Epidemiology, Detection, and Control of Foodborne Microbial Pathogens

Seventy-three samples of raw cow milk marketed at farm level (12 pre-filled bottles, 61 from vending machines) were investigated for their microbiological quality and the occurrence of bacterial foodborne pathogens. Total viable counts (TVC) were mainly (67.1 %) in the range from 103 to 105 CFU/ml, while Escherichia coli and coagulase-positive staphylococci were each detected in 30.1 % of the samples. TVC results for raw milk from vending machines (34.4 % above 105 CFU/ml) were clearly higher than those from pre-filled bottles, emphasizing the importance of ensuring correct cleaning and disinfection procedures of vending machines. Moreover, regular monitoring of the microbiological quality of raw milk from vending machines should be considered. With regard to foodborne pathogens in raw milk marketed at farm level, 24.7 % of all samples were positive for Staphylococcus aureus harboring staphylococcal ­enterotoxin (SE) genes. Genes for SEA, SEC, and SED were thereby also detected. On the other hand, Campylobacter spp., Listeria monocytogenes, and Shiga toxin-producing Escherichia coli were not detected. But because the occurrence of foodborne pathogens can never be ruled out, raw milk should always be properly heated before consumption.

DNA sequencing and other DNA-based methods are now broadly used for detection and identification of bacterial foodborne pathogens. For the identification of foodborne bacterial pathogens, taxonomic assignments must be made to the species or even subspecies level. Long-read DNA sequencing provides finer taxonomic resolution than short-read sequencing. Here, we demonstrate the potential of long-read shotgun sequencing obtained from the Oxford Nanopore Technologies (ONT) MinION single-molecule sequencer, in combination with the Basic Local Alignment Search Tool (BLAST) with custom sequence databases, for foodborne pathogen identification. A library of mixed DNA from strains of the "Super-7" Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, O145, and O157[:H7]) was sequenced using the ONT MinION resulting in 44,245 long-read sequences. The ONT MinION sequences were compared to a custom database composed of the E. coli O-antigen gene clusters. A vast majority of the sequence reads were from outside of the O-antigen cluster and did not align to any sequences in the O-antigen database. However, 58 sequences (0.13% of the total sequence reads) did align to a specific Super-7 O-antigen gene cluster, with each O-antigen cluster aligning to at least four sequence reads. BLAST analysis against a custom whole-genome database revealed that 5096 (11.5%) of the MinION sequence reads aligned to one and only one sequence in the database, of which 99.6% aligned to a sequence from a "Super-7" STEC. These results demonstrate the ability of the method to resolve STEC to the serogroup level and the potential general utility of the MinION for the detection and typing of foodborne pathogens.

Bacillus cereus is among the primary food‐poisoning pathogenic bacterium that causes diarrhea and emetic types of diseases throughout the world. Recent advances show that bacteriophages become important tools in detection and control of foodborne bacterial pathogens in foods. They gain the interest of researchers for the food industries mainly because they are host‐specific and harmless to humans. Studies showed that bacteriophages could be employed as natural or engineered, whole or part, and temperate or virulent type in designing a range of tools for the detection and control of foodborne bacterial pathogens. This article discusses the recent methods and advances in the utilization strategies of bacteriophages in detection and control of foodborne pathogens, with particular focus on B. cereus pathogen. Moreover, the article presents the latest and relevant information of B. cereus‐infecting phages with respect to their potential applications in foods to address food safety issues. It also reflects future research directions by indicating gap of studies on the area.

