The Centers for Disease Control and Prevention (CDC) label the rapid global growth of 51 drug-resistant pathogens "one of our most serious health threats" [1] . The World Health 52 Organization (WHO) also warns that "without urgent, coordinated action by many stakeholders, local, high doses of antibiotics that can be applied regularly to sustain antimicrobial delivery.
Topical delivery also helps maintain a moist wound bed, which facilitates the prevention of 95 tissue dehydration, accelerates angiogenesis, assists in the breakdown of necrotic tissue and/or 96 fibrin, and provides for the transport of cytokines and growth factors [36, 37] . 2) Development of 97 novel antimicrobial agents that address the current global threat of antibiotic resistance. 98 We tested the in vitro efficacy of a topical formulation, the active component of which is 99 a compound synthesized as part of a unique first-in-class series of antibiofilm agents (referred to 100 as CZ compounds). More specifically, CZs are designed and synthesized to specifically wise over 20 min. The solution was stirred for 16 h. NaBH 4 (0.95 g, 24.9, 1 equiv.) was added 151 portion-wise over 20 min and the reaction stirred for an additional 1 h. The solvent was 152 evaporated, and the crude solid partitioned between EtOAc (500 ml) and 10% NaOH (250 ml).
The NaOH phase was washed with EtOAc (500 ml), and the combined organics were dried over 154 Na 2 SO 4 . Column chromatography was performed using gradient conditions starting at (300 Approximately 800 mg of topical agent were spread in a thin layer, i.e., "buttered" on 198 sterile 2" x 2" cotton gauze. The "buttered" side of the gauze pad was placed in contact with the 199 discs such that all n=8 discs were covered completely. Three additional gauze pads were placed Cellulose discs were sterilely removed and placed individually into 1 mL of PBS.
Samples were vortexed for 1 min, sonicated for 10 min at 42 kHz and plated using a 10-fold Page 10 of 27 209 dilution series to quantify the CFU/disc that remained after treatment. Positive controls of growth 210 (n=8) with no treatment were also quantified for comparison.
The same growth protocol as outlined above was used to test efficacy of topical products 212 against polymicrobial biofilms. However, inocula concentrations were varied to grow MRSA 213 and P. aeruginosa as polymicrobial biofilms. When the two isolates were inoculated at 1:1 or 214 even 1:1,000 ratio, P. aeruginosa overwhelmed the MRSA isolate. As such, a 1:10,000 ratio was 215 used; MRSA was inoculated at a concentration 10,000 times higher than P. aeruginosa. Each 216 isolate was suspended to a turbidity of 10% using a nephelometer (concentration equated to ~1 x 217 10 9 CFU/mL). MRSA was diluted 1:1,000 (~1 x 10 6 CFU/mL) and P. aeruginosa was diluted 218 1:10,000,000 (~1 x 10 2 CFU/mL) using a 10-fold dilution series. Twenty-five μL of each 219 solution were pipetted onto cellulose discs for a total of 50 μL per sample. Polymicrobial biofilm 220 growth was quantified as described above to obtain a baseline of CFU/disc. were drilled half way through the rod (Fig. 1C) . HeliPlug™ collagen coupons were sterilely cut 248 to size (5 mm x 10 mm), and pressed into each bored-out cavity of a rod (Fig. 1C) . Assembly 249 was performed in a biosafety cabinet to maintain sterility. Five-hundred mL of brain heart infusion (BHI) broth were added to the reactor after it 251 was assembled. The broth was aseptically inoculated with 10 5 CFU/mL (adjusted from 0.5
McFarland standard) of MRSA or P. aeruginosa for monomicrobial biofilm growth. The reactor 253 was set on a hot plate at 34°C and a baffle rotation of 130 rpm. Bacteria were grown in batch phase for 24 h, after which a 10% solution of BHI was flowed through the reactor at a rate of 255 6.94 mL/min using a peristaltic pump (MasterFlex L/S Microbore, Cole Palmer, Vernon Hills, 256 IL) for an additional 24 h (Fig 1A) .
