Cetylpyridinium-chloride (CPC) and Miramistin (MST) compared to established antiseptics under protein challenge in-vitro - evaluating alternative agents for wound cleansing CURRENT STATUS: POSTED

Background: Due to rising numbers microbial resistance to established antibiotics and first described tolerance developments for local wound antimicrobials a continuous need for alternative antimicrobial agents exists. Due to complex conditions in the microenvironment of especially chronic wounds, such as high protein levels, novel antimicrobials need to meet advanced requirements. Aim: Compare the antimicrobial efficacy of Cetylpiridinium-chloride (CPC) and miramistin (MST) to established antimicrobials under protein-challenge in-vitro. Methods: Antimicrobial activity of octenidin-dihydrochloride, povidon-iodine, polyhexamethylene-biguanide hydrochloride, chlorhexidine, cetylpiridinium-chloride and Miramistin after 0.5, 1, 3, 5 and 10 min of exposure against S. aureus, P. aeruginosa, E. coli, E. faecium and C. albicans was tested, using a quantitative suspension method with either 0.3% or 3% bovine albumin challenge, based on DIN EN 13727 (‘Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of bactericidal activity in the medical area - Test method and requirements (phase 2, step 1)’). Results: CPC and MST demonstrated no inferiority to the established agents in-vitro. Especially CPC showed equal reduction rates as octenidin and povidon-iodine and achieved significantly higher reduction rates within shorter exposure times than polyhexanide and chlorhexidine (p ≤ 0.01) for S. aureus, P. aeruginosa, E. faecium and C. albicans. Both agents demonstrated no significant loss of efficacy under high protein-challenge (3% albumin). Conclusion: In terms of antimicrobial activity cetylpyridinium-chloride and miramistin proved to be at least equally effective as established agents. No protein error was detected in the tested concentrations. More complex in-vitro assays and comprehensive in-vivo and clinical studies will be needed to determine their clinical value.

Surgical site infections (SSI) account for on average 20% of hospital-acquired infections in Europe. [3][4][5][6] Antimicrobial wound cleansing and local antisepsis are key factors in the treatment of acute and chronic wound infections. Octenidin-dihydrochloride (OCT), povidon-iodine (PVP-I), polyhexamethylene-biguanide hydrochloride (PHMB; polyhexanide) and chlorhexidine (CHX) represent the main antimicrobial agents used in wound management. So far no single antimicrobial has proven to be generally superior regarding treatment of local wound infection or promotion of wound healing. [7,8] [9] With adverse effects on essential cells for skin regeneration, such as fibroblasts and keratinocytes, reported for each agent [10,11], antibiotic resistance on the rise [12] and first reports of developing tolerance to local antiseptics [13,14], investigations into alternative antimicrobial agents with comparable efficacy and low cytotoxic impact to human skin cells are necessary.
Two potential alternatives are the quaternary ammonium compounds Cetylpyridinium-chloride (CPC) and Miramistin (MST), belonging to the family of cationic surface-active agents. Based on first promising results regarding antimicrobial efficacy and cytotoxicity to human cells in-vitro [15], this study aimed to compare MST and CPC to OCT, PVP-I, PHMB and CHX in challenging conditions simulating a high-protein wound environment, with short exposure times (as in clinical practice) against common wound pathogens (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecium and Candida albicans).
Cetylpyridinium-chloride (CPC; Sigma-Aldrich, Schnelldorf, Germany) and Miramistin (MST; Farmhim, Shostka, Ukraine) were purchased as powders and also prepared in distilled water to reach best suited final test concentrations of 0.5% (w/v; CPC) and 0.05% (w/v; MST) according to previous experiments [15]. An overview as well as specifications for the tested products/substances is provided in table 1.

Test organisms and nutrient solutions
In this work, Pseudomonas aeruginosa (DSM-939), Staphylococcus aureus (DSM-799), Escherichia coli (DSM-11250), Enterococcus faecium (DSM-2146) and Candida albicans (DSM-1386; all DSMZ, Braunschweig, Germany) were used as bacterial test strains. As nutrient solution sterile casein/soy peptone broth (CSB) was prepared consisting of 15 mg ml-1 casein peptone, 5 mg ml-1 soy peptone and 5 mg ml-1 sodium chloride diluted in distilled water. The pH value was adjusted to 7.2 using 5N sodium hydroxide (all AppliChem, Darmstadt, Germany). One fresh colony of each bacterial strain was added to 50 ml CSB and incubated over night at 37°C under aerobic conditions and agitation.
Bacterial test solutions were adjusted using a spectrophotometer to result in initial counts of ~ 1.5 -3.0 x 108 CFU ml-1. Fungal test solutions were prepared in the same manner using malt/soy peptone broth (MEB) and malt agar (MEA). Initial microbial CFU ml-1 counts were controlled by spreading serial dilutions of untreated microbial test solutions of each experiment onto agar plates allowing exact calculations of reduction rates.

