Abstract

Global food safety problems emerge one after another, and people's requirements for quality of life are constantly improving. At this time, it is very important to put forward a method that can quickly and directly detect pathogenic bacteria. We hope that this project can strengthen people's attention to food safety and reduce the tragedies caused by food safety. We used phage receptor binding protein (RBP)-nanomagnetic bead complexes (RBP-MBs) combined with paper-based sensors to isolate and colorimetrically detect four types of foodborne pathogenic bacteria simultaneously. Compared with the existing detection technology, it is quicker, simpler and more efficient in identifying multiple foodborne pathogens at one time.

1   Microbial pathogens

According to the statistics of the World Health Organization in 2020, 600 million people (almost one in every 10 people) in the world suffer from diseases due to eating contaminated food every year, and 420,000 people die, resulting in a loss of 33 million healthy life years (WHO, 2022). pathogenic Escherichia coli (E. coli, EC), Staphylococcus aureus (S. aureus, SA), Vibrio parahaemolyticus (V. parahaemolyticus, VP) and Salmonella enteritidis (S. enteritidis, SE) are four common foodborne bacterial pathogens.

1.1 Pathogenic E. coli

E. coli are mostly harmless bacteria that live in the intestines of people and animals and contribute to intestinal health. However, eating or drinking food or water contaminated with certain types of E. coli can cause mild to severe gastrointestinal illness. Some types of pathogenic (illness-causing) E. coli, such as Shiga toxin-producing E. coli (STEC), can be life-threatening. Different types of E. coli tend to contaminate different types of foods and water. Previous U.S. outbreaks of pathogenic E. coli have included leafy greens, sprouts, raw milk and cheeses, and raw beef and poultry.

Some wildlife, livestock, and humans are occasional carriers of EC and can contaminate meats and food crops. Ruminant animals such as cattle, goats, sheep, deer or elk, as well as other animals such as pigs or birds are known carriers of EC, such as STEC, and are often the pathway as to how STEC is introduced into the environment.

Figure1. Recent outbreaks of E. Coli (https://www.fda.gov/news-events/public-health-focus/e-coli-and-foodborne-illness)

1.2 S. aureus

S. aureus is the third most prevalent microbial pathogen for food-borne illnesses and accounts for about 25% of microbial food poisoning events (Wei, 2011). Enterotoxin produced by SA in food can cause Staphylococcal food poisoning (SFP). Symptoms of SFP include an acute onset of nausea, severe vomiting, abdominal pain, and diarrhea (Tong, 2015). SA has a certain tolerance to high temperature (up to 80℃) and high salt concentrations (up to 15% concentration of NaCl).

Figure 2   SEM image of Staphylococcus aureus taken from a vancomycin intermediate resistant culture (CDC/ Matthew J. Arduino, DRPHPhoto Credit:Janice Haney Carr, Public domain, via Wikimedia Commons)

1.3 V. parahaemolyticus

V. parahaemolyticus is a type of marine bacteria, mainly found in fish, shrimp, crab, shellfish, seaweed and other seafood. Raw seafood is a high-risk food for VP infection. The clinical manifestations of food poisoning caused by VP are acute onset symptoms, mainly including abdominal pain, vomiting, diarrhea, and even shock and death (Ghenem, 2017). During 1990–2019, VP was responsible for more than 40 global outbreaks (Pazhani, 2021). According to the statistics released by the China National Center for Food Safety Risk Assessment (CFSA), in recent years, VP has become the leading microbial pathogen causing foodborne diseases in China (Wu, 2014). In 2020, a survey in China showed that the prevalence of VP in fish and shrimp reached 14.9% in summer and 7.3% in winter (Li, 2020).

Figure 3 SEM image of Vibrio parahaemolyticus (Sathiyamoorthi, 2021)

1.4 S. enteritidis

S. enteritidis is the most prevalent strain of Salmonella and the predominant cause of foodborne Salmonella diseases (Shah, 2017). In the United States, SE accounts for about 32% of Salmonella outbreaks (Coveny, 2022).

Salmonella infections are common to humans and animals, and are mainly caused by eating contaminated food. According to statistics, among all bacterial food poisoning in the world, Salmonella poisoning often ranks first (CFSA, 2020). Salmonella is also one of the most common bacterial foodborne pathogens in the inland areas of China (Lin, 2016). According to the Centers for Disease Control and Prevention (CDC), 9 multistate outbreaks of Salmonella food poisoning were reported in the United States in the year of 2021, causing more than 1,200 people sick and 250 Americans hospitalized (News desk, 2022).  Typical symptoms of Salmonella infections include fever, nausea, vomiting, diarrhea and abdominal colic (Lin, 2016). Salmonella can survive for 15 minutes at 60 ℃, 2-3 weeks in water and 3-4 months in refrigerator (Hebei Institute of Food Inspection, 2016)

Figure 4 Colorized SEM of SE. Blue is growth medium. Picture is colored in false colors to illustrate difference. (Photo by Jean Guard, U.S. Department of Agriculture, Public domain, via Wikimedia Commons)

2    Current detection approaches for microbial pathogens

Pathogen testing is important to ensure the safety of food being taken and monitor the hygiene quality of food processing and shelf-life stability.

