in regulating QS pathways in a number of human and foodborne pathogens. Methods to detect AI-2 in a rapid and highly sensitive manner can help in the early detection of bacterial infections. Herein, we describe a rapid, selective, and highly sensitive protein based biosensing system employing the Fluorescence Resonance Energy Transfer (FRET) between a protein fusion LuxP-EGFP and 7-diethylamino-3-[N-(2-maleimidoethyl)carbamoyl]coumarin (MDCC). The developed biosensing system, which can detect AI-2 at subnanomolar levels, was successfully applied to detect AI-2 in clinical samples such as saliva and blood serum.

Deciphering bacterial universal language by detecting the quorum sensing signal, autoinducer-2, with a whole-cell sensing system.

Bacteria communicate with neighboring bacteria of the same species or of other species by means of chemical signaling molecules. The concentration of such signaling molecules is proportional to the bacterial population size; upon reaching a threshold concentration, corresponding to a threshold cell density, certain specialized genes are expressed. This system of communication among bacteria is known as quorum sensing (QS). QS regulates diverse behaviors, such as formation of biofilms and production of pathogenic factors. Autoinducer-2 (AI-2) is a QS signaling molecule that is used for interspecies communication by both Gram-positive and Gram-negative bacteria. Bacteria are known to play an important role in many diseases, from infections to chronic inflammation. Therefore, QS is involved in a variety of disorders of bacterial origin or where bacteria play a crucial pathogenic role. One such condition is inflammatory bowel disease (IBD), a chronic inflammation of the gastrointestinal (GI) tract that includes debilitating diseases, such as ulcerative colitis (UC) and Crohn's disease (CD). To date, noninvasive methods are unavailable for the diagnosis and monitoring of IBD. We hypothesized that detection of QS molecules in physiological samples, specifically saliva and stool specimens, would provide with a method for the noninvasive, early diagnosis and monitoring of IBD conditions. To that end, we developed and optimized a whole-cell sensing system for AI-2, which is based on Vibrio harveyi strain BB170. Furthermore, we standardized and applied the biosensing system for the quantitative detection of AI-2 in saliva, stool, and intestinal samples from IBD patients.

Bioluminescence inhibition assay for the detection of hydroxylated polychlorinated biphenyls.

Hydroxylated polychlorinated biphenyls (OH-PCBs) are an important class of contaminants that mainly originate from polychlorinated biphenyl metabolism. They may conceivably be as dangerous and persistent as the parent compounds; most prominently, OH-PCBs are endocrine disruptors. Due to increasing evidence of the presence of OH-PCBs in the environment and in living organisms, including humans, and of their toxicity, methods of detection for OH-PCBs are needed in the environmental and medical fields. Herein, we describe the development and optimization of a protein-based inhibition assay for the quantification of OH-PCBs. Specifically, the photoprotein aequorin was utilized for the detection of OH-PCBs. We hypothesized that OH-PCBs interact with aequorin, and we established that OH-PCBs actually inhibit the bioluminescence of aequorin in a dose-dependent manner. We took advantage of this phenomenon to develop an assay that is capable of detecting a wide variety of OH-PCBs with a range of detection limits, the best detection limit being 11 nM for the compound 2-hydroxy-2',3,4',5',6-pentachorobiphenyl. The viability of this system for the screening of OH-PCBs in spiked biological and environmental samples was also established. We envision the implementation of this novel bioluminescence inhibition assay as a rapid, sensitive, and cost-effective method for monitoring OH-PCBs. Furthermore, to the best of our knowledge, this is the first time aequorin has been employed to detect an analyte by the inhibition of its bioluminescence reaction. Hence, this strategy may prove to be a general approach for the development of a new generation of protein-based inhibition assays.

Investigating the effect of antibiotics on quorum sensing with whole-cell biosensing systems.

