Preliminary Results of the Use of a Stabilized Hypochlorous Acid Solution in the Management of Ralstonia Pickettii Biofilm on Silicone Breast Implants

Preliminary Results of the Use of a Stabilized Hypochlorous Acid Solution in the Management of... Abstract Background Ralstonia Pickettii biofilms are associated with pocket infections following breast implant surgeries. Biofilm protects bacteria most topically applied antimicrobial irrigations. Objectives To evaluate the effectiveness of four antimicrobial solutions on the planktonic form and established biofilm of Ralstonia Pickettii grown on 3 different types of silicone breast implants. Methods Time kill assays at clinical concentrations of chlorhexidine gluconate, povidone iodine, triple-antibiotic solution, and a 0.025% hypochlorous acid solution stabilized in amber glass were evaluated. Normal saline was the control. Three types of silicone implants, two with a textured surface and one smooth surface, were selected. Planktonic assays were performed after implants were soaked for one, five, 30, and 120 minute time points. Biofilm assays were performed after 5 and 120 minutes of implant soak time. Both tests evaluated cell-forming units (CFU/mL). Results Triple antibiotic solution had no effect on R. pickettii and was dropped from the study. Remaining solutions showed total kill of planktonic bacteria at one minute. Saline control showed no significant effect on biofilm as anticipated. Stabilized hypochlorous acid was the only solution tested capable of eradicating R. pickettii biofilm on all implant surfaces tested within the first five minute soak time. Conclusions Noncytotoxic, 0.025% hypochlorous acid in normal saline, stabilized in amber glass, successfully eradicated Ralstonia pickettii in planktonic and mature biofilm on three types of silicone implants during initial five minute soak time and may be the preferred antimicrobial solution for pocket lavage. This preliminary study requires further investigation. Leaching and implant compatibility testing is currently in progress. Breast augmentation surgeries are one of the most common cosmetic procedures. A reported 10 million women have breast implants globally, with US statistics suggesting breast augmentation was the second most common aesthetic surgical procedure with 310,444 cases in 2016, contrasted with 43,181 breast implant explantation surgeries.1,2 The most common associated complications with breast augmentation surgery include capsular contraction, hematoma, and infection.3 Breast implant infections are reported at between 1.5% to 2.5% of cosmetic cases and 20% to 35% in reconstructive surgeries.4,5 A multicenter observational study of surgical practice during breast implantation in the United Kingdom demonstrated intersurgeon variability of prevention practices and suggested methods to prevent implant related infections (eradication of skin commensal organisms, antibiotic prophylaxis, environmental controls, and barrier precautions) were applied inconsistently.6 Bacterial biofilms are known to be the cause of chronic infections7 and the estimated financial impact of revision surgery following surgical device related biofilm infection in the United States nears $1 billion annually.8Ralstonia pickettii, Propionibacterium species, coagulase-negative staphylococci, and Corynebacterium species are associated with pocket infections following breast implant surgeries.9 Recently, Ralstonia pickettii infection has been associated with the development of breast implant-associated anaplastic large cell lymphoma (BIA-ALCL).10,11 In a study by Hu and colleagues, 22 patients diagnosed with BIA-ALCL were evaluated. Nineteen ALCL associated breast-implant samples were compared to 12 nontumor capsule specimens and three contralateral breast samples from ALCL positive patients. In both BIA-ALCL and contralateral breast samples, Ralsotnia spp. was seen in significant numbers (P < 0.05), whereas Staphylococcus spp. was more dominant in the nontumor capsule specimens (P < 0.001). Fluoresecent in situ hybridization analysis of BIA-ALCL samples confirmed Ralstonia spp. in 10 of 11 samples (91%). Researchers utilized scanning electron microscopy (SEM) to evaluate seven BIA-ALCL and contralateral samples, and described established bacterial biofilm on all samples. Additionally, 18 capsular samples similarly showed established bacterial biofilm under SEM. The importance of bacterial infection and biofilm formation leading to major complications of ALCL and capsular contraction after implant surgery warrants discussion and investigation of antimicrobial and antibiofilm approaches to prevention. A meta-analysis of ALCL identified 80 cases (50 in the United States) with an average patient age of 52 years and time from surgery until diagnosis to be 11 years.12 A global review of 40 federal implant databases identified textured implants as being more commonly associated with ALCL than smooth (40% vs 4.2%; P = 0.0001).13 The US Food and Drug Administration (FDA) subsequently released a safety communication warning of the small but increasing risk of developing ALCL in the scar capsule adjacent to the implant.14 A joint statement on best practices for BIA-ALCL prevention and management of BIA-ALCL from the American Society for Aesthetic Plastic Surgery and the American Society of Plastic Surgeons has been established. Ralstonia pickettii is a nonfermenting Gram-negative bacillus and has been associated with minor infections to sepsis, especially in immunocompromised individuals. The biofilm forming bacteria has been identified in a hospital water supplies, respiratory solutions, and water for injection.15R. pickettii biofilm based infections are particularly difficult to treat due to reports of a wide range of antibiotic resistance,16,17 as well as the documented ineffectiveness of popular disinfectants, particularly chlorhexidine gluconate.18 Current intraoperative pocket lavage practices vary based upon surgeon preference secondary to a lack of strong evidence to identify the most effective antimicrobial solution. Gowda and colleagues recently reported on the practice of a 1979 plastic surgeons, achieving a response of 12.8% (253/1979), on their current practice with a 20 question survey.19 The survey results indicated that among plastic surgeons (average years of practice of 21 ± 9 year; 34 ± 50 implant reconstructions per year) 52% utilized chlorhexidine gluconate, 50% triple antibiotic soak, and 44% povidone-iodine for pocket lavage, while 97% indicated Cefazolin was the preoperative systemic antibiotic of choice. Breast implant-associated pocket infections and capsular contracture have been associated with a variety of organisms including staphylococcal spp., multidrug resistant staphylococcus, Ralstonia pickettii, Propionibacterium species, coagulase-negative staphylococci, and Corynebacterium.9 Pure hypochlorous acid, stabilized in amber glass, in a variety of concentrations has demonstrated broad spectrum antimicrobial activity against a variety of bacteria, spores, viruses, and protozoa (Table 1), and demonstrated superior effectiveness in comparison to other antimicrobial cleansers and solutions.20 Additionally, its clinical effectiveness in vivo has been demonstrated in necrotizing fasciitis both in germicidal capacity and the ability to reduce inflammatory toxins.21,22 However, pure hypochlorous acid in amber glass has not been studied against Ralstonia spp either in planktonic or biofilm form. The purpose of this study was to evaluate stabilized hypochlorous acid 0.025% in amber glass, compared to three common antimicrobial irrigation solutions (chlorhexidine gluconate, povidone-iodine, and triple-antibiotic solution) on the planktonic form and biofilm formation of Ralstonia pickettii on three common silicone breast implants. Table 1. Organisms Tested in Solution Microbial species ATTC no. % reduction Log reduction Gram-positive  Staphylococcus aureus Methicillin-susceptible (MSSA)* 29213 >99.999 >5  Staphylococcus aureus Methicillin-resistant (MRSA)* 33591 >99.999 >5  Staphylococcus epidermidis 12228 >99.999 >5  Staphylococcus haemolyticus 29970 >99.99 >4  Staphylococcus hominis 27844 >99.99 >4  Staphylococcus saprophyticus 35552 >99.99 >4  Streptococcus pyogenes* 49399 >99.99 >4  Corynebacterium amycolatum 49368 >99.99 >4  Enterococcus faecium 51559 >99.99 >4  Bacillus oleronius 700005 >99.999 >5  Clostridium perfringens* 13124 >99.99 >4  Propionibacterium acnes 29399 >99.999 >5 Gram-negative  Acinetobacter baumannii 19606 >99.99 >4  Escherichia coli 8739 >99.99 >4  Enterobacter aerogenes 51697 >99.999 >5  Haemophilus influenzae 49144 >99.999 >5  Klebsiella pneumoniae* 10031 >99.999 >5  Moraxella catarrhalis 8176 >99.9 >3  Proteus mirabilis* 14153 >99.999 >5  Pseudomonas aeruginosa 27853 >99.9999 >6  Serratia marcescens 14756 >99.999 >5  Vibrio vulnificus* 27562 >99.999 >5  Bacteroides fragilis* 25285 >99.999 >5 Fungi  Candida albicans 10231 >99.99 >4  Aspergillus brasiliensis 16404 >99.99 >4 Microbial species ATTC no. % reduction Log reduction Gram-positive  Staphylococcus aureus Methicillin-susceptible (MSSA)* 29213 >99.999 >5  Staphylococcus aureus Methicillin-resistant (MRSA)* 33591 >99.999 >5  Staphylococcus epidermidis 12228 >99.999 >5  Staphylococcus haemolyticus 29970 >99.99 >4  Staphylococcus hominis 27844 >99.99 >4  Staphylococcus saprophyticus 35552 >99.99 >4  Streptococcus pyogenes* 49399 >99.99 >4  Corynebacterium amycolatum 49368 >99.99 >4  Enterococcus faecium 51559 >99.99 >4  Bacillus oleronius 700005 >99.999 >5  Clostridium perfringens* 13124 >99.99 >4  Propionibacterium acnes 29399 >99.999 >5 Gram-negative  Acinetobacter baumannii 19606 >99.99 >4  Escherichia coli 8739 >99.99 >4  Enterobacter aerogenes 51697 >99.999 >5  Haemophilus influenzae 49144 >99.999 >5  Klebsiella pneumoniae* 10031 >99.999 >5  Moraxella catarrhalis 8176 >99.9 >3  Proteus mirabilis* 14153 >99.999 >5  Pseudomonas aeruginosa 27853 >99.9999 >6  Serratia marcescens 14756 >99.999 >5  Vibrio vulnificus* 27562 >99.999 >5  Bacteroides fragilis* 25285 >99.999 >5 Fungi  Candida albicans 10231 >99.99 >4  Aspergillus brasiliensis 16404 >99.99 >4 *Microbial efficacy of 0.025% HOCL in amber glass. In vitro testing. These microorganims are implicated in various chronic (eg, pressure and diabetic ulcers) and acute (eg, necrotizing fasciitis) infection. View Large Table 1. Organisms Tested in Solution Microbial species ATTC no. % reduction Log reduction Gram-positive  Staphylococcus aureus Methicillin-susceptible (MSSA)* 29213 >99.999 >5  Staphylococcus aureus Methicillin-resistant (MRSA)* 33591 >99.999 >5  Staphylococcus epidermidis 12228 >99.999 >5  Staphylococcus haemolyticus 29970 >99.99 >4  Staphylococcus hominis 27844 >99.99 >4  Staphylococcus saprophyticus 35552 >99.99 >4  Streptococcus pyogenes* 49399 >99.99 >4  Corynebacterium amycolatum 49368 >99.99 >4  Enterococcus faecium 51559 >99.99 >4  Bacillus oleronius 700005 >99.999 >5  Clostridium perfringens* 13124 >99.99 >4  Propionibacterium acnes 29399 >99.999 >5 Gram-negative  Acinetobacter baumannii 19606 >99.99 >4  Escherichia coli 8739 >99.99 >4  Enterobacter aerogenes 51697 >99.999 >5  Haemophilus influenzae 49144 >99.999 >5  Klebsiella pneumoniae* 10031 >99.999 >5  Moraxella catarrhalis 8176 >99.9 >3  Proteus mirabilis* 14153 >99.999 >5  Pseudomonas aeruginosa 27853 >99.9999 >6  Serratia marcescens 14756 >99.999 >5  Vibrio vulnificus* 27562 >99.999 >5  Bacteroides fragilis* 25285 >99.999 >5 Fungi  Candida albicans 10231 >99.99 >4  Aspergillus brasiliensis 16404 >99.99 >4 Microbial species ATTC no. % reduction Log reduction Gram-positive  Staphylococcus aureus Methicillin-susceptible (MSSA)* 29213 >99.999 >5  Staphylococcus aureus Methicillin-resistant (MRSA)* 33591 >99.999 >5  Staphylococcus epidermidis 12228 >99.999 >5  Staphylococcus haemolyticus 29970 >99.99 >4  Staphylococcus hominis 27844 >99.99 >4  Staphylococcus saprophyticus 35552 >99.99 >4  Streptococcus pyogenes* 49399 >99.99 >4  Corynebacterium amycolatum 49368 >99.99 >4  Enterococcus faecium 51559 >99.99 >4  Bacillus oleronius 700005 >99.999 >5  Clostridium perfringens* 13124 >99.99 >4  Propionibacterium acnes 29399 >99.999 >5 Gram-negative  Acinetobacter baumannii 19606 >99.99 >4  Escherichia coli 8739 >99.99 >4  Enterobacter aerogenes 51697 >99.999 >5  Haemophilus influenzae 49144 >99.999 >5  Klebsiella pneumoniae* 10031 >99.999 >5  Moraxella catarrhalis 8176 >99.9 >3  Proteus mirabilis* 14153 >99.999 >5  Pseudomonas aeruginosa 27853 >99.9999 >6  Serratia marcescens 14756 >99.999 >5  Vibrio vulnificus* 27562 >99.999 >5  Bacteroides fragilis* 25285 >99.999 >5 Fungi  Candida albicans 10231 >99.99 >4  Aspergillus brasiliensis 16404 >99.99 >4 *Microbial efficacy of 0.025% HOCL in amber glass. In vitro testing. These microorganims are implicated in various chronic (eg, pressure and diabetic ulcers) and acute (eg, necrotizing fasciitis) infection. View Large METHODS The study was conducted from March to April 2017. Time kill assays at clinical concentrations of 0.05% chlorhexidine gluconate, 10% povidone-iodine, triple-antibiotic solution (1 g Cefazolin, 80 mg Gentamicin and 50,000 U of Bacitracin in 500 mL saline), and stabilized 0.025% hypochlorous acid solution stabilized in amber glass were evaluated against Ralstonia pickettii ATCC 27511. Normal saline was used as the control solution. Three separate silicone implant types, Smooth Surface (Mentor Worldwide, Irvine, CA); Siltex (Mentor Worldwide, Irvine, CA); Biocell (Allergan plc, Dublin, Ireland) representing both smooth and textured surface implants were selected (Figure 1). Breast implant shells were thoroughly cleaned with distilled water and 70% alcohol and cut into uniform circles of 0.495 inch diameter using a punch. The cut implant shells were then dry heat sterilized before each experiment. Planktonic assays were performed after implants were soaked for 1, 5, 30, and 120 minute time points. R. pickettii was grown by streaking onto nutrient agar and incubating for 18 to 20 hours at 37°C. The organisms will be suspended in phosphate buffered saline (PBS) and adjusted to an optical density (OD) of 0.8 to 1.0 in phosphate buffered saline (PBS). This OD is equivalent to approximately 108 CFU/mL which is 0.5 McFarland. A total of 1 mL of test article was added to a glass test tube, 1 mL of saline served as a control to determine inoculum prior to treatment with the test article, and 10 μL of adjusted inoculum were added to each corresponding test tube to achieve a starting inoculum of 105 CFU/mL. At 1 min, 5 min, 30 mins, and 2 hour, 200 μL aliquots were taken from each test tube and added to Dey and Engley (D/E) neutralizing broth (Hardy Diagnostics, Santa Maria, CA) to neutralize the test article. A 200 μL aliquot was also taken from control tube and added to phosphate buffered saline. Figure 1. View largeDownload slide Schematic representation of the experimental groups used in the study. Three types of implants; Mentor Smooth (1), Mentor Siltex (2), and Allergan Biocell (3) treated with (A) 0.9% saline; (B) 0.025% v/v Phase One; (C) 0.35% v/v Betadine; (D) 0.05% v/v Chlorhexidine; and (E) triple antibiotic solution (1 g Cefazolin, 80 mg Gentamicin and 50,000 U of Bacitracin in 500 mL saline). Figure 1. View largeDownload slide Schematic representation of the experimental groups used in the study. Three types of implants; Mentor Smooth (1), Mentor Siltex (2), and Allergan Biocell (3) treated with (A) 0.9% saline; (B) 0.025% v/v Phase One; (C) 0.35% v/v Betadine; (D) 0.05% v/v Chlorhexidine; and (E) triple antibiotic solution (1 g Cefazolin, 80 mg Gentamicin and 50,000 U of Bacitracin in 500 mL saline). Tenfold serial dilutions were performed for each sample tube using neutralizing buffer as the diluent. An amount of 100 μL of the appropriate dilutions of each sample were plated on a nutrient plate in duplicates and incubated overnight. The colonies were counted postincubation. If the test article is antimicrobial, there was a reduction in colony counts between the treatment groups. Data are evaluated by comparing the difference in CFU/mL for the 4 test articles and the blank control at 1 min, 5 min, 30 mins, and 2 hour time points. Cytotoxicity (CT50) Testing In vitro cytotoxicity of test articles were tested against the Vero cell line (ATCC CCL- 1) using the cell proliferation kit Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI). Briefly, cells were seeded into 96-well plates at a density of approximately 20,000 cells/well. The growth medium was RPMI 1640 medium (containing 10% Fetal Bovine Serum (FBS) and 2 mM L-glutamine and 100 IU/mL penicillin-100 µg/mL streptomycin). Cells wiere grown at 37°C with 5% CO2. After 24 hours, the media was removed from the wells by aspiration. The cells were then exposed to a series of 11 twofold dilutions of the test article in RPMI media for 24 hours at 37°C with 5% CO2 prior to measuring cell viability using the Cell Titer Proliferation assay. Biofilm Assay Biofilm assays were performed after 2 to 3 mL of 105 CFU/mL bacterial inoculum was added to each tube containing the respective implants and placed into a shaking incubator (250-300 rpm) at 30°C for 24 hours, allowing for formation of biofilm on implants. After 24 hours’ incubation, the implants having the biofilm were rinsed twice with Butterfields phosphate buffer. After the rinses, the implants were aseptically transferred to tubes containing 5 mL test articles for contact time points; 5 min and 2 hours. Postincubation, the implants were rinsed twice with Butterfields phosphate buffer, placed in 5 mL of sterile neutralizing buffer and sonicated at 50 to 60 Hz for 5 minutes. Tenfold serial dilutions were performed for each sample tube using neutralizing buffer as the diluent. A total of 100 μL of appropriate dilutions of each sample were plated on a nutrient agar plate in duplicates and incubated overnight. The colonies were counted postincubation. Data were evaluated by comparing the difference in CFU/mL for the implants and blank control at 5 min and 2 hours. RESULTS Figures 2-4 show the results of the test solutions and saline control against the planktonic form of the study bacteria on the three different types of implants. Triple antibiotic solution showed no effect on the study bacteria during planktonic assay and was therefore dropped from the study (Supplementary Table 5). All subsequent solutions showed total kill of planktonic bacteria at one minute soak times compared to saline control (Supplementary Tables 1-4). The results of these test solutions on the established R. pickettii biofilm are presented in Figures 5-7 based on the type of implant. Individual implant biofilm kill assays are graphically represented in Supplementary Table 6 respectively (Supplemental Tables 1-6 are available online as Supplementary Material at www.aestheticsurgeryjournal.com). Figure 2. View largeDownload slide Time kill curves against Ralstonia picketti ATCC 27511 for Mentor smooth breast implants when treated with saline, Phase One, Chlorhexidine, Betadine, and triple antibiotic irrigation solution at clinical conc. for 0 min, 1 min, 5 min, 30 min, and 2 hour contact times. The assay was carried out in triplicates. The implant was completely immersed in 106 to 107 CFU/mL of initial inoculum of Ralstonia picketti before treating with the four test articles. Figure 2. View largeDownload slide Time kill curves against Ralstonia picketti ATCC 27511 for Mentor smooth breast implants when treated with saline, Phase One, Chlorhexidine, Betadine, and triple antibiotic irrigation solution at clinical conc. for 0 min, 1 min, 5 min, 30 min, and 2 hour contact times. The assay was carried out in triplicates. The implant was completely immersed in 106 to 107 CFU/mL of initial inoculum of Ralstonia picketti before treating with the four test articles. Figure 3. View largeDownload slide Time kill curves against Ralstonia picketti ATCC 27511 for Mentor Siltex breast implants when treated with saline, Phase One, Chlorhexidine, Betadine, and triple antibiotic irrigation solution at clinical conc. for 0 min, 1 min, 5 min, 30 min, and 2 hour contact times. The assay was carried out in triplicates. The implant was completely immersed in 106 to 107 CFU/mL of initial inoculum of Ralstonia picketti before treating with the four test articles. Figure 3. View largeDownload slide Time kill curves against Ralstonia picketti ATCC 27511 for Mentor Siltex breast implants when treated with saline, Phase One, Chlorhexidine, Betadine, and triple antibiotic irrigation solution at clinical conc. for 0 min, 1 min, 5 min, 30 min, and 2 hour contact times. The assay was carried out in triplicates. The implant was completely immersed in 106 to 107 CFU/mL of initial inoculum of Ralstonia picketti before treating with the four test articles. Figure 4. View largeDownload slide Time kill curves against Ralstonia picketti ATCC 27511 for Allergan Biocell breast implants when treated with saline, Phase One, Chlorhexidine, Betadine, and triple antibiotic irrigation solution at clinical conc. for 0 min, 1 min, 5 min, 30 min, and 2 hour contact times. The assay was carried out in triplicates. The implant was completely immersed in 106 to 107 CFU/mL of initial inoculum of Ralstonia picketti before treating with the four test articles. Figure 4. View largeDownload slide Time kill curves against Ralstonia picketti ATCC 27511 for Allergan Biocell breast implants when treated with saline, Phase One, Chlorhexidine, Betadine, and triple antibiotic irrigation solution at clinical conc. for 0 min, 1 min, 5 min, 30 min, and 2 hour contact times. The assay was carried out in triplicates. The implant was completely immersed in 106 to 107 CFU/mL of initial inoculum of Ralstonia picketti before treating with the four test articles. Figure 5. View largeDownload slide Biofilm assay curves for Mentor Smooth. The implants were treated with saline, Phase One, Chlorhexidine, and Betadine at clinical conc. at 0 min, 5 min, and 2 hours. A biofilm with 106 to 107 CFU/mL of initial inoculum of Ralstonia picketti ATCC 27511 was grown over the three implants for 24 hours before treating with the three test articles. The assay was carried out in triplicates. Figure 5. View largeDownload slide Biofilm assay curves for Mentor Smooth. The implants were