Detection Of Foodborne Pathogens Research Articles

The food microbiological analyst: pairing tradition with the future

Choosing methods for the detection, isolation and identification of foodborne pathogens or spoilage microorganisms from foodstuffs is a complex task. Although there are standard food microbiology methods available around the world, many have changed little in decades, while some tests take many days to perform. The use of automated technologies, genomics and rapid methods are now replacing many traditional tests. Food microbiology analysts need to understand the underlying science and limitations of these methods for food analysis and the crucial importance of validation protocols for correct results reported to customers. This article discusses the current status of food microbiology analysis in commercial laboratories, what the future food microbiology analytic laboratory looks like, and the education changes that may be required to become a future food microbiology analyst.

On-Site Detection of Multiple Foodborne Pathogens Using a Paper-Based Isothermal Amplification Chip and a Smartphone-Based Portable Device

On-Site Detection of Multiple Foodborne Pathogens Using a Paper-Based Isothermal Amplification Chip and a Smartphone-Based Portable Device

Biofunctionalized Magnetic Nanoparticles with Multiplex Touchdown PCR for Simultaneous and Rapid Detection/Identification of Campylobacter jejuni and Campylobacter coli

The simple, accurate, and rapid detection of foodborne pathogens is essential for public health. Development of an immunomagnetic separation (IMS) multiplex touchdown PCR (IMS–multiplex TD–PCR) assay for simultaneous detection and distinguishing of C. jejuni and C. coli is reported herein. Polyclonal antibody (pAb) against multiepitope antigen (MEA) was conjugated to ferromagnetic nanoparticles (FMNs) to produce anti‐MEA FMNs. Optimal anti‐MEA FMNs loading yielded 26.7 μg of immunoglobulin G (IgG) molecules per mg of FMNs with an average size of 72 ± 9 nm, corresponding to an 83% rate of pAb conjugation. Anti‐MEA FMNs (20 μg) for IMS captured culturable C. jejuni cells at 3.54 × 102 colony‐forming unit (CFU)/mL in pure culture, while higher amounts (40 and 60 μg) reduced the recovery. The scanning electron microscope (SEM) analysis revealed the attachment of anti‐MEA FMNs to target bacteria, forming aggregated cells and magnetic nanoparticles in ellipse‐like shapes. The subsequent multiplex TD–PCR assay simultaneously detected and distinguished C. jejuni and C. coli at 104 CFU/mL in mixed culture and at 103 CFU/mL for each individual species. Furthermore, the limit of detection (LOD) of the IMS–multiplex TD–PCR assay was 104 CFU/g in spiked chicken breast samples. Specificity was 100% for both C. jejuni and C. coli as none of the amplicons were detected in control samples where Campylobacter was absent. This assay is able to detect and distinguish C. jejuni and C. coli simultaneously and is simple, accurate, and rapid with a time to result of 4 h without an enrichment step, making it a promising approach for rapid and culture‐free detection of Campylobacter in chicken products.

An ultra-fast, one-step RNA amplification method for the detection of Salmonella in seafood.

Salmonella is one of the most common pathogens associated with food-borne illness resulting from seafood consumption. Herein, an accelerated strand exchange amplification (ASEA) requiring only a pair of primers and one polymerase was first reported for ultra-fast, one-step RNA amplification detection of Salmonella in seafood. The ASEA method could detect Salmonella typhimurium DNA in dilutions as low as 10 copies per reaction and displayed good specificity for Salmonella under the interference of a variety of food-borne pathogens. In particular, ASEA could detect RNA in one step without additional reverse transcription. The detection limit for Salmonella in artificially contaminated oyster was 1 CFU mL-1 following 12 h of enrichment. Moreover, excellent performance of this assay was observed with 99.02% consistency relative to real-time PCR through actual sample detection. Combined with the rapid nucleic acid extraction method, the entire detection process could be completed within 20 min. Therefore, this assay opens up new prospects for the detection of food-borne pathogens in seafood with its rapidity, which would be very beneficial for food safety supervision and pathogen detection of high-throughput samples.

Retroreflection-based sandwich type affinity sensing of isothermal gene amplification products for foodborne pathogen detection.

