2Plessen Ophthalmology, 3006 Orange Grove, Christiansted, USVI 00820, USA
3Veloce BioPharma, LLC, 1007 N. Federal Hwy #E4, Fort Lauderdale, FL 33304, USA
Methods: Biofilms of multi-drug resistant Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Candida albicans were developed on solid surfaces using the Calgary Biofilm Device plate. Minimum biofilm eradication concentration was then determined for each test drug and for control samples of known antimicrobials, ciprofloxacin and itraconazole. Quality control fungal strains of Candida auris, Trichophton mentagrophytes, Microsporum canis, Candida albicans and Aspergillus fumigatus were grown on Sabouraud's Dextrose Agar plates as the growth medium for the anti-fungal susceptibility. Minimal Inhibitory Concentration was then determined for each test drug and for control samples of know antifungals, fluconazole and amphotericin B.
Results: The low-dose povidone-iodine formulations completely eliminated all biofilms of bacterial and fungal species in the test systems. Ciprofloxacin was able to eradicate one bacterial biofilm only at concentrations greater than 0.25 ug/mL. Fluconazole was ineffective against C. albicans, A. fumigatus and C. auris. Amphotericin B had good anti-fungal activity against fungal strains.
Conclusions: These novel dilute PVP-I formulations are effective anti-biofilm and anti-fungal /sporicidal agents in vitro. Further evaluation in living models is warranted.
Keywords: Biofilm; Povidone-Iodine; Wounds;
Biofilms are not just bacterial slime layers but rather represent complex biological systems. The comprising microorganisms are organized into a coordinated functional community. Biofilms are often attached to a surface and may include a single species or a diverse group of sessile microorganisms. These specialized heterogeneous communities are strengthened and protected by the biofilm in a manner that eventually leads to downstream deleterious gene expression and the manifestation of pathogenic factors. Chronic wounds have been shown to involve biofilm formation of both bacterial and fungal species [2,3]. It is in such a setting that biofilm microorganisms are able to share nutrients and are sheltered from harmful factors in the environment, such as desiccation, antibiotics, and the body's immune system [4,5].
More recently it has been noted that bacterial biofilms may impair cutaneous wound healing and reduce topical antibacterial efficiency in healing or treating infected skin wounds [6]. Chronic wounds are often a result of cutaneous surgery, vascular insufficiency or trauma, frequently requiring management by primary care physicians, dermatologists, plastic surgeons and wound care specialists. During normal wound healing, the process that leads to tissue regeneration results from a series of tightly regulated sequential events [7,8]. In the case of chronic, non-healing wounds, this process is disrupted, leading to a prolonged inflammatory response and stalled healing. It is hypothesized that microbial colonization and the formation of a biofilm within the wound bed is positively associated with the transition from the acute to chronic state even without classical signs of infection [9]. When a biofilm forms, the penetration of some antimicrobials is reduced, while some individual members shift their metabolism to a more dormant state that can render other antibiotics ineffective. Treatment strategies are further complicated by co-morbidities that affect circulation such as diabetes, poor perfusion, and malnutrition, increasing the risk of infection and reducing the success of orally administered antibiotics [9]. It follows therefore, that early detection of biofilms in wounds is crucial to successful chronic wound management.
Many of the treatments currently available are designed to treat acute wound infections, which, unlike chronic infections, tend to appear quickly and run their course over a short period of time. Planktonic bacteria typically respond to antibiotics and are easily exterminated by a healthy immune system. In contrast, chronic wounds are normally characterized by a tenacious and excessive inflammatory response when compared with acute wounds and are less susceptible to antibiotics [10]. Hence, the lack of antimicrobial effectiveness may be related to reduced or incomplete penetration of antimicrobials into the biofilm. A novel approach to the aforementioned problem is presented that employs the common antiseptic povidone-iodine at very low concentrations in a gel formulation to chronic wounds. Similar formulations have already been successfully used for indications for dermatology and ophthalmology [11,12]. An in vitro assay was performed to further characterize the anti-biofilm effect of this clinically successful treatment against organisms known to form biofilms in cutaneous wound infections.
