A combination of burn wound injury and Pseudomonas infection elicits unique gene expression that enhances bacterial pathogenicity.

Burns are a leading cause of morbidity and mortality worldwide with the most common cause of death resulting from sepsis, often from Pseudomonas aeruginosa. We previously reported that a non-lethal flame burn induced an altered host immune response. Using this model, gene expression in both the murine host and P. aeruginosa was measured using a NanoString custom probe panel. We observed differing patterns of gene expression in both host and P. aeruginosa in the skin, blood, liver, and spleen of mice that were burned and/or infected, compared to mice that were neither burned nor infected (i.e., Sham). In mice that were both burned and infected (B/I), we observed changes in gene expression in both the host and P. aeruginosa that were distinct from all other treatment conditions. These data suggest that the combination of the burned state and superimposed infection affects both host and pathogen gene expression to increase infection propensity. Gene transcription significantly changed from 6 to 24 h post-B/I in each tissue. Finally, inhibiting IL-10 signaling or co-administering arginine at the time of P. aeruginosa infection prolonged or restored survival in an otherwise 100% fatal burn and infection model. These findings suggest that disease states such as burns may differentially alter innate immune response gene expression in both a host- and pathogen-specific manner.IMPORTANCEThe interaction between an underlying disease process and a specific pathogen may lead to the unique expression of genes that affect bacterial pathogenesis. These genes may not be observed during infection in the absence of, or with a different underlying process or infection during the underlying process with a different pathogen. To test this hypothesis, we used Nanostring technology to compare gene transcription in a murine-burned wound infected with P. aeruginosa. The Nanostring probeset allowed the simultaneous direct comparison of immune response gene expression in both multiple host tissues and P. aeruginosa in conditions of burn alone, infection alone, and burn with infection. While RNA-Seq is used to discover novel transcripts, NanoString could be a technique to monitor specific changes in transcriptomes between samples and bypass the additional adjustments for multispecies sample processing or the need for the additional steps of alignment and assembly required for RNASeq. Using Nanostring, we identified arginine and IL-10 as important contributors to the lethal outcome of burned mice infected with P. aeruginosa. While other examples of altered gene transcription are in the literature, our study suggests that a more systematic comparison of gene expression in various underlying diseases during infection with specific bacterial pathogens may lead to the identification of unique host-pathogen interactions and result in more precise therapeutic interventions.

Genome sequencing of Pseudomonas aeruginosa strain M2 illuminates traits of an opportunistic pathogen of burn wounds.

Pseudomonas aeruginosa is a Gram-negative nosocomial pathogen and one of the most prevalent organisms isolated from burn wounds worldwide. Pseudomonas aeruginosa strain M2 (O5 serotype, type B flagella) is utilized for examining the murine model associated with burns. Pseudomonas aeruginosa M2 is similar in lethality to common laboratory P. aeruginosa strains when infecting CD-1 mice. Conversely, we recently showed that, relative to these strains, P. aeruginosa M2-infected mice are more susceptible to sepsis and demonstrate a 6-log reduction in LD50 from subcutaneous infection at the infection site directly after 10% total body surface area burn. To better understand this striking phenotypic difference from other P. aeruginosa strains employed in burn models, we sequenced the P. aeruginosa M2 genome. A total of 4,136,641 read pairs were obtained, providing an average genome coverage of 97.5X; subsequent assembly yielded a draft genome with 187 contigs comprising 6,360,304 bp with a G + C content of 66.45%. Genome-based phylogeny estimation of 92 P. aeruginosa strains placed P. aeruginosa M2 with P. aeruginosa-12-4-4(59), a nonairway clinical strain isolated from the blood culture of a burn patient. Phylogenomic analyses identified genes shared between P. aeruginosa M2 and P. aeruginosa 14, another strain exhibiting increased lethality in thermal tissues, as well as P. aeruginosa M2 unique genes with diverse functions like degradation of toxic aromatic compounds, iron scavenging, swarming motility and biofilm formation, defense against invasive DNA, and host assault. Predicted lateral gene transfers illuminate proteins heretofore uncharacterized for roles in P. aeruginosa biology. Our work yields a rich resource for assessing P. aeruginosa genes required for increased lethality in burn tissue seroma.

A Nonlethal Full-Thickness Flame Burn Produces a Seroma Beneath the Forming Eschar, Thereby Promoting Pseudomonas aeruginosa Sepsis in Mice.

The World Health Organization estimates ~180,000 deaths occur annually from burn-related injuries. Many victims who survive the initial burn trauma succumb to bacterial infections that lead to sepsis during treatment. Although advancements in burn care continue to improve in high-income countries due to their burn centers and advanced research, low and middle-income countries continue to see high frequencies of burn injuries and burn-related deaths due to secondary infections. Bacterial-derived sepsis is the most life-threatening danger for people that survive burn injuries. Here we provide evidence for the first time that a subeschar seroma forms postburn even in the absence of infection in mice. The seroma fills with a volume estimated at 500 µL of fluid, 25% of the blood supply, free of red blood cells. The seroma fluid supports robust Pseudomonas aeruginosa (PA) growth and contains inflammatory cytokines and chemokines, which recruit immature neutrophils and monocytes to the seroma in the absence of endothelial breakdown. These immune cells fail to contain PA expansion and dissemination. This recruitment of monocytes and immature neutrophils may result in sequestering these critical immune cells away from other tissues during a pivotal time during bacterial dissemination, promoting PA-mediated sepsis.

