Recent Advances in the Detection of Listeria monocytogenes

Abstract

Listeria monocytogenes is the third-most severe pathogen causing a yearly outbreak of food poisoning in the world that proliferates widely in the environment. Infants, pregnant mothers, and immuno-compromised people are at high risk. Its ability to grow in both biotic and abiotic environments leads to epidemics that infect 5 out of 10 people annually. Because of the epithelial adhesion (by E-cadherin binding), it can suppress immune cells and thrive in the gastrointestinal tract till the brain through blood flow (E-cadherin). Microbial culture is still used as a gold standard, but takes a long time and often yields false positive results due to incompetence and temperature variations. Therefore, in order to treat it rather than using broad spectrum antibiotics, a standardized time-saving and highly specific technology for early detection is very important. It has been observed that the production of a particular antibody is delaying (so does the detection process) as a result of the inadequate understanding of the pathophysiology of the bacteria. This book chapter provides a brief summary of a pathogen as well as the scientific advances that led to its identification more easily.

1. Introduction

Listeria monocytogenes is a gram-positive, rod-shaped, facultative anaerobic ubiquitous bacterium consisting of a mucopolysaccharide capsule, and has a low G + C content [1, 2, 3, 4, 5, 6, 7, 8]. It is motile between 10 and 25°C and measures about 0.4–1.5 μm in length [8], it does not produce spores. The genus Listeria includes six species: L. monocytogenes, L. ivanovii, L. seeligeri, L. innocua, L. welshimeri, and L. grayi. Among them, L. monocytogenes (frequently available), L. ivanovii (often), and L. seeligeri (rare) are potentially pathogenic [2, 6, 8, 9]. Consuming contaminated foods such as unpasteurized dairy products, raw meats, frozen foods, pre-packaged foods, environmental factors, sporadic cases of listeriosis, and outbreaks of the disease are the main causes of L. monocytogenes infection [1, 3, 7, 8, 9, 10, 11, 12, 13, 14]. L. monocytogenes infection can cause septicaemia, meningitis, encephalitis, spontaneous abortion, fever, and self-limiting gastroenteritis in immune-compromised conditions [1, 3, 6, 7, 8, 11, 13, 15, 16]. It is commonly accepted that bacteria, after passing the intestinal barrier, travel through the blood and lymph to the liver, where they reproduce in hepatocytes, as well as the spleen. After that, bacteria can spread via the bloodstream and reach the brain and placenta (Figure 1).

Figure 1.

Route of infection of Bacteria.

To detect it and reduce the growth of Listeria various immuno-based and molecular techniques has been developed and modified. Modifications were made according to the report published by researcher in different time intervals. Starting from the gold standard culture, ELISA, PCR, NMR, NGS to biosensor was used. Though there is high chance of false positive result, culture is given the preferences to carry forward other techniques to justify the research. The purpose of this book chapter was to give a quick overview of L. monocytogenes and the advancement in available detection techniques. From culture based technique, immuno-based technique, molecular technique till sensor development had been discovered by different scientists in different time intervals and their advancements had been made as per the understanding of pathophysiology and virulence factor taking part in infection.

