Key Requirements for Microbial Characterization

In general, EFSA mandates whole genome sequencing (WGS) for taxonomic identification and the detection of genes of concern, such as those conferring AMR or producing toxins. For GM microorganisms, detailed descriptions of inserted or deleted sequences, vector characteristics, and phenotypic changes are required.

The guidance also emphasizes the need for the depositing of strains in recognized culture collections and the use of curated databases for bioinformatic analyses.

Antimicrobial Resistance and Toxigenicity

The assessment of AMR is primarily performed via WGS analysis. When WGS identifies an AMR gene, it should be determined whether the gene is intrinsic or acquired, which may require further phenotypic testing and literature searches to distinguish. For yeasts and fungi, susceptibility to at least two antifungal classes must be demonstrated.

The guidance also calls for cytotoxicity testing in Bacillus and taxonomically related species, as well as WGS-based screening for virulence factors in Enterococcus and Bacillus cereus strains.

Environmental Risk Assessment

Environmental risk assessment (ERA) is required for both non-GM active agents considered to pose a risk, all GM active agents, and GM and non-GM biomasses considered to be a risk. Key risk areas include persistence and invasiveness; horizontal gene transfer; effects on non-target organisms, the environment, and biogeochemical processes; and the effect of management techniques.

GM active agents should undergo a comparative ERA against a non-GM parental strain or a suitable comparator. Biomasses are assessed primarily for the presence of residual DNA and the potential for horizontal gene transfer.

Safety Outcomes

Non-GM active agents are deemed low risk when microorganisms qualify for the Qualified Presumption of Safety (QPS) list, or:

• Lack genes of concern (e.g., acquired AMR) or are adequately susceptible to antifungal compounds
• Do not produce harmful metabolites or therapeutic antimicrobials
• Are non-pathogenic or do not cause adverse effects on non-target species
• Do not cause adverse effects on the environment.

These same safety conditions apply for products containing GM active agents that do not introduce genes of concern by their genetic modification.

However, the use of an active agent that does not fulfill one or more of the safety conditions required for its taxonomic unit (i.e, bacteria, yeast or fungi, microalgae or protist, or virus) is considered a risk for animals, humans, and/or the environment.

GM and non-GM biomasses and bacteriophages, and products produced using GM and non-GM microorganisms, are not considered to represent a hazard, and do not pose a risk to animals, humans, and the environment when:

• The strain qualifies for the QPS approach

Or, if the strain does not qualify for the QPS approach:

• It does not harbor genes of concern
• It does not produce harmful metabolites/therapeutic antimicrobials
• It is not pathogenic
• It does not cause adverse effects on the environment
• No viable cells/DNA of the host strain are detected in the final product (where relevant).

Specific safety conditions vary among taxonomic units.

Implications for Applicants

Applications for microorganisms and their derived products to be used in the food chain must include robust, up-to-date scientific data, tailored to the specific strain and product use, as outlined in the new guidance.

Several existing EFSA guidance documents will be superseded by the new guidance document, as outlined in Appendix A of the new guidance document.