Cooperation and competition between CRISPR- and omics-based technologies in foodborne pathogens detection: a state of the art review

Current California agricultural practices strive to comanage food safety and habitat conservation on farmland. However, the ecology of foodborne pathogens in wild bird populations, especially those avian species residing in proximity to fresh produce production fields, is not fully understood. In this repeated cross-sectional study, avifauna within agricultural lands in California were sampled over 1 year. Feces, oral swabs, and foot/feather swabs were cultured for zoonotic Salmonella spp., Escherichia coli O157:H7, and non-O157 Shiga toxin-producing E. coli (STEC) and characterized by serotyping and pulsed-field gel electrophoresis. Of 60 avian species sampled, 8 species (13.3%, bird groups of sparrows, icterids, geese, wrens, and kinglets) were positive for at least one of these foodborne pathogens. At the individual bird level, the detection of foodborne pathogens was infrequent in feces (n = 583; 0.5% Salmonella, 0.34% E. coli O157:H7, and 0.5% non-O157 STEC) and in feet/feathers (n = 401; 0.5% non-O157 STEC), and it was absent from oral swabs (n = 353). Several subtypes of public health importance were identified, including Salmonella enterica serotype Newport, E. coli O157:H7, and STEC serogroups O103 and O26. In late summer and autumn, the same STEC subtype was episodically found in several individuals of the same and different avian species, suggesting a common source of contamination in the environment. Sympatric free-range cattle shared subtypes of STEC O26 and O163 with wild geese. A limited rate of positive detection in wild birds provides insights into broad risk profile for contamination considerations but cannot preclude or predict risk on an individual farm.IMPORTANCE The shedding dynamics of foodborne pathogens by wild birds on farmland are not well characterized. This yearlong study sampled wild birds for foodborne pathogens within agricultural lands in northern California. There was a low prevalence of Salmonella spp., Escherichia coli O157:H7, and non-O157 Shiga-toxin producing E. coli (prevalence, 0.34% to 0.50%) identified in bird populations in this study. However, pathogens of public health importance (such as Salmonella Newport, E. coli O157:H7, and STEC O103 and O26) were identified in fecal samples, and two birds carried STEC on their feet or feathers. Identical pathogen strains were shared episodically among birds and between wild geese and free-range cattle. This result suggests a common source of contamination in the environment and potential transmission between species. These findings can be used to assess the risk posed by bird intrusions in produce fields and enhance policy decisions toward the comanagement of food safety and farmland habitat conservation.