2. History and epidemiology

The Judicial Commission on Bacteriological Nomenclature and Taxonomy approved its use in 1940 and gave it the generic name “Listeria” in the sixth edition of Bergey’s Manual of Determinative Bacteriology whereas Murray et al. proposed the name “monocytogenes” for the species, indicating that infected mono-gastric animals have a high concentration of monocytes in their peripheral blood [4]. Though the first culture was done from a meningitis patient in France in 1921; the official human infection was reported by Nyfeldt from Denmark in 1929 [8, 17]. In 1979 contaminated vegetables affected 23 patients admitted to Boston hospital, in 1981 consumption of contaminated coleslaw affect numerous people. Contaminated milk (1983), contaminated manure used to grow cabbage in 1989, similar cases were observed till 1990s. In 2000 consumption of raw food and in 2001 consumption of contaminated hot dog resulted in 21% mortality [7]. 333 Food recalls made by US, Department of Agriculture (USDA) with Food and Drug Administration from 2002 to 2006 revealed that 32.4% of the most leading cases are from Listeria monocytogenes [18]. Study carried out by European Center for Diseases Prevention and Control together with European Food and Safety Authority in 2004–-2006, revealed that most Listeria monocytogenes positive cases found in meat and fish products [19]. Another outbreak was reported from CANADA in 2008 where 57 confirmed cases and 22 deaths that were due to the consumption of delicatessen meat [20, 21]. In 2010, 23.6% deaths had observed as stated by survey carried out by world health Organization [17], besides 78% were hospitalized with no deaths whereas in 2011 66% died, in 2013 17% died which if counted on average was 18% from the time range of 1998–2016. In 2018, 2 people died due to listeriosis [22]. According to the Centre for Disease Control and Prevention (CDC) it was estimated that 1600 people get listeriosis each year, and about 260 die. The recent outbreak as of June 8, 2022 reported from Florida was associated with contaminated ice-cream [23]. And presently it is no longer only a problem to humans but also to animals and various food items were confirmed to transmit this pathogen [24]. To summarize the outbreaks of different time intervals after 2008 have been summarized in Table 1.

Serial no.	Source	Year and cases	Contaminated Brand	References
1	Cantaloupes	2011, out of 147 persons infected, 33 deaths and 1 miscarriage.	NA	CDC [23]
2	Ricotta Salata Cheese	2012, out of 22 infected, 4 death, and 1 miscarriage.	Frescolina Marte brand of Italy	CDC [23]
3	Cheese	2013, out of 6 infected, 1 miscarriage and 1 death	Les Frères, Petit Frère, and Petit Frère with Truffles cheeses made by Crave Brothers Farmstead Cheese Company of Waterloo, Wisconsin, U.S..A	CDC [23]
4	Dairy Product	2014, 8 infected among them 5 were pregnant and 1 death	Roos Foods, Kenton, Delaware, U.S.A	CDC [23]
5	Cheese	2014, out of 5 infected, 1 death and 1 transmitted to new born and 3 cases of pregnancy.	Oasis Brand, Inc. U.S.A	CDC [23]
6	Bean Sprout	2014, out of 5 infected and 2 deaths.	Wholesome Soy Products, Inc. Sprouts of Chicago	CDC [23]
7	Commercial Apple Caramel	2014, out of 35 infected 1 miscarriage, 11 face problem during pregnancy and 3 suffered from meningitis.	Bidart Bros. of Bakersfield, California, Happy Apples, California Snack Foods and Merb’s Candies	CDC [23]
8	Ice cream	2015, out of 10 infected, 3 death and 10 hospitalized.	Blue Bell Creameries Ice Cream	CDC [23]
9	Soft Cheese	2015, out of 30 infected, 3 death, 1 miscarriage and 28 hospitalized	Central Valley Cheese, Inc. Turlock, California. Karoun Diaries, Inc.	CDC [23]
10	Frozen vegetables	2016, out of 9 infected 3 death	CRF Frozen Foods of Pasco, Washington	CDC [23]
11	Raw Milk	2016, out of 2 cases reported, 1 death	Miller’s Organic Farm in Bird-In-Hand, Pennsylvania	CDC [23]
12	Packaged Salads	2016, out of 19 infected, 1 death and 19 hospitalized	Dole Ohio Facility	CDC [23]
13	Vulto Creamery Soft Raw Milk Cheese	2017, out of 8 infected 2 death and 8 hospitalized where 1 was newly born..	Vulto Creamery of Walton, New York	CDC [23]
14	Deli Ham	2018, out of 4 infected, 1 death and 4 hospitalized.	Johnston County Hams, Inc. in Smithfield, North Carolina	CDC [23]
15	Pork products	2018, out of 4 cases, 4 hospitalized with no deaths	C. Corporation of Houston, Texas, Long Phung Food Products	CDC [23]
16	Deli sliced meats and cheese	2019, out of 10 infected, 1 death and 10 hospitalized.	Deli Sliced products	CDC [23]
17	Hard boiled eggs	2019, out of 8 infected, 1 death and 5 hospitalized.	NA	CDC [23]
18	Enoki Mushrooms	2020, out of 36 infected, 4 death and 31 hospitalized.	H and C Food, Inc. Guan’s Mushroom Co. And Sun Hong Food. Inc.	CDC [23]
19	Deli Meats	2020, out of 12 infected, 1 death and 12 hospitalized.	Due to Italian style meat like salami, mortadella and prosciutto but supplier was not identified	CDC [23]
20	Queso Fresco	2021, out of 13 infected, 1 death and 12 hospitalized.	EI Abuelito Cheese Inc.	CDC [23]
21	Fully cooked chickens	2021, out of 3 infected, 1 death and 3 hospitalized.	Tyson Foods Inc., Jet’s Pizza, Casey’s General Store, Marco’s Pizza, Little Caesars, and Circle K.	CDC [23]
22	Fresh Express Packaged Salads	2021, out of 10 infected, 1 death and 10 hospitalized.	Fresh Express, U.S.A	CDC [23]
23	Dole Packaged Salads	2021, out of 18 infected, 3 death and 16 hospitalized.	Dole	CDC [23]
24	Ice Cream	2022, Out of 23 infect3ed 1 death and 22 hospitalized.	Big Olaf Creamery Ice Cream	CDC [23]

