Direct or DNA Extraction-Free Amplification and Quantification of Foodborne Pathogens.

The use of direct nucleic acid amplification of pathogens from food matrices has the potential to reduce time to results over DNA extraction-based approaches as well as traditional culture-based approaches. Here we describe protocols for assay design and experiments for direct amplification of foodborne pathogens in food sample matrices using loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). The examples provided include the detection of Escherichia coli in milk samples and Salmonella in pork meat samples. This protocol includes relevant reagents and methods including obtaining target sequences, assay design, sample processing, and amplification. These methods, though used for specific example matrices, could be applied to many other foodborne pathogens and sample types.

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

Foodborne pathogens continue to be a major health concern 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 the cost 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.

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

Foodborne pathogens are responsible for 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 matrix protects bacterial cells from industrial environmental stress factors such as cleaning and disinfection operations. Moreover, during these environmental stresses, many bacterial species can be entered in a viable but nonculturable (VBNC) state. VBNC cells are characterized by an active metabolism and a loss of cultivability on conventional bacteriological agar. This leads to an underestimation of total viable cells in environmental samples and thus may pose 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 combining propidium monoazide (PMA) and quantitative PCR (qPCR) and a fluorescence microscopic method associated with the LIVE/DEAD BacLight™ viability stain.

Application of MinION Long-Read Sequencer for Semi-targeted Detection of Foodborne Pathogens.

Foodborne pathogens remain a serious health issue in developed and developing countries. Safeness of food products has been assured for years with culture-based microbiological methods; however, these present several limitations such as turnaround time and extensive hands-on work, which have been typically address taking advantage of DNA-based methods such as real-time PCR (qPCR). These, and other similar techniques, are targeted assays, meaning that they are directed for the specific detection of one specific microbe. Even though reliable, this approach suffers from an important limitation that unless specific assays are design for every single pathogen potentially present, foods may be considered erroneously safe. To address this problem, next-generation sequencing (NGS) can be used as this is a nontargeted method; thus it has the capacity to detect every potential threat present. In this chapter, a protocol for the simultaneous detection and preliminary serotyping of Salmonella enterica serovar Enteritidis, Salmonella enterica serovar Typhimurium, Listeria monocytogenes, and Escherichia coli O157:H7 is described.

Method to Study the Survival Abilities of Foodborne Bacterial Pathogens Under Food Processing Conditions.

Properly using controllable atmospheric containers can facilitate investigations of the survival abilities and physiological states of key and emerging-foodborne pathogens under recreated applicable food processing environmental conditions. Notably, saturated salt solutions can efficiently control relative humidity in airtight containers. This chapter describes a practical experimental setup, with necessary prerequisites for exposing foodborne pathogens to simulated and relevant food processing environmental conditions. Subsequent analyses for studying cell physiology will also be suggested.

Integrating MALDI-TOF Mass Spectrometry with Machine Learning Techniques for Rapid Antimicrobial Resistance Screening of Foodborne Bacterial Pathogens.

Although MALDI-TOF mass spectrometry (MS) is considered as the gold standard for rapid and cost-effective identification of microorganisms in routine laboratory practices, its capability for antimicrobial resistance (AMR) detection has received limited focus. Nevertheless, recent studies explored the predictive performance of MALDI-TOF MS for detecting AMR in clinical pathogens when machine learning techniques are applied. This chapter describes a routine MALDI-TOF MS workflow for the rapid screening of AMR in foodborne pathogens, with Campylobacter spp. as a study model.

Characterization of Foodborne Pathogens in Biofilms: A Four-Dimensional Structural Dynamics Approach Using HCS-CLSM.

The functional properties of biofilms are intimately related to their spatial architecture. Structural data are therefore of prime importance to dissect the complex social and survival strategies of biofilms and ultimately to improve their control. Confocal laser scanning microscopy (CLSM) is the most widespread microscopic tool to decipher biofilm structure, enabling noninvasive three-dimensional investigation of their dynamics down to the single-cell scale. The emergence of fully automated high content screening (HCS) systems, associated with large-scale image analysis, has radically amplified the flow of available biofilm structural data. In this contribution, we present a HCS-CLSM protocol used to analyze biofilm four-dimensional structural dynamics at high throughput. Meta-analysis of the quantitative variables extracted from HCS-CLSM will contribute to a better biological understanding of biofilm traits.

Identifying Putative Biomarkers of Foodborne Pathogens Using a Metabolomic Approach.

