Selection of Participants
All study protocols were reviewed and approved by the University of California Davis (UCD) Institutional Review Board (IRB) and carried out in accordance with their regulations. Burn subjects were male or female adults (> 18 years old) enrolled on admission to the UC Davis Health (UCDH) burn intensive care unit (BICU) for treatment of thermal injury affecting ≥ 10% of the total body surface area (TBSA). Inclusion criteria required that the subject have burn and spared cutaneous sites bilaterally symmetrical to each other, with a spared site > 10 cm from affected tissue. Informed consent was obtained from the subject or a legally authorized representative in accordance with the UCD IRB policies. Subjects were excluded if injury occurred > 48 hours prior to admission or if wound debridement occurred prior to initial sample collection. Subjects remained on the study for the first 28 days of admission to the BICU or until discharge from the BICU, if occurring prior to 28 days. The course of clinical treatment was not altered for any subject on the study.
Healthy Volunteers: A cohort of 18 healthy adult (> 18 years old) volunteers from UCDH were consented, in accordance with UCD IRB policies, for a single timepoint skin swab collection on the right anterior forearm. This cohort served as a method validation for sequencing from premoistened swabs on healthy skin, and as a comparison to the burned cohort. The health status of volunteers was determined by the following criteria: no hospitalization in the last 30 days or antibiotic use in the last 14 days.
A cohort of five BICU nurses who actively participated in routine patient care were consented and enrolled for a single timepoint skin swab collection on the right anterior forearm. Volunteers were included if they were an active BICU nurse with no recent (< 14 days) history of antibiotic use.
Sample and Data Collection
Microbial Sampling: Bacterial populations were sampled using a sterile rayon swab (BD BBL Culture Swab, Media-free, BD, Franklin Lanes, NJ, USA) premoistened in sterile saline (Research Products International Corp, Mount Prospect, IL, USA). Burn subjects were sampled at the following four locations: spared skin, burn wound, perianal, rectal. Skin sampling was performed by spinning the swab for 30 seconds across a 4 cm2 area while applying moderate pressure11. Following collections, the swab tip was placed in a sterile 1.5ml microcentrifuge tube (Eppendorf™ Snap-Cap Microcentrifuge Biopur™ Safe-Lock™, Eppendorf, Hamburg, Germany) and stored at -70°C. Working surfaces were sterilized with 70% ethanol between each sample. Burn wound samples were collected during wound care following removal of dressings and topical treatments but prior to disinfection with dilute chlorohexidine gluconate (4.0% w/v). Perianal and rectal samples were collected by nurses according to UCDH protocol. The “burn wound” and “spared skin” sites were determined for each subject based on the following criteria (Figure S1):
Partial or full thickness burn present at the burn site
Healthy and burned skin sites were bilaterally symmetrical from each other across the sagittal plane of the body
Healthy skin was a minimum of 10cm from burned tissue
Neither site was expected to be obstructed by line placement or clinical equipment
The healthy site was not expected to be utilized as a donor site for autologous skin grafting
One swab from each of the four sites was collected on approximately day 0, 3, 7, 14, 21, and 28 post admission of the subject’s stay in the UCDH BICU (Fig. 1). Swabs were collected until day 28 or up to discharge, if prior to 28 day, with 48-hour variance from the target timepoint to accommodate clinical workflow.
Patient Demographics: The following metadata patient demographics were recorded for each subject throughout BICU admission: age, gender, percent TBSA, burn depth, mechanism of injury, time of injury, intubation status, topical and systemic antibiotic dosing, dressings applied during wound care, microbiology cultures, infection, sepsis, nutrition, graft adhesion or failure, wound care frequency and type, and mortality.
Library generation and Sequencing
DNA was isolated from swabs using the DNeasy PowerSoil Kit (Qiagen, Valencia, CA, USA) with modifications. Samples were pre-processed by adding solution C1 to the PowerBead tubes and vortexing. Three hundred microliters of this solution were pipetted into the sample collection tube containing the swab tip and incubated at 37°C for one hour. The collection tube was then centrifuged with a spin basket (Spin-X centrifuge tube filters, Corning Costar Corporation, Cambridge, MA, USA), and the flow through pipetted back into the PowerBead tube. Further steps followed the manufacturer’s protocol with the exception of adjusting the final elution volume to 50 microliters. All modifications were performed to maximize DNA yield as cutaneous sampling yield low microbial biomass.12 Extracted DNA was stored at -20°C.
