Longitudinal Profiling Of The Burn Patient Cutaneous And Gastrointestinal Microbiota

Longitudinal Profiling Of The Burn Patient Cutaneous And Gastrointestinal Microbiota | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Longitudinal Profiling Of The Burn Patient Cutaneous And Gastrointestinal Microbiota Kelly Lima, Ryan Davis, Stephenie Liu, David Greenhalgh, Nam Tran This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-240723/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Sepsis is a leading cause of morbidity and mortality in patients that have sustained a severe burn injury. Early detection and treatment of infections improves outcomes and understanding changes in the host microbiome following injury and during treatment may aid in burn care. The loss of functional barriers, systemic inflammation, and commensal community dysbiosis all contribute to a burn patient’s increased risk of infection. We sampled 10 burn patients to evaluate cutaneous microbial populations on the burn wound and corresponding spared skin on days 0, 3, 7, 14, 21, and 28 post-intensive care unit admission. In addition, skin samples were paired with perianal and rectal locations to evaluate changes in the burn patient gut microbiome following injury and treatment. We found significant ( P = 0.011 ) reduction in alpha diversity on the burn wound compared to spared skin throughout the sampling period as well as reduction in common skin commensal bacteria such as Propionibacterium acnes and Staphylococcus epidermitis . Compared to healthy volunteers ( n = 18 ), the burn patient spared skin also exhibited a significant reduction in diversity ( P = 0.001 ). Treatments such as systemic or topical antibiotic administration, skin grafting, and nutritional formulations also impact diversity and community composition at the sampling locations. When evaluating each subject individually, an increase in relative abundance of a clinically isolated bacteria could be seen in 5/9 infections detected among the burn patient cohort. Pathology Laboratory Diagnostics Surgery Microbiome wound 16S rRNA gene sequencing bacteria burn injury skin graft and infection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Thermal injuries are prevalent worldwide, affecting over 8 million people globally in 2017. 1 Notwithstanding the prevalence, survival from thermal injury has been improving with advances in care. Data from the Global Burden of Disease 2017 study show a 46.6% (-49.7 to -38.8%) decrease in age-standardized mortality from 1990 to 2017. 1 Despite improved outcomes, significant predictors of mortality include burn size, age, sex, inhalation injury, and infection. 2 Burn patients are at increased risk of developing sepsis from infection, which is the most common cause of morbidity in this population. 3 The infection risk is due to many factors including: hypermetabolic response to injury, systemic inflammation, immune dysfunction, epidermal barrier disruption, and loss of commensal bacterial protection. 4,5 Early detection and treatment of sepsis reduces morbidity and mortality, subsequently, burn patients are closely monitored for signs of infection using clinical guidelines. 3,6 Due to the unique variability in types and severity of burn injury, algorithms are in development to monitor predictors of sepsis. 7 Although early sepsis detection is crucial for appropriate treatment, clinical indicators only present once infection has led to a systemic response in the patient. Prevention of pathogenic microbial colonization is the primary tool employed in burn care. Topical and systemic antimicrobials are an integral part of sepsis prevention; however, commensal and pathogenic bacteria persist on the human skin and gut (two potential sites of bacterial seeding) despite use of antimicrobials. The mechanisms by which these populations experience dysbiosis and interact with the host during disease is continually being discovered. Few studies have been conducted evaluating microbial dysbiosis during treatment and recovery following severe thermal injury. 8 Immediate perturbations of cutaneous and gut populations after injury have not been explored. Currently, culture-independent methodologies by sequencing of the 16S ribosomal RNA gene or whole genome shotgun metagenomics are standard for microbiome assessment. 9,10 However, culture-independent microbial research of the microbiome is limited in the burn population. A knowledge gap exists between recognizing infection, judicial antimicrobial use, and understanding complex host-commensal microbial interactions following thermal injury. To this end, understanding microbial population dynamics over the course of treatment and early detection of infection is imperative for sepsis management in this at-risk population. There are three objectives for this study: 1) to provide preliminary data on longitudinal sampling of skin and gut microbiome in severely burned patients, 2) to understand changes in bacterial population dynamics over time and 3) to determine if culture-independent monitoring of these populations can predict bacterial overgrowth leading to infection or sepsis. Methods Selection of Participants Burn Patients 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. Burn Nurses 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 cm 2 area while applying moderate pressure 11 . 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 Longitudinal Design 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 Isolation 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. Control Samples 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 region 13 , generating a 550 base-pair amplicon : Forward Primer = 5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG and Reverse Primer = 5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC 11 . 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). Sequencing 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). Data Analysis Bioinformatics 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 2 17 (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‐classifier 20 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 Statistical Analysis 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 Results Subject Demographics The study dataset comprised of 10 male burn patients admitted to the UCDH BICU, 18 healthy volunteers working at UCDH but not in the BICU, and 5 female BICU nurses. Burn patients were aged 31 to 61 years (median 38 years) and suffered a median TBSA burn of 26.5% (range 10–76%). Of the 10 burn patients, 50% ( n = 5 ) required grafting of the wound sampling location, 50% ( n = 5 ) experienced bacterial and/or fungal wound infections, 20% ( n = 2 ) were diagnosed with culture positive bacterial pneumonia, and 20% ( n = 2 ) with blood culture positive septicemia. There were no instances of diagnosed inhalation injury (Table 1 ). Median time from BICU admission to the first sample collection was 5 hours (range 1–14 hours) and the median number of days on study was 21 (range 7–28 days). Table 1 Burn Patient Demographics. Abbreviations: B, Blood; R, Respiratory; W, Wound Subject ID Age (years) Total TBSA Burn (%) Race or Ethnicity Mechanism of Injury Time on Study (Days) Burn Depth at Collection Location Burn Site Grafted (y/n) Culture Positive (y/n) Day(s) of Culture(s) Organism(s) Cultured (Location) S1 37 36 Hispanic flame 21 partial-thickness n n NA NA S2 34 16 White flame 7 full-thickness y n NA NA S3 34 22 White flame 14 partial-thickness n y 3 MRSA (B/W) S4 61 18.5 Native American flame 21 partial-thickness n y 9 Klebsiella sp. (R) S5 39 20 White flame 14 full-thickness y y 8 MRSA (W) S6 31 40 Other flame 28 partial-thickness n y 3 12 14 Enterobacter cloacae complex (W) Lactobacillus sp. (W) Streptococcus pneumonia , Stenotrophomonas maltophilia (R) S7 36 76 Hispanic scald 28 partial-thickness n y 6 9 Lactobacillus sp.; yeast (W) Candida albicans (B) S8 43 31 Hispanic flame 28 full-thickness y n NA NA S9 58 40.5 White flame 14 full-thickness y n NA NA S10 43 10 White flame 28 full-thickness y y 7 Staphylococcus aureus (W) Sampling and Bioinformatic Filtering Of the total collected samples (n = 228), 210 were included in the dataset. Twelve samples were excluded pre-sequencing due to undetectable nucleic acid concentrations following extraction and six samples were excluded bioinformatically with < 20% of sequences passing quality filters and/or achieving a sampling depth of < 1156 (Table 2 ). Following recommendations from Salter et al., one potential contaminant amplicon sequence variant (ASV), a unique DNA variant in the dataset, mapping to Ralstonia species (sp.) was identified. 31 This genus has been reported as a reagent contaminant and appeared in all low abundance samples following implementation of a new extraction kit ( Figure S3 ). The ASV was removed from the dataset by filtering with q2-demux – all results reported are in exclusion of this taxon. Table 2 Total Samples Included in Analysis Collection Group Samples Collected Samples Post Extraction Total Included %Excluded Burn 48 46 44 8.3 Spared 49 44 44 10.2 Perianal 47 46 45 4.4 Rectal 48 48 46 4.2 Reagent Control 10 6 5 50 Air Control 3 3 3 0 Nurse 5 5 5 0 Healthy Volunteer 18 18 18 0 Total 228 216 210 7.9 Sample Group Diversity and Taxonomy Taxonomic Relative Abundance Sample group taxonomy was collapsed by collection location across all burn subjects and sampling timepoints with phylum level classification (Fig. 2 ). The four most prevalent genera sampled from the burn patient wound ( Staphylococcus , Burkholderia , Stenotrophomonas , and Propionibacterium ) were also predominant on burn patient spared skin in varying proportions ( Table 3 ). Analysis of differential feature abundance of features (q2-ALDEx2) found four ASVs with an effect size > 1 between burn wound and spared skin ( Figure S4 ). Of the four features identified by ALDEx2, three mapped by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) with 100% identity to strains of Propionibacterium acnes and one to Staphylococcus epidermitis , both are reduced on the burn wound. Perianal and rectal sampling locations did not exhibit significantly different taxa when compared by ANCOM and ALDEx2. Summary of taxonomic relative abundance consisting of the top 10 phyla sequenced from samples collected at four collection locations (burn wound, spared skin, perianal, and rectal) on the burn patient cohort. Phyla are listed descending from least to most abundant. Summary of the relative abundance of the top 10 phyla and genera level taxa on the healthy volunteer and BICU nurse cohorts. Phyla and genera are listed descending from least to most abundant. Table 3. Predominant Taxa by Sample Group In descending order, the top four most predominant phyla and genera as determined by percent relative abundance summed by sample group among burn patients, healthy volunteers, and BICU nurses. Similarly, sample group taxonomy was collapsed by healthy volunteer and BICU nurse cohorts by phylum and genus level classification (Fig. 3 ). Eleven ASVs exhibit differential relative abundances between BICU nurses and healthy volunteers. These ASVs mapped by NCBI BLAST to the following taxa by \(\ge\) 99.78% identity: Stenotrophomonas maltophilia , Escherichia coli , Stenotrophomonas sp. , Variovax paradoxus , Sphingomonas sp. , Staphylococcus epidermidis , Pseudomonas sp. , and Microbacterium sp. With the exception of Staphyloccus epidermidis , all are enriched on BICU nurse samples. Between-sample diversity, as measured by Shannon Diversity, is significantly different between all group comparisons ( p < 0.05 ) except between air controls/burn wound ( P = 0.487 ), air controls/nurses ( P = 0.052 ), and burn wound/nurses ( P = 0.325 ) (Fig. 4 a). Healthy volunteers have significantly higher mean Shannon diversity compared to cutaneous burn samples and nurses ( p < 0.01 ). Shannon diversity is not significantly different between the