Quorum sensing (QS) allows bacteria to communicate with one another by means of QS signaling molecules and control certain behaviors in a group-based manner, including pathogenicity and biofilm formation. Bacterial gut microflora may play a role in inflammatory bowel disease pathogenesis, and antibiotics are one of the available therapeutic options for Crohn's disease. In the present study, we employed genetically engineered bioluminescent bacterial whole-cell sensing systems as a tool to evaluate the ability of antibiotics commonly employed in the treatment of chronic inflammatory conditions to interfere with QS. We investigated the effect of ciprofloxacin, metronidazole, and tinidazole on quorum sensing. Several concentrations of individual antibiotics were allowed to interact with two different types of bacterial sensing cells, in both the presence and absence of a fixed concentration of N-acylhomoserine lactone (AHL) QS molecules. The antibiotic effect was then determined by monitoring the biosensor's bioluminescence response. Ciprofloxacin, metronidazole, and tinidazole exhibited a dose-dependent augmentation in the response of both bacterial sensing systems, thus showing an AHL-like effect. Additionally, such an augmentation was observed, in both the presence and absence of AHL. The data obtained indicate that ciprofloxacin, metronidazole, and tinidazole may interfere with bacterial communication systems. The results suggest that these antibiotics, at the concentrations tested, may themselves act as bacterial signaling molecules. The beneficial effect of these antibiotics in the treatment of intestinal inflammation may be due, at least in part, to their effect on QS-related bacterial behavior in the gut.

Engineered cells as biosensing systems in biomedical analysis.

Over the past two decades there have been great advances in biotechnology, including use of nucleic acids, proteins, and whole cells to develop a variety of molecular analytical tools for diagnostic, screening, and pharmaceutical applications. Through manipulation of bacterial plasmids and genomes, bacterial whole-cell sensing systems have been engineered that can serve as novel methods for analyte detection and characterization, and as more efficient and cost-effective alternatives to traditional analytical techniques. Bacterial cell-based sensing systems are typically sensitive, specific and selective, rapid, easy to use, low-cost, and amenable to multiplexing, high-throughput, and miniaturization for incorporation into portable devices. This critical review is intended to provide an overview of available bacterial whole-cell sensing systems for assessment of a variety of clinically relevant analytes. Specifically, we examine whole-cell sensing systems for detection of bacterial quorum sensing molecules, organic and inorganic toxic compounds, and drugs, and for screening of antibacterial compounds for identification of their mechanisms of action. Methods used in the design and development of whole-cell sensing systems are also reviewed.

A protein switch sensing system for the quantification of sulfate.

Protein engineering has generated versatile methods and technologies that have been instrumental in advancements in the fields of sensing, therapeutics, and diagnostics. Herein, we demonstrate the employment of rational design to engineer a unique bioluminescence-based protein switch. A fusion protein switch combines two totally unrelated proteins, with distinct characteristics, in a manner such that the function of one protein is dependent on another. Herein we report a protein switch sensing system by insertion of the sulfate-binding protein (SBP) into the structure of the photoprotein aequorin (AEQ). In the presence of sulfate, SBP undergoes a conformational change bringing the two segments of AEQ together, "turning on" bioluminescence in a dose-dependent fashion, thus allowing quantitative detection of sulfate. A calibration plot was obtained by correlating the amount of bioluminescence generated with the concentration of sulfate present. The switch demonstrated selectivity and reproducibility, and a detection limit of 1.6×10(-4)M for sulfate. Moreover, the sensing system was validated by performing sulfate detection in clinical and environmental samples, such as, serum, urine, and tap water. The detection limits and working ranges in all three samples fall within the average normal/recommended sulfate levels in the respective matrices.

Bacterial spores as platforms for bioanalytical and biomedical applications.

Genetically engineered bacteria-based sensing systems have been employed in a variety of analyses because of their selectivity, sensitivity, and ease of use. These systems, however, have found limited applications in the field because of the inability of bacteria to survive long term, especially under extreme environmental conditions. In nature, certain bacteria, such as those from Clostridium and Bacillus genera, when exposed to threatening environmental conditions are capable of cocooning themselves into a vegetative state known as spores. To overcome the aforementioned limitation of bacterial sensing systems, the use of microorganisms capable of sporulation has recently been proposed. The ability of spores to endow bacteria-based sensing systems with long lives, along with their ability to cycle between the vegetative spore state and the germinated living cell, contributes to their attractiveness as vehicles for cell-based biosensors. An additional application where spores have shown promise is in surface display systems. In that regard, spores expressing certain enzymes, proteins, or peptides on their surface have been presented as a stable, simple, and safe new tool for the biospecific recognition of target analytes, the biocatalytic production of chemicals, and the delivery of biomolecules of pharmaceutical relevance. This review focuses on the application of spores as a packaging method for whole-cell biosensors, surface display of recombinant proteins on spores for bioanalytical and biotechnological applications, and the use of spores as vehicles for vaccines and therapeutic agents.

