Unauthorized GM Bacillus subtilis production strain identified in a vitamin B2 feed additive

By Jane Byrne

- Last updated on GMT

© istock/jarun011
© istock/jarun011

Related tags Dna Antibiotic resistance China Eu

A genetically modified microorganism (GMM) integrating an antibiotic resistance gene was found in a riboflavin supplement from China aimed at feed use, finds newly published study. 

Regulators from Germany and Italy, who analyzed a vitamin B2​ product imported from China three years ago, just released their findings in Food Chemistry.​ 

The research was first reported on by Independent Science News​.

The tests followed the 2014 incident whereby a German enforcement laboratory alerted EU officials to illegal GE bacterial contamination in the imported feedstuff.

According to EU biosafety regulations, no GM bacterial strain or any DNA is allowed to be present in commercial food and feed supplements.

The officials said they performed tests to characterize the isolated strain. 

Discrepancy with Chinese reports

The analysis showed the illegal contaminating strain was not among those the Chinese manufacturer claimed to be using.

“The differences in the information provided by the Chinese company and the results of the molecular characterization strongly imply that the production strain must have been contaminated or switched before or during production.” 

In 2016, the EU Commission’s Food and Veterinary Office (FVO) carried out an audit in China in order to evaluate their control systems for GMO.

The review was in response to the detection of the presence of GMOs in rice products exported from China and to a case of Bt63 presence in a feed additive exported to the EU along with the case described in the study.

The FVO reported that the Chinese competent authorities carried out investigations at all vitamin B2 producers, but the Chinese officials did not identify any irregularities.

In addition, the tests indicated the viable strains of the GMO Bacillus subtilis strain contained DNA extrachromosomal plasmids with other antibiotic resistance genes. These conferred resistance to the antibiotics, ampicillin, kanamycin, bleomycin, tetracycline, and erythromycin, said the authors.

Vitamin B2 product (80% feed grade) imported from China was analyzed. Viable B. subtilis cells were identified and DNAs of two bacterial isolates (LHL and LGL) were subjected to three whole genome sequencing (WGS) runs with different devices (MiSeq, 454 or HiSeq system). WGS data revealed the integration of a chloramphenicol resistance gene, the deletion of the endogenous riboflavin (rib) operon and presence of four putative plasmids harboring rib operons.

“These results immediately highlighted that the product was not compliant with the European food and feed safety regulatory requirements for feed additives containing GMMs.”

Riboflavin or vitamin B2 is an essential vitamin in animal nutrition. Riboflavin supplements used to be chemically synthesized, but now, are often produced by commercial fermentation using overproducing strains of GE bacteria.

“Microbial synthesis of riboflavin involves genetically engineered selected strains of Escherichia (E.) coli, Bacillus (B.) subtilis, Ashbya (A.) gossypii, and Candida (C.) famata (Abbas & Sibirny, 2011).”

In 2015, a French laboratory also found one riboflavin sample also contaminated with what is likely to be an identical bacterial strain, again from China (Barbau-Piednoir et al., 2015).

The review of scientific literature on GMM strains in the context of riboflavin production revealed that antibiotic resistance genes are used as selection markers and thus potentially present in production strains, explained the researchers. 

Detection methods

“The lack of a specific detection method for a respective production strain made the analytical work quite challenging.”

The regulators, thus, said they also documented the next generation sequencing (NGS) tools they used in their tests to help EU food and feed competent enforcement authorities and official control laboratories in their investigations related to the presence of GMMs in biotechnological products.

“The approach described in our study allowed to provide a detailed molecular characterization of an unknown GMM and thereby facilitated the risk assessment and as well as the development of specific PCR methods for its detection.

“Nevertheless, the study showed that the bioinformatics analysis of the data produced by NGS is still a challenging task and would require the development of adapted bioinformatics tools in order to be implemented for routine data analysis and management in the frame of GMO characterization and detection.”

Source: Food Chemistry

Published online ahead of print:https://doi.org/10.1016/j.foodchem.2017.03.042

Title:Molecular characterization of an unauthorized genetically modified Bacillus subtilis production strain identified in a vitamin B2 feed additive

Authors:​ Paracchini, V, Petrillo, M, Reiting, R, Angers-Loustau, A, Wahler, D, Stolz, A, Pecoraro, S.

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