Point-of-Care Diagnostic System for Viable Salmonella Species via Improved Propidium Monoazide and Recombinase Polymerase Amplification Based Nucleic Acid Lateral Flow.

Salmonella species are prominent foodborne microbial pathogens transmitted through contaminated food or water and pose a significant threat to human health. Accurate and rapid point-of-care (POC) diagnosis is gaining attention in effectively preventing outbreaks of foodborne disease. However, the presence of dead bacteria can interfere with an accurate diagnosis, necessitating the development of methods for the rapid, simple, and efficient detection of viable bacteria only. Herein, we used an improved propidium monoazide (PMAxx) to develop a nucleic acid lateral flow (NALF) assay based on recombinase polymerase amplification (RPA) to differentiate viable Salmonella Typhimurium. We selected an RPA primer set targeting the invA gene and designed a probe for NALF. RPA-based NALF was optimized for temperature (30-43 °C), time (1-25 min), and endonuclease IV concentration (0.025-0.15 unit/µL). PMAxx successfully eliminated false-positive results from dead S. Typhimurium, enabling the accurate detection of viable S. Typhimurium with a detection limit of 1.11 × 102 CFU/mL in pure culture. The developed method was evaluated with spiked raw chicken breast and milk with analysis completed within 25 min at 39 °C. This study has potential as a tool for the POC diagnostics of viable foodborne pathogens with high specificity, sensitivity, rapidity, and cost-effectiveness.

Naked-eye detection with loop-mediated isothermal amplification for P. carotovorum subsp. carotovorum in agricultural products.

Pectobacterium carotovorum causing soft-rot disease requires on-site detection before the distribution of agricultural products. Loop-mediated isothermal amplification (LAMP), which is resistant to food inhibitors, is known for its high detection sensitivity for pathogens and when coupled with lateral flow immunoassay (LFA) enables visualizations. For detection of soft-rot disease, we developed a LAMP-LFA system targeting 16S ribosomal RNA, a partial sequence gene of P. carotovorum subsp. carotovorum. The LAMP-LFA was performed at 60 °C for 50 min followed by hybridization of digoxygenin-labeled LAMP amplicon and biotinylated probe. Detection sensitivity was 3.22 × 101 CFU/mL in pure culture, which specifically detected the target. In Chinese cabbage and potato, the target was detected up to low levels of 1.57 × 102 CFU/g and 1.29 × 102 CFU/g, respectively. This study showed potential applicability as a sensitive point-of-care system for soft-rot disease bacteria detection in agricultural products. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01315-z.

Quantitative risk assessment of foodborne Salmonella illness by estimating cooking effect on eggs from retail markets.

In this study, we performed a quantitative microbial risk assessment (QMRA) of Salmonella through intake of egg consumption after cooking (dry-heat, moist-heat, and raw consumption). Egg samples (n = 201) from retail markets were analyzed for the presence of Salmonella. In addition, temperature and time were investigated during egg transit, storage, and display. A predictive model was developed to characterize the kinetic behavior of Salmonella in eggs, and data on egg consumption and frequency were collected. Eventually, the data was simulated to estimate egg-related foodborne illnesses. Salmonella was not found in any of the 201 egg samples. Thus, the estimated initial contamination level was -4.0 Log CFU/g. With R2 values of 0.898 and 0.922, the constructed predictive models were adequate for describing the fate of Salmonella in eggs throughout distribution and storage. Eggs were consumed raw (1.5%, 39.2 g), dry-heated (57.5%, 43.0 g), and moist-heated (41%, 36.1 g). The probability of foodborne Salmonella illness from the consumption of cooked eggs was evaluated to be 6.8×10-10. Additionally, the probability of foodborne illness not applied cooking methods was 1.9×10-7, indicating that Salmonella can be reduced by cooking. Therefore, the risk of Salmonella infection through consumption of eggs after cooking might be low in S. Korea.

A review of mechanism analysis methods in multi-species biofilm of foodborne pathogens.

