Evolution in Food Pathogen detection: From culture media to Universal detection systems of multiple pathogens

It has been a long way from the traditional culture-based methods to the current objective of detecting multiple pathogens in a sample without the need of culturing the food samples.

 

Culturing methods

Traditional culture-based methods for detecting pathogens in food are based on the growth of viable bacteria in nutrient media. A general procedure includes homogenizing a food sample, enriching viable pathogens if the concentration of pathogens is low, and then separating the target pathogen in a selective medium followed by biochemical tests to ensure the target’s presence. Finally, sub-typing to identify specific targets.  

Culturing methods represent the first choice for many food testing laboratories because they are sensitive, inexpensive, and easy to use. Culture-based detection methods tend to be laborious, time-consuming, and slow to provide results. Such methods include unique enrichment methods, chromogenic plates, and film-based methods.

Immunological Methods

Immunological methods Include enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay, and immune separation methods such as immunomagnetic separation (IMS). For Immunological methods to work well, the absence of interfering molecules in the samples, such as non-targeted cells, DNA, or proteins, is required.

 

Immunoassays are based on a quantitative reaction of an antigen with its antibody. Therefore, they are suited for detecting microorganisms based on their production of specific antigens and for quantitative detection of bacterial toxins. The sensitivity and specificity of immunoassays are mainly determined by the antiserum used. The use of well-selected monoclonal antibodies can be advantageous.  Most immunoassays require a step or two of pre-enrichment.

 

Molecular methods

Molecular Detection Methods (nucleic acid-based methods) include simple polymerase chain reaction (PCR), multiplex PCR, real-time PCR (qPCR), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), and oligonucleotide DNA microarray.

 Nucleic acid‐based detection methods are faster than traditional methods, are specific, reproducible, and only require small amounts of target DNA. Nucleic acid-based methods are performed by detecting specific DNA or RNA sequences of the target pathogenic organism.

 

Polymerase chain reaction (PCR) is the most commonly used nucleic acid method for detecting pathogenic microorganisms. Many different advances of the original PCR methodology have been described over the last two decades. qPCR and isothermal amplification techniques, including LAMP and NASBA, are essential improvements in detecting pathogens. 

 

Messenger RNA (mRNA) is regarded as a better indicator than DNA to assure viability. Since mRNA is only present in metabolically active cells. Reverse transcription-PCR (RT-PCR) is one of the RNA-based most used molecular techniques.

 

Phage based methods

Phage (bacteriophages) presents a potential tool usable for bacterial detection. Phage can recognize their host and infect their target cells with an extraordinary specificity, and is usable with various rapid detection formats.

 

Most phage-based tests employ lytic phages as lysing agents, and detection of the new progeny phages released from target bacterial cells indicates cell viability. Phages are used to detect various pathogens such as Salmonella Typhimurium, Salmonella Enteritidis, Listeria monocytogenes, Escherichia coli 0157:H7, and Staphylococcus aureus.

 

A further step such as PCR might be added to gain specificity and identity of the organism. qPCR is one of the most promising options, yielding detection of pathogens in 8 hours. Combining phage amplification and lysis with PCR/qPCR, immunoassay, or enzyme assay approaches appears to provide a rapid alternative to cultural methods for detecting pathogens in food.

 

Biosensor devices

 A Biosensor is a device that consists of two main elements: a bioreceptor and a transducer. The bioreceptor is responsible for recognizing the target analyte. It can either be a biological material (enzymes, antibodies, nucleic acids, and cell receptors), biologically derived material, or Biomimic (imprinted polymers and synthetic catalysts). The transducer can convert the biological interactions into a measurable electrical signal that can be optical, electrochemical, mass-based, thermometric, micromechanical, or magnetic.

 

Biosensors are rapid, sensitive, and low‐cost detection devices that are designed for simple operation. Different sensing elements (transducer element) and configurations of biosensors have been reported in recent years for the direct detection of foodborne pathogens. Although much work still needs to be done to ensure their reliability, stability of biomaterials, and compatibility to in‐field tests, they are potential alternatives to conventional methods. 