EFSA Supporting PublicationsVolume 13, Issue 4 1017E Technical reportOpen Access Multi-country outbreak of Shiga toxin-producing Escherichia coli infection associated with haemolytic uraemic syndrome European Food Safety Authority and European Centre for Disease Prevention and Control, European Food Safety Authority and European Centre for Disease Prevention and ControlSearch for more papers by this author European Food Safety Authority and European Centre for Disease Prevention and Control, European Food Safety Authority and European Centre for Disease Prevention and ControlSearch for more papers by this author First published: 06 April 2016 https://doi.org/10.2903/sp.efsa.2016.EN-1017Citations: 3 Published date: 6 April 2016 Question number: EFSA-Q-2016-00232 AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References Luna-Gierke RE, Wymore K, Sadlowski J, Clogher P, Gierke RW, Tobin-D'Angelo M, et al. Multiple-Aetiology Enteric Infections Involving Non-O157 Shiga Toxin-Producing Escherichia coli - FoodNet, 2001–2010. Zoonoses Public Hlth. 2014 Nov; 61(7): 492– 8. European Centre for Disease Prevention and Control. Escherichia coiifactsheet Stockholm: ECDC; [cited 2016 March 29]. Available from: http://ecdc.europa.eu/en/healthtopics/escherichia_coli/basic_facts/Pages/basic_facts.aspx. Fauci A, Braunwald E, Kasper D, Hauser S, Longo D, Jameson J, et al. Harrison's Principles of internal medicine. MH Medical, editor: McGraw Hill Medical; 2008. 1811– 5 p. EFSA (European Food Safety Authority) and ECDC (European Centre for Disease Prevention and Control). The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2014. EFSA journal. 13(12): 4329,191. European Centre for Disease Prevention and Control. Surveillance Atlas of Infectious Diseases Stockholm: ECDC; 2016 [cited 2016 March 29]. Available from: http://atlas.ecdc.europa.eu/public/index.aspx. Espie E, Grimont F, Mariani-Kurkdjian P, Bouvet P, Haeghebaert S, Filliol I, et al. Surveillance of hemolytic uremic syndrome in children less than 15 years of age, a system to monitor O157 and non-O157 Shiga toxin-producing Escherichia coli infections in France, 1996–2006. Pediatr Infect Dis J. 2008 Jul; 27(7): 595– 601. Institut de Veille Sanitaire. Epidémie d'infections à E. coli producteurs de Shiga-toxines non 0157 liée à la consommation de camembert au lait cru, nord-ouest de la France, octobre-décembre 20052007 March 30. Available from: http://opac.invs.sante.fr/index.php2lvNnotice_display&id=9416. Ethelberg S, Smith B, Torpdahl M, Lisby M, Boel J, Jensen T, et al. Outbreak of non-O157 Shiga toxin-producing Escherichia coli infection from consumption of beef sausage. Clin Infect Dis. 2009 Apr 15; 48(8): e78– 81. Germinario C, Chironna M, CGallone M, Tafuri S, Desiante F, Calabrese G. Community-wide Outbreak of Haemolytic Uremic Syndrome Associated with Shiga Toxin 2-Producing Escherichia coli O26:H11 in Southern Italy, Summer 2013. Euro Surveill. 2016;(Forthcoming). International Organization for Standardization. ISO/TS 13136:2012 Microbiology of food and animal feed -Real-time polymerase chain reaction (PCR)-based method for the detection of food-borne pathogens -Horizontal method for the detection of Shiga toxin-producing Escherichia coli (STEC) and the determination of O157, O111, O26, O103 and O145 serogroups. Technical Specifications: ISO; 2012. p. 22. Peron E, Zaharia A, Zota L, Severi E, Mârdh O, Usein C, et al. Early findings in outbreak of haemolytic uraemic syndrome among young children caused by Shiga toxin-producing Escherichia coli, Romania, January to February 2016. Eurosurveillance. 2016; 21(11). Jenssen G, Hovland E, Bjerre A, Bangstad H-J, Nygard K, Vold L. Incidence and etiology of hemolytic-uremic syndrome in children in Norway, 1999–2008 - a retrospective study of hospital records to assess the sensitivity of surveillance. BMC Infectious Diseases. 2014; 14(1): 265. Nielsen EM, Andersen MT. Detection and characterization of verocytotoxin-producing Escherichia coli by automated 5' nuclease PCR assay. J Clin Microbiol. 2003 Jul; 41(7): 2884– 93. Citing Literature Volume13, Issue4April 20161017E ReferencesRelatedInformation

The Economics of Enteric Infections: Human Foodborne Disease Costs

Dairy manure, a by-product in the dairy industry, is also a potential source of nutrients for crops. However, improper application of biological soil amendments of animal origin can be a source of contamination with enteric foodborne pathogens. A 2-year field study was conducted to evaluate impacts of dairy manure fertilizer application on the microbial safety of red raspberry (Rubus idaeus L) production. Fertilizers, including a standard synthetic fertilizer (CON), straight lagoon raw manure (SL), anaerobically digested liquid effluent (DLE), compost (COM) and dairy manure-derived refined fertilizers including ammonium sulfate (AS) and phosphorous solid (PS), were randomly applied in quadruplicate to raspberry plots. Soil, fertilizer, foliar, and raspberry fruit samples were collected during the cropping season for the quantification of indicator microorganisms (total coliform and generic Escherichia coli) and detection of important foodborne pathogens (Shiga toxin-producing E. coli (STEC), Salmonella, and Listeria monocytogenes). Counts of total coliforms in soil were stable over the 2017 cropping season and were not impacted by fertilizer application. In 2018, total coliforms increased with season and soils treated with COM had a significantly higher coliform number than those treated with CON. Both total coliform and generic E. coli in raspberry fruit samples were below the detectable level (3 most probable number/g) regardless of fertilizer types. In both years, no STEC or L. monocytogenes was detected from any of the collected samples regardless of fertilizer treatments. However, Salmonella were detected in some of the fertilizers, including PS (2017), DLE (2018), and SL (2018), which were transferred to soil samples taken directly after application of these fertilizers. Salmonella were not detected in soil samples 2 or 4 months post fertilizer application, foliar, or raspberry fruit samples regardless of fertilizer applications. In summary, one-time application of raw dairy manure or dairy manure-derived fertilizers more than 4 months prior to harvest has no major impact on food safety of red raspberry (6 ft. tall) production in Lynden sandy loam under good agricultural practices.