Loop-mediated isothermal amplification (LAMP) is an outstanding method for molecular diagnostics, as the rapid, specific, and sensitive amplification of target genes is possible. However, it is necessary to measure fluorescence in the quantitative analysis of LAMP products, so a sophisticated optical setup is required. This study tried to develop a novel sensing method that can quantify target analytes with simple equipment, such as nonspectroscopic white light and a CMOS camera. To achieve this, a retroreflective Janus particle (RJP) as a probe and specially designed loop primers, fluorescein isothiocyanate (FITC)- and biotin-modified loop primers, were introduced into the LAMP system. By performing LAMP in the presence of designed primers, double-stranded amplicons possessing FITC and biotin labels at each end are generated in proportion to the quantity of the target pathogen. Using the anti-FITC antibody-modified sensing surface and streptavidin-conjugated RJP probes, the amplicons can be captured in sandwich-configuration and detected under nonspectroscopic conditions composed of white light and a camera. To confirm the feasibility of the sensing system, the invA gene of Salmonella was selected as the target. It was possible to quantitatively analyze the Salmonella concentration from 0 to 106 colony-forming units, sufficiently covering the required detection range. In addition, quantitative analyses of pathogens in contaminated food sources, including milk and chicken meat, were successfully conducted with a limit of detection of 10 CFU.

Bacterial Lighthouses-Real-Time Detection of Yersinia enterocolitica by Quorum Sensing.

Foodborne zoonotic pathogens have a severe impact on food safety. The demand for animal-based food products (meat, milk, and eggs) is increasing, and therefore faster methods are necessary to detect infected animals or contaminated food before products enter the market. However, conventional detection is based on time-consuming microbial cultivation methods. Here, the establishment of a quorum sensing-based method for detection of foodborne pathogens as Yersinia enterocolitica in a co-cultivation approach using a bacterial biosensor carrying a special sensor plasmid is described. We combined selective enrichment with the simultaneous detection of pathogens by recording autoinducer-1-induced bioluminescent response of the biosensor. This new approach enables real-time detection with a calculated sensitivity of one initial cell in a sample after 15.3 h of co-cultivation, while higher levels of initial contamination can be detected within less than half of the time. Our new method is substantially faster than conventional microbial cultivation and should be transferrable to other zoonotic foodborne pathogens. As we could demonstrate, quorum sensing is a promising platform for the development of sensitive assays in the area of food quality, safety, and hygiene.

A finger-actuated microfluidic biosensor for colorimetric detection of foodborne pathogens

A microfluidic biosensor was developed for rapid detection of Salmonella using a finger-actuated micropump, a finger-actuated micromixer, gold@platinum nanocatalysts (Au@PtNCs) and a smartphone App. First, immune magnetic nanobeads (MNBs), bacterial sample and immune Au@PtNCs were successively finger-actuated pumped into microfluidic chip. Then, they were fully mixed using finger-actuated micromixer to form MNB-Salmonella-Au@PtNC complexes. After hydrogen peroxide-tetramethylbenzidine was pumped into chip and catalyzed by nanocatalysts on complexes, resulting in color change from colorless to blue, the image of catalysate was collected and finally analyzed by self-developed smartphone App or directly compared with Pantone color card in chip to determinebacterial concentration. Experimental results showed this biosensor could quantitatively detect Salmonella from 3.5 × 102 to 3.5 × 105 CFU/mL in 1 h with lower detection limit of 350 CFU/mL. This biosensor has successfully integrated loading, mixing, incubation, washing, separation and detection onto a chip and might pave a promising way for bacterial detection.

“Lighting-up” methylene blue-embedded zirconium based organic framework triggered by Al3+ for advancing the sensitivity of E. coli O157:H7 analysis in dual-signal lateral flow immunochromatographic assay

