Biofilms from a total of 30 chronic wound patients were included in this survey study. A total of 30 chronic wounds were sampled and grouped into one of three categories of wounds: venous leg ulcers (V), diabetic foot ulcers (D), or pressure ulcers (P). To identify the microbial populations that occur in these types of wounds and to gain a preliminary understanding of their relative importance, we took advantage of three powerful molecular approaches that included PRADS [108
], FRACS [109
], and PRADS [110
]. These techniques allowed the bacterial diversity that occurs within these CWPB types to be evaluated.
Protocol for subject enrollment, DNA extraction, and Sample Preparation
Under the guidance of IBR protocols, chronic wounds of 30 subjects treated at the Southwest Regional Wound Care Center (Lubbock, Texas) were debrided as per standard of care; the debridement samples were collected with sterile tools into sterile collection tubes and frozen until processing for DNA extraction. Samples from 10 subjects with venous leg ulcers, 10 subjects with diabetic foot ulcers, and 10 subjects with pressure (decubitus) ulcers were included in this study. Debridement samples (300 mg ± 150 mg) were collected into Lysing Matrix E tubes from the FastDNA® SPIN for Soil Kit from MP Biomedicals LLC (Solon, OH). The tubes were frozen at -70°C until DNA extraction could be performed. When DNA extraction was performed, the samples were removed from the freezer and allowed to thaw at room temperature. Subsequently, the DNA extraction protocol for the kit was followed with the exception that human debridement samples were used in place of soil samples. The extracted sample DNA was stored at -70°C. After measuring the relative concentration of bacterial DNA present based upon 16s quantitative PCR using 16S Universal Eubacterial primers 530F (5'-GTG CCA GCM GCN GCG G) and 1100R (5'-GGG TTN CGN TCG TTG), the 10 samples from each of the three wound groups were pooled at equal bacterial DNA ratios to create three pools of DNA, each representing a major category of chronic wound.
Partial ribosomal amplification and pyrosequencing (PRAPS)
The modified 16S Eubacterial primers 530F and 1100R were used for amplifying the 600 bp region of 16S rRNA genes. The primer pair used for 454 Amplicon Sequencing was designed with special Fusion Primers at the 5' end of each primer as follows: 530F-A (5'-GCC TCC CTC GCG CCA TCA GGT GCC AGC MGC NGC GG) and 1100R-B (5'-GCC TTG CCA GCC CGC TCA GGG GTT NCG NTC GTT G). All wound DNA samples were diluted to 100 ng/μl. A 100 ng aliquot of sample DNA was used for a 50 μl PCR reaction. HotStarTaq Plus Master Mix Kit (QIAGEN, CA, USA) was used for PCR under the following conditions: 94°C for 3 minutes followed by 32 cycles of 94°C for 30 seconds; 60°C for 40 seconds and 72°C for 1 minute; and a final elongation step at 72°C for 5 minutes. PCR products were purified using the PSI Ψ Clone PCR 96 Kit (Princeton Separations Inc, Freehold, NJ). Following PCR amplification, samples were normalized in concentration and sent on dry ice to the Medical Biofilm Research Institute [111
] for pyrosequencing using genome sequencer FLX system's standard amplicon sequencing protocols (F. Hoffmann-La Roche Ltd, Basel, Switzerland).
Full ribosomal amplification, cloning and Sanger sequencing (FRACS)
The Eubacterial 16S primers 27F (5'-AGA GTT TGA TCM TGG CTC AG) and 1525R (5'-AAG GAG GTG WTC CAR CC) were synthesized by Integrated DNA Technologies (Coralville, IA). These primers amplify roughly 1500 bp spanning almost the entire 16S gene. A total of 100 ng of sample DNA was used for each 50 μl PCR reaction. HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, CA) was used for PCR using the following conditions: 94°C for 3 minutes followed by 35 cycles of 94°C for 30 seconds, 52°C for 40 seconds, and 72°C for 2 minutes. A final elongation step at 72°C for 10 minutes was also included. The amplified fragments were subcloned into the pGEM-T Easy Vector (Promega, Madison, WI) and transformed into a competent E. coli K12 strain. Following blue/white screening using standard methods, 200 clones from each library were isolated and subcultured. The plasmid DNA was extracted by using a QIAprep Spin Miniprep Kit (Qiagen, Valencia, CA). Bidirectional sequencing using T7 and SP6 primers was performed by Agencourt Technologies (Beverly, MA).
