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Published in final edited form as: J Orthop Res. 2020 Jun 9;38(12):2657–2663. doi: 10.1002/jor.24765

Direct antimicrobial activity of cationic amphipathic peptide WLBU2 against Staphylococcus aureus biofilms is enhanced in physiologic buffered saline

Jonathan B Mandell 1,3, John Koch 1, Berthony Deslouches 4, Kenneth L Urish 1,2,5,6,7
PMCID: PMC7665995  NIHMSID: NIHMS1615220  PMID: 32484998

Abstract

Periprosthetic joint infection of total knee arthroplasties represents a major challenge to the field of orthopaedic surgery. These infections are commonly associated with antibiotic-tolerant Staphylococcus aureus biofilms. Engineered cationic amphipathic peptide WLBU2 has shown the ability to kill antibiotic-resistant pathogens and drug-tolerant bacterial biofilms. The novelty of using WLBU2 during the direct irrigation and debridement of periprosthetic joint infections led our group to investigate the optimal washout conditions for treatment of S. aureus biofilms. S. aureus mature biofilms were grown on metal implant material and treated with WLBU2 dissolved in differing irrigation solvents. Mature biofilms were treated both in vitro as well as in a periprosthetic joint infection murine model. WLBU2 activity against S. aureus biofilms was increased when dissolved in dPBS with pH of 7.0 compared to normal saline with pH of 5.5. WLBU2 activity was decreased in acidic dPBS and increased in alkaline dPBS. WLBU2 activity could be decreased in hypertonic dPBS and increased in hypotonic dPBS. WLBU2 dissolved in less acidic dPBS displayed increased efficacy in treating PJI implants ex vivo. WLBU2 demonstrated the ability to eliminate PJI associated S. aureus biofilms on arthroplasty material. The efficacy of engineered cationic amphipathic peptide WLBU2 for intraoperative elimination of S. aureus biofilms can be further optimized when kept in a less acidic and more physiologic pH adjusted saline. Understanding optimal physical washout conditions are vital for the success of WLBU2 in treating S. aureus biofilms in PJI clinical trials going forward.

Keywords: biofilm, antimicrobial peptide

Introduction

Roughly 2 million hospital-associated infections occur annually in the United States 1. Staphylococcus aureus is a major organism responsible for these infections, which include surgical site and implant prosthesis related infections 1-3. Total knee arthroplasties (TKAs) are the largest major surgical procedures by volume in the US, with over 700,000 performed every year. An infected total knee arthroplasty, termed periprosthetic joint infection (PJI), occurs in 1.5–2% of patients undergoing joint replacement surgery 4,5. PJI treatment involves multiple subsequent surgical procedures and long-term antibiotic regimens. In acute PJI, debridement antibiotics and implant retention (DAIR) is a common approach. Treatment failure is over 60% and five-year mortality is approximately 25% 6-8. The majority of PJI’s are due to S. aureus 9. The high antibiotic tolerance of biofilms is increasingly recognized as a primary reason for these difficult-to-eradicate infections 10-12. Novel antimicrobials which have better activity against S. aureus biofilms are needed.

WLBU2 is a de novo-engineered cationic peptide inspired by the optimization of cationic helical amphipathic structures of the intracellular motifs (lentivirus lytic peptides) observed in the transmembrane protein gp41 of the human immunodeficiency virus (HIV)-1. Such helical motifs are also characteristic of many naturally occurring antimicrobial peptides, such as magainin, the cathelicidin LL37, and others 13-15. It takes a series of days for traditional antibiotics such as cefazolin and vancomycin to eliminate biofilm on an implant surfaces 12. WLBU2 has been demonstrated to eliminate biofilms and create culture negative implants within 30 minutes 16. Potential indications of its use include intraoperative delivery for irrigation and debridement in PJI based on its ability to rapidly eliminate antibiotic-tolerant biofilm from implant surfaces 16 and broad-spectrum activity against ESKAPE (Enterococus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens 13,17. Activity of cationic peptides are dependent on ionic strength and pH 18,19. Typical irrigation solutions utilized in the lavage of surgical wounds include normal saline or lactated ringers solution. These solutions have a more acidic pH. The effect of pH and ionic strength of these buffered solutions on the needed contact time for treatment of PJI with WLBU2 should be considered.

