Contaminated Food Samples Research Articles

Solvatochromism mechanism of perylene diimide radicals stabilized by low barrier hydrogen bond and detection of formaldehyde in real food samples

Formaldehyde (FA) is widely used for food preservation and whitening because of its preservative and bleaching effects. Excessive FA seriously endangers human health, so monitoring FA-treated food is of great significance for food safety. In this paper, perylene diimide radicals with the chirality (BTFPDI) and water-solubility (TFPDI-bPEI) were designed respectively based on our previous studies. Property of solvatochromism induced by these perylene diimide radicals aggregation was demonstrated by the concentration and temperature dependent absorption spectrum and the circular dichroism spectrum. Further the colorimetric monitoring of trace FA in gaseous and liquid was realized by the condensation reaction of FA with amine to induce TFPDI-bPEI aggregation. After the addition of FA, TFPDI-bPEI molecule aggregated in aqueous solution due to the specific reaction of a large number of –NH2 groups with FA, resulting in a blue-shift of the maximum absorption peak from 780 nm to 561 nm, and the detection limit as low as 0.86 μM to FA and the response time of only 20 s. This method could further detect FA in food with good recoveries and detect FA in contaminated food samples.

Fabrication of a Label-Free Immunosensor Using Surface-Engineered AuPt@GQD Core-Shell Nanocomposite for the Selective Detection of Trace Levels of Escherichia coli from Contaminated Food Samples.

Fabrication of label-free immunosensors is highly necessitated due to their simplicity, cost-effectiveness, and robustness. Herein, we report the facile development of a label-free, direct, rapid, capacitive immunosensor for ultrasensitive and rapid recognition of trace levels of Escherichia coli from contaminated food samples. This was achieved using gold platinum core-shell nanoparticles loaded with graphene quantum dots (AuPt@GQDs) that were utilized as electrode modifiers. The incorporation of GQDs to the surface of AuPt core-shell nanoparticles was performed using the "greener" probe-sonication method. The electrochemical properties of AuPt@GQDs, determined using cyclic voltammetry and electrochemical impedance spectroscopy, suggested the optimized loading concentration of AuPt to be 0.05% in the core-shell nanocomposite to exhibit the highest current response. Furthermore, immobilization of anti-E. coli monoclonal antibodies (anti-E. coli mAb) onto the surface of modified electrodes was performed using amine coupling. The high specific binding of E. coli cells onto the surface of the immuno-electrode was measured as a direct function of change in transient capacitance with time that was measured at low and high frequencies. The resultant immunosensor (bovine serum albumin/anti-E. coli mAb/AuPt0.05@GQDs/FTO) demonstrated a detection range (5 to 4.5 × 103 cells/mL), with the detection limit as low as 1.5 × 102 cells/mL, and an excellent sensitivity ∼171,281.40 μF-1 mL cells-1 cm-2 without the use of any labels (R2-0.99). These findings were further verified using real sample analysis wherein the immuno-electrode demonstrated outstanding sensitivity, the highest noticed so far. More interestingly, the high resuability ∼48 weeks (RSD-5.92%) and excellent reproducibility in detection results (RSD ∼ 9.5%) testify its potential use in a clinical setting. The results reveal the usefulness of the surface-engineered AuPt@GQDs core-shell nanocomposite as an electrode modifier that can be used for the development of newer on-site monitoring devices to estimate trace levels of pathogens present as contaminants in food samples.

Evaluation of the Increased Genetic Resolution and Utility for Source Tracking of a Recently Developed Method for Genotyping Cyclospora cayetanensis.

Cyclospora cayetanensis is a foodborne parasite that causes cyclosporiasis, an enteric illness in humans. Genotyping methods are used to genetically discriminate between specimens from cyclosporiasis cases and can complement source attribution investigations if the method is sufficiently sensitive for application to food items. A very sensitive targeted amplicon sequencing (TAS) assay for genotyping C. cayetanensis encompassing 52 loci was recently designed. In this study, we analyzed 66 genetically diverse clinical specimens to assess the change in phylogenetic resolution between the TAS assay and a currently employed eight-marker scheme. Of the 52 markers, ≥50 were successfully haplotyped for all specimens, and these results were used to generate a hierarchical cluster dendrogram. Using a previously described statistical approach to dissect hierarchical trees, the 66 specimens resolved into 24 and 27 distinct genetic clusters for the TAS and an 8-loci scheme, respectively. Although the specimen composition of 15 clusters was identical, there were substantial differences between the two dendrograms, highlighting the importance of both inclusion of additional genome coverage and choice of loci to target for genotyping. To evaluate the ability to genetically link contaminated food samples with clinical specimens, C. cayetanensis was genotyped from DNA extracted from raspberries inoculated with fecal specimens. The contaminated raspberry samples were assigned to clusters with the corresponding clinical specimen, demonstrating the utility of the TAS assay for traceback efforts.

