Isolation of deoxynivalenol-transforming bacteria from the chicken intestines using the approach of PCR-DGGE guided microbial selection

Abstract

Background: Contamination of grains with trichothecene mycotoxins, especially deoxynivalenol (DON), has been an ongoing problem for Canada and many other countries. Mycotoxin contamination creates food safety risks, reduces grain market values, threatens livestock industries, and limits agricultural produce exports. DON is a secondary metabolite produced by some Fusarium species of fungi. To date, there is a lack of effective and economical methods to significantly reduce the levels of trichothecene mycotoxins in food and feed, including the efforts to breed Fusarium pathogen-resistant crops and chemical/physical treatments to remove the mycotoxins. Biological approaches, such as the use of microorganisms to convert the toxins to non- or less toxic compounds, have become a preferred choice recently due to their high specificity, efficacy, and environmental soundness. However, such approaches are often limited by the availability of microbial agents with the ability to detoxify the mycotoxins. In the present study, an approach with PCR-DGGE guided microbial selection was developed and used to isolate DON -transforming bacteria from chicken intestines, which resulted in the successful isolation of several bacterial isolates that demonstrated the function to transform DON to its de-epoxy form, deepoxy-4-deoxynivalenol (DOM-1), a product much less toxic than DON.

Results: The use of conventional microbiological selection strategies guided by PCR-DGGE (denaturing gradient gel electrophoresis) bacterial profiles for isolating DON-transforming bacteria has significantly increased the efficiency of the bacterial selection. Ten isolates were identified and isolated from chicken intestines. They were all able to transform DON to DOM-1. Most isolates were potent in transforming DON and the activity was stable during subculturing. Sequence data of partial 16S rRNA genes indicate that the ten isolates belong to four different bacterial groups, Clostridiales, Anaerofilum, Collinsella, and Bacillus.

Conclusions: The approach with PCR-DGGE guided microbial selection was effective in isolating DON-transforming bacteria and the obtained bacterial isolates were able to transform DON.

Figure 1

Transformation of DON to DOM-1 by the subcultures of digesta samples . The digesta samples were from the large intestine of chickens fed clean or DON-contaminated wheat (10 μg g^-1 DON) during the in vivo enrichment experiment. The subcultures were grown in L10 broth containing 100 μg ml^-1 DON. Each subculture was incubated for 72 hours. n = 6.

Figure 2

Flow chart showing the process of selection for chicken intestinal bacteria with the ability to transform DON . *Selection criteria used in each step of the selection. Numbers in the parentheses indicate particular steps in the selection.

Figure 3

LC-MS chromatograms showing the biotransformation of DON to DOM-1 . A) DON (100 μg ml^-1) in L10 broth without any bacterial inoculum after 72 hr incubation. Selected ion monitoring at m/z 231, 249, 267, 279, and 297. B) Transformation of DON (100 μg ml^-1) to DOM-1 in L10 broth inoculated with isolate LS100 after 72 hr incubation. Selected ion monitoring at m/z 215, 233, 245, 251, 263, and 281.

Figure 4

PCR-DGGE bacterial profiles showing the richness of bacterial populations . A) Bacterial profiles before and after antibiotic treatments. Lane 1: large intestinal digesta sample (LIC); Lane 2: start culture that was the first subculture from the digesta (LIC) before lincomycin treatment; Lanes 3 and 4: same start culture after the treatment with lincomycin at 60 and 30 μg ml^-1, respectively; Lanes 5 and 6: same start culture after the treatment with tylosin at 80 and 40 μg ml^-1, respectively. B) Changes of PCR-DGGE bacterial profiles through the selection by antibiotics and AIM+CecExt medium. Lane 1: start culture (1^st subculture from the digesta) before antibiotic and AIM+CecExt treatments; Lane 2: the same culture (in Lane 1) after antibiotic and AIM+CecExt treatments; Lane 3: a pure culture of a single colony isolate with DON-transforming activity (Isolate LS-61). Note: Lane 1, lanes 2 - 4, and lanes 5 - 6 of Panel A were from three separate DGGE gels. The migration of their DNA bands was not identical among the different gels.