Detection Of Foodborne Bacterial Pathogens Research Articles

Microscopic identification of foodborne bacterial pathogens based on deep learning method

Accurate and rapid detection of foodborne bacterial pathogens is critical for food quality control. Nowadays, tracking morphological bacterial properties using microscope is still a priority at the grass-roots food supervision department due to its simplicity and low cost. However, the method requires highly professional personnel and there are certain misjudgments in the process of analysis. Automatically recognizing foodborne pathogen using deep learning algorithm to replace manual microscopy will not only reduce expert cost, artificial misjudgment, and operation time in detection, but also provide more objective and accurate identification. Here, we firstly constructed a high-quality and large-scale dataset of foodborne pathogenic bacteria, allowing the deep learning-based model to be efficiently trained and achieve accurate identification. The deep convolutional neural network-based model is capable of identifying six common foodborne pathogens, including Escherichia coli (O157:H7), Vibrio parahaemolyticus, Staphylococcus aureus, Bacillus cereus, Salmonella typhi, and Streptococcus hemolyticus, with accuracy rates of 90%–100%. This method can assist or replace the manual microscopic inspection step in traditional detection methods, and is promising to break through the traditional approach that heavily relies on manual judgment, greatly reduce the cost of experts and human errors, and provide rapid, accurate, and powerful discriminatory results in large quantities for detection.

Bacteriophage-Based Biosensors: A Platform for Detection of Foodborne Bacterial Pathogens from Food and Environment.

Foodborne microorganisms are an important cause of human illness worldwide. Two-thirds of human foodborne diseases are caused by bacterial pathogens throughout the globe, especially in developing nations. Despite enormous developments in conventional foodborne pathogen detection methods, progress is limited by the assay complexity and a prolonged time-to-result. The specificity and sensitivity of assays for live pathogen detection may also depend on the nature of the samples being analyzed and the immunological or molecular reagents used. Bacteriophage-based biosensors offer several benefits, including specificity to their host organism, the detection of only live pathogens, and resistance to extreme environmental factors such as organic solvents, high temperatures, and a wide pH range. Phage-based biosensors are receiving increasing attention owing to their high degree of accuracy, specificity, and reduced assay times. These characteristics, coupled with their abundant supply, make phages a novel bio-recognition molecule in assay development, including biosensors for the detection of foodborne bacterial pathogens to ensure food safety. This review provides comprehensive information about the different types of phage-based biosensor platforms, such as magnetoelastic sensors, quartz crystal microbalance, and electrochemical and surface plasmon resonance for the detection of several foodborne bacterial pathogens from various representative food matrices and environmental samples.

Metal-Ion-Responsive Chromogenic Probe for Rapid, On-Location Detection of Foodborne Bacterial Pathogens in Contaminated Food Items.

An amphiphilic chromogenic probe based on an oxidized di(indolyl)arylmethane backbone has been utilized for visual detection of both Cu2+ (detection limit = 8.5 ppb) and Hg2+ (detection limit = 10.2 ppb) ions via mutually independent sensing pathways. The Cu2+ ion binds to the carboxylate ends (donor site) and induces a color change from orange to yellow in the aqueous medium, while coordinating Hg2+ at the bisindolyl moiety (acceptor site) can result in the formation of a red-colored solution. Interestingly, by selecting the proper excitation channel, we can specifically excite either the monomer species or nanoaggregates. The addition of Hg2+ enhances the monomer fluorescence, while Cu2+ induces quenching. However, in both cases, metal-ion coordination triggers dissociation of a preformed self-assembled structure. Further, the in-situ-formed Cu(II) complex was utilized for rapid, on-location detection of food-borne pathogens, such as Escherichia coli (E. coli) in contaminated food items and water (detection limit = 52 CFU·mL-1). E. coli induces reduction of Cu2+ to Cu+ and transforms the yellow-colored solution into an orange-colored solution. Finally, low-cost, reusable paper strips were designed as an eco-friendly, sustainable strategy to detect bacterial pathogens.