A Nonlethal Murine Flame Burn Model Leads to a Transient Reduction in Host Defenses and Enhanced Susceptibility to Lethal Pseudomonas aeruginosa Infection.

Of the 486,000 burn injuries that required medical treatment in the United States in 2016, 40,000 people were hospitalized, with >3,000 fatalities. After burn injury, humans are at increased risk of sepsis and mortality from infections caused by Pseudomonas aeruginosa, an opportunistic pathogen. We hypothesize that systemic events were initiated from the burn that increased the host's susceptibility to P. aeruginosa. A nonlethal 10% total body surface area (TBSA), full-thickness flame burn was performed in CD-1 mice without and with subsequent P. aeruginosa (strain M2) infection. The 50% lethal dose for subcutaneous infection with P. aeruginosa M2 at the burn site immediately after the burn decreased by 6 log, with mortality occurring between 18 and 26 h, compared with P. aeruginosa-infected mice without burn injury. Bacteria in distal organs were detected by 18 h, concurrent with the onset of clinical symptoms. Serum proinflammatory cytokines (interleukin-6 [IL-6], IL-1ß, gamma interferon, and tumor necrosis factor alpha) and the anti-inflammatory cytokine IL-10 were first detected at 12 h postburn with infection and continued to increase until death. Directly after burn alone, serum levels of HMGB1, a danger-associated molecular pattern and TLR4 agonist, transiently increased to 50 ng/ml before returning to 20 ng/ml. Burn with P. aeruginosa infection increased serum HMGB1 concentrations >10-fold (250 ng/ml) at the time of death. This HMGB1-rich serum stimulated TLR4-mediated NF-κB activation in a TLR4 reporter cell line. Treatment of infected burned mice with P5779, a peptide inhibitor of HMGB1, increased the mean survival from 23 to 42 h (P < 0.0001). We conclude that the high level of serum HMGB1, which preceded the increase in proinflammatory cytokines, is associated with postburn mortality.

A Cross-sectional Analysis of Insurance Coverage of Extremity Contouring After Massive Weight Loss.

INTRODUCTION: After bariatric surgery, patients often experience redundant skin in the upper arms and medial thighs as sequelae of massive weight loss. Insurance companies have unpredictable criteria to determine the medical necessity of brachioplasty and thighplasty, which are often ascribed as cosmetic procedures. We evaluated current insurance coverage and characterized policy criteria for extremity contouring in the postbariatric population. METHODS: We conducted a cross-sectional analysis of insurance policies for coverage of brachioplasty and thighplasty in January 2019. Insurance companies were selected based on their state enrolment data and market share. A web-based search and direct calls were conducted to identify policies. A comprehensive list of standard criteria was compiled based on the policies that offered coverage. RESULTS: Of the 56 insurance companies assessed, half did not provide coverage for either procedure (n = 28). No single criterion featured universally across brachioplasty and thighplasty policies. Functional impairment was the most commonly cited condition for preapproval of brachioplasty and/or thighplasty (94%). Conversely, minimum weight loss was the least frequent criterion within the insurance policies (6%). Only 5% of the insurance companies (n = 3) would consider coverage of liposuction-assisted lipectomy as a modality for brachioplasty or thighplasty. CONCLUSIONS: We propose a comprehensive list of reporting recommendations to help optimize authorization of extremity contouring in the postbariatric population. There is great intercompany variation in preapproval criteria for brachioplasty and thighplasty, illustrating an absence of established recommendations or guidelines. High-level evidence and investigations are needed to ascertain validity of the limited coverage criteria in current use.

A Review of National Insurance Coverage of Post-bariatric Upper Body Lift.

INTRODUCTION: Recent years have seen an increased utilisation of upper body lift following massive weight loss. Although it is typically considered cosmetic, the recurrent skin conditions and decline in quality of life may warrant medical necessity. We evaluated current insurance coverage and characterised policy criteria for upper body lift in the post-bariatric population. METHODS: We defined upper body lift as a combination of mastopexy and upper back excision (UBE) and conducted a cross-sectional analysis of US insurance policies. Insurance companies were selected based on their enrolment data and market share. A web-based search and telephone interviews were conducted to identify the policy. Criteria were abstracted from the publicly available policies that offered coverage. RESULTS: Of the 56 insurance companies assessed, 5% would consider coverage of both procedures. Although fewer companies held established policies for UBE than mastopexy in the post-bariatric population (79% vs 96%, p = 0.0081), there were significantly more policies that offered pre-approval for UBE than for mastopexy (30% vs 5%, p = 0.0017). Three medical necessity criteria were common to both procedures: evidence of functional impairment, secondary skin conditions, and medical photographs. CONCLUSION: Policy criteria for coverage of mastopexy or UBE differ greatly between companies. Further evaluation of medical necessity criteria for post-bariatric mastopexy and UBE with the establishment of a standardised guideline is needed. We propose a comprehensive list of reporting recommendations to help optimise authorisation of upper body lift in the post-bariatric population, and we urge plastic surgeons to challenge current definition of "cosmetic" by insurance companies. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .