Antibodies against the DNABII protein integration host factor (IHF) inhibit sinus implant biofilms

Abstract

Objectives:

Chronic rhinosinusitis is a common, costly condition often treated with endoscopic sinus surgery and intraoperative placement of intranasal sinus implant materials. While these materials aid in post-operative healing, they also support bacterial biofilm formation and thus contribute to negative outcomes. This study examined pre-treatment of sinus implant materials with antibody against an essential bacterial biofilm structural component, the DNABII family of DNA-binding proteins, as a strategy to prevent biofilm formation.

Methods:

Sinus implant materials were equilibrated in IgG-enriched antiserum against the DNABII protein integration host factor (IHF), individually or in combination with amoxicillin-clavulanate prior to inoculation with nontypeable Haemophilus influenzae (NTHI), a predominant pathogen of chronic rhinosinusitis. After 16 hours, the bacterial burden was quantitated and compared to pre-treatment with saline, IgG-enriched naive serum or amoxicillin-clavulanate alone.

Results:

NTHI readily formed biofilms on all three materials in vitro. However, pre-treatment of each material with IgG-enriched anti-IHF resulted in a significant decrease in bacterial burden compared to controls (P≤0.05). Moreover, a significant and synergistic outcome was achieved with a cocktail of anti-IHF plus amoxicillin-clavulanate (P≤0.05) with complete inhibition of NTHI biofilm formation on all three materials.

Conclusions:

Biofilm formation was well-supported in vitro on three sinus implant materials that vary in composition and resorption characteristics, however pre-treatment of each with DNABII protein targeted antibodies, in combination with a previously ineffective antibiotic, was highly effective to prevent the formation NTHI biofilms. These data demonstrate the potential for clinical utility of pre-treatment of sinus implant and additional surgical materials with anti-DNABII antibodies.

INTRODUCTION

Chronic rhinosinusitis (CRS) is a common condition with an estimated prevalence of 4.5–12% of individuals in the US.¹ CRS carries a significant economic burden as overall disease-related healthcare costs range between $6.9–9.9 billion USD per year.² Patients report a significant decrease in general health-related quality of life domains, which include diminished cognition and mood, and increased fatigue. Thus, indirect costs associated with overall decreased productivity are estimated to be $13 billion USD per year.^1,2 As CRS contributes to the continued rise in healthcare costs, there is significant interest to develop methods to reduce or preferably prevent the aforementioned symptoms.

Although various definitions are reported, the disease process of CRS is widely characterized by mucosal inflammation of the paranasal sinuses that includes nasal obstruction and drainage that persists for at least 12 weeks.³ Whereas multiple theories exist to explain the pathogenesis of CRS, there is controversy as to potential etiologies, associated conditions and common inflammatory mediators.³ However, there is evidence that bacteria play a significant role in the inflammatory process, as reported by a national Otolaryngology-Head and Neck Surgery joint task force review.³ Furthermore, recent studies demonstrate the presence of bacterial biofilms in samples obtained from CRS patients, and identify nontypeable Haemophilus influenzae (NTHI) biofilms on over 75% of samples, which suggested a role for these recalcitrant bacterial communities in the pathogenesis and chronicity of this disease.^4–9

Part of the treatment algorithm for CRS is endoscopic sinus surgery (ESS). ESS is often combined with intraoperative use of sinus implants or nasal packing materials to aid in hemostasis, decrease synechiae and prevent lateralization of the middle turbinate. Use of sinus implant materials is controversial, as the materials can serve as a nidus for inflammation and infection, cause postoperative pain and discomfort, and induce mucosal trauma when removed from the nasal cavity.¹⁰ Moreover, several studies demonstrate lack of significant clinical benefit.^11,12 Confounding their value is that these porous, mesh-like materials provide an opportune environment for bacterial biofilm formation and persistence. Furthermore, clinicians often resort to numerous rounds of antibiotics to treat chronic bacterial infections post-ESS, which consequently contributes to the problematic rise in multiple antibiotic-resistant bacteria worldwide. These factors can contribute to continued mucosal inflammation, infection, persistent postoperative symptoms and antibiotic-resistant bacterial strains, which results in greater post-operative visits and healthcare costs.^13–15

