Rapid Identification Of Foodborne Pathogens Research Articles

Accelerated Detection of Foodborne Pathogens and Toxins: Techniques and Applications – A Review

Pathogens associated with food and their corresponding toxins, including mycotoxins, pose a sig¬nificant threat to public health, leading to various health issues worldwide, particularly affecting infants, young children, and the elderly. The rise in foodborne illnesses highlights the crucial importance of food safety in ensuring a reliable food supply and promoting health. Timely detection of foodborne pathogens, toxins, and mycotoxins is essential for reducing the incidence of foodborne diseases. This review aims to provide comprehensive insights into the methodology for rapid detection, detailing the principles, appli¬cations, advantages, and limitations to enhance current knowledge. Conventional culture-based methods for foodborne pathogen identification are selective and are hindered by significant time demands, labor intensity, challenges from complex sample preparation, delayed results, and the need for specialized per¬sonnel. In contrast, the new emerging detection techniques such as immunological, nucleic acid-based, and biosensor-based methods offer time efficiency, reduced labor, improved accuracy, sensitivity, specificity, and reliability, and are user-friendly. Recently, numerous novel techniques have been developed for the rapid identification of foodborne pathogens, toxins, and mycotoxins, transforming food safety practices. Providing rapid and accurate results is crucial for controlling and managing foodborne disease outbreaks while ensuring food safety. As the global food supply chain becomes more complex, advancing rapid and automated detection methods remains essential for safeguarding food safety and quality, as well as enhanc¬ing public health.

Raman-Activated Cell Ejection for Validating the Reliability of the Raman Fingerprint Database of Foodborne Pathogens

Raman spectroscopy for rapid identification of foodborne pathogens based on phenotype has attracted increasing attention, and the reliability of the Raman fingerprint database through genotypic determination is crucial. In the research, the classification model of four foodborne pathogens was established based on t-distributed stochastic neighbor embedding (t-SNE) and support vector machine (SVM); the recognition accuracy was 97.04%. The target bacteria named by the model were ejected through Raman-activated cell ejection (RACE), and then single-cell genomic DNA was amplified for species analysis. The accuracy of correct matches between the predicted phenotype and the actual genotype of the target cells was at least 83.3%. Furthermore, all anticipant sequencing results brought into correspondence with the species were predicted through the model. In sum, the Raman fingerprint database based on Raman spectroscopy combined with machine learning was reliable and promising in the field of rapid detection of foodborne pathogens.

Rapid Identification of Foodborne Pathogens in Limited Resources Settings Using a Handheld Raman Spectroscopy Device

Rapid and precise methods to detect pathogens are paramount in ensuring food safety and selecting appropriate disinfection treatments. Raman spectrometry is a promising technology being investigated for detecting pathogens and achieving rapid, culture-free, and label-free methods. Nonetheless, previous Raman techniques require additional steps, including the preparation of slides that could introduce significant variability. In this study, we investigated the capability of a Raman handheld device for rapid identification of monocultures of Listeria monocytogenes, Salmonella Typhimurium, Escherichia coli O157:H7, and Staphylococcus aureus, and the combination of co-cultures in BHI broth suspension by utilising principal component analysis (PCA) and support vector machine (SVM) classification of Raman spectra. The detection method accurately identified monocultures (0.93 ± 0.20), achieving good discrimination after 24 h of bacterial growth. However, the PCA–SVM system was less accurate for classifying co-cultures (0.67 ± 0.35). These results show that this method requires an isolation step followed by biomass enrichment (>8 log10 CFU/mL) for accurate identification. The advantage of this technology is its simplicity and low-cost preparation, achieving high accuracy in monocultures in a shorter time than conventional culture-dependent methods.