Development and Application of Four Foodborne Pathogens by TaqMan Multiplex Real-Time PCR.

A TaqMan multiplex real-time PCR (mRT-PCR) was developed to detect simultaneously Salmonella spp., Escherichia coli O157, Staphylococcus aureus, and Listeria monocytogenes in food samples. The method involves four sets of primers and probes tailored to the unique DNA sequences found in the invA, nuc, rfbE, and hly genes of each pathogen. The generated standard curves, correlating gene copy numbers with Ct values, demonstrated high accuracy (R2 > 0.99) and efficiency (92%-104%). Meanwhile, the limit of detection was 100 CFU/mL for the four target bacteria in artificially contaminated food samples after 6-8 h of enrichment. The assay's effectiveness was further verified by testing 80 naturally contaminated food samples, showing results largely in agreement with traditional culture methods. Overall, this newly developed TaqMan mRT-PCR, inclusive of a pre-enrichment step, proves to be a dependable and effective tool for detecting single or multiple pathogens in diverse food items, offering significant potential for in vitro diagnostics.

A cost-effective and field-deployable sensing system for chip-integrated detection of bacteria with the naked eye

To tailor the broadly applied polymerase chain reaction (PCR) technology to point-of-care settings, an intuitive, time-saving, and cost-effective method to determine the DNA amplification results has significant practical potential. Herein we present an economical and field-deployable sensing system that enables naked-eye readout of PCR results. The proposed system is powered by an amplification enhancer to greatly enhance the positive-to-background ratio and a palm-sized readout device to assist on-site applications. High specificity, as well as single-copy sensitivity, was achieved in detecting Salmonella enterica and other bacterial pathogens. Using the proposed system, we identified the pathogenic bacterial strains in artificially contaminated food samples easily, quickly, and reliably. Also crucial for point-of-care testing, this naked-eye readout system applies to microfluidic platforms with the sample volumes scaled down to 1 μL. The time savings, portability, and cost-effectiveness of the established sensing system make it transformative for on-chip bacteria detection in point-of-care and field settings, especially in resource-limited areas.

Development, evaluation and application of a polymerase spiral reaction (PSR) based assay on accurate detection of Vibrio parahaemolyticus viable cells and virulence factors from rice product

Vibrio parahaemolyticus is a predominate cause of microbiological food safety. This study aimed to develop a polymerase spiral reaction (PSR) based assay for rapid and visual detection on V. parahaemolyticus. Firstly, the specificity and conservativity of species-specific target tlh gene were determined. Secondly, targeting tlh and virulence factors tdh and trh, PSR assay was developed and optimized using constructed positive plasmid controls and standard strain. Visualized result determination using fluorescent dye was applied to avoid false positive issues. Thirdly, 44 strains were used for specificity evaluation. Fourthly, pure and mixed species artificial contamination systems were included to test its applicability. In addition, a propidium monoazide (PMA)-PSR assay was set up to determine viable but non-culturable (VBNC) cells. Target tlh gene acquired high specificity and PSR assay was optimized to carry out at 65ᵒC for 1 h. Detection limit of PSR assay in purified genomic DNA, plasmid and artificially contaminated food samples were 5.31–53.1 pg/μL, 100 copies, and 103 CFU/mL, respectively, demonstrating the sensitivity as 100-fold higher than regular PCR. With PMA-PSR assay, VBNC cells were accurately determined without the disruption of dead cells and food compositions. Thus, PSR is applicable in detection of V. parahaemolyticus viable cells and virulence factors.