days of collection for any burn patient sampling site; however, bacterial populations from the burn wound exhibit sustained reduced alpha diversity compared to spared skin for days 0–21 (Fig. 4 b). Bray Curtis dissimilarity distance of microbial abundance differed significantly between collection groups ( P = 0.001 PERMANOVA) (Fig. 4 c). Effects of Burn Treatments on Diversity Antimicrobials Systemic antimicrobials were administered to 100% of the burn study population at one or more timepoints. Samples collected following systemic antibiotic administration were clustered by collection location and compared to samples collected prior to any systemic antibiotic administration. The burn wound exhibits significantly reduced alpha diversity ( P = 0.023 ) following systemic antibiotic administration (Fig. 5 a). Analysis with ANCOM and ALDEx2 did not reveal any significantly different taxa on the burn wound samples following systemic antibiotic administration. However, the relative abundance of the phylum Firmicutes decreased (57.9–20.3%) following antibiotic administration while Proteobacteria increased (24.6–51.8%) (Fig. 5 b). Application of different topical antimicrobials during the wound care prior to sample collection did not differentially impact alpha diversity of the burn wound populations (Fig. 5 c) and were not significantly different from samples collected on admission, prior to the first wound care. Grafting Autologous skin grafting occurred at the sampling site in 50% of the burn study population ( n = 5 ) affecting 33.3% of all burn wound samples ( n = 15 ). Samples obtained from grafted skin exhibited reduced alpha diversity compared to non-grafted samples ( P = 0.036 ) while grafting had no significant effect on the alpha diversity at other collection locations (Fig. 6 a). When visualized longitudinally, alpha diversity trends towards recovery by day 28 (Fig. 6 b). Analysis by ALDEx2 did not classify any ASVs as significantly different between grafted and non-grafted wound samples. Analysis by ANCOM revealed the phylum Fusobacteria to be significantly reduced among the grafted wound samples (W = 14). The relative abundance of Proteobacteria increased from 25.1–66.2% following grafting while Firmicutes decreased from 45.3–25.4% (Fig. 6 c). Diet The effects of diet type (enteral, parenteral, or regular) were minimal on alpha diversity between collection locations (Fig. 7 a). Longitudinal analysis of Bray Curtis dissimilarity between diet types indicate that gut samples derived from patients receiving enteral nutrition become more compositionally dissimilar to those collected from patients consuming a regular diet over time (Fig. 7 b). Analysis of differential relative abundance by ANCOM found the genera Proteus , Enterococcus , and Methanobrevibacter to be significantly enriched in patients receiving enteral nutrition (W = 180, W = 174, W = 174, respectively) compared to a regular diet (Fig. 7 c and Figure S4) . ALDEx2 did not identify significantly different ASVs between the groups. ( 7a ) Mean Shannon Diversity between diet type that each patient received during the collection timepoint for each collection location (burn wound, spared skin, perianal, and rectal) sampled. ( 7b ) Bray Curtis dissimilarly by axis 2 of the PCOA plot graphed on a volatility plot to visualize the impact of time on compositional diversity. Axis 2 explained 6.605% of variation between groups. ( 7c ) Taxonomic summary of the top 10 phyla sequenced from the sum of perianal and rectal samples separated by diet type at the time of collection. The designation “NA” represents unclassified phyla. Detection of Clinically Identified Infections Six of 10 burn patients experienced culture positive infection at one or more timepoints during the study. Nine instances of bacterial infection were detected in wound, blood, and respiratory cultures occurring across these six patients. Burn wound samples were evaluated for the family or genus of the infecting organism and plotted by relative abundance to detect changes over time. Irrespective of culture location, 55.6% (5/9) of infections were detectable by an increase in relative abundance on the burn wound from the single sample site collected (Fig. 8 ). Notably, for Subject 6 (Fig. 8 d), Stenotrophomonas maltophilia was cultured from a respiratory sample on day 14 and clinically treated for pneumonia. On this subject’s wound, the genera Stenotrophomonas was undetectable on admission, however on day 14 consisted 15.1% of ASVs sequenced. Stenotrophomonas remained a dominant taxon on the wound comprising 60.6% and 72.3% of ASVs on days 21 and 28, respectively, despite systemic antibiotic administration for pneumonia on day 14. Discussion Previous studies have profiled the human skin microbiome reporting differential populations and diversity found on the human skin; however the majority still support one of the sentinel molecular 16S sequencing studies that found that the human forearm consists predominantly (94.6% of clones) of the phyla Actinobacteria , Firmicutes , and Proteobacteria . 32 These data are supported by our findings for the control populations of healthy volunteers and BICU nurses sampled on the right forearm as well as the burn patient wound and spared skin samples ( Table 3 ). Despite partial and full-thickness tissue destruction, the burn wound samples still have predominant phyla reflective of an uninjured skin microbiome. We found that burn wound microbial populations were significantly less diverse compared to spared skin, healthy volunteers, and BICU nurses (Fig. 4 a). Reduction in microbial population alpha diversity has been reported as a characteristic of non-healing wounds and could have implications for healing rates in the burn population. 33 We expected to see an initial reduction and subsequent recovery of alpha diversity from the wound sites, however this was not supported (Fig. 4 b). The sustained reduction in alpha diversity could be due to frequent routine application of topical antimicrobials or differential healing in our population, among other factors not controlled for in this study. Propionibacterium acnes and Staphylococcus epidermitis are two skin commensal organisms that were significantly reduced on the wound site, supporting that burn wounds experience perturbance of normal commensal bacteria. Interestingly, the diversity and composition of the BICU nurse microbiome was significantly different compared to healthy volunteers not working in the BICU. Relative enrichment of Stenotrophomonas sp. and reduction in the normal commensal Staphylococcus epidermitis suggest that nurses experience microbial dysbiosis of their cutaneous microbiota. Isolation protocols such as frequent sanitization of the hands and forearms could impact the bacterial populations. In addition, the patient population nurses interact with could influence the cutaneous microbiota, leading to disparities between nurses working in different units. Further research is warranted in this area as our study has a small sample size of five nurses. Our findings suggest that systemic antimicrobial administration significantly reduces microbial diversity at the burn wound but does not impact other microbial populations evaluated in the study. The relative decrease in Firmicutes and increase in Proteobacteria following administration could be a result of broad-spectrum coverage against Gram-positive organisms allowing for Gram-negative enrichment. Interestingly, the type of topical antimicrobial applied on the burn wound did not significantly impact alpha diversity or taxonomic composition at the wound. Grafting of a burn wound and the clinical process associated with the procedure, such as excision, anesthesia, and prophylactic antibiotics are all proposed to cause dysbiosis to microbial populations. We found that diversity is reduced at the wound site following grafting but may trend towards recovery by day 28 (Fig. 6 b) as the graft takes and the cutaneous microenvironment becomes more reflective of normal skin. A non-significant decrease in bacterial diversity seen at the spared skin following grafting could be due to the surgery and prophylactic antibiotics associated with the procedure (Fig. 6 a). Adequate nutrition is an essential component of burn care and patients often receive a variety of nutritional sources to meet high caloric needs. We investigated whether the type of nutrition a patient is receiving impacts gut microbial populations and found that enteral nutrition trends towards decreased gut diversity and increased compositional difference compared to samples obtained from patients receiving a regular diet (Fig. 7 ). These data are not significant and should be further investigated in both burn and non-burn ICU patients. The burn population is one that could greatly benefit from early detection of infection with the potential to reduce the incidence of sepsis and subsequent mortality. The ability to monitor common sites of infection, such as the burn wound, for colonization of pathogenic bacteria that could result in local infection or systemic spread would improve targeted antimicrobial therapies and patient outcome. It has been shown that machine learning can be used to predict disease states such as inflammatory bowel disease, type 2 diabetes, and liver cirrhosis from 16S microbial sequencing datasets. 34,35 Additionally, susceptibility to bloodstream infection following chemotherapy has been predicted by the pre-treatment gut microbiome. 36 To investigate this potential use in the burn population we retrospectively tracked changes in relative abundance of organisms over time that were cultured as part of the subjects’ clinical care. We found that local and systemic infections could be seen by the increased abundance of the inciting bacteria at the burn wound (Fig. 8 ). This patient population should be included in studies evaluating the clinical application of sequencing methodologies and machine learning to detect infection. Comprehensive sampling of multiple sites throughout a patient’s hospitalization could also help indicate the source of infection, which is often unknown in this population. Studies utilizing 16S microbial community analysis inherently contain limitations. Sample preparation variables such as collection and storage methodology have been shown to influence results. 37 Extraction and amplification of a single section of bacterial DNA can contribute bias to the dataset, as can the sequencing platform used and the bioinformatic algorithms performed during downstream analysis. 38 We aim to acknowledge and address these limitations through the transparency of our methodology and public sharing of the raw sequence data. An additional limitation specific to this study is the burn patient enrollment gender bias towards male participants. Gender was not selected for during enrollment and males are admitted to the ICU for burn injury more frequently than females. In 2006, a ten year review of United States hospital burn admissions found males to be overrepresented, accounting for 67.9%. 39 However, further research should aim for an even gender distribution, as females are at increased risk of death following burn injury and microbial populations have been shown to differ by gender. 