Fusion peptide P15-CSP shows antibiofilm activity and pro-osteogenic activity when deposited as a coating on hydrophilic but not hydrophobic surfaces.

In the context of porous bone void filler for oral bone reconstruction, peptides that suppress microbial growth and promote osteoblast function could be used to enhance the performance of a porous bone void filler. We tested the hypothesis that P15-CSP, a novel fusion peptide containing collagen-mimetic osteogenic peptide P15, and competence-stimulating peptide (CSP), a cationic antimicrobial peptide, has emerging properties not shared by P15 or CSP alone. Peptide-coated surfaces were tested for antimicrobial activity toward Streptoccocus mutans, and their ability to promote human mesenchymal stem cell (MSC) attachment, spreading, metabolism, and osteogenesis. In the osteogenesis assay, peptides were coated on tissue culture plastic and on thin films generated by plasma-enhanced chemical vapor deposition to have hydrophilic or hydrophobic character (water contact angles 63°, 42°, and 92°, respectively). S. mutans planktonic growth was specifically inhibited by CSP, whereas biofilm formation was inhibited by P15-CSP. MSC adhesion and actin stress fiber formation was strongly enhanced by CSP, P15-CSP, and fibronectin coatings and modestly enhanced by P15 versus uncoated surfaces. Metabolic assays revealed that CSP was slightly cytotoxic to MSCs. MSCs developed alkaline phosphatase activity on all surfaces, with or without peptide coatings, and consistently deposited the most biomineralized matrix on hydrophilic surfaces coated with P15-CSP. Hydrophobic thin films completely suppressed MSC biomineralization, consistent with previous findings of suppressed osteogenesis on hydrophobic bioplastics. Collective data in this study provide new evidence that P15-CSP has unique dual capacity to suppress biofilm formation, and to enhance osteogenic activity as a coating on hydrophilic surfaces.

Antibiofilm Efficacy of DispersinB(®) Wound Spray Used in Combination with a Silver Wound Dressing.

Chronic wounds including diabetic foot ulcers, pressure ulcers, and venous leg ulcers are a worldwide health problem. As the traditional methods of treatment have proven ineffective against chronic wounds involving biofilms, there is an unmet clinical need for developing products with an antibiofilm component that inhibits and/or disrupts biofilms and thus make the biofilm-embedded bacteria more susceptible to antimicrobial therapy. We developed a DispersinB® antibiofilm enzyme-based wound spray for treating chronic wounds in conjunction with an antimicrobial. Under in vitro conditions, the DispersinB® and Acticoat™ combination performed significantly better (P < 0.05) than Acticoat™ alone, indicating the synergy between the two compounds because of DispersinB® enhancing the antimicrobial activity of Acticoat™. Furthermore, DispersinB® wound spray enhanced the antimicrobial activity of Acticoat™ in a chronic wound mouse model of methicillin-resistant Staphylococcus aureus (MRSA) infection. Thus, this novel combination of DispersinB® and Acticoat™, an antimicrobial dressing, prompts clinical evaluation for potential applications in biofilm-based chronic wound management.

Recombinant human DNase I decreases biofilm and increases antimicrobial susceptibility in staphylococci.

Extracellular DNA is an adhesive component of staphylococcal biofilms. The aim of this study was to evaluate the antibiofilm activity of recombinant human DNase I (rhDNase) against Staphylococcus aureus and Staphylococcus epidermidis. Using a 96-well microtiter plate crystal-violet binding assay, we found that biofilm formation by S. aureus was efficiently inhibited by rhDNase at 1-4 µg l⁻¹, and preformed S. aureus biofilms were efficiently detached in 2 min by rhDNase at 1 mg l⁻¹. Pretreatment of S. aureus biofilms for 10 min with 10 mg l⁻¹ rhDNase increased their sensitivity to biocide killing by 4-5 log units. rhDNase at 10 mg l⁻¹ significantly inhibited biofilm formation by S. epidermidis in medium supplemented with sub-MICs of antibiotics. We also found that rhDNase significantly increased the survival of S. aureus-infected Caenorhabditis elegans nematodes treated with tobramycin compared with nematodes treated with tobramycin alone. We concluded that rhDNase exhibits potent antibiofilm and antimicrobial-sensitizing activities against S. aureus and S. epidermidis at clinically achievable concentrations. rhDNase, either alone or in combination with antimicrobial agents, may have applications in treating or preventing staphylococcal biofilm-related infections.

Characterization of the poly-ß-1,6-N-acetylglucosamine polysaccharide component of Burkholderia biofilms.

