Evaluation of the impact of six different DNA extraction methods for the representation of the microbial community associated with human chronic wound infections using a gel-based DNA profiling method

Ethical statement

The study was granted ethical approval from Ethics Review Committee of University of Sri Jayewardenepura (Ref. No: 12/16) and Colombo South Teaching Hospital (Ref. No: 506) in Sri Lanka.

Specimen collection and preparation

Wound tissue debridement specimens from chronic foot wounds were collected from ten patients undergoing surgical debridement at a Tertiary Care Hospital in Sri Lanka. The specimens were collected by a well-trained and qualified medical officer/surgeon after obtaining the informed consent from the patients.

The tissue debridement specimens were collected into sterile eppendorf tubes and immediately transported in a portable cooler and stored at −20 °C for a few weeks or −80 °C up to several months. The samples from each patient were kept separate and were not pooled. Each tissue debridement specimen was weighed and cut into 12 small pieces (each weighing 25 mg) using a sterile scalpel or a sharp sterile needle.

Twenty-five milligrams of tissue debridement specimen was subjected to each DNA extraction method. Each extraction was done in duplicate. The methods H1 and H2 involved heat treatment of tissue specimens (Asadzaheh et al. 2010; Sampath et al. 2016; Silva et al. 2012), BP1 and BP2 were based on bead beater–phenol chloroform extraction (Sampath et al. 2016; Walter et al. 2001; Weerasekera et al. 2013); method S was based on DNA precipitation at high salt concentration (Asadzaheh et al. 2010) and method K used a commercial DNA extraction kit (Oates et al. 2012).

Heating in distilled water inside a boiling water bath (H1 method)

In the procedure of heat treatment in Distilled water (H1 method), the weighed tissue debridement sample was thoroughly minced and suspended in 100 µl of sterile distilled water. The specimen was immersed for 10 min in a 100 °C water bath. Tubes were centrifuged at (13,000 rpm) 15,493×g for 10 min and the supernatant was removed to a sterile tube and stored at −20 °C. This method was carried out as described by Silva et al. in 2012 with modifications (Silva et al. 2012).

Heating in NaOH inside a boiling water bath (H2 method)

DNA extraction using the heat treatment in NaOH (H2 method) was carried out as described by Asadzaheh et al. (2010) with the modifications (Asadzaheh et al. 2010). Tissue debridement specimen was minced, suspended in 100 µl of 50 mM NaOH and incubated in a 100 °C water bath for 20 min. Subsequently 20 µl Tri-HCl (pH = 7.5) was added. The tube was gently mixed by inverting several times and centrifuged at (13,000 rpm) 15,493×g for 10 min. The upper aqueous phase was transferred into a sterile clean tube and stored at −20 °C until used.

Bead beater–phenol chloroform extraction method using STES buffer (BP1 Method)

Bead beater–phenol chloroform extraction method using STES buffer (BP1 method) was carried out according to the procedure described by Sampath et al. in 2016 (Sampath et al. 2016). A tissue debridement specimen was suspended in 100 µl STES buffer [200 mM Tris HCl (pH 7.6), 100 mM EDTA, 0.1% SDS] and 40 µl of TE buffer [10 mM Tris HCl (pH 8), 1 mM EDTA]. Further, 120 µl Phenol: Chloroform mixture (1:1 V/V) and 0.3 g sterile zirconium beads (0.1 mm diameter; Bio Spec-Products) were added to each tube. Then the specimens were homogenized using a mini bead beater (model 3110BX; Bio Spec Products) at 480 rpm for 5 min. The upper aqueous phase (100 µl) was transferred to a sterile eppendorf tube. Ten microliter of 3 M sodium acetate was added and DNA was precipitated in the presence of 220 µl cold ethanol (100%) at −20 °C for 4 h. The solution was then subjected for centrifugation at (13,000 rpm) 15,493×g for 12 min. Air dried DNA pellet was dissolved in 30 µl TE buffer and stored at −20 °C until used.

