Chronic Biofilm Research Articles

The relationship of biofilms to chronic rhinosinusitis

To provide an update on the state of biofilm research in otolaryngology. Chronic rhinosinusitis is a polymicrobial infection, which includes planktonic and biofilm infections with bacterial and fungal elements. The importance of genetic shift in microbes, when converting into a biofilm state, as well as the multiple phenotypes in each bacterial colony cannot be overemphasized. This creates a very sophisticated community of pathogens, some of which will likely survive a simple chemical treatment. Sinus cultures cannot be expected to provide a complete knowledge of the cause of chronic sinusitis. A new diagnostic method and innovative treatment plans will be necessary to provide a lasting treatment of chronic rhinosinusitis. Surgery combined with postoperative treatment is the most effective mean of controlling the majority of chronic rhinosinusitis infections. The challenges associated with the treatment of chronic rhinosinusitis patients may be met by focusing more on the community of microorganism present in the sinuses. The understanding of the implication of chronic biofilm infections is growing rapidly but will require an enormous effort to completely control chronic rhinosinusitis.

Survey of bacterial diversity in chronic wounds using Pyrosequencing, DGGE, and full ribosome shotgun sequencing

BackgroundChronic wound pathogenic biofilms are host-pathogen environments that colonize and exist as a cohabitation of many bacterial species. These bacterial populations cooperate to promote their own survival and the chronic nature of the infection. Few studies have performed extensive surveys of the bacterial populations that occur within different types of chronic wound biofilms. The use of 3 separate16S-based molecular amplifications followed by pyrosequencing, shotgun Sanger sequencing, and denaturing gradient gel electrophoresis were utilized to survey the major populations of bacteria that occur in the pathogenic biofilms of three types of chronic wound types: diabetic foot ulcers (D), venous leg ulcers (V), and pressure ulcers (P).ResultsThere are specific major populations of bacteria that were evident in the biofilms of all chronic wound types, including Staphylococcus, Pseudomonas, Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia, and Serratia spp. Each of the wound types reveals marked differences in bacterial populations, such as pressure ulcers in which 62% of the populations were identified as obligate anaerobes. There were also populations of bacteria that were identified but not recognized as wound pathogens, such as Abiotrophia para-adiacens and Rhodopseudomonas spp. Results of molecular analyses were also compared to those obtained using traditional culture-based diagnostics. Only in one wound type did culture methods correctly identify the primary bacterial population indicating the need for improved diagnostic methods.ConclusionIf clinicians can gain a better understanding of the wound's microbiota, it will give them a greater understanding of the wound's ecology and will allow them to better manage healing of the wound improving the prognosis of patients. This research highlights the necessity to begin evaluating, studying, and treating chronic wound pathogenic biofilms as multi-species entities in order to improve the outcomes of patients. This survey will also foster the pioneering and development of new molecular diagnostic tools, which can be used to identify the community compositions of chronic wound pathogenic biofilms and other medical biofilm infections.

The Evolution of Quorum Sensing in Bacterial Biofilms

Bacteria have fascinating and diverse social lives. They display coordinated group behaviors regulated by quorum-sensing systems that detect the density of other bacteria around them. A key example of such group behavior is biofilm formation, in which communities of cells attach to a surface and envelope themselves in secreted polymers. Curiously, after reaching high cell density, some bacterial species activate polymer secretion, whereas others terminate polymer secretion. Here, we investigate this striking variation in the first evolutionary model of quorum sensing in biofilms. We use detailed individual-based simulations to investigate evolutionary competitions between strains that differ in their polymer production and quorum-sensing phenotypes. The benefit of activating polymer secretion at high cell density is relatively straightforward: secretion starts upon biofilm formation, allowing strains to push their lineages into nutrient-rich areas and suffocate neighboring cells. But why use quorum sensing to terminate polymer secretion at high cell density? We find that deactivating polymer production in biofilms can yield an advantage by redirecting resources into growth, but that this advantage occurs only in a limited time window. We predict, therefore, that down-regulation of polymer secretion at high cell density will evolve when it can coincide with dispersal events, but it will be disfavored in long-lived (chronic) biofilms with sustained competition among strains. Our model suggests that the observed variation in quorum-sensing behavior can be linked to the differing requirements of bacteria in chronic versus acute biofilm infections. This is well illustrated by the case of Vibrio cholerae, which competes within biofilms by polymer secretion, terminates polymer secretion at high cell density, and induces an acute disease course that ends with mass dispersal from the host. More generally, this work shows that the balance of competition within and among biofilms can be pivotal in the evolution of quorum sensing.