 

Culture-independent methods

Several methods to detect pathogens that do not include bacterial pre-enrichment have been recently reported. The assays include molecular and phage-based methods. (Wang and JK Salazar Culture‐Independent Rapid Detection Methods for Bacterial Pathogens and Toxins in Food Matrices, Y, in Comprehensive Reviews in Food Science and Food Safety Volume15, Issue1, January 2016, Pages 183-205.

The figure compares culture‐independent detection (left) (including steps of centrifugation, filtration, and immunomagnetic separation (IMS)) to a conventional culture‐based method (right).

With the development of techniques with higher sensitivity, direct detection of low levels of foodborne pathogens in food samples that no longer require enrichment has become available. In addition, detection techniques based on noncultural methods for effective separation, concentration, and purification of pathogens significantly reduce the overall testing time. 

 

Detection of multiple pathogens in a single assay- universal detection

There is a large diversity of microbial pathogens; therefore, the development of a universal detection methodology is faced with many challenges. However, it is the ultimate goal in pathogen detection since it eliminates the need to know the particular pathogen, and if multiple pathogens are present, all will detect.

 

Several studies have been conducted to detect multiple pathogens in a sample with a single assay. Below are two examples. 

 

Dr. Dele Ogunremi and his team applied genomics and bioinformatics to develop a universal detection methodology for microbial organisms in food. The application of genomics and bioinformatics has led to the development of a unified protocol consisting of the following steps:

1. General amplification of the DNA of the metagenome derived from a food sample

2. Nanopore sequencing of amplified DNA   

 

3. Optimized bioinformatics detection of targets using the CosmosID proprietary technology.  

Ogunremi and his team inoculated lettuce (25 grams) with bacteria, viruses, and/or parasites at levels of 1 colony-forming unit of Salmonella, 5 oocysts of Cryptosporidium parvum, and 500 plaque-forming units of Hepatitis A virus.

 

The team used spiked food samples blindly submitted to the testing laboratory and naturally contaminated food samples that were the subjects of a regulatory food recall. Various combinations of bacteria, viruses, and parasites were tested successfully. They also were successful in detecting Salmonella in recalled foods.

 

 Ma et al. (Foods 2020, 9(3), 278) used recombinase polymerase amplification (RPA) combined with a lateral flow dipstick (LFD) to detect simultaneous Staphylococcus aureus, Vibrio parahaemolyticus, and Salmonella Enteritidis. The detection process, including amplification and reading, was finished in 15 min at 37 °C. The detection limits were 2.6 × 10¹ CFU/mL for Staphylococcus aureus, 7.6 × 10¹ CFU/mL for Vibrio parahaemolyticus, and 1.29 × 10¹ CFU/mL for Salmonella Enteritidis.

The multiplex LFD-RPA assay saves time, simple, sensitive, and specific, allowing visual and measurable analysis of three fragments in a single reaction. The assay can be applied in the field since the amplification could be completed in 10 min at 37 °C and could be monitored by the naked eye. The application of a test strip reader decreased the man-made influence and allowed quantification detection. It improved the reliability of the diagnosis. In summary, the multiplex LFD-RPA has practical significance for the rapid detection of foodborne pathogens in the field.

 

Final thought

 When I started to work in microbiology, traditional methods for pathogen detection in foods were all culture-based, slow, and labor-intensive. During my carrier, I managed to watch the evolution of pathogen detection methods from 100% plates and tubes to dominantly novel methods. It is hard to believe how much progress was made in rapid advanced methodology in this field. Many of the current methods are more versatile, easier to use, and yield much faster results. 

 

There is always a need for faster, more accurate, and easier-to-use methodologies that can be performed anywhere.  It will be interesting to observe where the pathogen test will bring us in the next decade.