Table 1.

Summarized table for the outbreaks of Listeria monocytogenes occurring from 2011 to 2022.

3. Classification

It is classified on the basis of its character devised on culture media in different time intervals by different scientists and shown in Table 2.

Serial number	Classification	References
Kingdom	Bacteria, Cavalier-Smith, 2002-bacteries, bacteria, bacterias	[25]
Subkingdom	Postbacterial, Cavaler-Smith, 2002
Phylum	Firmicutes corrig/Baccillota, Gibbons and Murray, 1978
Class	Bacili, Ludwig et al.,2010
Order	Bacillales, Prevot, 1953
Family	Listeriaceae, Ludwig et al., 2010
Genus	Listeria, Pirie, 1940
Species	L. monocytogenes (Murray et al., 1926) Pirie, 1940)

Table 2.

Classification of Listeria monocytogenes.

4. Serotyping

L. monocytogenes has thirteen distinct serotypes. The majority of sporadic cases of listeriosis are tied to just three L. monocytogenes serotypes: 1/2a, 1/2b, and 4b, whereas serotype 4b is linked to nearly all outbreaks [1, 3]. While serovars 1/2a, 1/2b, and 4b appear to be similarly dispersed in Canada and the United States, serovars 4b predominate throughout the majority of Europe [16]. Several molecular subtyping methods were used.

5. Virulence factor

The haemolysin (hly) gene was the first virulence factor discovered in Listeria’s cellular structure, providing new insight into the intracellular and host-pathogen interactions during listeriosis [8, 16]. With the help of internalin A expressed on the cell surface of the bacteria, and epithelial cadherin (E-cadherin) expressed on the surface of epithelial cells [26, 27], L. monocytogenes is able to adhere to the host cell, evade immune response, and spread by penetrating the epithelial layers of the inner lining of the digestive tract [1, 28]. The proteins actA, Phospholipases (PlcA and PlcB (discovered in 1962)) are required for intracellular actin-based motility and cell-to-cell dissemination where the invasion protein InlB, LLO and PlcA encourage escape from the phagocytic vacuole, and PlcB promotes invasion [8, 13, 29]. The 10-kb virulence locus contains a group of these genes.

The p60 protein, expressed by the iap gene, differs in each species of Listeria and contains 484 polypeptides but its specific role of it is not determined due to the fatality associated with the iap gene mutation. In one study it was found to be associated with intestinal invasion; binding with CaCO₂ and murine hydrolase [30]. In addition, L. monocytogenes contains metal ions that, when administered to infected mice in salt form, it will not only reduce the lethal dose but also increase bacterial proliferation rates in the liver and spleen and synthetic media (Figure 2) [8].

Figure 2.

Virulence factors of Listeria.