Integration of spore-based genetically engineered whole-cell sensing systems into portable centrifugal microfluidic platforms.

Bacterial whole-cell biosensing systems provide important information about the bioavailable amount of target analytes. They are characterized by high sensitivity and specificity/selectivity along with rapid response times and amenability to miniaturization as well as high-throughput analysis. Accordingly, they have been employed in various environmental and clinical applications. The use of spore-based sensing systems offers the unique advantage of long-term preservation of the sensing cells by taking advantage of the environmental resistance and ruggedness of bacterial spores. In this work, we have incorporated spore-based whole-cell sensing systems into centrifugal compact disk (CD) microfluidic platforms in order to develop a portable sensing system, which should enable the use of these hardy sensors for fast on-field analysis of compounds of interest. For that, we have employed two spore-based sensing systems for the detection of arsenite and zinc, respectively, and evaluated their analytical performance in the miniaturized microfluidic format. Furthermore, we have tested environmental and clinical samples on the CD microfluidic platforms using the spore-based sensors. Germination of spores and quantitative response to the analyte could be obtained in 2.5-3 h, depending on the sensing system, with detection limits of 1 x 10(-7) M for arsenite and 1 x 10(-6) M for zinc in both serum and fresh water samples. Incorporation of spore-based whole-cell biosensing systems on microfluidic platforms enabled the rapid and sensitive detection of the analytes and is expected to facilitate the on-site use of such sensing systems.

Packaging sensing cells in spores for long-term preservation of sensors: a tool for biomedical and environmental analysis.

Whole-cell sensing systems have successfully been employed for detection of various biologically and environmentally important analytes. A limitation to their use for on-field analysis is the paucity of preservation methods for long-term storage and transport. For that, we have previously developed spore-based genetically engineered whole-cell sensing systems that are able not only to maintain the activity of the sensing cells but also to preserve it for long periods of time in normal and extreme environmental conditions. Herein, we have employed these spore-based sensing systems for analysis of real samples, such as blood serum and freshwater. Spores were able to germinate in the presence of the sample matrix, and the minimum time required for the spores to germinate and generate vegetative sensing cells able to elicit a measurable response to target analytes resulted to be around 2 h. Of the two spore-based sensing systems selected to detect model analytes in real samples, one was able to detect arsenic concentrations as low as 1 x 10(-7) M in freshwater and serum samples, and the other one could sense down to 1 x 10(-6) M of zinc in serum. The analysis of human serum samples from healthy subjects for their zinc content proved the viability of spore-based sensing systems. The complete assays, including spore germination and analyte detection, were performed in 2.5 h or less for arsenic and zinc. Furthermore, the assay is inexpensive and simple to carry out and offers unique advantages for the incorporation of the spore-based sensing systems into portable analytical platforms, such as microfluidic devices, to be employed for on-site analysis.

Paper strip whole cell biosensors: a portable test for the semiquantitative detection of bacterial quorum signaling molecules.

Herein, we report the development of a novel, inexpensive, and portable filter-paper-based strip biosensor for the detection of bacterial quorum sensing signaling molecules, N-acylhomoserine lactones (AHLs). AHLs are generally employed by Gram-negative bacteria for their cell-cell communication to control expression of specialized genes, such as those involved in biofilm formation and production of virulence factors, in a population-density-dependent manner. First, a bacterial cell-based sensing system employing components of AHL-mediated QS regulatory system as recognition elements and beta-galactosidase as the reporter protein was designed and developed. The bacterial-sensing cells were then liquid-dried on strips of filter paper. beta-Galactosidase as the reporter allows for the visual monitoring of the analyte-induced signal when a colorimetric method of detection is applied. The paper strip biosensor was able to detect low AHL concentrations down to 1 x 10(-8) M. Furthermore, it was successfully applied to the detection of AHLs in physiological samples, such as saliva. The filter-paper-based sensing strips could provide reproducible results upon storage at 4 degrees C for at least 3 months. In conclusion, a filter-paper-based strip biosensor was developed that allows for visual, fast, and convenient detection of AHLs in a dose-dependent manner in a test sample. In addition, it does not require expensive equipment or trained personnel and allows ease of transportation and storage. Therefore, we envision that this biosensor will serve as a simple and economical portable field kit for on-site monitoring of AHL in a variety of clinical and environmental samples.

