Patients

Patients were recruited and enrolled at the NIH under clinical trial NCT03018275 (Beginning Assessment of Cutaneous Treatment Efficacy of Roseomonas in Atopic Dermatitis, Phase I/II; BACTERiAD I/II). Prior to enrollment all patients and/or legal guardians signed informed consent. Pediatric patients signed assents. SCORAD (an established scor ing algorithm for a topic d ermatitis) values were determined under standard approaches (20). Two investigators documented the surface areas involved and intensity of disease. One investigator was intentionally misinformed that the study was a placebo-controlled design and therefore was unaware that all patients were on active treatment with the investigational drug. Only scores from the blinded investigator are shown. Patients provided the subjective values for pruritus and sleep disturbance. To meet inclusion criteria, patients needed to have a SCORAD value of 10 or higher, have disease present on the antecubital fossae and/or forearms, and have previously attempted standard of care therapy. Values for the antecubital specific SCORAD were obtained by adding the intensity values for the antecubital region (score 0–3 for dryness, erythema, edema, oozing, excoriation, and lichenification) to the patient-reported subjective score (score 0–10) for pruritus of the antecubital region. Antecubital fossae were swabbed for the presence of Gram-negative bacteria as previously described (17, 29). S. aureus and CNS burden was determined by vortexing swabs in 2 ml of typsin broth (Remel) for 30 seconds and plating 100 μl on blood agar plates (Remel). The following day, the number of colonies was enumerated and multiplied by 20 to obtain the total CFU in the 2-ml collection volume and then averaging values between both arms. Relative abundance of S. aureus was obtained by dividing the colony numbers for S. aureus by those for CNS.

Screening blood work included complete blood count, chemistry panel, calcium, magnesium, phosphorus, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, bilirubin, albumin, and HIV antibodies for adults. Urine pregnancy testing was performed on all adult female patients. There were no restrictions on home medication use except for those specified in the exclusion criteria. Adult patients were instructed to make no changes in their home regimen. Pediatric patients were asked to maintain their standard approach; parents reported using daily emollients but reserving steroid treatments for flares. Parents were asked to recall average days/month of topical steroid use for the 3 months prior to treatment and then prospectively track steroid use during treatment. Transepidermal water loss was measured by VapoMeter (Delfin) per the manufacturer’s instructions and represents the average results of the bilateral antecubital fossae.

Inclusion criteria

Inclusion criteria were (a) age 18 years or older (adult cohort) or age 7–17 (pediatric cohort); (b) SCORAD of at least 10; (c) carry a physician diagnosis of AD with active involvement of the antecubital fossa; (d) willing to allow storage of blood for future research; (e) no history of other skin disease; (f) initiated or attempted standard of care therapy at least 6 months prior to enrollment; and (g) agreement to use adequate contraception if indicated.

Exclusion criteria

Exclusion criteria were (a) presence of an indwelling venous or arterial catheter; (b) individuals living with anyone with a diagnosed immunodeficiency, cardiac valvular disease, and/or indwelling catheter; (c) presence of allergies to amikacin, ciprofloxacin, gentamicin, levofloxacin, and tobramycin (which would preclude treatment of any unexpected infection); (d) history of cardiac valvular disease; (e) any history of grade 2 or higher neutropenia or leukopenia; (f) clinical suspicion of immunodeficiency, liver disorder, kidney disorder, and/or HIV; (g) pregnant or breastfeeding; (h) any history of anti–tumor necrosis factor (TNF) treatment; (i) inability to demonstrate proper bacteria administration procedure despite coaching and training; (j) use of any antibiotics within 4 weeks of enrollment; (k) use of oral steroids within 4 weeks of enrollment; and (l) any condition that, in the opinion of the investigator, contraindicates participation in this study.

Endpoints

Primary endpoints were (a) frequency of solicited and unsolicited adverse events, serious adverse events, and death; and (b) 50% reduction in regional or total SCORAD.

Secondary endpoints (applicable only to the pediatric cohort) were (a) 30% improvement in the Children’s Dermatology Life Quality Index (CDLQI); and (b) 30% improvement in the Family Dermatology Life Quality Index (FDLQI).

Qualitative analysis

Patient statements at intake and follow-up visits were documented and examined for thematic consistencies. Commentary on body sites treated, duration of benefit, ease of use, doses missed, overall satisfaction, and adverse reactions were compiled and analyzed.

Sample size derivations

Safety. Sample size calculations demonstrated that with 15 total patients in the dose escalation portion (10 adults, 5 children), there would be a probability of 0.14 of observing 1 or more serious adverse events (SAEs) or AEs (grade 2 or higher as defined by the DAIDS toxicity table) if the true rate were 0.01, and a probability of 0.8 of observing 1 or higher if the true rate were 0.1 by binomial distribution. An independent safety monitor assessed all clinical and laboratory data.