Metabolomics is the study of low molecular weight biochemical molecules (typically <1500 Da) in a defined biological organism or system. In case of food systems, the term "food metabolomics" is often used. Food metabolomics has been widely explored and applied in various fields including food analysis, food intake, food traceability, and food safety. Food safety applications focusing on the identification of pathogen-specific biomarkers have been promising. This chapter describes a nontargeted metabolite profiling workflow using gas chromatography coupled with mass spectrometry (GC-MS) for characterizing three globally important foodborne pathogens, Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica, from selective enrichment liquid culture media. The workflow involves a detailed description of food spiking experiments followed by procedures for the extraction of polar metabolites from media, the analysis of the extracts using GC-MS, and finally chemometric data analysis using univariate and multivariate statistical tools to identify potential pathogen-specific biomarkers.

Using GWAS and Machine Learning to Identify and Predict Genetic Variants Associated with Foodborne Bacteria Phenotypic Traits.

One of the main challenges in food microbiology is to prevent the risk of outbreaks by avoiding the distribution of food contaminated by bacteria. This requires constant monitoring of the circulating strains throughout the food production chain. Bacterial genomes contain signatures of natural evolution and adaptive markers that can be exploited to better understand the behavior of pathogen in the food industry. The monitoring of foodborne strains can therefore be facilitated by the use of these genomic markers capable of rapidly providing essential information on isolated strains, such as the source of contamination, risk of illness, potential for biofilm formation, and tolerance or resistance to biocides. The increasing availability of large genome datasets is enhancing the understanding of the genetic basis of complex traits such as host adaptation, virulence, and persistence. Genome-wide association studies have shown very promising results in the discovery of genomic markers that can be integrated into rapid detection tools. In addition, machine learning has successfully predicted phenotypes and classified important traits. Genome-wide association and machine learning tools have therefore the potential to support decision-making circuits intending at reducing the burden of foodborne diseases. The aim of this chapter review is to provide knowledge on the use of these two methods in food microbiology and to recommend their use in the field.

A critical review on metal organic frameworks (MOFs)-based sensors for foodborne pathogenic bacteria detection.

The pervasive threat of foodborne pathogenic bacteria necessitates advancements in rapid and reliable detection methods. Traditional approaches suffer from significant limitations including prolonged processing times, limited sensitivity and specificity. This review comprehensively examines the integration of metal organic frameworks (MOFs) with sensor technologies for the enhanced detection of foodborne pathogens. MOFs, with their unique properties such as high porosity, tunable pore sizes, and ease of functionalization, offer new avenues for sensor enhancement. This paper provides a comprehensive analysis of recent developments in MOFs-based sensors, particularly focusing on electrochemical, fluorescence, colorimetric, and surface-enhanced Raman spectroscopy sensors. We have provided a detailed introduction for the operational principles of these sensors, highlighting the role of MOFs play in enhancing their performance. Comparative analyses demonstrate MOFs' superior capabilities in enhancing signal response, reducing response time, and expanding detection limits. This review culminates in presenting MOFs as transformative materials in the detection of foodborne pathogens, paving the way for their broader application in ensuring food safety.

Vibrio parahaemolyticus Foodborne Illness Associated with Oysters, Australia, 2021-2022.

The bacterium Vibrio parahaemolyticus is ubiquitous in tropical and temperate waters throughout the world and causes infections in humans resulting from water exposure and from ingestion of contaminated raw or undercooked seafood, such as oysters. We describe a nationwide outbreak of enteric infections caused by Vibrio parahaemolyticus in Australia during September 2021-January 2022. A total of 268 persons were linked with the outbreak, 97% of whom reported consuming Australia-grown oysters. Cases were reported from all states and territories of Australia. The outbreak comprised 2 distinct strains of V. parahaemolyticus, sequence types 417 and 50. We traced oysters with V. parahaemolyticus proliferation back to a common growing region within the state of South Australia. The outbreak prompted a national recall of oysters and subsequent improvements in postharvest processing of the shellfish.

Empowering consumers to purchase safe ready-to-eat chicken from street restaurants in Ouagadougou, Burkina Faso: impact of a multi-media behavior change campaign.