Biofilms are known to contribute to the chronicity of many bacterial diseases within the otolaryngologic field such as chronic and recurrent otitis media, otitis media with effusion, tonsillitis, and post-tympanostomy otorrhea.^16–20 Biofilms are communities of bacteria, encased in a self-produced extracellular polymeric substance (EPS) which provides protection for resident bacteria from both antimicrobials and host immune effectors.²¹ This EPS is typically comprised of proteins and polysaccharides, however a very common major component of the biofilm EPS is extracellular DNA, arranged in a lattice-like structure.²² At each crossed strand of DNA is a member of the DNABII family of DNA-binding proteins, of which there are only two members: integration host factor (IHF) and histone-like protein (HU).²³ Antibodies directed against DNABII proteins induce biofilm collapse of all 21 bacterial species tested to date, which includes the high-risk ESKAPE pathogens in vitro.^24–28 Moreover, resolution of biofilms is shown in multiple experimental models of disease and include periodontitis/peri-implantitis,²⁹ NTHI-induced otitis media^25,28,30 and Pseudomonas aeruginosa-induced lung infection.³⁰ DNABII proteins are also detected in multi-species clinical specimens from patients with cystic fibrosis,³¹ otitis media with effusion,³² and within skin at the site of surgical incision after Cesarean delivery.³³

Specific to biofilm formation on sinus packing materials, we’ve shown that NTHI readily forms a community of adherent bacteria that fulfill the definition of a ‘biofilm’ on Nasopore® in vitro and that these biofilms are eradicated by incubation with antibody against the DNABII proteins but not by use of antibiotic alone.³⁴ However, prevention of biofilm formation on sinus implant materials is not yet described. Thus, the objective of this study was to determine if pre-treatment of Nasopore®, Posisep®X and Merocel® with antibody against DNABII proteins prevented NTHI biofilm formation, and if when combined with a commonly-prescribed antibiotic for CRS, amoxicillin-clavulanate, a synergistic reduction or complete eradication of the biofilm would result.

MATERIALS AND METHODS

Pretreatment of Sinus Implant Materials

All sinus implant materials were removed from packaging and sectioned under sterile conditions. Nasopore®, Posisep®X and Merocel® were sectioned into 2.0×2.0×1.5mm cubes with a sterile scalpel, then placed into individual wells of 8-chambered coverglass slides. Pre-treatments included sterile Dulbecco’s phosphate-buffered saline (DPBS), 15 μg IgG-enriched naive rabbit serum (NRS), 15 μg IgG-enriched rabbit anti-IHF_NTHI, 0.1 μg amoxicillin-clavulanate/ml, 0.25 μg amoxicillin-clavulanate/ml, 0.5 μg amoxicillin-clavulanate/ml, 1.0 μg amoxicillin-clavulanate/ml or IgG-enriched antibody plus antibiotic for 30 minutes at 25°C.

Bacterial Strain and Culture Conditions

NTHI 86–028NP is a clinical isolate recovered from the nasopharynx of a child with chronic otitis media with effusion, used herein due to its clinical relevance and extensive use in in vitro and in vivo studies. NTHI that constitutively expresses green fluorescent protein (NTHI strain 86–028NP/pRSM2211)³⁵ was grown on chocolate agar supplemented with 20 μg kanamycin/ml and incubated at 37°C, 5% CO₂, in a humidified atmosphere for 18 hours. Colonies were inoculated into brain-heart infusion (BHI) broth supplemented with 2 μg each of β-NAD and heme per ml (sBHI) and incubated to achieve mid-log phase growth. One-thousand CFU NTHI were added to 200 μl of either DPBS, IgG-enriched NRS, anti-IHF_NTHI, amoxicillin-clavulanate or antibody plus antibiotic. This entire bacterial suspension was applied to the pretreated sinus implant cube and incubated static for 16 hours at 37°C, 5% CO₂, humidified atmosphere.

Quantitation of Bacteria in Bioresorbable Sinus Implant Materials

After 16 hours of incubation as described, bioresorbable materials (Nasopore® and Posisep®X) were transferred into sterile microcentrifuge tubes, washed with DPBS to remove non-adherent bacteria, then crushed with Dounce homogenizers and sonicated to release biofilm-resident bacteria. The liquid-cube homogenate was then serially diluted and plated on chocolate agar for quantitation of bacterial load. All assays described herein were repeated a minimum of 3 times.