Subinhibitory Concentrations of Azithromycin Decrease Nontypeable Haemophilus influenzae Biofilm Formation and Diminish Established Biofilms

Nontypeable Haemophilus influenzae (NTHi) commonly causes otitis media, chronic bronchitis in emphysema, and early airway infections in cystic fibrosis. Long-term, low-dose azithromycin has been shown to improve clinical outcomes in chronic lung diseases, although the mechanism of action remains unclear. The inhibition of bacterial biofilms by azithromycin has been postulated to be one mechanism mediating these effects. We hypothesized that subinhibitory concentrations of azithromycin would affect NTHi biofilm formation. Laboratory strains of NTHi expressing green fluorescent protein and azithromycin-resistant clinical isolates were grown in flow-cell and static-culture biofilm models. Using a range of concentrations of azithromycin and gentamicin, we measured the degree to which these antibiotics inhibited biofilm formation and persistence. Large biofilms formed over 2 to 4 days in a flow cell, displaying complex structures, including towers and channels. Subinhibitory concentrations of azithromycin significantly decreased biomass and maximal thickness in both forming and established NTHi biofilms. In contrast, subinhibitory concentrations of gentamicin had no effect on biofilm formation. Furthermore, established NTHi biofilms became resistant to gentamicin at concentrations far above the MIC. Biofilm formation of highly resistant clinical NTHi isolates (azithromycin MIC of > 64 microg/ml) was similarly decreased at subinhibitory azithromycin concentrations. Clinically obtainable azithromycin concentrations inhibited biofilms in all but the most highly resistant isolates. These data show that subinhibitory concentrations of azithromycin have antibiofilm properties, provide mechanistic insights, and supply an additional rationale for the use of azithromycin in chronic biofilm infections involving H. influenzae.

Silver against Pseudomonas aeruginosa biofilms

Silver has been recognized for its antimicrobial properties for centuries. Most studies on the antibacterial efficacy of silver, with particular emphasis on wound healing, have been performed on planktonic bacteria. Our recent studies, however, strongly suggest that colonization of wounds involves bacteria in both the planktonic and biofilm modes of growth. The action of silver on mature in vitro biofilms of Pseudomonas aeruginosa, a primary pathogen of chronic infected wounds, was investigated. The results show that silver is very effective against mature biofilms of P. aeruginosa, but that the silver concentration is important. A concentration of 5-10 mug/mL silver sulfadiazine eradicated the biofilm whereas a lower concentration (1 mug/mL) had no effect. The bactericidal concentration of silver required to eradicate the bacterial biofilm was 10-100 times higher than that used to eradicate planktonic bacteria. These observations strongly indicate that the concentration of silver in currently available wound dressings is much too low for treatment of chronic biofilm wounds. It is suggested that clinicians and manufacturers of the said wound dressings consider whether they are treating wounds primarily colonized either by biofilm-forming or planktonic bacteria.

PR24 EFFECTS OF COMBINED TOPICAL NEGATIVE PRESSURE (TNP) AND ANTISEPTIC INSTILLATION ON PSEUDOMONAS BIOFILM

Purpose TNP in the form of V.A.C. dressings have been successfully used to enhance chronic wound healing. An in vitro model of the chronic wound biofilm was created to test the effects of TNP with and without concurrent antimicrobial drug instillation on biofilms.Method A P.aeruginosa biofilm model was constructed in vitro. Continuous TNP and TNP combined with varying frequencies of betadine instillation were tested on the biofilm model for 24 hours. Outcome parameters measured included viable bacterial count, fluorescent microscopy, confocal laser scanning microscopy, and electron microscopy. Statistical analysis was performed using ANOVA and MANOVA in SPSS 13.0.Results Bacterial biofilms, when exposed to TNP alone, showed a 42% decrease in viable count; while exposure to betadine alone showed a reduction of up to 84%. Combined TNP and antiseptic instillation increased the bacteriocidal effect to 99.7% (p < 0.001).Exposure to TNP also resulted in a decrease in biofilm thickness, and diffusion distance, with an increase in surface area‐to‐volume ratio (p = 0.005, 0.002, and 0.009 respectively).Conclusion TNP alone showed a modest effect on the reduction of bacterial biofilm. More importantly it appeared to compress and fragment the overall biofilm architecture. This physical deformity in turn led to more effective drug penetration into the highly resistant biofilm community. The result was an approximate 100 fold enhancement of bacteriocidal effect when betadine instillation was combined with TNP as compared to betadine alone.

A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels

Pseudomonas aeruginosa causes chronic biofilm infections, and its ability to attach to surfaces and other cells is important for biofilm formation and maintenance. Mutations in a gene called wspF, part of a putative chemosensory signal-transduction operon, have been shown to result in cell aggregation and altered colony morphology. The WspF phenotypes depend on the presence of WspR, which is a member of a family of signal transduction proteins known as response regulators. It is likely that the effect of the wspF mutation is to cause constitutive activation of WspR by phosphorylation. WspR contains a GGDEF domain known to catalyze formation of a cytoplasmic signaling molecule cyclic diguanylate (c-diGMP). We determined that purified WspR catalyzed the formation of c-diGMP in vitro and phosphorylation stimulated this activity. We observed increased cellular levels of c-diGMP and increased biofilm formation in a wspF mutant. Expression of a protein predicted to catalyze degradation of c-diGMP reversed the phenotypes of a wspF mutant and inhibited biofilm initiation by wild-type cells, indicating that the presence of c-diGMP is necessary for biofilm formation. A transcriptome analysis showed that expression levels of at least 560 genes were affected by a wspF deletion. The psl and pel operons, which are involved in exopolysaccharide production and biofilm formation, were expressed at high levels in a wspF mutant. Together, the data suggest that the wsp signal transduction pathway regulates biofilm formation through modulation of cyclic diguanylate levels.