Foodborne disease resulting from food sold at urban informal markets is a major public health challenge in Sub-Saharan Africa. This study assessed the impact of an innovative nine-months multi-media campaign engaging a key influencer, aimed at empowering consumers to choose safer ready-to-eat chicken meat at informal street restaurants in Ouagadougou, Burkina Faso. A two-wave panel study assessed associations between recall of TV, radio, billboard, and social media advertisements, and self-reported behavior regarding purchasing and consumption of ready-to-eat chicken at outlets, consumer intentions, knowledge, attitudes, norms, and agency. A panel of randomly selected adult consumers (n = 852) were interviewed pre- and post-campaign, 12 months apart. 60% recalled at least one of the campaign channels when prompted. Mixed-effects models showed associations between prompted recall and feeling better informed about food safety (aOR 1.449) and increased knowledge (0.132 unit increase on total score). Social media recall was associated with higher perceived access to information (aOR 1.449) and knowledge. Billboard recall increased odds of higher perceived health benefits of paying attention to food safety behaviors when purchasing chicken (aOR 2.046). TV ad recall was associated with a 0.159 unit decrease in the gap between consumers' intentions and behavior. An engaging consumer food safety multimedia campaign that engages key influencers improved food safety awareness and knowledge, ultimately supporting consumers to choose safer chicken at markets.

Impact of non-pharmaceutical interventions during the COVID-19 pandemic on pathogens transmitted via food in the Netherlands.

The COVID-19 pandemic impacted the transmission of many pathogens. The aim was to determine the effect of non-pharmaceutical interventions on the incidence of diseases transmitted via food. Weekly incidence rates for nine foodborne pathogens were collected from national surveillance registries. Weekly pathogen incidence during lockdown weeks of 2020 and 2021 were compared with corresponding weeks in 2015-2019. The same analyses were performed to determine the effect of self-defined expected impact levels of measures (low, intermediate and high). Eight out of 9 diseases showed a significant decrease in case number in 2020, except for listeriosis, which remained unchanged. The largest decrease was observed for rotavirus gastronteritis A (-81%), norovirus gastroenteritis (-78%), hepatitis A (-75%) and shigellosis (-72). In 2021, lower case numbers were observed for 6 out of 9 diseases compared with 2015-2019, with the largest decrease for shigellosis (-5/%) and hepatitis E (-47%). No significant change was observed for listeriosis, STEC infection and rotavirus gastroenteritis. Overall, measures with increased expected impact level did not result in a larger decrease in number of cases, except for Campylobacter, and norovirus and rotavirus gastroenteritis. Disease transmitted via food significantly decreased during the COVID-19 pandemic, with a more pronounced effect during 2020 than 2021.

Incidence of foodborne diseases in Ecuador. / Incidencia de las enfermedades transmitidas por alimentos en Ecuador.

BACKGROUND: Motivation for the study. In Ecuador, foodborne disease (FBD) incidence rates adjusted for population size have not been estimated, which will serve to identify priority geographic areas. BACKGROUND: Main findings. Between 2015-2020, 113,695 cases of FBD were identified, with "other food poisoning" and hepatitis A being most common. The highest incidence rates were found in the Amazon region. There is marked variability by geographic region in the incidence rates during the study period. BACKGROUND: Public health implications. It is necessary to optimize the registry system, establish detection and treatment protocols, analyze the causes related to the higher incidence of FBD in the Amazon region, and design a health promotion program focused on preventing contamination and establishing diagnostic and treatment protocols. BACKGROUND: In order to describe the incidence rates per 100 000 population of foodborne disease (FBD) cases during the period 2015-2020 in Ecuador, we carried out a secondary analysis of epidemiological surveillance records and population projections from the National Institute of Statistics and Census. A total of 113,695 cases were reported with an incidence of more than 100 cases per 100 000 population (2015-2019). In 2020, the records are considerably lower than those reported in previous years. Most cases were reported as "other food poisoning". The highest incidence rates of FBD were found in the Amazon region. In general, there is a marked annual variability in the incidence of FBD according to the geographic regions of Ecuador. In conclusion, FBD represent a public health problem in Ecuador. Comprehensive preventive strategies should be designed with special emphasis on the Amazon region.