The sensitive detection of foodborne pathogens is of great significance for ensuring food safety and quality. Herein, on the basis of methylene blue-embedded zirconium based organic framework (UIO@MB) as the remarkable capture carrier and signal indicator, with the Al3+-assisted the fluorescent signal response, we developed a label-free and dual-signal lateral flow immunochromatographic assay (LDLFIA) for sensitive detection of Escherichia coli (E. coli) O157:H7. The UIO@MB sensing carrier without monoclonal antibodies (mAbs) was manufactured, which adhered to bacteria to form the UIO@MB-E. coli O157:H7 conjugate, resulting in visible blue band. Then the fluorescent response of the OH-rich UIO@MB was excited by introducing Al3+, arising from capturing of Al3+ by -OH through coordination and electrostatic affinity, thus generating a green fluorescent band. Impressively, a smartphone-based portable reading system was developed that can reflect the test results of UIO@MB-LDLFIA immediately. Under optimum conditions, UIO@MB-LDLFIA can complete colorimetric and fluorescent mode detection within 90 min, with a detection sensitivity of 103 CFU/mL, which were 100 times lower than traditional gold nanoparticles-based LFIA and polymerase chain reaction (PCR). Moreover, the feasibility of the method was further evaluated by the determination of E. coli O157: H7 in drinking water and cabbage with average recoveries of 85.1-123.0%.

Development of modified enrichment broth for short enrichment and recovery of filter-injured Salmonella Typhimurium

The filter concentration method facilitates the rapid detection of foodborne pathogens. The filter concentration method lowered the limit of detection (LOD) of artificially inoculated cabbage with Salmonella Typhimurium; however, the procedure injured foodborne pathogens during filtering procedure. Thus, to detect injured pathogens under the detection limit, an enrichment broth promoting pathogen resuscitation and growth is required. To rapidly recover, cultivate and lower the time to result (TTR) of S. Typhimurium detection after filter concentration method, a brain heart infusion (BHI) broth-based modified enrichment broth (MEB) was developed. The MEB was developed by fitting growth curves to a modified Gompertz model; 1.00 g/L of sodium pyruvate, 0.20 g/L proline and 2.0 g/L magnesium sulphate additives were optimized as additional components to rapidly grow filter-injured S. Typhimurium. As a result, the rate of filter-injured S. Typhimurium went from 100% to 0.0% using MEB within 3.5 h. In contrast, BHI required 4 h and buffered peptone water (BPW) required more than 4 h to decrease the injury rate to 0.0%. Using MEB, BHI and BPW, filter-injured S. Typhimurium in cabbages were enriched to 4.056 ± 0.026 Log CFU/25 g, 3.571 ± 0.187 Log CFU/25 g and 3.708 ± 0.156 Log CFU/25 g, respectively. Additionally, 1–9 CFU/mL S. Typhimurium in cabbage was detected within 3.0 h, including 1 h enrichment with MEB, whereas 5.0 h was required for BHI and BPW. Thus, the MEB developed in this study showed great potential as a short enrichment broth for the rapid detection of filter-injured S. Typhimurium.

가정간편식(Home Meal Replacement)의 일반세균 및 식중독세균 오염현황 분석과 위생관리의 중요성

The market size of the home meal replacement (HMR) is continuously increasing. However, there are high concerns about safety such as poor hygiene of companies and detection of foodborne pathogens in HMR. This study analyzed the contamination level of total aerobic bacteria and 5 types of foodborne pathogens in 30 Ready-to-Eat foods (20 Gimbap, 5 sandwiches and 5 hamburgers). As a result, it was confirmed that total aerobic bacteria were contaminated with at least 67 CFU/g and up to 412 CFU/g in 12 samples. Staphylococcus aureus (11-132 CFU/g) and Bacillus cereus (41-233 CFU/g) were contaminated in 9 samples. The microbial contamination level was the highest in Gimbap. Small amounts of foodborne pathogens can multiply during inappropriate distribution and storage, causing foodborne illness. Therefore, to expand and growth of HMR industry further, it is considered necessary to secure microbiological safety through continuous monitoring of HMR and hygiene management of companies.

A label-free electrochemical immunosensor for rapid detection of salmonella in milk by using CoFe-MOFs-graphene modified electrode

The presence of Salmonella in food has been a serious threat to human health all over the world. Herein, a novel electrochemical immunosensor based on CoFe-MOFs-graphene nanocomposite was constructed to detect Salmonella in milk. The synergy of the large specific surface area and high porosity of CoFe-MOFs and the good electrical conductivity of graphene materials greatly enhanced the current signal of the nanocomposite. Gold nanoparticles were modified on CoFe-MOFs-graphene hybrids by a simple electrodeposition method. Anti-Salmonella antibody was immobilized onto the surface of the constructed electrode through the formation of a stable Au–NH2 bond. Scanning electron microscopy was employed to reveal the structure of the CoFe-MOFs-graphene nanocomposite, and the elements of the nanocomposite were observed by energy-dispersive X-ray spectroscopy. In addition, Fourier transform infrared (FT-IR) and X-ray powder diffraction (XRD) were used to analyze the structure of related materials. Under optimal experimental parameters, the constructed immunosensor detected a wide linear range of Salmonella from 2.4 × 102 CFU mL−1 to 2.4 × 108 CFU mL−1 with a detection limit of 1.2 × 102 CFU mL−1 (S/N = 3). The immunosensor showed good stability, selectivity, and reproducibility. The designed immunosensor provides a new strategy for the rapid detection of foodborne pathogens.

