Evaluation of different methods for extracting extracellular DNA from the biofilm matrix

Abstract

The occurrence of high concentrations of extracellular DNA (eDNA) in the extracellular matrices of biofilms plays an important role in biofilm formation and development and possibly in horizontal gene transfer through natural transformation. Studies have been conducted to characterize the nature of eDNA and its potential function in biofilm development, but it is difficult to extract eDNA from the extracellular matrices of biofilms without any contamination from genomic DNA released by cell lysis during the extraction process. In this report, we compared several different extraction methods in order to obtain highly pure eDNA from different biofilm samples. After different extraction methods were explored, it was concluded that using chemical treatment or enzymatic treatment of biofilm samples may obtain larger amounts of eDNA than using the simple filtration method. There was no detectable cell lysis when the enzymatic treatment methods were used, but substantial cell lysis was observed when the chemical treatment methods were used. These data suggest that eDNA may bind to other extracellular polymers in the biofilm matrix and that enzymatic treatment methods are effective and favorable for extracting eDNA from biofilm samples. Moreover, randomly amplified polymorphic DNA analysis of eDNA in Acinetobacter sp. biofilms and Acinetobacter sp. genomic DNA and DNA sequencing analysis revealed that eDNA originated from genomic DNA but was not structurally identical to the genomic DNA.

FIG. 1.

Micrographs of biofilm samples treated with different extractants and stained with PI and SYTO-9. Biofilm samples without treatment (A) or treated with CER (B), EDTA (C), SDS (D), NaOH (E), N-glycanase (F), dispersin B (G), or proteinase K (H) are included. All biofilm samples were homogenized before microscopic observation. Bar, 20 μm.

FIG. 2.

Yields of eDNA isolated from 4-day biofilms after biofilm samples were treated with different enzymes and combinations. Biofilms formed by Acinetobacter sp. (A. baylyi) strain AC811, E. coli K-12, P. aeruginosa PAO1, and S. aureus ATCC 25923 were used for eDNA extraction. Biofilm samples were treated by filtration (ck, control); with N-glycanase, dispersin B, or proteinase K; or with various combinations (G + K, combination of N-glycanase and proteinase K; D + K, combination of dispersin B and proteinase K; G + D + K, combination of all three enzymes). Bars represent means and standard deviations from three replicates. Statistical analysis was done by using Student's t test. Different letters above the bars indicate significant differences (P < 0.05).

FIG. 3.

Comparative RAPD analysis, done using two sets of primers (A, primer F1; B, primer T7), of Acinetobacter sp. strain AC811 genomic DNA (lane 10) and eDNA obtained from biofilm samples by using the following different extractants: control (lane 1), CER (lane 2), EDTA (lane 3), SDS (lane 4), NaOH (lane 5), N-glycanase (lane 6), dispersin B (lane 7), proteinase K (lane 8), and a combination of dispersin B and proteinase K (lane 9). Unique bands (B1 to B4, marked with arrows) were excised and sequenced.