The objective of this study was to understand the influence of pH and ionic strength of typical buffer solutions used in the operating room on the antimicrobial activity and needed contact time of WLBU2 to eliminate S. aureus biofilms. PJI implants from our murine model will be tested with WLBU2 directly during irrigation of implants. We hypothesized that the typical solutions used for irrigation in the operating room, normal saline and lactated ringers, would result in loss of activity based on lower pH as compared to a more physiologic buffered solution. The effects of pH and ionic strength on altering contact time of WLBU2 to reduce S. aureus biofilm burden remains unknown. This would have important formulary implications to minimize needed contact time in direct topical application of this novel antimicrobial peptide.

Methods

Bacterial strains and culture

S. aureus SH100025 was used for in vitro assays and the ex vivo PJI animal model. SH1000 was inoculated in Tryptic Soy Broth (TSB, Bectin Dickinson and Company) overnight at 37 °C with shaking at 250 rpm. Strains were diluted in Mueller Hinton Broth (MHB; Bectin Dickinson and Company) to a final concentration of 0.5 × 106 CFU/ml using the 0.5 MacFarland Standard (GFS Chemicals) and an Infinite M200 Spectrophotometer (Tecan). WLBU2 (PLG0206) was supplied by Peptilogics (San Jose, California). All experiments were performed in triplicate at three separate times with freshly inoculated cultures.

S. aureus biofilm implant in vitro killing assays

Implant material was prepared from 0.6 mm diameter stainless steel Kirschner wire (Synthes) and cut into 6 mm length, autoclaved, and plated in wells along with SH1000 at 1 × 106 CFU/ml. After plating, fresh MHB media was exchanged at 24 hours. At 48 hours, wire with mature biofilms were either placed into normal saline, lactated ringers, or dPBS with fold dilutions of WLBU2 at 62, 125, 250, 500, and 1000 μg/ml. After treatment, Kirschner wires were placed in 1ml sonication fluid consisting of 1% Tween 20 in dPBS and sonicated for 10 minutes. Sonicate was serially diluted and plated on TSA II with 5% sheep blood CS100 plates blood agar for colony forming unit (CFU) analysis. For pH analysis, prior to WLBU2 addition, dPBS was adjusted to more acidic pH using hydrochloric acid and a more alkaline pH using ammonium hydroxide. pH of dPBS was measured using an Orion Star A111 Benchtop pH Meter (Thermo-Scientific) before peptide addition and biofilm treatment and not monitored continuously during treatment. Infected implant pieces were tested with WLBU2 at both 0.5 and 1.0 mg/ml in PBS adjusted to pH of 6.5, 6.8, 7.0, 7.2, 7.4, and 8.0. For ionic strength analysis, dPBS was adjusted to hypertonic conditions by addition of NaCl to dPBS (0.3 M) and hypotonic conditions by addition of deionized water to dPBS (0.08 M). Biofilm implant pieces were treated with WLBU2 at 0.12, 0.25, 0.5 and 1.0 mg/ml for 2.5, 5, 7.5, 10, 15, and 20 minutes, then CFU analysis was performed. A 99.9% reduction in CFUs was determined from the average of CFUs from untreated Kirschner wires after biofilms that were grown over 48 hours and after a dPBS wash prior placing in sonication fluid.

Periprosthetic Joint Infection Ex vivo Washout Model

All experiments were performed under approved IACUC animal protocol in University of Pittsburgh Division of Laboratory Animal Resources. Twelve-week-old immunocompetent B57BL/6 J female mice (Jackson), weight of 17–20 grams, were used for all experiments. Mice where anesthetized by 2% isoflurane, hair was removed from leg and treated with betadine. With a scalpel, a medial parapatellar incision was made, and lateral displacement of the quadriceps-patellar complex allowed for visualization of the femoral intercondylar notch. With a 25-gauge needle, the femoral intramedullary canal was manually reamed. Mature S. aureus biofilm was previously established on a 0.6 mm wide/6 mm long Kirschner wire (Synthes) like the in vitro experiments, was inserted into the reamed canal, and sutured closed. Mice received buprenorphine for pain. 48 hours later, mice were euthanized and the infected Kirschner wire implant were extracted, placed in WLBU2 at 1.0 mg/ml in PBS previously pH adjusted to 6.5, 7.0, 7.2, or 7.4 for 10 minutes, and then placed 1% Tween 20 on ice. Implants were sonicated for 10 minutes. Samples were serially diluted and plated on TSA II with 5% sheep blood CS100 plates for CFU analysis. CFU analysis on blood agar plates was performed in a blinded manner. The primary experimental outcome was determined as the quantity of biofilm burden remaining on implant pieces after WLBU2 treatment. Experimental groups were PJI implant pieces treated with WLBU2 at pH of 6.5, 7.0, 7.2, and 7.4, while control groups were PJI implant pieces treated with dPBS only. A total of six mice were used for control and experimental groups.