Recent Advancements in the Technologies Detecting Food Spoiling Agents.

To match the current life-style, there is a huge demand and market for the processed food whose manufacturing requires multiple steps. The mounting demand increases the pressure on the producers and the regulatory bodies to provide sensitive, facile, and cost-effective methods to safeguard consumers’ health. In the multistep process of food processing, there are several chances that the food-spoiling microbes or contaminants could enter the supply chain. In this contest, there is a dire necessity to comprehend, implement, and monitor the levels of contaminants by utilizing various available methods, such as single-cell droplet microfluidic system, DNA biosensor, nanobiosensor, smartphone-based biosensor, aptasensor, and DNA microarray-based methods. The current review focuses on the advancements in these methods for the detection of food-borne contaminants and pathogens.

A Short- and Long-Range Fluorescence Resonance Energy Transfer-Cofunctionalized Fluorescence Quenching Collapsar Probe Regulates Amplified and Accelerated Detection of Salmonella.

Accurate and rapid quantification of foodborne pathogens is of great significance for food safety and human health. In this work, we have successfully constructed a fluorescence quenching collapsar probe (FQCP) on the basis of a conventional aptamer-encoded molecular beacon (AEMB) and applied it for the detection of Salmonella. In structure, the FQCP is assembled by AEMBs in fours via specific streptavidin and biotin binding. Such a simple format makes the FQCP cofunctionalized with short- and long-range fluorescence resonance energy transfer (FRET) effects, thereby leading to a significantly suppressed inherent background fluorescence that is much lower than that of the conventional AEMB. Moreover, the FQCP exhibits superior biostability because of the blocking of its 3' terminal. The reaction kinetics of the FQCP for Salmonella recognition is obviously improved since the probe designed with four binding sites increases the probability to react with Salmonella. As a result, the FQCP-based sensing platform can rapidly output the target detection signal within 30 min associated with a greatly improved signal-to-noise ratio up to 32.4. The system was also demonstrated with a well antimatrix effect for ultrasensitive detection of Salmonella from tap water, milk, red bull, green tea, orange juice, and Coca-Cola. Our study provides insights into the facile tailoring of functional nucleic acids for amplified and mix-to-answer detection of foodborne pathogens, which could become a powerful analytical tool for straightforward sensing of pathogens in the fields of food safety analysis, clinical diagnostics, and environmental monitoring.

Highly sensitive smartphone-based detection of Listeria monocytogenes using SYTO9

Listeriosis is caused by Listeria monocytogenes (LM) and is currently considered to be one of the leading food-borne diseases worldwide, with mortality rate of 20%∼30%. Currently, detection methods for LM are time-consuming with low sensitivity, and delayed detection results. SYTO9 has a high affinity for DNA and exhibits enhanced fluorescence upon binding. Therefore, this study used SYTO9 staining and image processing to develop a rapid loop mediated isothermal amplification (LAMP) detection method for LM. Smartphone was successfully used for detecting the color change in different concentrations of LM. Besides, the optimized LAMP reaction temperature was 63 °C by color identification, and the limit of detection for LM was 6 copies/µL in the green channel. So, the developed method, based on image processing, is simple, sensitive and rapid, which provides a new idea and method for rapid detection of LM and other food-borne bacterial pathogens.

Towards Real-Time and Affordable Strain-Level Metagenomics-Based Foodborne Outbreak Investigations Using Oxford Nanopore Sequencing Technologies.