Biofilms are an aggregation of microorganisms that have high resistance to antimicrobial agents. In the food industry, it has been widely studied that foodborne pathogens on both food surfaces and food-contact surfaces can form biofilms thereby threatening the safety of the food. In the natural environment, multi-species biofilms formed by more than two different microorganisms are abundant. In addition, the resistance of multi-species biofilms to antimicrobial agents is higher than that of mono-species biofilms. Therefore, studies to elucidate the mechanisms of multi-species biofilms formed by foodborne pathogens are still required in the food industry. In this review paper, we summarized the novel analytical methods studied to evaluate the mechanisms of multi-species biofilms formed by foodborne pathogens by dividing them into four categories: spatial distribution, bacterial interaction, extracellular polymeric substance production and quorum sensing analytical methods.

Diverse methods of reducing and confirming false-positive results of loop-mediated isothermal amplification assays: A review.

Loop-mediated isothermal amplification (LAMP), a rapid and sensitive isothermal nucleic acid amplification method, is a promising alternative to other molecular amplification techniques due to its superior specificity and sensitivity. However, due to primer dimerization, LAMP results in nonspecific and nontemplate amplification. And during the amplification confirmation process, there is carry-over contamination. These factors can result in false-positive results that overestimate the amount of DNA, preventing accurate detection. This review outlined several techniques for reducing false-positive LAMP results before amplification and confirming false-positive results after amplification. Before the amplification step, DNA polymerase activity can be decreased with organic additives such as dimethyl sulfoxide, betaine, and pullulan to prevent nonspecific amplification. The enzyme uracil-DNA-glycosylase (UDG) can eliminate false-positive results caused by carry-over contamination, and the hot-start effect with gold nanoparticles can reduce nonspecific amplification. When confirming false-positive results using clustered regularly interspaced short palindromic repeats, guide RNA accurately detects LAMP amplification, allowing differentiation from nonspecific amplification. By confirming amplification, the colorimetric change in the deoxyribozyme (DNAzyme) formed by the reaction of the G-quadruplex sequence of the LAMP amplicon and hemin can distinguish false-positive results. Lateral flow immunoassay can distinguish false-positive results by accurately recognizing hybridized probes to LAMP amplicons.

Correction to: Electrochemical impedimetric biosensors for food safety.

[This corrects the article DOI: 10.1007/s10068-020-00776-w.].

Salmonella Risk Assessment in Poultry Meat from Farm to Consumer in Korea.

This study predicted Salmonella outbreak risk from eating cooked poultry in various methods. The incidence of Salmonella in poultry meat and the environment from farm to home for consumption was investigated. To develop the predictive models, Salmonella growth data were collected at 4-25 °C during storage and fitted with the Baranyi model. The effects of cooking on cell counts in poultry meat were investigated. Temperature, duration, and consumption patterns were all searched. A simulation in @Risk was run using these data to estimate the probability of foodborne Salmonella disease. In farm, Salmonella was detected from only fecal samples (8.5%; 56/660). In slaughterhouses, Salmonella was detected from feces 16.0% (38/237) for chicken and 19.5% (82/420) for duck) and from carcasses of each step (scalding, defeathering, and chilling) by cross contamination. In chicken (n = 270) and duck (n = 205), Salmonella was detected in 5 chicken (1.9%) and 16 duck meat samples (7.8%). Salmonella contamination levels were initially estimated to be -3.1 Log CFU/g and -2.5 Log CFU/g, respectively. With R2 values between 0.862 and 0.924, the predictive models were suitable for describing the fate of Salmonella in poultry meat with of 0.862 and 0.924. The Salmonella was not detected when poultry meat cooks completely. However, if poultry meat contaminated with Salmonella were cooked incompletely, Salmonella remained on the food surface. The risk of foodborne Salmonella disease from poultry consumption after cooking was 3.0 × 10-10/person/day and 8.8 × 10-11/person/day in South Korea, indicating a low risk.

Thermophilic helicase-dependent amplification-based CRISPR/Cas12a system: Detection of stx2 in Escherichia coli O157:H7 by controlling primer dimers.