Statistical Analysis

All statistical methods were performed using Prism 7.0 (GraphPad, La Jolla CA). Multiple groups were compared using a Kruskal-Wallis test with a Dunn’s Multiple Comparisons post-test. In all cases, p < 0.05 (*), p < 0.005 (**), p < 0.0005 (***), and p < 0.0001 (****) was considered significant.

Results

WLBU2 activity is decreased in typical buffered solutions used in the clinic.

Activity of WLBU2 was tested in common clinically used buffered solutions for irrigation of a PJI in the operating room, normal saline and lactated ringers, as a function of dose. These results were compared to a physiologic Phosphate Buffered Saline a typical culture washing media used in microbiology laboratories. Normal saline with a measured pH of 5.8 displayed nearly 99% reduction in bacterial biofilm CFUs with WLBU2 at 62–1000 μg/ml (Fig 1). In comparison, lactated ringers and dPBS with pH of 6.5 and 7.0 respectively displayed over 99.9% reduction in bacterial biofilm with WLBU at 62–1000 μg/ml. WLBU2 had a higher efficacy at reducing biofilm mass on an implant surface in higher pH solutions. A WLBU2 concentration dependent effect was not observed in treating biofilms. All three washout solutions have distinct range of pH but also contain differing amounts of buffers, which result in slightly different osmolarity and ionic strengths (normal saline- 308 mOsm/L, 0.15 M; lactated ringers- 274 mOsm/L, 0.14 M; dPBS- 299 mOsm/L, 0.16 M).

Figure 1. WLBU2 in dPBS displays improved killing of S. aureus biofilms.

Figure 1.

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SH1000 mature biofilms were grown on stainless steel Kirschner wire implant pieces over 48 hours in MHB. Biofilms were washed with dPBS and placed into MHB with fold dilutions of WLBU2. Biofilms were treated with WLBU2 for 10 minutes, in normal saline, lactated ringers, and dPBS. Treated biofilms were washed with PBS, placed into 1% Tween 20 in dPBS sonication solution and sonicated for 10 minutes. Colony forming unit (CFU) quantification on blood agar plates was performed to determine biofilm burden present after treatment. The dotted black line represents a 99.9% decrease compared to untreated implant CFU burden.

Physiologic pH enhances WLBU2 activity against S. aureus biofilms.

After observing large differences in WLBU2 activity in different buffered solutions, we questioned if needed contact time for therapeutic treatment would be altered by pH and ionic strength of washout solution. Mature biofilms were again cultured on surgical implant material and treated for 2.5, 5, 7.5, 10, 12.5, 15, and 20 minutes with multiple doses of WLBU2. CFU quantification on blood agar plates was performed to determine a three-log reduction from untreated controls (Fig 2A). We observed a clear reduction in contact time needed to obtain a three-log reduction as the pH was increased to more alkaline conditions. At 1.0 mg/ml WLBU2 in 6.5 pH PBS needed 15 minutes to achieve a three-log reduction while WLBU2 in 8.0 pH PBS only needed 2.5 minutes (Fig 2A). Additionally, CFU analysis revealed that WLBU2 treatments with PBS at more physiologic values of 7.4 and 8.0 were able to obtain 0 CFU or (culture sterile) samples (Fig 2B).

Figure 2. pH adjusted dPBS enhances WLBU2 activity against S. aureus biofilms.

Figure 2.

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Biofilm implant pieces were treated for 2.5, 5, 7.5, 10, 12.5, 15, and 20 minutes with WLBU2 in dPBS. dPBS pH was adjusted from 6.5 to 8.0 before peptide addition and biofilm treatment. Contact time needed to obtain a three-log reduction in biofilm CFU was reduced with increasing pH (A). Additionally, CFU analysis displayed WLBU2 treatment with dPBS at alkaline values were able to obtain 0 CFU (culture sterile) samples after sonication (B). The dotted black line displays smallest (2.5 min) and largest (20 min) WLBU2 contact time recorded values above 20 min were unable to achieve either 3 log reduction or 0 CFU (culture sterile) implant pieces. A 99.9% reduction in CFUs was analyzed from the average of CFUs from untreated Kirschner wires after biofilm after a dPBS wash prior placing in sonication media.