The current routine laboratory practices to investigate food samples in case of foodborne outbreaks still rely on attempts to isolate the pathogen in order to characterize it. We present in this study a proof of concept using Shiga toxin-producing Escherichia coli spiked food samples for a strain-level metagenomics foodborne outbreak investigation method using the MinION and Flongle flow cells from Oxford Nanopore Technologies, and we compared this to Illumina short-read-based metagenomics. After 12 h of MinION sequencing, strain-level characterization could be achieved, linking the food containing a pathogen to the related human isolate of the affected patient, by means of a single-nucleotide polymorphism (SNP)-based phylogeny. The inferred strain harbored the same virulence genes as the spiked isolate and could be serotyped. This was achieved by applying a bioinformatics method on the long reads using reference-based classification. The same result could be obtained after 24-h sequencing on the more recent lower output Flongle flow cell, on an extract treated with eukaryotic host DNA removal. Moreover, an alternative approach based on in silico DNA walking allowed to obtain rapid confirmation of the presence of a putative pathogen in the food sample. The DNA fragment harboring characteristic virulence genes could be matched to the E. coli genus after sequencing only 1 h with the MinION, 1 h with the Flongle if using a host DNA removal extraction, or 5 h with the Flongle with a classical DNA extraction. This paves the way towards the use of metagenomics as a rapid, simple, one-step method for foodborne pathogen detection and for fast outbreak investigation that can be implemented in routine laboratories on samples prepared with the current standard practices.

Evaluation of whole-genome sequencing-based subtyping methods for the surveillance of Shigella spp. and the confounding effect of mobile genetic elements in long-term outbreaks.

Many public health laboratories across the world have implemented whole-genome sequencing (WGS) for the surveillance and outbreak detection of foodborne pathogens. PulseNet-affiliated laboratories have determined that most single-strain foodborne outbreaks are contained within 0–10 multi-locus sequence typing (MLST)-based allele differences and/or core genome single-nucleotide variants (SNVs). In addition to being a food- and travel-associated outbreak pathogen, most Shigella spp. cases occur through continuous person-to-person transmission, predominantly involving men who have sex with men (MSM), leading to long-term and recurrent outbreaks. Continuous transmission patterns coupled to genetic evolution under antibiotic treatment pressure require an assessment of existing WGS-based subtyping methods and interpretation criteria for cluster inclusion/exclusion. An evaluation of 4 WGS-based subtyping methods [SNVPhyl, coreMLST, core genome MLST (cgMLST) and whole-genome MLST (wgMLST)] was performed on 9 foodborne-, travel- and MSM-related retrospective outbreaks from a collection of 91 Shigella flexneri and 232 Shigella sonnei isolates to determine the methods’ epidemiological concordance, discriminatory power, robustness and ability to generate stable interpretation criteria. The discriminatory powers were ranked as follows: coreMLST<SNVPhyl<cgMLST<wgMLST (range: 0.970–1.000). The genetic differences observed for non-MSM-related Shigella spp. outbreaks respect the standard 0–10 allele/SNV guideline; however, mobile genetic element (MGE)-encoded loci caused inflated genetic variation and discrepant phylogenies for prolonged MSM-related S. sonnei outbreaks via wgMLST. The S. sonnei correlation coefficients of wgMLST were also the lowest at 0.680, 0.703 and 0.712 for SNVPhyl, coreMLST and cgMLST, respectively. Plasmid maintenance, mobilization and conjugation-associated genes were found to be the main source of genetic distance inflation in addition to prophage-related genes. Duplicated alleles arising from the repeated nature of IS elements were also responsible for many false cg/wgMLST differences. The coreMLST approach was shown to be the most robust, followed by SNVPhyl and wgMLST for inter-laboratory comparability. Our results highlight the need for validating species-specific subtyping methods based on microbial genome plasticity and outbreak dynamics in addition to the importance of filtering confounding MGEs for cluster detection.

Direct Detection of Foodborne Pathogens via a Proximal DNA Probe-Based CRISPR-Cas12 Assay.

