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

Abstract

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.

Clinical investigation of biofilm in non-healing wounds by high resolution microscopy techniques

“…17,31,32 This product is recommended for use on highly exudating wounds because it is purported to absorb exudate. However, in this clinical setting it did not demonstrate length of absorptive or antibiofilm action within a routine of daily dressing changes.…”

“…This supports what is understood about the protective properties of the EPS matrix to antimicrobial chemotherapeutic onslaught. 31,32 Interestingly, one case revealed a putative yeast biofilm in a sample collected from a CNHW on a patient with history of recurrent antibiotics.…”

Combinatorial Effect of D-Aminoacids and Tetracycline Against Pseudomonas Aeruginosa Biofilm

“…Porcine skin model for bacterial biofilm attachment was selected because it fulfills the requirements of an ex vivo model due to its similarity with human skin physiology. The other advantage of using such a model is that a large number of trials can be conducted economically [23]. Our study clearly elucidated that a single application of 5 mmol D-tryptophan-0.5 MIC tetracycline combination was capable of reducing P. aeruginosa biofilm attachment to porcine skin explants.…”

“…Ex-vivo porcine skin model was prepared according to the protocol described earlier [21,23]. Hides from carcasses of freshly killed adult pigs obtained from a local slaughter house were thoroughly washed and de-haired.…”

Biofilms and Wounds: An Identification Algorithm and Potential Treatment Options

“…18 However, as previously mentioned biofilms are not confluent on a surface, it is highly probable that they will not be recovered or identified in biopsies of many chronic wounds. Furthermore, many microbes in biofilms reside in a viable, but nonculturable state, impairing their recovery.…”