# |
Organism |
Isolate # |
Phenotype |
Source |
1 |
Staphylococcus aureus |
ATCC 33591 |
Multi-drug resistant |
ATCC |
2 |
Klebsiella pneumoniae |
ATCC BAA-2473TM |
Multi-drug resistant |
ATCC |
3 |
Pseudomonas aeruginosa |
1674623 |
Multi-drug resistant |
Eurofins |
4 |
Candida albicans* |
3288194 |
Multi-drug resistant |
Eurofins |
5 |
Candida auris |
CDC 0389 |
Multi-drug resistant |
ATCC |
6 |
Trichophton mentagrophytes |
MYA4439 |
Quality Control |
ATCC |
7 |
Microsporum canis |
ATCC 26299 |
Quality Control |
ATCC |
8 |
Candida albicans** |
ATCC 90028 |
Quality Control |
ATCC |
9 |
Aspergillus fumigatus |
MYA3626 |
Quality Control |
ATCC |
**Strain tested for Minimal Inhibitory Concentration
The cation-adjusted Mueller Hinton Broth (CAMHB)/ RPMI (for C. albicans) were used as negative control. The CBD plate with the pegs containing a robust biofilm was first rinsed in PBS and then transferred to the treatment plate containing the test articles and control. The plate was incubated for 24 hours at 35°C and then read for determination of MIC values. After incubation, the pegs were rinsed in PBS twice and transferred to a recovery plate containing fresh culture media. The pegs were sonicated in a water bath sonicator for 30 minutes to detach any remaining adherent biofilm. The plate was incubated overnight at 35°C to evaluate growth and the MBEC values were determined.
MIC - Broth microdilution assays were performed according to the procedures detailed in CLSI document M38-A2 and document M27-A3 [1 - 2]. Briefly, 2X stock solutions of test articles were prepared at 128 μg/mL for Amphotericin B and Fluconazole. Two-fold serial dilutions of the stock solutions were performed. Diluted stock solutions of the fungal spores were adjusted (to be equivalent to 8 x 104 CFU/mL). 1X final concentration of the antifungals or antibiotics was obtained when they were combined with the fungal spore broth in the microtiter plate. The final concentration of the fungal spores in the microtiter plate was 4 x 104 CFU/mL. Plates were incubated for 7 days at 30±2°C for M. canis and 48 h at 27 - 30°C for all the other strains in an ambient air incubator. MIC values recorded are the minimum concentration of the test articles that inhibited the visible growth as observed by unaided eye. The MIC values were expressed in v/v % for the test article and in μg/mL for the control antibiotics (Table 2). The assay was performed in duplicates.
Strains |
Isolate ID |
Phenotype |
MBEC (% PVP-I and µg/mL Abx) |
|||
1% PVP-I Solution |
0.25% PVP-I Gel |
Ciprofloxacin |
Itraconazole |
|||
S. aureus |
ATCC 33591 |
MDR |
25 |
100 |
0.25 |
NA |
K. pneumoniae |
BAA-2473 |
MDR |
25 |
100 |
>128 |
NA |
P. aeruginosa |
1674623 |
MDR |
25 |
100 |
>128 |
NA |
C. albicans |
3288194 |
MDR |
25 |
100 |
NA |
>1024 |
MIC assay was carried to determine if the the lose dose PVP-I test article had anti-fungal activity. At serially diluted concentrations as low as 6.25% and 3.125%, PVP-I solutions showed anti-fungal activity against all the test strains. Fluconazole was ineffective against C. albicans, A. fumigatus and C. auris. Amphotericin B had good anti-fungal activity against all the strains tested (Table 3).