Rapid, on-site and yes-or-no detection of Salmonella typhimurium in foods using Argonaute-enabled assay

Early detection and control of foodborne diseases are crucial for ensuring food safety and consumer health. Therefore, there is a need for faster and more sensitive detection methods. We developed a rapid, portable and visible lateral flow biosensor (LFB) for foodborne pathogenic bacteria detection powered by a Pyrococcus furiosus Argonaute nuclease (PfAgo), named as PfAgo-LFB. LAMP (Loop-mediated isothermal amplification) was used to amplify the target nucleic acid. By using PfAgo as the “lighting up” technology, we can overcome the limitations of false-positive results and lack of effective endpoint monitoring in LAMP, greatly enhancing its applicability. It turned out that PfAgo-LFB was able to provide yes-or-no (positive or negative) results with a limit of detection (LOD) as low as 1 CFU/mL for S. typhimurium and the detection range was between 100 to 108 CFU/mL. It was able to secure a sample-to-answer test for contaminated food samples in less than 45 min and demonstrated satisfactory selectivity. In summary, we designed a PfAgo-LFB that rendered a novel, rapid, accurate, easy-to-deploy, on-site and yes or no detection of S. typhimurium, which could be generalized for building a “one-for-all” biosensing platform.

Digital PCR assay for the specific detection and estimation of Salmonella contamination levels in poultry rinse

Strains of Salmonella are a frequent cause of foodborne illness and are known to contaminate poultry products. Most Salmonella testing methods can qualitatively detect Salmonella and cannot quantify or estimate the Salmonella load in samples. Therefore, the aim of this study was to standardize and validate a partitioned-based digital PCR (dPCR) assay for the detection and estimation of Salmonella contamination levels in poultry rinses. Pure culture Salmonella strains were cultured, enumerated, cold-stressed for 48 h, and used to inoculate whole carcass chicken rinse (WCCR) at 1–4 log CFU/30 mL and enriched at 37 °C for 5 h. Undiluted DNA samples with primer and probes targeting the Salmonella-specific invA gene were used for the dPCR assay. The dPCR assay was highly specific, with a limit of detection of 0.001 ng/μL and a limit of quantification of 0.01 ng/μL. The dPCR assay further showed no PCR reaction inhibition up to 5 μg of crude DNA extract. The assays accurately detected all cold-stressed Salmonella in inoculated WCCR samples following a 5-h enrichment. Most importantly, when converted to log, the dPCR copies/μL values accurately estimated the inoculated Salmonella levels. The dPCR assay standardized in this study is a robust method for the detection and estimation of Salmonella concentration in contaminated food samples. This approach can allow same-day decision-making for poultry processors attempting to maintain limits and controls on Salmonella contamination.

Occurrence and molecular characterization of Salmonella Typhimurium and Salmonella Enteritidis isolates from contaminated food samples from Palestine

Salmonella is one of the most frequently isolated foodborne pathogens. It is of major public health concern worldwide. Poultry meat and eggs represent an important source of Salmonellae organism for consumer health. This study aimed to evaluate the occurrence of Salmonella enterica serotype Typhimurium and Enteritidis using multiplex PCR (mPCR) among isolates collected from the local market and to assess genetic relationships between isolates of S. Typhimurium, which was the only serotype isolated from the tested food samples. This was done using virulence factors profiling and fingerprint profiling by random amplified polymorphic DNA (RAPD-PCR) and repetitive sequence PCR (REP-PCR) using enterobacterial repetitive intergenic consensus (ERIC-PCR) and interspersed repetitive DNA sequence BOXAIR-PCR.
 The overall occurrence percentage of S. Typhimurium and S. Enteritidis out of 51 isolates was 54.9% and 0.0%, respectively. Only 13 out of 17 virulence genes were detected in these isolates. The occurrence of the detected virulence genes among these isolates was 100%, 50.0%,46.4%, 39.3%, 35.7%, 35.7%, 32.1%, 25.0%, 25.0%, 17.6%, 14.3%, 14.3%, 3.6% for invA, sopB, prgH, sitC, pefA, tolC, cdtB, msgA, sifA, iroN, spiA, ipfC and pagC, respectively. The remaining virulence genes were absent in all of the isolates. Based on the combination of the presence and absence of virulence genes, eight profiles were detected among these isolates, the most common genetic profile was V5 (each 32.1%). Based on this genetic profile at cut-off point 96.0%, both ERIC and BOX primers allowed for discrimination into 4 and 6 clusters or clones of 16 S. Typhimurium isolates, respectively. Results of PCR typing methods showed that, three strains clustered together using both ERIC-PCR and BOX-PCR typing methods and they had the same virulotype (V1), while other four strains also clustered together by both typing methods and had the same virulotype (V8).
 Contamination of food with Salmonellae especially with S. Typhimurium was high and indicated a bad microbiological quality of food. This emphasizes the need for rigorous public health and food safety methods to lower the human health hazard and risk associated with Salmonellae infection.