6. Pathogenesis

Bacterial entry into cells and intra-cytosolic replication serve as a precursor to diffusion throughout tissues as well as a mechanism for evading numerous antibacterial host responses and replicating in a protected environment. Bacteria escape the internal vacuole of the host cell by releasing toxin listerolysin O after pore formation on host cells due to which it gains the ability of haemolysis [31, 32]. On the other hand, actA starts intracellular proliferation; E-cadherin present on the goblet cell’s epithelial lining of intestinal villi [33] serves as a receptor for internalin, while InlB receptors include hepatocytes growth factor receptor, and GAGs (glycosaminoglycans) for the entry as depicted in Figure 3. Listeria was the only pathogen to use E-cadherin as a receptor for entrance till the discovery of fungus Candida. The fungus Candida albicans causes oropharyngeal and hematogenously disseminated candidiasis with an ability to penetrate oral epithelial cells and endothelium cells in vitro. Als₃ is a surface protein that permits fungi to enter host cells. It interacts with both E- and N-cadherin on epithelial and endothelial cells [34]. The three-dimensional structure of the N-terminal of ecto-domain of E-cadherin revealed the molecular details of the interaction between internalin and human E-cadherin. This structure demonstrated that internalin interacts via its leucine-rich repeats in a completely distinct form, thus forming the homotypic E-cadherin interactions [35].

Figure 3.

Cellular signaling in Listeriosis.

The previously unrecognized crucial position of amino acid 16 [36, 37] proline showed specificity to E-cadherin as in guinea pig [38]. Hence, the cytoplasmic domain Ecad links via beta and alpha catenin [37]. Based on this structure, a “murinized” internalin has recently been designed and developed [39] to display the specific interaction with murine E-cadherin. The initial InlB receptor to be identified was gC1qR/p32 [40]. This intriguing molecule, which is very acidic and capable of trimerization, “sits” on a membrane [41]. It is mainly located in the mitochondria but can also be found in the nucleus and on the cell surface. In another case, it was found that gC1qR/p32 interacts with Plasmodium falciparum-infected red blood cells in contact with endothelial cells [42], InlB receptor, MET, a tyrosine kinase and the hepatocyte growth factor receptor (HGFR) [43]. HGFR and MET have been proposed crucial for Plasmodium invasion. Plasmodium invasion-induced hepatocyte injury results in the opening of cell structure to invasion. Met would then act as a signaling mediator [44]. Also, it was reported that the curved face of the InlB leucine-rich repeat region interacts with the first immunoglobulin-like domain of the MET stalk [a domain that does not bind HGF/S that allows InlB to get fixed for minute signal transmission [45]. MET activation requires C-terminal domains of InlB for heparin-mediated receptor clustering and robust signaling. In terms of structure, InlB differs from HGF. For InlB, the bacterium uses functional mimicry rather than structural imitation to take advantage of the properties of its receptors.

As the translocation of L. monocytogenes is fixed listerolysin O (LLO) triggered the host-tissue response allowing it to cross the intestinal barrier in an InlA dependent manner [33] where the absence of response to Listeria becomes consistent. After this, Listeria reaches the liver via the portal vein and got phagocytized by Kupffer cell including early necrostain-1-s-dependent death [46, 47]. Soon then, the monocytes get recruited and the type-1, type-2 inflammation takes place with the expression of Interleukin 33 and Interleukin 4. This phenomenon results in the restoration of liver haemolysis [48, 49] promoting the bacteria to flourish.

6.1 Factor affecting Listeria monocytogenes

In the case of L. monocytogenes, the main factors that contribute to its development and inhibition are pH, the composition of the food product, natural antimicrobial components, biological structures, temperature, atmosphere, competitive flora [2], metal ions, high salt [50], and water activity [51] which is discussed in detail in Table 3.

Serial no.	Factor affecting the growth of L. monocytogenes	References
1.	pH; suitable pH for its growth ranges from 4 to 9.4	[3, 51, 52, 53, 54]
2.	Water activity; 0.90 lower the water lower is the chance of growth	[3, 51, 52, 53]
3.	Natural antimicrobial components; higher the quantity higher is the chance of growth of Listeria	[3, 52, 53]
4	Atmosphere; vacuum commission on pack for an aerobiosis, increased carbon Microbiological dioxide etc. extend the lag and generation times	[51, 52, 53]
5.	Metal/ Mineral ions; increase in concentration of metal ions decrease in growth of Listeria to no growth	[53]
6.	Temperature; −0.4°C to 45°C	[3, 51, 52, 53, 54]
7.	High salt; NaNo3, NaCl inhibit the growth of bacteria but can survive in salty environment	[3, 51, 52, 53, 55]

Table 3.

