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 pre-formed S. aureus biofilms were efficiently detached in 2 min by rhDNase at 1 mg l−1. Pre-treatment 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 subminimal inhibitory concentrations of antibiotics. We also also found rhDNase significantly increased the survival of S. aureus-infected C. elegans nematodes treated with tobramycin compared to 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.

A spectrofluorometric assay was developed for quantification of bacterial biofilms grown on a microtiter plate. The method involved staining biofilms formed by gram-negative and gram-positive bacteria with wheat germ agglutinin-Alexa Fluor 488 conjugate, which selectively binds to N-acetylglucosamine residues in biofilms. The fluorescence of stained biofilms was measured with a fluorescent plate reader. This method was compared with a widely used microplate colorimetric assay involving crystal violet staining of biofilms formed by both gram-negative and gram-positive bacteria. A strong linear association existed between the two methods (r (2)=0.99/0.94). Being more sensitive and specific as compared to colorimetric method, the spectrofluorometric assay provides a better alternative for quantification and characterization of bacterial biofilms.

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-Dglucosamine-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 M. 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 oPDMplus-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 antiinfective coatings for medical devices, including urinary catheters.Microorganisms can attach to and colonize any biomaterial surface, putting patients at risk for local and systemic infections. More than 900,000 episodes of catheter-associated urinary tract infections occur annually in acute-care hospitals in the United States, accounting for 40% of all nosocomial infections and involving between 10 and 30% of patients with indwelling urinary catheters (30). Catheter-associated urinary tract infection prolongs the hospital stay between an estimated 2.4 and 4.5 days, with resultant increased healthcare costs (15,16). Recent studies have shown that a wide range of persistent catheter-related infections may be related to the ability of bacteria to form biofilms (6, 28). Treatment of device-related infections with conventional antimicrobial agents frequently fails because microorganisms growing in biofilms are more tolerant or phenotypically resistant to antimicrobial agents than planktonic cells (24). The insensitivity of biofilm bacteria to antibiotics is a function of cell wall composition, surface structure, and phenotypic variation in enzymatic activity (8,14). It has also been suggested that the negatively charged exopolysaccharide is very effective in protecting bacterial cells from cationic antibiotics by restricting their permeation (2...

Aim:  To determine the effect of sodium bicarbonate (SB), sodium metaperiodate (SMP) and sodium dodecyl sulfate (SDS) combination on biofilm formation and dispersal in dental unit waterline (DUWL)‐associated bacteria and yeast. Methods and Results:  The in vitro effect of SB, SMP and SDS alone and in combination on biofilm formation and dispersal in Pseudomonas aeruginosa, Klebsiella pneumoniae, Actinomyces naeslundii, and Candida albicans was investigated using a 96‐well microtitre plate biofilm assay. The combination showed a broad‐spectrum inhibitory effect on growth as well as biofilm formation of both gram‐negative and gram‐positive bacteria, and yeast. In addition, the SB + SMP + SDS combination was significantly more effective in dispersing biofilm than the individual compounds. The combination dispersed more than 90% of P. aeruginosa biofilm whereas the commercial products, Oxygenal 6, Sterilex Ultra, and PeraSafe showed no biofilm dispersal activity. Conclusion:  The composition comprising SB, SMP, and SDS was effective in inhibiting as well as dispersing biofilms in DUWL‐associated bacteria and yeast. Significance and Impact of the Study:  This study shows that a composition comprising environmentally friendly and biologically safe compounds such as SB, SMP, and SDS has a potential application in reducing DUWL‐associated acquired infections in dental clinics.

We demonstrated the production of poly-␤-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 ⌬pgaBC 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 TRXWMG⌬C (⌬pgaC) 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.