Quantitation of Bacteria in Non-bioresorbable Sinus Implant Materials

For the non-bioresorbable material tested herein (Merocel®), after 16 hours the inoculated sinus implant cubes were transferred to a microcentrifuge tube, washed with DPBS to remove non-adherent bacteria, and incubated for an additional 24 hours in sBHI, static, 37°C, 5% CO₂, humidified atmosphere to allow biofilm-resident bacteria to multiply. Cubes were then crushed with Dounce homogenizers, sonicated and serially diluted and plated on chocolate agar as above.

Imaging Bacterial in Sinus Implant Materials

A set of replicate cubes were prepared specifically for image analysis to support bacterial load determinations. As such, cubes were cut, pretreated and inoculated as detailed above. A NTHI luminescent reporter was used (NTHI 86–028NP/pKMLN-1)³⁶ to overcome the non-specific autofluorescence of these materials. After 16 hours, Nasopore® cubes were transferred, washed and homogenized as mentioned above, and the liquid-cube suspension transferred to an 8-well chambered coverglass. Posisep®X cubes were transferred to chambered coverglass and washed as before; this material does not spontaneously dissolve overnight, therefore biofilms were directly imaged within the intact cube. Merocel® cubes were transferred to chambered coverglass, washed and incubated for an additional 24 hours in sBHI as mentioned previously. Each cube was then imaged via IVIS® Spectrum imaging system to detect the luminescent signal within each homogenate/cube.

Quantitation of Luminescence

Images were analyzed with Living Image® software and average radiance due to luminescence measured on a scale to allow for direct comparison between replicate assays. A region of interest (ROI) of identical size was selected for each cube. Non-specific background luminescent signal from a sterile cube pretreated with DPBS (no bacteria) was subtracted from each average radiance reading.

Statistical Analyses

Statistical significance among treatments was determined by One-way ANOVA with Dunnet’s multiple comparisons test using GraphPad Prism® 8 software (GraphPad Software, Inc). A P≤0.05 was considered significant.

RESULTS

Titration of Antibody with Nasopore® as a Test Material

Nasopore® was used as a test material, since it is one of the most commonly used materials used after endoscopic sinus surgery and is a representative material to establish a working model for other materials used post-operative recovery. Four concentrations of IgG-enriched anti-IHF_NTHI were tested in an effort to establish the optimal antibody concentration that would prevent biofilm formation on the material. At each concentration tested, anti-IHF_NTHI significantly prevented NTHI biofilm formation compared to negative controls (P≤ 0.05)[Fig. 1]. Pre-treatment with 15 μg of IgG-enriched anti-IHF_NTHI was deemed optimal to consistently yield a significant reduction in bacterial load (ideally ~50%) on all three implant materials to be tested when compared to material maintained in DPBS (P≤0.001) or IgG from NRS (P≤ 0.05), yet still allow for some growth so we could later assess the synergy between antibody pre-treatment plus use of a commonly-used, broad-spectrum antibiotic. These results demonstrated the effectiveness of anti-IHF pre-treatment to prevent NTHI biofilm formation at all concentrations tested herein.

Figure 1:

Pre-treatment with IgG-enriched anti-IHF_NTHI at four titrated concentrations significantly prevented biofilm formation on Nasopore® compared to pre-treatment with DPBS or IgG from NRS. While each concentration of IgG-enriched anti-IHF_NTHI tested was effective to prevent biofilm formation, 15 μg was selected for the studies presented herein, as this dose allowed for the ability to demonstrate synergy between IgG-enriched anti-IHF_NTHI plus antibiotic. *, P≤0.05; **P≤0.01; ****P≤0.0001.