Bead beater–phenol chloroform extraction method using TN150 buffer (BP2 method)

The process of bead beater–phenol chloroform extraction method using TN150 buffer (BP2 method) was carried out according to the procedure described by Walter et al. (2001) with modifications (Walter et al. 2001). The weighed tissue debridement specimen was suspended in 1 ml of sterile TN150 buffer and 0.3 g of sterile zirconium beads (diameter, 0.1 mm) were added. The tube was placed in a mini-bead beater (model 3110BX; Bio Spec Products), shaken at 480 rpm for 3 min, and stored on ice. It was subjected for centrifugation at (13,000 rpm) 15,493×g for 5 min. The upper phase (300 µl) was transferred into a new sterile eppendorf tube. Two hundred microliters of saturated phenol and 200 µl of chloroform-isomyl alcohol (24:1) were then added. The tube was inverted several times and centrifuged at (13,000 rpm) 15,493×g for 12 min. The supernatant was transferred into a sterile new micro-centrifuge tube. Fifty microliters of 3 M sodium acetate and 1 ml ice cold ethanol was added to the tube, mixed by inverting and stored at −20 °C for 4 h for precipitation of DNA. Following centrifugation at (13,000 rpm) 15,493×g for 12 min, supernatant was discarded and the DNA pellet was allowed to air dry. After all the traces of alcohol had evaporated, DNA was dissolved in 30 µl of sterile TE buffer [10 mM Tris HCl (pH 8), 1 mM EDTA] and stored at −20 °C until used.

Salting out method (S method)

Salting out method (S method) was performed according to the procedure described by Asadzaheh et al. (2010) with several modifications (Asadzaheh et al. 2010). The weighed tissue debridement sample was suspended in 600 µl of sterile TNES buffer [10 mM Tris–HCl pH = 7.5, 400 mM NaCl, 100 mM EDTA, 0.5% SDS] and 20 µl of proteinase K and mixed by inverting. The mixture was thoroughly mixed by vortex mixing and incubated at 50 °C inside a water bath until the tissue was completely lysed. After tissue debris was completely lysed, 200 µl 5 M NaCl was added and mixed vigorously for 20 s. The tube was centrifuged at (13,000 rpm) 15,493×g for 10 min and the supernatant was removed into a new sterile eppendorf tube. Equal volume of cold 100% ethanol was added, mixed, and kept overnight at −20 °C. The supernatant was discarded, after centrifugation at (13,000 rpm) 15,493×g for 12 min. The pellet was washed sequentially using 500 µl of 100% ethanol, 70% ethanol and allowed to air dry. The DNA was re-suspended with 30 µl TE buffer and stored at −20 °C until used.

DNeasy blood and tissue kit (K method)

The DNA extraction using the DNeasy blood and tissue kit [Qiagen Ltd., West Sussex, United Kingdom] (K method) was carried out following the manufactures’ instructions. The weighed tissue debridement sample was dissected into small pieces and placed in a 1.5 ml micro-centrifuge tube. The dissected pieces of tissues were suspended in 180 µl of buffer ALT and 20 µl proteinase K. The mixture was thoroughly mixed by vortex mixing and incubated at 56 °C until the tissue was completely lysed. Following vortex mixing for 5 min, buffer AL (200 µl) was added to the sample and mixed. Two hundred microliter of ethanol was added. Mixture was pipetted into the DNeasy Mini Spin column placed inside a 2 ml collection tube and centrifuged at (8000 rpm) 5875×g for 1 min. The DNeasy Mini Spin column was then placed in a new collection tube and 500 µl of buffer, AW1 was added and centrifuged. This step was repeated with the buffer AW2 and a high spin was given to dry the column membrane. The DNeasy Mini Spin column was subsequently placed in a clean micro-centrifuge tube and 100 µl of buffer AE was added directly onto the membrane. Following centrifugation, DNA was eluted and stored at −20 °C until used.

DNA quantification

DNA yield and DNA purity were determined using Nano drop 2000/200C spectrophotometer (Thermo Fisher Scientific, USA). The absorbance ratios; A260/280 nm and A260/230 nm were measured to assess DNA purity: A260/280 nm for protein contamination and A260/230 nm for salt and phenol contamination. DNA is known to absorb light at 260 nm and the A260/280 ratio; 1.8–2.0 and A260/230 ratio; >1.8 indicating that the sample was of good purity with little or no contamination (Vesty et al. 2017).