BACKGROUND: Con el objetivo de describir las tasas de incidencia por cada 100 000 habitantes de los casos de enfermedades transmitidas por alimentos (ETA) durante el periodo 2015-2020 en Ecuador se realizó un análisis secundario de los registros de vigilancia epidemiológica y de las proyecciones poblacionales del Instituto Nacional de Estadística y Censos. Se reportaron 113 695 casos con una incidencia superior a los 100 casos por cada 100 000 habitantes (2015-2019). En el 2020 los registros son considerablemente inferiores a los reportes de años anteriores. La mayoría de los casos se reportaron como «otras intoxicaciones alimentarias¼. Las tasas de incidencia de ETA más altas se observaron en la región amazónica. En general existe una marcada variabilidad anual en la incidencia de las ETA según las regiones geográficas del Ecuador. En conclusión, las ETA representan un problema de salud pública en el Ecuador. Se deben diseñar estrategias preventivas integrales con especial énfasis en la región amazónica. BACKGROUND: Motivación para realizar el estudio. En Ecuador, no se ha estimado las tasas de incidencia de enfermedades transmitidas por alimentos (ETA) ajustadas al tamaño de la población, lo que servirá para identificar áreas geográficas prioritarias. BACKGROUND: Principales hallazgos. Entre 2015-2020 se identificaron 113 695 casos de ETA, siendo más comunes las «otras intoxicaciones alimentarias¼ y la hepatitis A. Las tasas de incidencia más altas se observaron en la región amazónica. Existe una marcada variabilidad por región geográfica en las tasas de incidencia reportadas durante el periodo de estudio. BACKGROUND: Implicancias en la salud pública. Es necesario optimizar el sistema de registro, instaurar protocolos de detección y tratamiento, así como analizar las causas relacionadas con la mayor incidencia de ETA en la región amazónica y diseñar un programa de promoción de salud enfocado en prevenir contaminación y establecer protocolos de diagnóstico y tratamientos.

Risk factors of foodborne parasitic diseases still remain in China: a WeChat-based knowledge, attitudes, and practices study.

OBJECTIVE: With the progress of economic globalization and food diversification, foodborne parasitic diseases pose a significant public health challenge in China. This study aimed to explore the knowledge, attitudes, and practices regarding foodborne parasitic diseases among the Chinese population via WeChat, which is a promising tool for disease surveillance and health education. METHODS: Using a questionnaire, this cross-sectional study was conducted on September 25, 2023. Participants completed a structured questionnaire by scanning a QR code provided in a tweet from the WeChat official account of the Jiangsu Institute of Parasite Disease. Multivariable logistic regression analysis was employed to explore potential independent determinants of adequate knowledge of foodborne parasitic diseases, and the positive attitude and good practice rates of the participants were calculated. RESULTS: In total, 5,675 valid questionnaires were collected via the WeChat official account. Most participants (79.91%) fell within the age range of 20-40 years, with a higher representation of males (53.18%), and 89.80% were of Han Chinese ethnicity. It was found that 76.65% of the participants had adequate level of knowledge. Multivariable logistic regression analysis revealed that individuals of Hui nationality (OR = 0.57, 95% CI: 0.40-0.81, p = 0.002), clinicians (OR = 0.13, 95% CI: 0.09-0.19, p < 0.001), teachers (OR = 0.48, 95% CI: 0.34-0.69, p < 0.001), and government staff (OR = 0.40, 95% CI: 0.30-0.53, p < 0.001) had significantly higher levels of knowledge. Among the participants, 33.9% reported consuming raw fish or drunken shrimp, 10.6% would still try to consume raw fish despite the risk of parasitic infection, and 84.1% did not use separate cutting boards for raw and cooked foods in their kitchens. CONCLUSIONS: Although majority of the Chinese public (76.65%) demonstrates adequate level of knowledge on foodborne parasitic diseases, there is a need to enhance personal hygiene practices and dietary habits, particularly the utilization of distinct cutting boards and the consumption of raw fish. A WeChat official account is an accessible tool for spreading foodborne parasitic diseases related health information to the public. WeChat-based health education should be implemented to enhance public awareness regarding the prevention and control of foodborne parasitic diseases.

Fresh produce-associated foodborne disease outbreaks in Australia, 2001 to 2017.

Abstract: Fresh produce is an important source of foodborne outbreaks in Australia. Using descriptive analysis, we examined confirmed and suspected foodborne outbreaks associated with fresh produce in Australia recorded in the OzFoodNet outbreak register from 2001 to 2017. The outbreak register contains reports of foodborne disease outbreaks collected by OzFoodNet epidemiologists and public health officials. A fresh produce outbreak was defined as the occurrence of two or more cases of the same illness in which the investigation had implicated a common food and this food contained fresh produce. A total of 92 fresh produce outbreaks were reported, encompassing 3,422 reported illnesses, 446 hospitalisations and four deaths. Of these outbreaks, 76.1% (70/92) were caused by a known pathogen, with the majority caused by either Salmonella enterica (n = 30) or Norovirus (n = 29). Most outbreaks (77.2%; 71/92) were associated with consumption of foods containing multiple ingredients, some of which were not fresh produce. The largest outbreaks associated with a single fresh produce item included bean sprouts contaminated with S. enterica serovar Saintpaul (419 illnesses and 76 hospitalisations) and semi-dried tomatoes contaminated with Hepatitis A (372 illnesses and 169 hospitalisations). Restaurants (45.7%; n = 42/92) and commercial catering (15.2%: n = 14/92) were common settings for fresh produce outbreaks. Outbreaks occurred in all states and territories of Australia and in all seasons, with an increased frequency in the warmer months (September-May). Although the number of fresh produce-associated outbreaks did not seem to be increasing in Australia, integrated surveillance is needed to rapidly identify sources of infection due to the propensity of these outbreaks to be large and widespread.

New primary production and processing standards developed to reduce foodborne illness risks.

Abstract: This editorial summarises a set of three new standards developed by Food Standards Australia New Zealand, which respectively address food safety requirements for the commodities of berries, leafy vegetables, and melons.

Listeria monocytogenes in the seafood industry: Exploring contamination sources, outbreaks, antibiotic susceptibility and genetic diversity.

Fish and seafood are rich sources of protein, vitamins, and minerals, significantly contributing to individual health. A global increase in consumption has been observed. Listeria monocytogenes is a known problem in food processing environments and is found in various seafood forms, including raw, smoked, salted, and ready-to-eat. Without heat treatment and given L. monocytogenes' ability to multiply under refrigerated conditions, consuming seafood poses a substantial health hazard, particularly to immunocompromised individuals. Numerous global outbreaks of listeriosis have been linked to various fish products, underscoring the importance of studying L. monocytogenes. Different strains exhibit varying disease-causing abilities, making it crucial to understand and monitor the organism's virulence and resistance aspects for food safety. This paper aims to highlight the genetic diversity of L. monocytogenes found in fish products globally and to enhance understanding of contamination routes from raw fish to the final product.

Surveillance of Food and Waterborne Pathogens in North-East India: Protocol for a Laboratory-Based Sentinel Surveillance Study.

BACKGROUND: Food safety is a global concern, which is often underestimated owing to challenges in investigating foodborne diseases. These challenges arise from the increased globalization of the food trade, advancements in agricultural practices, and shifts in environmental factors. In North-East India, common diarrheal outbreaks from fermented foods pose significant health risks. Despite these challenges, systematic data on foodborne pathogens is lacking in India, highlighting a crucial gap in understanding food safety issues. OBJECTIVE: The aim of this research protocol is to establish an integrated surveillance system to identify enteric pathogens circulating within humans, food animals, and the environment through a health approach in North-East India, and to conduct outbreak investigations. METHODS: The Indian Council of Medical Research (ICMR) initiated a surveillance study across all 8 North-East states in India, employing a centralized digital database for data collation. The project aims to enhance the infrastructure for microbial culture, antibiotic sensitivity testing, and molecular epidemiological studies. The study involves laboratory-based surveillance of foodborne pathogens in market foods, hospitalized diarrheal patients, poultry and animal farms, slaughterhouses, butcher shops, and diarrheal outbreaks. A standardized case report form ensures consistent data collection of age, sex, signs, symptoms, and admission dates for diarrheal cases. Stool and rectal swabs will undergo testing for pathogen identification and antimicrobial resistance. Similarly, samples of market foods, food animals, and the environment will be collected. Outbreaks confirmed by the Integrated Disease Surveillance Project (IDSP) will be thoroughly investigated following standardized guidelines. RESULTS: In phase I, 5 surveillance centers were established across 4 states (ie, Assam [Dibrugarh and Guwahati], Tripura, Sikkim, and Arunachal Pradesh) in 2020. Following an interim phase I data assessment and the successful establishment of a streamlined system for data procurement, investigation, recording, and analysis, along with the implementation of regular training and monitoring programs, phase II expansion was initiated in 2023-24. This includes the addition of 7 more centers (including 3 veterinary centers) in the remaining 4 states (ie, Manipur, Meghalaya, Mizoram, and Nagaland), eventually covering the entire North-Eastern Region of India. CONCLUSIONS: Food and waterborne diseases are a constant public health problem in many countries. Key challenges to the enhancement of food safety policy include the paucity of systematic data and awareness. With this background, ICMR's initiative is the first systematic surveillance study in the country to adopt a single health approach. Data obtained from this project will help to understand the risk of acquiring food and waterborne pathogens, their transmission pathways, and antimicrobial resistance patterns. The scientific evidence generated through this project will be helpful in formulating and strengthening food safety policy and in initiating government programs to protect the health of the nation. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/56469.