treated with saline, Phase One, Chlorhexidine, and Betadine at clinical conc. at 0 min, 5 min, and 2 hours. A biofilm with 106 to 107 CFU/mL of initial inoculum of Ralstonia picketti ATCC 27511 was grown over the three implants for 24 hours before treating with the three test articles. The assay was carried out in triplicates. Figure 6. View largeDownload slide Biofilm assay curves for Mentor Siltex. The implants were treated with saline, Phase One, Chlorhexidine, and Betadine at clinical conc. at 0 min, 5 min, and 2 hours. A biofilm with 106 to 107 CFU/mL of initial inoculum of Ralstonia picketti ATCC 27511 was grown over the three implants for 24 hours before treating with the three test articles. The assay was carried out in triplicates. Figure 6. View largeDownload slide Biofilm assay curves for Mentor Siltex. The implants were treated with saline, Phase One, Chlorhexidine, and Betadine at clinical conc. at 0 min, 5 min, and 2 hours. A biofilm with 106 to 107 CFU/mL of initial inoculum of Ralstonia picketti ATCC 27511 was grown over the three implants for 24 hours before treating with the three test articles. The assay was carried out in triplicates. Figure 7. View largeDownload slide Biofilm assay curves for Allergan Biocell. The implants were treated with saline, Phase One, Chlorhexidine, and Betadine at clinical conc. at 0 min, 5 min, and 2 hours. A biofilm with 106 to 107 CFU/mL of initial inoculum of Ralstonia picketti ATCC 27511 was grown over the three implants for 24 hours before treating with the three test articles. The assay was carried out in triplicates. Figure 7. View largeDownload slide Biofilm assay curves for Allergan Biocell. The implants were treated with saline, Phase One, Chlorhexidine, and Betadine at clinical conc. at 0 min, 5 min, and 2 hours. A biofilm with 106 to 107 CFU/mL of initial inoculum of Ralstonia picketti ATCC 27511 was grown over the three implants for 24 hours before treating with the three test articles. The assay was carried out in triplicates. Biofilm assays showed differentiated penetration and impact of solutions on mature biofilm grown on the silicone implants. Saline control showed no significant effect on the biofilm for any of the implants, as anticipated. Chlorhexidine gluconate (Irrisept 0.05%, Irrimax Corporation, Lawrenceville, GA) did not have antibiofilm activity at Ralstonia biofilm-associated organisms at 5 minutes or at 2 hours for Siltex or Biocell, but did produce limited reduction at 5 minutes, and complete eradication at 2 hours for the smooth implant only. Povidone-Iodine 10% (Betadine, Purdue Pharma L.P., Stamford, CT) effectively eradicated biofilm on the smooth and Biocell implant at 5 minutes but required 2 hours of soak time to demonstrate complete biofilm eradication on Siltex. Pure HOCl (PhaseOne, Integrated Healing Technologies, Nashville, TN) demonstrated superior effectiveness in eradicating R. Pickettii biofilm on all three implant surfaces tested within the first five minute soak time. DISCUSSION Biofilm phenotype bacteria such as R. pickettii, have five primary methods by which they evade host and antimicrobial destruction, leading to localized and systemic infection.23,24 Biofilm phenotype bacteria secrete up to 800 proteins to create an exopolymeric structure (EPS). This matrix, consists of polysaccharides and proteinaceous material created by the bacteria and deposited during the inflammatory reaction of the host and serves as a “biological force-field” to shield bacteria. This prevents host immunologic defenses such as phagocytosis both through a physical barrier and by bacteria specific production of glycoproteins which impair the action of polymorphic neutrophils.25 Their product of strong adhesion molecules prevent displacement during routine irrigation and cleansing and generally require more aggressive forms of sharp debridement or explant of implant to remove these structures.26 Attachment may be strengthened by the use of effector proteins which may promote host cell senescence and improve adherence. To directly avoid the principle mode of action of most systemic and topical antibiotics (eg, the disruption of mitosis), biofilm bacteria become sessile within the base of the biofilm. Most topical antimicrobials are unable to penetrate mature biofilm and are typically used as an adjunct to sharp debridement to slow biofilm reformation.26 Finding an antimicrobial that could penetrate a mature EPS may benefit care by eradicating established biofilm on surgical implants before implantation, following explant of biofilm based infections, during dressing changes in chronic wounds between weekly or biweekly debridement and for wounds where sharp debridement is contraindicated. Challenges to diffusion of antimicrobials and antibiotics through a mature exopolymeric structure include:27 Host and bacterial DNA and proteins deposited on EPS surface which may inactivate these products. Biofilm specific gene expression which activates efflux pumps in the bacterial cell membrane to enhance removal of antimicrobial agents. Secretion of specific enzymes that bind or inactivate antimicrobial agents. High negative charge of biofilm EPS limiting diffusion of charged solutions. Chlorhexidine (CHG) is a bisbiguanide which binds to bacterial cell walls and alters osmotic equilibrium.28 While CHG has demonstrated the ability to decrease hospital acquired infection, especially as a skin disinfectant, multiple studies have identified concerns over CHG resistance, especially in staphylococci and methicillin-resistant staphylococci.29-32 The use of chlorhexidine gluconate 0.05% in this study was found to be ineffective in penetrating mature biofilm on the silicone implants tested. Previous studies in total joint arthroplasty identified that a 2% CHG concentration was required for biofilm effective penetration.33 However, a major downside to using CHG as an irrigation is the fact that concentrations as low as 0.02% were found to be cytotoxic to fibroblasts, which may prolong healing.34 While effective against planktonic bacterium, CHG’s inability to penetrate mature biofilm is further challenged by recent FDA warnings related to allergic reactions, including 52 cases of anaphylaxis.35 Despite its current popularity, CHG’s concerns over cytotoxicity, bacterial resistance, potential for allergic reaction, and ineffectiveness against biofilm warrant consideration for alternative antimicrobials during intraoperative irrigation. Additionally, bleach-based hypochlorite solutions have poor biofilm penetration due to neutralization of the active chlorine in the outermost regions of the biofilm,36 while disinfectant molecules react with the EPS impacting concentration and preventing penetration.27 Besides antimicrobial solutions, various antibiofilm modalities have been suggested in the literature, including ultrasound therapy, instillation negative pressure wound therapy, topical cadexomer iodine, hydrotherapy, and antibiofilm agents.37,38 These approaches vary in associated evidence to support their use and are often suggested in chronic wound management, not during acute surgical procedures. Therefore, the effectiveness of intraoperative preventive measures, such as the use of antimicrobial irrigation, were investigated in this study and identified that traditional solutions may not be as effective as stabilized hypochlorous acid in amber glass. Hypochlorous acid (HOCl) has long been known to demonstrate powerful antimicrobial activity. In human immunity, the polymorphic neutrophil creates the uncharged HOCl molecule following conversion of intracellular oxygen by nicotinamide adenine dinucleotide phosphate (NADPH) to hydrogen peroxide, which is then converted to HOCl via myeloperoxidase.39 The microbicidal effect of hypochlorous solution is dependent on HOCL concentration and is more effective than disassociated bleach based chlorine compounds such as hypochlorite.15 Bacterial and spore wall disruption by stabilized HOCl is related to oxidation of sulfhydryl enzymes, ring chlorination of amino acids, inhibition of protein synthesis, breaks in DNA, and DNA synthesis and enhanced by molecular neutrality supporting passive diffusion.39,40 This results in rapid, in-solution inactivation of selected Gram-positive and Gram-negative species and fungi. Stabilized hypochlorous acid has effective biofilm impairment capabilities and has been shown at 1/32 dilution (6 ppm) to penetrate biofilm while enhancing fibroblast migration.39 Additionally, in vitro studies suggest that HOCl may additionally provide anti-inflammatory benefits from the inactivation of bacterial endotoxins and other matrix degrading enzymes.21,22 Given the extreme oxidative activity of the molecule however, commercial solutions in the pure HOCl form have not been available given its inability to be bottled and stored in plastic or other common containers. Temperature and storage materials determine the stability, antimicrobial properties, and rate of chlorine decline in prepared solutions.41 Improper storage may lead to the production of harmful chemical byproducts. However, a proprietary method for stabilizing the HOCl molecule in a potent, noncytotoxic form has been developed (PhaseOne, 0.025% Integrated Healing Technologies, Nashville, TN). Packaged in amber glass with proprietary methods for its production, the HOCl solution has been shown to produce more rapid 4-log reduction of bacterial colony counts than other HOCL-bleach based solutions, polyhexamethylene biguanide and surfactant based cleansers and remain noncytotoxic to host fibroblasts.20,39,42 No comparison studies of this pure HOCl solution to commercially available surgical antimicrobial irrigations are available, to compare both rates of infection and development of capsular contracture after augmentation surgery. Drinane and colleagues compared triple antibiotic solution to saline irrigation in a retrospective cohort analysis. In 55 patients, no difference was found in the incidence or severity of capsular contraction between the two solutions (3.6% saline vs 3.7% triple antibiotic; P = 0.97).43 To date, comparison studies of this pure HOCl solution to commercially available surgical antimicrobial irrigations are unavailable, leaving surgeons a lack of guidance as to which solutions are best selected for both planktonic bacteria and potential biofilm prevention on selected implants. The results of the reported study herein suggest that PhaseOne, pure HOCl in amber glass, may be a preferred antimicrobial solution due to its rapid, safe, and effective action against both planktonic and biofilm bacteria, without the cytotoxicity or concerns over hypersensitivity or allergic reaction associated with commonly used antimicrobials. Limitations There were limitations associated with this study. First, the in vitro nature of the study requires further patient trials to verify the comparative effectiveness of these agents on the prevention of surgical site infection in patients undergoing breast implantation. Further, the reports in this study are limited to the testing of R. pickettii. Infections associated with breast implants following augmentation