PCR amplification of bacterial DNA

The V2–V3 region of the bacterial 16S ribosomal DNA (rDNA) was PCR amplified using previously published universal eubacterium-specific primers HDA1 (with additional GC clamp) (5′CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG GAC TCC TAC GGG AGG CAG CAG T 3′) (Forward primer) and HDA2 (5′ GTA TTA CCG CGG CTG CTG GCA C 3′) (Reverse primer) (Anukam and Reid 2007; Oates et al. 2012; Walter et al. 2001). This primer amplifies a DNA fragment having the nucleotide position between 339 and 539 (E. coli 16S rRNA gene). The GC-clamp, which is a sequence that is rich in guanine and cytosine, is added to the 5′ end of the forward primer in order to prevent DNA from being completely denatured into single strands and to improve band resolution in denaturing gels.

Amplification reactions were performed in 200 µl thin wall PCR^® tubes (BIOLOGIX, USA). For direct PCR reactions using HDA 1 (GC clamped) and HDA 2 primers, 50.0 µl reaction mixture consisted of 5.0 µl of 10× PCR buffer containing 25 mmol/l MgCl₂ (Promega, USA); 1.0 µl of 10 mM dNTPs containing dATP, dGTP, dCTP and dTTP (Promega, USA); 1.0 µl of each primer [10 mM] (IDT, USA); 0.25 µl of Go Taq DNA polymerase (Promega, USA).

PCR amplification was done using GeneAmp PCR systems 9700 (Applied Bio systems). PCR reaction consisted of initial denaturation at 94 °C for 1 min, followed by 30 cycles consisting of 94 °C for 30 s for denaturation, 56 °C for 30 s for annealing and 72 °C for 30 s for extension, a final extension at 68 °C for 7 min with final hold at 4 °C. All PCR experiments included a negative (no template) control and a positive control. Resulting PCR products were separated by electrophoresis using 1× TAE [40 mM Tris HCl (pH 8), 20 mM acetic acid, 1 mM EDTA] on a 1.5% (w/v) agarose gel, stained with ethidium bromide and viewed by UV trans-illuminator [Vilber Lourmat, QUANTUM ST4].

Denaturant gradient gel electrophoresis

Denaturant gradient gel electrophoresis of amplified PCR products which were generated from DNA extracted from wound tissue debridement samples were performed on acrylamide gels in a DCode™ universal mutation detection system (Bio-Rad) according to the conditions described by Rasiah et al. (2005). The gels were prepared using 8% acrylamide (acrylamide to bis-acrylamide, 37.5:1) with a 30–55% gradient of urea and formamide. The gels were run using 1x TAE buffer [40 mM Tris HCl (pH 8), 20 mM acetic acid, 1 mM EDTA] at a constant voltage of 130 V at 60 °C for 4 h. Electrophoresis buffer (1× TAE) was maintained throughout at 60 °C. Gels were stained with ethidium bromide, visualized and photographed on a UV trans-illuminator (Vilber Lourmat, QUANTUM ST4).

Statistical analysis

All extractions were performed in duplicate to account for analytical variability. Means of DNA yield were analyzed using SPSS (version 20.0; Inc. Chicago) by one-way ANOVA with Welch correction. Data were expressed as mean ± SD. The extraction methods which have a significant difference between its mean values were grouped and multiple comparison was done using the Games–Howell. Differences were considered as significant when p value was <0.05.

Quality of DNA extracted each DNA extractions method

Evaluation of DNA integrity by agarose gel electrophoresis

The integrity of extracted DNA samples was analyzed by agarose gel electrophoresis. A high molecular weight band indicated the presence of genomic DNA (Fig. 1). All six DNA extraction methods showed low intense bands on agarose gel (Fig. 1). However variation in the yield and purity of DNA were observed among different methods.

When the bead beater–phenol chloroform extraction methods were occupied (BP1 and BP2 method) BP1 method yielded maximum DNA yield and high A260/280 nm ratios (1.8–2.0) in each specimen. Of the ten specimens, only four yielded A260/280 nm ratios >1.8. Comparatively to BP1 method, BP2 method yielded low DNA amount with a little contamination by the respective substances.

