Chronic Biofilm Infections Research Articles

Trimeric Form of Intracellular ATP Synthase Subunit β of Aggregatibacter actinomycetemcomitans Binds Human Interleukin-1β

Bacterial biofilms resist host defenses and antibiotics partly because of their decreased metabolism. Some bacteria use proinflammatory cytokines, such as interleukin (IL)-1β, as cues to promote biofilm formation and to alter virulence. Although one potential bacterial IL-1β receptor has been identified, current knowledge of the bacterial IL-1β sensing mechanism is limited. In chronic biofilm infection, periodontitis, Aggregatibacter actinomycetemcomitans requires tight adherence (tad)-locus to form biofilms, and tissue destroying active lesions contain more IL-1β than inactive ones. The effect of IL-1β on the metabolic activity of A. actinomycetemcomitans biofilm was tested using alamarBlue™. The binding of IL-1β to A. actinomycetemcomitans cells was investigated using transmission electron microscopy and flow cytometry. To identify the proteins which interacted with IL-1β, different protein fractions from A. actinomycetemcomitans were run in native-PAGE and blotted using biotinylated IL-1β and avidin-HRP, and identified using mass spectroscopy. We show that although IL-1β slightly increases the biofilm formation of A. actinomycetemcomitans, it reduces the metabolic activity of the biofilm. A similar reduction was observed with all tad-locus mutants except the secretin mutant, although all tested mutant strains as well as wild type strains bound IL-1β. Our results suggest that IL-1β might be transported into the A. actinomycetemcomitans cells, and the trimeric form of intracellular ATP synthase subunit β interacted with IL-1β, possibly explaining the decreased metabolic activity. Because ATP synthase is highly conserved, it might universally enhance biofilm resistance to host defense by binding IL-1β during inflammation.

New methods for the detection of orthopedic and other biofilm infections

The detection and identification of bacteria present in natural and industrial ecosystems is now entirely based on molecular systems that detect microbial RNA or DNA. Culture methods were abandoned, in the 1980s, because direct observations showed that <1% of the bacteria in these systems grew on laboratory media. Culture methods comprise the backbone of the Food and Drug Administration-approved diagnostic systems used in hospital laboratories, with some molecular methods being approved for the detection of specific pathogens that are difficult to grow in vitro. In several medical specialties, the reaction to negative cultures in cases in which overt signs of infection clearly exist has produced a spreading skepticism concerning the sensitivity and accuracy of traditional culture methods. We summarize evidence from the field of orthopedic surgery, and from other medical specialties, that support the contention that culture techniques are especially insensitive and inaccurate in the detection of chronic biofilm infections. We examine the plethora of molecular techniques that could replace cultures in the diagnosis of bacterial diseases, and we identify the new Ibis technique that is based on base ratios (not base sequences), as the molecular system most likely to fulfill the requirements of routine diagnosis in orthopedic surgery.

A multitask biosensor for micro-volumetric detection of N-3-oxo-dodecanoyl-homoserine lactone quorum sensing signal

N-3-oxo-dodecanoyl-homoserine lactone (3OC 12-HSL) is the main quorum sensing (QS) signal produced by the human pathogen Pseudomonas aeruginosa, a major cause of hard-to-treat nosocomial infections and years-lasting chronic biofilm infections in the lungs of cystic fibrosis (CF) patients. 3OC 12-HSL-dependent QS is considered a promising target for novel anti-pseudomonads drugs. However, the screening systems employed to date for the identification of QS inhibitors (QSI) were aimed at the identification of inhibitors of 3OC 12-HSL signaling rather than of the synthesis or the export of this molecule. Moreover, the low concentration of 3OC 12-HSL in CF sputum has hampered large scale studies aimed at addressing the role of this molecule in the CF lung infection. Here we describe the construction and characterization of PA14-R3, a new whole-cell biosensor for the quantitative detection of 3OC 12-HSL. PA14-R3 provides fast and direct quantification of 3OC 12-HSL over a wide range of concentrations (from pM to μM), and proved to be an easy-to-handle, cost-effective and reliable biosensor for high-throughput screening of 3OC 12-HSL levels in samples of different origin, including CF sputum. Moreover, the specific features of PA14-R3 made it possible to develop and validate a novel high-throughput screening system for QSI based on the co-cultivation of PA14-R3 with the PA14 wild-type strain. With respect to previous screening systems for QSI, this approach has the advantage of being cost-effective and allowing the identification of compounds targeting, besides 3OC 12-HSL signaling, any cellular process critical for QS response, including 3OC 12-HSL synthesis and secretion.