Prevention of NTHI Biofilms on Nasopore®

We next determined whether IgG-enriched anti-IHF_NTHI would be effective to prevent biofilm formation on Nasopore® and further, whether the antibody pretreatment would be preferential to antibiotic treatment alone. In fact, pretreatment of Nasopore® with IgG-enriched anti-IHF_NTHI significantly reduced biofilm formation 2-log fold compared to pretreatment with DPBS or IgG-enriched NRS, which each allowed growth of ≥10⁵ CFU NTHI/cube (P≤0.05)[Fig. 2]. Additionally, pretreatment with amoxicillin-clavulanate yielded a slight reduction in bacterial load, whereas a significant and synergistic effect was shown by pretreatment with IgG-enriched anti-IHF_NTHI plus 0.5 μg antibiotic/ml H₂O(P≤0.01). Notably, complete prevention of biofilm formation was achieved by pretreatment of Nasopore® with IgG-enriched anti-IHF_NTHI plus 1.0 μg amoxicillin-clavulanate/ml water(P≤0.05). These results highlighted the efficacy of anti-IHF pre-treatment to prevent NTHI biofilm formation and demonstrated a synergistic effect of anti-IHF plus amoxicillin-clavulanate pre-treatment to completely inhibit biofilm formation.

Figure 2:

Synergy between 15 μg IgG-enriched anti-IHF_NTHI plus amoxicillin-clavulanate prevented NTHI biofilm formation on Nasopore® at two different tested antibiotic concentrations; 0.5 and 1.0 μg amoxicillin-clavulanate /ml. At both concentrations, synergy was observed to prevent of biofilm formation on Nasopore® compared to either DPBS or IgG-enriched NRS, each delivered with antibiotic. 15 μg IgG-enriched anti-IHF_NTHI alone significantly prevented biofilm formation on Nasopore® compared to DPBS or IgG from NRS, which demonstrated the effectiveness of anti-IHF_NTHI to prevent biofilm formation, even without antibiotic present. When combined with antibiotic, anti-IHF pre-treatment completely inhibited biofilm formation, whereas antibiotic alone was ineffective. *, P≤0.05; **P≤0.01.

Prevention of NTHI Biofilms on Posisep®X

Similarly, we wanted to consider other sinus implant materials to see if a comparable outcome effect could be achieved. Once again, biofilm formation was prevented when Posisep®X was pretreated with IgG-enriched anti-IHF_NTHI, which reduced biofilm formation 2-log fold and was significant compared to pretreatment with either DPBS or IgG-enriched NRS (P≤0.01)[Fig. 3]. Furthermore, there was a significant and synergistic effect observed when Posisep®X was pretreated with IgG-enriched anti-IHF_NTHI plus 0.1 μg or 0.25 μg amoxicillin-clavulanate/ml H₂O compared to antibiotic alone (P≤0.001 or P≤0.05, respectively). These latter pre-treatments completely prevented biofilm formation and reduced the bacterial burden 3-log fold compared to negative controls on Posisep®X. These data demonstrated that pre-treatment of Posisep®X with anti-IHF alone is effective to prevent biofilm formation and that this antiserum acted synergistically with antibiotic to completely prevent biofilm formation on an additional commonly-used sinus implant material.

Figure 3:

Synergy between 15 μg IgG-enriched anti-IHF_NTHI and amoxicillin-clavulanate to prevent NTHI biofilm formation on Posisep®X at two different tested antibiotic concentrations; 0.1 and 0.25 μg amoxicillin-clavulanate/ml. At both concentrations, synergy was observed to completely prevent biofilm formation on Posisep®X compared to DPBS plus antibiotic or IgG-enriched NRS plus antibiotic. 15 μg IgG-enriched anti-IHF_NTHI alone significantly prevented biofilm formation on Posisep®X compared to DPBS or NRS, which showed the effectiveness of anti-IHF_NTHI pre-treatment, even without antibiotic. Furthermore, antibiotic alone was ineffective to prevent biofilm formation, whereas pre-treatment with anti-IHF_NTHI plus amoxicillin-clavulanate completely inhibited biofilm formation. *, P≤0.05; **, P≤0.01; ***, P≤ 0.001; ****P≤0.0001.

