The specific and sensitive detection of bacterial pathogens within 4 h using bacteriophage amplification

This paper describes a novel approach, termed the 'phage amplification assay', for the rapid detection and identification of specific bacteria. The technique is based on the phage lytic cycle with plaque formation as the assay end-point. It is highly sensitive, quantitative and gives results typically within 4 h. The assay comprises four main stages : (1) phage infection of target bacterium ; (2) destruction of exogenous phage ; (3) amplification of phage within infected host and (4) plaque formation from infected host with the aid of helper bacteria. A key component of this assay is a potent virucidal agent derived from natural plant extracts, pomegranate rind extract (PRE). In combination with ferrous sulphate PRE can bring about an 11 log-cycle reduction in phage titre within 3 min. This is achieved without any injury to the infected target bacteria. Subsequently, any resulting plaques are derived only from infected target organisms. Data are presented for a range of bacterial hosts including Pseudomonas aeruginosa, Salmonella typhimurium and Staphylococcus aureus. The detection limit for Ps. aeruginosa was 40 bacteria ml⁻¹ in a time of 4 h and 600 bacteria m⁻¹ for Salm. typhimurium. Application of the principles of this technology to other bacterial genera is discussed.     

Inactivation of human pathogens and spoilage bacteria on the surface and internalized within fresh produce by using a combination of ultraviolet light and hydrogen peroxide

Aims: To evaluate the efficacy of ultraviolet (UV) light (254 nm) combined with hydrogen peroxide (H₂O₂) to inactivate bacteria on and within fresh produce. Methods and Results: The produce was steep inoculated in bacterial cell suspension followed by vacuum infiltration. The inoculated samples were sprayed with H₂O₂ under constant UV illumination. The log count reduction (LCR) of Salmonella on and within lettuce was dependent on the H₂O₂ concentration, temperature and treatment time with UV intensity being less significant. By using the optimized parameters (1·5% H₂O₂ at 50°C, UV dose of 37·8 mJ cm^?2), the surface Salmonella were reduced by 4·12 ± 0·45 and internal counts by 2·84 ± 0·34 log CFU, which was significantly higher compared with H₂O₂ or UV alone. Higher LCR of Escherichia coli O157:H7, Pectobacterium carotovora, Pseudomonas fluorescens and Salmonella were achieved on leafy vegetables compared with produce, such as cauliflower. In all cases, the surface LCR were significantly higher compared with the samples treated with 200 ppm hypochlorite. UV–H₂O₂‐treated lettuce did not develop brown discolouration during storage but growth of residual survivors occurred with samples held at 25°C. Conclusions: UV–H₂O₂ reduce the bacterial populations on and within fresh produce without affecting the shelf‐life stability. Significance of the Study: UV–H₂O₂ represent an alternative to hypochlorite washes to decontaminate fresh produce.     

Detection of foodborne pathogens using DNA probes and a dipstick format

The detection of foodborne microorganisms has traditionally been done using microbiologically based methods. Such “gold standard” methods are generally reliable but have the disadvantages of being labor intensive, subjective, and time consuming. Over the last several years, the development of DNA probe-based methods has simplified the methods used to detect organisms such asSalmonella, Listeria, andE. coli by targeting the unique DNA or RNA sequences of these organisms using DNA probes and nonradioactive detection.     

Application of bacteriophages for detection and control of foodborne pathogens

The incidence of foodborne infectious diseases is stable or has even increased in many countries. Consequently, our awareness regarding hygiene measures in food production has also increased dramatically over the last decades. However, even today’s modern production techniques and intensive food-monitoring programs have not been able to effectively control the problem. At the same time, increased production volumes are distributed to more consumers, and if contaminated, potentially cause mass epidemics. Accordingly, research directed to improve food safety has also been taken forward, also exploring novel methods and technologies. Such an approach is represented by the use of bacteriophage for specific killing of unwanted bacteria. The extreme specificity of phages renders them ideal candidates for applications designed to increase food safety during the production process. Phages are the natural enemies of bacteria, and can be used for biocontrol of bacteria without interfering with the natural microflora or the cultures in fermented products. Moreover, phages or phage-derived proteins can also be used to detect the presence of unwanted pathogens in food or the production environments, which allows quick and specific identification of viable cells. This review intends to briefly summarize and explain the principles and current standing of these approaches.     