Multicellular signalling and growth of Pseudomonas aeruginosa

The main reason for Pseudomonas aeruginosa persistence within the lungs of chronically infected cystic fibrosis patients seems to be that the bacteria reside within biofilm structures which protect them from diverse adverse environmental conditions, antibiotic treatment, and from the host immune response. Thereby, it may be of great significance that the human defence systems do not face single bacteria but multicellular communities in which the high level of diversity of both phenotypes and genotypes provides insurance for bacterial survival. Indeed, in the last decade, the orthodox view of bacterial populations as being a homogeneous collection of sibling cells has been abandoned, and there is accumulating evidence of bacterial interactiveness within biofilm structures, which is supported by the identification of signalling molecules that mediate cooperative traits and a coordinated behaviour. A detailed understanding of the molecular mechanisms that contribute to multicellular development in bacterial biofilms will be pivotal for the identification of novel targets as the basis for the development of new alternative treatment strategies directed against chronic persistent biofilm infections.

Biofilms in chronic infections – a matter of opportunity – monospecies biofilms in multispecies infections

It has become evident that aggregation or biofilm formation is an important survival mechanism for bacteria in almost any environment. In this review, we summarize recent visualizations of bacterial aggregates in several chronic infections (chronic otitis media, cystic fibrosis, infection due to permanent tissue fillers and chronic wounds) both as to distribution (such as where in the wound bed) and organization (monospecies or multispecies microcolonies). We correlate these biofilm observations to observations of commensal biofilms (dental and intestine) and biofilms in natural ecosystems (soil). The observations of the chronic biofilm infections point toward a trend of low bacterial diversity and sovereign monospecies biofilm aggregates even though the infection in which they reside are multispecies. In contrast to this, commensal and natural biofilm aggregates contain multiple species that are believed to coexist, interact and form biofilms with high bacterial and niche diversity. We discuss these differences from both the diagnostic and the scientific point of view.

Bacterial biofilm on monofilament suture and porcine xenograft after inguinal herniorrhaphy

Bacterial biofilms have been implicated in multiple clinical scenarios involving infection of implanted foreign bodies, but have been little studied after hernia repair. We now report a case of revision inguinal herniorrhaphy complicated by chronic pain at the operated site without any external indication of infection. Computed tomographic imaging revealed a contrast-enhancing process in the left groin. Subsequent surgical exploration found an inflammatory focus centered on implanted porcine xenograft material and nonabsorbable monofilament sutures placed at the previous surgery. Confocal microscopic examination of these materials with Live/Dead staining demonstrated abundant viable bacteria in biofilm configuration. The removal of these materials and direct closure of the recurrent hernia defect eliminated the infection and resolved the patient's complaints. These results demonstrate that implanted monofilament suture and xenograft material can provide the substratum for a chronic biofilm infection.

A 96-well-plate–based optical method for the quantitative and qualitative evaluation of Pseudomonas aeruginosa biofilm formation and its application to susceptibility testing

A major reason for bacterial persistence during chronic infections is the survival of bacteria within biofilm structures, which protect cells from environmental stresses, host immune responses and antimicrobial therapy. Thus, there is concern that laboratory methods developed to measure the antibiotic susceptibility of planktonic bacteria may not be relevant to chronic biofilm infections, and it has been suggested that alternative methods should test antibiotic susceptibility within a biofilm. In this paper, we describe a fast and reliable protocol for using 96-well microtiter plates for the formation of Pseudomonas aeruginosa biofilms; the method is easily adaptable for antimicrobial susceptibility testing. This method is based on bacterial viability staining in combination with automated confocal laser scanning microscopy. The procedure simplifies qualitative and quantitative evaluation of biofilms and has proven to be effective for standardized determination of antibiotic efficiency on P. aeruginosa biofilms. The protocol can be performed within approximately 60 h.

AlgK Is a TPR-Containing Protein and the Periplasmic Component of a Novel Exopolysaccharide Secretin

The opportunistic pathogen Pseudomonas aeruginosa causes chronic biofilm infections in cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by overproduction of the exopolysaccharide alginate. Here we show that AlgK, a protein essential for production of high molecular weight alginate, is an outer membrane lipoprotein that contributes to the correct localization of the porin AlgE. Our 2.5 A structure shows AlgK is composed of 9.5 tetratricopeptide-like repeats, and three putative sites of protein-protein interaction have been identified. Bioinformatics analysis suggests that BcsA, PgaA, and PelB, involved in the production and export of cellulose, poly-beta-1,6-N-Acetyl-D-glucosamine, and Pel exopolysaccharide, respectively, share the same topology as AlgK/E. Together, our data suggest that AlgK plays a role in the assembly of the alginate biosynthetic complex and represents the periplasmic component of a new type of outer membrane secretin that differs from canonical bacterial capsular polysaccharide secretion systems.