Challenge and neutralisation solutions
To simulate a challenging wound environment and determine possible decrease in efficacy of the tested antiseptics especially under high-protein conditions, bovine albumin (Carl Roth, Karlsruhe, Germany) was added to the experimental setup (as recommended in the standard DIN EN 13727 by the german institute for standardization (DIN)). [16] Challenge solutions contained either 3 mg ml-1 (0.3%) or 30 mg ml-1 (3%) albumin and were prepared using an autoclaved casein/natriumchloride solution dissolved in distilled water. The challenge solution was subsequently aliquoted and stored at -18°C until usage.
To prevent further antimicrobial activity beyond sampling time points, a neutralisation solution was used in accordance with DIN EN 13727, comprising 3 g l-1 sodium thiosulfate, 30 g l-1 saponine, 30 g l-1 polysorbate 80 (Tween 80), 3 g l-1 lecithin, 1 g l-1 L-histidine and 1 g l-1 L-cysteine (all Carl Roth, Karlsruhe, Germany) diluted in distilled water and autoclaved for sterility. Both, challenge and neutralization solution, were individually validated regarding their purpose and showed no antimicrobial effects of its own.

Quantitative suspension method
The test method for evaluation of antimicrobial efficacy was based on DIN EN 13727 and slightly adjusted to fit the purpose. [16] Briefly, 1 ml microbial and 1 ml challenge solution (0.3% or 3% bovine albumin) were carefully mixed for two minutes and 8 ml of antimicrobial test solution was added. After 0.5 , 1, 3, 5 and 10 min of exposure 1 ml of the resulting solution was transferred into the prepared neutralisation solution (8 ml neutralizer and 1 ml distilled water) and continuously agitated. After 10 sec, 0.5 ml was again transferred into 4.5 ml neutralisation solution to thoroughly block further antimicrobial activity. Subsequently, this sample was serially tenfold diluted in either CSB (for bacteria) or MEB (for yeast), 25 l of every dilution step were seeded on either CSA or MEA plates and incubated at 37°C under aerobic conditions overnight. Surviving microorganisms (in CFU ml-1) were counted. Experiments were performed threefold at different times and in duplicates for each tested antiseptic and microorganism challenged with either 3 mg ml-1 or 30 mg ml-1 albumin solution.

Statistical analysis
Reduction rates were calculated for all tested antimicrobials (in Δlog10 CFU ml-1). For bacteria, a high antimicrobial efficacy (reducing at least 99.999% of initial bacterial counts) is indicated by a reduction of at least 5 log10 reduction steps within 1 min as specified in DIN EN 13727. [16] For yeast the cutoff for a high efficacy is considered at least 4 log10 reduction steps as specified in DIN EN 13624.
Mean values and SEM were calculated and differences considered statistically significant at p < 0.05.
Statistical evaluations of the antimicrobial efficacy were performed using two-way repeated measures ANOVA with Tukey's HSD test as post-hoc analysis for multiple comparisons. The statistic package GraphPad PRISM (GraphPad Software, Inc., La Jolla, United States of America) was used for statistical analysis.

Antimicrobial efficacy on tested pathogens
An overview on the reduction rates (in Δlog10 CFU ml-1 ± SEM reduction steps compared to initial CFU ml-1) of the tested antimicrobial agents is given in table 2.

Octenidin-dihydrochloride/phenoxyethanol (OCT) and povidone-iodine (PVP-I)
OCT and PVP-I achieved significant and complete eradication against all tested microorganism within 0.5 min of exposure (p < 0.0001). Increased protein challenge of 0.3% or 3% albumin did not exert a negative effect on their antimicrobial activity ( fig. 1 -5).

Polyhexamethylene-biguanide hydrochloride (PHMB)
PHMB showed to be less effective in the conducted experiments. After 0.5 min of exposure with 0.3% protein challenge, PHMB achieved significantly lower reduction rates compared to other tested antimicrobials, such as OCT or PVP-I (p ≤ 0.01). A significant and strong antimicrobial efficacy (> 5 log10 steps within 1 min; p < 0.0001) was achieved against all investigated microorganism, but for complete eradication extended exposure times were necessary: 3 min for S. aureus
Under 3% protein challenge CHX achieved comparable results, demonstrating no significant difference in efficacy between a 0.3% and 3% challenge. Only for S. aureus an extended exposure time was needed to achieve the required cut off (3 min; 5.991.99, fig. 1). Also, compared to the lower challenge, CHX achieved a complete eradication of C. albicans within 3 min of exposure ( fig. 5).