Development of rapid on-site detection of Salmonella Enteritidis, S. Typhimurium, and S. Thompson in food samples using an ultrafast PCR system

The conventional Salmonella serotyping methods are labor intensive and time-consuming; therefore, rapid identification method is needed to detect outbreaks early and prevent their spread. In this study, we developed for the first time a detection method for the rapid and accurate on-site serotyping of Salmonella enterica subsp. enterica serovar Enteritidis, Thompson, and Typhimurium, all of which have epidemiological importance worldwide. This method, which is based on specific marker genes, was constructed with portable equipment and a simple DNA extraction step. For the specific detection of the three serovars, genetic markers obtained via a bioinformatics analysis were selected and showed high specificity for 97 strains. The ultrafast polymerase chain reaction (PCR) showed high linearity (≥0.997) in both pure culture and a spiked food sample, with a detection specificity of 102 colony forming unit (CFU)/ml; these results are consistent with real-time PCR. Moreover, ultrafast PCR was further improved for developing direct ultrafast PCR method for field detection, the entire procedure of which could be completed within 25 min. The test results of an artificially contaminated food sample showed that the three serovars could be detected using the direct ultrafast PCR with a very short (3 h) enrichment step when inoculated at 1 CFU/g. This method exhibited various advantages, such as its ultrafast and labor-saving characteristics, which can be applied to the diagnosis of Salmonella serovar in the field.

The survival and enterotoxin gene expression of Staphylococcus aureus planktonic and biofilm cells in quick-frozen food

Food safety issues caused by food-borne pathogenic microorganisms during low-temperature food storage have attracted broad attention. In the current study, Staphylococcus aureus cells in planktonic and different stages of biofilm formation were inoculated into four types of quick-frozen rice and flour products. The changes in culturable cell number of S. aureus in the artificially contaminated food samples stored at 4 °C and −20 °C were recorded for 60 days. Next, the PMA-qPCR method was used to explore the viable cell number and determine whether low temperature induced S. aureus enter into the viable but non-culturable (VBNC) state. Finally, the expression changes of enterotoxin gene seb in S. aureus were determined by RT-qPCR during food sample storage. Results showed that the culturable cell number of S. aureus was not significantly changed during 60 days except for the decreased culturable cell number in the 8 h biofilm cells in MF stored at −20 °C. In addition, S. aureus cells did not enter into the VBNC state during low temperature storage. Moreover, the seb gene was continuously expressed in most samples although with difference in expression level. The findings provided an alert for the risk of S. aureus contamination in quick-frozen rice and flour products.

Colorimetric aptasensor for detection of Bacillus cytotoxicus spores in milk and ready-to-use food

The high incidence of foodborne diseases caused by pathogenic bacteria raises concerns worldwide and imposes considerable public healthcare challenges. This is especially observed with dormant spores of Bacilli, which can often survive treatments used by the food industry to kill growing bacteria. The early and rapid detection of bacterial spores is essential to ensure food safety. Commercial availability of such a test will present a high potential for food sector. We present a point-of-need colorimetric assay for detection of Bacillus cytotoxicus spores in food. The detection principle is based on spore-enhanced peroxidase-like catalytic activity of gold nanoparticles. The sensing platform consists of a microtube containing gold nanoparticles (AuNPs), and magnetic particles (MPs), both conjugated with specific aptamer BAS6R that recognize B. cytotoxicus spores. Upon the addition of the sample, spores were determined as present by the enhanced color change of the solution, due to the oxidation of tetramethylbenidine (TMB) with H2O2. The assay was evaluated by the naked eye (on/off) and quantitatively with use of a spectrophotometer. BAS6R@AuNPs aptasensor coupled to BAS6R@MPs proved to be highly sensitive, achieving the naked-eye limit of detection as low as 102 cfu/mL in water and milk, and 104 cfu/mL in mashed potatoes. Moreover, discrimination between spores of B. cytotoxicus and B. subtilis as well as bacterial vegetative cells was achieved in contaminated food samples, providing a good selectivity. This work provides a promising proof of concept for the development of instrument-free, low-cost and rapid assay for Bacillus cytotoxicus spore detection, which is able to compete in sensitivity with conventional costly and time-consuming laboratory analyses.