39,40 Conclusion This study profiled the microbial populations found on the burn wound, spared skin, perianal, and rectal locations for the first 28 days of 10 burn patients admitted to the UCDH BICU. Our data support the idea that microbial dysbiosis occurs at the burn wound and spared skin following injury compared to healthy volunteers, and that BICU nurses may also experience community dysbiosis from cutaneous populations sampled from the forearm. Treatments such as antibiotics, grafting, and nutritional formulations can impact the diversity and community compositions sampled during the patient’s ICU stay. Surprisingly, the topical antimicrobial applied at the wound did not result in a significant difference of relative abundance in any AVSs. In addition, it is possible to detect changes in relative abundance of clinically identified infectious bacteria at the burn wound, even if it is not the site of the culture location. Due to the impact that sepsis can have on patient outcome, further research in establishing burn wound bacterial profiles can aid in the development of tools to detect pathogenic changes. Declarations ACKNOWLEDGEMENTS We kindly acknowledge the UCDH BICU physicians Dr. Tina Palmieri, Dr. Kathleen Romanowski, and Dr. Soman Sen as well as the entire BICU nursing staff for their participation in subject identification and sample collection. Thank you to the ARCS Foundation for funding this project. The Genomics Shared Resource is supported by the UC Davis Comprehensive Cancer Center Support Grant (CCSG) awarded by the National Cancer Institute (NCI P30CA093373). The DNA Technologies and Expression Analysis Core at the UC Davis Genome Center is supported by NIH Shared Instrumentation Grant 1S10OD010786-01. AUTHOR CONTRIBUTIONS Kelly Lima (K.L): K.L performed subject consent, sample collection, wrote the manuscript in its entirety, performed bioinformatic and statistical analysis of the study data, and generated Figures and Tables for the manuscript. Ryan Davis (R.D.): R.D. provided technical support and expertise during DNA extraction and completed 16S library generation in its entirety. R.D. also provided technical support during bioinformatic analysis of sequence data and reviewed and edited the paper. Stephenie Liu (S.L.): S.L. provided technical support and expertise during DNA extraction and sample submission for sequencing. S.L. also significantly contributed to the review and editing of the manuscript. David Greenhalgh (D.G.): D.G is co-investigator for the study who provided significant input on the study design and significantly contributed to the review and editing of the manuscript. Nam Tran (N.T.): N.T. serves as study PI for the study. Significantly contributed to study design and significantly contributed to the review and editing of the manuscript with co-authors. ADDITIONAL INFORMATION Data Availability The datasets generated during and/or analyzed during the current study are available in the NCBI Sequence Read Archive (SRA) repository, [PRJNA701230], and upon request. Competing Interests There is NO competing interest. Declarations James, S. L. et al. Epidemiology of injuries from fire, heat and hot substances: global, regional and national morbidity and mortality estimates from the Global Burden of Disease 2017 study. Inj. Prev. 26 , i36–i45 (2020). Capek, K. D. et al. Contemporary Burn Survival. J. Am. Coll. Surg. 226 , 453–463 (2018). Greenhalgh, D. G. et al. American Burn Association consensus conference to define sepsis and infection in burns. J. Burn Care Res. Off. Publ. Am. Burn Assoc. 28 , 776–790 (2007). Deitch, E. A. & Berg, R. Bacterial Translocation from the GutA Mechanism of Infection. J. Burn Care Rehabil. 8 , 475–482 (1987). Beckmann, N., Pugh, A. M. & Caldwell, C. C. Burn injury alters the intestinal microbiome’s taxonomic composition and functional gene expression. PLOS ONE 13 , e0205307 (2018). The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) | Critical Care Medicine | JAMA | JAMA Network. https://jamanetwork.com/journals/jama/fullarticle/2492881. Tran, N. K. et al. Novel application of an automated-machine learning development tool for predicting burn sepsis: proof of concept. Sci. Rep. 10 , 12354 (2020). Plichta, J. K. et al. Cutaneous Burn Injury Promotes Shifts in the Bacterial Microbiome in Autologous Donor Skin: Implications for Skin Grafting Outcomes. Shock Augusta Ga 48 , 441–448 (2017). Turnbaugh, P. J. et al. The Human Microbiome Project. Nature 449 , 804–810 (2007). Jovel, J. et al. Characterization of the Gut Microbiome Using 16S or Shotgun Metagenomics. Front. Microbiol. 7 , (2016). MCutting. Manual of Procedures for Human Microbiome Project Core Microbiome Sampling Protocol A HMP Protocol # 07-001. (2010). Jo, J.-H., Kennedy, E. A. & Kong, H. H. Research Techniques Made Simple: Bacterial 16S Ribosomal RNA Gene Sequencing in Cutaneous Research. J. Invest. Dermatol. 136 , e23-7 (2016). Klindworth, A. et al. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res. 41 , e1–e1 (2013). Bolyen, E. et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat. Biotechnol. 37 , 852–857 (2019). Callahan, B. J. et al. DADA2: High resolution sample inference from Illumina amplicon data. Nat. Methods 13 , 581–583 (2016). Katoh, K. & Standley, D. M. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Mol. Biol. Evol. 30 , 772–780 (2013). Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree 2--approximately maximum-likelihood trees for large alignments. PloS One 5 , e9490 (2010). Shannon, C. E. A Mathematical Theory of Communication. Bell Syst. Tech. J. 27 , 379–423 (1948). Bray, J. R. & Curtis, J. T. An Ordination of the Upland Forest Communities of Southern Wisconsin. Ecol. Monogr. 27 , 326–349 (1957). Bokulich, N. A. et al. Optimizing taxonomic classification of marker-gene amplicon sequences with QIIME 2’s q2-feature-classifier plugin. Microbiome 6 , 90 (2018). McDonald, D. et al. An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea. ISME J. 6 , 610–618 (2012). Mandal, S. et al. Analysis of composition of microbiomes: a novel method for studying microbial composition. Microb. Ecol. Health Dis. 26 , 27663 (2015). Fernandes, A. D., Macklaim, J. M., Linn, T. G., Reid, G. & Gloor, G. B. ANOVA-Like Differential Expression (ALDEx) Analysis for Mixed Population RNA-Seq. PLOS ONE 8 , e67019 (2013). Bokulich, N. A. et al. q2-longitudinal: Longitudinal and Paired-Sample Analyses of Microbiome Data. mSystems 3 , e00219-18 (2018). Kruskal, W. H. & Wallis, W. A. Use of Ranks in One-Criterion Variance Analysis. J. Am. Stat. Assoc. 47 , 583 (1952). Bisanz, Jordan E. qiime2R: Importing QIIME2 artifacts and associated data into R sessions . (2018). Anderson, M. J. Permutational Multivariate Analysis of Variance (PERMANOVA). in Wiley StatsRef: Statistics Reference Online 1–15 (American Cancer Society, 2017). doi:10.1002/9781118445112.stat07841. Mohammadi, T., Reesink, H. W., Vandenbroucke-Grauls, C. M. J. E. & Savelkoul, P. H. M. Removal of contaminating DNA from commercial nucleic acid extraction kit reagents. J. Microbiol. Methods 61 , 285–288 (2005). Barton, H. A., Taylor, N. M., Lubbers, B. R. & Pemberton, A. C. DNA extraction from low-biomass carbonate rock: An improved method with reduced contamination and the low-biomass contaminant database. J. Microbiol. Methods 66 , 21–31 (2006). McDonald, D. et al. The Biological Observation Matrix (BIOM) format or: how I learned to stop worrying and love the ome-ome. GigaScience 1 , 7 (2012). Salter, S. J. et al. Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol. 12 , 87 (2014). Gao, Z., Tseng, C., Pei, Z. & Blaser, M. J. Molecular analysis of human forearm superficial skin bacterial biota. Proc. Natl. Acad. Sci. 104 , 2927–2932 (2007). Gardiner, M. et al. A longitudinal study of the diabetic skin and wound microbiome. PeerJ 5 , e3543 (2017). Oh, M. & Zhang, L. DeepMicro: deep representation learning for disease prediction based on microbiome data. Sci. Rep. 10 , 6026 (2020). Pasolli, E., Truong, D. T., Malik, F., Waldron, L. & Segata, N. Machine Learning Meta-analysis of Large Metagenomic Datasets: Tools and Biological Insights. PLOS Comput. Biol. 12 , e1004977 (2016). Montassier, E. et al. Pretreatment gut microbiome predicts chemotherapy-related bloodstream infection. Genome Med. 8 , 49 (2016). Choo, J. M., Leong, L. E. & Rogers, G. B. Sample storage conditions significantly influence faecal microbiome profiles. Sci. Rep. 5 , 16350 (2015). Poretsky, R., Rodriguez-R, L. M., Luo, C., Tsementzi, D. & Konstantinidis, K. T. Strengths and Limitations of 16S rRNA Gene Amplicon Sequencing in Revealing Temporal Microbial Community Dynamics. PLOS ONE 9 , e93827 (2014). Latenser, B. A. et al. National Burn Repository 2006: A Ten-Year Review. J. Burn Care Res. 28 , 635–658 (2007). Fierer, N., Hamady, M., Lauber, C. L. & Knight, R. The influence of sex, handedness, and washing on the diversity of hand surface bacteria. Proc. Natl. Acad. Sci. 105 , 17994–17999 (2008). Additional Declarations No competing interests reported. Supplementary Files SupplementalMaterial.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 08 Mar, 2021 Reviews received at journal 22 Feb, 2021 Reviewers agreed at journal 22 Feb, 2021 Reviewers invited by journal 22 Feb, 2021 Editor assigned by journal 22 Feb, 2021 Editor invited by journal 18 Feb, 2021 Submission checks completed at journal 17 Feb, 2021 First submitted to journal 13 Feb, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-240723","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":12447165,"identity":"74441235-43d5-4896-9214-2fe6d5ad45b7","order_by":0,"name":"Kelly Lima","email":"","orcid":"","institution":"University of California, Davis","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kelly","middleName":"","lastName":"Lima","suffix":""},{"id":12447166,"identity":"9a259995-0aff-4365-9aea-0c55dee2d848","order_by":1,"name":"Ryan Davis","email":"","orcid":"","institution":"University of California, Davis","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ryan","middleName":"","lastName":"Davis","suffix":""},{"id":12447167,"identity":"0e4d4a8c-4177-4f67-927f-8d015b5e2a0f","order_by":2,"name":"Stephenie Liu","email":"","orcid":"","institution":"University of California, Davis","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Stephenie","middleName":"","lastName":"Liu","suffix":""},{"id":12447168,"identity":"f7b926f8-e7e5-4d2a-a1e0-80c363fe30b5","order_by":3,"name":"David Greenhalgh","email":"","orcid":"","institution":"University of California, Davis","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Greenhalgh","suffix":""},{"id":12447169,"identity":"34e3f843-8b86-42fd-ad52-c0c293ea4bd1","order_by":4,"name":"Nam Tran","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYDACHjBpA8QJYMTAcICgFmYQmUa6lsMMUPVEaDHnOX/sw88d5xP725OfSTz4wyDHdyMBvxbL3mbmmb1nbifOOPPMTCKxjcFYkpAWg/PMzAy8bbcTN0gkGBskNjAkbiBGC+PftnNALemfDRL+MNQT1nK2mZmZt+0AUEuO4YMENoYEA4Jazhw2ZpZtSzaeceZN4YPENgnDmWceENKS+JjxbZudbH97+oaDP/7YyPMdJ2ALOpAgTfkoGAWjYBSMAuwAAJwDSG0oKj5iAAAAAElFTkSuQmCC","orcid":"","institution":"University of California, Davis","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Nam","middleName":"","lastName":"Tran","suffix":""}],"badges":[],"createdAt":"2021-02-14 03:14:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-240723/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-240723/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":6260478,"identity":"5b517c7f-a196-4864-a6e7-8170fbf6ff1d","added_by":"auto","created_at":"2021-02-23 15:37:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6061,"visible":true,"origin":"","legend":"Longitudinal Sampling Schedule","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-240723/v1/9a127ace1540abb7f670dfd0.png"},{"id":6259969,"identity":"0c15478d-f4c6-414d-9abd-108ef76098a3","added_by":"auto","created_at":"2021-02-23 15:31:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":83727,"visible":true,"origin":"","legend":"Burn Patient Taxonomic Summary ","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-240723/v1/58c4b77f721b6f9039ae3a0c.png"},{"id":6260223,"identity":"ef188157-db8a-4d4e-bcf1-148f482de572","added_by":"auto","created_at":"2021-02-23 15:34:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":57736,"visible":true,"origin":"","legend":"Healthy Volunteer and BICU Nurse Taxonomic Summary","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-240723/v1/2bd30f3534a50b5c6745f8a9.png"},{"id":6259973,"identity":"f091df68-ebf9-465f-b527-a00857fc51e2","added_by":"auto","created_at":"2021-02-23 15:31:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":41496,"visible":true,"origin":"","legend":"Diversity and Taxonomy of Skin Microbiota.