Methods To Improve Molecular Detection of Salmonella in Complex Herbal Matrices Containing Inhibitors

Salmonella is one of the main causes of foodborne diseases worldwide. Molecular tests such as the PCR assay are rapid and sensitive and are increasingly becoming the preferred method for pathogen detection. However, the presence in the analyzed samples of substances that reduce the sensitivity of the assay or totally inhibit PCR amplification might result in failure of pathogen detection. Using a multiplex real-time PCR assay, I investigated the detection of Salmonella enterica serovar Typhimurium in three herbal matrices containing inhibiting substances: (i) chamomile (Matricaria recutita), (ii) sage (Salvia officinalis), and (iii) mint (Menthae piperitae). Internal positive controls in the multiplex PCR reactions indicated the degree of inhibition. All three herbs inhibited PCR amplification at the standard matrix concentration (10% suspension). I applied and compared four approaches for overcoming the negative effect of the matrices on the PCR detection of Salmonella. The efficiency strongly depended on the matrix and the method used for removing the inhibitory substances. By using a series of centrifugation steps combined with a direct PCR, I removed the PCR inhibitors and successfully detected the pathogen in each of the tested matrices. This approach did not significantly decrease the sensitivity of the PCR assay, and the detection of the pathogen was with a quantification cycle delay of only 1.48 ± 1.05 cycles compared with the control. Thus, the proposed simple, efficient, reliable, quick, and cost-effective method allowed for removal of PCR inhibitors and subsequent detection of foodborne bacterial pathogens in complex matrices containing PCR inhibitors.

Rapid detection of foodborne bacterial pathogens using visual high‐throughput microfluidic chip

AbstractBACKGROUNDFoodborne diseases are a major global public health concern. It is urgent to monitor foodborne pathogens from contaminated foods based on multi‐target detection.RESULTSIn this work, a self‐priming compartmentalization (SPC) microdevice made of poly(methyl methacrylate) (PMMA) was developed and integrated into a loop‐mediated isothermal amplification (LAMP) system for performing high‐throughput visual detection. The microfluidic chip is a self‐filling microwell array which can introduce reaction solution only through an access hole in a vacuum pumping system. The independent and closed wells can avoid cross‐contamination and ensure the accuracy of the results. The approach was able to perform the simultaneous identification of six foodborne pathogens (Salmonella typhimurium, Bacillus cereus, Staphylococcus aureus, Yersinia enterocolitica, Vibrio parahaemolyticus and Listeria monocytogenes) within 1 h. The results demonstrated that the method could specifically recognize the six foodborne pathogens.CONCLUSIONThe use of a LAMP‐based microdevice can be a faster alternative to conventional methods to detect pathogens in food samples, especially for those foods in which no pathogen is allowed (S. aureus), or foods for infant use. The simplicity and portability of the device can provide an alternative platform for foodborne pathogens where food safety needs accurate and timely detection confirmation. © 2020 Society of Chemical Industry

Viability Detection of Foodborne Bacterial Pathogens in Food Environment by PMA-qPCR and by Microscopic Observation.

Foodborne pathogens are responsible of foodborne diseases and food poisoning and thus pose a great threat to food safety. These microorganisms can adhere to surface and form a biofilm composed of an extracellular matrix. This extracellular matrix protects bacterial cells from industrial environmental stress factors such as cleaning and disinfection operations. Moreover, during these environmental stresses, many bacterial species can enter a viable but nonculturable (VBNC) state. VBNC cells are characterized by a loss of cultivability on conventional bacteriological agar. This leads to an underestimation of total viable cells in environmental samples, and thus poses a risk for public health. In this chapter, we present a method to detect viable population of foodborne pathogens in industrial environmental samples using a molecular method with a combination of propidium monoazide (PMA) and quantitative PCR (qPCR) and a fluorescence microscopic method associated with the LIVE/DEAD BacLight™ viability stain.

The Use of Multiplex Real-Time PCR for the Simultaneous Detection of Foodborne Bacterial Pathogens.

Foodborne pathogens continue to be a major health issue worldwide. Culture-dependent methodologies are still considered the gold-standard to perform pathogen detection and quantification. These methods present several drawbacks, such as being time-consuming and labor-intensive. The implementation of real-time PCR has allowed to overcome these limitations and even reduce costs associated with the analyses, due to the possibility of simultaneously and accurately detecting several pathogens in one single assay, with results comparable to those obtained by classical approaches. In this chapter a protocol for the simultaneous detection of two of the most important foodborne pathogens, Salmonella spp. and Listeria monocytogenes, is described.

