Laura Nistico

Characterization of biofilm matrix, degradation by DNase treatment and evidence of capsule downregulation in Streptococcus pneumoniae clinical isolates

BackgroundStreptococcus pneumoniae is a common respiratory pathogen and a major causative agent of respiratory infections, including otitis media (OM). Pneumococcal biofilms have been demonstrated on biopsies of the middle ear mucosa in children receiving tympanostomy tubes, supporting the hypothesis that chronic OM may involve biofilm development by pathogenic bacteria as part of the infectious process. To better understand pneumococcal biofilm formation six low-passage encapsulated nasopharyngeal isolates of S. pneumoniae were assessed over a six-eight day period in vitro.ResultsMultiparametric analysis divided the strains into two groups. Those with a high biofilm forming index (BFI) were structurally complex, exhibited greater lectin colocalization and were more resistant to azithromycin. Those with a low BFI developed less extensive biofilms and were more susceptible to azithromycin. dsDNA was present in the S. pneumoniae biofilm matrix in all strains and treatment with DNase I significantly reduced biofilm biomass. Since capsule expression has been hypothesized to be associated with decreased biofilm development, we also examined expression of cpsA, the first gene in the pneumococcal capsule operon. Interestingly, cpsA was downregulated in biofilms in both high and low BFI strains.ConclusionAll pneumococcal strains developed biofilms that exhibited extracellular dsDNA in the biofilm matrix, however strains with a high BFI correlated with greater carbohydrate-associated structural complexity and antibiotic resistance. Furthermore, all strains of S. pneumoniae showed downregulation of the cpsA gene during biofilm growth compared to planktonic culture, regardless of BFI ranking, suggesting downregulation of capsule expression occurs generally during adherent growth.

The role of biofilms in otolaryngologic infections: update 2007.

Purpose of reviewBiofilms have been shown to play a role in otitis media, sinusitis, cholesteatoma, tonsillitis, adenoiditis, and device infections. This article is written to review recent advances in the field. Recent findingsThe role of biofilms in the persistence of chronic, mucosal-based ENT-related infections was first recognized in otitis media. Definitive proof was lacking until the demonstration of bacterial biofilms on the middle-ear mucosa of children, not only with chronic otitis media with effusion, but also with recurrent otitis media. Strains of Pseudomonas aeruginosa isolated from cholesteatoma are avid biofilm formers. Biofilms have been reported in the adenoids of children with chronic rhinosinusitis, helping to explain the clinical observation that adenoidectomy can be beneficial to children with chronic otitis or chronic rhinosinusiti. Additional studies have confirmed the presence of biofilms in chronic tonsillitis. Biofilms have also been shown to be involved in infected cochlear implants and tracheotomy tubes. SummaryThe recognition that chronic otolaryngologic bacterial infections are biofilm related has been the impetus for the development of new technologies for the study of biofilms and their prevention and treatment. Understanding that chronic bacterial infections are biofilm related is fundamental to developing rationale strategies for treatment and prevention.

Direct Demonstration of Viable Staphylococcus aureus Biofilms in an Infected Total Joint Arthroplasty: A Case Report

Infection following total joint arthroplasty is difficult to diagnose and treat; a nascent body of evidence from studies of prosthetic joint infections suggests that biofilm bacteria are the underlying cause. We describe the case of a patient who had chronic recurring symptoms of infection that persisted for years following total elbow arthroplasty despite numerous medical and surgical interventions. Confocal microscopy performed on fluid, tissue, and cement at the final surgical revision demonstrated viable bacteria in biofilm aggregates. Reverse transcriptase-polymerase chain reaction analysis confirmed the presence of metabolically active Staphylococcus aureus. These observations comprise compelling evidence that viable biofilm bacteria play an important role in refractory infection following joint arthroplasty.Chronic infection following joint replacement is increasingly thought to result from the presence of bacterial biofilm communities attached to the implant. Biofilm bacteria are vastly more resistant to conventional antibiotic therapy than are their single planktonic counterparts (unattached solitary bacteria living freely) and are typically difficult to culture by conventional microbiological methods. The biofilm paradigm can explain contradictory signs and symptoms that suggest infection but are often associated with negative cultures. Moreover, biofilm infections are difficult to detect by simple Gram stain and culture techniques but can persist as a nidus of infection from which recurrent acute exacerbations may arise through episodic planktonic “showering”.