The rapid and accurate detection of foodborne pathogens is paramount for safeguarding public health and ensuring the safety of food supplies. In this study, we introduce a groundbreaking approach in the form of a 3D printed multiplex colorimetric genetic analysis microchip tailored specifically for point-of-care analysis of foodborne pathogens. This microchip represents a significant advancement in genetic analysis technology, offering a streamlined and efficient solution for on-site detection.Conventional methods for fabricating genetic analysis chips often involve photolithography, which can be time-consuming, expensive, and technically demanding. In contrast, our microchip is fabricated using stereolithography-digital light processing (SL-DLP) technology, which offers several distinct advantages. SL-DLP is known for its convenience, reproducibility, and cost-effectiveness, making it an ideal choice for the rapid and scalable production of genetic analysis chips. By leveraging this innovative fabrication technique, we have overcome many of the limitations associated with traditional manufacturing methods, paving the way for enhanced accessibility and widespread adoption of genetic analysis technologies.The genetic analysis microchip consists of several key components, each meticulously designed to facilitate efficient and accurate detection of foodborne pathogens. Central to its functionality is the inclusion of a cell sample solution inlet, which allows for the introduction of the sample into the microchip. This inlet is strategically positioned to ensure uniform distribution of the sample throughout the microchip, maximizing the efficiency of the detection process. Additionally, the microchip features four reaction chambers, each dedicated to the isothermal amplification of a different target pathogen. This multiplexing capability enables simultaneous detection of multiple pathogens, greatly enhancing the efficiency and throughput of the analysis process.To further streamline the detection process, the microchip incorporates four loop-mediated isothermal reaction (LAMP) reagent inlets, allowing for the pre-loading of lyophilized reagents into each reaction chamber. This pre-loading step eliminates the need for manual addition of reagents, reducing the risk of contamination and human error. Furthermore, the microchip is fabricated using a photocurable resin composed of poly(ethylene glycol) diacrylate (PEG-DA) (MW=258), which offers several advantages. Notably, this resin enables the printing of high-resolution and transparent structures, facilitating clear observation of color changes indicative of successful gene amplification.To enable colorimetric genetic analysis, Eriochrome Black T (EBT) is added to the LAMP reagent, inducing a distinctive color change upon amplification of the target gene. This colorimetric signal serves as a visual indicator of pathogen presence, allowing for rapid and intuitive interpretation of results. In our experiments, we successfully detected four common foodborne pathogens - Escherichia coli O157:H7, Salmonella enterica, Vibrio parahaemolyticus, and Listeria monocytogenes - within a remarkably short timeframe of 20 minutes. This rapid turnaround time is critical for timely intervention and mitigation of foodborne illness outbreaks.To assess the sensitivity of our microchip, we conducted a limit of detection (LOD) test using samples containing varying concentrations of the target pathogen. Remarkably, we were able to detect as few as 10 cells of the pathogen with a high degree of accuracy, underscoring the sensitivity and reliability of our approach. Furthermore, in multiplex detection tests, each target gene was independently detected within dedicated LAMP chambers, demonstrating the microchip's exceptional specificity and its ability to accurately distinguish between different pathogens.Importantly, the simplicity of fabrication and the readiness for on-site analysis make our microchip highly versatile and adaptable to a wide range of applications beyond food safety. In addition to foodborne pathogen detection, our microchip holds promise for use in clinical diagnosis, forensic analysis, and environmental monitoring. By enabling rapid and accurate genetic analysis in diverse settings, our microchip has the potential to revolutionize the field of molecular diagnostics and significantly improve public health outcomes.In summary, we have developed a novel 3D printed multiplex colorimetric genetic analysis microchip that represents a significant advancement in point-of-care diagnostic technology. By combining state-of-the-art fabrication techniques with innovative design principles, we have created a powerful tool for the rapid and reliable detection of foodborne pathogens. Moving forward, we envision widespread adoption of our microchip as a cornerstone of food safety protocols, facilitating proactive measures to protect consumer health and prevent foodborne illness outbreaks.

