Priscilla Phillips

Antimicrobial dressing efficacy against mature Pseudomonas aeruginosa biofilm on porcine skin explants.

An ex vivo porcine skin explant biofilm model that preserves key properties of biofilm attached to skin at different levels of maturity (0–3 days) was used to assess the efficacy of commercially available antimicrobial dressings and topical treatments. Assays were also performed on the subpopulation of antibiotic tolerant biofilm generated by 24 hours of pre‐treatment with gentamicin (120× minimal inhibitory concentration) prior to agent exposure. Five types of antimicrobial agents (iodine, silver, polyhexamethylene biguanide, honey and ethanol) and four types of moisture dressings (cotton gauze, sodium carboxymethylcellulose fibre, calcium alginate fibre and cadexomer beads) were assessed. Time‐release silver gel and cadexomer iodine dressings were the most effective in reducing mature biofilm [between 5 and 7 logarithmic (log) of 7‐log total], whereas all other dressing formulations reduced biofilm between 0·3 and 2 log in 24 or 72 hours with a single exposure. Similar results were found after 24‐hour exposure to silver release dressings using an in vivo pig burn wound model, demonstrating correlation between the ex vivo and in vivo models. Results of this study indicate that commonly used microbicidal wound dressings vary widely in their ability to kill mature biofilm and the efficacy is influenced by time of exposure, number of applications, moisture level and agent formulation (sustained release).

The effect of negative pressure wound therapy with periodic instillation using antimicrobial solutions on Pseudomonas aeruginosa biofilm on porcine skin explants

Negative pressure wound therapy with instillation (NPWTi) is increasingly used as an adjunct therapy for a wide variety of infected wounds. However, the effect of NPWTi on mature biofilm in wounds has not been determined. This study assessed the effects of NPWTi using saline or various antimicrobial solutions on mature Pseudomonas aeruginosa biofilm using an ex vivo porcine skin explant biofilm model. Treatment consisted of six cycles with 10‐minute exposure to instillation solution followed by 4 hours of negative pressure at −125 mm Hg over a 24‐hour period. NPWTi using saline reduced bacterial levels by 1‐log (logarithmic) of 7‐log total colony‐forming units (CFUs). In contrast, instillation of 1% povidone iodine (2‐log), L‐solution (3‐log), 0·05% chlorhexidine gluconate (3‐log), 0·1% polyhexamethylene biguanide (4‐log), 0·2% polydiallyldimethylammonium chloride (4‐log) and 10% povidone iodine (5‐log), all significantly reduced (P < 0·001) total CFUs. Scanning electron micrographs showed disrupted exopolymeric matrix of biofilms and damaged bacterial cells that correlated with CFU levels. Compared with previous studies assessing microbicidal effects of topical antimicrobial dressings on biofilms cultured on porcine skin explants, these ex vivo model data suggest that NPWTi with delivery of active antimicrobial agents enhances the reduction of CFUs by increasing destruction and removal of biofilm bacteria. These results must be confirmed in human studies.

Development of a novel ex vivo porcine skin explant model for the assessment of mature bacterial biofilms

Bacterial biofilms have been proposed to be a major factor contributing to the failure of chronic wounds to heal because of their increased tolerance to antimicrobial agents and the prolonged inflammation they cause. Phenotypic characteristics of bacterial biofilms vary depending on the substratum to which they attach, the nutritional environment, and the microorganisms within the biofilm community. To develop an ex vivo biofilm model that more closely mimics biofilms in chronic skin wounds, we developed an optimal procedure to grow mature biofilms on a central partial‐thickness wound in 12‐mm porcine skin explants. Chlorine gas produced optimal sterilization of explants while preserving histological properties of the epidermis and dermis. Pseudomonas aeruginosa and Staphylococcus aureus developed mature biofilms after 3 days that had dramatically increased tolerance to gentamicin and oxacillin (∼100× and 8,000× minimal inhibitory concentration, respectively) and to sodium hypochlorite (0.6% active chlorine). Scanning electron microscopy and confocal microscopy verified extensive exopolymeric biofilm structures on the explants. Despite a significant delay, a ΔlasI quorum‐sensing mutant of P. aeruginosa developed biofilm as antibiotic‐tolerant as wild‐type after 3 days. This ex vivo model simulates growth of biofilms on skin wounds and provides an accurate model to assess effects of antimicrobial agents on mature biofilms.

Chronic Wounds, Biofilms and Use of Medicinal Larvae

Chronic wounds are a significant health problem in the United States, with annual associated costs exceeding

Expression of Porphyromonas gingivalis small RNA in response to hemin availability identified using microarray and RNA-seq analysis

20 billion annually. Traditional wound care consists of surgical debridement, manual irrigation, moisture retentive dressings, and topical and/or systemic antimicrobial therapy. However, despite progress in the science of wound healing, the prevalence and incidence of chronic wounds and their complications are escalating. The presence & complexity of bacterial biofilms in chronic wounds has recently been recognized as a key aspect of non-healing wounds. Bacterial biofilms are sessile colonies of polymicrobial organisms (bacteria, fungus, etc.) enclosed within a self-produced exopolymeric matrix that provides high levels of tolerance to host defenses, antibiotics and antiseptics. Thus, there is a need for alternative therapies to reduce biofilms in chronic wounds. In this report, we present initial findings from in vitro experiments which show that larval debridement therapy with disinfected blow fly larvae (Phaenicia sericata) reduced total CFUs (6-logs) of planktonic and mature biofilms of Pseudomonas aeruginosa or Staphylococcus aureus grown on dermal pig skin explants by 5-logs after 24 hours of exposure, and eliminated biofilms (no measurable CFUs) after 48 hours of exposure.