We demonstrated the production of poly-ß-1,6-N-acetylglucosamine (PNAG) polysaccharide in the biofilms of Burkholderia multivorans, Burkholderia vietnamiensis, Burkholderia ambifaria, Burkholderia cepacia, and Burkholderia cenocepacia using an immunoblot assay for PNAG. These results were confirmed by further studies, which showed that the PNAG hydrolase, dispersin B, eliminated immunoreactivity of extracts from the species that were tested (B. cenocepacia and B. multivorans). Dispersin B also inhibited biofilm formation and dispersed preformed biofilms of Burkholderia species. These results imply a role for PNAG in the maintenance of Burkholderia biofilm integrity. While PNAG was present in biofilms of all of the wild-type test organisms, a ΔpgaBC mutant of B. multivorans (Mu5) produced no detectable PNAG, indicating that these genes are needed for Burkholderia PNAG formation. Furthermore, restoration of PNAG production in PNAG negative E. coli TRXWMGΔC (ΔpgaC) by complementation with B. multivorans pgaBCD confirmed the involvement of these genes in Burkholderia PNAG production. While the confocal scanning laser microscopy of untreated wild-type B. multivorans showed thick, multilayered biofilm, Mu5 and dispersin B-treated wild-type biofilms were thin, poorly developed, and disrupted, confirming the involvement of PNAG in B. multivorans biofilm formation. Thus, PNAG appears to be an important component of Burkholderia biofilms, potentially contributing to its resistance to multiple antibiotics and persistence during chronic infections, including cystic fibrosis-associated infection.

Antimicrobial and antibiofilm activity of quorum sensing peptides and Peptide analogues against oral biofilm bacteria.

Widespread antibiotic resistance is a major incentive for the investigation of novel ways to treat or prevent infections. Much effort has been put into the discovery of peptides in nature accompanied by manipulation of natural peptides to improve activity and decrease toxicity. The ever increasing knowledge about bacteria and the discovery of quorum sensing have presented itself as another mechanism to disrupt the infection process. We have shown that the natural quorum sensing (QS) peptide, competence-stimulating peptide (CSP), used by the caries causing bacteria Streptococcus mutans when used in higher than normally present concentrations can actually contribute to cell death in S. mutans. Using an analogue of this quorum sensing peptide (KBI-3221), we have shown it to be beneficial at decreasing biofilm of various Streptococcus species. This chapter looks at a number of assay methods to test the inhibitory effects of quorum sensing peptides and their analogues on the growth and biofilm formation of oral bacteria.

Enhanced expression of engineered ACA-less beta-1, 6-N-acetylglucosaminidase (dispersin B) in Escherichia coli.

beta-1,6-N-Acetylglucosaminidase (dispersin B), which cleaves poly-ss-(1,6)-linked N-acetylglucosamine, is encoded by dspB of Aggregatibacter actinomycetemcomitans. To enhance the production of dispersin B, we engineered dspB to transcribe mRNAs devoid of the trinucleotide ACA. Transcription and translation levels of ACA-less and wild-type dspB expressed in Escherichia coli (E. coli) under T5 and T7 promoters were analyzed by real-time RT-PCR and protein quantification, respectively. The ACA-less dspB mRNA level was significantly higher (P < 0.01) and produced 77.6 and 34.9% more dispersin B than wild-type dspB expressed under T7 and T5 promoters, respectively. Dispersin B expression under T7 promoter caused a 98-99.5% drop in the glyceraldehyde-3-phosphate dehydrogenase (gapA) mRNA level, which was not observed with T5 promoter. Fusion of green fluorescent protein (GFP) with dispersin B allowed rapid quantification of dispersin B production by measuring fluorescence intensity in culture broth. Although the cultures containing 0.1% glucose showed sustained increase in dispersin B-GFP production until 12 h, no significant increase in dispersin B activity was observed beyond 4 and 6 h after induction when expressed under T7 and T5 promoters, respectively. This study demonstrates the effectiveness of ACA-less mRNA and the advantage of GFP tagging for enhanced dispersin B production and quantification, which could be adapted for improving the production of other commercially important proteins in E. coli.

Antibiofilm activity of GlmU enzyme inhibitors against catheter-associated uropathogens.

The colonization of uropathogenic bacteria on urinary catheters resulting in biofilm formation frequently leads to the infection of surrounding tissue and often requires removal of the catheter. Infections associated with biofilms are difficult to treat since they may be more than 1,000 times more resistant to antibiotics than their planktonic counterparts. We have developed an antibiofilm composition comprising an N-acetyl-D-glucosamine-1-phosphate acetyltransferase (GlmU) inhibitor and protamine sulfate, a cationic polypeptide. The antibiofilm activity of GlmU inhibitors, such as iodoacetamide (IDA), N-ethyl maleimide (NEM), and NEM analogs, including N-phenyl maleimide, N,N'-(1,2-phenylene)dimaleimide (oPDM), and N-(1-pyrenyl)maleimide (PyrM), was tested against that of catheter-associated uropathogens. Both IDA and NEM inhibited biofilm formation in Escherichia coli. All NEM analogs showed antibiofilm activity against clinical isolates of E. coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus epidermidis, and Enterococcus faecalis. The combination of oPDM with protamine sulfate (PS) enhanced its antibiofilm activity and reduced its effective concentration to as low as 12.5 microM. In addition, we found that the in vitro inhibitory activity of oPDM-plus-PS-coated silicone catheters against P. aeruginosa and S. epidermidis colonization was superior to that of catheters coated with silver hydrogel. Confocal scanning laser microscopy further confirmed that the oPDM-plus-PS-coated silicone catheters were almost free from bacterial colonization. Thus, a broad-spectrum antibiofilm composition comprising a GlmU inhibitor and protamine sulfate shows promise for use in anti-infective coatings for medical devices, including urinary catheters.