Prevention of NTHI Biofilms on Merocel®

Lastly, we wanted to test the effectiveness of pre-treatment with IgG-enriched anti-IHF_NTHI on a non-bioresorbable material, such as Merocel® and again, determine if this pretreatment would synergize with antibiotic to prevent biofilm formation. Here however, the effect was even more marked as pretreatment of Merocel® with IgG-enriched anti-IHF_NTHI alone reduced biofilm formation 4-log fold compared to pretreatment of DPBS or IgG-enriched NRS (P≤0.05)[Fig. 4]. Similarly, pretreatment of Merocel® with IgG-enriched anti-IHF_NTHI plus 0.5 μg amoxicillin-clavulanate/ml H₂O reduced biofilm formation 5-log fold and completely prevented any biofilm formation whereas antibiotic alone had failed. These results showed the efficacy of pre-treatment of anti-IHF to prevent biofilm formation and the ability of anti-IHF pre-treatment to act synergistically with antibiotic to completely prevent biofilm formation on a third clinically-relevant, but compositionally different, sinus implant material.

Figure 4:

Synergy between 15 μg IgG-enriched anti-IHF_NTHI and amoxicillin-clavulanate to prevent NTHI biofilm formation on Merocel® with 0.5 μg amoxicillin-clavulanate/ml. Synergy was observed to completely prevent biofilm formation on Merocel® compared to DPBS plus antibiotic or IgG-enriched NRS plus antibiotic. 15 μg IgG-enriched anti-IHF_NTHI alone significantly reduced biofilm formation on Merocel® compared to DPBS or NRS, which demonstrates the utility of anti-DNABII pre-treatment to prevent biofilm formation without the use of an antibiotic. *, P≤0.05.

Imaging of NTHI Biofilms on Nasopore®

To support quantitative data with that derived via image analysis, we also visualized biofilm formation on each pretreated Nasopore® cube after cubes were inoculated with a luminescent reporter isolate of NTHI followed by assay with an IVIS® imaging system. Imaging data show Nasopore® cubes pretreated with 15 μg of IgG-enriched anti-IHF_NTHI had a significant 4-fold reduced average radiance compared to DPBS or IgG-enriched NRS-pretreated cubes(P≤0.05), which complemented prior data[Fig. 5]. Similarly, pretreatment of Nasopore® cubes with a combination of 15 μg of IgG-enriched anti-IHF_NTHI plus 1.0 μg of amoxicillin-clavulanate/ml H₂O, completely prevented biofilm formation, which compared to use of antibiotic alone, was significant (P≤0.05). These results further supported quantitative data to show pre-treatment with anti-IHF alone was effective to prevent biofilm formation on Nasopore® and when combined with amoxicillin-clavulanate, completely prevented biofilm growth on the material.

Figure 5:

Luminescent image of homogenized Nasopore® cubes pretreated with either DPBS, 15 μg IgG-enriched NRS or 15 μg IgG-enriched anti-IHF_NTHI alone (A, B, and C, respectively) or plus 1.0 μg amoxicillin-clavulanate/ml (E, F and G, respectively). A cube pretreated with DPBS (no bacteria) was used as a negative control to subtract out background luminescent signal (D). Quantitation of luminescent signal (H). Cubes pretreated with 15 μg IgG-enriched anti-IHF_NTHI both with or without antibiotic had significantly less luminescent signal compared to negative controls or use of antibiotic alone. We detected no luminescent signal from cubes treated with IgG-enriched anti-IHF_NTHI plus amoxicillin-clavulanate, which showed anti-IHF plus antibiotic acted synergistically to prevent biofilm formation. These results showed pre-treatment with anti-IHF_NTHI alone is effective to prevent biofilm formation, and when combined with antibiotic, completely inhibited biofilm formation on this material. *, P≤0.05; **, P≤0.01.

Imaging of NTHI Biofilms on Posisep® X

To capture representative images to support the count data findings for Posisep®X above, cubes were images as described. Image analysis showed that pretreatment with 15 μg of IgG-enriched anti-IHF_NTHI significantly prevented biofilm formation in Posisep® X cubes compared to negative control DPBS or NRS as represented by a 5-fold reduction in average radiance (P≤0.05)[Fig. 6]. Furthermore, the combination of 15 μg of IgG-enriched anti-IHF_NTHI plus 0.1 μg of amoxicillin-clavulanate/ml H₂O completely prevented biofilm formation on the material and had a 4-fold reduction in average radiance signal compared to antibiotic alone (P≤0.05). These results supported our earlier quantitative data and visually demonstrated that pre-treatment with anti-IHF alone significantly prevented biofilm formation and when combined with antibiotic, completely inhibits biofilm formation.