Ionic strength alters WLBU2 activity against S. aureus biofilms.

Ionic strength has been demonstrated to alter the activity of cationic peptides 20. After we observed the ability of pH to alter the needed contact time to eliminate biofilms, we questioned if ionic strength had a similar ability to alter contact time needed to eliminate biofilms. There was a clear linear relationship between ionic strength and contact time needed to eliminate biofilms. WLBU2 in hypotonic dPBS of 0.08 M displayed less time needed to obtain a three-log reduction (Fig 3A) and 0 CFU (culture sterile) samples (Fig 3B) compared to hypertonic dPBS.

Figure 3. Ionic strength adjusted dPBS enhances WLBU2 activity against S. aureus biofilms.

Figure 3.

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Biofilm implant pieces were treated for 2.5, 5, 7.5, 10, 12.5, 15, and 20 minutes with WLBU2 in PBS. Biofilms were similarly treated with WLBU2 in ionic strength adjusted dPBS and contact time needed for 3 log reduction (A) and 0 CFU (culture sterile) implant pieces (B). The dotted black line displays smallest (2.5 min) and largest (20 min) WLBU2 contact time recorded values above 20 min were unable to achieve either 3 log reduction or 0 CFU (culture sterile) implant pieces. A 99.9% reduction in CFUs was analyzed from the average of CFUs from untreated Kirschner wires after biofilm after a dPBS wash prior placing in sonication media.

Physiologic pH adjusted WLBU2 washout improves PJI implant biofilm treatment

We confirmed the dependence of pH and buffered solution on WLBU2 activity in our murine PJI animal model. During 48 hours of infection, mice did not display a significant loss in body weight. CFU analysis was performed on infected implant pieces with WLBU2 washout using pH adjusted PBS at 6.5, 7.0, 7.2, and 7.4 pH as well as implant pieces treated with PBS at 7.0 with no WLBU2 (No Drug). Implants were tested at a fixed timepoint of 10 minutes, which was determined from conditions which resulted in culture sterile implants in vitro. WLBU2 washout using pH adjusted to 7.4 displayed significant reduction in biofilm CFU compared to no WLBU2 (No Drug) (n=6, **P=0.005) (Fig 4). Additionally, WLBU2 washout with 7.4 pH displayed significant reduction of biofilm CFU compared to WLBU2 washout with 6.5 pH (n=6, *P=0.04)

Figure 4. Physiologic pH adjusted dPBS irrigation improves WLBU2 treatment of PJI biofilms.

Figure 4.

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PJI implants treated with WLBU2 in PBS with pH of 7.4 displayed a significant reduction in biofilm CFU compared to untreated control (No Drug) (n=6, **P=0.005). WLBU2 washout in 7.4 pH PBS displayed significantly less CFU implant burden compared to WLBU2 washout in 6.5 pH PBS (n=6, *P=0.04). Black line represents a 99.9% decrease compared to PBS washout only (No Drug).

Discussion

WLBU2 is a synthetically engineered cationic peptide with strong anti-biofilm activity currently undergoing a Federal Drug Agency (FDA) phase I clinical study (ACTRN 12618001920280). As the activity of naturally occurring cationic peptides have a known dependence on pH and ionic strength, the objective of this study was to demonstrate the effects of pH and solution tonicity on the activity of WLBU2 against S. aureus biofilms. Here, we demonstrated that pH and ionic strength altered the contact time needed for WLBU2 to achieve culture sterile implants or a clinically relevant reduction in biofilm mass. Contact time to achieve these parameters increased with ionic strength and was inversely related to the pH of the washout solution used. These effects were additionally observed in our murine periprosthetic infection model, as physiologic pH adjusted dPBS washout solution enhanced WLBU2 activity against S. aureus biofilms. This is the first report to our knowledge demonstrating pH and ionic strength can alter contact times needed to successfully treat biofilm related infection. These would be important formulation parameters in further FDA phase II and III clinical studies.