Disruption of Staphylococcus Epidermidis Biofilms by Medicinal Maggot Lucilia Sericata Excretions/Secretions

Chronic infections are commonly associated with biofilms formed by bacteria such as Staphylococcus epidermidis. With the increase in antibiotic resistant bacteria, maggot debridement therapy has been reintroduced for the treatment of chronic wounds. Studies have shown that the excretion/ secretions (ES) of Lucilia sericata larvae (maggots) contain many bioactive compounds which may contribute to the efficacy of maggot therapy. The present study evaluates the effect of L. sericata ES on the formation and disruption of S. epidermidis 1457 and 5179-R1 biofilms. These strains employ either polysaccharide intercellular adhesin (PIA) or accumulation associated protein (Aap) for intercellular adhesion. A semiquantitative biofilm assay was used to measure the formation/disruption of S. epidermidis 1457 and 5179-R1 biofilms by ES. ES activity was characterized according to concentration, incubation time and temperature, thermal stability, and size. Immunofluorescence microscopy was used to ascertain the effect of ES on PIA and Aap. In the presence of ES, S. epidermidis 1457 and 5179-R1 nascent biofilm formation was inhibited, and pre-formed biofilms disrupted. ES activity was temperature and time dependent, inactivated by heat treatment, and disruption depended on the mechanism of intercellular adhesion. The molecule(s) responsible was >10 kDa in size and appeared to have protease or glucosaminidase activity. ES interferes with S. epidermidis biofilm formation, specifically degrading factors employed in biofilm accumulation, which would increase bacterial susceptibility to antibiotics and the host's immune system. In purified form, ES-factors may have general applicability for the treatment or prevention of chronic biofilm infections caused by staphylococci.

Cutaneous Fistula from the Gastric Remnant Resulting from a Chronic Suture-associated Biofilm Infection

A 53-year-old woman developed three chronic draining sinuses after Roux-en-Y gastric bypass; these persisted for almost 1 year despite antibiotics and local wound care. At approximately 1 year post-operatively, the drainage from the most superior sinus increased significantly and assumed a greenish hue, prompting concerns for gastrocutaneous fistula despite negative radiologic evaluation. At surgery, the patient was found to have a retained permanent multifilament suture at the base of each sinus, with associated inflammatory and fibrous tissue and a "slimy" matrix. Confocal laser scanning microscopy of both the explanted sutures and investing soft tissue revealed extensive bacterial biofilm formation. Also at surgery, a frank fistulous track was noted communicating the most superior suture/sinus to the gastric remnant, necessitating laparotomy and remnant gastrectomy in addition to removal of the foreign bodies (sutures) and concomitant panniculectomy. The patient has subsequently been free of complaint or finding for over 3 years.

Polymicrobial Nature of Chronic Diabetic Foot Ulcer Biofilm Infections Determined Using Bacterial Tag Encoded FLX Amplicon Pyrosequencing (bTEFAP)

BackgroundDiabetic extremity ulcers are associated with chronic infections. Such ulcer infections are too often followed by amputation because there is little or no understanding of the ecology of such infections or how to control or eliminate this type of chronic infection. A primary impediment to the healing of chronic wounds is biofilm phenotype infections. Diabetic foot ulcers are the most common, disabling, and costly complications of diabetes. Here we seek to derive a better understanding of the polymicrobial nature of chronic diabetic extremity ulcer infections.Methods and FindingsUsing a new bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP) approach we have evaluated the bacterial diversity of 40 chronic diabetic foot ulcers from different patients. The most prevalent bacterial genus associated with diabetic chronic wounds was Corynebacterium spp. Findings also show that obligate anaerobes including Bacteroides, Peptoniphilus, Fingoldia, Anaerococcus, and Peptostreptococcus spp. are ubiquitous in diabetic ulcers, comprising a significant portion of the wound biofilm communities. Other major components of the bacterial communities included commonly cultured genera such as Streptococcus, Serratia, Staphylococcus and Enterococcus spp.ConclusionsIn this article, we highlight the patterns of population diversity observed in the samples and introduce preliminary evidence to support the concept of functional equivalent pathogroups (FEP). Here we introduce FEP as consortia of genotypically distinct bacteria that symbiotically produce a pathogenic community. According to this hypothesis, individual members of these communities when they occur alone may not cause disease but when they coaggregate or consort together into a FEP the synergistic effect provides the functional equivalence of well-known pathogens, such as Staphylococcus aureus, giving the biofilm community the factors necessary to maintain chronic biofilm infections. Further work is definitely warranted and needed in order to prove whether the FEPs concept is a viable hypothesis. The findings here also suggest that traditional culturing methods may be extremely biased as a diagnostic tool as they select for easily cultured organisms such as Staphylococcus aureus and against difficult to culture bacteria such as anaerobes. While PCR methods also have bias, further work is now needed in comparing traditional culture results to high-resolution molecular diagnostic methods such as bTEFAP.

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.

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.

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

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