Different factors affecting the growth of Listeria monocytogenes.

6.2 Recent advances in the detection of Listeria monocytogenes

It was said that to be infectious there must be 100 CFU/mL/g of Listeria in food. Due to delayed and non-specified symptoms, it is difficult to detect at an early stage. In Australia, it was observed that the 10 CFU in 25 g of packaged food cause listeriosis and 100 CFU/mL lead to the reoccurrence. So, scientists had developed several techniques to fulfill the need for a robust, sensitive and reproducible technique to detect L. monocytogenes. The most applicable and available detection techniques developed so, far are discussed below:

6.2.1 Culture-based techniques

According to a 2007 report by Lorber, the time-consuming yet precise cold enrichment method was created in the 1990s [56]. The FDA approved the Ottaviani et al., 1997 proposal to employ chromogenic medium (ALOA) for the identification of Listeria species [57]. Lecithin was hydrolysed, and the blue/green colonies appeared as a result of the dissociation of a chromogenic substrate by an enzyme β-D-glucosidase. Their colonies looked hazy halos. Following the confirmation of the bacteria, it was re-suspended in non-selective media and prepared for the 4–5 days long biochemical test. In addition, there used to be a high probability of false positive results, a need for several chemicals, media, and reagents [USA old 9], as well as a requirement of time and effort [58]. Hitchins and Valimaa et al. used the FDA-BAM technique to identify Listeria from dairy and seafood whose LOD was validated to be less than 1 CFU/mL in 2013 [59] and 2015 [60], respectively. Valimaa et al. obtained comparable results from ISO 11290-1 method developed in 2004 where the LOD was 1 CFU/g. Later, Valimaa et al. discovered that the LOD was 1 CFU/g using the USDA-FSIS approach developed in 2013 [60]. The most probable number technique [61] was more sensitive than a chromogenic medium, according to Dwivedi et al. [61]. In order to quick identification of Listeria, demonstrated that the MPN-PCR technique was more promising than previous approaches [62].

6.2.2 Immuno-based techniques

Antigen-antibody biochemistry appears to be promising for the screening and diagnosis of a disease. To light this, Gasanov et al., reported in 2005 that an immunological technique had sensitivity greater than that of the conventional method, which is 10⁵ cells/mL [6]. However, Diaz-Amigo, reported in 2010 that DNA is the most dependable basis to carry forward the immunological diagnosis, adding that the method is time-consuming for antibody preparation [63].

6.2.2.1 ELISA

In 2005, Bell and Kyriakides, discovered that the sandwich ELISA method was superior than culture to detect Listeria in food samples [64]. The LOD was found to be 10⁵–10⁶ CFU/mL when the anti-antigen was used to target the Listeria antigen [64]. Ueda and Kuwabara used the enzyme linked fluorescence assay, or ELFA, to analyze food samples in 2010 [65]. Based on the food sample’s acidity and basicity, a LOD of 10⁵–10⁶ CFU/mL was found to be reliable. Malla et al. tested sera samples at a dilution of 1:200 for listeriosis using an indirect ELISA. Positive P/N ratios were set to greater than 2. Synthetic LLO-2 peptide (0.40 g/well) and rLLO (0.50 g/well) were used as antigens during this method [66].

6.2.2.2 Immuno-magnetic separation

In 2006, Amagliani et al. introduced a method utilizing nanoparticles to combine a magnetic field with a concentrated amount of bacterial cells. This was done to boost the sensitivity of the detection technique [67]. In 2006 Yang et al., designed a prototype combining real time PCR to immuno based approach using rabbit anti-Listeria with immuno-magnetic nanoparticle coated beads for the detection of hlyA gene in milk sample and the LOD observed was >10² CFU/0.5 mL [68]. Similarly, in 2010 Walcher et al. used paramagnetic beads coated with endolysin-derived cell wall domain of Listeria from tainted raw milk. The LOD lies between 10² and 10³ CFU/mL [69].