Microfluidic devices for sample preparation and rapid detection of foodborne pathogens

Rapid detection of foodborne pathogens at an early stage is imperative for preventing the outbreak of foodborne diseases, known as serious threats to human health. Conventional bacterial culturing methods for foodborne pathogen detection are time consuming, laborious, and with poor pathogen diagnosis competences. This has prompted researchers to call the current status of detection approaches into question and leverage new technologies for superior pathogen sensing outcomes. Novel strategies mainly rely on incorporating all the steps from sample preparation to detection in miniaturized devices for online monitoring of pathogens with high accuracy and sensitivity in a time-saving and cost effective manner. Lab on chip is a blooming area in diagnosis, which exploits different mechanical and biological techniques to detect very low concentrations of pathogens in food samples. This is achieved through streamlining the sample handling and concentrating procedures, which will subsequently reduce human errors and enhance the accuracy of the sensing methods. Integration of sample preparation techniques into these devices can effectively minimize the impact of complex food matrix on pathogen diagnosis and improve the limit of detections. Integration of pathogen capturing bio-receptors on microfluidic devices is a crucial step, which can facilitate recognition abilities in harsh chemical and physical conditions, offering a great commercial benefit to the food-manufacturing sector. This article reviews recent advances in current state-of-the-art of sample preparation and concentration from food matrices with focus on bacterial capturing methods and sensing technologies, along with their advantages and limitations when integrated into microfluidic devices for online rapid detection of pathogens in foods and food production line.     

Development of antibody array for simultaneous detection of foodborne pathogens

Pathogenic bacterial contaminations present serious problems for food industry and public health. Rapid, accurate and affordable assays are needed. In this study, antibody arrays to simultaneously detect two foodborne pathogenic bacteria (Escherichia coli O157:H7 and Salmonella spp.) have been developed using chemiluminescent detecting system. Solid supports using nitrocellulose membrane and poly-l-lysine (PLL) glass slide were compared and optimized for antibody array construction. Many parameters including optimal concentrations of antibodies, blocking reagents, assay time, storage time, sensitivity and cross-reactivity were considered during optimization. This study revealed that the PLL slide was a more suitable support due to highly accurate results and the absence of non-specific background. Phosphate-buffered saline (PBS, pH 7.2) and 3% skim milk in PBS buffer were optimal spotting and blocking reagents, respectively. With the same sensitivity for bacterial detection as in a conventional ELISA (10(5)-10(6)CFU/ml for the E. coli O157:H7 and 10(6)-10(7)CFU/ml for Salmonella detections), this antibody array has advantages of a much shorter assay time of 1h and much lower required amounts of antibodies. Moreover, there was no cross-reactivity in the detection among bacteria tested in this study. Bacteria detection in food sample was feasible as demonstrated using bacteria-added milk.     

Impact of molecular biology on the detection of foodborne pathogens

Molecular biological methods that use antibodies and nucleic acids to detect specific foodborne bacterial pathogens were scarcely known a decade and a half ago. Few scientists could have predicted that these tools of basic research would come to dominate the field of food diagnostics. Today, a large number of cleverly designed assay formats using these technologies are available commercially for the detection in foods of practically all major established pathogens and toxins, as well as of many emerging pathogens. These tests range from very simple antibody-bound latex agglutination assays to very sophisticated DNA amplification methods. Although molecular biological assays are more specific, sensitive, and faster than conventional (often cultural) microbiological methods, the complexities of food matrices continue to offer unique challenges that may preclude the direct application of these molecular biological methods. Consequently, a short cultural enrichment period is still required for food samples prior to analysis with these assays. The greater detection sensitivity of molecular biological methods may also affect existing microbiological specifications for foods; this undoubtedly will have repercussions on the regulatory agencies, food manufacturers, and also consumers. The US government has the right to retain a nonexclusive royalty-free license in and to any copyright covering this article. Use of trade names is for identification only and does not imply an endorsement by the US FDA.     

Development and evaluation of a multiplex PCR for simultaneous detection of five foodborne pathogens

Aims: To develop a rapid multiplex PCR method for simultaneous detection of five major foodborne pathogens (Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, Salmonella Enteritidis and Shigella flexneri, respectively). Methods and Results: Amplification by PCR was optimized to obtain high efficiency. Sensitivity and specificity assays were investigated by testing different strains. With a multipathogen enrichment, multiplex PCR assay was able to simultaneously detect all of the five organisms in artificially contaminated pork samples. The developed method was further applied to retail meat samples, of which 80% were found to be positive for one or more of these five organisms. All the samples were confirmed by traditional culture methods for each individual species. Conclusions: This study reported a rapid multiplex PCR assay using five primers sets for detection of multiple pathogens. Higher consistency was obtained between the results of multiplex PCR and traditional culture methods. Significance and Impact of the Study: This work has developed a reliable, useful and cost‐effective multiplex PCR method. The assay performed equally as well as the traditional cultural method and facilitated the sensitive detection both in artificially contaminated and naturally contaminated samples.     