Preparation of an AgNPs@Polydimethylsiloxane (PDMS) multi-hole filter membrane chip for the rapid identification of food-borne pathogens by surface-enhanced Raman spectroscopy

The consumption of food infected with food-borne pathogens has become a global public health problem. Therefore, it is monitor food-borne infections to avoid health and financial consequences. The rapid detection and differentiation of bacteria for biomedical and food safety applications continues to be a significant challenge. Herein, we present a label-free surface-enhanced Raman scattering approach for separating harmful bacteria from food. The method relies on the ascorbic acid reduction method to synthesize silver nanoparticles (AgNPs) and a polydimethylsiloxane (PDMS) multi-hole filter membrane chip (AgNPs@PDMS multi-hole filter membrane chip). Surface-enhanced Raman spectroscopy (SERS) was used, followed by multivariate statistical analysis to differentiate five important food-borne pathogens, including Staphylococcus aureus, Salmonella typhimurium, Listeria monocytogenes, Clostridium difficiles and Clostridium perfringens. The results demonstrated that compared to normal Raman signals, the intensity of the SERS signal was greatly enhanced with an analytical enhancement factor of 5.2 × 103. The spectral ranges of 400–1800 cm−1 were analyzed using principal component analysis (PCA) and stepwise linear discriminant analysis (SWLDA) were used to determine the optimal parameters for the discrimination of food-borne pathogens. The first three principal components (PC1, PC2, and PC3) accounted for 87.3% of the total variance in the spectra. The established SWLDA model had 100% accuracy and cross-validation accuracy, which accurately distinguished the SERS spectra of the five species. In conclusion, the SERS technology based on the AgNPs@PDMS multi-hole filter membrane chip was useful for the rapid identification of food-borne pathogens and can be employed for food quality management.

One-Day Molecular Detection of Salmonella and Campylobacter in Chicken Meat: A Pilot Study.

Salmonella and Campylobacter ssp. are bacterial pathogens responsible for most foodborne infections in EU countries. Poultry serves as a reservoir for these pathogens, and its important role in the meat industry makes it essential to develop a rapid detection assay able to provide results in one day. Indeed, the rapid identification of foodborne pathogens is an important instrument for the monitoring and prevention of epidemic outbreaks. To date, Salmonella and Campylobacter screening is mainly conducted through molecular methods (PCR or real-time PCR) performed after 18–24 h long enrichments. In this study, we evaluated short enrichments (0, 2, 4, and 6 h) combined with a colorimetric loop-mediated isothermal AMPlification (LAMP) or real-time PCR to detect Salmonella and Campylobacter in poultry meat contaminated at different concentration levels (101, 103, and 105 CFU/g). Our results show that real-time PCR allows the detection of Salmonella and Campylobacter, even after shorter enrichment times than prescribed by ISO references; particularly, it detected Salmonella down to 101 CFU/g since T0 and Campylobacter from 103 CFU/g since T0. Detection with LAMP was comparable to real-time PCR without the requirement of a thermal cycler and with shorter execution times. These characteristics make colorimetric LAMP a valid alternative when one-day results are needed, improving the timely identification of positive meat batches, even in the absence of specialized instrumentation.

Ultrafast bacterial cell lysis using a handheld corona treater and loop-mediated isothermal amplification for rapid detection of foodborne pathogens

Bacterial foodborne diseases have become a growing global health concern, and nucleic acid-based analysis is considered as a promising approach for rapid identification of foodborne pathogens. Accordingly, nucleic acid isolation becomes the prerequisite first step for subsequent bioanalysis. However, current nucleic acid extraction suffers from operation time delayed, amplification inhibited by chemicals, or bulky instruments required. Here, we presented an ultrafast bacterial cell lysis induced by corona discharge within 40 s and combined it with loop-mediated isothermal amplification (LAMP) for the first time to achieve rapid and convenient detection of foodborne pathogens. Firstly, the viability and morphological changes revealed that corona discharge could destabilize bacterial cell envelope and eliminate more than 90% of Salmonella enterica (S. enterica) or Staphylococcus aureus (S. aureus) within 40 s. More surprisingly, more DNA were released from 100-103 CFU of electrical lysed S. enterica than equivalent thermal lysed ones, manifesting advantages of corona discharge on shorter preparation time and greater yields of DNA. Furthermore, the advantages of this ultrafast bacterial cell lysis were leveraged when collaborating with colorimetric LAMP. After conducting colorimetric LAMP in a water bath at 63 °C for 1 h, we could visually detect released DNA from 100 CFU of electrical lysed S. enterica and 103 CFU of electrical lysed S. aureus, respectively. Finally, we validated the applicability of the electrical lysis combined with LAMP reaction to detect (viable) S. enterica and S. aureus in artificially spiked pork samples. Therefore, we provided a rapid and convenient bioanalysis platform integrating an ultrafast electrical lysis and LAMP detection, opening up its extensive applications for versatile pathogens in resource-limited settings.