6.2.3 Molecular methods of detections

6.2.3.1 DNA microarrays

The Listeria virulence genes inlB, plcA, plcB, and clpE were discovered by Volkhov et al. in 2002 using DNA microarray. He reported that the Listeria result was positive using this method [70]. In 2003, Borucki and Call investigated serotype-specific probe differentiation by combining 585 genomic DNA (10 samples) mixed probes and found that it was successful for 29 probes [71]. After that, it was used as a confirmatory technique by Brehm-Stecher and Johnson to check the specificity of polymorphism and PCR amplification. With a detection limit of 8 log CFU/mL [72], Bang et al. reported that 9/16 of the microarrays used to test artificially infected milk were positive. He emphasized that this approach was accurate and reliable. Despite being encouraged, it needs patience and has the potential to cross-hybridize, which could lead to a false test result [73].

6.2.3.2 PCR based methods

PCR has been widely used in molecular diagnostics as a potential tool for minute sample detection. A heat cycle in PCR required a set of specialized primers for the target amplification. The results are then analyzed using gel electrophoresis. The modifications that were made in order to detect Listeria using PCR are discussed below:

6.2.3.2.1 Conventional PCR

The use of primers in PCR makes it a potential method for identifying pathogens in a sample. Aznar and Alacron reported that the result obtained from PCR was positive for 56 out of 217 cases in naturally infected samples with a limit of detection of 1 CFU/g where only 17 was observed to be positive during culture. They employed primers designed to target the genes hlyA, iap, inlB, inlA, 16S, and 23S rRNA, as well as the proteins phospholipase C, fibronectin-binding protein, and hypersensitivity protein for the detection [74].

In cases of non-viable DNA amplification, Klein and Juneja [75] reported that the PCR method was a false positive. Reverse transcriptase PCR (RT-PCR) was used because mRNA has a short half-life and immediately disintegrates after cell death [72], to target the hly, prfA, and iap gene transcripts rather than DNA. The detection threshold was between (2.5 × 10⁶ and 3 × 10⁶) CFU/mL. In order to validate the technique, they used cooked beef that had been intentionally infected. They found that the analysis was sensitive to 1 CFU/g. However, Pan and Breidt used real-time PCR and achieved success with propidium monoazide and ethidium monoazide in amplification of dead cells, arguing against it as an efficient method for detecting bacteria in low numbers whose LOD was 10³ CFU/mL [76].

6.2.3.3 Multiplex PCR

Multiplex PCR was described by Alarcon et al. [77] as a reliable, efficient, and time-saving technique for simultaneously detecting several organisms in contaminated samples. Samples with varying LOD as 57 CFU/ml of Salmonella spp., 79 CFU/ml of L. monocytogenes, and 260 CFU/ml of S. aureus [73]. This strategy was employed by Lei et al. to identify six common food-borne pathogens in RTE food with LODs of 1–100 CFU/ml [78]. The MPCR method was introduced by Zhang et al., in 2009 and targets the hly gene of L. monocytogenes, the nuc gene of S. aureus, the invA gene of S. enterica, the stx gene of E. coli, and the intimin gene of E. coli, with a detection limit of 1 CFU/mL [79]. In 2006, Mustapha and Li stated that MPCR as non-specific for the similar sized amplicon and optimization [80].

