Use of prokaryotic environmental isolates in genetic engineering work

In its updated statement, the ZKBS specifies criteria and testing methods for classifying wild-type isolates of bacteria and archaea from environmental samples into a risk group. This classification is necessary if bacteria and Archaea are to be used as donor or recipient organisms in genetic engineering without having been clearly identified to the species level beforehand. Environmental isolates should first be reliably characterized using modern biochemical and molecular biological or taxonomic methods. However, if these taxonomic studies do not result in a clear assignment to a known species, the principles and procedures described in the statement should be applied. Detailed investigation of an environmental isolate may be unnecessary if the taxonomic and physiological studies allow its assignment to certain larger taxonomic units, metabolic types or the microflora of extreme locations, which, due to certain characteristics, contain only organisms of risk group 1. This is the case, for example, with acidophilic, alkaliphilic, psychrophilic and thermophilic as well as autotrophic prokaryotes. Archaea Until recently, Methanobrevibacter oralis were generally considered non-pathogenic. However, there is now evidence that Methanobrevibacter oralis may be involved in opportunistic infections in humans. Therefore, the ZKBS has classified M. oralis as risk group 2 as a precautionary measure (ref. 45241.0223, September 2021). Risk assessment criteria

1. Origin of the isolate If a location typical for the microorganism is characterized by extreme living conditions (e.g. high salt content or high temperatures), pathogenicity for humans or animals cannot be assumed.
2. Growth conditions: Isolates that reproduce only below 25 °C or only above 42 °C are generally considered non-pathogenic to humans; the same applies if the pH value in the culture medium for reproduction must be below 5.5 or above 8.5.
3. Nutrient requirements Organisms that grow very poorly or not at all on complex media can usually be excluded as pathogens.

The criteria listed under 1.-3. may already be sufficient to carry out a risk assessment of an isolate of unclear taxonomic position. However, more often, further investigations are required to determine the pathogenic potential of a new isolate may be necessary. These may include: 4. Animal testing Animal experiments can be conducted to assess pathogenic potential, for example to determine the lethal dose 50 (LD50) in a mouse model. 5. Cytotoxicity studies By incubating established eukaryotic cell lines with culture supernatants or culture filtrates, a possible cytotoxicity or cytopathic effect of environmental isolates can be tested. 6. Adhesion tests Co-culture experiments can be used to test whether an isolate forms adhesins and can thus colonize eukaryotic cells.

According to the criteria listed above, environmental isolates from extremophilic, halophilic, or autotrophic organism groups do not pose a hazard. Isolates that cannot be assigned to any known species and belong to these groups of organisms can be used as donor or recipient organisms in genetic engineering work. Risk group 1 without having been previously classified into a risk group by the ZKBS. The risk group assignment of an isolate that cannot be assigned to a known species or belongs to the extremophilic, halophilic, or autotrophic organism groups is a case-by-case decision. This must be made by the ZKBS before the organisms are used as donor or recipient organisms in genetic engineering work.

**Note: ** When classifying prokaryotes with phytopathogenic potential, the statement of the ZKBS on criteria for the evaluation and classification of plant viruses, phytopathogenic fungi and phytopathogenic bacteria as donor and recipient organisms for genetic engineering work (ref. 6790-10-53) must also be taken into account.

The updated ZKBS statement can be found at File number 6790-10-43 can be retrieved.