\n(4a) Mean Shannon Diversity Index from cutaneous sampling locations and control swabs. (4b) Volatility plot displaying Shannon Diversity over sampling days 0, 3, 7, 14, 21, and 28 for samples collected at the burn wound and spared skin of the burn patient cohort. (4c) Compositional community analysis by Bray Curtis dissimilarity demonstrate clustering by sample type (P=0.001) between skin and control samples. Each color-coded point on the graph correlated with a subject body site or control type. Abbreviations: RC, Reagent Control.\n","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-240723/v1/29ef14f184aa8c339dee5cd7.png"},{"id":6259977,"identity":"ded1fe8f-b6c1-4c90-ba0c-5d0507c85db4","added_by":"auto","created_at":"2021-02-23 15:31:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":55985,"visible":true,"origin":"","legend":"Impact of Systemic and Topical Antibiotics on Microbial Diversity and Taxonomy \n(5a) Mean Shannon Diversity of samples collected prior to systemic antibiotic administration and post for each collection location across the burn patient cohort. Microbial diversity at the burn wound is significantly reduced (p\u003c0.05) following systemic antibiotic administration. The spared skin, rectal, and perianal collection locations were not significantly altered. (5b) Taxonomic summary of the top 10 phyla sequenced from burn wound samples collected pre- and post-systemic antibiotic administration. SR1 and TM7 indicate candidate phyla that have been identified by sequencing but have no cultivated examples. (5c) Mean Shannon Diversity of burn wound samples collected after clinical application of differential topical antimicrobials.\n","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-240723/v1/bb313a4eda90b92c39669191.png"},{"id":6260479,"identity":"253d295e-fbd3-43bc-b497-76312cea5d43","added_by":"auto","created_at":"2021-02-23 15:37:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":69288,"visible":true,"origin":"","legend":"Effects of Grafting on Burn Wound Alpha Diversity and Taxonomy\n(6a) Mean Shannon Diversity from each sampling location (burn wound, spared skin, perianal, and rectal) clustered by presence or absence of grafting at the wound sampling location. Diversity at the burn wound is significantly reduced when grafted. (6b) Volatility plot of mean Shannon Diversity from the burn wound samples over time clustered by grafting status. Longitudinal display of the data indicates potential recovery of diversity by day 28 (n=4). (6c) Taxonomic summary of the top 10 phyla sequenced at the burn wound and clustered by wound grafting status. Abbreviations: NS, not significant.\n","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-240723/v1/a97730b109968e7a501cfdb2.png"},{"id":6260237,"identity":"71a83c7c-65f5-4139-a0b5-6acf4e12d71a","added_by":"auto","created_at":"2021-02-23 15:34:03","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":74971,"visible":true,"origin":"","legend":"Nutritional Impacts on Burn Patient Microbial Diversity and Taxonomy. \n(7a) Mean Shannon Diversity between diet type that each patient received during the collection timepoint for each collection location (burn wound, spared skin, perianal, and rectal) sampled. (7b) Bray Curtis dissimilarly by axis 2 of the PCOA plot graphed on a volatility plot to visualize the impact of time on compositional diversity. Axis 2 explained 6.605% of variation between groups. (7c) Taxonomic summary of the top 10 phyla sequenced from the sum of perianal and rectal samples separated by diet type at the time of collection. The designation “NA” represents unclassified phyla.\n","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-240723/v1/6aab38b77427ef1834fef08b.png"},{"id":6260236,"identity":"e5d5145a-6d00-4961-b0b0-885bc5ab5958","added_by":"auto","created_at":"2021-02-23 15:34:03","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":69769,"visible":true,"origin":"","legend":"Longitudinal Visualization of Culture Isolated Bacteria from the Burn Wound\n(8a-g) Volatility plots display in orange the longitudinal change relative abundance of a bacterial ASV from the order or genus that was identified by culture for individual burn subjects. The thick blue line depicts the average of the relative abundance of the same ASV from the remaining subjects. Thin blue lines are representative of individual relative abundances of the ASV from each of the remaining subjects. A black triangle indicates the day a culture was collected for a clinically suspected infection for each subject while on the study. Subjects 3, 4, 5, 7, and 10 had one culture and Subject 6 had two. Abbreviations: MRSA, Methicillin Resistant Staphylococcus aureus; RS, remaining subjects.\n","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-240723/v1/a8d0f047509b7293f2c3c001.png"},{"id":13669431,"identity":"3cded8eb-da05-4b8b-89f0-477949f1e725","added_by":"auto","created_at":"2021-09-17 11:00:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2202967,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-240723/v1/7561fd9d-063b-4c46-a447-defc612bf996.pdf"},{"id":6260231,"identity":"d9ac15ff-e311-4dad-8d6e-4442575f7d6e","added_by":"auto","created_at":"2021-02-23 15:34:03","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":504802,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-240723/v1/e974d08a41666149613ceb28.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eLongitudinal Profiling Of The Burn Patient Cutaneous And Gastrointestinal Microbiota\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eThermal injuries are prevalent worldwide, affecting over 8\u0026nbsp;million people globally in 2017.\u003csup\u003e1\u003c/sup\u003e Notwithstanding the prevalence, survival from thermal injury has been improving with advances in care. Data from the Global Burden of Disease 2017 study show a 46.6% (-49.7 to -38.8%) decrease in age-standardized mortality from 1990 to 2017.\u003csup\u003e1\u003c/sup\u003e Despite improved outcomes, significant predictors of mortality include burn size, age, sex, inhalation injury, and infection.\u003csup\u003e2\u003c/sup\u003e Burn patients are at increased risk of developing sepsis from infection, which is the most common cause of morbidity in this population.\u003csup\u003e3\u003c/sup\u003e The infection risk is due to many factors including: hypermetabolic response to injury, systemic inflammation, immune dysfunction, epidermal barrier disruption, and loss of commensal bacterial protection.\u003csup\u003e4,5\u003c/sup\u003e Early detection and treatment of sepsis reduces morbidity and mortality, subsequently, burn patients are closely monitored for signs of infection using clinical guidelines.\u003csup\u003e3,6\u003c/sup\u003e Due to the unique variability in types and severity of burn injury, algorithms are in development to monitor predictors of sepsis.\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eAlthough early sepsis detection is crucial for appropriate treatment, clinical indicators only present once infection has led to a systemic response in the patient. Prevention of pathogenic microbial colonization is the primary tool employed in burn care. Topical and systemic antimicrobials are an integral part of sepsis prevention; however, commensal and pathogenic bacteria persist on the human skin and gut (two potential sites of bacterial seeding) despite use of antimicrobials. The mechanisms by which these populations experience dysbiosis and interact with the host during disease is continually being discovered. Few studies have been conducted evaluating microbial dysbiosis during treatment and recovery following severe thermal injury.\u003csup\u003e8\u003c/sup\u003e Immediate perturbations of cutaneous and gut populations after injury have not been explored. Currently, culture-independent methodologies by sequencing of the 16S ribosomal RNA gene or whole genome shotgun metagenomics are standard for microbiome assessment.\u003csup\u003e9,10\u003c/sup\u003e However, culture-independent microbial research of the microbiome is limited in the burn population.\u003c/p\u003e\u003cp\u003eA knowledge gap exists between recognizing infection, judicial antimicrobial use, and understanding complex host-commensal microbial interactions following thermal injury. To this end, understanding microbial population dynamics over the course of treatment and early detection of infection is imperative for sepsis management in this at-risk population. \u003cem\u003eThere are three objectives for this study: 1) to provide preliminary data on longitudinal sampling of skin and gut microbiome in severely burned patients, 2) to understand changes in bacterial population dynamics over time and 3) to determine if culture-independent monitoring of these populations can predict bacterial overgrowth leading to infection or sepsis.\u003c/em\u003e\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSelection of Participants\u003c/h2\u003e\u003cp\u003e\u003cstrong\u003eBurn Patients\u003c/strong\u003e\u003cp\u003eAll 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 (\u0026gt;\u0026thinsp;18 years old) enrolled on admission to the UC Davis Health (UCDH) burn intensive care unit (BICU) for treatment of thermal injury affecting\u0026thinsp;\u0026ge;\u0026thinsp;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\u0026thinsp;\u0026gt;\u0026thinsp;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\u0026thinsp;\u0026gt;\u0026thinsp;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.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eHealthy Volunteers\u003c/b\u003e: A cohort of 18 healthy adult (\u0026gt;\u0026thinsp;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.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eBurn Nurses\u003c/strong\u003e\u003cp\u003e 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 (\u0026lt;\u0026thinsp;14 days) history of antibiotic use.\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003eSample and Data Collection\u003c/h2\u003e\u003cp\u003e\u003cb\u003eMicrobial Sampling\u003c/b\u003e: 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 cm\u003csup\u003e2\u003c/sup\u003e area while applying moderate pressure\u003csup\u003e11\u003c/sup\u003e. Following collections, the swab tip was placed in a sterile 1.5ml microcentrifuge tube (Eppendorf\u0026trade; Snap-Cap Microcentrifuge Biopur\u0026trade; Safe-Lock\u0026trade;, Eppendorf, Hamburg, Germany) and stored at -70\u0026deg;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 \u0026ldquo;burn wound\u0026rdquo; and \u0026ldquo;spared skin\u0026rdquo; sites were determined for each subject based on the following criteria (\u003cb\u003eFigure S1\u003c/b\u003e):\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003ePartial or full thickness burn present at the burn site\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eHealthy and burned skin sites were bilaterally symmetrical from each other across the sagittal plane of the body\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eHealthy skin was a minimum of 10cm from burned tissue\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eNeither site was expected to be obstructed by line placement or clinical equipment\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eThe healthy site was not expected to be utilized as a donor site for autologous skin grafting\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eLongitudinal Design\u003c/strong\u003e\u003cp\u003eOne swab from each of the four sites was collected on approximately day 0, 3, 7, 14, 21, and 28 post admission of the subject\u0026rsquo;s stay in the UCDH BICU (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). 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.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePatient Demographics\u003c/b\u003e: 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.