Local Antibiotic Delivery Systems

Local Antibiotic Delivery Systems

Role of biofilm-associated protein bap in the pathogenesis of bovine Staphylococcus aureus.

Staphylococcus aureus is a common cause of intramammary infections, which frequently become chronic, associated with the ability of the bacteria to produce biofilm. Here, we report a relationship between the ability to produce chronic bovine mastitis and biofilm formation. We have classified bovine mastitis S. aureus isolates into three groups based on the presence of particular genetic elements required for biofilm formation: group 1 (ica(+) bap(+)), group 2 (ica(+), bap negative), and group 3 (ica negative, bap negative). Overall, animals naturally infected with group 1 and 2 isolates had a lower milk somatic cell count than those infected with isolates of group 3. In addition, Bap-positive isolates were significantly more able to colonize and persist in the bovine mammary gland in vivo and were less susceptible to antibiotic treatments when forming biofilms in vitro. Analysis of the structural bap gene revealed the existence of alternate forms of expression of the Bap protein in S. aureus isolates obtained under field conditions throughout the animal's life. The presence of anti-Bap antibodies in serum samples taken from animals with confirmed S. aureus infections indicated the production of Bap during infection. Furthermore, disruption of the ica operon in a bap-positive strain had no effect on in vitro biofilm formation, a finding which strongly suggested that Bap could compensate for the deficiency of the PIA/PNAG product (a biofilm matrix polysaccharide). Altogether, these results demonstrate that, in the bovine intramammary gland, the presence of Bap may facilitate a biofilm formation connected with the persistence of S. aureus.

Rapid direct method for monitoring antibiotics in a mouse model of bacterial biofilm infection.

We have developed a rapid, continuous method for monitoring the effectiveness of several antibacterial agents in real time, noninvasively, by using a recently described mouse model of chronic biofilm infection (J. L. Kadurugamuwa et al., Infect. Immun. 71:882-890, 2003), which relies on biophotonic imaging of bioluminescent bacteria. To facilitate real-time monitoring of infection, we used a Staphylococcus aureus isolate that was made bioluminescent by inserting a modified lux operon into the bacterial chromosome. This bioluminescent reporter bacterium was used to study the antimicrobial effects of several antibiotics belonging to different molecular families. Treatment with rifampin, tobramycin, and ciprofloxacin was started 7 days after subcutaneous implantation of catheters precolonized with 10(4) CFU of S. aureus. Three different doses of antibiotics were administered twice a day for 4 consecutive days. The number of metabolically active bacteria in untreated mice and the tobramycin- and ciprofloxacin-treated groups remained relatively unchanged over the 4-week observation period, indicating poor efficacies for tobramycin and ciprofloxacin. A rapid dose-dependent decline in metabolic activity in rifampin-treated groups was observed, with almost a 90% reduction after two doses and nearly undetectable levels after three doses. The disappearance of light emission correlated with colony counts. After the final treatment, cell numbers rebounded as a function of concentration in a time-dependent manner. The staphylococci isolated from the catheters of mice treated with rifampin were uniformly resistant to rifampin but retained their in vitro susceptibilities to tobramycin and ciprofloxacin. Since the metabolic activities of viable cells and a postantibiotic effect could be detected directly on the support matrix nondestructively and noninvasively, the methodology is specifically appealing for investigating the effects of antibiotics on biofilms in vivo. Moreover, our study points to the possible use of biophotonic imaging for the detection of the development of resistance to therapeutic agents during treatment of chronic infections in vivo.

Targeting Quorum Sensing for Treatment of Chronic Bacterial Biofilm Infections

Targeting Quorum Sensing for Treatment of Chronic Bacterial Biofilm Infections

Efficacy of erythromycin inhalation in chronic respiratory infection caused by Pseudomonas aeruginosa

The prognosis of chronic respiratory tract infection with Pseudomonas aeruginosad considered to be poor. However, low-dose and long-term 14 membered macrolide, such as erythromycin or clarithromycin, treatment has been reported as effective clinically in chronic lower respiratory tract disease. There was no report to investigate the effect of macrolide on chronic biofilm related Pseudomonas aeruginosa respiratory tract infection in vivo. In a newly established murine model of chronic Pseudomonas aeruginosa respiratory infection mimicking diffuse panbronchiolitis (DPB), we investigated the effect of erythromcin inhalation on viable bacteria in the lungs. Infection was produced by placement of a plastic tube in the bronchus with inoculating. Pseudomonas aeruginosa suspended in saline was also inoculated in bronchus after intubation of the tube. Viable bacteria were constantly isolated at 10(4) 10(6) cfu-specimen from the lungs for more than 30 days. Treatment with erythromycin inhalation for four days reduced the number of viable bacteria in the lungs with statistically significant. Our result indicated that our new model of chronic respiratory tract infection is simple and provides a useful tool to study the pathogenic process and treatment of such infection. Our results also suggest that the erythromycin inhalation is effective on chronic Pseudomons aeruginosa respiratory infection.