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Page 4 of 11 Matiasek et al. Plast Aesthet Res 2018;5:36 I http://dx.doi.org/10.20517/2347-9264.2018.50

gluteal/ischial or trochanteric, respectively). Whereas the first 3 patients were assessed in 2014, another 10
patients were treated between 2015 and 2017 in a multicentric approach.

METHODS
Sampling of wound exudates
To simulate realistic wound conditions, wound exudates from patients with leg ulcers, treated with
conventional NPWT, were obtained. This occurred when the foam dressings in the wounds were replaced.
At this point they were transferred into sterile containers, then soaked with 5 mL of a protease inhibitor
solution (Complete Protease Inhibitor Cocktail Tablets, Roche, Germany). To collect wound exudates, the
dressings were cut into appropriate pieces using sterile scissors and then squeezed through a sterile press. The
wound exudates were collected and captured in 50 mL centrifuge tubes and stored at -20 °C until processing
continued or at 6 °C when processed on the same day as the exudates were collected.

Microbiological activity test methods
Comparative data was obtained by also testing Serasept® (containing polyhexamethylene biguanide 0.04%;
Serag-Wiessner GmbH & Co. KG, Germany) and Prontosan® (containing polyhexamethylene biguanide 0.1%;
Braun Melsungen AG, Germany) rinsing solutions. A negative control was provided using saline solution.

Quantitative suspension tests were performed according to the dilution-neutralisation method described in
DIN prEN 13727:2009. The tests conformed to the conditions for low organic load (0.3 g/L of bovine serum
albumin) and for high organic load (0.3 g/L of bovine serum albumin and 3.0 mol/L of ovine erythrocytes).
This method was fine tuned depending on the volume of the extracted wound exudates. One part of load
solution was mixed with one part of bacterial suspension. After 2 min, 8 parts of the negative control (0.9%
saline solution) or one of the following rinsing solutions - octenilin® Wound Irrigation Solution (containing
octenidine dihydrochloride 0.05%), Serasept® 2 or Prontosan® were added and the test mixture was completely
blended using a vortex mixer. At the predetermined exposure times - 0.5 and 2 min - the solution was stirred
again and 1 part was added to 9 parts of neutralisation solution. After the neutralisation time was complete, a
serial dilution up to 10-2 was prepared and the appropriate amount of solution was placed on agar plates.

Bacterial count from exudates
The wound exudates were diluted in a sterile dilution series with tryptone sodium chloride (Tryptone-NaCl)
and plated on Casein Soy Peptone Agar (CSA) according to the dilution levels. All visible colony forming units
(CFU) were counted, regardless of their species. Additionally, the exudate samples were spiked with the test
organism Staphylococcus aureus ATCC 33592 (MRSA strain) to further increase the microbiological load.
Storage and cultivation of the test organism was conducted in accordance with legal regulations (prDIN EN
12353:2011).

The agar plate count and the analysis were conducted in accordance with the legal regulation DIN prEN
13727:2009. The logarithmic germ reduction factor (lg RF) was calculated by applying the following formula:
lg RF = lg N -lg N 2
0
lg RF reduction of live cell count after exposure time t, given as decadic logarithm
lg N live cell count per ml in sample solution at the beginning of exposure time t
0
lg N live cell count per ml in sample solution at the end of exposure time t
2
According to the requirements given in DIN prEN 13727:2009, antiseptic agents must reduce the live cell count
by five orders of magnitude. This reduction is considered to be a sufficient antibacterial effect.

Patients
Initially 3 patients with category 4 gluteal PUs were treated with NPWTi in combination with octenilin®