Methods for the detection of microbes in foods based on the pathogenic properties of the microbe are highly desirable since they can accurately estimate the real risk associated with the microbe. Cell-based biosensor (CBBS), are functional bioassays that rapidly quantify the interaction of pathogens or toxins with mammalian cells and have the potential to be used as a screening tool for a broad range of chemical or biological hazards.

By Professor Arun Bhunia

Foodborne pathogens and safety concerns
Rapid and accurate screening of food products for possible contamination by harmful microbial agents will expedite the timely release of products for retail distribution and will also allow authorities to take necessary action to mitigate human illness, death or economic crises.

Foodborne outbreaks continue to be a major concern in both US and Europe. Each year in the US no fewer than 76 million Americans are affected by foodborne outbreaks, of which 62 million are caused by unknown etiologic agents [1]. The annual cost in the US of foodborne outbreaks is about $152 billion of which $95.8 billion is due to unknown pathogens [2]. The annual cost for five major foodborne pathogens; Listeria monocytogenes, Salmonella (non-typhoidal serotypes), E. coli O157:H7, E. coli non-O157 STEC (Shiga-toxin producing E. coli) and Campylobacter is about $43.2 billion. The data for Europe are similar. In addition to human illness or even death, routine food recalls have severe economic consequences, the extent and severity of which is immediately felt whenever a suspect product is withdrawn from the market place. Such economic effects can often force the food producer to
declare bankruptcy.

The need for a rapid screening tool
The overall food production system is complex, with the particular degree of complexity depending on the nature of operation. For example, production systems for livestock, dairy herd, or aquaculture production are markedly different from those for fruit and vegetables since each system has its own unique on-farm practices, harvesting strategies, and processing sequences. Again, the storage, handling, and distribution chains for each type of operation are different.
Pathogen contamination can occur in any point during food production. The numbers and nature of pathogens are vast. Hence, to ensure product safety at each stage of production in the food supply chain, a suitable broad screening tool would be of great importance [3,4].

Conventional culture-based methods coupled with antibody or nucleic acid are reliable but are labour-intensive, time-consuming (they take at least 2-10 days to yield a result), expensive, and target only specific pathogens or toxins [5]. Thus such approaches may not be economically feasible, and may actually even discourage food producers from using them. Producers often take enormous risk by releasing products without thorough testing.

From a food safety point of view, it does not make economic sense to use expensive test kits when most test samples are expected to be negative. Thus a cost-effective rapid pre-screening tool that allows testing of multiple samples for a wide array of pathogens or toxins would be highly desirable. Products that are free of pathogens can be released immediately, particularly perishable products with limited shelf-life. Products showing positive results warrant rigorous testing with pathogen-specific probes. Another important consideration when developing or recommending methods for product testing is that the methods must be able to detect unknown agents. As mentioned above, of the reported 76 million cases in the US, alarmingly, 62 million were caused by unknown agents. That means current methods are ineffective in monitoring the presence of unknown biohazards. Moreover, with the potential escalation of bio-terrorism through food and water contamination, deployment of a broadband rapid detection technology to serve as a first line of defense is very attractive.

Mammalian cell-based functional bioassay
It is becoming increasingly clear that conventional methods may be ineffective in dealing with the changing scenario of food safety and biosecurity issues [4,6]. Hence, it is imperative that future detection methods must be appropriate for the foreseeable challenges, such as the detection of emerging pathogens, genetically-altered microbes and unknown or little known biohazards or allergens [3,7]. Mammalian cell-based “functional” assays have the potential to meet such needs because these assays, unlike others, rapidly interprets the inherent interaction of mammalian cells with biologically active compounds in a physiologically relevant manner [3,7-10].
The hallmark of the pathogenesis by a pathogen is the disruption of normal physiological function [Figure 1]. Pathogen-induced cellular damage thus often bears a detectable signature of that agent. Common outcomes of mammalian cell injury include altered cell shape, membrane damage, inhibition of protein synthesis, cell death via apoptosis or necrosis, lysis, and detachment of cells from the substrata [11]. Cell-Based BioSensor Systems (CBBS) can also provide insight into the mechanism of action, which in turn facilitates agent detection and classification [12]. For example, toxins can be classified according to their mode of action such as, (a) membrane pore-forming toxin (hemolysin), (b) diarrheagenic toxin (cholera toxin), (c) superantigen that activate the immune response (staphylococcal enterotoxin B), (d) neurotoxin (botulinum toxin) and (e) protein synthesis inhibitory toxin (Shiga-like toxin).

There are several advantages of CBBS:

• Fast sample–to–result: Results can be obtained in a few minutes to hours
• Screening of viable/active agents: CBBS responds to only viable or active pathogens or toxins
• Multiplexing: CBBS can be formatted to detect multiple biohazards and even unknown or genetically-altered pathogens
• Limit of detection: CBBS can detect 100 or fewer cells or nanogram quantities of toxin
• Broad application: CBBS could be used as a first response to assess the nature of toxicants or threat agents to address food safety or food defense needs

Current status of cell-based assay systems
Our research group has developed a cell-based biosensor utilizing a mouse lymphocyte cell line, Ped-2E9, which is highly sensitive to a broad range of toxins: listeriolysin O (L. monocytogenes), enterotoxin (Bacillus spp.), α-hemolysin (Staphylococcus aureus), phospholipase C (Clostridium perfringens) and cytolysin from sea anemone (Stoichactis helianthus) at nanogram levels [13-15]. Toxins injure Ped-2E9 cell membranes to release alkaline phosphatase that is detected rapidly (within 1-6 h) [Figure 1]. In a more recent development, the Ped-2E9 cells have been embedded in collagen matrix to create a 3D scaffold in a multi-well plate for high throughput screening and detection of toxins at nanogram quantities and bacteria at 100-1000 cells/g/ml from food and beverages [13,16]. The scaffold maintains a predetermined porosity to permit selective permeation of the pathogen to the Ped-2E9 cells while keeping coarse food particles away from the cells. Advances in CBS include the use of engineered mammalian cells carrying specific receptors on their surface to respond to a specific pathogen or toxin [8,17,18].

Conclusion and future prospects
The functional biosensor is unique since it responds to active harmful biological agents but not to harmless or dead microbes or inactive toxins. Depending on the type of cell lines used, the specificity of CBBS can vary. Though CBBS development is still at an early stage, it shows great promise as a screening tool for specific pathogens or a broad range of biological agents, allowing food producers to make quick decision about product release.  CBBS can also be utilized by civil authorities or food inspection agencies to identify and prevent the spread of dangerous pathogens and toxins that may have been inadvertently or intentionally introduced into the food supply or into water sources. Knowledge about the type of toxin administered will enable the agencies to quickly make vital decisions regarding the counter measures that could potentially save hundreds of lives.

References
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The author
Professor Arun Bhunia
Professor of Food Microbiology
Molecular Food Microbiology Laboratory
Department of Food Science
Purdue University
West Lafayette, IN 47906-2009,
USA
e-mail: bhunia@purdue.edu