Activity. A 50% reduction in SCORAD or regional SCORAD (SCORAD-50) was used as per prior publications (21, 24, 47, 48). The placebo response in SCORAD was calculated from placebo control data as approximately 5%–30% over studies lasting 1–12 months (22, 24, 49–51). The natural history of AD suggests that 50%–90% of patents will self-resolve by approximately 12–15 years of age (6, 7, 25). The pace at which a cohort more than 7 years of age would be expected to outgrow their disease is less than 5% per year based on previous reports (6, 7, 25). Thus, we calculated no more than 2 of our cohort to self-resolve over the course of a year; <2% of a cohort would be expected to self-resolve during the 6- to 16-week treatment timeframe. With 15 total participants, if 4 successes were observed at the end of the study then there would be a 0.9 probability (90% power) of concluding the treatment were active if the true activity rate were 0.4. These calculations were based on the binomial distribution.

R. mucosa pharmaceutical formulation

Preparations of R. mucosa for clinical use were produced under investigational new drug (IND) application 17303 from the Food and Drug Administration (FDA). Three isolates of R. mucosa taken from 3 HVs were grown in minimal media (R2A broth, Teknova; or Hanks Buffered Salt Solution, HBSS, Gibco) for 24–48 hours. Isolates were selected based on their ability to inhibit the growth of S. aureus, activate vitamin D pathways in human keratinocytes, and improve outcomes in mouse models of AD (17). Genomic sequencing was performed on all strains to verify that no transmittable, clinically significant antibiotic resistance genes were present. SNP-level assessment of strains is ongoing. The bacterial cells were washed 3 times in PBS (Gibco) and resuspended into 10%–15% sucrose in water for a concentration of 109 CFU/ml based on previously reported growth parameters (29). Serial dilutions were performed in 10%–15% sucrose to generate stocks of 104, 105, and 106 per ml. Aliquots of diluted bacterial samples were plated on R2A agar (Remel) and incubated at 32°C for 48–72 hours to enumerate prelyophilization CFU concentration. Starting CFU values were 90%–105% of expected concentrations. Eight hundred microliters (adult) or 1.5 ml (pediatric) of bacterial solution was frozen in 1.5-ml amber glass vials (Wheaton; adult) or a 3-ml self-contained sprayer system (Discount Vials; pediatrics) prior to lyophilization (Labconco). Vials/sprayers were sealed, labeled, and stored at –70°C until dispensed to the patients. Three aliquots per batch were reconstituted in sterile water and plated after serial dilution to enumerate postlyophilization CFU concentration. Survival was 93%–99% of starting CFU after lyophilization. These aliquots were also plated on soybean-casein digest agar (BD Bioscience), Sabouraud dextrose agar (Remel), MacConkey agar (BD Bioscience), xylose lysine agar (Remel), charcoal agar (BD Bioscience), and mannitol salt agar (Remel) and assessed for the presence of contaminating bacteria as per USP 61/62. No contamination was found in any batches of Roseomonas treatment. To avoid batch effects, all doses were derived and vialed in one session.

R. mucosa dosing and application

Initial dosing was based on the lowest dose with activity in prior mouse models (17). Twice weekly doses were selected due to the slow growth to R. mucosa and a desire to avoid applying new bacteria before the prior dose would reach stationary growth phase (29). Adult patients received vials of 2 ml of sterile water (Wheaton). Adults were trained on aspirating 800 μl of water using a 1-ml syringe (BD Bioscience). The 800 μl of water was injected into vials of the lyophilized bacteria, the stopper was replaced, and the vials were gently shaken for approximately 1 minute. The patients then aspirated 200–250 μl of the reconstituted bacterial solution and placed an atomizer tip on the syringe (MAD300, Teleflex). The solution was sprayed on the antecubital/forearm region of one arm, then repeated on the opposing side. The patients were allowed to use the remaining 300–400 μl of solution on additional body surface areas so long as they documented which areas they treated. In total, patients administered 1 application, twice per week, for a total of 6 weeks. During the initial 2 weeks, patients reconstituted vials of 104 CFU/ml; therefore, application of 200–250 μl indicated a treatment dose of 2 × 103 to 2.5 × 103 total bacterial colonies per surface area. After remote follow-up was performed to assure no complications with treatment, the patients progressed to 2 weeks of treatment with 2 × 104 to 2.5 × 104 bacterial colonies per surface area, then finally 2 × 105 to 2.5 × 105 total bacterial colonies per surface area. Patients returned for clinical assessment after completion of the full 6 weeks of therapy and were contacted remotely 30 days after treatment. Patients received hands-on training prior to dispensing the medication and were provided with an instructional video for later reference (https://youtu.be/LkWavevg22w).