The two heating methods including H1 and H2 yielded considerable amount of DNA with more contamination of proteins and other impurities. DNA extraction using S method could yield maximum amount of DNA from most of the specimens but, their purity was very low. The reason for the lower DNA purity may be due to possible high salt contamination. DNA extraction using K method yielded considerably high DNA yield with a maximum A260/280 and A260/230 ratios indicating minimal DNA contaminants.

Following the Welch statistics, the extraction methods which have significant differences between the mean values of their DNA yield were grouped and multiple comparison was done using the Games–Howell. According to the multiple comparison, it was found that there was not a significant difference (at 95% confident level) between the means of DNA yield produced by each method. However, it was found that there was a significant difference (at 95% confident level) between the means of A260/280 ratio (p = 0.000): BP1 method and H1, H2 methods, BP2 and H1, H2 methods; H2 and K methods; S and H1, H2 methods. Further, a significant difference (at 95% confident level) was found between the means of A260/230 ratios (p = 0.000): BP1 method and H1, H2, S methods; BP2 method and H1, H2, S, K methods; K method and H1, H2, S methods.

PCR amplification of V2–V3 region of 16S rDNA

In order to evaluate the effect of DNA extraction method on the quality of DNA, the DNA extracted from all six methods were used for the determination of the microbial biodiversity in wound specimens. PCR amplification of the V2–V3 region of extracted bacterial 16S rRNA gene was carried out and the products were run on agarose gel (Fig. 2). The agarose gel electrophoresis of PCR product showed well separated and consistent bands with no impurity bands, with a product size of approximately 200 bp indicating the presence of well amplified V2–V3 region of 16S rRNA gene of bacterial species (Anukam and Reid 2007; Oates et al. 2012; Walter et al. 2001).

Comparative analysis of DGGE fingerprint profiles

Highly diverse DGGE banding patterns were observed in each specimen on 30–55% denaturing gradient. Further intra- and inter-subject variations were readily observed in the specimens following different extraction methods (Figs. 3, 4). The differences in DGGE profiles were reflected by the number and intensity of the DNA bands in comparisons of bacterial profiles of the same sample using different DNA extraction methods (Figs. 3, 4). Each DGGE band was assumed to represent a single species (Muyzer et al. 1999). Therefore, the number of bands from each lane was counted and average number of bands per specimen was calculated (Table 3).

DNA extraction method	Average number of bands in each specimen
	S/no: 1	2	3	4	5	6	7	8	9	10
STES buffer and phenol chloroform extraction method (BP1)	14	10	06	08	16	11	06	04	04	08
TN150 buffer and phenol chloroform extraction method (BP2)	14	12	07	09	13	12	07	05	08	09
DNA extraction tissue kit (K)	15	06	07	10	11	13	07	04	05	06
Boiling in distilled water (H1)	13	09	05	08	06	08	05	05	08	05
Boiling in aqueous NaOH (H2)	14	11	06	11	10	12	07	06	08	07
Salting out method (S)	14	09	05	07	10	12	05	05	08	06

S/No specimen number

The observed multiple bands in the DGGE gel are suggestive of a polymicrobial nature of the chronic wound infection. Theoretically, all profiles should have been identical since DNA was extracted from the tissues obtained from same chronic wound specimen. However, there was a variation in intensity of bands and number of the bands in the same specimens which were subjected for different DNA extraction methods. This observation suggests that the different DNA extraction methods may have had varying sensitivity for the different bacteria.

Excising of selected DGGE bands, PCR amplification, and sequencing

Selected predominant DGGE bands from the DGGE gel were excised, PCR amplified, purified and sequenced using Sanger method. According to the sequencing results, Enterococcus feacalis, Group A Streptococcus spp., Providencia spp., E.coli and Vellionella spp. were found in the sample no. 1 following six extraction methods (Fig. 1). However the resulting intensity of these DGGE bands differed in different extraction methods. Pseudomonas aeruginosa was found from six extraction methods done for specimen No. 2 (Fig. 2). When considering the specimen No. 2, Providencia spp. could only be identified from the BP1 method while other methods did not yield the specific DNA band (Fig. 2).