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eLibrary generation and Sequencing\u003c/h2\u003e\u003cp\u003e\u003cstrong\u003eDNA Isolation\u003c/strong\u003e\u003cp\u003eDNA 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\u0026deg;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\u0026rsquo;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.\u003csup\u003e12\u003c/sup\u003e Extracted DNA was stored at -20\u0026deg;C.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eControl Samples\u003c/strong\u003e\u003cp\u003eOne control mock specimen, designated \u0026ldquo;reagent control\u0026rdquo;, was processed in parallel with each set of subject\u0026rsquo;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 \u0026ldquo;air control\u0026rdquo; 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.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e16S Amplicon Library Generation\u003c/b\u003e: 16S metagenomic sequencing libraries were prepared at the Genomics Shared Resource (Sacramento, CA, USA) according to Illumina\u0026rsquo;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 region\u003csup\u003e13\u003c/sup\u003e, generating a 550 base-pair amplicon : Forward Primer\u0026thinsp;=\u0026thinsp;5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG and Reverse Primer\u0026thinsp;=\u0026thinsp;5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC\u003csup\u003e11\u003c/sup\u003e. 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\u0026trade; dsDNA HS Assay Kit (Life Technologies, Carlsbad, CA, USA).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSequencing\u003c/strong\u003e\u003cp\u003eLibraries 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).\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eData Analysis\u003c/h2\u003e\u003cp\u003e\u003cstrong\u003eBioinformatics\u003c/strong\u003e\u003cp\u003eBacterial 16S rRNA gene sequence data were analyzed with the open source software Quantitative Insights Into Microbial Ecology 2 (QIIME 2) version 2020.2.\u003csup\u003e14\u003c/sup\u003e 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).\u003csup\u003e15\u003c/sup\u003e Sequence alignment was performed using Multiple Sequence Alignment Software Version 7 (MAFFT)\u003csup\u003e16\u003c/sup\u003e (via q2-alignment) and a rooted phylogeny generated with FastTree 2\u003csup\u003e17\u003c/sup\u003e (via q2‐phylogeny).\u003c/p\u003e\u003c/p\u003e\u003cp\u003eDiversity 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.\u003csup\u003e18,19\u003c/sup\u003e Taxonomy was assigned using the q2‐feature‐classifier\u003csup\u003e20\u003c/sup\u003e classify‐sklearn Na\u0026iuml;ve Bayes taxonomy classifier against the Greengenes 13_8 99% Operational Taxonomic Units full length reference sequences.\u003csup\u003e21\u003c/sup\u003e 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.\u003csup\u003e22,23\u003c/sup\u003e 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.\u003csup\u003e24\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003cp\u003eNon-parametric Kruskal-Wallis test performed in QIIME 2 determined alpha diversity significance (via q2-diversity) between subject groups, sampling location, and metadata parameters.\u003csup\u003e25\u003c/sup\u003e Alpha diversity visualizations were generated in QIIME 2 and QIIME 2R.\u003csup\u003e26\u003c/sup\u003e Principal coordinate analysis (PCoA) plots were generated as visualizers for beta diversity measures and statistical analysis performed with Permutational Multivariate Analysis of Variance (PERMANOVA)\u003csup\u003e27\u003c/sup\u003e test for between metric significance. Differential taxonomic abundance determined by ALDEx2 was considered significant with a false discovery rate adjusted p-value of \u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eContaminant Identification and Removal\u003c/strong\u003e\u003cp\u003eContaminants can arise during collection, nucleic acid isolation, PCR amplification, and during sequencing by lane cross contamination.\u003csup\u003e28,29\u003c/sup\u003e 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).\u003csup\u003e14,30\u003c/sup\u003e\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSubject Demographics\u003c/strong\u003e\u003cp\u003eThe study dataset comprised of 10 male burn patients admitted to the UCDH BICU, 18 healthy volunteers working at UCDH but not in the BICU, and 5 female BICU nurses. Burn patients were aged 31 to 61 years (median 38 years) and suffered a median TBSA burn of 26.5% (range 10\u0026ndash;76%). Of the 10 burn patients, 50% (\u003cem\u003en\u0026thinsp;=\u0026thinsp;5\u003c/em\u003e) required grafting of the wound sampling location, 50% (\u003cem\u003en\u0026thinsp;=\u0026thinsp;5\u003c/em\u003e) experienced bacterial and/or fungal wound infections, 20% (\u003cem\u003en\u0026thinsp;=\u0026thinsp;2\u003c/em\u003e) were diagnosed with culture positive bacterial pneumonia, and 20% (\u003cem\u003en\u0026thinsp;=\u0026thinsp;2\u003c/em\u003e) with blood culture positive septicemia. There were no instances of diagnosed inhalation injury (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Median time from BICU admission to the first sample collection was 5 hours (range 1\u0026ndash;14 hours) and the median number of days on study was 21 (range 7\u0026ndash;28 days).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBurn Patient Demographics. Abbreviations: B, Blood; R, Respiratory; W, Wound\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"11\"\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSubject ID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAge (years)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTotal TBSA Burn (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRace or Ethnicity\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMechanism of Injury\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTime on Study (Days)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eBurn Depth at Collection Location\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eBurn Site Grafted\u003c/p\u003e\u003cp\u003e(y/n)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eCulture Positive\u003c/p\u003e\u003cp\u003e(y/n)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eDay(s) of Culture(s)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003eOrganism(s) Cultured\u003c/p\u003e\u003cp\u003e(Location)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHispanic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eflame\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003epartial-thickness\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003en\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003en\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWhite\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eflame\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003efull-thickness\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003ey\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003en\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWhite\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eflame\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003epartial-thickness\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003en\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003ey\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003eMRSA (B/W)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNative American\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eflame\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003epartial-thickness\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003en\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003ey\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e\u003cem\u003eKlebsiella sp.\u003c/em\u003e (R)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWhite\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eflame\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003efull-thickness\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003ey\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003ey\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003eMRSA (W)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOther\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eflame\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003epartial-thickness\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003en\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003ey\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e3\u003c/p\u003e\u003cp\u003e12\u003c/p\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003eEnterobacter cloacae complex (W)\u003c/p\u003e\u003cp\u003e\u003cem\u003eLactobacillus sp.\u003c/em\u003e (W)\u003c/p\u003e\u003cp\u003e\u003cem\u003eStreptococcus pneumonia\u003c/em\u003e, \u003cem\u003eStenotrophomonas maltophilia\u003c/em\u003e (R)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHispanic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003escald\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003epartial-thickness\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003en\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003ey\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e6\u003c/p\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e\u003cem\u003eLactobacillus sp.;\u003c/em\u003e yeast (W)\u003c/p\u003e\u003cp\u003e\u003cem\u003eCandida albicans\u003c/em\u003e (B)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHispanic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eflame\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003efull-thickness\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003ey\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003en\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWhite\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eflame\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003efull-thickness\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003ey\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003en\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWhite\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eflame\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003efull-thickness\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003ey\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003ey\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e (W)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSampling and Bioinformatic Filtering\u003c/strong\u003e\u003cp\u003eOf the total collected samples (n\u0026thinsp;=\u0026thinsp;228), 210 were included in the dataset. Twelve samples were excluded pre-sequencing due to undetectable nucleic acid concentrations following extraction and six samples were excluded bioinformatically with \u0026lt;\u0026thinsp;20% of sequences passing quality filters and/or achieving a sampling depth of \u0026lt;\u0026thinsp;1156 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Following recommendations from Salter et al., one potential contaminant amplicon sequence variant (ASV), a unique DNA variant in the dataset, mapping to \u003cem\u003eRalstonia\u003c/em\u003e species (sp.) was identified.\u003csup\u003e31\u003c/sup\u003e This genus has been reported as a reagent contaminant and appeared in all low abundance samples following implementation of a new extraction kit (\u003cb\u003eFigure S3\u003c/b\u003e). The ASV was removed from the dataset by filtering with q2-demux \u0026ndash; all results reported are in exclusion of this taxon.