Rapid Detection of Microbial Mass Spectra VITEK-MS for Campylobacter jejuni and Listeria monocytogenes

A matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) method were established to detect Campylobacter jejuni and Listeria monocytogenes from a chicken farm and samples from commercial live chickens. Suspected isolates were pretreated and crystallized by matrix solutions consisting of ethanol, acetonitrile, and water with different proportions. The effect of matrix composition was evaluated by examining the differences in characteristic peak values and relative abundances. Significantly different matrix solutions were needed for the detection of C. jejuni and L. monocytogenes: the former did not require lysis by formic acid and was sensitive to trifluoroacetic acid (TFA), but the latter was just the opposite. Control of the water content in matrix solution, TFA concentration, and matrix concentration will enhance the number and relative abundance of characteristic peaks and improve the detection rate. An optimized pretreatment enables the rapid identification of foodborne pathogens, which will make it possible to improve the timeliness of disease prevention and foodborne risk monitoring.

SUPERIORITY OF PARTIAL 16S rRNA GENE SEQUENCING FOR IDENTIFICATION OF BACTERIAL STRAINS ISOLATED from MEAT PRODUCTS

Unlike phenotypic methods of bacterial identification, 16S rRNA gene sequencing is an innovative DNA-based technique for the rapid identification of foodborne pathogens. In this study, the ability of both partial 16S rRNA gene sequencing and the API Rapid20E, for identification of 48 different bacterial strains isolated from meat products, plus 2 control bacterial strains, was compared. Genotypically, the partial (770 bp) 16S rRNA gene sequencing was able to identify 38 (76%) isolates to the species level and 11 (22%) isolates to the genus level, whereas only one (2%) isolate could not be correctly identified. Phenotypically, the API Rapid20E could identify 24 (48%) isolates to the specie level, and 14 (28%) isolates to the genus level, while 12 (24%) isolates could not be discriminated at any taxonomic level. The results revealed that partial 16S rRNA gene sequencing was useful in ascertaining the relevance of tested strains; hence, it can be used in food microbiology laboratory for correct and rapid identification of foodborne pathogens.

Development of a Novel Hexa-plex PCR Method for Identification and Serotyping of Salmonella Species

Salmonella is one of the most important foodborne pathogens, which causes a huge economic burden worldwide. To detect Salmonella rapidly is very meaningful in preventing salmonellosis and decreasing economic losses. Currently, isolation of Salmonella is confirmed by biochemical and serobased serotyping methods, which are time consuming, labor intensive, and complicated. To solve this problem, a hexa-plex polymerase chain reaction (PCR) method was developed using comparative genomics analysis and multiplex PCR technology to detect Salmonella and Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Agona, Salmonella Choleraesuis, and Salmonella Pullorum simultaneously. The accuracy of this method was tested by a collection of 142 Salmonella. Furthermore, the strategy described in this article to mine serovar-specific fragments for Salmonella could be used to find specific fragments for other Salmonella serotypes and bacteria. The combination of this strategy and multiplex PCR is promising in the rapid identification of foodborne pathogens.

Safety Assessment of Fresh and Processed Seafood Products by MALDI-TOF Mass Fingerprinting

Foodborne intoxications caused by the consumption of fish and other products of marine origin contaminated with bacterial pathogens are an ever-present threat, either due to bacteria and/or its metabolites. In addition, the rapid spoilage of seafood due to microbial activity, results in high economic losses. The development of the microbiota in seafood products depends on the microbiological ambience of capture, processing and storage, and the applied preservation method. Thus, pathogenic and spoilage bacterial species in seafood may come from the indigenous microbiota of the aquatic ambience or are introduced by contamination during processing. Rapid and accurate bacterial species identification is essential for an effective control program to ensure safety and quality of either processed or minimally processed seafood. In the present work, matrix-assisted laser desorption ionization-time of flight mass spectrometry was successfully applied to identify 26 bacterial strains isolated from fresh fish and processed seafood samples. The approach was based on the comparison of unknown spectra to a reference spectral library and demonstrated to be a fast and accurate technique for bacterial species differentiation, which can be used for the rapid identification of foodborne pathogens and spoilage bacteria potentially present in products of marine origin.

