Antibiotic resistance marker gene used in genetically modified crops found in bacteria isolated from all China's rivers Dr Eva Sirinathsinghji
A new study conducted in China finds 6 out of 6 major rivers tested positive for ampicillin antibiotic resistant bacteria . Sequencing of the gene responsible, the blá gene, shows it is a synthetic version derived from a lab and different from the wild type. This suggests to the researchers that synthetic plasmid vectors from genetic engineering applications may be the source of the ampicillin resistance, which is affecting the human population. The blá gene confers resistance to a wide range of therapeutic antibiotics and the widespread environment pollution with blá resistant bacteria is a major public health concern.
The development of antibiotic resistant pathogens, commonly dubbed “superbugs”, are increasingly common due to the overuse of antibiotics in medical and veterinary practices, and the ever-increasing application of genetic engineering to industrial processes including agriculture, biofuel fermentation and environmental remediation on top of laboratory research. Previously, genetic engineering experiments were confined to the laboratory, but with industrial and agricultural applications becoming more common over the last decade, the chances of uncontrolled discharge as well as deliberate release into the environment has widened. One prime example is the planting of genetically modified (GM) crops, many of which carry antibiotic resistant genes.
Genetic engineering uses plasmids - extra-chromosomal DNA molecules that naturally exist in bacteria and other unicellular species - for propagating and manipulating DNA sequences in research and in genetic modification of plants and animals. Plasmids often carry antibiotic resistance marker genes to allow selection with antibiotics for the modified DNA or cells carrying the gene of interest (see  (FAQ on Genetic Engineering, ISIS Tutorial). The presence of these antibiotic resistance genes and plasmids in the environment leaves open the possibility of the genes being taken up and transferred into the genetic material of unrelated species of bacteria, some of which may well be serious pathogens.
The transfer of genes directly into the genetic material of cells, bypassing normal reproduction, is referred to as horizontal gene transfer, to distinguish it from the usual vertical gene transfer that occurs in natural reproduction within the same species or in some cases between closely related species.
Scientists including those in I-SIS have issued repeated warnings since the 1990s on the dangers of horizontal gene transfer associated with genetic engineering and GM plants and animals that are released into the open environment [3-6] (Gene Technology and Gene Ecology of Infectious Diseases, I-SIS scientific publication; Horizontal Gene Transfer - The Hidden Hazards of Genetic Engineering, ISIS/TWN report; GM DNA Does Jump Species, SiS 47; Scientists Discover New Route for GM-gene 'Escape', SiS 50), only to be met with denial and dismissal from the proponents and from our regulators.
The new study led by Jun Wen Li at Sechuan University reveals widespread contamination of 6 out of 6 major urban rivers (the Sungari, Haihe, Yellow, Yangtze, Huangpu and Pearl Rivers) with bacteria carrying a synthetic version of the blá gene . The blá gene confers resistance to the most common class of antibiotics called β-lactams, which includes besides ampicillin (a beta-lactam), the penicillin derivatives (penams), cephalosporins (cephems), monobactams, and carbapenems.
The researchers took samples from the rivers, extracted plasmids from bacteria present, and used PCR (polymerase chain reaction) and quantitative real-time PCR to assess the presence of blá DNA. The assay was specific for the blá gene that comprises most recombinant plasmid strains, such as pBR322 and pUC19, both widely used for research and genetic modification. The detection rate varied from 21.9 % (in the Hai He River samples) to 36.4 % (in the Yangtze River samples). The Pearl and Hai He rivers showed the widest range of cephalosporin resistance from the blá gene present in bacterial samples, extending to 3rd- and 4th-generation drugs like cefotaxime and cefoperazone, while the range was narrower (e.g., cefalotin, cephazolin, cefmetazole, and cefoxitin) in samples from the other rivers tested. Analysis confirmed that sequences “neighbouring” the blá sequences “most frequently represented artificial or synthetic constructs, including cloning, expression, shuttle, gene-fusion, and gene trap vectors” derived from recombinant laboratory plasmid vectors, identifying most strongly with pBR322; and confirming the artificial origin of the DNA that does not naturally exist in nature.
Metagenomic technology, which involves transforming environmental genomic DNA into a laboratory recipient strain, is a unique way to study complex genetic samples from ecosystems without purifying the strains. As this study concerned plasmids within environmental microbes, the procedure was modified so that the plasmids were extracted and electro-transformed directly into the laboratory strains. Antibiotics selection was used to identify clones expressing resistant plasmids, which were then isolated and analyzed. A plasmid metagenomic library of 205 environmental plasmid-carrying E. coli HB101 strains was constructed, which showed a positive blá rate of 27.3%. Furthermore, samples from all 6 rivers are also resistant to tetracycline. In addition, some transformants are resistant to other antibiotics such as gentamicin and sulfanilamides. With this technique focusing on plasmids, it is worth noting that plasmid sequences integrated into the bacterial genome were not investigated, and if measured, would likely increase the rate of antibiotic resistant gene contamination further.
The rivers sampled are in highly industrial areas, and the Pearl River in particular was previously reported the most polluted with antibiotics, though the study did not attempt to determine the source of the pollution. What is clear is that once recombinant (GM) plasmids or plasmid sequences are discharged into the environment, the DNA can spread to wild bacteria through the process of horizontal gene transfer. Thus, researchers suggest that horizontal gene transfer of genetically engineered plasmids to microbes in the soil or from lactic acid bacteria to human and animal gut microbes is a likely consequence of such pollution, and may well underlie the rise in antibiotic resistance in animals as well as humans.
But there is another likely major source of GM antibiotic resistance, and that is from GM crops planted in the fields.
The majority of GM crops already released commercially or field trialled in the open environment carry antibiotic resistant genes derived from the synthetic plasmids that were used for genetic modification. China both grows and imports GM foods and trees, many of which harbour the blá gene including: Syngenta’s Bt11 Yieldgard Maize and Bt176 NaturGard Knockout Maize, Monsanto’s Mon21 Roundup Ready Maize and Bayer’s ZM003 Liberty Link Maize. China has also been developing many GM crops, including rice . Bt ‘Shanyou’63, was already the subject of controversy since 2005; the unapproved variety (both in China and other countries) illegally sold and planted in Hubei province, contaminated Chinese rice products exported to Europe and Japan, and has been detected in China and various countries since then. Bt63 was developed in Huazhong Agriculture University in Wuhan, Hubei Province. As recently as July 2009, the European Union called on China to tighten export controls on rice products because shipments might contain traces of the Bt 63 strain, which is not authorized in the European Union . Perhaps it is not a coincidence that the Yangtze River, one of those tested in the study, runs through the Hubei province.
This study is the first to address the potential pollution of our environment with antibiotic resistant genes from genetic engineering experiments. It provides the first comprehensive and direct evidence of horizontal gene transfer from genetic engineering and genetic modification. It can be predicted that similar findings will emerge elsewhere, if the appropriate molecular probes are used with the most sensitive PCR assays, which hitherto has not been done.
Article first published 13/02/13
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Robert Schneider Comment left 2nd March 2013 19:07:24
I'm sorry that I can't contribute to what I feel is a great and informative site. I do, however, make your presence known whenever I can, as I feel trusting and refreshed, like drinking good water, from the information you share. Thank you all.
Jake Winston Comment left 11th July 2014 22:10:10
Thank you so much for this eye-opening study. I wish it would get more exposure.