Fluorescent and bioluminescent cell-based sensors: strategies for their preservation.

Luminescent whole-cell biosensing systems have been developed for a variety of analytes of environmental, clinical, and biological interest. These analytical tools allow for sensitive, rapid, simple, and inexpensive quantitative detection of target analytes. Furthermore, they can be designed to be nonspecific, semispecific, or highly specific/selective. A notable feature of such sensing systems employing living cells is that they provide information on the analyte bioavailability and activity. These characteristics, along with their suitability to miniaturization, make cell-based sensors ideal for field applications. However, a major limitation to on-site use is their "shelf-life." To address this problem, various methods for preservation of sensing cells have been reported, including freeze-drying, immobilization in different types of matrices, and formation of spores. Among these, the use of spores emerged as a promising strategy for long-term storage of whole-cell sensing systems at room temperature as well as in extreme environmental conditions.

Detection of bacterial quorum sensing N-acyl homoserine lactones in clinical samples.

Bacteria communicate among themselves using certain chemical signaling molecules. These signaling molecules generally are N-acyl homoserine lactones (AHLs) in Gram-negative bacteria and oligopeptides in Gram-positive bacteria. In addition, both Gram-positive and Gram-negative bacteria produce a family of signaling molecules known as autoinducer-2 that they employ for their communications. Bacteria coordinate their behavior by releasing and responding to the chemical signaling molecules present in proportion to their population density. This phenomenon is known as quorum sensing. The role of bacteria in the pathogenesis of several diseases, including gastrointestinal (GI) disorders, is well established. Moreover, rather recently bacterial quorum sensing has been implicated in the onset of bacterial pathogenicity. Thus, we hypothesized that the signaling molecules involved in bacterial communication may serve as potential biomarkers for the diagnosis and management of several bacteria-related diseases. For that, we previously developed a method based on genetically engineered whole-cell sensing systems for the rapid, sensitive, cost-effective and quantitative detection of AHLs in biological samples, such as saliva and stool, from both healthy and diseased individuals with GI disorders. Although various analytical methods, based on physical-chemical techniques and bacterial whole-cell biosensors, have been developed for the detection of AHLs in the supernatants of bacterial cultures, only a few of them have been applied to AHL monitoring in real samples. In this paper, we report work performed in our laboratory and review that from others that describes the detection of AHLs in biological, clinical samples, and report some of our recent experimental results.

Construction of spores for portable bacterial whole-cell biosensing systems.

Whole-cell sensing systems based on living genetically engineered bacteria are known to have high sensitivity, selectivity, and rapid response times. Although these systems have found applications in biomedical and environmental analyses, their limited shelf life and transportability still restrict their use for on-site monitoring of analytes. To that end, we have developed a new method for the long-term preservation, storage, and transport of whole-cell biosensing systems that is based on bacterial spores, a dormant form of life. Specifically, we have employed spore-forming bacteria such as Bacillus subtilis and Bacillus megaterium for development of luminescent sensing systems for two model analytes, namely, arsenic and zinc. These sensing cells were converted to spores, which can then be "revived" (germinated) at a later time to generate viable and metabolically active cells. Herein, we demonstrate that these spore-based sensing systems retained their analytical performance, in terms of detection limit, dynamic range, and reproducibility, after storage at room temperature for at least 6 and 8 months, respectively, as well as after three cycles where the cells alternated between being dormant or active, i.e., sporulation-germination cycles. The ability to cycle the sensing cells between active and dormant states prolongs the cell's lifetimes and increases their robustness and ruggedness, thus making them more amenable for field applications. In addition, the small size of spores allows for their easy transport and incorporation in miniaturized portable devices. Finally, we envision that this novel strategy could expand the use of whole-cell biosensors for on-site sensing not only in mild environments but also in harsh environments and locations where there is no easy access to a laboratory, e.g., in developing countries.