surgery have also been linked to multiple bacteria. Barbieri and colleagues identified varying biofilm producing capabilities of staphylococcus epidermidis and S. aureus in isolates from breast cancer patients following mastectomy and reconstruction. Infections were found to be related to differing strains of bacterium with specific phenotypic and genotypic expression of biofilm producing genes.44 Researchers have similarly identified propionbacterium acnes,45 coagulase-negative staphylococci, methicillin-resistant S. aureus (MRSA), streptococcus pyogenes, diphtheroids, and bacillus species as potential pathogens of interest.46 Finally, more research is needed to determine if management of planktonic bacteria at initial surgery, or management of bacterial biofilm during recurrent surgery is a greater determinant of overall success in the prevention against surgical site infection. By their nature, biofilm phenotype bacteria may begin in a planktonic form and then convert to a biofilm producing organism in response to environmental stress. It remains unknown whether this occurs during the immediate intraoperative period or develops over time. Pure HOCl in amber glass has been shown to be effective against other established bacterial biofilms including, pseudomonas aeruginosa,22 staphylococcus aureus, and candida albicans.39 CONCLUSIONS Prevention of breast implant related bacterial infections requires control of multiple variables within the perioperative and surgical theatres. Environmental factors, antibiotic selection and administration, attention to appropriate skin disinfection, barrier measures, and surgical technique are a few examples of practice considerations. The decision to irrigate the implant pocket and implant prior to placement and closure with an irrigation solution may influence infection rates. While many solutions are capable of managing broad spectrum control of planktonic bacteria, the ability to eradicate mature biofilm on the implant or from within the pocket is essential. Many commonly used antimicrobial irrigation solutions are incapable of penetrating the EPS shield of biofilm encased bacteria. However, many disinfectants and antimicrobials may be cytotoxic, including to fibroblasts and keratinocytes thereby delaying the wound healing process.47 Principle factors in selecting an antimicrobial irrigation are not only their microbicidal and antibiofilm properties, but also the consideration of surgical time commitment required to allow the solutions to dwell for maximum effect. In some cases, long dwell times may prolong surgery, which is not reasonable or feasible in the operative environment. Therefore, the goal in selecting such an irrigation solution would be its impact on planktonic and biofilm bacteria, the relative speed of its mode of action and the safety of the solution to prevent host cell cytotoxicity. In this preliminary study, 0.025% hypochlorous acid in normal saline solution (1/32 dilution), stabilized in amber glass, successfully eradicated planktonic Ralstonia pickettii in 60 seconds and R. pickettii biofilm grown on all three silicone implants during an initial five minute soak time in vitro. Povidone iodine showed the potential of eradicating biofilm, however required 120 minute soak time compared to the five minute soak time of PhaseOne. Chlorhexidine gluconate 0.05% was unable to penetrate established biofilm after two hours and triple antibiotic was removed from the study due to inability to show impact on even planktonic forms of studied bacteria. Pure, 0.025% hypochlorous acid stabilized in amber glass (PhaseOne) may be the preferred antimicrobial solution to manage both planktonic bacteria and established biofilm phenotype bacteria associated with silicone breast implant infections, given its rapid action, chemical stability, and safety profile. This preliminary study requires further investigation of implant leachability and compatibility tests with PhaseOne, which are currently in progress. Supplementary Material This article contains supplementary material located online at www.aestheticsurgeryjournal.com. Disclosures Dr Brindle is the Chief Clinical Officer of Integrated Healing Technologies and has equity incentive ownership with the company. Dr Porter is the Chief Scientific Advisor for Integrated Healing Technologies; Chief Scientific Officer, Dragon Bio-Consultants, Ltd.; Chairman, President, and CEO of VDDI Pharmaceuticals; and President PharmacoTherapy Consultants, LLC. Dr Arumugam is the Director and Dr Bijlani is Associate Director of Cell Microbiology at Emery Pharmaceuticals. Dr Najafi is the Chief Executive Officer at Emery Pharmaceuticals. Dr Fisher is a Founder, Chief Medical Officer, and Owner at Integrated Healing Technologies. Laboratory analysis by Emery Pharmaceuticals was funded by Integrated Healing Technologies. Experimental methodology, testing and data analysis were performed independently of Integrated Healing Technologies employees (full report on file). Funding This article was supported by Integrated Healing Technologies LLC (Nashville, TN), who co-funded the development of this supplement. REFERENCES 1. Doren EL , Miranda RN , Selber JC et al. U.S. Epidemiology of breast implant-associated anaplastic large cell lymphoma . Plast Reconstr Surg . 2017 ; 139 ( 5 ): 1042 - 1050 . Google Scholar CrossRef Search ADS PubMed 2. Cosmetic surgery national data bank statistics . Aesthet Surg J . 2017 ; 37 ( suppl 2 ): 1 - 29 . 3. Gupta V , Yeslev M , Winocour J et al. Aesthetic breast surgery and concomitant procedures: incidence and risk factors for major complications in 73,608 cases . Aesthet Surg J . 2017 ; 37 ( 5 ): 515 - 527 . Google Scholar CrossRef Search ADS PubMed 4. Feldman EM , Kontoyiannis DP , Sharabi SE , Lee E , Kaufman Y , Heller L . Breast implant infections: is cefazolin enough ? Plast Reconstr Surg . 2010 ; 126 ( 3 ): 779 - 785 . Google Scholar CrossRef Search ADS PubMed 5. Franchelli S , Pesce M , Savaia S et al. Clinical and microbiological characterization of late breast implant infections after reconstructive breast cancer surgery . Surg Infect (Larchmt) . 2015 ; 16 ( 5 ): 636 - 644 . Google Scholar CrossRef Search ADS PubMed 6. Henderson JR , Kandola S , Hignett SP et al. Infection prophylaxis for breast implant surgery: could we do better ? Eplasty . 2017 ; 17 : e19 . Google Scholar PubMed 7. Wolcott RD . Biofilms cause chronic infections . J Wound Care . 2017 ; 26 ( 8 ): 423 - 425 . Google Scholar CrossRef Search ADS PubMed 8. Deva AK , Adams WP Jr , Vickery K . The role of bacterial biofilms in device-associated infection . Plast Reconstr Surg . 2013 ; 132 ( 5 ): 1319 - 1328 . Google Scholar CrossRef Search ADS PubMed 9. Del Pozo JL , Tran NV , Petty PM et al. Pilot study of association of bacteria on breast implants with capsular contracture . J Clin Microbiol . 2009 ; 47 ( 5 ): 1333 - 1337 . Google Scholar CrossRef Search ADS PubMed 10. Hu H , Johani K , Almatroudi A et al. Bacterial biofilm infection detected in breast implant-associated anaplastic large-cell lymphoma . Plast Reconstr Surg . 2016 ; 137 ( 6 ): 1659 - 1669 . Google Scholar CrossRef Search ADS PubMed 11. Hu H , Jacombs A , Vickery K , Merten SL , Pennington DG , Deva AK . Chronic biofilm infection in breast implants is associated with an increased T-cell lymphocytic infiltrate: implications for breast implant-associated lymphoma . Plast Reconstr Surg . 2015 ; 135 ( 2 ): 319 - 329 . Google Scholar CrossRef Search ADS PubMed 12. Ramos-Gallardo G , Cuenca-Pardo J , Rodríguez-Olivares E et al. Breast implant and anaplastic large cell lymphoma meta-analysis . J Invest Surg . 2017 ; 30 ( 1 ): 56 - 65 . Google Scholar CrossRef Search ADS PubMed 13. Srinivasa DR , Miranda RN , Kaura A et al. Global adverse event reports of breast implant-associated ALCL: an international review of 40 government authority databases . Plast Reconstr Surg . 2017 ; 139 ( 5 ): 1029 - 1039 . Google Scholar CrossRef Search ADS PubMed 14. Health C for D and R . Breast Implants—Anaplastic Large Cell Lymphoma (ALCL) In Women with Breast Implants: Preliminary FDA Findings and Analyses . https://www.fda.gov/MedicalDevices/ProductsandMedicalProcedures/ImplantsandProsthetics/BreastImplants/ucm239996.htm. Accessed August 20, 2017 . 15. Ryan MP , Pembroke JT , Adley CC . Ralstonia pickettii: a persistent gram-negative nosocomial infectious organism . J Hosp Infect . 2006 ; 62 ( 3 ): 278 - 284 . Google Scholar CrossRef Search ADS PubMed 16. Zellweger C , Bodmer T , Täuber MG , Mühlemann K . Failure of ceftriaxone in an intravenous drug user with invasive infection due to Ralstonia pickettii . Infection . 2004 ; 32 ( 4 ): 246 - 248 . Google Scholar CrossRef Search ADS PubMed 17. Ryan MP , Adley CC . The antibiotic susceptibility of water-based bacteria Ralstonia pickettii and Ralstonia insidiosa . J Med Microbiol . 2013 ; 62 ( Pt 7 ): 1025 - 1031 . Google Scholar CrossRef Search ADS PubMed 18. Adley C , Saieb F . Microbials: biofilm formation in high-purity water: Ralstonia pickettii a special case for analysis . Ultrapure Water . 2005 ; 22 : 14 - 19 . 19. Gowda AU , Chopra K , Brown EN , Slezak S , Rasko Y . Preventing breast implant contamination in breast reconstruction: a national survey of current practice . Ann Plast Surg . 2017 ; 78 ( 2 ): 153 - 156 . Google Scholar CrossRef Search ADS PubMed 20. Rani SA , Hoon R , Najafi RR , Khosrovi B , Wang L , Debabov D . The in vitro antimicrobial activity of wound and skin cleansers at nontoxic concentrations . Adv Skin Wound Care . 2014 ; 27 ( 2 ): 65 - 69 . Google Scholar CrossRef Search ADS PubMed 21. Crew JR , Thibodeaux KT , Speyrer MS et al. Flow-through instillation of hypochlorous acid in the treatment of necrotizing fasciitis . Wounds . 2016 ; 28 ( 2 ): 40 - 47 . Google Scholar PubMed 22. Crew J , Varilla R , Rocas TA et al. NeutroPhase(®) in chronic non-healing wounds . Int J Burns Trauma . 2012 ; 2 ( 3 ): 126 - 134 . Google Scholar PubMed 23. Phillips P , Sampson E , Yang Q , Antonelli P , Progulske-Fox A , Schultz G . Bacterial biofilms in wounds . Wound Heal South Afr . 2009 ; 1 ( 2 ): 10 - 12 . 24. Percival SL , McCarty SM , Lipsky B . Biofilms and wounds: an overview of the evidence . Adv Wound Care (New Rochelle) . 2015 ; 4 ( 7 ): 373 - 381 . Google Scholar CrossRef Search ADS PubMed 25. Jensen PØ , Bjarnsholt T , Phipps R et al. Rapid necrotic killing of polymorphonuclear leukocytes is caused by quorum-sensing-controlled production of rhamnolipid by Pseudomonas aeruginosa . Microbiology . 2007 ; 153 ( Pt 5 ): 1329 - 1338 . Google Scholar CrossRef Search ADS PubMed 26. Wolcott RD , Rumbaugh KP , James G et al. Biofilm maturity studies indicate sharp debridement opens a time- dependent therapeutic window . J Wound Care . 2010 ; 19 ( 8 ): 320 - 328 . Google Scholar CrossRef Search ADS PubMed 27. Bjarnsholt T , Cooper R , Fletcher J et al. World Union of wound Healing Societies (WUWHS), Florence Congress, Position Statement. Management of Biofilm . Wounds International ; 2016 . 28. George J , Klika AK , Higuera CA . Use of chlorhexidine preparations in total joint arthroplasty . J Bone Jt Infect . 