Lack of detection of ampicillin resistance gene transfer from Bt176 transgenic corn to culturable bacteria under field conditions.

Population levels of total and ampicillin-resistant culturable bacteria and the putative horizontal bla gene acquirement from Bt-corn were studied in commercial fields of transgenic corn in Spain during the years 2000-2003. Commercial fields consisting of conventional corn (Dracma) and Bt176 transgenic corn (Compa CB) were located in three climatic regions. The effect of corn type, plant material, field location, stage of sampling and year of study were studied on total and ampicillin resistant bacterial population levels, on median effective dose and on the slope of the dose-response curve to ampicillin. None of the parameters measured were significantly different (P<0.01) between transgenic and non-transgenic cornfields under the diverse conditions studied. However, in population levels of ampicillin resistant bacteria, the minimum difference between sample means to be significant with a likelihood of 80% was 8.9%. Specific detection of putative bacteria harbouring bla TEM-1 ampicillin resistance genes acquired from Bt176 corn was performed with a method based on the extraction of DNA from the culturable bacterial fraction and with PCR. Primers for PCR were targeted to the bla gene and the corresponding flanking regions present in the pUC18 cloning vector or the Bt176 construct. The culturable bacterial fraction of 144 field samples (up to 864 analysis, including ampicillin enrichments) was analysed by PCR. The estimated total number of bacteria analysed was 10(8). The level of detection of a transfer event according to the sensitivity of the methods used was 10(-6). Four samples of transgenic and five of non-transgenic corn gave positive signals. However, the amplification products did not correspond to the ones expected from Bt176 or pUC18. The limitations of the sampling design and of the methods used are discussed.