Foodborne pathogens can cause illnesses. Existing tools for detecting foodborne pathogens are typically time-consuming or require complex protocols. Here, we report an assay to directly analyze pathogenic genes based on CRISPR-Cas12. This new test, termed proximal DNA probe-based CRISPR-Cas12 (PPCas12), facilitates the detection of foodborne pathogens without amplification steps. The elimination of the nucleic acid amplification process dramatically reduced the processing time, complexity, and costs in the analysis of foodborne pathogens. The substitution of the frequently used dually labeled DNA reporter with a proximal DNA probe in the PPCas12 assay led to a 4-fold sensitivity enhancement. PPCas12 offered a limit of detection of 619 colony-forming units in the detection of Salmonella enterica (S. enterica) without the nucleic acid amplification process. The specific recognition of genes via PPCas12 allowed distinguishing S. enterica from other foodborne pathogens. The PPCas12 assay was applied in the screening of S. enterica contamination on fresh eggs with high precision. Hence, the new PPCas12 assay will be a valuable tool for on-site monitoring of foodborne pathogens.

Monitoring Food Contamination in Real Time Using Printable Dnazyme Sensors

Foodborne illnesses have a pronounced impact on the global community, with cases numbering into the hundreds of millions annually and associated treatments costing tens of billions of dollars. Our inability to commercially detect food contamination in real-time is at the center of this crisis, as it places emphasis on static expiry dates that lack accuracy. These predicted dates not only fail to convey contamination, but also result in the disposal of mass amounts of edible food products. When contaminants are retroactively detected, measures such as food recalls are logistically complex and have massive economic ramifications. Existing methods for real-time detection of contaminants are expensive, require sophisticated equipment or offer poor detection capabilities, thus preventing commercialization. Additionally, detection requires packaging to be opened, making it unfeasible to monitor food products on an individual basis. Here, we propose a detection system centered upon the covalent immobilization of pathogen-specific RNA-cleaving fluorescent deoxyribozymes (RFDs) onto food wraps for real-time pathogen detection. These biomolecules exhibit a significant increase in fluorescence when exposed to their respective target. By functionalizing food wrap materials through plasma treatment, suitable crosslinkers can be used to bind amine modified RFDs onto the bulk wrap material. In particular, carbon dioxide plasma paired with carbodiimide chemistry presents itself as a well-studied immobilization approach that is applicable within the proposed sensing platform. Additionally, the use of microcontact printing to pattern RFD-crosslinker solutions onto food wraps presents a strategy suitable for industry scale-up. Given the transparency of food wrap materials and the printed sensor, this platform also overcomes the current need to open packaging for pathogen detection. Ultimately, we envision a patterned RFD barcode that provides multiplex detection of common foodborne pathogens. Use of a handheld fluorescence reader would allow for inexpensive, portable detection of food contamination along the entirety of the food production pipeline.

Recent trends and developments of PCR-based methods for the detection of food-borne Salmonella bacteria and Norovirus.

In recent years, rapid detection methods such as polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) have been continuously developed to improve the detection of food-borne pathogens in food samples. The recent developments of PCR and qPCR in the detection and identification of these food-borne pathogens are described and elaborated throughout this review. Specifically, further developments and improvements of qPCR are discussed in detecting Salmonella and norovirus. Promising advances in these molecular detection methods have been widely used to prevent human food-borne illnesses and death caused by the food-borne pathogens. In addition, this review presents the limitations and challenges of the detection methods which include conventional culture method and conventional PCR method in detecting Salmonella and norovirus. Furthermore, several advances of qPCR such as viability PCR (vPCR) and digital PCR (dPCR) have been discussed in the detection of Salmonella and norovirus. Good practice of analysis of the food-borne pathogens and other contaminants in the food industry as well as the advancement of molecular detection methods will help improve and ensure food safety and food quality.

Advances in Nanomaterials-Based Electrochemical Biosensors for Foodborne Pathogen Detection.

Electrochemical biosensors utilizing nanomaterials have received widespread attention in pathogen detection and monitoring. Here, the potential of different nanomaterials and electrochemical technologies is reviewed for the development of novel diagnostic devices for the detection of foodborne pathogens and their biomarkers. The overview covers basic electrochemical methods and means for electrode functionalization, utilization of nanomaterials that include quantum dots, gold, silver and magnetic nanoparticles, carbon nanomaterials (carbon and graphene quantum dots, carbon nanotubes, graphene and reduced graphene oxide, graphene nanoplatelets, laser-induced graphene), metal oxides (nanoparticles, 2D and 3D nanostructures) and other 2D nanomaterials. Moreover, the current and future landscape of synergic effects of nanocomposites combining different nanomaterials is provided to illustrate how the limitations of traditional technologies can be overcome to design rapid, ultrasensitive, specific and affordable biosensors.