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Comparative analysis of single-species and polybacterial wound biofilms using a quantitative, in vivo, rabbit ear model.

ABSTRACT: INTRODUCTION:The recent literature suggests that chronic wound biofilms often consist of multiple bacterial species. However, without appropriate in vivo, polybacterial biofilm models, our understanding of these complex infections remains limited. We evaluate and compare the effect of single- and mixed-species biofilm infections on host wound healing dynamics using a quantitative, in vivo, rabbit ear model. METHODS:Six-mm dermal punch wounds in New Zealand rabbit ears were inoculated with Staphylococcus aureus strain UAMS-1, Pseudomonas aeruginosa strain PAO1, or both, totaling 10/6 colony-forming units/wound. Bacterial proliferation and maintenance in vivo were done using procedures from our previously published model. Wounds were harvested for histological measurement of wound healing, viable bacterial counts using selective media, or inflammatory cytokine (IL-1?, TNF-?) expression via quantitative reverse-transcription PCR. Biofilm structure was studied using scanning electron microscopy (SEM). For comparison, biofilm deficient mutant UAMS-929 replaced strain UAMS-1 in some mixed-species infections. RESULTS:Bacterial counts verified the presence of both strains UAMS-1 and PAO1 in polybacterial wounds. Over time, strain PAO1 became predominant (p<0.001). SEM showed colocalization of both species within an extracellular matrix at multiple time-points. Compared to each monospecies infection, polybacterial biofilms impaired all wound healing parameters (p<0.01), and increased expression of IL-1? and TNF-? (p<0.05). In contrast, mixed-species infections using biofilm-deficient mutant UAMS-929 instead of wild-type strain UAMS-1 showed less wound impairment (p<0.01) with decreased host cytokine expression (p<0.01), despite a bacterial burden and distribution comparable to that of mixed-wild-type wounds. CONCLUSIONS:This study reveals that mixed-species biofilms have a greater impact on wound healing dynamics than their monospecies counterparts. The increased virulence of polybacterial biofilm appears dependent on the combined pathogenicity of each species, verified using a mutant strain. These data suggest that individual bacterial species can interact synergistically within a single biofilm structure.

SUBMITTER: Seth AK

PROVIDER: S-EPMC3414496 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Comparative analysis of single-species and polybacterial wound biofilms using a quantitative, in vivo, rabbit ear model.

Seth Akhil K AK Geringer Matthew R MR Hong Seok J SJ Leung Kai P KP Galiano Robert D RD Mustoe Thomas A TA

PloS one 20120808 8

<h4>Introduction</h4>The recent literature suggests that chronic wound biofilms often consist of multiple bacterial species. However, without appropriate in vivo, polybacterial biofilm models, our understanding of these complex infections remains limited. We evaluate and compare the effect of single- and mixed-species biofilm infections on host wound healing dynamics using a quantitative, in vivo, rabbit ear model.<h4>Methods</h4>Six-mm dermal punch wounds in New Zealand rabbit ears were inocula ...[more]