Partial Ribosomal Amplification, DGGE, and Sanger sequencing (PRADS)
The 16S Eubacterial primers 1070F (5'-ATG GCT GTC GTC AGC T) and 1492R+GC (5'-GCC GCC TGC AGC CCG CGC CCC CCG TGC CCC CGC CCC GCC GCC GGC CCG GGC GCC TTA CCC TTG TTA CGA CTT) were synthesized by Integrated DNA Technologies (Coralville, IA). These primers produced an approximately 450 bp 16S ribosomal DNA (rDNA) amplicon with a GC clamp to be analyzed by denaturing gradient gel electrophoresis (DGGE). PCR reactions (50 μl of sample DNA) were performed using 2× PCR Master Mix (Promega, Madison, WI). Each reaction mixture consisted of 1.5 mM MgCl2, 200 μM of each dNTP, 0.5 μM of both the 1070F and 1492R+GC primers, 0.025 U/μl Taq DNA polymerase, and 100 ng template DNA.
Denaturing gradient gel electrophoresis (DGGE) was performed on the 16S amplicons described above using the DCode™ DGGE system (Bio-rad). A 40%–70% denaturing gradient was optimal for separation of the approximately 450 bp 16S amplicons, where 7 M urea and 40% formamide is defined as 100%. Gels also contained an 8%–12% acrylamide gradient with a 12% native stacking gel. Different volumes of each sample were loaded for optimal visualization of bands with varying intensities. The gel was run at 60 V for 20 hours and was then stained with SYBR Gold® (Molecular Probes, Invitrogen, Carlsbad, CA) and visualized with a FluorChem™ 8800 fluorescence imager (Alpha Innotech Inc. San Leandro, CA). Nineteen predominant bands that could be visualized by eye were excised using a sterile, disposable scalpel and placed in 20 μl TE buffer (Figure ). This included 4 bands from the venous leg ulcer group (group V), 8 from the diabetic foot ulcer group (group D), and 6 from the pressure ulcer group (group P).
The TOPO TA Cloning® kit (Invitrogen Inc. Carlsbad, CA) was used to clone the DNA from the excised DGGE bands (pCR® 2.1-TOPO® vector and One Shot Chemically Competent E. coli cells). The maximum amount of DNA (4 μl diffused DNA in TE buffer) was used in each of the cloning reactions following the manufacture's instructions. Twelve clones per band were selected and grown overnight in 250 μl LB broth containing 50 μg/ml kanamycin in 96 well plates. The same 50 μl PCR reaction was prepared as described above using the M13F and M13R primer set instead of 1070F and 1492R+GC and 5 μl of the overnight culture was added to each 50 μl PCR reaction as the template DNA. The initial 96°C denaturing step was sufficient to rupture the E. coli cells, releasing its DNA as the starting template. These PCR products were then sequenced by the University of Washington's High-Throughput Genomics Unit (Seattle, WA).
Samples were not collected in parallel with samples collected for the PRAPS, FRACS, or PRADS; but culture results were instead collected retrospectively from the subjects' medical records. All samples were collected under IBR protocols. This information is provided as a contrast to the type of data normally collected from wounds. Results were examined from each subject's medical record. The culture results were collected usually within a week of the samples that was collected for the PRAPS, FRACS, and PRADS analyses. Sharp debridement from the subject's wound was placed in thioglycolate broth with indicator (Hardy Diagnostics, Santa Maria, CA) and incubated at 35°C for up to 24 hours with the lid tightly closed before being transferred to a CLIA certified microbiology laboratory for thorough aerobic and anaerobic culture analyses. Bacteria were identified using Gram stains, non-selective and selective/differential media, and biochemical tests.
Assembly, including vector scanning, quality analysis, and consensus calling, of sequencing data was performed using Seq-Man Pro assembler (DNAstar Madison, WI). Sequences were assembled using SeqMan Pro Assembler at 96% similarity, match size of 25, match spacing of 50, minimum sequence length of 100, 0.0 and 0.7 gap and gap extension penalties, and a minimum mismatch of 8 at end bases. Multiple alignments were performed with MegAlign (DNAstar Madison, WI). BLAST analyses were performed using WND.BLAST [112
] and a custom 16S ribosomal database derived from RDPII version 9 [113
]. The database was first parsed with a custom script to provide genus and species names as hit definitions following BLAST analyses. For FRACS E-values < 10E-100 were considered acceptable for determining genus, while hits of E = 0.0 and associated full alignments > 1400 bp were considered acceptable for determining genus and species. For PRADS and PRAPS all species determinations are considered putative, E-values of 10–100 are considered acceptable for determining genus, while E-values better than 10–140, along with full input sequence alignments, and genus/species agreement of all similarly scoring top hits were required to list a putative species.