2017 ; 2 ( 1 ): 15 - 22 . Google Scholar CrossRef Search ADS PubMed 29. Wang JT , Sheng WH , Wang JL et al. Longitudinal analysis of chlorhexidine susceptibilities of nosocomial methicillin-resistant Staphylococcus aureus isolates at a teaching hospital in Taiwan . J Antimicrob Chemother . 2008 ; 62 ( 3 ): 514 - 517 . Google Scholar CrossRef Search ADS PubMed 30. Sheng WH , Wang JT , Lauderdale TL , Weng CM , Chen D , Chang SC . Epidemiology and susceptibilities of methicillin-resistant Staphylococcus aureus in Taiwan: emphasis on chlorhexidine susceptibility . Diagn Microbiol Infect Dis . 2009 ; 63 ( 3 ): 309 - 313 . Google Scholar CrossRef Search ADS PubMed 31. Horner C , Mawer D , Wilcox M . Reduced susceptibility to chlorhexidine in staphylococci: is it increasing and does it matter ? J Antimicrob Chemother . 2012 ; 67 ( 11 ): 2547 - 2559 . Google Scholar CrossRef Search ADS PubMed 32. Cookson BD , Bolton MC , Platt JH . Chlorhexidine resistance in methicillin-resistant Staphylococcus aureus or just an elevated MIC? An in vitro and in vivo assessment . Antimicrob Agents Chemother . 1991 ; 35 ( 10 ): 1997 - 2002 . Google Scholar CrossRef Search ADS PubMed 33. Smith DC , Maiman R , Schwechter EM , Kim SJ , Hirsh DM . Optimal irrigation and debridement of infected total joint implants with chlorhexidine gluconate . J Arthroplasty . 2015 ; 30 ( 10 ): 1820 - 1822 . Google Scholar CrossRef Search ADS PubMed 34. van Meurs SJ , Gawlitta D , Heemstra KA , Poolman RW , Vogely HC , Kruyt MC . Selection of an optimal antiseptic solution for intraoperative irrigation: an in vitro study . J Bone Joint Surg Am . 2014 ; 96 ( 4 ): 285 - 291 . Google Scholar CrossRef Search ADS PubMed 35. Drug Safety and Availability—FDA Drug Safety Communication . FDA warns about rare but serious allergic reactions with the skin antiseptic chlorhexidine gluconate . https://www.fda.gov/Drugs/DrugSafety/ucm530975.htm. Accessed September 25, 2017 . 36. Stewart PS , Rayner J , Roe F , Rees WM . Biofilm penetration and disinfection efficacy of alkaline hypochlorite and chlorosulfamates . J Appl Microbiol . 2001 ; 91 ( 3 ): 525 - 532 . Google Scholar CrossRef Search ADS PubMed 37. Attinger C , Wolcott R . Clinically addressing biofilm in chronic wounds . Adv Wound Care (New Rochelle) . 2012 ; 1 ( 3 ): 127 - 132 . Google Scholar CrossRef Search ADS PubMed 38. Snyder RJ , Bohn G , Hanft J et al. Wound biofilm: current perspectives and strategies on biofilm disruption and treatments . Wounds . 2017 ; 29 ( 6 ): S1 - S17 . Google Scholar PubMed 39. Sakarya S , Gunay N , Karakulak M , Ozturk B , Ertugrul B . Hypochlorous acid: an ideal wound care agent with powerful microbicidal, antibiofilm, and wound healing potency . Wounds . 2014 ; 26 ( 12 ): 342 - 350 . Google Scholar PubMed 40. Ono T , Yamashita K , Murayama T , Sato T . Microbicidal effect of weak acid hypochlorous solution on various microorganisms . Biocontrol Sci . 2012 ; 17 ( 3 ): 129 - 133 . Google Scholar CrossRef Search ADS PubMed 41. Robinson G , Thorn R , Reynolds D . The effect of long-term storage on the physiochemical and bactericidal properties of electrochemically activated solutions . Int J Mol Sci . 2012 ; 14 ( 1 ): 457 - 469 . Google Scholar CrossRef Search ADS PubMed 42. Robson MC , Payne WG , Ko F et al. Hypochlorous acid as a potential wound care agent: Part II. Stabilized hypochlorous acid: its role in decreasing tissue bacterial bioburden and overcoming the inhibition of infection on wound healing . J Burns Wounds . 2007 ; 6 : e6 . Google Scholar PubMed 43. Drinane JJ , Kortes MJ , Bergman RS , Folkers BL . Evaluation of antibiotic irrigation versus saline irrigation in reducing the long-term incidence and severity of capsular contraction after primary augmentation mammoplasty . Ann Plast Surg . 2016 ; 77 ( 1 ): 32 - 36 . Google Scholar CrossRef Search ADS PubMed 44. Barbieri R , Pesce M , Franchelli S , Baldelli I , De Maria A , Marchese A . Phenotypic and genotypic characterization of Staphylococci causing breast peri-implant infections in oncologic patients . BMC Microbiol . 2015 ; 15 : 26 . Google Scholar CrossRef Search ADS PubMed 45. Aubin GG , Portillo ME , Trampuz A , Corvec S . Propionibacterium acnes, an emerging pathogen: from acne to implant-infections, from phylotype to resistance . Med Mal Infect . 2014 ; 44 ( 6 ): 241 - 250 . Google Scholar CrossRef Search ADS PubMed 46. Rubino C , Brongo S , Pagliara D et al. Infections in breast implants: a review with a focus on developing countries . J Infect Dev Ctries . 2014 ; 8 ( 9 ): 1089 - 1095 . Google Scholar CrossRef Search ADS PubMed 47. Ortega-Peña S , Hidalgo-González C , Robson MC , Krötzsch E . In vitro microbicidal, anti-biofilm and cytotoxic effects of different commercial antiseptics . Int Wound J . 2017 ; 14 ( 3 ): 470 - 479 . Google Scholar CrossRef Search ADS PubMed © 2017 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Aesthetic Surgery Journal Oxford University Press

Preliminary Results of the Use of a Stabilized Hypochlorous Acid Solution in the Management of Ralstonia Pickettii Biofilm on Silicone Breast Implants

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Mosby Inc.
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© 2017 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com
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1090-820X
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1527-330X
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10.1093/asj/sjx229
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Abstract

Abstract Background Ralstonia Pickettii biofilms are associated with pocket infections following breast implant surgeries. Biofilm protects bacteria most topically applied antimicrobial irrigations. Objectives To evaluate the effectiveness of four antimicrobial solutions on the planktonic form and established biofilm of Ralstonia Pickettii grown on 3 different types of silicone breast implants. Methods Time kill assays at clinical concentrations of chlorhexidine gluconate, povidone iodine, triple-antibiotic solution, and a 0.025% hypochlorous acid solution stabilized in amber glass were evaluated. Normal saline was the control. Three types of silicone implants, two with a textured surface and one smooth surface, were selected. Planktonic assays were performed after implants were soaked for one, five, 30, and 120 minute time points. Biofilm assays were performed after 5 and 120 minutes of implant soak time. Both tests evaluated cell-forming units (CFU/mL). Results Triple antibiotic solution had no effect on R. pickettii and was dropped from the study. Remaining solutions showed total kill of planktonic bacteria at one minute. Saline control showed no significant effect on biofilm as anticipated. Stabilized hypochlorous acid was the only solution tested capable of eradicating R. pickettii biofilm on all implant surfaces tested within the first five minute soak time. Conclusions Noncytotoxic, 0.025% hypochlorous acid in normal saline, stabilized in amber glass, successfully eradicated Ralstonia pickettii in planktonic and mature biofilm on three types of silicone implants during initial five minute soak time and may be the preferred antimicrobial solution for pocket lavage. This preliminary study requires further investigation. Leaching and implant compatibility testing is currently in progress. Breast augmentation surgeries are one of the most common cosmetic procedures. A reported 10 million women have breast implants globally, with US statistics suggesting breast augmentation was the second most common aesthetic surgical procedure with 310,444 cases in 2016, contrasted with 43,181 breast implant explantation surgeries.1,2 The most common associated complications with breast augmentation surgery include capsular contraction, hematoma, and infection.3 Breast implant infections are reported at between 1.5% to 2.5% of cosmetic cases and 20% to 35% in reconstructive surgeries.4,5 A multicenter observational study of surgical practice during breast implantation in the United Kingdom demonstrated intersurgeon variability of prevention practices and suggested methods to prevent implant related infections (eradication of skin commensal organisms, antibiotic prophylaxis, environmental controls, and barrier precautions) were applied inconsistently.6 Bacterial biofilms are known to be the cause of chronic infections7 and the estimated financial impact of revision surgery following surgical device related biofilm infection in the United States nears $1 billion annually.8Ralstonia pickettii, Propionibacterium species, coagulase-negative staphylococci, and Corynebacterium species are associated with pocket infections following breast implant surgeries.9 Recently, Ralstonia pickettii infection has been associated with the development of breast implant-associated anaplastic large cell lymphoma (BIA-ALCL).10,11 In a study by Hu and colleagues, 22 patients diagnosed with BIA-ALCL were evaluated. Nineteen ALCL associated breast-implant samples were compared to 12 nontumor capsule specimens and three contralateral breast samples from ALCL positive patients. In both BIA-ALCL and contralateral breast samples, Ralsotnia spp. was seen in significant numbers (P < 0.05), whereas Staphylococcus spp. was more dominant in the nontumor capsule specimens (P < 0.001). Fluoresecent in situ hybridization analysis of BIA-ALCL samples confirmed Ralstonia spp. in 10 of 11 samples (91%). Researchers utilized scanning electron microscopy (SEM) to evaluate seven BIA-ALCL and contralateral samples, and described established bacterial biofilm on all samples. Additionally, 18 capsular samples similarly showed established bacterial biofilm under SEM. The importance of bacterial infection and biofilm formation leading to major complications of ALCL and capsular contraction after implant surgery warrants discussion and investigation of antimicrobial and antibiofilm approaches to prevention. A meta-analysis of ALCL identified 80 cases (50 in the United States) with an average patient age of 52 years and time from surgery until diagnosis to be 11 years.12 A global review of 40 federal implant databases identified textured implants as being more commonly associated with ALCL than smooth (40% vs 4.2%; P = 0.0001).13 The US Food and Drug Administration (FDA) subsequently released a safety communication warning of the small but increasing risk of developing ALCL in the scar capsule adjacent to the implant.14 A joint statement on best practices for BIA-ALCL prevention and management of BIA-ALCL from the American Society for Aesthetic Plastic Surgery and the American Society of Plastic Surgeons has been established. Ralstonia pickettii is a nonfermenting Gram-negative bacillus and has been associated with minor infections to sepsis, especially in immunocompromised individuals. The biofilm forming bacteria has been identified in a hospital water supplies, respiratory solutions, and water for injection.15R. pickettii biofilm based infections are particularly difficult to treat due to reports of a wide range of antibiotic resistance,16,17 as well as the documented ineffectiveness of popular disinfectants, particularly chlorhexidine gluconate.18 