The golden standard method for food-borne pathogen detection is the conventional microbiological test based on bacteria culturing. It is reliable and accurate, but it takes heavy workload and needs 2-3 days to get results and another 7-10 days for confirmation (Adzitey, 2013).

Figure 5  Representative culture media for Salmonella Typhi strains on: (a) XLD agar; (b) SS agar (Salman, 2021)

Immunodiagnostics such as Enzyme-Linked Immune Sorbent Assay (ELISA) uses antibodies to specifically identify pathogens. It has high specificity and sensitivity, and can be used in large scale. Though it is relatively fast compared with the conventional culturing tests, it is still not in “real-time”. In addition, low sensitivity and affinity of the antibody to the pathogens can occur and contaminants may interfere with the results (Umesha, 2018).

Polymerase chain reaction (PCR) technology targets nucleic acid like DNA of the pathogens. PCR is sensitive, specific, accurate, and can sense low level DNA in the sample over the above two conventional methods. PCR takes hours to over a day in processing. The reaction specificity is affected by conditions such as magnesium concentration and cross contamination. Multiplex PCR (mPCR) and broad-range PCR assays are developed based on PCR to simultaneously detect multiple pathogens. Since they are multitasking, they can save time and labor work compared with PCR when testing for several pathogens (Umesha, 2018).

Metabolomic approaches, such as liquid chromatography mass spectrometry (LCMS), gas chromatography mass spectrometry (GC-MS), matrix assisted laser desorption/ionization tandem time of flight mass spectrometry (MALDI-TOF-MS) and nuclear magnetic resonance (NMR), are used to detect the metabolites of pathogens. These methods are currently used more in pathogenic fungi than bacteria (Oyedeji, 2021). The cost for equipment and maintenance are high for these methods and the processing is time-consuming.

Due to the complexity of the testing methods above, they are incapable to give results in real time, require laboratory equipment and professional staff together with laboratory management and quality control measures. Testing costs are not cheap.

ATP bioluminescence technology is a fast (within minutes), portable and convenient tool commonly used to analyze the overall quantity of microorganisms in food via ATP detection in cells. But it has the disadvantages of low sensitivity and inability to detect specific strains. In addition, the results are affected a lot by factors like temperature change (Sun, 2022).

3    Aim of our project

Our project aims to develop a sensitive and rapid detection tool for four microbial pathogens, S. aureus (SA), V. parahaemolyticus (VP), pathogenic E. coli (EC), and S. enteritidis (SE) in food. Compared with other available approaches, our tool is sensitive, rapid, cheap, portable and easy to handle, with no requirements for devices, laboratory setup, and professional personnel. Therefore, our tool can be widely used in various conditions such as household settings and is especially friendly in remote areas with poor economic condition and laboratory resources.

4    Overall design of our tool

Our detection tool consists two major components.

• Four receptor binding protein (RBP)-nanomagnetic bead complexes (RBP-MBs): The RBP side of the complexes can bind specifically to SA, VP, EC, and SE, respectively and the nanomagnetic bead side can be attracted by magnets. The complexes can isolate the four strains of interest from samples rapidly and easily while maintaining the viability of the bacteria.
• Paper-based sensors: The sensors contain bacterial cell lysis reagent and four different chromogenic substrates. It shows different color signals when in contact with the pathogens respectively.

Figure 6 Overall design of our detection tool for SA, VP, EC and SE strains in food (images from https://pixabay.com)

To develop our tool, we used three steps: Construction of plasmid vectors for RBP production, formation of RBP-nanomagnetic bead complexes and development of paper-based sensors.

5    Components of our tool

5.1 RBPs

RBPs are special tail proteins of bacteriophages. The phage-specific recognition of its host bacteria is realized by RBPs. Different RBPs can specifically bind to different bacteria strains. RBPs have been used to develop various diagnostic tools for its efficiency and sensitivity in identifying bacterial pathogens.