Development of a 3-plex droplet digital PCR for identification and absolute quantification of Salmonella and its two important serovars in various food samples

Salmonella is one of the most common foodborne pathogens that cause diarrhea in human, in which Enteritidis and Typhimurium are the top serovars frequently isolated from foodstuffs. A novel 3-plex droplet digital PCR (ddPCR) was successfully developed in this study for the simultaneous identification and absolute quantification of Salmonella and its two important serovars (Enteritidis and Typhimurium). The specificity of all the primers and probes was validated by testing 24 strains of Salmonella and 10 strains of other bacteria. Meanwhile, the anti-interference ability of this method was also evaluated using high proportion of non-target strains or various bacterial combinations. The detection limit of this 3-plex ddPCR was as low as 5 fg/μL for gDNA and 101 CFU/mL for pure cultures, respectively. Moreover, compared with quantitative PCR (qPCR), the detection limit of this ddPCR was much lower when it was applied to the detection of artificially contaminated food samples, which was 101 CFU/mL for lettuce, and 102 CFU/mL for milk and chicken juice samples, respectively. Additionally, a total of 100 retailed food samples were tested by this validated 3-plex ddPCR, which was also compared with traditional cultivation method. Notably, 19 of 20 samples showed consistent positive results by both methods, while the rest with the lowest concentration of Salmonella cells was detectable only by ddPCR. These results demonstrated that the 3-plex ddPCR established in this study presented highly specific, sensitive, and absolute quantitative capability, which had the potential to identify Salmonella, and to differentiate the two important serovars Enteritidis and Typhimurium in a wide variety of food samples.

Antimicrobial Edible Cellulose-Based (CB) Films and Coatings for Enhancing Microbial Safety of White Cheese During Storage

In this research, the antioxidant and antibacterial characteristics of edible cellulose-based film (CBEF) coating incorporated with β-carotene, Hesperedin and Propolis on the quality of the white cheese at 4°C were investigated. Samples evaluated were: Control, CBE films with 8 different contents containing natural or non-natural antimicrobial agents for 15 days at 5 day intervals. The antimicrobial effects of edible cellulosic films prepared with antimicrobial agents against Staphylococcus aureus and Escherichia coli, which are important food pathogens, were directly evaluated with artificially contaminated food samples under cold storage conditions. Fresh white cheese samples, which are one of the semi-hard traditional Turkish cheeses, were used for artificial contamination. The antimicrobial activity of the tested antimicrobial substances was measured by the paper disc method. The DPPH free radical scavenging activities, the total phenolic content, and the characteristics (FT-IR, DSC and TGA) of the CBEFs were investigated. All of the samples significantly reduced E. coli, S. aureus and total viable counts, as compared with control during the storage time. Specifically, it was determined that hesperidin sprayed films showed an antimicrobial effect on E.coli growth and carotene sprayed films showed an antimicrobial effect on S. aureus growth. Growth of both bacterial species was highly inhibited (>50%) in the presence of 20% propolis. It was concluded that β-carotene, hesperidin and propolis can be substituted as natural preservatives in white cheese. With this advanced packaging technology, food safety can be ensured, the shelf life of the product can be extended and food losses in this sector can be reduced.

Development and Evaluation of a Next-Generation Sequencing Panel for the Multiple Detection and Identification of Pathogens in Fermented Foods.

