7. Microbiomes, a food safety perspective

Microbiomes are a complex and dynamic network of microorganisms (bacteria, viruses, fungi, archaea) that adapt and live in a functional relationship with their specific habitats (e.g. human, soil, plant, water, animals, production sites along the food chain) (Berg et al., 2020). Neighbouring microbiome ecosystems exert mutual influences, even when physically separated (e.g. animal and soil) (Flandroy et al., 2018). In addition, microbiomes are very sensitive to environmental conditions and exposure to substances of different nature. In humans, various factors (e.g. genetics, diet, drugs, lifestyle, oxygen, pH) contribute to shaping the microbiomes’ subpopulations along the various sections of the gastrointestinal tract (Shetty et al., 2017).

Why is the gut microbiome gaining interest?

There is an increasing amount of scientific information associating – or to a lesser extent demonstrating – that the gut microbiome has the potential to influence human health. For example, the microbiome influences the development of the immune system, has a protective role (first line of defence in the gut), and synthesizes metabolites essential for maintaining human homeostasis (vitamin D and shortchain fatty acids). In addition, microbiome imbalances have also been associated with some non-communicable disorders (NCD), including inflammatory and metabolic diseases (obesity, diabetes, inflammatory bowel disease) (Lynch and Pedersen, 2016). Non-human microbiomes have also been associated with the health status of other ecosystems, e.g. soil and plant (Flandroy et al., 2018). Such microbial populations present in the different environments along the food chain are also contributors to food quality and food safety (Weimer et al., 2016).

Since the microbiome may play a role in human homeostasis, it can be used as a target for different dietary interventions to maintain and promote health (e.g. optimizing fibre intake, administration of pre- and probiotics) (Wilson et al., 2020). On the other hand, microbiome disruptors (e.g. imbalanced diet) are also attracting attention as they may lead to dysbiosis,²³ which can eventually result in adverse effects on human health (Das and Nair, 2019). Much of the current interest gathers around the capacity of food additives, chemical residues (pesticides and veterinary drugs), antibiotics or other environmental pollutants to lead to biologically relevant microbiome perturbations (Cao et al., 2020; Chiu et al., 2020).

Technological advancements enhancing our understanding of microbiomes

Until recently, traditional microbiology has focused on the individual identification of microorganisms and related functions in, for instance, food production (e.g. fermentation), promoting health status (e.g. probiotic gut bacteria) or as contributors to disease (e.g. pathogens). New technological advances in the omics and bioinformatic fields have enabled the holistic study of microbial community structures (microbiomes) and their functional activity within a given environment (Galloway-Pena and Hanson, 2020). Metagenomics tools sequence the DNA material and provide information on the taxonomical composition of the microbiome and gene diversity, which provides an indication of the potential microbial functions. Other omics technologies target the microbiome activity. They indicate active metabolic pathways through the analysis of gene expression by RNA sequencing (transcriptomics and metatranscriptomics), the protein resulting from such expression (proteomics) or final end-products or metabolites (metabolomics). The science supporting the microbiome study is relatively new and still evolving. It is still lacking standardization and generates immense amounts of data that cannot yet be interpreted. Therefore, our understanding of microbiomes and interactions with their ecosystems is still limited.

What are the food safety implications to be considered?

The study of the microbiome from farm to fork has the potential to improve our understanding of hazards and health risks. Within the context of food safety, different microbiomes can be exploited for different purposes.

The microbiome is not a food safety risk per se. Until recently, the microbiome has been an unexplored contributor to food safety and quality. The holistic understanding of the microbiome-environment-host interactions, and their influence on human exposure to different types of biotic or abiotic factors, open a new avenue to better understand hazards and health risks, and therefore microbiological and chemical assessments.

Microbiomes in food production chains as indicator for hazards

Now that microbial populations can be evaluated in a holistic manner by using omics technologies (e.g. complete DNA or RNA analysis by culture-independent deep shotgun metagenomics or metatranscriptomics, respectively, proteomics or metabolomics), it will be possible to monitor for the full potential or the presence of microbial hazards (e.g. pathogens, pathogenic factors and antimicrobial resistance) up and downstream in food production, not only in food and food ingredients, but also in the environment of production sites (Beck et al., 2021; De Filippis et al., 2021). It will also improve our understanding of factors (processing steps, microbiomes in the environment, storage) influencing the pathogenic potential at a given location and the acquisition and flow of antimicrobial resistance along the production chain. Therefore, the study of the microbiomes will bring a new perspective to the characterization of microbial hazards. Moreover, it will provide the basis for the development of suitable and effective preventive measures. There are numerous scenarios where the microbiome and microbial compounds can be used as hazard indicators of food safety and quality (Weimer et al., 2016). The following are some examples: selection of safe starter cultures and their monitoring during product manufacturing, evaluation of air microbiome in age-drying chambers to minimize the potential transfer of pathogens from the environment to the product, and the influence of storage conditions in the composition and production of compounds affecting the food safety and quality.