Low-fouling surface plasmon resonance biosensor for multi-step detection of foodborne bacterial pathogens in complex food samples

Recent outbreaks of foodborne illnesses have shown that foodborne bacterial pathogens present a significant threat to public health, resulting in an increased need for technologies capable of fast and reliable screening of food commodities. The optimal method of pathogen detection in foods should: (i) be rapid, specific, and sensitive; (ii) require minimum sample preparation; and (iii) be robust and cost-effective, thus enabling use in the field. Here we report the use of a SPR biosensor based on ultra-low fouling and functionalizable poly(carboxybetaine acrylamide) (pCBAA) brushes for the rapid and sensitive detection of bacterial pathogens in crude food samples utilizing a three-step detection assay. We studied both the surface resistance to fouling and the functional capabilities of these brushes with respect to each step of the assay, namely: (I) incubation of the sensor with crude food samples, resulting in the capture of bacteria by antibodies immobilized to the pCBAA coating, (II) binding of secondary biotinylated antibody (Ab2) to previously captured bacteria, and (III) binding of streptavidin-coated gold nanoparticles to the biotinylated Ab2 in order to enhance the sensor response. We also investigated the effects of the brush thickness on the biorecognition capabilities of the gold-grafted functionalized pCBAA coatings. We demonstrate that pCBAA-compared to standard low-fouling OEG-based alkanethiolate self-assemabled monolayers-exhibits superior surface resistance regarding both fouling from complex food samples as well as the non-specific binding of S-AuNPs. We further demonstrate that a SPR biosensor based on a pCBAA brush with a thickness as low as 20nm was capable of detecting E. coli O157:H7 and Salmonella sp. in complex hamburger and cucumber samples with extraordinary sensitivity and specificity. The limits of detection for the two bacteria in cucumber and hamburger extracts were determined to be 57CFU/mL and 17CFU/mL for E. coli and 7.4×103CFU/mL and 11.7×103CFU/mL for Salmonella sp., respectively. In addition, we demonstrate the simultaneous detection of E. coli and Salmonella sp. in hamburger sample using a multichannel SPR biosensor having appropriate functional coatings.

On-chip electrical detection of parallel loop-mediated isothermal amplification with DG-BioFETs for the detection of foodborne bacterial pathogens

Over one million DG-BioFETs are used for the parallel electrical detection of LAMP reactions identifying the presence of bacterial pathogens, demonstrating a miniaturized DNA-based screening platform.

Electrical/ Electrochemical Impedance Measurement for Biological Applications

As a principle of transduction, the impedance technique has been applied in the field of biological study ranging from microbiology to cancer cell/tissue studies. This paper summarizes the applications of electrical/electrochemical impedance spectroscopy that have been practiced in our research in the past several years, mainly including two distinct fields--the detection of foodborne bacterial pathogens and the study of oral cancer cells. Foodborne diseases caused by foodborne pathogens have been a serious threat to public health for decades and remain one of the major concerns of our society. The development of rapid, sensitive, and specific methods is central to implementing effective practice to ensure food safety and security. We have been using impedance technique as a means to detect and/or quantify foodborne pathogenic bacteria. This paper will particular introduce the recent significant development in this field, including the use of microfabricated microelectrodes, microfluidic chip-based devices, nanoparticles and integration with other techniques such as dielectrophoresis. Based on the fundamental impedance microbiology, which is a technique based on the measurement of changes in electrical impedance of a culture medium or a reaction solution resulting from the bacterial growth, we have added some new aspects to this subject that have made the impedance technique a more valuable technique for detection of foodborne bacterial pathogens. These aspects include the use of different electrode systems and the analysis of impedance components using equivalent circuits for better improvement on the detection systems. The advances in microfabrication technologies have launched the use of microfabricated microarray electrodes in impedance detection and the miniaturization of impedance microbiology into a chip format, which has shown great promising in rapid detection of bacterial growth. Further, the integration of impedance technique with biosensor technology has led to the development of impedance biosensors that is expending rapidly for bacteria detection in recent years. Impedance biosensors for bacteria detection are based on the analysis of electrical properties of bacterial cells when they are attached to or associated with the electrodes. Such impedance biosensor methods have substantially reduced the assay time down to 30 min~2 h compared with growth-based impedance methods. The dimensional compatibility of those microfabricated biosensors with the target bacterial cells has enabled them to detect the binding of bacterial cells on its surface without any amplification step. On the other hand, electrical impedance spectroscopy (EIS) enables direct sensing of cellular activities occurring on an electrode surface by measuring the induced capacitance and/or resistance changes by cells attached on the electrode. Particular, EIS is capable of distinguishing different types of cells based on the cellular activity-induced electrical signals. We have demonstrated the use of real-time impedance technique to study the cellular activities of oral cancer cells in a label-free manner, including cell proliferation, adhesion, spreading, and apoptosis induced by drug treatments. Furthermore, it is known that during transformation from non-cancer cells to caner cells, the cellular morphology, structure, and function are altered, which may includes, but are not limited to changes in the nuclei, composition of the cytoplasm, structure of the cell membrane, changes in the nuclei, composition of the cytoplasm, structure of the cell membrane, and expression levels of ion channels, etc. These changes may result in alterations in cancer cell properties that could be detected by the EIS well before these morphological features that normally used in detection can be seen or biomarkers are developed. We have explored the use of real-time impedance measurements to distinguish oral cancer cells from normal oral epithelial cells in label-free manner by studying the kinetics of cell spreading and attachment of the two cell types.

Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.

Detection of foodborne bacterial pathogens from individual filth flies.

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.

Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations.

The incidence of foodborne diseases has increased over the years and resulted in major public health problem globally. Foodborne pathogens can be found in various foods and it is important to detect foodborne pathogens to provide safe food supply and to prevent foodborne diseases. The conventional methods used to detect foodborne pathogen are time consuming and laborious. Hence, a variety of methods have been developed for rapid detection of foodborne pathogens as it is required in many food analyses. Rapid detection methods can be categorized into nucleic acid-based, biosensor-based and immunological-based methods. This review emphasizes on the principles and application of recent rapid methods for the detection of foodborne bacterial pathogens. Detection methods included are simple polymerase chain reaction (PCR), multiplex PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP) and oligonucleotide DNA microarray which classified as nucleic acid-based methods; optical, electrochemical and mass-based biosensors which classified as biosensor-based methods; enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay which classified as immunological-based methods. In general, rapid detection methods are generally time-efficient, sensitive, specific and labor-saving. The developments of rapid detection methods are vital in prevention and treatment of foodborne diseases.

DNA extraction protocol for rapid PCR detection of pathogenic bacteria

Twelve reagents were evaluated to develop a direct DNA extraction method suitable for PCR detection of foodborne bacterial pathogens. Many reagents exhibited strong PCR inhibition, requiring significant dilution of the extract with a corresponding reduction in sensitivity. Most reagents also exhibited much lower recovery of DNA from the gram-positive test organism (Listeria monocytogenes) than from the gram-negative organism (Escherichia coli O157:H7), preventing unbiased detection and quantitation of both organisms. The 5× HotSHOT+Tween reagent exhibited minimal inhibition and high extraction efficiency for both test organisms, providing a 15-min single-tube DNA-extraction protocol suitable for highly sensitive quantitative PCR assays.

Multichannel oscillatory-flow multiplex PCR microfluidics for high-throughput and fast detection of foodborne bacterial pathogens

In the field of continuous-flow PCR, the amplification throughput in a single reaction solution is low and the single-plex PCR is often used. In this work, we reported a flow-based multiplex PCR microfluidic system capable of performing high-throughput and fast DNA amplification for detection of foodborne bacterial pathogens. As a demonstration, the mixture of DNA targets associated with three different foodborne pathogens was included in a single PCR solution. Then, the solution flowed through microchannels incorporated onto three temperature zones in an oscillatory manner. The effect factors of this oscillatory-flow multiplex PCR thermocycling have been demonstrated, including effects of polymerase concentration, cycling times, number of cycles, and DNA template concentration. The experimental results have shown that the oscillatory-flow multiplex PCR, with a volume of only 5 μl, could be completed in about 13 min after 35 cycles (25 cycles) at 100 μl/min (70 μl/min), which is about one-sixth of the time required on the conventional machine (70 min). By using the presently designed DNA sample model, the minimum target concentration that could be detected at 30 μl/min was 9.8 × 10(-2) ng/μl (278-bp, S. enterica), 11.2 × 10(-2) ng/μl (168-bp, E. coli O157: H7), and 2.88 × 10(-2) ng/μl (106-bp, L. monocytogenes), which corresponds to approximately 3.72 × 10(4) copies/μl, 3.58 × 10(4) copies/μl, and 1.79 × 10(4) copies/μl, respectively. This level of speed and sensitivity is comparable to that achievable in most other continuous-flow PCR systems. In addition, the four individual channels were used to achieve multi-target PCR analysis of three different DNA samples from different food sources in parallel, thereby achieving another level of multiplexing.