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTotal Samples Included in Analysis\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCollection Group\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSamples Collected\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSamples Post Extraction\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTotal Included\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e%Excluded\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBurn\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpared\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePerianal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRectal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eReagent Control\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAir Control\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNurse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHealthy Volunteer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e228\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e216\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e210\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e7.9\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eSample Group Diversity and Taxonomy\u003c/h2\u003e\u003cp\u003e\u003cstrong\u003eTaxonomic Relative Abundance\u003c/strong\u003e\u003cp\u003eSample group taxonomy was collapsed by collection location across all burn subjects and sampling timepoints with phylum level classification (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The four most prevalent genera sampled from the burn patient wound (\u003cem\u003eStaphylococcus\u003c/em\u003e, \u003cem\u003eBurkholderia\u003c/em\u003e, \u003cem\u003eStenotrophomonas\u003c/em\u003e, and \u003cem\u003ePropionibacterium\u003c/em\u003e) were also predominant on burn patient spared skin in varying proportions (\u003cb\u003eTable\u0026nbsp;3\u003c/b\u003e). Analysis of differential feature abundance of features (q2-ALDEx2) found four ASVs with an effect size\u0026thinsp;\u0026gt;\u0026thinsp;1 between burn wound and spared skin (\u003cb\u003eFigure S4\u003c/b\u003e). Of the four features identified by ALDEx2, three mapped by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) with 100% identity to strains of \u003cem\u003ePropionibacterium acnes\u003c/em\u003e and one to \u003cem\u003eStaphylococcus epidermitis\u003c/em\u003e, both are reduced on the burn wound. Perianal and rectal sampling locations did not exhibit significantly different taxa when compared by ANCOM and ALDEx2.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSummary of taxonomic relative abundance consisting of the top 10 phyla sequenced from samples collected at four collection locations (burn wound, spared skin, perianal, and rectal) on the burn patient cohort. Phyla are listed descending from least to most abundant.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSummary of the relative abundance of the top 10 phyla and genera level taxa on the healthy volunteer and BICU nurse cohorts. Phyla and genera are listed descending from least to most abundant.\u003c/p\u003e \u003cp\u003e\u003cstrong\u003eTable 3. \u003c/strong\u003ePredominant Taxa by Sample Group\u003c/p\u003e\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e \u003cp\u003eIn descending order, the top four most predominant phyla and genera as determined by percent relative abundance summed by sample group among burn patients, healthy volunteers, and BICU nurses.\u003c/p\u003e\u003cp\u003eSimilarly, sample group taxonomy was collapsed by healthy volunteer and BICU nurse cohorts by phylum and genus level classification (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Eleven ASVs exhibit differential relative abundances between BICU nurses and healthy volunteers. These ASVs mapped by NCBI BLAST to the following taxa by \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\ge\\)\u003c/span\u003e\u003c/span\u003e99.78% identity: \u003cem\u003eStenotrophomonas maltophilia\u003c/em\u003e, \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eStenotrophomonas sp.\u003c/em\u003e, \u003cem\u003eVariovax paradoxus\u003c/em\u003e, \u003cem\u003eSphingomonas sp.\u003c/em\u003e, \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e, \u003cem\u003ePseudomonas sp.\u003c/em\u003e, and \u003cem\u003eMicrobacterium sp.\u003c/em\u003e With the exception of \u003cem\u003eStaphyloccus epidermidis\u003c/em\u003e, all are enriched on BICU nurse samples.\u003c/p\u003e\u003cp\u003eBetween-sample diversity, as measured by Shannon Diversity, is significantly different between all group comparisons (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) except between air controls/burn wound (\u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.487\u003c/em\u003e), air controls/nurses (\u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.052\u003c/em\u003e), and burn wound/nurses (\u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.325\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Healthy volunteers have significantly higher mean Shannon diversity compared to cutaneous burn samples and nurses (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e). Shannon diversity is not significantly different between the days of collection for any burn patient sampling site; however, bacterial populations from the burn wound exhibit sustained reduced alpha diversity compared to spared skin for days 0\u0026ndash;21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Bray Curtis dissimilarity distance of microbial abundance differed significantly between collection groups (\u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.001\u003c/em\u003e PERMANOVA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e \u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eEffects of Burn Treatments on Diversity\u003c/h2\u003e\u003cp\u003e\u003cstrong\u003eAntimicrobials\u003c/strong\u003e\u003cp\u003eSystemic antimicrobials were administered to 100% of the burn study population at one or more timepoints. Samples collected following systemic antibiotic administration were clustered by collection location and compared to samples collected prior to any systemic antibiotic administration. The burn wound exhibits significantly reduced alpha diversity (\u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.023\u003c/em\u003e) following systemic antibiotic administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Analysis with ANCOM and ALDEx2 did not reveal any significantly different taxa on the burn wound samples following systemic antibiotic administration. However, the relative abundance of the phylum Firmicutes decreased (57.9\u0026ndash;20.3%) following antibiotic administration while Proteobacteria increased (24.6\u0026ndash;51.8%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Application of different topical antimicrobials during the wound care prior to sample collection did not differentially impact alpha diversity of the burn wound populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec) and were not significantly different from samples collected on admission, prior to the first wound care.\u003c/p\u003e\u003c/p\u003e \u003cp\u003e\u003cstrong\u003eGrafting\u003c/strong\u003e\u003cp\u003eAutologous skin grafting occurred at the sampling site in 50% of the burn study population (\u003cem\u003en\u0026thinsp;=\u0026thinsp;5\u003c/em\u003e) affecting 33.3% of all burn wound samples (\u003cem\u003en\u0026thinsp;=\u0026thinsp;15\u003c/em\u003e). Samples obtained from grafted skin exhibited reduced alpha diversity compared to non-grafted samples (\u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.036\u003c/em\u003e) while grafting had no significant effect on the alpha diversity at other collection locations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). When visualized longitudinally, alpha diversity trends towards recovery by day 28 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Analysis by ALDEx2 did not classify any ASVs as significantly different between grafted and non-grafted wound samples. Analysis by ANCOM revealed the phylum Fusobacteria to be significantly reduced among the grafted wound samples (W\u0026thinsp;=\u0026thinsp;14). The relative abundance of Proteobacteria increased from 25.1\u0026ndash;66.2% following grafting while Firmicutes decreased from 45.3\u0026ndash;25.4% (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec).\u003c/p\u003e\u003c/p\u003e \u003cp\u003e\u003cstrong\u003eDiet\u003c/strong\u003e\u003cp\u003eThe effects of diet type (enteral, parenteral, or regular) were minimal on alpha diversity between collection locations (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Longitudinal analysis of Bray Curtis dissimilarity between diet types indicate that gut samples derived from patients receiving enteral nutrition become more compositionally dissimilar to those collected from patients consuming a regular diet over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). Analysis of differential relative abundance by ANCOM found the genera \u003cem\u003eProteus\u003c/em\u003e, \u003cem\u003eEnterococcus\u003c/em\u003e, and \u003cem\u003eMethanobrevibacter\u003c/em\u003e to be significantly enriched in patients receiving enteral nutrition (W\u0026thinsp;=\u0026thinsp;180, W\u0026thinsp;=\u0026thinsp;174, W\u0026thinsp;=\u0026thinsp;174, respectively) compared to a regular diet (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec \u003cb\u003eand Figure S4)\u003c/b\u003e. ALDEx2 did not identify significantly different ASVs between the groups.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e(\u003cb\u003e7a\u003c/b\u003e) Mean Shannon Diversity between diet type that each patient received during the collection timepoint for each collection location (burn wound, spared skin, perianal, and rectal) sampled. (\u003cb\u003e7b\u003c/b\u003e) Bray Curtis dissimilarly by axis 2 of the PCOA plot graphed on a volatility plot to visualize the impact of time on compositional diversity. Axis 2 explained 6.605% of variation between groups. (\u003cb\u003e7c\u003c/b\u003e) Taxonomic summary of the top 10 phyla sequenced from the sum of perianal and rectal samples separated by diet type at the time of collection. The designation \u0026ldquo;NA\u0026rdquo; represents unclassified phyla.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eDetection of Clinically Identified Infections\u003c/h2\u003e\u003cp\u003eSix of 10 burn patients experienced culture positive infection at one or more timepoints during the study. Nine instances of bacterial infection were detected in wound, blood, and respiratory cultures occurring across these six patients. Burn wound samples were evaluated for the family or genus of the infecting organism and plotted by relative abundance to detect changes over time. Irrespective of culture location, 55.6% (5/9) of infections were detectable by an increase in relative abundance on the burn wound from the single sample site collected (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Notably, for Subject 6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed), \u003cem\u003eStenotrophomonas maltophilia\u003c/em\u003e was cultured from a respiratory sample on day 14 and clinically treated for pneumonia. On this subject\u0026rsquo;s wound, the genera \u003cem\u003eStenotrophomonas\u003c/em\u003e was undetectable on admission, however on day 14 consisted 15.1% of ASVs sequenced. \u003cem\u003eStenotrophomonas\u003c/em\u003e remained a dominant taxon on the wound comprising 60.6% and 72.3% of ASVs on days 21 and 28, respectively, despite systemic antibiotic administration for pneumonia on day 14.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrevious studies have profiled the human skin microbiome reporting differential populations and diversity found on the human skin; however the majority still support one of the sentinel molecular 16S sequencing studies that found that the human forearm consists predominantly (94.6% of clones) of the phyla \u003cem\u003eActinobacteria\u003c/em\u003e, \u003cem\u003eFirmicutes\u003c/em\u003e, and \u003cem\u003eProteobacteria\u003c/em\u003e.