Strains |
Isolate ID |
Phenotype |
MIC (% PVP-I and µg/mL Antifungal) |
|||||
1% PVP-I Solution |
Amphotericin B |
Fluconazole |
||||||
C. albicans |
ATCC 90028 |
QC |
6.25 |
6.25 |
0.5 |
0.5 |
>64 |
>64 |
A. fumigatus |
ATCC MYA 3626 |
QC |
6.25 |
6.25 |
1 |
1 |
>64 |
>64 |
M. canis |
ATCC 36299 |
QC |
3.125 |
3.125 |
0.125 |
0.125 |
8 |
8 |
T. mentagrophytes |
ATCC MYA 4439 |
QC |
3.125 |
3.125 |
0.25 |
0.25 |
16 |
16 |
C. auris |
CDC 0389 |
QC |
3.125 |
3.125 |
2 |
2 |
>64 |
>64 |
Biofilms are found in approximately 60% of chronic wounds and 6% of acute wounds. Eradication of the resident bacteria is an ongoing challenge in the treatment of these cases. Effective antiseptics for wound healing should ideally address both inflammation and biofilm formation associated with chronic wounds [22]. In vitro evidence suggests that iodine not only has broad spectrum antibacterial effects, but also counteracts inflammation elicited by both pathogens and the host response. These anti-inflammatory effects appear to be multifactorial and have been shown to be clinically relevant [23]. Antiseptics, as an alternative for topical wound treatment, tend to be microbiocidal and have a broader spectrum of antimicrobial activity than antibiotics. Furthermore, in comparison to most antibiotics and antifungals, antiseptics reduce the likelihood of resistance emerging due to their multiple mechanisms of action targeting various aspects of cell biology in microbes; hence the use of topical antibiotics and antifungals should be discouraged if appropriate antiseptics are available [24]. Although amphotericin B was effective against all fungal strains, and the other control antibiotics and antifungals in this study were partially effective, they all share to propensity for this development of resistance as none are broad spectrum antimicrobials, nor are they produced in topical formulations. Comparison of the antimicrobial spectra of the most commonly used antiseptics (Povidone-iodine 10%, Polihexanide, Chlorhexidine, Octenidine, Ethanol 70%) against Gram-positive, Gram-negative, Actinobacteria, spores, fungi and viruses demonstrates the strong microbiocidal activity of Povidone-Iodine 10% against all classes of microorganisms. The remaining categories of antiseptics were not strongly biocidal across all categories [24].
The use of PVP-I for chronic wounds has been well documented in the literature. In vivo human studies conducted in varying settings have established the efficacy of PVP-I in reducing the bacterial load in both acute and chronic wounds [25-31]. There have been concerns about perceived cytotoxicity with PVP-I, and the potential detrimental effect of PVP-I on wound healing, and therefore biofilms, has been widely argued, with several in vitro studies demonstrating the dose-dependent cytotoxicity of PVP-I on cultures of granulocytes, monocytes, keratinocytes, and fibroblasts [32-34]. In vivo studies have failed to demonstrate detrimental effects on wound healing and the much lower concentrations of PVP-I used in this study reduce this potential effect greatly. Low dose PVP-I formulations for a variety of diseases have recently gained interest in the eye, ear and skin without any reported toxicity [11,12,35].
Also of note is the efficacy of low dose PVP-I against a biofilm comprised of C. auris, which has proven to be multi-drug resistant and difficult to culture emerging pathogen which can be causative in both systemic and wound infections. To our knowledge, this is the first topical approach to C. auris. The limitations of this study include utilization of the MIC assay for fungal organisms, rather than the MBEC assay. The MBEC is a more comprehensive test and better simulator of in vivo biofilm conditions; however this assay is not currently developed for fungi as they are much less prevalent as causative agents for biofilms. Another limitation is the study performed was in vitro and may not mimic in vivo conditions.
