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. Whereas these materials aid in postoperative healing, they also support bacterial biofilm formation and thus contribute to negative outcomes. This study examined pretreatment 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 immunoglobulin G (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 pretreatment with saline, IgG-enriched naive serum, or amoxicillin-clavulanate alone.

Results: NTHI readily formed biofilms on all three materials in vitro. However, pretreatment 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, pretreatment 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 pretreatment of sinus implant and additional surgical materials with anti-DNABII antibodies.

Level of evidence: NA Laryngoscope, 130:1364-1371, 2020.

Keywords: Chronic rhinosinusitis; antibiotics; endoscopic sinus surgery; nontypeable Haemophilus influenzae; sinus implant.

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.

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.

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.

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.

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.

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.

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.