\u003csup\u003e32\u003c/sup\u003e These data are supported by our findings for the control populations of healthy volunteers and BICU nurses sampled on the right forearm as well as the burn patient wound and spared skin samples (\u003cb\u003eTable\u0026nbsp;3\u003c/b\u003e). Despite partial and full-thickness tissue destruction, the burn wound samples still have predominant phyla reflective of an uninjured skin microbiome. We found that burn wound microbial populations were significantly less diverse compared to spared skin, healthy volunteers, and BICU nurses (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Reduction in microbial population alpha diversity has been reported as a characteristic of non-healing wounds and could have implications for healing rates in the burn population.\u003csup\u003e33\u003c/sup\u003e We expected to see an initial reduction and subsequent recovery of alpha diversity from the wound sites, however this was not supported (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The sustained reduction in alpha diversity could be due to frequent routine application of topical antimicrobials or differential healing in our population, among other factors not controlled for in this study. \u003cem\u003ePropionibacterium acnes\u003c/em\u003e and \u003cem\u003eStaphylococcus epidermitis\u003c/em\u003e are two skin commensal organisms that were significantly reduced on the wound site, supporting that burn wounds experience perturbance of normal commensal bacteria.\u003c/p\u003e\u003cp\u003eInterestingly, the diversity and composition of the BICU nurse microbiome was significantly different compared to healthy volunteers not working in the BICU. Relative enrichment of \u003cem\u003eStenotrophomonas sp.\u003c/em\u003e and reduction in the normal commensal \u003cem\u003eStaphylococcus epidermitis\u003c/em\u003e suggest that nurses experience microbial dysbiosis of their cutaneous microbiota. Isolation protocols such as frequent sanitization of the hands and forearms could impact the bacterial populations. In addition, the patient population nurses interact with could influence the cutaneous microbiota, leading to disparities between nurses working in different units. Further research is warranted in this area as our study has a small sample size of five nurses.\u003c/p\u003e\u003cp\u003eOur findings suggest that systemic antimicrobial administration significantly reduces microbial diversity at the burn wound but does not impact other microbial populations evaluated in the study. The relative decrease in Firmicutes and increase in Proteobacteria following administration could be a result of broad-spectrum coverage against Gram-positive organisms allowing for Gram-negative enrichment. Interestingly, the type of topical antimicrobial applied on the burn wound did not significantly impact alpha diversity or taxonomic composition at the wound. Grafting of a burn wound and the clinical process associated with the procedure, such as excision, anesthesia, and prophylactic antibiotics are all proposed to cause dysbiosis to microbial populations. We found that diversity is reduced at the wound site following grafting but may trend towards recovery by day 28 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb) as the graft takes and the cutaneous microenvironment becomes more reflective of normal skin. A non-significant decrease in bacterial diversity seen at the spared skin following grafting could be due to the surgery and prophylactic antibiotics associated with the procedure (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003eAdequate nutrition is an essential component of burn care and patients often receive a variety of nutritional sources to meet high caloric needs. We investigated whether the type of nutrition a patient is receiving impacts gut microbial populations and found that enteral nutrition trends towards decreased gut diversity and increased compositional difference compared to samples obtained from patients receiving a regular diet (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These data are not significant and should be further investigated in both burn and non-burn ICU patients.\u003c/p\u003e\u003cp\u003eThe burn population is one that could greatly benefit from early detection of infection with the potential to reduce the incidence of sepsis and subsequent mortality. The ability to monitor common sites of infection, such as the burn wound, for colonization of pathogenic bacteria that could result in local infection or systemic spread would improve targeted antimicrobial therapies and patient outcome. It has been shown that machine learning can be used to predict disease states such as inflammatory bowel disease, type 2 diabetes, and liver cirrhosis from 16S microbial sequencing datasets.\u003csup\u003e34,35\u003c/sup\u003e Additionally, susceptibility to bloodstream infection following chemotherapy has been predicted by the pre-treatment gut microbiome.\u003csup\u003e36\u003c/sup\u003e To investigate this potential use in the burn population we retrospectively tracked changes in relative abundance of organisms over time that were cultured as part of the subjects\u0026rsquo; clinical care. We found that local and systemic infections could be seen by the increased abundance of the inciting bacteria at the burn wound (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This patient population should be included in studies evaluating the clinical application of sequencing methodologies and machine learning to detect infection. Comprehensive sampling of multiple sites throughout a patient\u0026rsquo;s hospitalization could also help indicate the source of infection, which is often unknown in this population.\u003c/p\u003e\u003cp\u003eStudies utilizing 16S microbial community analysis inherently contain limitations. Sample preparation variables such as collection and storage methodology have been shown to influence results.\u003csup\u003e37\u003c/sup\u003e Extraction and amplification of a single section of bacterial DNA can contribute bias to the dataset, as can the sequencing platform used and the bioinformatic algorithms performed during downstream analysis.\u003csup\u003e38\u003c/sup\u003e We aim to acknowledge and address these limitations through the transparency of our methodology and public sharing of the raw sequence data. An additional limitation specific to this study is the burn patient enrollment gender bias towards male participants. Gender was not selected for during enrollment and males are admitted to the ICU for burn injury more frequently than females. In 2006, a ten year review of United States hospital burn admissions found males to be overrepresented, accounting for 67.9%.\u003csup\u003e39\u003c/sup\u003e However, further research should aim for an even gender distribution, as females are at increased risk of death following burn injury and microbial populations have been shown to differ by gender.\u003csup\u003e39,40\u003c/sup\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study profiled the microbial populations found on the burn wound, spared skin, perianal, and rectal locations for the first 28 days of 10 burn patients admitted to the UCDH BICU. Our data support the idea that microbial dysbiosis occurs at the burn wound and spared skin following injury compared to healthy volunteers, and that BICU nurses may also experience community dysbiosis from cutaneous populations sampled from the forearm. Treatments such as antibiotics, grafting, and nutritional formulations can impact the diversity and community compositions sampled during the patient\u0026rsquo;s ICU stay. Surprisingly, the topical antimicrobial applied at the wound did not result in a significant difference of relative abundance in any AVSs. In addition, it is possible to detect changes in relative abundance of clinically identified infectious bacteria at the burn wound, even if it is not the site of the culture location. Due to the impact that sepsis can have on patient outcome, further research in establishing burn wound bacterial profiles can aid in the development of tools to detect pathogenic changes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe kindly acknowledge the UCDH BICU physicians Dr. Tina Palmieri, Dr. Kathleen Romanowski, and Dr. Soman Sen as well as the entire BICU nursing staff for their participation in subject identification and sample collection. Thank you to the ARCS Foundation for funding this project. The Genomics Shared Resource is supported by the UC Davis Comprehensive Cancer Center Support Grant (CCSG) awarded by the National Cancer Institute (NCI P30CA093373). The DNA Technologies and Expression Analysis Core at the UC Davis Genome Center is supported by NIH Shared Instrumentation Grant 1S10OD010786-01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKelly Lima (K.L): K.L performed subject consent, sample collection, wrote the manuscript in its entirety, performed bioinformatic and statistical analysis of the study data, and generated Figures and Tables for the manuscript.\u003c/p\u003e\n\u003cp\u003eRyan Davis (R.D.): R.D. provided technical support and expertise during DNA extraction and completed 16S library generation in its entirety. R.D. also provided technical support during bioinformatic analysis of sequence data and reviewed and edited the paper.\u003c/p\u003e\n\u003cp\u003eStephenie Liu (S.L.): S.L. provided technical support and expertise during DNA extraction and sample submission for sequencing. S.L. also significantly contributed to the review and editing of the manuscript.\u003c/p\u003e\n\u003cp\u003eDavid Greenhalgh (D.G.): D.G is co-investigator for the study who provided significant input on the study design and significantly contributed to the review and editing of the manuscript.\u003c/p\u003e\n\u003cp\u003eNam Tran (N.T.): N.T. serves as study PI for the study. Significantly contributed to study design and significantly contributed to the review and editing of the manuscript with co-authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eADDITIONAL INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available in the NCBI Sequence Read Archive (SRA) repository, [PRJNA701230], and upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is NO competing interest.\u003c/p\u003e"},{"header":"Declarations","content":"\u003col\u003e\n\u003cli\u003eJames, S. L. \u003cem\u003eet al.\u003c/em\u003e Epidemiology of injuries from fire, heat and hot substances: global, regional and national morbidity and mortality estimates from the Global Burden of Disease 2017 study. \u003cem\u003eInj. Prev.\u003c/em\u003e\u003cstrong\u003e26\u003c/strong\u003e, i36\u0026ndash;i45 (2020).\u003c/li\u003e\n\u003cli\u003eCapek, K. D. \u003cem\u003eet al.\u003c/em\u003e Contemporary Burn Survival. \u003cem\u003eJ. Am. Coll. Surg.\u003c/em\u003e\u003cstrong\u003e226\u003c/strong\u003e, 453\u0026ndash;463 (2018).\u003c/li\u003e\n\u003cli\u003eGreenhalgh, D. G. \u003cem\u003eet al.\u003c/em\u003e American Burn Association consensus conference to define sepsis and infection in burns. \u003cem\u003eJ. Burn Care Res. Off. Publ. Am. Burn Assoc.\u003c/em\u003e\u003cstrong\u003e28\u003c/strong\u003e, 776\u0026ndash;790 (2007).\u003c/li\u003e\n\u003cli\u003eDeitch, E. A. \u0026amp; Berg, R. Bacterial Translocation from the GutA Mechanism of Infection. \u003cem\u003eJ. Burn Care Rehabil.\u003c/em\u003e\u003cstrong\u003e8\u003c/strong\u003e, 475\u0026ndash;482 (1987).\u003c/li\u003e\n\u003cli\u003eBeckmann, N., Pugh, A. M. \u0026amp; Caldwell, C. C. Burn injury alters the intestinal microbiome\u0026rsquo;s taxonomic composition and functional gene expression. \u003cem\u003ePLOS ONE\u003c/em\u003e\u003cstrong\u003e13\u003c/strong\u003e, e0205307 (2018).\u003c/li\u003e\n\u003cli\u003eThe Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) | Critical Care Medicine | JAMA | JAMA Network. https://jamanetwork.com/journals/jama/fullarticle/2492881.\u003c/li\u003e\n\u003cli\u003eTran, N. K. \u003cem\u003eet al.\u003c/em\u003e Novel application of an automated-machine learning development tool for predicting burn sepsis: proof of concept. \u003cem\u003eSci. Rep.\u003c/em\u003e\u003cstrong\u003e10\u003c/strong\u003e, 12354 (2020).