In our project, four RBPs, gp13, gp15, CBD(C-terminal cell-binding domain), and TSP (tailspike protein), are initially selected to detect EHEC O157:H7（a typical strain of pathogenic E. coli）, VP, SA, and SE, respectively, due to their high specificity in binding with the corresponding bacteria strains. After expert advice, our project abandoned the EHEC O157:H7 strain for strain testing for safety concerns, and replaced the relatively safe E.coli strain K12 MG1655 before entering the laboratory for a formal experiment.

gp13 is a putative RBP within the genome of Phage EP335. Researches have demonstrated that GFP-gp13 bind strongly and evenly to the cell surfaces of E. coli O157 strains prone to EP335 infection (Witte, 2021). Because we changed the EC strain into K12 MG1655, we replaced gp13 with a more broad-spectrum RBP protein TFP (tail fiber protein) that can bind to multiple pathogenic E.coli strains.

Figure 7 Fluorescence and phase contrast images of GFP-gp13 cell binding to different E. coli O157 strains such as TW01286, 396, 999/1 and 777/1. Scale bars represent 5 mm (Witte, 2021).

TFP is a receptor binding protein (RBP) from the genome of Escherichia coli (EC) phage T7. Researches have demonstrated that GFP-TFP can bind to the cell surfaces of several E. coli strains such as the pathogenic E.coli O157 and K12 MG1655.

gp15 is an RBP found in the genome of the vB_VpaP_GHSM17 phage. vB_VpaP_GHSM17 is a recently isolated phage that infects VP (Liang, 2022). gp15 is a protein of the phage that can specifically identify VP.

CBD is the C-terminal part of the endolysins from gram-positive phages. Studies have shown that CBDs of the bacteriophages adhere to the bacterial cell wall and grant endolysins access into the bacteria (Stone, 2019). CBDs can bind specifically with target bacteria with great affinity, and can be easily manufactured in an E. coli expression system. Therefore, CBDs are widely investigated in biosensor development for bacteria detection. The CBD used in this project is from the genome of the phage P108 which infects SA. It has showed broad-spectrum detecting capability toward SA strains (Wang, 2020).

Figure 8 Characterization and purification of endolysin LysP108 derived from phage P108 of SA strain XN108. (A) Schematic illustration of structure of phage endolysin LysP108. (B) 3D structure of endolysin protein LysP108. (Lu, 2021)

Tailspike protein (TSP, also named as gp9) is an RBP from the genome of bacteriophage Salmonella P22. TSP is used by the P22 phage to attach to the lipopolysaccharides of its target bacteria (Seul,2014). TSPs play an important role in the initial stage of phage P22’s infection of Salmonella enterica serovar Typhimurium, which help in the formation of a channel to translocate viral genome into the cytoplasm (Wang, 2019).

Figure 9 Phage P22 binds obliquely to the cell surface of Salmonella enterica serovar Typhimurium. PG: peptidoglycan cell wall; OM: outer membrane of the bacteria; gp9: TSP (Wang, 2019).

We used the pET28a plasmid as the expression vector for the RBPs. And we constructed plasmid vectors of pET28a-TFP, pET28a-gp15, pET28a-CBD, and pET28a-TSP via seamless cloning to produce TFP, gp15, CBD and TSP proteins, respectively. To verify the binding ability of these proteins with the target bacteria, plasmid vectors of pET28a-GFP-TFP, pET28a-GFP-gp15, pET28a-GFP-CBD and pET28a-GFP-TSP are constructed to express green fluorescent protein (GFP) fusion proteins of TFP, gp15, CBD and TSP. Under the confocal laser scanning microscope, green fluorescence on the cell surface of the target bacteria after incubation with GFP fusion proteins indicates specific binding of the proteins to viable bacteria.

Figure 10 Plasmid vectors of pET28a-TFP and pET28a-GFP-TFP

In our project, we chose RBP(TFP, gp15, CBD, TSP) from several phages to accurately capture the specific target bacteria (EC,VP,SA,SE).Through the construction of GFP-RBPs (GFP-TFP, GFP-gp15, GFP-CBD, and GFP-TSP), the four RBPs were proven to be effective in our experiment. RBPs were constructed as part number BBa_K4430000~BBa_K4430003,and GFP-RBPs were constructed as part number BBa_K4430005~BBa_K4430008.

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5.2 RBP-nanomagnetic bead complexes

Carboxyl magnetic beads (MBs) contain carboxyl functional groups on their surface and are superparamagnetic nanomagnetic beads. These beads can be covalently conjugated with primary amine from proteins, nucleic acids and other molecules via stable amide bond. The conjugated nanomagnetic bead complex has magnetic property and can be separated by magnets.

In our project, the RBPs, TFP, gp15, CBD and TSP, produced by the plasmid vectors are conjugated to the nanomagnetic beads and four types of RBP-nanomagnetic bead complexes (RBP-MBs) are formed. The RBP side of an RBP-MB binds specifically to the target bacteria and the MB side can be attracted by a magnet. Therefore, in a solution with the target bacteria, the bacteria can be screened and captured by the RBP-MBs and can be isolated via magnetic separation. Throughout the process, the purified bacteria remain viable, and thus can be used for further testing requirements of various kinds such as tests for drug resistance and genetic information.

