Modernization of Control of Pathogenic Micro-Organisms in the Food-Chain Requires a Durable Role for Immunoaffinity-Based Detection Methodology-A Review

Abstract

Food microbiology is deluged by a vastly growing plethora of analytical methods. This review endeavors to color the context into which methodology has to fit and underlines the importance of sampling and sample treatment. The context is that the highest risk of food contamination is through the animal and human fecal route with a majority of foodborne infections originating from sources in mass and domestic kitchens at the end of the food-chain. Containment requires easy-to-use, failsafe, single-use tests giving an overall risk score in situ. Conversely, progressive food-safety systems are relying increasingly on early assessment of batches and groups involving risk-based sampling, monitoring environment and herd/flock health status, and (historic) food-chain information. Accordingly, responsible field laboratories prefer specificity, multi-analyte, and high-throughput procedures. Under certain etiological and epidemiological circumstances, indirect antigen immunoaffinity assays outperform the diagnostic sensitivity and diagnostic specificity of e.g., nucleic acid sequence-based assays. The current bulk of testing involves therefore ante- and post-mortem probing of humoral response to several pathogens. In this review, the inclusion of immunoglobulins against additional invasive micro-organisms indicating the level of hygiene and ergo public health risks in tests is advocated. Immunomagnetic separation, immunochromatography, immunosensor, microsphere array, lab-on-a-chip/disc platforms increasingly in combination with nanotechnologies, are discussed. The heuristic development of portable and ambulant microfluidic devices is intriguing and promising. Tant pis, many new platforms seem unattainable as the industry standard. Comparability of results with those of reference methods hinders the implementation of new technologies. Whatever the scientific and technological excellence and incentives, the decision-maker determines this implementation after weighing mainly costs and business risks.

Keywords: food microbiology; immunoaffinity assays; immunoagglutination; immunochromatographic testing; immunomagnetic separation; immunosensors; one health; pathogenic micro-organisms; responsive monitoring; review.

Figure 1

Food and water-borne outbreaks, i.e., cases in which ≥2 persons fell ill from the same food and the same agent, per causative pathogen in 2018 in the EU and associated countries. The explanation of “other” relevant for this paper is as follows. Other bacteria include Aeromonas hydrophila, (enteroinvasive (EIEC) or enterotoxigenic (ETEC)) Escherichia coli, Enterococcus, Leptospira spp., Shigella spp., Shigella flexneri, Shigella sonnei, Yersinia enterocolitica, and unspecified bacteria. Other viruses include adenovirus, flavivirus, hepatitis E virus, rotavirus, and unspecified viruses. Other parasites include Giardia intestinalis (lamblia), Giardia spp., Taenia saginata, and unspecified parasites. Figure courtesy of EFSA from [18].

Figure 2

When, what, and how to screen in the animal food production chain (dark blue color) to secure food safety with respect to microbial hazards. Hazard critical point analysis will reveal optimal sampling moments. The sectors (purple color) which determine the type of sampling (red color) and testing (green and light blue colors) possible are indicated. The primary sector includes the pre-harvest stage until slaughter and comprises reproduction, egg and milk, fattening, transport, and slaughter phases. The secondary sector includes all food-processing steps converting milk, eggs, and meat into complex products. The tertiary and quaternary sectors include wholesale, street vendors, catering, institutional kitchens, and finally private kitchens and (domestic) consumption.

Figure 3

A simplified overview of “handles” of a Gram-negative bacterium analytically available to find a bacterial cell among all other food components. Flagella, H-antigen, are only present on motile cells and when present, it can be a single flagellum or multiple flagella organized mono-/lopho-/amphi-/peritrichously [57]. Lipopolysaccharides, O-antigens, form the outside of the outer membrane of the bacterial cell. Pili or fimbriae, F-antigens, are of little relevance to food microbiologists. Capsular polysaccharides (CPS), K-antigen, can be formed in both Gram-negative and Gram-positive bacteria. A special subtype of the K-antigen is the Vi-antigen in Salmonella. The activity of excreted enzymes is partly specific and used as a marker to identify a bacterium. Analysis of excreted toxins can also be used to trace and identify a pathogenic bacterial cell. The depicted lists of enzymes and toxins are not exhaustive.

Figure 4

Impression of analytical methods and approaches to find and determine (pathogenic) micro-organisms in the food-chain. It should be noted that electrochemical immunosensors exploit many different detection principles, including amperometry, impedimetry, field-effect transistor (FET), potentiometry. This overview is far from complete. ATP, adenosine triphosphate; AgBP, antigen-binding proteins (not immunoglobulin related); CPA, cross-priming amplification; EIA, enzyme immunoassay; ELFA, enzyme-linked immunofluorescent assay; ELISA, enzyme-linked immunosorbent assay; ESI, electro-spray ionization; Ig, immunoglobulin; IR, infra-red; GC, gas-chromatography; ICT, immunochromatographic test; (RT-)LAMP, (reverse transcription) loop-mediated isothermal amplification; LFIA, lateral flow immunoassays; MALDI-TOF, matrix-assisted laser-desorption ionization time-of-flight; MS, mass-spectrometry; MS ⁿ, multi-stage MS; NASBA, nucleic acid sequence-based amplification; NMR, nuclear magnetic resonance; qPCR, quantitative (real-time) polymerase chain reaction; RCA, rolling circle amplification; rec. binding fragments, parts of immunoglobulins obtained by recombinant DNA protein engineering; RPA, recombinase polymerase amplification; rt-PCR, reverse transcription-polymerase chain reaction; SDA, strand displacement amplification; SERS, surface-enhanced Raman-spectroscopy.

Figure 5

Illustration of an example of a bead or microsphere array used in BBA assays. In the left panel beads are discriminatory in two dimensions: a gradient of an internal color (Y-axis) and diameter of the microsphere (X-axis). Each bead is thus identifiable and can be coated distinctively (right panel). In this case, one of the beads picked from the series in the left panel, is carrying an antigen (black-colored rod shape) to which an immunoglobulin from the analytical matrix (grey-colored Y-shaped structure) is specifically bound. To enable detection of the sample-derived and captured antibody in a flow-cytometer, a secondary, labeled binder is allowed to attach (dark-red-colored Y-shaped structure with reddish orb). The assay is usually developed in Microtiter trays. In the instrument, bead-size, bead fluorescence and label fluorescence of each bead in the suspension are measured and reported. Note that many variations on this test principle are deployed.

Figure 6

Number of publications per annum returned by PubMed when using search terms “biosensor” and “bacteria” [181].