Cetylpyridinium-chloride (CPC)
Regardless of the protein challenge, CPC achieved a strong antimicrobial efficacy, as indicated by the

Discussion
Due to the globally rising challenge of antibiotic resistance and tolerance development in microorganim [12], advancements to counteract such challenges and investigations into new local antimicrobial agents are indispensable. Based on previously published data on the basic efficacy of CPC and MST as alternative antimicrobials and their cytotoxicity to human skin cells (keratinocytes and fibroblasts) [15], as relevant side-effects in wound management, this study extended the previous work to evaluate CPC and MST compared to established antiseptics. To especially account for the high-protein environment of wounds, bovine albumin was used in the experimental setup as a challenge substance to evaluate the protein-error of the tested agents, capable of reducing an antimicrobials efficacy. [17,18] As expected, the established agents OCT, PVP-I, CHX and PHMB managed the broad range of tested microorganism well and achieved a high antimicrobial efficacy, as reported in several previous studies [11,19,20]. All investigated agents, except PHMB and CHX, achieved the required reduction of > 5 Therefore, in this study CPC, OCT and PVP-I showed no, MST a slight and CHX as well as PHMB a comparable higher protein-error in the tested concentrations. Generally, the reported results indicate a certain extend of protein error in a complex high-protein microenvironment of the wound, depending on the concentration of the used antimicrobial, the amount of challenging total protein as well as the class of antimicrobials. For biguanides such as CHX and PHMB high protein challenge seems to be more relevant than for other antimicrobials. Especially in the case of PHMB the results have to be interpreted with regard to the used lower antimicrobial concentration available as commercial product (0.02%), so that higher concentrations of PHMB are unlikely to yield a relevant protein error.
In light of the presented results, compared to established antimicrobials, the general antimicrobial efficacy of CPC and MST, as well as challenged with a higher protein concentration (3%), proved to be at least equal, even superior in certain cases, against a broad range of microorganisms encountered in wound management. Especially CPC demonstrated the same efficacy as the highly potential antiseptics OCT and PVP-I with no protein error. MST proved to be only slightly less effective, yet still significantly more effective than the biguanides PHMB and CHX. Additionally, in the context of biocompatibility to human wound cells, which should be given equal consideration as previously described by Mueller & Kramer [20], the alternative agents demonstrated promising toxicity profiles in earlier published in-vitro evaluations. [15] For OCT and PVP-I severe cytotoxic effects on human keratinocytes and fibroblasts in-vitro have been reported [20], even stating dilutions of commercially available products as low as 12.5% (OCT) and 7.5% (PVP-I) to reduce cell viability and proliferation of fibroblasts and keratinocytes to 0% [24]. MST (0.05%) in comparison only demonstrated a reduction of cell vitality to about 30% for keratinocytes and fibroblasts in a previous study [15], being still highly toxic but more biocompatible than OCT or PVP-I. CPC on the contrary proved even less toxic, showing only a 50% reduced cell vitality within 60 min of exposure [15], ranging its toxicity towards human skin cells considerably lower than OCT or PVP-I while achieving the same antimicrobial efficacy.

Conclusion
Overall, CPC and MST feature a high potential as alternative antimicrobials based on their in-vitro efficacy profiles compared to established antiseptics. Especially CPC proved to be at least equally, in case of PHMB and CHX even significantly (p < 0.0001) more effective against tested microorganisms than established antiseptics. Neither CPC nor MST suffered a significant loss in efficacy under challenging high-protein (3% albumin) conditions. Considering that the here tested concentration of The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare, that they have no competing interests.  Tables   Table 1 -Specifications on test substances. Information have been obtained from the manufacturer.
The investigated antimicrobial agent/product is stated in the first row, followed by corresponding chemical group, manufacturer, composition as well as final test concentration of the antimicrobial agent tested in this study (commercially available products were used in undiluted formulation as provided by manufacturer).  Table 2 is only available as a download in the supplemental files section.   protein-challenge to achieve full reduction. After 3 min, PHMB also managed a complete eradication of P. aeruginosa (significant reductions over time are indicated as *p < 0.05, **p < 0.01, ***p <0.001, ****p < 0.0001).  min. CHX did not achieve full reduction of C. albicans within 10 min (significant reductions over time are indicated as *p < 0.05, **p < 0.01, ***p <0.001, ****p < 0.0001).

Supplementary Files
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