MXene, owing to its high electrical conductivity, large specific surface area, and abundant surface functional groups, has been widely applied in the detection of foodborne pathogens. Therefore, it is necessary to review recent developments in the emerging material MXene for the detection and killing of foodborne pathogens, which is expected to facilitate the further development and utilization of MXene. This work comprehensively reviews advances in MXene applications for detecting and killing foodborne pathogens. Firstly, applications of MXene in electrochemical sensors, surface-enhanced Raman scattering (SERS), fluorescence platforms, and fluorescence–electrochemical dual-mode sensing systems are introduced. Subsequently, the sterilization mechanisms of MXene are described, followed by a detailed explanation of its practical applications in active food packaging, surface modification of food processing equipment, and instant sterilization techniques. Finally, conclusions, challenges, and future prospects in the area of MXene for the detection and killing of foodborne pathogens are discussed in depth. Significantly, this review uniquely summarizes applications of MXene in the detection and sterilization of foodborne pathogens, offering new perspectives on its use in food safety.

The increased consumption of minimally processed vegetables (MPV) in various countries is related to the continued interest of consumers in seeking practical and healthy food items. Due to multiple processing steps, MPV can be contaminated by several foodborne pathogens that pose significant health risks to consumers. The use of rapid techniques to detect pathogens in ready-to-eat (RTE) foods such as MPV is therefore essential to provide high quality and safe products. This review aims to provide a comprehensive description of molecular-based techniques for rapid detection of pathogenic bacteria in MPV, and their occurrence reported in studies published in the last 10 years. The main pathogens detected using rapid methods were Salmonella spp., Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, Shigella spp., and Campylobacter jejuni. Molecular-based techniques included real-time polymerase chain reaction (PCR), multiplex PCR, matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS), and denaturing gradient gel electrophoresis (DGGE). The data indicate high incidences of pathogenic bacteria in MPV, stressing the need for their rapid detection in these products to prevent associated health risks. Further studies should be carried out to increase the sensitivity of molecular-based techniques and prevent false positives due to undesirable non-specific PCR amplifications.

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.