Porphyromonas gingivalis and adverse pregnancy outcome

There is a significant body of work suggesting that sRNA-mediated post-transcriptional regulation is a conserved mechanism among pathogenic bacteria to modulate bacterial virulence and survival. Porphyromonas gingivalis is recognized as an etiological agent of periodontitis and implicated in contributing to the development of multiple inflammatory diseases including cardiovascular disease. Using NimbleGen microarray analysis and a strand-specific method to sequence cDNA libraries of small RNA-enriched P. gingivalis transcripts using Illuminas high-throughput sequencing technology, we identified putative sRNA and generated sRNA expression profiles in response to growth phase, hemin availability after hemin starvation, or both. We identified transcripts that mapped to intergenic sequences as well as antisense transcripts that mapped to open reading frames of the annotated genome. Overall, this approach provided a comprehensive way to survey transcriptional activity to discover functionally linked RNA transcripts, responding to specific environmental cues, that merit further investigation.

Biofilm detection by wound blotting can predict slough development in pressure ulcers: A prospective observational study.

ABSTRACT Porphyromonas gingivalis is a Gram-negative, anaerobic bacterium considered to be an important pathogen of periodontal disease that is also implicated in adverse pregnancy outcome (APO). Until recently, our understanding of the role of P. gingivalis in APO has been limited and sometimes contradictory. The purpose of this review is to provide an overview of past and current research on P. gingivalis that addresses some of the controversies concerning the role of this organism in the pathogenesis of APO.

Porphyromonas gingivalis strain-dependent inhibition of uterine spiral artery remodeling in the pregnant rat†

Bacteria have been found to form multicellular aggregates which have collectively been termed “biofilms.” It is hypothesized that biofilm formation is a means to protect bacterial cells including protection form the immune response of humans. This protective mechanism is believed to explain persistent chronic wound infections. At times, the biofilms are abundant enough to see, and remove by simple wiping. However, recent evidence has shown that the removal of these visible portions are not sufficient, and that biofilms can continue to form even with daily wiping. In this work, we tested an approach to detect the biofilms which are present after clinically wiping or sharp wound debridement. Our method is based on a variation of impression cytology in which a nitrocellulose membrane was used to collect surface biofilm components, which were then differentially stained. In this prospective study, members of an interdisciplinary pressure ulcer team at a university hospital tested our methods ability to predict the generation of wound slough in the week that followed each blotting. A total of 70 blots collected from 23 pressure ulcers produced 27 wounds negative for staining and 43 positive. In the negative blots 55.6% were found to have decreased wound slough, while 81.4% with positive staining had either increase or unchanged wound slough generation. These results lead to an odds ratio of positive blotting cases of 9.37 (95% confidence intervals: 2.47–35.5, p = 0.001) for slough formation; suggesting that the changes in wound slough formation can be predicted clinically using a non‐invasive wound blotting method.

Biofilm maturity studies indicate sharp debridement opens a time-dependent therapeutic window

Abstract Porphyromonas gingivalis (Pg) is an important periodontal pathogen that is also implicated in pregnancy complications involving defective deep placentation (DDP). We hypothesized that Pg invasion of the placental bed promotes DDP. Pregnant rats were intravenously inoculated with sterile vehicle, Pg strain W83, or A7436 at gestation day (GD) 14 (acute cohort). Nonpregnant rats received repeated oral inoculations for 3 months before breeding (chronic cohort). Tissues and/or sera were collected at GD18 for analysis. Pg infection status was determined by seroconversion (chronic cohort) and by presence of Pg antigen in utero-placental tissues processed for histology and morphometric assessment of spiral artery remodeling. Mesometrial tissues from seropositive dams were analyzed for expression of interleukin 1β, 6, and 10, TNF, TGF-β, follistatin-related protein 3, and inhibin beta A chain since these genes regulate extravillous trophoblast invasion. The in situ distribution of W83 and A7436 antigen in utero-placental tissues was similar in both cohorts. In the acute cohort, mesometrial stromal necrosis was more common with W83, but arteritiswas more common with A7436 infection (P < 0.05). Increased vascular necrosiswas seen in mesometrium of chronically infected groups (P < 0.05). Only A7436-infected animals had increased fetal deaths, reduced spiral artery remodeling, reduced inhibin beta A expression, and an increased proportion of FSLT3 positive extravillous trophoblasts within spiral arteries. While infection with both Pg strains produced varying pathology of the deep placental bed, only infection with strain A7436 resulted in impaired spiral artery remodeling. Summary Sentence Invasion of the placental bed by Porphyromonas gingivalis impairs spiral artery remodeling and increases fetal loss in a microbial strain-dependent manner.