BACKGROUND: s: To overcome the limitation of polymerase chain reaction (PCR), isothermal amplification methods such as thermophilic helicase-dependent amplification (tHDA) have been developed. However, formation of primer dimer due to the single amplification temperature are major problems of tHDA. When cross-dimerization of forward and reverse primer occurred, false-positive results can be found on the lateral flow assay (LFA) which is one of the major detection methods widely used as a point of care diagnosis. Therefore, specific method of detecting only the target amplicon is required. RESULTS: In this study, a tHDA-based CRISPR/Cas12a system was developed to detect low levels of Escherichia coli O157:H7 in fresh salad mix without the false-positive results produced by primer dimers. For the comparison of the effect in eliminating false-positive results by CRISPR/Cas12a system, LFA was also evaluated. The tHDA-based CRISPR/Cas12a system detected as low as 101 CFU/mL E. coli O157:H7 in bacterial pure culture. In LFA false-positive results were produced due to the primer dimer, whereas the primer dimer produced by tHDA was not detected in the CRISPR/Cas12a system. These results indicated that the CRISPR/Cas12a system eliminated the formation of primer dimer. In fresh salad mix, the tHDA-based CRISPR/Cas12a system combined with the filter concentration method detected 103 CFU/g E. coli O157:H7. CONCLUSION: This study was the first to amplify stx2 of E. coli O157:H7 with tHDA as an isothermal amplification method and detected the amplicon without false-positive results by combining tHDA with CRISPR/Cas12a. Therefore, this study showed great potential for detecting low levels of E. coli O157:H7 present in fresh salad mix.

Combination of filtration and immunomagnetic separation based on real-time PCR to detect foodborne pathogens in fresh-cut apple.

Rapid detection methods require pre-enrichment culture in order to detect low levels of foodborne pathogens. To rapidly detect foodborne pathogens, enrichment culture processes could be replaced. Filtration and immunomagnetic separation methods have been identified to effectively concentrate and separate target pathogens from foods. In this study, a combination of filtration and immunomagnetic separation (IMS) has enabled the rapid and sensitive detection of foodborne pathogens. The pretreatment method, including separation and concentration procedures, increased sensitivity 10-100-fold. The sensitivity of a combination method using filtration and IMS to detect Escherichia coli O157:H7 and Salmonella enterica subsp. enterica serovar Typhimurium was 100-101 CFU/10 mL. In fresh-cut apples, IMS combined with filtration effectively improved the detection limit of real-time PCR to 2.70 × 101 CFU/g in E. coli O157:H7 and 1.80 × 102 CFU/g in Salmonella. The filtration simplified processing of large-volumes (250 mL) and effectively concentrated pathogens while decreasing immunomagnetic beads used in IMS. Bacterial concentration by IMS combined with filtration increased sensitivity 10-100-fold compared with control. In addition, the application of IMS effectively removed concentrated residual food material (10-15 mg/mL) after filtration, improving relative sensitivity. In conclusion, this method may detect foodborne pathogen in foods such as fresh-cut fruits in a more rapid and sensitive fashion than traditional culture-based methods.

Antibacterial activities of polyphenols against foodborne pathogens and their application as antibacterial agents.

Polyphenols are secondary metabolites produced in higher plants. They are known to possess various functional properties in the human body. Polyphenols also exhibit antibacterial activities against foodborne pathogens. Their antibacterial mechanism is based on inhibiting bacterial biofilm formation or inactivating enzymes. Food-derived polyphenols with such antibacterial activity are natural preservatives and can be used as an alternative to synthetic preservatives that can cause side effects, such as allergies, asthma, skin irritation, and cancer. Studies have reported that polyphenols have positive effects, such as decreasing harmful bacteria and increasing beneficial bacteria in the human gut microbiota. Polyphenols can also be used as natural antibacterial agents in food packaging system in the form of emitting sachets, absorbent pads, and edible coatings. We summarized the antibacterial activities, mechanisms and applications of polyphenols as antibacterial agents against foodborne bacteria.

A Review of Isothermal Amplification Methods and Food-Origin Inhibitors against Detecting Food-Borne Pathogens.