The pH of the solution is an important factor in the membrane interactive properties of antimicrobial peptides. The majority of characterized naturally occurring antimicrobial peptides at physiologic pH of 7.4 are positively charged, although some display neutral or negative charges depending on their primary amino acid composition 18-20. Pathologic conditions observed in cystic fibrosis results in a lower pH of the airway epithelial surface fluid, leading to decreased antimicrobial activity of the innate antimicrobial peptide LL-37 and other positively charged antimicrobial peptides produced by the host defense against S. aureus and P. aeruginosa 21,22. We observed increasing the pH of the irrigation solution to above 7.4 resulted in a reduction in the contact time needed for positively charged WLBU2 to obtain a 3-log reduction in S. aureus biofilms. Two common irrigation solutions used in the operating room include lactated ringers and normal saline which have pH of approximately 6.5 and 5.5 respectively. Formulating WLBU2 in these solutions would dramatically decrease the efficacy of WLBU2 by increasing the needed contact time for removal of biofilms. The solution pH has been known to alter antimicrobial peptide activity, and here we show that solution pH alters the needed contact time during local delivery.

Naturally occurring antimicrobial peptide activity can be altered by increasing ionic strength 23,24. Certain antimicrobial peptides have significantly increased activity under high ionic strength compared to other peptides with similar structure 25. In cystic fibrosis, airway epithelial surface fluid contains increased NaCl concentrations, and this has been shown to decrease innate immune peptide activity 26,27. The rationally designed antimicrobial peptide WLBU2 with optimized charge, hydrophobicity, and amphiphilicity for prokaryotic as compared to eukaryotic membranes has shown more resilience performance under variable environments. WLBU2 activity against P. aeruginosa and S. aureus has demonstrated more resilience to increasing NaCl concentrations compared to the cationic innate immune peptide LL-37 18,28,29. Our study displays WLBU2 activity against S. aureus biofilms is enhanced when dissolved in hypotonic dPBS solution with ionic strength below physiological levels. These conditions minimized the needed contact time to eliminate S. aureus biofilm. The solution’s ionic strength has been known to alter antimicrobial peptide activity, and here we show that solution ionic strength alters the contact time needed to be efficacious during local delivery.

There are several limitations of this study including evaluation of local peptide tissue toxicity in our PJI model and comparison of WLBU2 against other clinically used antiseptics. We tested washout solutions with WLBU2 with implants ex vivo after they were removed from the animal. As such, we did not evaluate the toxicity of the amphipathic antimicrobial peptide to the knee joint and surrounding tissue using histologic analysis. However, WLBU2 has been tested for toxicity in both small and large animals, as part of FDA regulations for the completion of preclinical development. Because the safety data follow these regulations, the drug has been approved by the FDA for phase I clinical trial. Additionally, this is an imperfect model of PJI since our implant is neither porous coated nor weight bearing and therefore lacking some important clinical considerations. Further testing will be focused on testing efficacy at eliminating biofilms in vivo locally at the site of PJI. We did not directly measure pH of PBS solutions when altering ionic strength before biofilm treatment. Our work displays that WLBU2 could be used similarly to already clinically utilized intraoperative antimicrobials like betadine and chlorohexidine. Although we did not compare WLBU2 to these agents in this study, the superiority of WLBU2 over traditional antibiotics in biofilm mode of growth has been demonstrated both in vitro and in vivo 13,16.

Ionic strength and pH are known to alter antimicrobial peptide activity based on protein stability and folding. Here, we demonstrate that pH and ionic strength are also critical variables that alter the antimicrobial peptide contact time needed in S. aureus biofilm clearance during local delivery. This is important in the formulation of antimicrobial peptides for topical, intraoperative, or other types of local delivery. From an intraoperative or local irrigation perspective, suspending WLBU2 in a typical clinical irrigation solution such as normal saline or lactated ringers would result in prolonged contact times beyond 15 minutes that are difficult to reasonably obtain in the operating room. Our data suggest that WLBU2 needs to be suspended in a slightly physiologic alkaline and hypotonic buffered saline solution for optimal results. Using these optimized conditions in the formulation of WLBU2, we demonstrated that the contact time can be minimized to less than 10 to 15 minutes. Determination of the optimal irrigation solvent in which WLBU2 can eliminate S. aureus biofilms in a reasonable amount of time is essential if used intraoperatively in the context of PJI.

Acknowledgements

The study was supported in part by the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS K08AR071494), the National Center for Advancing Translational Science (NCATS KL2TR0001856), the Orthopaedic Research and Education Foundation, and the Musculoskeletal Tissue Foundation. The first and senior author have a pending patent application related to data presented in the manuscript.

Footnotes

This study was performed at the Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA

All authors have been involved with the research design, acquisition, analysis, or interpretation of results. All authors either drafted or substantially revised manuscript and approved final submission.

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