- Otto M. Staphylococcal infections: mechanisms of biofilm maturation and detachment as critical determinants of pathogenicity. Annu Rev Med. 2013;64:175-188. doi: 10.1146/annurev-med-042711-140023
- Jamal M, Ahmad W, Andleeb S, Jalil F, Imran M, Nawaz MA, et al. Bacterial biofilm and associated infections. J Chin Med Assoc. 2018;81(1):7-11. doi: 10.1016/j.jcma.2017.07.012
- Zacchino SA, Butassi E, Cordisco E, Svetaz LA. Hybrid combinations containing natural products and antimicrobial drugs that interfere with bacterial and fungal biofilms. Phytomedicine. 2017;37:14-26. doi: 10.1016/j.phymed.2017.10.021
- Joo HS, Otto M. Mechanisms of resistance to antimicrobial peptides in staphylococci. Biochim Biophys Acta. 2015;1848(11 Pt B):3055-3061. doi: 10.1016/j.bbamem.2015.02.009
- Vuong, C, Voyich JM, Fischer ER, Braughton KR, Whitney AR, DeLeo FR, et al. Polysaccharide intercellular adhesin (PIA) protects Staphylococcus epidermidis against major components of the human innate immune system. Cell Microbiol. 2004;6(3):269-275.
- Davis SC, Ricotti C, Cazzaniga A, Welsh E, Eaglstein WH, Mertz PM. Microscopic and physiologic evidence for biofilm-associated wound colonization in vivo. Wound Repair Regen. 2008;16(1):23-29. doi:10.1111/j.1524-475X.2007.00303.x
- Takeo M, Lee W, Ito M. Wound healing and skin regeneration. Cold Spring Harb Perspect Med. 2015;5(1):a023267-a023267. doi: 10.1101/cshperspect.a023267
- Coletti D, Teodori L, Lin Z, Beranudin JF, Adamo S. Restoration versus reconstruction: cellular mechanisms of skin, nerve and muscle regeneration compared. Regen Med Res. 2013;1(1):4. doi: 10.1186/2050-490X-1-4
- Swanson T, Angel D, Sussman G, Cooper R, Haesler E, Ousey K, et al. Wound infection in clinical practice: principles of best practice. Wounds International 2016. 2016.
- Omar A, Wright JB, Schultz G, Burrell R, Nadworthy P. Microbial Biofilms and Chronic Wounds. Microorganisms. 2017;5(1): pii: E9. doi: 10.3390/microorganisms5010009
- Capriotti K, Stewart KP, Pelletier JS, Capriotti JA. Molluscum Contagiosum treated With Dilute Povidone-iodine: A Series of Cases. J Clin Aesth Derm. 2017;10(3):41-45.
- Pelletier JS, Stewart KP, Capriotti K, Capriotti JA. Rosacea Blepharitis Treated with A Novel Preparation of Dilute Povidone-Iodine and Dimethylsulfoxide: A Case Report and Review of the Literature. Ophthalm Ther. 2015;4:143-150. doi: 10.1007/s40123-015-0040-4
- Clinical and Laboratory Standards Institute (CLSI). Reference method for broth dilution antifungal susceptibility testing of filamentous fungi: approved standard-Second Edition CLSI document M38-A2. 2008.
- Clinical and Laboratory Standards Institute (CLSI). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard-Third Edition. CLSI document M27-A3. 2008.
- Ceri H, Olson M, Morck D, Storey D, Read R, Buret A, Olson B. The MBEC Assay System: multiple equivalent biofilms for antibiotic and biocide susceptibility testing. Methods Enzymol. 2001;337:377-385.
- Jayaraja Kumar K, Jayachandran E, Hemanth Kumar Reddy C, Gunashakaran V, Ramesh Y, Kalayan Babu P, et al. Application of broad spectrum antiseptic povidone iodine as powerful action: a review. J Pharm Sci Technol. 2009;1(2):48-58.
- Ripa S, Bruno R, Reder R. Clinical applications of Povidone-Iodine as a topical antimicrobial. Handbook of Topical Antimicrobials Industrial Applications, Industrial applications in consumer products and Pharmaceuticals: CRC Press, 2002. 2002.