Current intraoperative pocket lavage practices vary based upon surgeon preference secondary to a lack of strong evidence to identify the most effective antimicrobial solution. Gowda and colleagues recently reported on the practice of a 1979 plastic surgeons, achieving a response of 12.8% (253/1979), on their current practice with a 20 question survey.19 The survey results indicated that among plastic surgeons (average years of practice of 21 ± 9 year; 34 ± 50 implant reconstructions per year) 52% utilized chlorhexidine gluconate, 50% triple antibiotic soak, and 44% povidone-iodine for pocket lavage, while 97% indicated Cefazolin was the preoperative systemic antibiotic of choice. Breast implant-associated pocket infections and capsular contracture have been associated with a variety of organisms including staphylococcal spp., multidrug resistant staphylococcus, Ralstonia pickettii, Propionibacterium species, coagulase-negative staphylococci, and Corynebacterium.9 Pure hypochlorous acid, stabilized in amber glass, in a variety of concentrations has demonstrated broad spectrum antimicrobial activity against a variety of bacteria, spores, viruses, and protozoa (Table 1), and demonstrated superior effectiveness in comparison to other antimicrobial cleansers and solutions.20 Additionally, its clinical effectiveness in vivo has been demonstrated in necrotizing fasciitis both in germicidal capacity and the ability to reduce inflammatory toxins.21,22 However, pure hypochlorous acid in amber glass has not been studied against Ralstonia spp either in planktonic or biofilm form. The purpose of this study was to evaluate stabilized hypochlorous acid 0.025% in amber glass, compared to three common antimicrobial irrigation solutions (chlorhexidine gluconate, povidone-iodine, and triple-antibiotic solution) on the planktonic form and biofilm formation of Ralstonia pickettii on three common silicone breast implants. Table 1. Organisms Tested in Solution Microbial species ATTC no. % reduction Log reduction Gram-positive  Staphylococcus aureus Methicillin-susceptible (MSSA)* 29213 >99.999 >5  Staphylococcus aureus Methicillin-resistant (MRSA)* 33591 >99.999 >5  Staphylococcus epidermidis 12228 >99.999 >5  Staphylococcus haemolyticus 29970 >99.99 >4  Staphylococcus hominis 27844 >99.99 >4  Staphylococcus saprophyticus 35552 >99.99 >4  Streptococcus pyogenes* 49399 >99.99 >4  Corynebacterium amycolatum 49368 >99.99 >4  Enterococcus faecium 51559 >99.99 >4  Bacillus oleronius 700005 >99.999 >5  Clostridium perfringens* 13124 >99.99 >4  Propionibacterium acnes 29399 >99.999 >5 Gram-negative  Acinetobacter baumannii 19606 >99.99 >4  Escherichia coli 8739 >99.99 >4  Enterobacter aerogenes 51697 >99.999 >5  Haemophilus influenzae 49144 >99.999 >5  Klebsiella pneumoniae* 10031 >99.999 >5  Moraxella catarrhalis 8176 >99.9 >3  Proteus mirabilis* 14153 >99.999 >5  Pseudomonas aeruginosa 27853 >99.9999 >6  Serratia marcescens 14756 >99.999 >5  Vibrio vulnificus* 27562 >99.999 >5  Bacteroides fragilis* 25285 >99.999 >5 Fungi  Candida albicans 10231 >99.99 >4  Aspergillus brasiliensis 16404 >99.99 >4 Microbial species ATTC no. % reduction Log reduction Gram-positive  Staphylococcus aureus Methicillin-susceptible (MSSA)* 29213 >99.999 >5  Staphylococcus aureus Methicillin-resistant (MRSA)* 33591 >99.999 >5  Staphylococcus epidermidis 12228 >99.999 >5  Staphylococcus haemolyticus 29970 >99.99 >4  Staphylococcus hominis 27844 >99.99 >4  Staphylococcus saprophyticus 35552 >99.99 >4  Streptococcus pyogenes* 49399 >99.99 >4  Corynebacterium amycolatum 49368 >99.99 >4  Enterococcus faecium 51559 >99.99 >4  Bacillus oleronius 700005 >99.999 >5  Clostridium perfringens* 13124 >99.99 >4  Propionibacterium acnes 29399 >99.999 >5 Gram-negative  Acinetobacter baumannii 19606 >99.99 >4  Escherichia coli 8739 >99.99 >4  Enterobacter aerogenes 51697 >99.999 >5  Haemophilus influenzae 49144 >99.999 >5  Klebsiella pneumoniae* 10031 >99.999 >5  Moraxella catarrhalis 8176 >99.9 >3  Proteus mirabilis* 14153 >99.999 >5  Pseudomonas aeruginosa 27853 >99.9999 >6  Serratia marcescens 14756 >99.999 >5  Vibrio vulnificus* 27562 >99.999 >5  Bacteroides fragilis* 25285 >99.999 >5 Fungi  Candida albicans 10231 >99.99 >4  Aspergillus brasiliensis 16404 >99.99 >4 *Microbial efficacy of 0.025% HOCL in amber glass. In vitro testing. These microorganims are implicated in various chronic (eg, pressure and diabetic ulcers) and acute (eg, necrotizing fasciitis) infection. View Large Table 1. Organisms Tested in Solution Microbial species ATTC no. % reduction Log reduction Gram-positive  Staphylococcus aureus Methicillin-susceptible (MSSA)* 29213 >99.999 >5  Staphylococcus aureus Methicillin-resistant (MRSA)* 33591 >99.999 >5  Staphylococcus epidermidis 12228 >99.999 >5  Staphylococcus haemolyticus 29970 >99.99 >4  Staphylococcus hominis 27844 >99.99 >4  Staphylococcus saprophyticus 35552 >99.99 >4  Streptococcus pyogenes* 49399 >99.99 >4  Corynebacterium amycolatum 49368 >99.99 >4  Enterococcus faecium 51559 >99.99 >4  Bacillus oleronius 700005 >99.999 >5  Clostridium perfringens* 13124 >99.99 >4  Propionibacterium acnes 29399 >99.999 >5 Gram-negative  Acinetobacter baumannii 19606 >99.99 >4  Escherichia coli 8739 >99.99 >4  Enterobacter aerogenes 51697 >99.999 >5  Haemophilus influenzae 49144 >99.999 >5  Klebsiella pneumoniae* 10031 >99.999 >5  Moraxella catarrhalis 8176 >99.9 >3  Proteus mirabilis* 14153 >99.999 >5  Pseudomonas aeruginosa 27853 >99.9999 >6  Serratia marcescens 14756 >99.999 >5  Vibrio vulnificus* 27562 >99.999 >5  Bacteroides fragilis* 25285 >99.999 >5 Fungi  Candida albicans 10231 >99.99 >4  Aspergillus brasiliensis 16404 >99.99 >4 Microbial species ATTC no. % reduction Log reduction Gram-positive  Staphylococcus aureus Methicillin-susceptible (MSSA)* 29213 >99.999 >5  Staphylococcus aureus Methicillin-resistant (MRSA)* 33591 >99.999 >5  Staphylococcus epidermidis 12228 >99.999 >5  Staphylococcus haemolyticus 29970 >99.99 >4  Staphylococcus hominis 27844 >99.99 >4  Staphylococcus saprophyticus 35552 >99.99 >4  Streptococcus pyogenes* 49399 >99.99 >4  Corynebacterium amycolatum 49368 >99.99 >4  Enterococcus faecium 51559 >99.99 >4  Bacillus oleronius 700005 >99.999 >5  Clostridium perfringens* 13124 >99.99 >4  Propionibacterium acnes 29399 >99.999 >5 Gram-negative  Acinetobacter baumannii 19606 >99.99 >4  Escherichia coli 8739 >99.99 >4  Enterobacter aerogenes 51697 >99.999 >5  Haemophilus influenzae 49144 >99.999 >5  Klebsiella pneumoniae* 10031 >99.999 >5  Moraxella catarrhalis 8176 >99.9 >3  Proteus mirabilis* 14153 >99.999 >5  Pseudomonas aeruginosa 27853 >99.9999 >6  Serratia marcescens 14756 >99.999 >5  Vibrio vulnificus* 27562 >99.999 >5  Bacteroides fragilis* 25285 >99.999 >5 Fungi  Candida albicans 10231 >99.99 >4  Aspergillus brasiliensis 16404 >99.99 >4 *Microbial efficacy of 0.025% HOCL in amber glass. In vitro testing. These microorganims are implicated in various chronic (eg, pressure and diabetic ulcers) and acute (eg, necrotizing fasciitis) infection. View Large METHODS The study was conducted from March to April 2017. Time kill assays at clinical concentrations of 0.05% chlorhexidine gluconate, 10% povidone-iodine, triple-antibiotic solution (1 g Cefazolin, 80 mg Gentamicin and 50,000 U of Bacitracin in 500 mL saline), and stabilized 0.025% hypochlorous acid solution stabilized in amber glass were evaluated against Ralstonia pickettii ATCC 27511. Normal saline was used as the control solution. Three separate silicone implant types, Smooth Surface (Mentor Worldwide, Irvine, CA); Siltex (Mentor Worldwide, Irvine, CA); Biocell (Allergan plc, Dublin, Ireland) representing both smooth and textured surface implants were selected (Figure 1). Breast implant shells were thoroughly cleaned with distilled water and 70% alcohol and cut into uniform circles of 0.495 inch diameter using a punch. The cut implant shells were then dry heat sterilized before each experiment. Planktonic assays were performed after implants were soaked for 1, 5, 30, and 120 minute time points. R. pickettii was grown by streaking onto nutrient agar and incubating for 18 to 20 hours at 37°C. The organisms will be suspended in phosphate buffered saline (PBS) and adjusted to an optical density (OD) of 0.8 to 1.0 in phosphate buffered saline (PBS). This OD is equivalent to approximately 108 CFU/mL which is 0.5 McFarland. A total of 1 mL of test article was added to a glass test tube, 1 mL of saline served as a control to determine inoculum prior to treatment with the test article, and 10 μL of adjusted inoculum were added to each corresponding test tube to achieve a starting inoculum of 105 CFU/mL. At 1 min, 5 min, 30 mins, and 2 hour, 200 μL aliquots were taken from each test tube and added to Dey and Engley (D/E) neutralizing broth (Hardy Diagnostics, Santa Maria, CA) to neutralize the test article. A 200 μL aliquot was also taken from control tube and added to phosphate buffered saline. Figure 1. View largeDownload slide Schematic representation of the experimental groups used in the study. Three types of implants; Mentor Smooth (1), Mentor Siltex (2), and Allergan Biocell (3) treated with (A) 0.9% saline; (B) 0.025% v/v Phase One; (C) 0.35% v/v Betadine; (D) 0.05% v/v Chlorhexidine; and (E) triple antibiotic solution (1 g Cefazolin, 80 mg Gentamicin and 50,000 U of Bacitracin in 500 mL saline). Figure 1. View largeDownload slide Schematic representation of the experimental groups used in the study. Three types of implants; Mentor Smooth (1), Mentor Siltex (2), and Allergan Biocell (3) treated with (A) 0.9% saline; (B) 0.025% v/v Phase One; (C) 0.35% v/v Betadine; (D) 0.05% v/v Chlorhexidine; and (E) triple antibiotic solution (1 g Cefazolin, 80 mg Gentamicin and 50,000 U of Bacitracin in 500 mL saline). Tenfold serial dilutions were performed for each sample tube using neutralizing buffer as the diluent. An amount of 100 μL of the appropriate dilutions of each sample were plated on a nutrient plate in duplicates and incubated overnight. The colonies were counted postincubation. If the test article is antimicrobial, there was a reduction in colony counts between the treatment groups. Data are evaluated by comparing the difference in CFU/mL for the 4 test articles and the blank control at 1 min, 5 min, 30 mins, and 2 hour time points. Cytotoxicity (CT50) Testing In vitro cytotoxicity of test articles were tested against the Vero cell line (ATCC CCL- 1) using the cell proliferation kit Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI). Briefly, cells were seeded into 96-well plates at a density of approximately 20,000 cells/well. The growth medium was RPMI 1640 medium (containing 10% Fetal Bovine Serum (FBS) and 2 mM L-glutamine and 100 IU/mL penicillin-100 µg/mL streptomycin). Cells wiere grown at 37°C with 5% CO2. After 24 hours, the media was removed from the wells by aspiration. The cells were then exposed to a series of 11 twofold dilutions of the test article in RPMI media for 24 hours at 37°C with 5% CO2 prior to measuring cell viability using the Cell Titer Proliferation assay. Biofilm Assay Biofilm assays were performed after 2 to 3 mL of 105 CFU/mL bacterial inoculum was added