Antibody-Based Sensors: Principles, Problems and Potential for Detection of Pathogens and Associated Toxins

Antibody-based sensors permit the rapid and sensitive analysis of a range of pathogens and associated toxins. A critical assessment of the implementation of such formats is provided, with reference to their principles, problems and potential for ‘on-site’ analysis. Particular emphasis is placed on the detection of foodborne bacterial pathogens, such as Escherichia coli and Listeria monocytogenes, and additional examples relating to the monitoring of fungal pathogens, viruses, mycotoxins, marine toxins and parasites are also provided.

Development and application of an oligonucleotide microarray for the detection of food-borne bacterial pathogens

The rapid and accurate detection and identification of food-borne pathogenic bacteria is critical for food safety. In this paper, we describe a rapid (<4 h) high-throughput detection and identification system that uses universal polymerase chain reaction (PCR) primers to amplify a variable region of bacterial the 16S rRNA gene, followed by reverse hybridization of the products to species-specific oligonucleotide probes on a chip. This procedure was successful in discriminating 204 strains of bacteria from pure culture belonging to 13 genera of bacteria. When this method was applied directly to 115 strains of bacteria isolated from foods, 112/115 (97.4%) were correctly identified; two strains were indistinguishable due to weak signal, while one failed to produce a PCR product. The array was used to detect and successfully identify two strains of bacteria from food poisoning outbreak samples, giving results through hybridization that were identical to those obtained by traditional methods. The sensitivity of the microarray assay was 10(2) CFU of bacteria. Thus, the oligonucleotide microarray is a powerful tool for the detection and identification of pathogens from foods.

Improved template preparation for PCR-based assays for detection of food-borne bacterial pathogens.

Shigella flexneri, Salmonella enterica serotype Typhimurium, and Listeria monocytogenes were applied to FTA filters, and the filters were used directly as templates to demonstrate their sensitivity and applicability in PCR-based detection assays. With pure cultures, the sensitivities of detection by FTA filter-based PCR were 30 to 50 and 200 CFU for the gram-negative enterics and Listeria, respectively. Different numbers of S. flexneri cells were used in controlled contamination experiments with several different foods (produce, beef, and apple cider). Aliquots from concentrated food washes subsequently spotted onto FTA filters and assayed by PCR gave consistently positive results and detection limits similar to those observed with pure-culture dilutions. This universal method for PCR template preparation from bacterial cells is rapid and highly sensitive and reduces interference from food-associated inhibitors of PCR. In addition, its broad applicability eliminates the need for multiple methods for analysis of food matrices.

Quantitative immunocapture PCR assay for detection of Campylobacter jejuni in foods.

The rapid detection of food-borne bacterial pathogens as part of a quality control program is necessary for the maintenance of a safe food supply. In this report, we present our findings for an immunocapture PCR method for the detection of Campylobacter jejuni in foods. The method permits direct detection of the pathogen without an enrichment step and can be performed in approximately 8 h. Assay results are quantitative, and one cell in a milliliter sample can be detected. Application of the method to spiked milk samples and chicken skin washes did not affect the sensitivity of the assay.

DNA-based methods for detection of food-borne bacterial pathogens

Food-borne diseases are mainly caused by pathogenic bacteria, which are transmitted to humans from the animal reservoir via food or which contaminates the food on the processing line. Detection of the pathogenic bacteria constitutes a challenge, as the bacteria are often present in low numbers, masked by the food matrix and outnumbered by high numbers of indigenous bacteria. Traditional detection methods, therefore, include enrichment and often pre-enrichment steps, which are time and labour consuming. Many different techniques have been developed to speed up detection of pathogenic bacteria and to increase the sensitivity of the detection. In the current paper, the state of the art for DNA-based detection methods are reviewed with a view to their limitations in relation to food microbiology.