Figure 6:

Luminescent image of Posisep®X cubes pretreated with DPBS, 15 μg IgG-enriched NRS or 15 μg IgG-enriched anti-IHF_NTHI alone (A, B, and C, respectively) or plus 0.1 μg amoxicillin-clavulanate/ml (E, F and G, respectively). Quantitation of luminescent signal (H). A cube pretreated with DPBS and no bacteria was used as a negative control to subtract out background luminescent signal (D). Cubes pretreated with 15 μg IgG-enriched anti-IHF_NTHI both with or without antibiotic had significantly less luminescent signal compared to negative controls or antibiotic alone. Synergy between IgG-enriched anti-IHF_NTHI plus amoxicillin-clavulanate showed no luminescent signal, which demonstrated complete inhibition biofilm formation. These data demonstrated the efficacy of anti-DNABII pre-treatment to significantly prevent biofilm formation on sinus implant materials and showed pre-treatment with anti-IHF plus antibiotic completely inhibited biofilm formation. *, P≤0.05; **, P≤0.01.

Imaging of NTHI Biofilms on Merocel®

Upon similar image analysis of Merocel®, we found pretreatment with 15 μg of IgG-enriched anti-IHF_NTHI significantly decreased bacterial burden on the material with a 2-fold reduction in average radiance signal compared to DPBS or IgG-enriched NRS pre-treatments(P≤0.05)[Fig. 7]. No luminescent signal was detected in the Merocel® cube pretreated with 15 μg of IgG-enriched anti-IHF plus 0.5 μg amoxicillin-clavulanate/ml H₂O compared to amoxicillin-clavulanate alone, wherein we were still able to detect a radiance signal(P≤0.05). These results supported our quantitative data and highlighted the efficacy of pre-treatment with anti-IHF to significantly prevent biofilm formation and demonstrate the synergy of anti-IHF and antibiotic to completely inhibit biofilm growth.

Figure 7:

Luminescent image of Merocel® cubes pretreated with either DPBS, 15 μg IgG-enriched NRS or 15 μg IgG-enriched anti-IHF_NTHI alone (A, B, and C, respectively) or plus 0.5 μg amoxicillin-clavulanate/ml (E, F and G, respectively). A cube pretreated with DPBS and no bacteria was used as a negative control to subtract out background luminescent signal (D). Quantitation of luminescent signal (H). There was less luminescent signal in cubes pretreated with 15 μg IgG-enriched anti-IHF_NTHI both with or without antibiotic compared to negative controls or antibiotic alone. Synergy between IgG-enriched anti-IHF_NTHI plus amoxicillin-clavulanate showed no luminescent signal, which demonstrated anti-IHF_NTHI plus antibiotic completely prevented biofilm formation on the material. These results highlighted the effectiveness of anti-DNABII pre-treatment to prevent biofilm formation on various sinus implant materials and therefore improve patient outcomes post-surgically. *, P≤0.05; **, P≤0.01.

Comparisons among materials tested

NTHI biofilms readily formed on Nasopore®, Posisep®X and Merocel® sinus implant materials. Pretreatment of each material with IgG-enriched anti-IHF_NTHI that targets a critical structural component of biofilms, was highly effective to prevent biofilm formation on all three materials. A significant and synergistic preventative outcome was achieved by pre-treatment of each material with IgG-enriched anti-IHF plus amoxicillin-clavulanate, whereas antibiotic alone was ineffective. Notably, when 15 μg of anti-IHF was combined with amoxicillin-clavulanate for pre-treatment of Nasopore®, Posisep®X or Merocel® (amoxicillin-clavulanate used at 1.0, 0.1 or 0.5 μg/ml H₂O, respectively), there was complete inhibition of biofilm formation.