to each tube containing the respective implants and placed into a shaking incubator (250-300 rpm) at 30°C for 24 hours, allowing for formation of biofilm on implants. After 24 hours’ incubation, the implants having the biofilm were rinsed twice with Butterfields phosphate buffer. After the rinses, the implants were aseptically transferred to tubes containing 5 mL test articles for contact time points; 5 min and 2 hours. Postincubation, the implants were rinsed twice with Butterfields phosphate buffer, placed in 5 mL of sterile neutralizing buffer and sonicated at 50 to 60 Hz for 5 minutes. Tenfold serial dilutions were performed for each sample tube using neutralizing buffer as the diluent. A total of 100 μL of appropriate dilutions of each sample were plated on a nutrient agar plate in duplicates and incubated overnight. The colonies were counted postincubation. Data were evaluated by comparing the difference in CFU/mL for the implants and blank control at 5 min and 2 hours. RESULTS Figures 2-4 show the results of the test solutions and saline control against the planktonic form of the study bacteria on the three different types of implants. Triple antibiotic solution showed no effect on the study bacteria during planktonic assay and was therefore dropped from the study (Supplementary Table 5). All subsequent solutions showed total kill of planktonic bacteria at one minute soak times compared to saline control (Supplementary Tables 1-4). The results of these test solutions on the established R. pickettii biofilm are presented in Figures 5-7 based on the type of implant. Individual implant biofilm kill assays are graphically represented in Supplementary Table 6 respectively (Supplemental Tables 1-6 are available online as Supplementary Material at www.aestheticsurgeryjournal.com). Figure 2. View largeDownload slide Time kill curves against Ralstonia picketti ATCC 27511 for Mentor smooth breast implants when treated with saline, Phase One, Chlorhexidine, Betadine, and triple antibiotic irrigation solution at clinical conc. for 0 min, 1 min, 5 min, 30 min, and 2 hour contact times. The assay was carried out in triplicates. The implant was completely immersed in 106 to 107 CFU/mL of initial inoculum of Ralstonia picketti before treating with the four test articles. Figure 2. View largeDownload slide Time kill curves against Ralstonia picketti ATCC 27511 for Mentor smooth breast implants when treated with saline, Phase One, Chlorhexidine, Betadine, and triple antibiotic irrigation solution at clinical conc. for 0 min, 1 min, 5 min, 30 min, and 2 hour contact times. The assay was carried out in triplicates. The implant was completely immersed in 106 to 107 CFU/mL of initial inoculum of Ralstonia picketti before treating with the four test articles. Figure 3. View largeDownload slide Time kill curves against Ralstonia picketti ATCC 27511 for Mentor Siltex breast implants when treated with saline, Phase One, Chlorhexidine, Betadine, and triple antibiotic irrigation solution at clinical conc. for 0 min, 1 min, 5 min, 30 min, and 2 hour contact times. The assay was carried out in triplicates. The implant was completely immersed in 106 to 107 CFU/mL of initial inoculum of Ralstonia picketti before treating with the four test articles. Figure 3. View largeDownload slide Time kill curves against Ralstonia picketti ATCC 27511 for Mentor Siltex breast implants when treated with saline, Phase One, Chlorhexidine, Betadine, and triple antibiotic irrigation solution at clinical conc. for 0 min, 1 min, 5 min, 30 min, and 2 hour contact times. The assay was carried out in triplicates. The implant was completely immersed in 106 to 107 CFU/mL of initial inoculum of Ralstonia picketti before treating with the four test articles. Figure 4. View largeDownload slide Time kill curves against Ralstonia picketti ATCC 27511 for Allergan Biocell breast implants when treated with saline, Phase One, Chlorhexidine, Betadine, and triple antibiotic irrigation solution at clinical conc. for 0 min, 1 min, 5 min, 30 min, and 2 hour contact times. The assay was carried out in triplicates. The implant was completely immersed in 106 to 107 CFU/mL of initial inoculum of Ralstonia picketti before treating with the four test articles. Figure 4. View largeDownload slide Time kill curves against Ralstonia picketti ATCC 27511 for Allergan Biocell breast implants when treated with saline, Phase One, Chlorhexidine, Betadine, and triple antibiotic irrigation solution at clinical conc. for 0 min, 1 min, 5 min, 30 min, and 2 hour contact times. The assay was carried out in triplicates. The implant was completely immersed in 106 to 107 CFU/mL of initial inoculum of Ralstonia picketti before treating with the four test articles. Figure 5. View largeDownload slide Biofilm assay curves for Mentor Smooth. The implants were treated with saline, Phase One, Chlorhexidine, and Betadine at clinical conc. at 0 min, 5 min, and 2 hours. A biofilm with 106 to 107 CFU/mL of initial inoculum of Ralstonia picketti ATCC 27511 was grown over the three implants for 24 hours before treating with the three test articles. The assay was carried out in triplicates. Figure 5. View largeDownload slide Biofilm assay curves for Mentor Smooth. The implants were treated with saline, Phase One, Chlorhexidine, and Betadine at clinical conc. at 0 min, 5 min, and 2 hours. A biofilm with 106 to 107 CFU/mL of initial inoculum of Ralstonia picketti ATCC 27511 was grown over the three implants for 24 hours before treating with the three test articles. The assay was carried out in triplicates. Figure 6. View largeDownload slide Biofilm assay curves for Mentor Siltex. The implants were treated with saline, Phase One, Chlorhexidine, and Betadine at clinical conc. at 0 min, 5 min, and 2 hours. A biofilm with 106 to 107 CFU/mL of initial inoculum of Ralstonia picketti ATCC 27511 was grown over the three implants for 24 hours before treating with the three test articles. The assay was carried out in triplicates. Figure 6. View largeDownload slide Biofilm assay curves for Mentor Siltex. The implants were treated with saline, Phase One, Chlorhexidine, and Betadine at clinical conc. at 0 min, 5 min, and 2 hours. A biofilm with 106 to 107 CFU/mL of initial inoculum of Ralstonia picketti ATCC 27511 was grown over the three implants for 24 hours before treating with the three test articles. The assay was carried out in triplicates. Figure 7. View largeDownload slide Biofilm assay curves for Allergan Biocell. The implants were treated with saline, Phase One, Chlorhexidine, and Betadine at clinical conc. at 0 min, 5 min, and 2 hours. A biofilm with 106 to 107 CFU/mL of initial inoculum of Ralstonia picketti ATCC 27511 was grown over the three implants for 24 hours before treating with the three test articles. The assay was carried out in triplicates. Figure 7. View largeDownload slide Biofilm assay curves for Allergan Biocell. The implants were treated with saline, Phase One, Chlorhexidine, and Betadine at clinical conc. at 0 min, 5 min, and 2 hours. A biofilm with 106 to 107 CFU/mL of initial inoculum of Ralstonia picketti ATCC 27511 was grown over the three implants for 24 hours before treating with the three test articles. The assay was carried out in triplicates. Biofilm assays showed differentiated penetration and impact of solutions on mature biofilm grown on the silicone implants. Saline control showed no significant effect on the biofilm for any of the implants, as anticipated. Chlorhexidine gluconate (Irrisept 0.05%, Irrimax Corporation, Lawrenceville, GA) did not have antibiofilm activity at Ralstonia biofilm-associated organisms at 5 minutes or at 2 hours for Siltex or Biocell, but did produce limited reduction at 5 minutes, and complete eradication at 2 hours for the smooth implant only. Povidone-Iodine 10% (Betadine, Purdue Pharma L.P., Stamford, CT) effectively eradicated biofilm on the smooth and Biocell implant at 5 minutes but required 2 hours of soak time to demonstrate complete biofilm eradication on Siltex. Pure HOCl (PhaseOne, Integrated Healing Technologies, Nashville, TN) demonstrated superior effectiveness in eradicating R. Pickettii biofilm on all three implant surfaces tested within the first five minute soak time. DISCUSSION Biofilm phenotype bacteria such as R. pickettii, have five primary methods by which they evade host and antimicrobial destruction, leading to localized and systemic infection.23,24 Biofilm phenotype bacteria secrete up to 800 proteins to create an exopolymeric structure (EPS). This matrix, consists of polysaccharides and proteinaceous material created by the bacteria and deposited during the inflammatory reaction of the host and serves as a “biological force-field” to shield bacteria. This prevents host immunologic defenses such as phagocytosis both through a physical barrier and by bacteria specific production of glycoproteins which impair the action of polymorphic neutrophils.25 Their product of strong adhesion molecules prevent displacement during routine irrigation and cleansing and generally require more aggressive forms of sharp debridement or explant of implant to remove these structures.26 Attachment may be strengthened by the use of effector proteins which may promote host cell senescence and improve adherence. To directly avoid the principle mode of action of most systemic and topical antibiotics (eg, the disruption of mitosis), biofilm bacteria become sessile within the base of the biofilm. Most topical antimicrobials are unable to penetrate mature biofilm and are typically used as an adjunct to sharp debridement to slow biofilm reformation.26 Finding an antimicrobial that could penetrate a mature EPS may benefit care by eradicating established biofilm on surgical implants before implantation, following explant of biofilm based infections, during dressing changes in chronic wounds between weekly or biweekly debridement and for wounds where sharp debridement is contraindicated. Challenges to diffusion of antimicrobials and antibiotics through a mature exopolymeric structure include:27 Host and bacterial DNA and proteins deposited on EPS surface which may inactivate these products. Biofilm specific gene expression which activates efflux pumps in the bacterial cell membrane to enhance removal of antimicrobial agents. Secretion of specific enzymes that bind or inactivate antimicrobial agents. High negative charge of biofilm EPS limiting diffusion of charged solutions. Chlorhexidine (CHG) is a bisbiguanide which binds to bacterial cell walls and alters osmotic equilibrium.28 While CHG has demonstrated the ability to decrease hospital acquired infection, especially as a skin disinfectant, multiple studies have identified concerns over CHG resistance, especially in staphylococci and methicillin-resistant staphylococci.29-32 The use of chlorhexidine gluconate 0.05% in this study was found to be ineffective in penetrating mature biofilm on the silicone implants tested. Previous studies in total joint arthroplasty identified that a 2% CHG concentration was required for biofilm effective penetration.33 However, a major downside to using CHG as an irrigation is the fact