Figure 11 Illustration of magnetic isolation of analyte using nanomagnetic beads

5.3 Paper-based sensors

Paper-based sensors (e. g. pH values and pregnancy test paper) are currently the most promising sensors in the chemical/bioanalysis-related fields, as analysts prefer to perform field and real-time /visual detection without the help of analytical instruments. Sensors built with paper as a substrate are low-cost and flexible with short response time. Moreover, they are biodegradable and suitable for mass deployment in resource-limited areas and can be easily used by unskilled operators. Paper is also a great medium for immobilization and trapping and, in some cases, for binding with biomolecules. Its porous structure with large connected pores composed of cellulose fibers allows transporting liquid by means of capillary forces that result in short response time. The porous structure of paper also allows any nano- and microparticles to remain immobilized in the paper structure. Paper can be functionalized with certain materials such as nitrocellulose paper used for immobilizing nucleic acids for selective sensing. Paper-based potentiometric sensors are reported for detecting many ions and proteins. Paper-based pathogen and virus sensors are also easy to incinerate. They can be used as one-time-use front end in sensor systems that can be peeled off and replaced (Dolai, 2020）.

Figure 12 Office paper platform for bioelectrochromic detection of electrochemically active bacteria using tungsten trioxide nanoprobes (Marques, 2015)

Our team designed paper-based sensors that can show different visual signals for a solution sample containing EC, VP, SA, and SE. To develop the sensor, we prepared a reaction system with the optimal reaction concentrations of a lysing agent and one of the four chromogenic substrates, chlorophenol red-β-D-galactopyranoside (CPRG), X-β-glu, 4-nitrophenyl β-D-glucopyanoside (pNPG), and magenta caprylate on a paper-based carrier. When drops of resuspension buffer of the target bacteria purified by RBP-MBs are added to the sensor, bacteria are lysed and enzymes within the cells are released into the system: β-galactosidase (β-gal) released from EC, β-glucosidase (β-glu) from VP, α-glucosidase (α-glu) from SA, and esterase from SE, respectively. The products of the catalytic reactions of the chromogenic substrates by these enzymes give different visual signals that can be detected by naked eye.

5.4 Tools used for color detection of the paper-based sensor

Snipaste, a software application from Windows system for screen capturing, and the screen capture tool from Wechat are both appropriate tools for the analysis of visual signals of the paper-based sensors. They can list out RGB (red, green, blue) color values, which distinguish subtle color differences that naked eyes can’t detect. These tools make our paper-based sensors more sensitive in detecting low concentrations of target bacteria. In order to precisely analyze the color signals of our paper-based sensors, our modeling group developed a software called Image Colorimetric Detection (ICD) with the help of the research team from South China University of Technology. Bacteria concentration can be calculated by the data given by ICD.

6 Application of our tool

Canned foods and meat products are the two most common food groups that cause foodborne illnesses. Main reasons of microbial contamination of canned foods include lax quality control of production process (such as poor sealing, failed sterilization, and improper cleaning process of equipment), low acidity of canned fruit, prolonged storage of opened food, large temperature fluctuations, and microbial contamination of raw and auxiliary materials (China Canned Food Industry Association, 2022). Meat is rich in nutrients, such as sugar, protein and water, which are needed for microbial growth (Nan，2004). Canned meat foods are especially vulnerable for bacterial contamination. Our product can be used to detect EC, VP, SA and SE in canned meat foods.

Figure 13 A meat can (Ll1324, CC0, via Wikimedia Commons)

To apply our tool for detection of pathogens in canned foods, a food sample is added into a tube with buffer. RBP-MBs are added. After 30-minute incubation at 37℃, magnets are applied outside the bottom of the tube for 30s to isolate the target bacteria. The supernatant is removed and the purified viable target bacteria are resuspended. A drop of the resuspension solution is added on to a paper-based sensor and a visual signal can be detected by naked eye. The purified bacteria can also be used for further tests of various kinds if necessary.

Compared with other methods, our product has several advantages. First of all, it is very accurate to detect the target bacteria strains because of the high selectivity of bacteriophage RBPs. It is also a rapid method with the whole process in less than 1 hour (Li, 2010). Moreover, it is very cheap and portable since the tool only uses tubes, magnets, reagents and a paper-based sensor. In addition, the procedure is easy and convenient to perform, so no professional staff is required and the training for its usage is simple. This tool can be used in all regions including remote areas with poor economic condition and laboratory setup and by ordinary non-professional personnel. Therefore, our product can be applied widely and cheaply for potentially contaminated food and helps to reduce the incidence of foodborne diseases in all regions.