DISCUSSION

CRS is a highly prevalent, costly, and challenging condition to treat. The pathogenesis that underlies CRS is controversial and continues to be investigated. Biofilms are present in CRS⁷ and are highly likely to contribute a significant portion to the pathophysiology, chronicity and recurrence of CRS given the recalcitrant biology of bacterial biofilms,³⁷ which correlates to the resistance of CRS to traditional antibiotic therapies.³⁸ Additionally, antibiotic resistance continues to rise, which causes complications in medical therapeutic options.^39,40 ESS has been shown to have positive quality of life and cost-effective outcomes^{1, 2} and provides an alternative to chronic medical therapy. As technology and knowledge surrounding ESS continues to improve, this advancement in our understanding will support the development of novel and powerful adjunctive therapies to enhance postoperative outcomes.

Sinus implant materials are widely marketed to reduce postoperative edema, adhesions, and bleeding. However, many studies show a lack of significant differences compared to traditional nasal saline irrigations.^2,11,41 Additionally, we have shown that these materials also support the formation of communities of bacteria that fit the criteria of a ‘biofilm’ and are not simply adherent to the surface of this material,³⁴ likely due to the porous, mesh-like material, which facilitates penetration of the material with development of 3D communities of bacteria. Thus, to insert a foreign material that supports biofilm formation into a patient with a predisposition to chronic inflammation and infection, we speculated the potential for undesirable consequences to the sinonasal environment due to the ability of these materials to further perpetuate ongoing inflammation and infection.

This study demonstrated prevention of biofilm formation on three sinus implant materials of varying composition and resorption character and expanded upon our prior work.³⁴ Furthermore, these data are the first to show that anti-IHF, when combined with antibiotics, completely prevents the formation of biofilms on these materials. Moreover, we are the first to demonstrate that pre-treatment of several diverse sinus implant materials with anti-DNABII protein prevented biofilm formation, even without additional treatment with an antibiotic.^25,34 While anti-DNABII treatment is highly effective to prevent biofilm formation and disrupt preexisting biofilms, this treatment does not kill the bacteria.²⁵ Therefore, a combinational approach of anti-DNABII treatment plus antibiotic might be most prudent for clinical use in post-surgical sites.

Our results highlight the potential clinical benefit of either pre-treatment or coating of surgical materials with antiserum directed against DNABII proteins to prevent the formation of biofilms, which would thereby decrease the likelihood of post-surgical complications and postoperative visits after endonasal surgical procedures. Ultimately, pre-treatment of sinus implant materials with anti-DNABII proteins could decrease healthcare utilization and healthcare costs associated with CRS, which are substantial socioeconomic burdens to healthcare worldwide. Whereas here we used an antibody directed against a DNABII protein derived from a model organism for CRS, our prior work has shown its effectiveness against numerous other species of bacterial biofilms, which demonstrates this treatment as a general anti-biofilm therapeutic for many biofilm-related disease states.^24–28 To further expand on the application of our results, we speculate there may be a role for anti-DNABII protein-coating for prevention of biofilm formation on other medical devices predisposed to infection such as urinary catheters, intravascular catheters, and prosthetics,⁴² among others. The development of novel therapeutic and/or preventative agent(s) that targets DNABII proteins as essential structural elements of the pathogenic biofilms that contribute to a number of chronic and recurrent disease states, could have a significant clinical impact and as such are the focus of continued development by our lab.

CONCLUSION

CRS is a recalcitrant, costly disease process often treated with ESS and sinus implant materials. These materials serve as an ideal nidus for biofilm formation and persistence which can contribute to negative post-operative outcomes. Pre-treatment with antibody directed against DNABII proteins was superior to antibiotic alone to prevent the formation of NTHI biofilms on three materially-different, clinically-relevant sinus implant materials when tested in vitro. In all cases, anti-IHF_NTHI plus antibiotic acted synergistically to inhibit and/or completely inhibit the formation of NTHI biofilms which suggests that with further dose optimization complete inhibition could be achieved for all tested sinus implant materials. It remains to be shown if such efficacy can also be achieved in vivo, where we acknowledge that additional challenges to development are likely to be encountered. However, these findings are highly encouraging to our ongoing efforts to attempt to vastly improve patient post-operative recovery after sinus surgery and pave the path for anti-DNABII targeted approaches as a clinically-beneficial adjunct to use of various surgical materials throughout the body.