The inoculation protocol to grow polymicrobial biofilms on collagen was similar to 258 cellulose; each isolate was suspended to a 0.5 McFarland Standard (~1 x 10 8 CFU/mL). MRSA 259 was diluted 1:1,000 (~1 x 10 5 CFU/mL) and P. aeruginosa was diluted 1:10,000,000 (~1 x 10 260 CFU/mL). However, we determined experimentally that polymicrobial biofilms grew more Polymicrobial biofilms were otherwise grown as described for monomicrobial growth. The 48-h biofilm growth protocol for cellulose produced well-established, mature 295 monomicrobial and polymicrobial biofilms of both MRSA and P. aeruginosa ( Fig. 2A-2C ).
However, quantification outcomes following efficacy analyses were highly variable, in particular 297 with the clinically-relevant products (data not shown as it was sporadic at best). We established a 298 sub-hypothesis after observing the inconsistent outcomes: we hypothesized that topical 299 treatments failed to reach the biofilms that formed on the underside of the cellulose disc (immediately adjacent to the surface of the agar), and the lack of exposure in that region led to 301 highly variable quantification data. 
To test the sub-hypothesis, biofilms were grown on cellulose following the same growth 313 protocol outlined above. SEM and CLSM imaging was performed to determine: 1) if biofilms 314 formed on the underside of the cellulose fiber network that was in apposition to the agar surface, 315 and 2) if those biofilms on the underside of cellulose discs were still viable following the topical 316 product delivery protocol.
SEM imaging confirmed the presence of biofilms on, within, and between the interstices 318 of the fibers on the underside of cellulose discs (Figs. 2 and 3) . Live/Dead imaging also indicated Page 15 of 27 319 that biofilms were viable on all surfaces of cellulose discs, but only surfaces in direct contact 320 with topical agents showed cell death; bacteria on the underside and center of cellulose discs 321 stained green (living), supporting our sub-hypothesis that bacteria on untreated surfaces were 322 still viable and were not exposed to topical product treatments (Figs. 4 and 5) [43]. 
Despite the limitation of this method, Live/Dead staining provided some useful 356 information on topical efficacy. Confocal imaging and staining indicated that CZ-01179 was 357 highly effective against well-established biofilms that were exposed to the formulated gel, 358 whereas clinical products had limited efficacy (Fig 5) . These outcomes provided rationale for 359 performing analysis on collagen coupons. Biofilms on collagen grew to maturity (Fig. 2) , and SEM images indicated more robust 362 biofilm formation compared to cellulose discs. Quantification of positive controls supported this 363 observation with ~1 log 10 more CFU/coupon compared to cellulose discs for both isolates.
Quantification data from efficacy testing against biofilms on collagen are reported in 365 Table 1 . Outcomes indicated that of the clinical standards of care, gentamicin was most effective 366 against both monomicrobial and polymicrobial biofilms of MRSA and of P. aeruginosa (Fig. 6   367 and Table 1 ). Gentamicin showed a log 10 reduction of 3.56 CFU/collagen in monomicrobial 368 biofilms of MRSA, and against polymicrobial biofilms it was effective against MRSA with a 369 log 10 reduction of 5.21 CFU/collagen. Against both monomicrobial and polymicrobial biofilms, 370 gentamicin showed complete eradication of P. aeruginosa, with no detectable growth (Table 1) .
At all three concentrations (0.5%, 1%, 2%) CZ-01179 reduced all monomicrobial and 372 polymicrobial biofilms to below detectable levels ( Fig. 6 and Table 1 (Table 1) . Retapamulin-treated MRSA biofilms had a log 10 reduction 383 of 1.62 CFU/collagen (Table 1) . respectively. The same was observed for polymicrobial biofilms; silver sulfadiazine showed 3.74 395 log 10 reductions and Neosporin® showed no reduction (Table 1 and Fig. 6 ). These data are of topical products this led to variable outcomes, complicating data interpretation. We conclude 411 that this method may not be ideal for assessing efficacy of topical products unless it is modified 412 to control for the lack of exposure to biofilms that are adjacent to the agar surface.
CZ-01179 gels (all three concentrations) had equal efficacy against P. aeruginosa 414 biofilms as gentamicin in the collagen test (Fig. 6) . CZ-01179 gels were more efficacious at 415 eradicating biofilms in all other cases when compared to the standard of care topicals in the 416 collagen tests (Fig. 6) . Gentamicin had the greatest log 10 reduction against monomicrobial 417 biofilms of P. aeruginosa and polymicrobial biofilms amongst the clinical standards of care.
These data were promising, but broader-scale consideration is given in clinical context; 