6.2.3.4 RT-PCR (Real-Time PCR)

A three-day PCR-based assay was developed by Kaclikova et al. (2003) with a detection limit of 10° CFU per 25 g of food, which is equivalent to the standard EN ISO 11290-1 or ISO 10560 methods of Listeria detection. LOD obtained was 1 × 10⁴ CFU/mL [81]. Bhagwat et al. reported that the total viable count detected was 1.35 ± 2 × 10⁸ in the salad and 0.35 ± 1.9 × 10⁸ in broccoli. In which the limit of detection of L. monocytogenes was 1.74 ± 1.1 × 10⁶ in salad, 6.37 ± 1.2 × 10³ in broccoli and overall less than 1000 cells/mL [82]. A hly-IAC Q-PCR assay to detect Listeria was created in 2005 by Rodriguer and Lazaro et al., utilizing different concentrations to spike the sample, and the detection limit was determined to be 8 [83]. To expand the scope of the technique Berrada et al. [84], developed RTQ-PCR to quantify the fluorescence emitted by the spiked sample. The obtained LOD was 10–10⁵ CFU/mL [84]. In 2006, Fairchild et al. developed a detection method using SYBR green which shows in presence of non-target DNA and primer-dimer formation [85, 86, 87, 88, 89]. O’Grady et al. found that targeting the ssrA gene in naturally and artificially contaminated foods (milk products, meat, and veggies) resulted in a detection limit of 1–5 CFU/25 g/mL [90]. Therefore, he came to the conclusion that it was a smart strategy for the particular sample. In 2010 Suo et al. reported the result of a qRT-PCR assay with the detection limit of 18 CFU/10 g on naturally and artificially contaminated ground beef, chicken, turkey and pork [91].

6.2.4 Biosensor based techniques

A biosensor is the biological specimen analyzer using analyte as an object and an electrochemical set up as a transducer generating readable data. In 2004, BIA3000 was first used by Leonard et al., as a biosensor where he passed the antibody over a biosensor chip immobilized on polyclonal goat anti-rabbit Fab antibodies to detect L. monocytogenes [92]. Advancing the sensor platform Poltronieri et al. reported that the use of surface plasmon resonance to detect L. monocytogenes was promising with a detection limit of 10² CFU/mL [93]. In this platform Au-labeled secondary antibodies were used. On further advancement Banerjee and Bhunia reported the use of mammalian B-lymphocyte Ped-2E9 cell merged in collagen matrix as a sensing platform to detect listeriolysin O from the contaminated food sample with a detection limit of 10²–10⁴ CFU/g [94].

In 2015, Lui et al. [95] developed a paper-based micro fluidic device that detects long DNA amplicons on the basis of hybridization reactions with a covalently immobilized DNA probe and biotin-labeled signal DNA strands, and chemiluminescent (CL) reactions catalyzed by a horseradish peroxidase (HRP) streptavidin conjugate. In this DNA biosensor, CL signals generated using a HRP-luminol-H2O2 system were heightened with p-iodine phenol (PIP) and detected with a CCD system. Under optical conditions, a linear range of 1.94 × 10^–1 pmol/L to 1.94 × 10⁴ pmol/L was achieved and the limit of detection was found to be 6.3 × 10^–2 pmol/L [91]. Then, in 2022 Zhang et al. [96] and his team from China develop a portable paper-based multi-biocatalyst platform to identify L. monocytogenes by detecting multiple biomarkers at different levels: gene hly (nucleic acid), acetoin (small molecule metabolite), and LLO (protein). The integrating detections of the three biomarkers were successfully performed by two different modified working electrodes on a single paper-based multi-biocatalyst platform. The sensitive and reliable identification of L. monocytogenes was achieved using the portable paper-based multi-biocatalyst platform with a wider detection range (from 1.0 × 10⁴ to 1.0 × 10⁹ CFU mL⁻¹) and lower detection limit (10⁴ CFU mL⁻¹) [97]. Similarly, in 2022 to further advance the sensing technology Du et al., developed a fluorescence-based dual recognition assembly using Fe₃O₄ @ZIF-8. The linear range of the detection of pure culture ranged from 1.4 × 10¹ to 1.4 × 10⁷ CFU/mL, with the detection limit of 0.88 CFU/mL.

Out of these methods, culture-based tests are usually preferred because of their availability, sensitivity, cost-effective and the ‘gold standards’ compared with other methods that are validated. To summarize the availability and development of detection methods is presented in Table 4.