The isothermal amplification method, a molecular-based diagnostic technology, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), is widely used as an alternative to the time-consuming and labor-intensive culture-based detection method. However, food matrices or other compounds can inhibit molecular-based diagnostic technologies, causing reduced detection efficiencies, and false-negative results. These inhibitors originating from food are polysaccharides and polyphenolic compounds in berries, seafood, and vegetables. Additionally, magnesium ions needed for amplification reactions can also inhibit molecular-based diagnostics. The successful removal of inhibitors originating from food and molecular amplification reaction is therefore proposed to enhance the efficiency of molecular-based diagnostics and allow accurate detection of food-borne pathogens. Among molecular-based diagnostics, PCR inhibitors have been reported. Nevertheless, reports on the mechanism and removal of isothermal amplification method inhibitors are insufficient. Therefore, this review describes inhibitors originating from food and some compounds inhibiting the detection of food-borne pathogens during isothermal amplification.

Filtration-based LAMP-CRISPR/Cas12a system for the rapid, sensitive and visualized detection of Escherichia coli O157:H7.

Escherichia coli O157:H7 is a major cause of fresh vegetable-associated infections that can threaten human health. A method for rapidly detecting food-borne pathogens should be developed for safe food management. A clustered regularly interspaced short palindromic repeats (CRISPR)-based detection method has the potential to greatly advance biosensing technology through its high sensitivity and specificity. In this study, we developed a rapid, sensitive, and visualized method of detecting E. coli O157:H7 (stx2 gene) based on a loop-mediated isothermal amplification (LAMP)-CRISPR/Cas12a system. The developed method was able to rectify the common false-negative results produced by LAMP, and the detection limit was 1.22 × 100 CFU/mL in pure culture. Furthermore, the LAMP-CRISPR/Cas12a system using filtration enrichment successfully detected 4.80 × 100 CFU/g of E. coli O157:H7 in romaine lettuce without pre-microbial enrichment culture. Consequently, the LAMP-CRISPR/Cas12a system is a useful technique for rapid and sensitive detection of E. coli O157:H7 in fresh products.

Review of multi-species biofilm formation from foodborne pathogens: multi-species biofilms and removal methodology.

Multi-species biofilms are ubiquitous worldwide and are a concern in the food industry. Multi-species biofilms have a higher resistance to antimicrobial therapies than mono-species biofilms. In addition, multi-species biofilms can cause severe foodborne diseases. To remove multi-species biofilms, controlling the formation process of extracellular polymeric substances (EPS) and quorum sensing (QS) effects is essential. EPS disruption, inhibition of QS, and disinfection have been utilized to remove multi-species biofilms. This review presents information on the formation and novel removal methods for multi-species biofilms.

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.

Enhancing safety and quality of shrimp by nanoparticles of sodium alginate-based edible coating containing grapefruit seed extract.

Edible coatings are safe and effective in extending the shelf life of foods. In this study, a nanoparticle-based edible coating solution was prepared, containing alginate as a coating agent and grapefruit seed extract as an antibacterial agent to improve the safety and quality of shrimp during storage. Shrimp coated with this formulation were maintained at 4°C for 8 days, and periodically analyzed for changes in sensory, chemical [total volatile basic nitrogen (TVB-N) and pH] and microbial parameters. The uncoated shrimp exceeded the microbiological limits at 7.87 log CFU/g on Day 4 of storage, whereas the nanoparticle-based coated shrimp did not exceed the limit by Day 8 of storage. In addition, uncoated shrimp tended to maintain their quality, while uncoated shrimp deteriorated due to increased TVB-N values, pH values, and off-flavors. Nanoparticles are easily dispersed in food to minimize flavor impact and enhance diffusion and bioactivity. We concluded that the nanoparticles coating extended the shelf life of shrimp by more than 5 days. Therefore, the use of nanoparticle-based coatings could be a new and effective way to maintain shrimp quality.

Development of enhanced selective media for detection of Vibrio parahaemolyticus in oysters.

This study was undertaken to develop enhanced selective media for detection of Vibrio parahaemolyticus in oysters. Primarily, tryptic soy agar (TSA) was supplemented with 4.5-5% NaCl, 0.1-0.5% oxgall, and/or 1-2% sodium citrate, and adjusted to pH 8-9. A total of 21 Vibrio spp., 24 indicators, and 26 food-borne isolates were streaked on the modified media, followed by 24 h of incubation at 37 °C. While all the indicators and isolates failed to grow on TSA containing 5% NaCl, 0.5% oxgall, and 2% sodium citrate (TSAOSS1; pH 9), V. parahaemolyticus was culturable on this selective medium. Particularly, the ability of TSAOSS1 to quantify V. parahaemolyticus in oysters was superior to thiosulphate citrate bile salts sucrose (TCBS) agar. V. parahaemolyticus distinctly produced its white-yellowish, round, and edge-pointed colony on TSAOSS1. TSAOSS1 with high selectivity potentials over TCBS may be a promising alternative for detection of V. parahaemolyticus in seafoods or natural reservoirs. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at (10.1007/s10068-021-00877-0).