Direct detection of bacterial pathogens in representative dairy products using a combined bacterial concentration-PCR approach

AIMS: To develop a simple, rapid method to concentrate and purify bacteria and their nucleic acids from complex dairy food matrices in preparation for direct pathogen detection using polymerase chain reaction (PCR). METHODS AND RESULTS: Plain non-fat yogurt and cheddar cheese were each seeded with Listeria monocytogenes or Salmonella enterica serovar. Enteritidis in the range of 10(1)-10(6) CFU per 11-g sample. Samples were then processed for bacterial concentration using high-speed centrifugation (9700 g) followed by DNA extraction, PCR amplification, and amplicon confirmation by hybridization. Bacterial recoveries after centrifugation ranged from 53 to >100% and 71 to >100% for serovar. Enteritidis and L. monocytogenes, respectively, in the non-fat yogurt samples; and from 77 to >100% and 69 to >100% for serovar. Enteritidis and L. monocytogenes, respectively, in the cheddar cheese samples. There were no significant differences in recovery efficiency at different inocula levels, and losses to discarded supernatants were always <5%, regardless of dairy product or pathogen. CONCLUSIONS: When followed by pathogen detection using PCR and confirmation by amplicon hybridization, detection limits of 10(3) and 10(1) CFU per 11-g sample were achieved for L. monocytogenes and serovar. Enteritidis, respectively, in both product types and without prior cultural enrichment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents progress toward the rapid and efficient direct detection of pathogens from complex food matrices at detection limits approaching those that might be anticipated in naturally contaminated products.     

Optimization and evaluation of a multiplex PCR for simultaneous detection of prominent foodborne pathogens of Enterobacteriaceae

Members of the family Enterobacteriaceae are major pathogens associated with gastrointestinal disorders caused by the consumption of contaminated foods. We have developed a multiplex PCR (mPCR) targeting specific genes for simultaneous detection and differentiation of five major Enterobacteriaceae members, namely, Salmonella sp. (invA), Escherichia coli (uidA), Shigella sp. (ipaH), Klebsiella pneumoniae (khe) and Citrobacter freundii (tpl), from both pure cultures and contaminated food samples, along with an internal amplification control (IAC). Simultaneous amplification of these five genes was optimized using reference strains and further evaluated on large number of isolates recovered from clinical and environmental sources. The mPCR assay showed high sensitivity for detecting 10 CFU/PCR for the above-mentioned pathogens directly from serially diluted overnight cultures. The mPCR assay was also able to detect all five pathogens spiked at an initial count of 10 CFU/g of meat and rice samples following an enrichment of 10 h in Brain Heart Infusion broth. To assess the practical application of this mPCR assay, we evaluated its efficacy for detecting possible contamination on natural samples, such as meat, fish, pastries and water. Based on the results, we suggest that this mPCR assay would be of immense help in detecting low counts of important Enterobacteriaceae pathogens inexpensively and thus can be used for the regular monitoring of food quality.     

Direct detection of isotopically labeled metabolites bound to a protein microarray using a charge-coupled device

A charge-coupled device (CCD) was used to quantitatively detect isotope-labeled ligands bound to a protein microarray. Protein microarrays with protein dots, 10-50 microm in diameter, were fabricated on an aluminized Mylar film using an electrospray deposition technique. Proteins in dots were immobilized by cross-linking in glutaraldehyde vapor. After contact with solutions of isotope-labeled metabolites, the protein microarrays were washed, dried and placed face down onto the surface of a standard B/W video CCD chip with the protective window removed. We show here that such a simple inexpensive CCD detector can be used to quantify distribution of 14C and other radioactive isotopes on microarrays.     

Amyloid detection using a Peltier-based device

Amyloid aggregation of polypeptides is related to a growing number of pathologic states known as amyloid disorders. At present, it is clear that any proteins submitted to appropriate physicochemical environment can acquire fibrilar conformation. Fourier transform infrared spectroscopy (FTIR) has been a widely used technique to study temperature- induced amyloid-fibrils formation in vitro. In this way, strict changes and temperature controls are required to characterize the physicochemical basis of the amyloid-fibrils formation. In this article, the development of a highly efficient and accurate Peltier-based system to improve FTIR measurements is presented (see An Old Physics Phenomenon Applied to a Serious Biomedical Pathology. The accuracy of the thermostatic control was tested with biophysical parameters on biological samples probing its reproducibility. The design of the present device contributes to maintain the FTIR environment stable, which represents a real contribution to improve the spectral quality and thus, the reliability of the results.     