Rapid and reliable detection of 11 food-borne pathogens using thin-film biosensor chips

Traditional methods for identifying food-borne pathogens are time-consuming and laborious, so it is necessary to develop innovative methods for the rapid identification of food-borne pathogens. Here, we report the development of silicon-based optical thin-film biosensor chips for sensitive detection of 11 food-borne pathogens. Briefly, aldehyde-labeled probes were arrayed and covalently attached to a hydrazine-derivatized chip surface, and then, biotinylated polymerase chain reaction (PCR) amplicons were hybridized with the probes. After washing and brief incubation with an antibiotin immunoglobulin G-horseradish peroxidase conjugate and a precipitable horseradish peroxidase substrate, biotinylated chains bound to the probes were visualized as a color change on the chip surface (gold to blue/purple). Highly sensitive and accurate examination of PCR fragment targets can be completed within 30 min. This assay is extremely robust, sensitive, specific, and economical and can be adapted to different throughputs. Thus, a rapid, sensitive, and reliable technique for detecting 11 food-borne pathogens was successfully developed.

Rapid detection of food-borne pathogens by using molecular techniques

Traditional methods of identification of food-borne pathogens, which cause disease in humans, are time-consuming and laborious, so there is a need for the development of innovative methods for the rapid identification of food-borne pathogens. Recent advances in molecular cloning and recombinant DNA techniques have revolutionized the detection of pathogens in foods. In this study the development of a PCR-based technique for the rapid identification of the food-borne pathogens Salmonella and Escherichia coli was undertaken. Suitable primers were designed based on specific gene fimA of Salmonella and gene afa of pathogenic E. coli for amplification. Agarose gel electrophoresis and subsequent staining with ethidium bromide were used for the identification of PCR products. The size of the amplified product was 120 bp as shown by comparison with marker DNA. These studies have established that fimA and afa primers were specific for detecting Salmonella and pathogenic E. coli, respectively, in the environmental samples. Thus a rapid, sensitive and reliable technique for the detection of Salmonella and pathogenic E. coli was developed.

Identification of type A, B, E, and F botulinum neurotoxin genes and of botulinum neurotoxigenic clostridia by denaturing high-performance liquid chromatography.

Denaturing high-performance liquid chromatography (DHPLC) is a recently developed technique for rapid screening of nucleotide polymorphisms in PCR products. We used this technique for the identification of type A, B, E, and F botulinum neurotoxin genes. PCR products amplified from a conserved region of the type A, B, E, and F botulinum toxin genes from Clostridium botulinum, neurotoxigenic C. butyricum type E, and C. baratii type F strains were subjected to both DHPLC analysis and sequencing. Unique DHPLC peak profiles were obtained with each different type of botulinum toxin gene fragment, consistent with nucleotide differences observed in the related sequences. We then evaluated the ability of this technique to identify botulinal neurotoxigenic organisms at the genus and species level. A specific short region of the 16S rRNA gene which contains genus-specific and in some cases species-specific heterogeneity was amplified from botulinum neurotoxigenic clostridia and from different food-borne pathogens and subjected to DHPLC analysis. Different peak profiles were obtained for each genus and species, demonstrating that the technique could be a reliable alternative to sequencing for the rapid identification of food-borne pathogens, specifically of botulinal neurotoxigenic clostridia most frequently implicated in human botulism.

DETECTION OF SALMONELLAE IN CHICKEN AND FISH SAMPLES USING DNA PROBE

ABSTRACT The use of DNA probes for rapid identification of foodborne pathogens is an emerging field. We have standardized a method for the detection of Salmonella in food samples by using a specific insertion sequence (IS‐200) of Salmonella chromosomal DNA as the probe. The procedure for colony hybridization, and washing conditions for the stringency were established. All the ten Salmonella strains, belonging to various serotypes tested were positive while 18 other bacterial cultures, mainly belonging to the family Enterobacteriaceae such as E. coli Shigella, Proteus etc., were negative in colony hybridization. The sensitivity of the detection was found to be 105 cfu using 32P‐labeled probe. We surveyed 16 chicken and 6 fish samples from local market using IS‐200 probe after preenrichment and selective enrichment. All the samples tested were found positive with DNA probe. The major strains of Salmonella isolated from these samples were S. gallinarum, S. typhimurium, S. enteritidis, S. choleraesuis and S. paratyphi A. These results were confirmed by standard Salmonella identification method.