Adenoid Reservoir for Pathogenic Biofilm Bacteria

ABSTRACT Biofilms of pathogenic bacteria are present on the middle ear mucosa of children with chronic otitis media (COM) and may contribute to the persistence of pathogens and the recalcitrance of COM to antibiotic treatment. Controlled studies indicate that adenoidectomy is effective in the treatment of COM, suggesting that the adenoids may act as a reservoir for COM pathogens. To investigate the bacterial community in the adenoid, samples were obtained from 35 children undergoing adenoidectomy for chronic OM or obstructive sleep apnea. We used a novel, culture-independent molecular diagnostic methodology, followed by confocal microscopy, to investigate the in situ distribution and organization of pathogens in the adenoids to determine whether pathogenic bacteria exhibited criteria characteristic of biofilms. The Ibis T5000 Universal Biosensor System was used to interrogate the extent of the microbial diversity within adenoid biopsy specimens. Using a suite of 16 broad-range bacterial primers, we demonstrated that adenoids from both diagnostic groups were colonized with polymicrobial biofilms. Haemophilus influenzae was present in more adenoids from the COM group (P = 0.005), but there was no significant difference between the two patient groups for Streptococcus pneumoniae or Staphylococcus aureus. Fluorescence in situ hybridization, lectin binding, and the use of antibodies specific for host epithelial cells demonstrated that pathogens were aggregated, surrounded by a carbohydrate matrix, and localized on and within the epithelial cell surface, which is consistent with criteria for bacterial biofilms.

Molecular and imaging techniques for bacterial biofilms in joint arthroplasty infections

Biofilm formation on surfaces is an ancient and integral strategy for bacterial survival. Billions of years of adaptation provide microbes with the ability to colonize any surface, including those used in orthopaedic surgery. Although remarkable progress has been made in the treatment of orthopaedic diseases with implanted prostheses, infection rates remain between 1% and 2%, and are higher for revision surgeries. The chronic nature of implant infections, their nonresponsiveness to antibiotics, and their frequent culture negativity can be explained by the biofilm paradigm of infectious disease. However, the role of biofilms in orthopaedic implant infections and aseptic loosening is controversial. To address these issues, we developed molecular diagnostic and confocal imaging techniques to identify and characterize biofilms associated with infected implants. We designed PCR and reverse transcription (RT)-PCR-based assays that can be used to detect bacterial infections associated with culture-negative joint effusions that distinguish between physiologically active Staphylococcus aureus and Staphylococcus epidermidis. Using clinical isolates of Pseudomonas aeruginosa, we constructed a series of reporter strains expressing colored fluorescent proteins to observe biofilms growing on 316L stainless steel and titanium orthopaedic screws. Three-dimensional structures of Pseudomonas aeruginosa and staphylococci biofilms growing on the screws were documented using confocal microscopy. The application of these tools for clinical diagnosis and biofilm research in animal and in vitro models is discussed.

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.

Identification of adenoid biofilms with middle ear pathogens in otitis-prone children utilizing SEM and FISH

OBJECTIVES Biofilms have been implicated in the development of several chronic infections. We sought to demonstrate middle ear pathogens in adenoid biofilms using scanning electron microscopy (SEM) and fluorescent in situ hybridization (FISH) with confocal laser scanning microscopy (CLSM). METHODS Comparative micro-anatomic investigation of adenoid mucosa using SEM and FISH with confocal scanning laser microscopic (CLSM) imaging from patients with recurrent acute otitis media (RAOM). RESULTS All otitis-prone children demonstrated biofilm surface area presence greater than 85% by SEM. FISH accompanied by CLSM imaging also demonstrated patchy biofilms All biofilms contained middle ear pathogens and were frequent in polymicrobial distributions: 4 of 6, 4 of 6 and 3 of 6 samples contained Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis, respectively. CONCLUSIONS Dense adenoid biofilms may act as a reservoir for reinfection of the tubotympanum. Aspiration of planktonic middle ear pathogens existing in resistant adenoid biofilms during a viral upper respiratory tract infection may be an important event in the development of RAOM.