An overview of transducers as platform for the rapid detection of foodborne pathogens

The driving advent of portable, integrated biosensing ways for pathogen detection methods offers increased sensitivity and specificity over traditional microbiological techniques. The miniaturization and automation of integrated detection systems present a significant advantage for rapid, portable detection of foodborne microbes. In this review, we have highlighted current developments and directions in foodborne pathogen detection systems. Recent progress in the biosensor protocols toward the detection of specific microbes has been elaborated in detail. It also includes strategies and challenges for the implementation of a portable platform toward rapid foodborne sensing systems.     

Direct and sensitive detection of a human virus by rupture event scanning

We have developed a sensitive, economical method that directly detects viruses by making use of the interaction between type 1 herpes simplex virus (HSV1) and specific antibodies covalently attached to the oscillating surface of a quartz crystal microbalance (QCM). The virions were detached from the surface by monotonously increasing the amplitude of oscillation of the QCM, while using the QCM to sensitively detect the acoustic noise produced when the interactions were broken. We term this process rupture event scanning (REVS). The method is quantitative over at least six orders of magnitude, and its sensitivity approaches detection of a single virus particle.     

Development of a DNA macroarray for simultaneous detection of multiple foodborne pathogenic bacteria in fresh chicken meat

A DNA macroarray was developed to provide the ability to detect multiple foodborne pathogens in fresh chicken meat. Probes targeted to the 16S rRNA and genus- and species-specific genes, including fimY, ipaH, prfA, and uspA, were selected for the specific detection of Salmonella spp., Shigella spp., Listeria monocytogenes, and Escherichia coli, respectively. The combination of target gene amplification by PCR and a DNA macroarray in our system was able to distinguish all target bacteria from pure cultures with a detection sensitivity of 10⁵ c.f.u. ml^?1. The DNA macroarray was also applied to 10 fresh chicken meat samples. The assay validation demonstrated that by combining the enrichment steps for the target bacteria and the DNA macroarray, all 4 target bacteria could be detected simultaneously from the fresh chicken samples. The sensitivity of L. monocytogenes and Shigella boydii detection in the fresh chicken samples was at least 10 and 3 c.f.u. of the initial contamination in 25 g samples, respectively. The advantages of our developed protocol are high accuracy and time reduction when compared to conventional culture. The macroarray developed in our investigation was cost effective compared to modern oligonucleotide microarray techniques because there was no expensive equipment required for the detection of multiple foodborne pathogens.     

A universal protocol for PCR detection of 13 species of foodborne pathogens in foods

A universal protocol for PCR detection of 13 species of foodborne pathogens in foods wasdeveloped. The protocol used a universal culture medium and the same PCR conditions with 13sets of specific primers. The 13 species of foodborne pathogens examined were Escherichiacoli, E. coli- ETEC, E. coli -O157:H7, Shigella spp. , Salmonella spp. , Yersinia enterocolitica, Y. pseudotuberculosis, Vibrio cholerae, V.parahaemolyticus, V. vulnificus , Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus . No interference was observed using the PCR assay when foodsample was artificially inoculated with each individual bacterial species. Twelve different seafoodsamples and two soft cheese samples without artificial inoculation were examined by thisprotocol. Vibrio vulnificus, Salmonella spp. , E. coli,Listeria monocytogenes and Bacillus cereus were detected in some foods.Internal probe hybridization and nested PCR procedures were used to confirm the above findings.     

Chemiluminescence detection of Escherichia coli in fresh produce obtained from different sources

A chemiluminescence‐based assay is developed for the rapid detection of Escherichia coli in fresh produce. The assay was based on the reaction of β‐galactosidase enzyme from E. coli with a phenylgalactosidase‐substituted dioxetane substrate. Light emitted from the reaction was measured in a luminometer and data correlated with counts of E. coli enumerated on sorbitol–MacConkey agar plates. A strain of E. coli O157:H7 was used to inoculate samples of fresh produce to differentiate the inoculum from the natural E. coli potentially present on the produce. Fresh market samples were tested for generic E. coli and E. coli O157:H7. Signi?cant differences in light emission were found in samples with high initial E. coli counts when market samples were compared to respective heat‐treated samples. The assay was able to detect E. coli in all produce tested, particularly at higher contamination or inoculation levels. The sensitivity of the assay ranged between 10²–10⁵ CFU within 30 min. The chemiluminescence assay provides a simple and rapid method for detection of viable E. coli, an important step towards enhancing food safety. Copyright © 2004 John Wiley & Sons, Ltd.