Molecular Subtyping and the Transformation of Public Health

Milk and milk products can harbour multiple varieties of foodborne pathogens, such as Staphylococcus aureus, Shigella like toxin producing E. coli, Salmonella and Bacillus cereus.Many food-borne outbreaks have been associated with dairy products as main vehicles for transmission.In this study, a total of (n=310) samples were collected from different places dairies, gaushalsa, local shops and vendors of Mathura region.Swabs were taken from milking buckets (50), dippers (64), canes (56) and raw milk (140) from cows.Samples were screened for S. aureus and Shiga toxin producing E. coli (STEC) by streaking on selective agar and molecularly characterized for housekeeping nuc and stx gene by m-PCR.Overall prevalence of S. aureus and STEC was revealed 80.64% and 7.41%, respectively.Confirmed S. aureus and STEC isolates were screened for biofilm formation capability by phenotypic method methods viz Congo red agar (CRA) assay, Tube Method (TM) and Tissue Culture Plate method (TCP).On CRA 9.2 % isolates of S. aureus were positive and 90.8% were negative while TM revealed 79.2%, 12.4% and 8.4% strong, moderate and weak biofilm formers.In TCP method, 91.6% isolates were strong, 5.6% moderate and 2.8% weak biofilm producers.Among STEC isolates, 34.78 % and65.22%were positive and negative on CRA while by TM, 43.47%, 26.08% and 30.43% were strong, moderate and weak biofilm formers.In another TCP assay, 52.17%, 30.43% and 17.39 % isolates were strong, moderate and weak biofilm producers, respectively.Among these three methods TCP was found more sensitive for S aureus as well as STEC.Under Scanning electron microscopy, the 3D structure of biofilms of S. aureus and STEC revealed and the biofilms were well organized, with intact cell-to-cell connections.STEC produced better biofilm than S aureus.This study revealed that biofilm forming S. aureus and STEC were obtained from dairy utensils and raw milk so, may be a sustainable source of contamination of dairy products.So, there is need of paying more attention to the cleaning and sanitizing processes of food contact surfaces to ensure the public health.

The explosive increase since the beginning of the 1990s in the number of publications reporting PCR-based methods for detection or molecular typing of foodborne pathogens has attracted the attention of end-user laboratories. However, the well-recognized difficulties in reproducing published tests because of variation in performance of PCR thermal cyclers (Schoder et al. 2003), in efficiency of different DNA polymerases, and in the presence of PCR inhibitors in the sample matrix, have hampered implementation in end-user laboratories. This particularly applies to laboratories with quality-assurance programmes. It is necessary to have PCR-based methods available as internationally recognized standards (Hoorfar and Cook 2002). Currently, lack of international standards often forces end-user laboratories to spend substantial resources on adaptation of the published tests. Although many commercial PCR kits are available, it is important that end-users and reference laboratories have access to open-formula, noncommercial and nonproprietary PCRs in which the information on target gene and reagents are fully available. The prerequisite for a PCR, published in the scientific literature, to be adopted as a standard is that it has to be nonproprietary, and to have been validated through multicentre collaborative trial according to the international criteria (Anon. 2001, 2002b; Hoorfar and Cook 2002). Multicentre trial validation of noncommercial PCRs for detection of zoonotic pathogens has been performed by a European validation and standardization project (FOOD-PCR: http://www.pcr.dk) involving 35 laboratories from 21 countries (Hoorfar 1999; Malorny et al. 2003). A major drawback of most published PCRs, surprisingly even to date, is that they do not contain an internal amplification control (IAC). An IAC is a nontarget DNA sequence present in the same sample reaction tube, which is co-amplified simultaneously with the target sequence. In a PCR without an IAC, a negative response (no band or signal) can mean that there was no target sequence present in the reaction. But, it could also mean that the reaction was inhibited, as a result of malfunction of thermal cycler, incorrect PCR mixture, poor polymerase activity and, not least, the presence of inhibitory substances in the sample matrix. Conversely, in a PCR with an IAC, a control signal will always be produced when there is no target sequence present. When neither IAC signal nor target signal is produced, the PCR reaction fails. Thus, when using a PCR-based method in routine analysis, an IAC, if the concentration adjusted correctly, will indicate false-negative results. It is the false-negative results that turn a risk into a threat for the population, whereas a false-positive result merely leads to a clarification of the presumptive results by re-testing the sample. The European Standardization Committee (CEN), in collaboration with International Standard Organization (ISO) has proposed a general guideline for PCR testing that requires the presence of IAC in the reaction mixture (Anon. 2002a). Therefore, only IAC-containing PCRs may undergo multicentre collaborative trials, which is a prerequisite for standardization. Scientific journals must provide the source of new PCR-based methods suitable for standardization. Therefore, we propose that henceforward the editorial boards of applied microbiology journals require inclusion of an IAC in diagnostic PCR reported in submitted manuscripts. This could be performed by providing a specific section devoted to PCR in their Instruction to Authors.