One control mock specimen, designated “reagent control”, was processed in parallel with each set of subject’s samples. Reagent controls were prepared at the laboratory immediately prior to DNA isolation by aseptically clipping a sterile swab into a collection tube and proceeding with extraction. Additionally, three “air control” samples were obtained during the wound care of burn subjects on day 3 prior to study sample collection. Air control swabs were spun in the air for 30 seconds to sample potentially aerosolized bacteria post dressing removal.
16S Amplicon Library Generation: 16S metagenomic sequencing libraries were prepared at the Genomics Shared Resource (Sacramento, CA, USA) according to Illumina’s (Illumina, San Diego, CA, USA) 16S Metagenomic Sequencing Library Preparation protocol (Part# 15044223 Rev. B). A universal primer set was used to amplify the V3-V4 hypervariable region13, generating a 550 base-pair amplicon : Forward Primer = 5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG and Reverse Primer = 5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC11. Primer pairs obtained from the Access Array Barcode Library for Illumina Sequencers were hybridized to the amplicons by a second round of PCR. Nucleic acid yields were measured by fluorometric quantification using the Qubit 2.0 with the Qubit™ dsDNA HS Assay Kit (Life Technologies, Carlsbad, CA, USA).
Libraries were pooled to a concentration of 118 ng/uL and volume of 40 uL for sequencing on the Illumina MiSeq platform (Illumina, San Diego, CA, USA). These libraries were sequenced using MiSeq v3 chemistry with 300-bp paired end reads at the UC Davis DNA Technologies and Expression Analysis Core (Davis, CA, USA).
Bacterial 16S rRNA gene sequence data were analyzed with the open source software Quantitative Insights Into Microbial Ecology 2 (QIIME 2) version 2020.2.14 Raw sequence data were demultiplexed (via q2-demux) and paired end reads were quality filtered, trimmed to a minimum median quality score of 20, and chimeric sequences removed using the Divisive Amplicon Denoising Algorithm 2 (DADA2).15 Sequence alignment was performed using Multiple Sequence Alignment Software Version 7 (MAFFT)16 (via q2-alignment) and a rooted phylogeny generated with FastTree 217 (via q2‐phylogeny).
Diversity analysis was performed using q2-diversity following rarefication by subsampling without replacement to 1156 sequences per sample. Alpha and beta diversity metrics were calculated by Shannon Diversity and Bray‐Curtis dissimilarity, respectively.18,19 Taxonomy was assigned using the q2‐feature‐classifier20 classify‐sklearn Naïve Bayes taxonomy classifier against the Greengenes 13_8 99% Operational Taxonomic Units full length reference sequences.21 This classifier was trained to the hypervariable V3-V4 region using the primers previously described. Differential abundance was determined by Analysis of Composition of Microbiomes (ANCOM) and q2-ALDEx2.22,23 Two methods for determining enriched or depleted taxa are reported due to the current lack of consensus on which multivariate analytical practice is most appropriate for microbial data. The QIIME2 Longitudinal (q2-longtitudinal) plugin compared site-specific subject sampling over time for metrics of alpha diversity, beta diversity, and taxonomy.24
Non-parametric Kruskal-Wallis test performed in QIIME 2 determined alpha diversity significance (via q2-diversity) between subject groups, sampling location, and metadata parameters.25 Alpha diversity visualizations were generated in QIIME 2 and QIIME 2R.26 Principal coordinate analysis (PCoA) plots were generated as visualizers for beta diversity measures and statistical analysis performed with Permutational Multivariate Analysis of Variance (PERMANOVA)27 test for between metric significance. Differential taxonomic abundance determined by ALDEx2 was considered significant with a false discovery rate adjusted p-value of < 0.05.
Contaminant Identification and Removal
Contaminants can arise during collection, nucleic acid isolation, PCR amplification, and during sequencing by lane cross contamination.28,29 We employed the recommendations established by Salter et. al to reduce and identify contaminants. Any sequence meeting these guidelines was filtered out from analysis (via q2-demux).14,30