that concentrations as low as 0.02% were found to be cytotoxic to fibroblasts, which may prolong healing.34 While effective against planktonic bacterium, CHG’s inability to penetrate mature biofilm is further challenged by recent FDA warnings related to allergic reactions, including 52 cases of anaphylaxis.35 Despite its current popularity, CHG’s concerns over cytotoxicity, bacterial resistance, potential for allergic reaction, and ineffectiveness against biofilm warrant consideration for alternative antimicrobials during intraoperative irrigation. Additionally, bleach-based hypochlorite solutions have poor biofilm penetration due to neutralization of the active chlorine in the outermost regions of the biofilm,36 while disinfectant molecules react with the EPS impacting concentration and preventing penetration.27 Besides antimicrobial solutions, various antibiofilm modalities have been suggested in the literature, including ultrasound therapy, instillation negative pressure wound therapy, topical cadexomer iodine, hydrotherapy, and antibiofilm agents.37,38 These approaches vary in associated evidence to support their use and are often suggested in chronic wound management, not during acute surgical procedures. Therefore, the effectiveness of intraoperative preventive measures, such as the use of antimicrobial irrigation, were investigated in this study and identified that traditional solutions may not be as effective as stabilized hypochlorous acid in amber glass. Hypochlorous acid (HOCl) has long been known to demonstrate powerful antimicrobial activity. In human immunity, the polymorphic neutrophil creates the uncharged HOCl molecule following conversion of intracellular oxygen by nicotinamide adenine dinucleotide phosphate (NADPH) to hydrogen peroxide, which is then converted to HOCl via myeloperoxidase.39 The microbicidal effect of hypochlorous solution is dependent on HOCL concentration and is more effective than disassociated bleach based chlorine compounds such as hypochlorite.15 Bacterial and spore wall disruption by stabilized HOCl is related to oxidation of sulfhydryl enzymes, ring chlorination of amino acids, inhibition of protein synthesis, breaks in DNA, and DNA synthesis and enhanced by molecular neutrality supporting passive diffusion.39,40 This results in rapid, in-solution inactivation of selected Gram-positive and Gram-negative species and fungi. Stabilized hypochlorous acid has effective biofilm impairment capabilities and has been shown at 1/32 dilution (6 ppm) to penetrate biofilm while enhancing fibroblast migration.39 Additionally, in vitro studies suggest that HOCl may additionally provide anti-inflammatory benefits from the inactivation of bacterial endotoxins and other matrix degrading enzymes.21,22 Given the extreme oxidative activity of the molecule however, commercial solutions in the pure HOCl form have not been available given its inability to be bottled and stored in plastic or other common containers. Temperature and storage materials determine the stability, antimicrobial properties, and rate of chlorine decline in prepared solutions.41 Improper storage may lead to the production of harmful chemical byproducts. However, a proprietary method for stabilizing the HOCl molecule in a potent, noncytotoxic form has been developed (PhaseOne, 0.025% Integrated Healing Technologies, Nashville, TN). Packaged in amber glass with proprietary methods for its production, the HOCl solution has been shown to produce more rapid 4-log reduction of bacterial colony counts than other HOCL-bleach based solutions, polyhexamethylene biguanide and surfactant based cleansers and remain noncytotoxic to host fibroblasts.20,39,42 No comparison studies of this pure HOCl solution to commercially available surgical antimicrobial irrigations are available, to compare both rates of infection and development of capsular contracture after augmentation surgery. Drinane and colleagues compared triple antibiotic solution to saline irrigation in a retrospective cohort analysis. In 55 patients, no difference was found in the incidence or severity of capsular contraction between the two solutions (3.6% saline vs 3.7% triple antibiotic; P = 0.97).43 To date, comparison studies of this pure HOCl solution to commercially available surgical antimicrobial irrigations are unavailable, leaving surgeons a lack of guidance as to which solutions are best selected for both planktonic bacteria and potential biofilm prevention on selected implants. The results of the reported study herein suggest that PhaseOne, pure HOCl in amber glass, may be a preferred antimicrobial solution due to its rapid, safe, and effective action against both planktonic and biofilm bacteria, without the cytotoxicity or concerns over hypersensitivity or allergic reaction associated with commonly used antimicrobials. Limitations There were limitations associated with this study. First, the in vitro nature of the study requires further patient trials to verify the comparative effectiveness of these agents on the prevention of surgical site infection in patients undergoing breast implantation. Further, the reports in this study are limited to the testing of R. pickettii. Infections associated with breast implants following augmentation surgery have also been linked to multiple bacteria. Barbieri and colleagues identified varying biofilm producing capabilities of staphylococcus epidermidis and S. aureus in isolates from breast cancer patients following mastectomy and reconstruction. Infections were found to be related to differing strains of bacterium with specific phenotypic and genotypic expression of biofilm producing genes.44 Researchers have similarly identified propionbacterium acnes,45 coagulase-negative staphylococci, methicillin-resistant S. aureus (MRSA), streptococcus pyogenes, diphtheroids, and bacillus species as potential pathogens of interest.46 Finally, more research is needed to determine if management of planktonic bacteria at initial surgery, or management of bacterial biofilm during recurrent surgery is a greater determinant of overall success in the prevention against surgical site infection. By their nature, biofilm phenotype bacteria may begin in a planktonic form and then convert to a biofilm producing organism in response to environmental stress. It remains unknown whether this occurs during the immediate intraoperative period or develops over time. Pure HOCl in amber glass has been shown to be effective against other established bacterial biofilms including, pseudomonas aeruginosa,22 staphylococcus aureus, and candida albicans.39 CONCLUSIONS Prevention of breast implant related bacterial infections requires control of multiple variables within the perioperative and surgical theatres. Environmental factors, antibiotic selection and administration, attention to appropriate skin disinfection, barrier measures, and surgical technique are a few examples of practice considerations. The decision to irrigate the implant pocket and implant prior to placement and closure with an irrigation solution may influence infection rates. While many solutions are capable of managing broad spectrum control of planktonic bacteria, the ability to eradicate mature biofilm on the implant or from within the pocket is essential. Many commonly used antimicrobial irrigation solutions are incapable of penetrating the EPS shield of biofilm encased bacteria. However, many disinfectants and antimicrobials may be cytotoxic, including to fibroblasts and keratinocytes thereby delaying the wound healing process.47 Principle factors in selecting an antimicrobial irrigation are not only their microbicidal and antibiofilm properties, but also the consideration of surgical time commitment required to allow the solutions to dwell for maximum effect. In some cases, long dwell times may prolong surgery, which is not reasonable or feasible in the operative environment. Therefore, the goal in selecting such an irrigation solution would be its impact on planktonic and biofilm bacteria, the relative speed of its mode of action and the safety of the solution to prevent host cell cytotoxicity. In this preliminary study, 0.025% hypochlorous acid in normal saline solution (1/32 dilution), stabilized in amber glass, successfully eradicated planktonic Ralstonia pickettii in 60 seconds and R. pickettii biofilm grown on all three silicone implants during an initial five minute soak time in vitro. Povidone iodine showed the potential of eradicating biofilm, however required 120 minute soak time compared to the five minute soak time of PhaseOne. Chlorhexidine gluconate 0.05% was unable to penetrate established biofilm after two hours and triple antibiotic was removed from the study due to inability to show impact on even planktonic forms of studied bacteria. Pure, 0.025% hypochlorous acid stabilized in amber glass (PhaseOne) may be the preferred antimicrobial solution to manage both planktonic bacteria and established biofilm phenotype bacteria associated with silicone breast implant infections, given its rapid action, chemical stability, and safety profile. This preliminary study requires further investigation of implant leachability and compatibility tests with PhaseOne, which are currently in progress. Supplementary Material This article contains supplementary material located online at www.aestheticsurgeryjournal.com. Disclosures Dr Brindle is the Chief Clinical Officer of Integrated Healing Technologies and has equity incentive ownership with the company. Dr Porter is the Chief Scientific Advisor for Integrated Healing Technologies; Chief Scientific Officer, Dragon Bio-Consultants, Ltd.; Chairman, President, and CEO of VDDI Pharmaceuticals; and President PharmacoTherapy Consultants, LLC. Dr Arumugam is the Director and Dr Bijlani is Associate Director of Cell Microbiology at Emery Pharmaceuticals. Dr Najafi is the Chief Executive Officer at Emery Pharmaceuticals. Dr Fisher is a Founder, Chief Medical Officer, and Owner at Integrated Healing Technologies. Laboratory analysis by Emery Pharmaceuticals was funded by Integrated Healing Technologies. Experimental methodology, testing and data analysis were performed independently of Integrated Healing Technologies employees (full report on file). Funding This article was supported by Integrated Healing Technologies LLC (Nashville, TN), who co-funded the development of this supplement. REFERENCES 1. Doren EL , Miranda RN , Selber JC et al. U.S. Epidemiology of breast implant-associated anaplastic large cell lymphoma . Plast Reconstr Surg . 2017 ; 139 ( 5 ): 1042 - 1050 . Google Scholar CrossRef Search ADS PubMed 2. Cosmetic surgery national data bank statistics . Aesthet Surg J . 2017 ; 37 ( suppl 2 ): 1 - 29 . 3. Gupta V , Yeslev M , Winocour J et al. Aesthetic breast surgery and concomitant procedures: incidence and risk factors for major complications in 73,608 cases . Aesthet Surg J . 2017 ; 37 ( 5 ): 515 - 527 . Google Scholar CrossRef Search ADS PubMed 4. 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Journal

Aesthetic Surgery JournalOxford University Press

Published: Dec 12, 2017

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