Development of Pseudomonas aeruginosa Biofilms in Partial-Thickness Burn Wounds Using a Sprague-Dawley Rat Model

Karna

et al. 2019

Sci Rep

Self Cite

Using Sprague-Dawley rats (350–450 g; n = 61) and the recently updated Walker-Mason rat scald burn model, we demonstrated that Pseudomonas aeruginosa readily formed biofilms within full-thickness burn wounds. Following the burn, wounds were surface-inoculated with P. aeruginosa in phosphate-buffered saline (PBS), while sterile PBS was used for controls. On post-burn days 1, 3, 7, and 11, animals were euthanized and samples collected for quantitative bacteriology, bacterial gene expression, complete blood cell counts, histology, and myeloperoxidase activity. Robust biofilm infections developed in the full-thickness burn wounds inoculated with 1 × 104 CFU of P. aeruginosa. Both histology and scanning electron microscopy showed the pathogen throughout the histologic cross-sections of burned skin. Quantigene analysis revealed significant upregulation of alginate and pellicle biofilm matrix genes of P. aeruginosa within the burn eschar. Additionally, expression of P. aeruginosa proteases and siderophores increased significantly in the burn wound environment. Interestingly, the host’s neutrophil response to the pathogen was not elevated in either the eschar or circulating blood when compared to the control burn. This new full-thickness burn biofilm infection model will be used to test new anti-biofilm therapies that may be deployed with soldiers in combat for immediate use at the site of burn injury on the battlefield.

Section: Introductionmentioning

confidence: 99%

Formation of Pseudomonas aeruginosa Biofilms in Full-thickness Scald Burn Wounds in Rats

Karna

et al. 2019

Sci Rep

Self Cite

“…While PNA-FISH staining techniques for tissue have been utilized by many groups in the research field, it is not used extensively in the clinical community. 25,30–32 Limitations to this type of sampling include burn wound integrity, as well as slower turnaround times (about 1–2 days), which are both mitigated by the swab technique. However, if it is possible to collect an intact sample, it may provide further insights into not only the types of pathogens present, but also the depth (ie, tissue penetration) and degree of the infection.…”

Section: Discussionmentioning

confidence: 99%

“…In vivo samples were collected from infected burn wounds of male Sprague-Dawley ( n = 3) rats using a previously described model of deep-partial thickness burn infection that was modified to investigate a tri-species infection. 25 In brief, a 10% total body surface area burn (based on Meeh’s formula) 26 was induced on the dorsum of anesthetized rats, followed by immediate surface inoculation of 10 5 CFU/wound of each microorganism ( S. aureus , P. aeruginosa , and C. albicans ; grown to mid-log state) suspended in 1× PBS. On day 3 post-injury, the rats were euthanized and the wound was excised and subjected to biopsy punches, tissue sectioning (histology), and complete swabbing of the entire remaining wound bed as part of sample collection approach.…”

Section: Methodsmentioning

confidence: 99%

Clinical Utility of PNA-FISH for Burn Wound Diagnostics: A Noninvasive, Culture-Independent Technique for Rapid Identification of Pathogenic Organisms in Burn Wounds

Journal of Burn Care &Amp; Research

Sanjar

et al. 2019

Self Cite

Burn injury results in an immediate compromised skin state, which puts the affected patient at an immediate risk for infection, including sepsis. For burn patients that develop infections, it is critical to rapidly identify the etiology so that an appropriate treatment can be administered. Current clinical standards rely heavily on culture-based methods for local and systemic infection testing, which can often take days to complete. While more advanced methods (ie, MALDI or NAAT) have improved turnaround times, they may still suffer from either the need for pure culture or sensitivity and specificity issues. Peptide nucleic acid fluorescent in situ hybridization (PNA-FISH) offers a way to reduce this time from days to hours and provide species-specific identification. While PNA-FISH has had great utility in research, its use in clinical microbiology diagnostics has been minimal (including burn wound diagnostics). This work describes a nonculture-based identification technique using commercial available U.S. FDA-approved PNA-FISH probes for the identification of common clinical pathogens, Pseudomonas aeruginosa and Staphylococcus aureus , present in burn wound infections. Additionally, calcofluor white was included for identification of Candida albicans . All three pathogens were identified from a tri-species infected deep-partial thickness rat burn wound model. These species were clearly identifiable in swab and tissue samples that were collected, with minimal autofluorescence from any species. Although autofluorescence of the tissue was present, it did not interfere or was otherwise minimized through sample preparation and analysis. The methodology developed was done so with patient care and diagnostic laboratories in mind that it might be easily transferred to the clinical setting.

“…A DPT or FT scald burn was inflicted on the dorsum of anesthetized rats by methods previously described (Brandenburg et al, 2019b). In brief, the day preceding the burn, male Sprague-Dawley rats weighing 350-450 g were anesthetized with isoflurane (Forane, Baxter Healthcare Corporation, Deerfield, IL, USA) prior to the dorsum being shaved and depilated with Nair (Church & Dwight Co., Inc., Ewing, NJ, USA).…”

Section: Overview Of Scald Burn Modelmentioning

confidence: 99%

“…Following inoculation, wounds were covered with a non-adherent interface dressing, followed by Tegaderm TM Film (3M Health Care, St. Paul, MN). Rats were placed in custom rat jackets (Brandenburg et al, 2019b) to prevent tampering with the wound throughout the study, and monitored over an 11 days period.…”

Section: Overview Of Scald Burn Modelmentioning

confidence: 99%

Comparative Analysis of the Host Response in a Rat Model of Deep-Partial and Full-Thickness Burn Wounds With Pseudomonas aeruginosa Infection

Front. Cell. Infect. Microbiol.

Smith

et al. 2020

Burn wound injury affects soldiers and civilians alike, often resulting in a dynamic, but un-orchestrated, host response that can lead to infection, scarring, and potentially death. To mitigate these factors, it is important to have a clinically relevant model of burn wound infection that can be utilized for advancing burn wound treatments. Our previous reports have demonstrated the ability of Pseudomonas aeruginosa to generate a biofilm infection within a modified Walker-Mason rat burn model of deep-partial (DPT) and full-thickness (FT) burn wounds (10% total body surface area) in male Sprague-Dawley rats (350-450 g). Here, we further define this model with respect to the host response when challenged with P. aeruginosa infection between the two burn types. Following burn injury and immediate surface exposure to P. aeruginosa, inflammation at the local and systemic levels were monitored for an 11 days period. Compared to burn-only groups, infection with P. aeruginosa further promoted local inflammation in both DPT and FT burn wounds, which was evident by enhanced cellular influx (including neutrophils and monocytes), increased levels of several pro-inflammatory cytokines (IL-1β, IL-6, GRO/KC, andMIP-1α), and reduced IL-10. Systemically, only minor changes were seen in circulating white blood cells and cytokines; however, increases in high mobility group box-1 (HMGB-1) and hyaluronan, as well as decreases in fibronectin were noted particularly in FT burns. Compared to the burn-only group, P. aeruginosa infection resulted in sustained and/or higher levels of HMGB-1 and hyaluronan. Combined with our previous work that defined the burn depth and development of P. aeruginosa biofilms within the wound, this study further establishes this model by defining the host response to the burn and biofilm-infection. Furthermore, this characterization shows several similarities to what is clinically seen and establishes this model for future use in the development and testing of novel therapeutics for burn wound treatment at home and on the battlefield.