The interaction between the gut microbiome and exogenous compounds and implications in human health

In addition to macro and micronutrients, the gut microbiome can enter in contact with other compounds through food and water consumption. These can be intentionally introduced in the product formulation (i.e. food additives) or result from upstream activities in the food chain (e.g. residues of veterinary drugs and pesticides) or be present inadvertently in the diet (e.g. environmental or industrial contaminants). The gut microbiome can metabolize and transform compounds, alter their bioavailability and modify their toxic potential (Claus, Guillou and Ellero-Simatos, 2016). Therefore, microbial activities can modify human exposure to such substances. Moreover, exogenous compounds also have the potential to induce changes in the composition and activity of the microbiome and lead to dysbiosis (Abdelsalam et al., 2020). Such microbial imbalance could eventually have implications for human health.

A yoghurt production plant.
©Shutterstock/branislavpudar

Exogenous compounds such as food additives, residues of veterinary drugs and pesticides or microplastics are very heterogeneous groups of chemical compounds. Out of this broad chemical spectrum, research has only been conducted on a limited number of compounds and show their potential to disturb the gut microbiota. Most studies, which differ in design and methodologies, are generally conducted at levels that exceed those found in a normal diet (Roca-Saavedra et al., 2018). Therefore, this information is of limited use from the perspective of food safety risk assessment as it does not reflect a realistic (low-level) dietary exposure. However, the limited number of studies specifically designed to evaluate exogenous compounds at low residue levels provides an indication that effects of exogenous compounds on the microbiome follow a dose-dependent relationship (Piñeiro and Cerniglia, 2021). Although some associations are made between microbiota alterations and adverse health effects observed in laboratory animals, the causal role of microbial disturbances specifically induced by these exogenous compounds on host alterations remains unclear (Walter et al., 2020). Microbiome science is a quickly developing field of research. Food safety risk assessment bodies are closely watching the emerging research regarding its significance for food safety risk assessment (Merten et al., 2020; National Academies of Sciences and Medicine, 2018; Piñeiro and Cerniglia, 2021). However, the science available to date does not provide enough consensus, mechanistic understanding and has not established sufficient repeatability (Sutherland et al., 2020), so that no clear conclusions can be drawn at the moment.

The role of the gut microbiome against foodborne pathogens

The gut microbiome contributes to the resistance against the colonization by foodborne pathogens and the proliferation of commensal opportunistic pathogens (Pilmis, Le Monnier and Zahar, 2020). Pathogen colonization does not only depend on the infective dose and the host’s immune system but also on the health status of the gut microbiota. Alterations on the structure and function of the gut microbiome, which may be caused by dietary imbalances or exposures to certain substances, can offer a window of opportunity for pathogens to break the gut barrier. Colonization resistance is one of the endpoints used to determine the microbiological Acceptable Daily Intake (mADI) in the assessment of veterinary drug residues (VICH, 2019).

Antimicrobial resistance (AMR)

In 2015, within the context of One Health approach, the World Health Organization (WHO) developed a Global Action Plan (GAP) on AMR (WHO, 2015). It acknowledges the role of the food and agriculture sectors in the global fight against AMR (Cahill et al., 2017). The food chain offers favourable conditions for AMR transmission, which link the animals, humans, food and environmental microbiome ecosystems (Cahill et al., 2017). The gut microbiota has been described as a reservoir of antimicrobial resistance (Hu and Zhu, 2016) and the high microbial density in the gut, especially in the large intestine, makes it highly susceptible to the transfer of genetic material (Smillie et al., 2011). In fact, the gastrointestinal tract is constantly exposed to new bacteria coming from the environment, including food, which may carry and potentially transfer antimicrobial resistance genes to members of the gut microbiome (Economou and Gousia, 2015; Penders et al., 2013). Metagenomics has enabled monitoring the resulting resistome (Hendriksen et al., 2019; Kim and Cha, 2021).²⁴ This holistic approach allows to study of the prevalence, distribution and trends of antibiotic resistance genes in a population, the co-resistance to antibiotic and non-antibiotic compounds, and the potential for horizontal transfer (Feng et al., 2018; Hendriksen et al., 2019).

©Shutterstock/Vadim Petrakov

What are the regulatory implications of microbiome research?

Chemical risk assessments aim at evaluating the safety of food additives, chemical residues in food and environmental pollutants and contaminants, and serve as the basis to establish health-based guidance values (e.g. Acceptable Daily Intake ADI, Acute Reference Dose ARfD). With the further step of exposure assessment, a risk can be characterized, and this serves as a basis to set up regulatory levels such as, for example, Maximum Residue Limits (MRLs) for veterinary drugs and pesticides, Maximum Levels (MLs) for contaminants, and Maximum Use Level (MUL) for food additives. Although the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and the Joint Meeting on Pesticide Residues (JMPR) have advanced their procedures to complement toxicological chemical assessments with a microbiological component (FAO and WHO, 2009), there are many ongoing discussions among risk assessors about further integrating the microbiome in chemical risk assessment (Merten et al., 2020; National Academies of Sciences and Medicine, 2018; Piñeiro and Cerniglia, 2021).