\u003c/li\u003e\n\u003cli\u003ePlichta, J. K. \u003cem\u003eet al.\u003c/em\u003e Cutaneous Burn Injury Promotes Shifts in the Bacterial Microbiome in Autologous Donor Skin: Implications for Skin Grafting Outcomes. \u003cem\u003eShock Augusta Ga\u003c/em\u003e\u003cstrong\u003e48\u003c/strong\u003e, 441\u0026ndash;448 (2017).\u003c/li\u003e\n\u003cli\u003eTurnbaugh, P. J. \u003cem\u003eet al.\u003c/em\u003e The Human Microbiome Project. \u003cem\u003eNature\u003c/em\u003e\u003cstrong\u003e449\u003c/strong\u003e, 804\u0026ndash;810 (2007).\u003c/li\u003e\n\u003cli\u003eJovel, J. \u003cem\u003eet al.\u003c/em\u003e Characterization of the Gut Microbiome Using 16S or Shotgun Metagenomics. \u003cem\u003eFront. Microbiol.\u003c/em\u003e\u003cstrong\u003e7\u003c/strong\u003e, (2016).\u003c/li\u003e\n\u003cli\u003eMCutting. Manual of Procedures for Human Microbiome Project Core Microbiome Sampling Protocol A HMP Protocol # 07-001. (2010).\u003c/li\u003e\n\u003cli\u003eJo, J.-H., Kennedy, E. A. \u0026amp; Kong, H. H. Research Techniques Made Simple: Bacterial 16S Ribosomal RNA Gene Sequencing in Cutaneous Research. \u003cem\u003eJ. Invest. Dermatol.\u003c/em\u003e\u003cstrong\u003e136\u003c/strong\u003e, e23-7 (2016).\u003c/li\u003e\n\u003cli\u003eKlindworth, A. \u003cem\u003eet al.\u003c/em\u003e Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e\u003cstrong\u003e41\u003c/strong\u003e, e1\u0026ndash;e1 (2013).\u003c/li\u003e\n\u003cli\u003eBolyen, E. \u003cem\u003eet al.\u003c/em\u003e Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. \u003cem\u003eNat. Biotechnol.\u003c/em\u003e\u003cstrong\u003e37\u003c/strong\u003e, 852\u0026ndash;857 (2019).\u003c/li\u003e\n\u003cli\u003eCallahan, B. J. \u003cem\u003eet al.\u003c/em\u003e DADA2: High resolution sample inference from Illumina amplicon data. \u003cem\u003eNat. Methods\u003c/em\u003e\u003cstrong\u003e13\u003c/strong\u003e, 581\u0026ndash;583 (2016).\u003c/li\u003e\n\u003cli\u003eKatoh, K. \u0026amp; Standley, D. M. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. \u003cem\u003eMol. Biol. Evol.\u003c/em\u003e\u003cstrong\u003e30\u003c/strong\u003e, 772\u0026ndash;780 (2013).\u003c/li\u003e\n\u003cli\u003ePrice, M. N., Dehal, P. S. \u0026amp; Arkin, A. P. FastTree 2--approximately maximum-likelihood trees for large alignments. \u003cem\u003ePloS One\u003c/em\u003e\u003cstrong\u003e5\u003c/strong\u003e, e9490 (2010).\u003c/li\u003e\n\u003cli\u003eShannon, C. E. A Mathematical Theory of Communication. \u003cem\u003eBell Syst. Tech. J.\u003c/em\u003e\u003cstrong\u003e27\u003c/strong\u003e, 379\u0026ndash;423 (1948).\u003c/li\u003e\n\u003cli\u003eBray, J. R. \u0026amp; Curtis, J. T. An Ordination of the Upland Forest Communities of Southern Wisconsin. \u003cem\u003eEcol. Monogr.\u003c/em\u003e\u003cstrong\u003e27\u003c/strong\u003e, 326\u0026ndash;349 (1957).\u003c/li\u003e\n\u003cli\u003eBokulich, N. A. \u003cem\u003eet al.\u003c/em\u003e Optimizing taxonomic classification of marker-gene amplicon sequences with QIIME 2\u0026rsquo;s q2-feature-classifier plugin. \u003cem\u003eMicrobiome\u003c/em\u003e\u003cstrong\u003e6\u003c/strong\u003e, 90 (2018).\u003c/li\u003e\n\u003cli\u003eMcDonald, D. \u003cem\u003eet al.\u003c/em\u003e An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea. \u003cem\u003eISME J.\u003c/em\u003e\u003cstrong\u003e6\u003c/strong\u003e, 610\u0026ndash;618 (2012).\u003c/li\u003e\n\u003cli\u003eMandal, S. \u003cem\u003eet al.\u003c/em\u003e Analysis of composition of microbiomes: a novel method for studying microbial composition. \u003cem\u003eMicrob. Ecol. Health Dis.\u003c/em\u003e\u003cstrong\u003e26\u003c/strong\u003e, 27663 (2015).\u003c/li\u003e\n\u003cli\u003eFernandes, A. D., Macklaim, J. M., Linn, T. G., Reid, G. \u0026amp; Gloor, G. B. ANOVA-Like Differential Expression (ALDEx) Analysis for Mixed Population RNA-Seq. \u003cem\u003ePLOS ONE\u003c/em\u003e\u003cstrong\u003e8\u003c/strong\u003e, e67019 (2013).\u003c/li\u003e\n\u003cli\u003eBokulich, N. A. \u003cem\u003eet al.\u003c/em\u003e q2-longitudinal: Longitudinal and Paired-Sample Analyses of Microbiome Data. \u003cem\u003emSystems\u003c/em\u003e\u003cstrong\u003e3\u003c/strong\u003e, e00219-18 (2018).\u003c/li\u003e\n\u003cli\u003eKruskal, W. H. \u0026amp; Wallis, W. A. Use of Ranks in One-Criterion Variance Analysis. \u003cem\u003eJ. Am. Stat. Assoc.\u003c/em\u003e\u003cstrong\u003e47\u003c/strong\u003e, 583 (1952).\u003c/li\u003e\n\u003cli\u003eBisanz, Jordan E. \u003cem\u003eqiime2R: Importing QIIME2 artifacts and associated data into R sessions\u003c/em\u003e. (2018).\u003c/li\u003e\n\u003cli\u003eAnderson, M. J. Permutational Multivariate Analysis of Variance (PERMANOVA). in \u003cem\u003eWiley StatsRef: Statistics Reference Online\u003c/em\u003e 1\u0026ndash;15 (American Cancer Society, 2017). doi:10.1002/9781118445112.stat07841.\u003c/li\u003e\n\u003cli\u003eMohammadi, T., Reesink, H. W., Vandenbroucke-Grauls, C. M. J. E. \u0026amp; Savelkoul, P. H. M. Removal of contaminating DNA from commercial nucleic acid extraction kit reagents. \u003cem\u003eJ. Microbiol. Methods\u003c/em\u003e\u003cstrong\u003e61\u003c/strong\u003e, 285\u0026ndash;288 (2005).\u003c/li\u003e\n\u003cli\u003eBarton, H. A., Taylor, N. M., Lubbers, B. R. \u0026amp; Pemberton, A. C. DNA extraction from low-biomass carbonate rock: An improved method with reduced contamination and the low-biomass contaminant database. \u003cem\u003eJ. Microbiol. Methods\u003c/em\u003e\u003cstrong\u003e66\u003c/strong\u003e, 21\u0026ndash;31 (2006).\u003c/li\u003e\n\u003cli\u003eMcDonald, D. \u003cem\u003eet al.\u003c/em\u003e The Biological Observation Matrix (BIOM) format or: how I learned to stop worrying and love the ome-ome. \u003cem\u003eGigaScience\u003c/em\u003e\u003cstrong\u003e1\u003c/strong\u003e, 7 (2012).\u003c/li\u003e\n\u003cli\u003eSalter, S. J. \u003cem\u003eet al.\u003c/em\u003e Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. \u003cem\u003eBMC Biol.\u003c/em\u003e\u003cstrong\u003e12\u003c/strong\u003e, 87 (2014).\u003c/li\u003e\n\u003cli\u003eGao, Z., Tseng, C., Pei, Z. \u0026amp; Blaser, M. J. Molecular analysis of human forearm superficial skin bacterial biota. \u003cem\u003eProc. Natl. Acad. Sci.\u003c/em\u003e\u003cstrong\u003e104\u003c/strong\u003e, 2927\u0026ndash;2932 (2007).\u003c/li\u003e\n\u003cli\u003eGardiner, M. \u003cem\u003eet al.\u003c/em\u003e A longitudinal study of the diabetic skin and wound microbiome. \u003cem\u003ePeerJ\u003c/em\u003e\u003cstrong\u003e5\u003c/strong\u003e, e3543 (2017).\u003c/li\u003e\n\u003cli\u003eOh, M. \u0026amp; Zhang, L. DeepMicro: deep representation learning for disease prediction based on microbiome data. \u003cem\u003eSci. Rep.\u003c/em\u003e\u003cstrong\u003e10\u003c/strong\u003e, 6026 (2020).\u003c/li\u003e\n\u003cli\u003ePasolli, E., Truong, D. T., Malik, F., Waldron, L. \u0026amp; Segata, N. Machine Learning Meta-analysis of Large Metagenomic Datasets: Tools and Biological Insights. \u003cem\u003ePLOS Comput. Biol.\u003c/em\u003e\u003cstrong\u003e12\u003c/strong\u003e, e1004977 (2016).\u003c/li\u003e\n\u003cli\u003eMontassier, E. \u003cem\u003eet al.\u003c/em\u003e Pretreatment gut microbiome predicts chemotherapy-related bloodstream infection. \u003cem\u003eGenome Med.\u003c/em\u003e\u003cstrong\u003e8\u003c/strong\u003e, 49 (2016).\u003c/li\u003e\n\u003cli\u003eChoo, J. M., Leong, L. E. \u0026amp; Rogers, G. B. Sample storage conditions significantly influence faecal microbiome profiles. \u003cem\u003eSci. Rep.\u003c/em\u003e\u003cstrong\u003e5\u003c/strong\u003e, 16350 (2015).\u003c/li\u003e\n\u003cli\u003ePoretsky, R., Rodriguez-R, L. M., Luo, C., Tsementzi, D. \u0026amp; Konstantinidis, K. T. Strengths and Limitations of 16S rRNA Gene Amplicon Sequencing in Revealing Temporal Microbial Community Dynamics. \u003cem\u003ePLOS ONE\u003c/em\u003e\u003cstrong\u003e9\u003c/strong\u003e, e93827 (2014).\u003c/li\u003e\n\u003cli\u003eLatenser, B. A. \u003cem\u003eet al.\u003c/em\u003e National Burn Repository 2006: A Ten-Year Review. \u003cem\u003eJ. Burn Care Res.\u003c/em\u003e\u003cstrong\u003e28\u003c/strong\u003e, 635\u0026ndash;658 (2007).\u003c/li\u003e\n\u003cli\u003eFierer, N., Hamady, M., Lauber, C. L. \u0026amp; Knight, R. The influence of sex, handedness, and washing on the diversity of hand surface bacteria. \u003cem\u003eProc. Natl. Acad. Sci.\u003c/em\u003e\u003cstrong\u003e105\u003c/strong\u003e, 17994\u0026ndash;17999 (2008).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Microbiome, wound, 16S rRNA gene sequencing, bacteria, burn injury, skin graft, and infection","lastPublishedDoi":"10.21203/rs.3.rs-240723/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-240723/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSepsis is a leading cause of morbidity and mortality in patients that have sustained a severe burn injury. Early detection and treatment of infections improves outcomes and understanding changes in the host microbiome following injury and during treatment may aid in burn care. The loss of functional barriers, systemic inflammation, and commensal community dysbiosis all contribute to a burn patient\u0026rsquo;s increased risk of infection. We sampled 10 burn patients to evaluate cutaneous microbial populations on the burn wound and corresponding spared skin on days 0, 3, 7, 14, 21, and 28 post-intensive care unit admission. In addition, skin samples were paired with perianal and rectal locations to evaluate changes in the burn patient gut microbiome following injury and treatment. We found significant (\u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.011\u003c/em\u003e) reduction in alpha diversity on the burn wound compared to spared skin throughout the sampling period as well as reduction in common skin commensal bacteria such as \u003cem\u003ePropionibacterium acnes\u003c/em\u003e and \u003cem\u003eStaphylococcus epidermitis\u003c/em\u003e. Compared to healthy volunteers (\u003cem\u003en\u0026thinsp;=\u0026thinsp;18\u003c/em\u003e), the burn patient spared skin also exhibited a significant reduction in diversity (\u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.001\u003c/em\u003e). Treatments such as systemic or topical antibiotic administration, skin grafting, and nutritional formulations also impact diversity and community composition at the sampling locations. When evaluating each subject individually, an increase in relative abundance of a clinically isolated bacteria could be seen in 5/9 infections detected among the burn patient cohort.\u003c/p\u003e","manuscriptTitle":"Longitudinal Profiling Of The Burn Patient Cutaneous And Gastrointestinal Microbiota","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-02-23 15:31:01","doi":"10.21203/rs.3.rs-240723/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-03-08T07:27:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-02-22T16:51:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"cbaedab8-ed7e-45c3-b633-ff1e5de6e795","date":"2021-02-22T14:17:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-02-22T10:12:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-02-22T10:08:36+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-02-18T23:27:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-02-18T00:10:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2021-02-14T03:06:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e9bde819-f79d-4b56-b6ad-5111f8003d79","owner":[],"postedDate":"February 23rd, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":2541073,"name":"Pathology"},{"id":2541074,"name":"Laboratory Diagnostics"},{"id":2541075,"name":"Surgery"}],"tags":[],"updatedAt":"2021-04-15T06:14:08+00:00","versionOfRecord":[],"versionCreatedAt":"2021-02-23 15:31:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-240723","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-240723","identity":"rs-240723","version":["v1"]},"buildId":"ehx78VzkSd0WSzXnipQa-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}