Pediatric patients were provided bacteria lyophilized in a self-contained sprayer system (Discount Vials). Eyedroppers (United States Plastic Corp) of 1.5 ml sterile water were provided. For each dose, patients or their parents were instructed to empty the contents of the eyedropper into the sprayer vial, wait 2–5 minutes for reconstitution, and then spray. Sprayers were metered so that 3 pump sprays mirrored the 250 μl applied in the adult trial. Dose concentrations of CFU/ml were identical to adult dosing. Similar to adults pediatric patients applied the treatment twice weekly for the first 3 months of treatment, then every other day for the final month. Dose escalations were every 4 weeks after safety assessments.

Bacterial growth assessment

For patch assay evaluation, 109 CFU of R. mucosa or S. aureus were plated on R2A agar (Remel). Individual TRUETest (SmartPractice) plastic challenge squares were removed from the adhesive backing with sterile forceps under sterile conditions. The squares were placed in the center of a plate immediately after plating of bacteria. Plates for R. mucosa were incubated at 32°C for 48 hours before measuring resultant zone of inhibition with the electronic caliper (Mitutoyo America); plates of S. aureus were incubated at 32°C overnight prior to measurement. For studies evaluating parabens individually, methyl-, ethyl-, propyl, butyl-, and benzyl-4-hydroxybutyrate (Sigma-Aldrich) were suspended in 100% ethanol. To mirror the 1 mg/patch of the standardized disk, each individual paraben was diluted to a final concentration of 200 μg/10 μl. With a sterile pipette, 10 μl of each mix was placed in the center of an R2A agar plate coated with 109 CFU of R. mucosa or S. aureus. Blanched as well as potentially paraben-resistant isolates were subcultured by taking a sterile loop and plating on R2A agar — viability was assessed by presence of growth by 48 hours. Studies using commercial topical emollients were performed by placing 70 μg of each product onto a sterile circular coverslip and then placing the coverslip onto the agar culture as above. Colloidal oatmeal was diluted to 300 mg/ml, 20 μl was placed in the center of the agar plate and covered with a 12-mm cover slip as above. Pictures were taken with a Nikon D5300; contrast and brightness were enhanced evenly across all plates to aide visualization.

Bacterial metabolomics

The 3 strains of R. mucosa used in the clinical treatment formulation as well as 3 strains from patients with AD were isolated and enumerated as previously described (17, 29). Frozen pellets of 109 CFU were sent to The Proteomics & Mass Spectrometry Facility at the Danforth Plant Science Center as arranged through Science Exchange. Samples were analyzed using LC-MS by HILIC chromatography on a 0.5 × 150 mm Zic-pHILIC column using 10 mM NH 4 HCO 3 in water (A) and 10 mM NH 4 HCO 3 in 95% acetonitrile (B) as solvents and by RPLC chromatography using the same solvents and a 0.5 × 100 mm PLRPS column. The samples were extracted with 200 μl of 80% methanol by vortexing for 10 minutes. The solids were collected by centrifugation, and then the supernatant was filtered through a 0.8-μm spin filter. Samples were injected onto the LC-MS and data were acquired in polarity switching mode with data-dependent acquisition of MS/MS spectra. Data analysis was performed on Elements for metabolomics (Proteome Software Inc.). The ID Score was set to 0.8, the Log 10 Intensity threshold was set to 7, the Minimum Number of Samples, was set to 3. After the thresholds were established, the statistical differences between the healthy and disease samples were tested with an analysis of variance (ANOVA) among the precursor intensities of the identified metabolites. The ANOVA used a standard Benjamini-Hochberg procedure to control for the false discovery rate (FDR), at a significance level of q = 0.05. Then, a FWER correction was applied.

Mice

Experiments were performed in both male and female mice, but age and sex matched within each experiment. C57BL/6 mice were purchased from Jackson Laboratories. Intravenous injection with saline diluent or R. mucosa was performed when mice were 7–8 weeks of age. Weights were taken on days 0, 3, 6, and 10. On day 10, mice were sacrificed and the right kidney, spleen, and liver were removed. Tissues were processed, sectioned, and stained with hematoxylin and eosin (H&E) (HistoServ, Inc.). All tissues were evaluated by a board-certified veterinary pathologist and photomicrographs were taken using an Olympus BX51 microscope and Olympus DP73 camera.

Statistics

Significance was calculated by 2-tailed paired Student’s t test with Prism software (GraphPad). Calculations were repeated using nonparametric assumptions under Wilcoxon’s matched-pairs test assumptions. The larger P value from these calculations is presented. A P value of less than 0.05 was considered significant.

Study approval

Studies in humans were conducted under registered clinical trial NCT03018275 after approval from the NIAID institutional review board. All subjects were provided informed consent prior to their participation in the study. All murine experiments were done in compliance with the guidelines of the NIAID Institutional Animal Care and Use Committee.