S. no.	Methods used	Source	Year	Specification	Limit of detection	References
1.	Bacterial culture	Food	1997	Blue/Green colonies of Listeria due to the dissociation of chromogenic substrate by an enzyme β-D-glucosidase.	4–5 days for the confirmation.	Ottavianii et al., [57]
2.	FDA-BAM	Dairy and Sea food	2013&2015	Specific to Listeria detection	1 CFU/mL	Valimaa et al. [59]
3.	ELISA	Food	2005	Able to detect other food pathogens too.	10^5–10^6 CFU/mL	Bell and Kyriakides [64]
4.	Immuno-magnetic separation	Milk	2007	Anti-listeria rabbit antibody was used with immuno-magnetic nanoparticles coated beads to detect hlyA gene	10^4 CFU/mL	Yang et al. [68]
5.	DNA Microarrays	Milk	2007	Antigen-labeled probe was used.	10^8 CFU/mL	Brehm-Stetcher et al. [72]
6.	Conventional PCR	Patient sample	2002 and 2003	Primers were used targeting hlyA, iap, inlB, inlA, 16S, and 23S rRNA, as well as the proteins phospholipase C, fibronectin-binding protein, and hypersensitivity protein.	101 CFU/mL	Aznar and Alacron [74]
7.	Multiplex PCR	Human	2004	Could detect other food-borne pathogen too. It was not limited to Listeria.	5.7 × 10^1 CFU/mL of Salmonella spp., 7.9 × 10^1 CFU/mL of L. monocytogenes, and 2.6 × 10^2 CFU/mL of S. aureus	Alarcon et al. [77]
8.	Real Time PCR equivalent to standard EN ISO 11290-1 method	Food	2003	3 days PCR-based assays were developed to detect Listeria from 25 g of food.	10^4 CFU/mL	Kaclikova et al. [81]
9.	RT-PCR	Food	2008	Target ssrA gene	1–5 CFU/mL	O’Grady et al. [90]
10.	qRT-PCR	Beef, Chicken, Pork	2010	qRT-PCR assay	10^2 CFU/mL	Suo et al. [91]
11.	BIA3000-Biosensor	Bacterial antigen	2004	Assembly was prepared immobilizing polyclonal goat anti-rabbit Fab antibodies	Resulted in minute detection	Leonard et al. [92]
12.	Surface plasmon resonance	Bacterial antigen	2009	Assembly was prepared immobilizing Au-labeled secondary antibodies	10^2 CFU/mL	Poltronieri et al. [93]
13.	Immuno-based sensor	mammalian B-lymphocyte	2010	Assembly was prepared with mammalian B-lymphocyte Ped-2E9 cell merged in collagen matrix.	10^2–10^4 CFU/mL	Banerjee and Bhunia [94]
14.	Single paper-based multi-biocatalyst	Bacteria	2022	Assembly was developed to identify L. monocytogenes by detecting multiple biomarkers at different levels: gene hly (nucleic acid), acetoin (small molecule metabolite), and LLO (protein).	10^4 CFU/mL	Zhang et al. [96]
15.	Dual recognition and highly sensitive detection of Listeria monocytogenes	Food	2022	Fe O @ZIF-8@aptamer	0.88 CFU/mL	Du et al. [97]

Table 4.

Available diagnostics methods for the detection of Listeria monocytogenes.

7. Conclusion

Pregnant women, young children, and elderly individuals with suppressed immune systems are mostly affected by Listeria monocytogenes, a common and third-most lethal food-borne illness. The worldwide death rate is substantially worsened by its outbreak. Despite the use of suitable food processing methods, outbreaks spurred on by eating foods infected with L. monocytogenes inevitably occur. There is not a commercially available vaccination to prevent listeriosis at present. The molecular processes, interspecies interactions, and cross-domain interactions that affect L. monocytogenes’ virulence, AMR, metabolic activity, and survival under stress as seen in the environment and host are not well explored. Numerous methodologies have been used until this point, notably DNA microarrays, PCR, immuno-based methods, culture-based methods, and ELISA. However, these methods have limits when it comes to the identification of this virus. Thereafter, it is necessary to put into effect a novel researcher’s suggestion for a quick detection approach, such as biosensor-based techniques, which have high sensitivity and specificity as well as time savings and more research is required to determine the possible significance of bacterial communication in the control of the stress response in L. monocytogenes.