Regulatory decisions require careful consideration, given their impact on the food system. For this reason, the science supporting risk assessment needs to be robust, reproducible and based on suitable and well-defined endpoints. However, although the microbiome’s potential as a component of risk assessment is widely recognized, critical technical limitations and knowledge gaps need to be addressed before the evaluation of microbiome interactions with food additives, pesticide and veterinary drug residues and other food contaminants are incorporated into regulatory activities.

What is the way forward?

Future perspectives on the topic

Latest trends have placed the microbiome as the target of dietary interventions to promote health or as a mediator in human disease due to alterations caused by different types of compounds (e.g. food additives, residues of veterinary drugs and pesticides, and environmental pollutants). However, the information supporting any claim requires careful and critical interpretation. While for the vast majority of cases research has only provided statistical associations between microbiome disturbances and health or disease, there is a need to prove causality (in other words, demonstrate the causal link between changes in the microbiome composition and function and physiopathological alterations in the subject). This would be the demonstration that, indeed, the microbiome contributes to either maintaining or disrupting human homeostasis. Moreover, further investigation about causal links could also indicate that microbial changes are a consequence of disease, not the cause. Once proven, it will be necessary to understand the dimension of such contribution.

Understanding the relative role and underlying mechanisms of the microbiome in health and disease will enable the update of chemical risk assessments and the development of evidence-based methodologies and frameworks to evaluate microbiome-related data.

Now that we have the possibility to evaluate the dynamics of microbial ecosystems, we also have significant potential to study the microbiome in food systems (ingredients, and foods and the different environments along the food production chain). These can include:

• establishing food or ingredient microbiome fingerprints at different production stages;
• early identification of abnormal shifts in starter cultures, products, and the environments of food production sites; and
• up and downstream monitoring of pathogenicity signatures and the resistome.

Scope for future research

One of the most basic and still essential need in microbiome science is the lack of a consensus for the definition of a healthy microbiome. However, establishing what constitutes a healthy microbiome is challenging. Factors such as diet, lifestyle, genetics and surrounding environments influence how the microbiomes evolve, resulting in high interindividual variability. Also important is to define dysbiosis and distinguish normal fluctuations in microbial composition and function from alterations of concern.

Still evolving analytical technologies and experimental methodologies need standardization and best practice guidelines to provide consistent, comparable and reproducible results. Moreover, for chemical risk assessment, there is a need to define fit-for-purpose experimental models, including the use of appropriate low-doses of test compounds (e.g. food additives, residues of veterinary drugs and pesticides) and exposure periods.

Although most research has targeted the bacterial component of the microbiome primarily, additional efforts are needed to study non-bacterial members such as viruses, fungi, archaea and protozoa. Further research is also necessary to elucidate all the substantial amount of generated data by omics technologies. It includes identifying new microbiome members, characterization of genes, metabolic pathways, proteins and metabolites.

To connect the microbiome with health and disease, it is critical to demonstrate causality and characterize biologically relevant microbiome disturbances. This will require the identification and validation of suitable microbiome-related biomarkers and endpoints.

These are knowledge gaps limiting the capacity to fully exploit the microbiome as a tool to promote food quality, and improve food safety processes, including incorporating microbiome data in chemical risk assessment and informing regulatory decisions.

Collaborations are key to moving forward

Microbiome science is inherently a multidisciplinary field. Most of the breakthrough advances have been possible thanks to coordinated efforts in the form of big projects with the participation of large multinational consortia (e.g. Human Microbiome Project).

To address the food microbiome as an additional component in chemical risk assessment and define possible framework, it would be necessary to convene a multidisciplinary group of experts (risk assessors, microbiome scientists, and regulators).

The current and strong interest in the microbiome is sometimes leading to overstatements, conveying the idea that it is a universal solution for almost everything. However, such statements needs a stronger scientific basis. Therefore, it is necessary to promote evidence-based, consistent and accurate communication strategies considering the status quo on microbiome knowledge and associated uncertainties. This is not only a challenging task, but also an opportunity to engage the public with stakeholders within the agrifood systems.

Due to the broad number of potential applications of the microbiome in agrifood systems, the complexities of the topic, and the need for consensus approaches, it would be beneficial and productive to involve all stakeholders, including academia, research organizations, industry and regulatory bodies. Many activities can be derived from such interactions, including the definition of topic-specific research needs, promotion of research collaborations, development of best practices, development and implementation of food safety applications (e.g. HACCP programs), and capacity development and so on. Given the consensus-driven nature and mission of FAO, the organization has the capacity to promote engagement activities and serve as a driving force in the dialogue about the microbiome in agrifood systems. As a first step forward, FAO is reviewing the scientific literature to define the status quo and knowledge gaps in understanding the interrelations of food additives, microplastics, residues of veterinary drugs and pesticides, the gut microbiome and human health. FAO’s intention is to update and expand the literature research to other relevant substances and microorganisms as new information becomes available. Recognizing research needs and areas of improvement will ultimately allow laying down the path towards defining and implementing microbiome-related applications to support food systems and policy activities