PMID: 22905182

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Project description:BackgroundBacterial infections of wounds impair healing and worsen scarring. We hypothesized that transcriptome analysis of wounds infected with Klebsiella pneumoniae (K.p.) or Pseudomonas aeruginosa (P.a.) would indicate host-responses associated with the worse healing of P.a.- than K.p.-infected wounds.MethodsWounds created on post-operative day (POD) 0 were infected during the inflammatory phase of healing on POD3 and were harvested on POD4 for microarray and transcriptome analysis. Other wounds received topical antibiotic after infection for 24 hours to promote biofilm development, and were harvested on POD6 or POD12.ResultsWounds infected for 24 hours, relative to uninfected wounds, elevated transcripts of immune-response functions characteristic of infiltrating leukocytes. But P.a.-infected wounds elevated many more transcripts and to higher levels than K.p.-infected wounds. Coincidently, suppressed transcripts of both wounds enriched into stress-response pathways, including EIF2 signaling; however, this was more extensive for P.a.-infected wounds, including many-fold more transcripts enriching in the 'cell death' annotation, suggesting resident cutaneous cell toxicity in response to a more damaging P.a. inflammatory milieu. The POD6 wounds were colonized with biofilm but expressed magnitudes fewer immune-response transcripts with no stress-response enrichments. However, elevated transcripts of P.a.-infected wounds were inferred to be regulated by type I interferons, similar to a network unique to P.a.-infected wounds on POD4. On POD12, transcripts that were more elevated in K.p.-infected wounds suggested healing, while transcripts more elevated in P.a.-infected wounds indicated inflammation.ConclusionsAn extensive inflammatory response of wounds was evident from upregulated transcripts 24 hours after infection with either bacterium, but the response was more intense for P.a.- than K.p.-infected wounds. Coincidently, more extensive down-regulated transcripts of P.a.-infected wounds indicated a stronger "integrated stress response" to the inflammatory milieu that tipped more toward cutaneous cell death. Unique to P.a.-infected wounds on POD4 and POD6 were networks inferred to be regulated by interferons, which may result from intracellular replication of P.a. These data point to specific downregulated transcripts of cells resident to the wound as well as upregulated transcripts characteristic of infiltrating leukocytes that could be useful markers of poorly healing wounds and indicators of wound-specific treatments for improving outcomes.

Project description:ImportanceThe prominent ear is a common external ear anomaly that is usually corrected through surgery. Electromechanical reshaping (EMR) may provide the means to reshape cartilage through the use of direct current (in milliamperes) applied percutaneously with needle electrodes and thus to reduce reliance on open surgery.ObjectiveTo determine the long-term outcomes (shape change, cell viability, and histology) of a more refined EMR voltage and time settings for reshaping rabbit auricle.Design, setting, and subjectsThe intact ears of 14 New Zealand white rabbits were divided into 2 groups. Group 1 received 4 V for 5 minutes (5 ears), 5 V for 4 minutes (5 ears), or no voltage for 5 minutes (control; 4 ears). Group 2 received an adjusted treatment of 4 V for 4 minutes (7 ears) or 5 V for 3 minutes (7 ears). A custom mold with platinum electrodes was used to bend the pinna and to perform EMR. Pinnae were splinted for 6 months along the region of the bend. Rabbits were killed humanely and the ears were harvested the day after splint removal. Data were collected from March 14, 2013, to July 8, 2014, and analyzed from August 29, 2013, to March 1, 2015.Main outcomes and measuresBend angle and mechanical behavior via palpation were recorded through photography and videography. Tissue was sectioned for histologic examination and confocal microscopy to assess changes to microscopic structure and cell viability.ResultsRabbits ranged in age from 6 to 8 months and weighed 3.8 to 4.0 g. The mean (SD) bend angles were 81° (45°) for the controls and, in the 5 EMR groups, 72° (29°) for 4 V for 4 minutes, 101° (19°) for 4 V for 5 minutes, 78° (18°) for 5 V for 3 minutes, and 126° (21°) for 5 V for 4 minutes. At 5 V, an increase in application time from 3 to 4 minutes provided significant shape change (78° [18°] and 126° [21°], respectively; P = .003). Pinnae stained with hematoxylin-eosin displayed localized areas of cell injury and fibrosis in and around electrode insertion sites. This circumferential zone of injury (range, 1.3-2.1 mm) corresponded to absence of red florescence on the cell viability assay.Conclusions and relevanceIn this in vivo study, EMR produces shape changes in the intact pinnae of rabbits. A short application of 4 V or 5 V can achieve adequate reshaping of the pinnae. Tissue injury around the electrodes is modest in spatial distribution. This study provides a more optimal set of EMR variables and a critical step toward evaluation of EMR in clinical trials.Level of evidenceNA.

Dataset Information

Comparative analysis of single-species and polybacterial wound biofilms using a quantitative, in vivo, rabbit ear model.

Publications

Comparative analysis of single-species and polybacterial wound biofilms using a quantitative, in vivo, rabbit ear model.

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