These days, bacterial detection methods have some limitations in sensitivity, specificity, and multiple detection. To overcome these, novel detection and identification method is necessary to be developed. Recently, NGS panel method has been suggested to screen, detect, and even identify specific foodborne pathogens in one reaction. In this study, new NGS panel primer sets were developed to target 13 specific virulence factor genes from five types of pathogenic Escherichia coli, Listeria monocytogenes, and Salmonella enterica serovar Typhimurium, respectively. Evaluation of the primer sets using singleplex PCR, crosscheck PCR and multiplex PCR revealed high specificity and selectivity without interference of primers or genomic DNAs. Subsequent NGS panel analysis with six artificially contaminated food samples using those primer sets showed that all target genes were multi-detected in one reaction at 108-105 CFU of target strains. However, a few false-positive results were shown at 106-105 CFU. To validate this NGS panel analysis, three sets of qPCR analyses were independently performed with the same contaminated food samples, showing the similar specificity and selectivity for detection and identification. While this NGS panel still has some issues for detection and identification of specific foodborne pathogens, it has much more advantages, especially multiple detection and identification in one reaction, and it could be improved by further optimized NGS panel primer sets and even by application of a new real-time NGS sequencing technology. Therefore, this study suggests the efficiency and usability of NGS panel for rapid determination of origin strain in various foodborne outbreaks in one reaction.

Targeted next generation sequencing of Cyclospora cayetanensis mitochondrial genomes from seeded fresh produce and other seeded food samples

Cyclospora cayetanensis, a coccidian apicomplexan parasite, causes large outbreaks of foodborne diarrheal disease globally. Tracking the source of C. cayetanensis oocyst contamination in food items is essential to reduce, even prevent outbreaks. We previously showed that a genotyping method based on mitochondrial single nucleotide polymorphism (SNP) profiles had discriminatory power in classifying C. cayetanensis clinical isolates. In food specimens, low level contamination by oocysts and difficulties in DNA extraction present significant challenges in genotyping method development. Here, we report the development of a highly sensitive, custom-designed, targeted sequencing method based on the Illumina AmpliSeq platform; our method was capable of consistently generating near-complete mitochondrial genome sequences of C. cayetanensis from foods with low levels of contamination. To simulate environmentally observed contamination levels in foods, we seeded various food matrices, such as fresh produce and prepared dishes, with known quantities of oocysts, and isolated genomic DNA from washed food samples. Using the Ampliseq Targeted Sequencing method, we obtained near-complete mitochondrial genome sequences of C. cayetanensis from food samples seeded with as low as five to ten oocysts and used the data in downstream analysis. The flexibility of the AmpliSeq platform could potentially allow for more genomic targets to be added to achieve higher discriminatory power. This level of sensitivity in capturing high resolution genome data from contaminated food samples is a critical milestone towards the potential development of a comprehensive genotyping method for C. cayetanensis.

CRISPR Cas12a-based "sweet" biosensor coupled with personal glucose meter readout for the point-of-care testing of Salmonella.

Salmonella is a pathogen that comes from different animal-originated foods and poses a significant threat to human health. The present detection methods for Salmonella are time-consuming and labor-intensive and requires skilled workers and specialized instruments. In this study, the conservative invA was selected as the target gene, and a quantitative detection method for Salmonella with wide availability and user-friendliness was established based on CRISPR Cas12a and a personal glucose meter (PGM). The indirect signal transformation from the original target DNA to the final glucose signal was achieved through RAA, CRISPR Cas12a reaction, enzymic reaction, and glucose signal reading by a PGM (accu-chek type from Roche). This PGMs-CRISPR assay showed a detection sensitivity of Salmonella as low as 5 colony-forming units (CFU)/reaction in either pure culture or artificially contaminated food samples and exhibited specificity between Salmonella isolates and non-Salmonella isolates. Furthermore, quantitative detection of Salmonella in spiked milk samples was also achieved within the range from 1 to 1 × 103 CFU/reaction. Subsequently, the correlation and consistency between the PGMs-CRISPR assay and quantitative polymerase chain reaction (qPCR) in detection of Salmonella in spiked milk samples were achieved. Therefore, a highly sensitive, portable, quantitative, and user-friendly detection method based on CRISPR Cas12a and PGMs was developed in this study for Salmonella detection and identification. PRACTICAL APPLICATION: A sensitive, rapid, user-friendly, and quantitative detection method based on CRISPR Cas12a for Salmonella in food has been developed in this study, which is of great significance to food safety supervision and management.

Comparative virulence of the worldwide ST19 and emergent ST213 genotypes of Salmonella enterica serotype Typhimurium strains isolated from food

Salmonella enterica Typhimurium represents one of the most frequent causal agents of food contamination associated to gastroenteritis. The sequence type ST19 is the founder and worldwide prevalent genotype within this serotype, but its replacement by emerging genotypes has been recently reported. Particularly, the ST213 genotype has replaced it as the most prevalent in clinical and contaminated food samples in Mexico and has been recently reported in several countries. In this study, the in vitro and in vivo virulence of ST213 and ST19 strains isolated from food samples in Mexico was evaluated. Three out of the five analyzed ST213 strains, showed a greater internalization capacity and increased secretion of interleukins IL-8 and IL-6 of Caco-2 cells than the ST19 strains. Microbiological counts in feces and tissues showed the ability of all strains tested to establish infection in the rat model. The ST213 strains also caused histopathological damage, characteristic of gastroenteritis in Wistar rats. In contrast to the in vitro result, one of the ST19 strains showed marked damage in the test animals. The ST213 genotype strains showed in vitro and in vivo virulence variability, but significantly higher than the observed in the ST19 genotype strains, thus such emergent genotype represents a public health concern.

Establishment and Validation of a Two-Step LAMP Assay for Detection of Bacillus cereus-Group Isolates in Food and Their Possibility of Non-haemolytic Enterotoxin Production.

The closely related members of the Bacillus cereus-group can mainly only be differentiated by whole genome sequencing. Among them, there are potentially toxin-producing bacteria. When consumed with food, these can cause vomiting or diarrhea and abdominal cramps. To date, although no EU-wide threshold exists, a bacterial count of 105 CFU/g can be regarded as critical. Specific and rapid detection of the bacteria is difficult due to their close relationship, and no loop-mediated isothermal amplification (LAMP) assay has been developed so far to detect potentially toxin-producing members of the B. cereus-group. Aim of this study was to develop a LAMP method to detect critical cell counts specifically and rapidly of potentially non-haemolytic enterotoxin (NHE)-producing cells of this group. A two-step LAMP assay was developed. First, the target sequence groEL was used to determine the representatives of the B. cereus-group. Second, since bacteria in which nheB is present are basically capable of producing enterotoxins, this gene was chosen for detection. The specificity of the developed assay was 100% for B. cereus-group isolates and 93.7% for the detection of nheB. The analytical sensitivity was 0.1 pg DNA/μl. Using simplified DNA extraction by boiling, cell-based sensitivity was determined. Targeting groEL and nheB, 11.35–27.05 CFU/reaction and 11.35–270.5 CFU/reaction were detectable, respectively. Artificially contaminated samples were investigated to prove the application in foods. Direct detection of the critical value of B. cereus-group cells was possible in 83.3% of samples and detecting the toxin-gene 50% thereof. After a 6-h incubation period, the detection rate increased to 100 and 91.7%, respectively. Additionally, 100 natively contaminated food samples were tested, also quantitatively and culturally. Samples with relevant contamination levels were reliably detected using groEL-LAMP. After a 6-h incubation period, isolates bearing the toxin gene nheB could also be reliably detected. In addition, colony material was boiled and used as a LAMP template for simple detection. Specificity for the B. cereus-group was 100 and 93.22% detecting nheB. The study demonstrated that screening of food samples with the groEL/nheB-LAMP assay can be performed within 1 day, making it possible to detect critical levels of potentially NHE-toxin-producing cells of the B. cereus-group.

Phage long tail fiber protein-immobilized magnetic nanoparticles for rapid and ultrasensitive detection of Salmonella

There is an urgent need to develop fast and sensitive detection methods for foodborne pathogens. But the conventional culture method that typically requires 2–3 days is not ideal for the rapid analysis. Food samples demonstrate a great challenge for direct detection due to the complex matrix. Hence, we present a new method based on the phage long-tail-fiber proteins (LTF4-a) immobilized magnetic nanoparticles (MNPs) for specific separation and concentration of Salmonella. The LTF4-a-MNP was prepared via the coupling of recombinant LTF4-a with MNPs and used to isolate and enrich Salmonella cells from contaminated food samples. The captured material was further integrated with the direct PCR program for accurate detection of Salmonella. Our study successfully established a new method for detecting contaminated food samples of Salmonella, the overall approach took no more than 3 h, which allowed a detection limit of 7 CFU/mL, demonstrating a promising alternative to the immunomagnetic separation method by replacing antibodies or aptamers, that is compatible with downstream analysis.