- Fleischer W, Reimer K. Povidone-iodine in antisepsis state of the art. Dermatology. 1997;195 Suppl 2:3-9.
- Rackur H. New aspects of mechanism of action of povidone-iodine.J Hosp Infect. 1985;6 Suppl A:13-23.
- Berkelman RL, Holland BW, Anderson RL. Increased Bactericidal Activity of Dilute Preparations of Povidone-Iodine Solutions. J Clin Microbiol. 1982;15(4):635-639.
- European Wound Management Association (EWMA). Position Document: Management of Wound Infection. 2006.
- Leaper DJ, Schultz G, Carville K, Fletcher J, Swanson T, Drake R. Extending the TIME concept: what have we learned in the past 10 years?(*). Int Wound J. 2012;9 Suppl 2:1-19. doi: 10.1111/j.1742-481X.2012.01097.x
- Bigliardi PL, Alsagoff SA, El-Kafrawi HY, Pyon JK, Wa CT, Villa MA. Povidone iodine in wound healing: A review of current concepts and practices. Int J Surg. 2017;44:260-268. doi: 10.1016/j.ijsu.2017.06.073
- Babu PA, Kumar PS, Padmaja P, Roa TK, Chitti S. MIC database: A collection of antimicrobial compounds from literature. Bioinformation. 2009;4(2):75-77.
- Fumal I, Braham C, Paquet P, Piérard- Franchimont C, Piérard GE. The beneficial toxicity paradox of antimicrobials in leg ulcer healing impaired by a polymicrobial flora: a proof-of-concept study. Dermatology. 2002;204 Suppl 1:70-74.
- Daròczy J. Antiseptic efficacy of local disinfecting povidone-iodine (Betadine) therapy in chronic wounds of lymphedematous patients. Dermatology. 2002;204 Suppl 1:75-78.
- Woo KY. Management of non-healable or maintenance wounds with topical povidone iodine. Int Wound J. 2014;11(6):622-626. doi: 10.1111/iwj.12017
- Han KH, Maitra AK. Management of partial skin thickness burn wounds with Iodine dressings. Burns. 1989;15(6):399-402.
- Homann HH, Rosbach O, Moll W, Vogt PM, Germann G, Hopp M, et al. A liposome hydrogel with polyvinyl-pyrrolidone iodine in the local treatment of partial-thickness burn wounds. Ann Plast Surg. 2007;59(4):423-427.
- Vogt PM, Hauser J, Rossbach O, Bosse B, Fleischer W, Steinau HU, et al. Polyvinyl pyr- rolidone-iodine liposome hydrogel improves epithelialization by combining moisture and antisepsis: a new concept in wound therapy. Wound Repair Regen. 2001;9(2):116-122.
- Vogt PM, Reimer K, Hauser J, Rossbach O, Steinau HU, Bosse B, et al. PVP-iodine in hydro- somes and hydrogel-A novel concept in wound therapy leads to enhanced epithelialization and reduced loss of skin grafts. Burns. 2006;32(6):698-705.
- Lineaweaver W, Howard R, Soucy D, McMorris S, Freeman J, Crain C, et al. Topical antimicrobial toxicity. Arch Surg. 1985;120(3):267-270.
- Burks RI. Povidone-iodine solution in wound treatment. Phys Ther. 1998;78(2):212-218.
- Van den Broek PJ, Buys LF, Van Furth R. Interaction of povidone-iodine compounds, phagocytic cells, and microorganisms. Anti-microb Agents Chemother. 1982;22(4):593-597.
- Tessema B, Wycherly B, Arumugam S, Capriotti K, Pelletier J, Barone S, Capriotti J. Efficacy of Dilute Povidone-Iodine Preparations Against Multi-Drug Resistant Biofilms of Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Candida albicans. Indian Jour of Research. 2018;7(1):376-377.