Optimization of resuscitation-promoting broths for the revival of Vibrio parahaemolyticus from a viable but nonculturable state.

This study was conducted to examine the effect of formulated resuscitation-promoting broths on the revival of viable but nonculturable Vibrio parahaemolyticus induced by cold and starvation stresses. Vibrio parahaemolyticus was incubated in artificial sea water at 4 °C for more than 8 months until this bacterium became undetectable, while retaining its intact cell count of more than 105 CFU/field over time. On day 250, V. parahaemolyticus was collected and enriched in tryptic soy broth supplemented with 3% NaCl, 10,000 U/mg catalase, 2% sodium pyruvate, 20 mM MgSO4, 5 mM EDTA, and a cell-free supernatant taken from V. parahaemolyticus ATCC 17802 in the stationary phase (pH 8). V. parahaemolyticus returned partially to a culturable state with a maximal cell density of 7.91 log CFU/mL in this formulated medium following 7 days of enrichment at 25 °C. In contrast, no V. parahaemolyticus was resuscitated when enriched in alkaline peptone water and tryptic soy broth.

Rapid detection of E. coli O157:H7 by a novel access with combination of improved sample preparation and real-time PCR.

Foodborne bacteria are typically present at very low concentrations in food. This study describes a quick and simple method for concentrating E. coli O157:H7 present in lettuce and cabbage, without microbial enrichment culture. This method involved reducing the extraction buffer and DNA elution volumes. The extraction buffer volume was adjusted to 225, 100, 50, 25, and 12.5 mL to isolate E. coli O157:H7 from 25 g of lettuce or cabbage. DNA was concentrated and compared using real-time PCR. When using 12.5 mL of buffer, < 4 CFU/g of E. coli O157:H7 could be detected within 2 h without enrichment. This result is 100-fold sensitive than pretreatment with of the conventional method using 225 mL. It is suggested that this method could contribute to the prevention of food poisoning accidents in institutional catering settings, such as schools or military facilities, by the rapid and sensitive detection of pathogens without special equipment prior to food consumption stages.

Electrochemical impedimetric biosensors for food safety.

Electrochemical impedimetric biosensors (EIBs) have a simple structure and can be used to rapidly and sensitively detect and measure hazards in food. EIBs detect and measure target molecules by transducing biochemical reactions on their surface to electrical signal outputs responding to a sinusoidal electrical signal input. Due to their structural simplicity and analytical sensitivity, EIBs are regarded as the most potent method of food hazard monitoring that can be implemented in the food supply chain. This paper discusses the theoretical background, structure, and construction of EIB and its applications in food safety.

Rapid and sensitive detection of E. coli O157:H7 and S. Typhimurium in iceberg lettuce and cabbage using filtration, DNA concentration, and qPCR without enrichment.

The outbreaks due to the low number of foodborne pathogens present in ready-to-eat products can be prevented by rapid and sensitive detection method. However, as a conventional detection method, it is impossible to monitor foodborne bacteria existing which is less than 50 cfu/25 g in a food. This study was designed to investigate the possibility of detecting 1 cfu in the short-term through filtration, DNA concentration, and qPCR. As a result of the filtration + DNA concentration method, the recovery concentrations of Escherichia coli O157:H7 and Salmonella Typhimurium was not significantly different from initial inoculation (>7 cfu/25 g). In iceberg lettuce and cabbage, this method was able to detect 7 and 7 cfu/25 g of E. coli and 68 and 5 cfu/25 g of S. Typhimurium. We demonstrated the potential of the filtration + DNA concentration method as a shorter time alternative to conventional enrichment-based rapid detection in vegetables.