Hydroxylated polychlorinated biphenyl detection based on a genetically engineered bioluminescent whole-cell sensing system.

The metabolites of polychlorinated biphenyls (PCBs), such as hydroxylated PCBs (OH-PCBs), have been identified as environmental contaminants. Various studies have shown that some OH-PCBs can potentially contribute to health problems. Detection of these compounds in environmental and biological samples could provide useful information about their levels and lead to a better understanding of their apparent toxicity. To that end, we have developed a whole-cell sensing system for the detection of OH-PCBs by taking advantage of the recognition of a group of related compounds, i.e., hydroxylated biphenyls, by the product of the hbpR gene in the hbp operon from Pseudomonas azelaica strain HBP1. By fusing the luxAB genes, encoding the reporter protein bacterial luciferase, to the hbp regulator-promoter sequence, a whole-cell sensing system was developed. Here, we describe the optimization and application of this whole-cell sensing system for the detection of a model compound, 2-hydroxy-3',4'-dichlorobiphenyl. A detection limit of 1 x 10(-8) M was achieved using this system. The detection of a broad range of individual OH-PCBs as well as an OH-PCB mixture was investigated. The system can detect OH-PCBs in whole serum samples in a trace amount, which is comparable to the detection of these analytes in medium alone. We envision that the method developed can potentially be employed as a rapid and sensitive way to monitor OH-PCBs for toxicological study in the laboratory, as well as a useful tool to evaluate the presence of bioavailable OH-PCBs in natural environments.

Biosensing systems for the detection of bacterial quorum signaling molecules.

Bacterial quorum sensing (QS) is a cell-to-cell communication phenomenon that allows bacteria to control the expression of certain specialized genes depending on their cell population size. Signaling molecules such N-acylhomoserine lactones (AHLs) mediate the communication, and their concentration reflects the bacterial population density. Quorum sensing regulates several processes including bacterial pathogenicity. We developed a method for the rapid, sensitive, and quantitative detection of AHLs in biological samples such as saliva and stools. The method is based on whole-cell sensing systems that employ QS regulatory systems as recognition elements and the luxCDABE gene cassette as a reporter. The method proved to be reproducible when applied to real samples and was able to detect low analyte concentrations down to 1 x 10(-9) M without requiring extensive sample preparation. We envision that this novel biosensing system could be employed in the diagnosis and management of various bacteria-related disorders, thus supporting the use of quorum sensing molecules as potential biomarkers of disease. Due to cost-effectiveness and high throughput, these biosensing systems could be successfully employed as a new tool for the screening of novel drugs that target quorum sensing mechanisms.

Regulation of ileal bile acid-binding protein expression in Caco-2 cells by ursodeoxycholic acid: role of the farnesoid X receptor.

Ursodeoxycholic acid (UDCA) is beneficial in cholestatic diseases but its molecular mechanisms of action remain to be clearly elucidated. Other bile acids, such as chenodeoxycholic (CDCA), are agonists for the nuclear farnesoid X receptor (FXR) and regulate the expression of genes relevant for bile acid and cholesterol homeostasis. In ileal cells CDCA, through the FXR, up-regulates the expression of the ileal bile acid-binding protein (IBABP), implicated in the enterohepatic circulation of bile acids. We report that UDCA (100 and 200 microM) induced a moderate increase of IBABP mRNA (approximately 10% of the effect elicited by 50 microM CDCA) in enterocyte-like Caco-2 cells and approximately halved the potent effect of CDCA (50 microM). On the contrary, UDCA reduced by 80-90% CDCA-induced IBABP transcription in hepatocarcinoma derived HepG2 cells. We confirmed that these effects on IBABP transcription required the FXR by employing a cell-based transactivation assay. Finally, in a receptor binding assay, we found that UDCA binds to FXR expressed in CHO-K1 cells (K(d)=37.7 microM). Thus, UDCA may regulate IBABP in Caco-2 cells, which express it constitutively, by acting as a partial agonist through a FXR mediated mechanism. The observation that in HepG2 cells, which do not express constitutively IBABP, UDCA was able to almost completely prevent CDCA-induced activation of IBABP promoter, suggests that tissue-specific factors, other than FXR, may be required for bile acid regulation of FXR target genes.