Chronic Surgical Site Infection Due to Suture-Associated Polymicrobial Biofilm

BACKGROUND Surgical site infection (SSI) is a common surgical complication; culture-negative SSI presents a particular problem in management. METHODS Examination of explanted foreign bodies (sutures) using confocal laser scanning microscopy (CLSM) and fluorescent in situ hybridization (FISH) after surgical exploration of a chronic culture-negative SSI. RESULTS Confocal microscopy (CM) demonstrated bacilli and cocci attached to the surface of the explanted sutures in a mixed biofilm. Fluorescent in situ hybridization confirmed that Staphylococci were components of the mixed biofilm. Removal of the foreign bodies (sutures) resolved the chronic infection. CONCLUSION Chronic SSI can arise from underlying bacterial biofilms, which can invest implanted foreign bodies and associated soft tissue surfaces.

Characterization of a mixed MRSA/MRSE biofilm in an explanted total ankle arthroplasty

Bacterial biofilms have been observed in many prosthesis-related infections, and this mode of growth renders the infection both difficult to treat and especially difficult to detect and diagnose using standard culture methods. We (1) tested a novel coupled PCR-mass spectrometric (PCR-MS) assay (the Ibis T5000) on an ankle arthroplasty that was culture negative on preoperative aspiration and then (2) confirmed that the Ibis assay had in fact detected a viable multispecies biofilm by further micrographic and molecular examinations, including confocal microscopy using Live/Dead stain, bacterial FISH, and reverse-transcriptase-PCR (RT-PCR) assay for bacterial mRNA. The Ibis technology detected Staphylococcus aureus, Staphylococcus epidermidis, and the methicillin resistance gene mecA in soft tissues associated with the explanted hardware. Viable S. aureus were confirmed using RT-PCR, and viable cocci in the biofilm configuration were detected microscopically on both tissue and hardware. Species-specific bacterial FISH confirmed a polymicrobial biofilm containing S. aureus. A novel culture method recovered S. aureus and S. epidermidis (both methicillin resistant) from the tibial metal component. These observations suggest that molecular methods, particularly the new Ibis methodology, may be a useful adjunct to routine cultures in the detection of biofilm bacteria in prosthetic joint infection.

Real-time microsensor measurement of local metabolic activities in ex vivo dental biofilms exposed to sucrose and treated with chlorhexidine.

ABSTRACT Dental biofilms are characterized by structural and functional heterogeneity. Due to bacterial metabolism, gradients develop and diverse ecological microniches exist. The aims of this study were (i) to determine the metabolic activity of microorganisms in naturally grown dental biofilms ex vivo by measuring dissolved oxygen (DO) and pH profiles with microelectrodes with high spatial resolution and (ii) to analyze the impact of an antimicrobial chlorhexidine (CHX) treatment on microbial physiology during stimulation by sucrose in real time. Biofilms were cultivated on standardized human enamel surfaces in vivo. DO and pH profiles were measured in a flow cell system in sterile human saliva, after sucrose addition (10%), again after alternative treatment of the sucrose exposed biofilms with CHX (0.2%) for 1 or 10 min or after being killed with paraformaldehyde (4%). Biofilm structure was visualized by vitality staining with confocal microscopy. With saliva as the sole nutrient source oxygen consumption was high within the superficial biofilm layers rendering deeper layers (>220 μm) anoxic. Sucrose addition induced the thickness of the anaerobic zone to increase with a concurrent decrease in pH (7.1 to 4.4). CHX exposure reduced metabolic activity and microbial viability at the biofilm surface and drove metabolic activity deeper into the biofilm. CHX treatment led to a reduced viability at the biofilm surface with minor influence on overall biofilm physiology after 1 min; even after 10 min there was measurable respiration and fermentation inside the biofilm. However, the local microenvironment was more aerated, less acidogenic, and presumably less pathogenic.